Ultra-long-acting insulin analog, preparation method therefor, and use thereof

Abstract

Provided are an ultra-long-acting insulin analog, a preparation method therefor, and a use thereof. The present invention comprises modification of an amino acid sequence of natural human insulin, and by means of dual fatty acid modification at a suitable site, prolongs the acting time of an insulin analog, thereby improving patient compliance. The present invention solves the technical problem in the prior art that the drug acting time of insulin analogs is unsatisfactory, uses a recombinant expression-based preparation method, and has a simple process route and low costs.

Description

Ultra-long-acting insulin analogue, preparation method and use thereof Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to an ultra-long-acting insulin analogue, a preparation method thereof, and uses thereof. Background Art

[0002] Insulin is a protein hormone secreted by pancreatic beta cells in the pancreas in response to endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is the only hormone in the body that lowers blood sugar and promotes the synthesis of glycogen, fat, and protein. Human insulin is composed of two peptide chains, A and B. The A chain of human insulin (Insulin Human) has 11 types of 21 amino acids, and the B chain has 15 types of 30 amino acids, for a total of 51 amino acids. Among them, A7 (Cys)-B7 (Cys), A 20 (Cys)-B 19 The sulfhydryl groups of the four cysteines (Cys) form two disulfide bonds to connect the A and B chains. In addition, A6 (Cys) in the A chain and A 11 To improve the effect and duration of insulin action, researchers have conducted extensive research on the amino acid composition and modification methods of insulin.

[0003] Insulin degludec was developed by Novo Nordisk and launched in Japan in October 2012 for the treatment of type 1 and type 2 diabetes. Insulin degludec is based on human insulin with the B 30 Threonine at position B is linked to a 16-carbon fatty acid via an L-γ-glutamic acid linker. 29 An ultra-long-acting basal insulin analogue obtained by modifying the amino acid residue at the lysine residue at the 2nd position of the basal lysine residue. This unique molecular structure allows it to exist in the formulation as a stable, soluble, double hexamer prior to injection. Insulin degludec has a limited duration of action in vivo and requires relatively frequent dosing, which may lead to side effects and reduce patient compliance, failing to meet the needs of insulin clinical applications. Further research into the structure and modification of insulin analogues is urgently needed to develop insulin analogues with longer durations of action and less frequent dosing. Summary of the Invention

[0004] The present invention aims to provide an ultra-long-acting insulin analogue to solve the technical problem of unsatisfactory drug action time of insulin analogues in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] An ultra-long-acting insulin analog comprising an A chain: GIVEQCCTSICSLX1QLENYCX2X3X4, and a B chain: FVX5QHLCGSHLVEALX6LVCGERGFX7X8X9X 10 X 11 X 12 ;

[0007] Wherein, X1 is Y or E or D; X2 is G or N or K; X3 is K or does not exist; X4 is A or G or does not exist;

[0008] X5 is N or D; X6 is Y or H or E; X7 is F or H or I; X8 is K or Y; X9 is T or G or does not exist; 10 P or G or K or not present; X 11 K or P or not present; X 12 is T or does not exist;

[0009] In X8 or X 10 or X 11 The amino acid residues are modified with fatty acids.

[0010] This technical solution also provides a method for preparing an ultra-long-acting insulin analog, comprising the following steps performed in sequence:

[0011] S1: Obtain insulin analog A precursor;

[0012] S2: Fatty acid modification of insulin analogs.

[0013] This technical solution also provides an application of an ultra-long-acting insulin analog in the preparation of a drug for treating diabetes or a drug for lowering blood sugar.

[0014] Furthermore, the amino acid residue of X3 is also modified with a fatty acid.

[0015] Furthermore, the fatty acid includes a C 18 Modifier, structural formula is shown in formula (3) 20 Modifier; the structural formula is shown in formula (4) C 22 modifiers;

[0016] Formula (2)

[0017] Formula (3)

[0018] Formula (4).

[0019] Furthermore, X1 is E; X2 is N or K; X3 is K or absent; X4 is absent; X5 is N; X6 is H; X7 is H; X8 is Y; X9 is T or G; X 10 P or G; X 11 K; X 12 Does not exist; in X 11 The amino acid residue of X2 or X3 is modified with a fatty acid.

[0020] Furthermore, 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.

[0021] Furthermore, the A chain and the B chain are linked by a disulfide bond.

[0022] Furthermore, in S1, recombinant expression is performed using Escherichia coli or yeast to obtain the insulin analog A precursor.

[0023] This technical solution can be obtained through expression in engineered bacteria (E. coli and yeast), in addition to chemical synthesis. The above-mentioned insulin analogs are obtained by conventional means in the prior art, and can be synthesized / expressed in-house or commissioned to a biotechnology company.

[0024] Furthermore, the molar ratio of the modifier to the insulin analog is 3:1-6:1.

[0025] The process of fatty acid modification is as follows: dissolving the modifier in N-methylpyrrolidone to obtain a modifier solution; dissolving the insulin analog in sodium carbonate to obtain an insulin analog solution; and reacting and purifying the insulin analog solution and the modifier solution to obtain a fatty acid-modified insulin analog.

[0026] In summary, the technical principles and beneficial effects of this technical solution are:

[0027] This technical solution uses a dual-modified structure to extend the duration of action of insulin analogs and improve patient compliance. The dual-modified structure is effective and has a prolonged effect, and its higher stability makes the compound highly promising for oral formulation. The insulin of this solution can be expressed using recombinant methods, which has a simple process route, low cost, and certain economic advantages. Through appropriate insulin amino acid design and corresponding fatty acid modification, this technical solution greatly improves the duration of the drug's blood sugar-lowering effect in vivo.

[0028] More specifically, the present technical solution is in the A of insulin 22 A new lysine (K) is introduced at position A 21The amino acid residue at position A1 is replaced with lysine (K), and the above site is used to connect the fatty acid modifier to achieve fatty acid modification of the A chain. Compared with the conventional modification of the G residue at the A1 site, the operation mode of this technical solution (A 21 A 22 This technical solution can also be used in the B of insulin 26 position (need to replace the amino acid residue (Y) with lysine (K)), B 28 position (need to replace amino acid residue (P) with lysine (K)), B 29 The fatty acid modification of the A chain and the B chain can be carried out at the same time (A chain and B chain can be modified with fatty acids). 21 A 22 Choose one of the fatty acid modifications at position B; and 26 Position, B 28 Position, B 29 Choose one of the two fatty acid modifications), and increase the duration of drug action in vivo through double modification. 27 The threonine (T) at position B was replaced by glycine (G). 28 The proline (P) at the position is replaced by glycine (G). The changes in the amino acid residues of the above peptide chain can further increase the duration of drug action in vivo based on fatty acid modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 shows the mass spectrometry results of analogue 2 of Example 4.

[0030] FIG2 shows the mass spectrometry results of analog 5 of Example 4.

[0031] FIG3 shows the mass spectrometry detection results of analog 6 of Example 4.

[0032] FIG4 shows the mass spectrometry detection results of analog 10 of Example 4.

[0033] FIG5 shows the mass spectrometry results of analog 11 of Example 4. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, 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, etc. used are all commercially available.

[0035] The structure of the ultra-long-acting insulin analog in this scheme is shown in formula (1):

[0036] S formula (1)

[0037] In formula (1), code X1-X 12 See Table 1 for the amino acids represented. A represents the insulin A chain, and B represents the insulin B chain. The amino acid sequence of the A chain 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 and B chains are connected by a disulfide bond, a common connection between insulin subunits, and will not be detailed here.

[0038] The sequence of human insulin is as follows: the amino acid sequence of the A chain is shown in SEQ ID NO. 3: GIVEQCCTSICSLYQLENYCN; the amino acid sequence of the B chain is shown in SEQ ID NO. 4: FVNQHLCGSHLVEALYLVCGERGFFYTPKT.

[0039] Table 1: Composition of ultra-long-acting insulin analogs

[0040] Site code amino acid of natural human insulin amino acid A of ultra-long-acting insulin analog 14 X1YY or E or DA 21 X2NG or N or KA 22 X3 does not have K or A 23 X4 does not exist A or G or does not exist B3X5NN or DB 16 X6YY or H or EB 25 X7FF or H or IB 26 X8YK or YB 27 X9TT or G or no B 28 X 10 PP or G or K or no B 29 X 11 KK or P or no B 30 X 12 TT or does not exist

[0041] The double modification sites are the K residues at the ends of chain A, X2 and X3, and the residues at the ends of chain B, X8 and X 10 、X 11 K residue, the modifier can be selected C 18 、C 20 、C 22 Equivalent fatty acid modification, that is, fatty acid modification at two sites on the insulin A chain and B chain. 18 、C 20 、C 22 The structural formulas of the fatty acid modifiers are shown in Formula (2), Formula (3) and Formula (4), respectively.

[0042] Formula (2)

[0043] Formula (3)

[0044] Formula (4)

[0045] Example 1: Hypoglycemic Effect in Normal Mice

[0046] The insulin analogs shown in Tables 2 and 3 were tested for the duration of their hypoglycemic effect in normal mice after a single dose. The testing process was as follows: SPF-grade KM mice, 10 weeks old and weighing approximately 35g, were used and given ample food and water for free access. After one week of acclimatization, the experiment began. The mice were randomly divided into a saline group and a drug test group, with 6 mice in each group. All mice were injected at a dose of 25U / kg. The saline group was also given 0.1ml / mouse as a control. At different times after administration, blood was collected from the tails of the mice and blood glucose was measured using a Johnson & Johnson blood glucose meter. By comparing blood glucose values ​​(comparison of blood glucose values ​​in the experimental group and the control group), the duration of the hypoglycemic effect of the insulin analogs in mice was determined. The test results are shown in Table 3.

[0047] Table 2: Base composition of insulin analogs to be tested (the sequence of the analogs refers to formula (1), and the specific amino acid residue selection refers to this table; " / " indicates the absence of an amino acid residue)

[0048] Site A 14 A 21 A 22 A 23 B3B 16 B 25 B 26 B 27 B 28 B 29 B 30 Code X1X2X3X4X5X6X7X8X9X 10 X 11 X 12Human insulin YN / / NYFYTPKT analogue 1EN / / NHHYTPK / analogue 2EN / / NHHYTPK / analogue 3ENK / NHHYTPK / analogue 4ENK / NHHYTPK / analogue 5ENK / NHHYTPK / analogue 6ENK / NHHYTPK / analogue 7ENK / NHHYTPK / analogue 8ENK / NHHYTPK / analogue 9ENK / NHHYGGK / analogue 10ENK / NHHYGGK / analogue 11ENK / NHHYGGK / analogue 12ENK / NHHYGGK / analogue 13EN / / NHHYTPK / analogue 14ENK / NHHYTPK /

[0049] Table 3: Fatty acid modification, dosage, and in vivo activity of the insulin analogs tested (see Table 2 for amino acid composition of the analogs; “ / ” indicates no modification site selected or fatty acid modification performed)

[0050] surface

[0051] From Table 2 and Table 3, we can see that analogue 1 (B 29 Single modification), analogue 2 (A1, B 29 The amino acid sequences of analog 13 (the unmodified precursor) are identical: the A chain sequence 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 analog 2 was fatty acid-modified at this amino residue.

[0052] Analogs 3-8 and 14 share the same amino acid sequence. Their A-chain sequences are shown in SEQ ID NO. 7: GIVEQCCTSICSLEQLENYCNK; their B-chain sequences are shown in SEQ ID NO. 6: FVNQHLCGSHLVEALHLVCGERGFHYTPK. In terms of amino acid sequence, analogs 3-8 and 14, compared to analog 1 (analogs 2 and 13), have a newly introduced lysine (K) at position A22. In analog 14, "no Glu" means that the fatty acid chain does not contain L-α-glutamic acid (Glu). The remaining fatty acid chains all contain L-α-glutamic acid (Glu). There are no significant differences in the synthetic processes. The specific structure of the fatty acid used for modification is shown in Formula (5).

[0053] Formula (5)

[0054] The amino acid sequences of analogs 9-12 are consistent, and their A chain sequence is shown in SEQ ID NO.7: GIVEQCCTSICSLEQLENYCNK; their B chain sequence is shown in SEQ ID NO.8: FVNQHLCGSHLVEALHLVCGERGFHYGGK. In terms of amino acid sequence, analogs 9-12 have three amino acid substitutions compared to analog 1 (analog 2, analog 13): A 22 Lysine (K), B 27 The threonine (T) at position B was replaced by glycine (G). 28 The proline (P) at position A was replaced by glycine (G).

[0055] The results of the in vivo activity test of insulin analogs were analyzed as follows:

[0056] (1) Insulin analogs (i.e., precursors) that are not modified with fatty acids have a short duration of glucose lowering in vivo

[0057] Among them, analogues 8 (A chain SEQ ID NO. 7, B chain SEQ ID NO. 6), 9 (A chain SEQ ID NO. 7, B chain SEQ ID NO. 8), and 13 (A chain SEQ ID NO. 5, B chain SEQ ID NO. 6) are all unmodified insulin analogues. At the same dose (25 U / kg), the three insulin analogues showed essentially identical in vivo activity in mice (2 hours), suggesting that the glucose-lowering duration of the precursor insulin analogues is short and requires fatty acid modification to extend this duration.

[0058] (2) Fatty acid modification on the K residues of the A and B chains can shorten the duration of the drug's blood sugar-lowering effect in vivo.

[0059] In order to increase the duration of insulin analog activity, the insulin represented by analog 1 attempted to add a C to the K residue on the B chain of analog 13. 20 Modification. This modification plays a very critical role in improving the duration of blood sugar reduction. The inventors then studied other fatty acid modification sites, and first tried to modify the A1 of the A chain SEQ ID NO.5 with fatty acids, which can further improve the duration of blood sugar reduction in the body of the drug, from 40h to 48h relative to analog 1. If the amino acid residue K is added to the end of the A chain SEQ ID NO.5 to form a new A chain sequence SEQ ID NO.7, fatty acid modification is performed on the residue K. A class of insulin analogs with A chain SEQ ID NO.7 + B chain SEQ ID NO.6 were studied (i.e., analogs 3-8, analog 14). Comparing analog 2 and analog 6, the difference between the two is that A22 C of K residue at position 20 Modification of the C residue and the G residue at position A1 20 The latter can increase the blood sugar lowering effect of the drug by 8 hours compared with the former. 22 C of K residue at position 20 Modification can effectively enhance the activity of drugs in mice and prolong the duration of activity. 22 The inventors first attempted to replace the site with a K residue and modify the residue with fatty acids, which achieved significant results in increasing the duration of drug action.

[0060] (3) The type of fatty acid modification has a significant effect on the duration of the drug's blood sugar-lowering effect in vivo

[0061] Analog 14 uses C 20 The modification only means that the fatty acid chain does not contain L-α-glutamic acid (Glu). Compared with analog 6, the C 20 The hypoglycemic effect of analogue 14 in mice was maintained for only 25 hours, which was more than twice the 56 hours of analogue 6.

[0062] The carbon chain length of the fatty acid also has a significant impact on the duration of the drug's hypoglycemic effect in vivo. Generally speaking, the larger the molecular weight of the modified molecule, the longer the drug's circulation time in the body, thereby extending the duration of the drug's action. However, in this technical solution, for the analogues of A chain SEQ ID NO.7 and B chain SEQ ID NO.6, C 20 (Analog 6) can significantly increase the duration of blood sugar reduction in vivo (56h), while C 22 (analog 7) and C 18 (Analog 5) modification can only achieve 35h of in vivo hypoglycemic maintenance time. Only two carbon changes can bring about a 60% improvement in effect, which was not predicted by the inventors before the experiment. For the analogs of A chain SEQ ID NO.7 and B chain SEQ ID NO.8, C 20 (Analog 10) modification can make the analogue's hypoglycemic duration in vivo reach 80h, while C 22 The duration of glucose reduction in vivo by analogue 11 was only 40 hours. The change of only two carbon atoms could bring about a 50% improvement in effect, which was not predicted by the inventors before the experiment. The experimental data showed that after screening a variety of fatty acid modifiers, it was found that C 20 The modification effect of modification (containing Glu) is the most ideal.

[0063] (4) The amino acid sequence of the B chain has a significant effect on the duration of glucose lowering in vivo after fatty acid modification

[0064] The inventors made adjustments to the B chain and obtained an analogue of the A chain SEQ ID NO.7 + the B chain SEQ ID NO.8, and attempted to modify the fatty acid. That is, the B chain was based on SEQ ID NO.6, but the two amino acid residues TP were replaced by GG. 20 Under the same C 22 After modification, analogue 7 (amino acid residue TP) maintained blood sugar reduction for 35 hours in vivo; analogue 11 (amino acid residue GG) maintained blood sugar reduction for 40 hours in vivo. Comparing analogue 10 with analogue 6, it can be seen that the replacement of two amino acid residues greatly improved the blood sugar reduction time in vivo, by 24 hours, with an improvement rate of 43%, which was not expected by the inventors before the experiment. Comparing analogue 11 with analogue 7, for C 22 Modification, the amino acid sequence of A chain SEQ ID NO.7 + B chain SEQ ID NO.8 is more advantageous, although the replacement of two amino acid residues did not produce a better result than C 20 The modification showed a more significant change, but the duration of blood sugar reduction in the body was increased by 5 hours, with an improvement rate of 14%. 29 Compared with analogue 4 (amino acid residue TP), analogue 12 (amino acid residue GG) showed a longer duration of drug action, which was increased by 8 hours, with an improvement rate of 20%. It can be seen that the analogues of A chain SEQ ID NO.7 + B chain SEQ ID NO.6 and A chain SEQ ID NO.7 + B chain SEQ ID NO.8 only differ in the two amino acids at the end of the B chain, but the effects of the two types of analogues after fatty acid modification are very different, especially for C. 20 Double modification of fatty acids, B chain SEQ ID NO.8 achieved unexpected technical effects.

[0065] In summary, analogues 9-12 have three amino acid substitutions compared with analogue 1 (analogue 2, analogue 13): A 22 Lysine (K), B 27 The threonine (T) at position B was replaced by glycine (G). 28 The proline (P) at position B is replaced by glycine (G). 27 、B 28 After the amino acid replacement, B 29 The activity time of the analogues with single modification of the site was significantly prolonged, and 22 and B 29The activity time of the double-modified analogues was significantly prolonged. 22 Lysine (K) at the site can provide a new modification site, A 22 and B 29 The double modification activity time of the site is longer than that of A1 and B 29 Double modification of the site.

[0066] In addition to the above sequences (analogs 9-12), the A chain terminal X2 (A 21 )N residue is replaced by K residue, and the end of B chain X8 (B 26 ) Y residue is replaced by K residue, and the terminal X of chain B is replaced by 10 (B 28 ) P residues were replaced with K residues, thereby introducing a dual modification site (K residue serves as the attachment site for the fatty acid modifier), forming new insulin sequences. Dual fatty acid modification based on these new insulin sequences also yielded effects similar to those of analogue 10.

[0067] Example 2: Recombinant expression of insulin analogs in Escherichia coli

[0068] Based on the amino acid sequence of insulin analog A (see Table 1 and Table 2 for amino acid sequences of insulin analogs containing both A and B chains), a cDNA sequence was designed according to the codon preference of Escherichia coli. The entire gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized cDNA fragment (inserted into the pET-26b(+) plasmid) was digested with Nde I and Hind III and recovered. The recombinant fusion expression pET-26b(+) plasmid was also digested with Nde I and Hind III and the large fragment was recovered. The insulin analog A gene fragment and pET-26b(+) were ligated with T4 ligase and transformed into the TOP10 strain. The recombinant plasmid containing the insulin analog A gene was selected by plate selection and named pET-26b-A. The recombinant expression strain was then transformed into the expression host strain BL21(DE3) using the CaCl2 method. The recombinant expression strain was then induced with 1 mmol / L IPTG to produce the insulin analog A fusion protein. The fusion protein is recovered from inclusion bodies, renatured, and subjected to ion exchange chromatography followed by enzymatic digestion. Finally, it is purified by reverse-phase chromatography and lyophilized to obtain insulin analog A precursor. The preparation process for insulin analog A precursor (i.e., the unmodified insulin analog) is as follows: After recombinant expression in E. coli, the cells are collected by high-speed centrifugation, suspended in water, disrupted at 60 MPa, and centrifuged to obtain insulin analog A inclusion bodies. The insulin analog inclusion bodies were dissolved in a dissolving solution (8 mol / L urea, 20 mmol / L β-mercaptoethanol, 20 mol / L Tris, pH 10.0) at a mass-to-volume ratio of 1:10, stirred until the precipitate was completely dissolved, and then centrifuged to obtain the insulin analog A fusion protein solution. The solution was diluted with 2-8°C pre-chilled water at a volume ratio of 1:10, allowed to stand at 2-8°C for renaturation for 40 hours, adjusted to pH 5.0-6.0 with dilute hydrochloric acid, allowed to stand for 2 hours, and centrifuged to obtain the insulin analog A fusion protein. The solution was dissolved in 50 mmol / L Tris, pH 9.0, and digested at 25°C for 8 hours. The insulin analog A precursor was obtained by gradient elution using a C8 reverse-phase chromatography column with mobile phase A: 20% acetonitrile, 0.1% TFA and mobile phase B: 60% acetonitrile.

[0069] Example 3: Recombinant expression of insulin analogs in Pichia pastoris

[0070] Based on the amino acid sequence of insulin analog A and the preferred codons of Pichia pastoris, a cDNA sequence was designed. The entire gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The synthesized cDNA fragment (inserted into the pPICZαA plasmid) was digested with Not I and Xho I and recovered. The recombinant fusion expression plasmid pPICZαA was also digested with the same enzymes to recover the larger fragment. The insulin analog A gene fragment and pPICZαA were ligated with T4 ligase and transformed into the TOP10 strain. The recombinant plasmid containing the insulin analog A gene was selected by plate selection and named pPICZαA-A. The recombinant expression strain was then transformed into the expression host strain X-33 by electroporation. The strain was then induced to express the insulin analog A fusion protein using a methanol-glycerol mixture. The fusion protein was purified by ion exchange chromatography, followed by enzyme digestion, and finally by reverse-phase chromatography to obtain the recombinant insulin analog A, which was lyophilized into a dry powder. After recombinant expression in Pichia pastoris, the supernatant was obtained by high-speed centrifugation and ion-purified using cation exchange chromatography with mobile phase A: 20 mmol / L sodium acetate, pH 3.0 and mobile phase B: 20 ​​mmol / L sodium acetate, 0.5 M NaCl, pH 3.0 to obtain the insulin analog A fusion protein. Enzymatic digestion was performed at 25°C, pH 8.0 for 8 hours, and gradient elution was performed using a C8 reverse-phase column with mobile phase A: 20% acetonitrile, 0.1% TFA and mobile phase B: 60% acetonitrile to obtain the insulin analog A precursor. This example describes a general method for obtaining the insulin analog A precursor. The obtained precursor was then subjected to subsequent modification.

[0071] Example 4: Modification of insulin analog precursors by modifiers

[0072] Single-modified and double-modified samples are mainly obtained by controlling the modification ratio, which is a conventional method in the prior art. Under the condition of pH 9.5-11.5, the modifier will preferentially modify A 22 and B 29 At the modification ratio of 1:4 (insulin analog precursor: modifier), A 22 With B 29 The double-modified samples were recovered by reverse-phase chromatography. To obtain the A22 monomodified analog (analog 3), the insulin analog precursor:modifier molar ratio was controlled between 1:1.5 and 1:2.5. To obtain the B29 monomodified analogs (analogs 4 and 12), the insulin analog precursor:modifier ratio was controlled between 1:1.5 and 1:2.5. The target samples were then separated by reverse-phase chromatography.

[0073] The modifier (C 18 、C 20 or C 22) was dissolved in N-methylpyrrolidone at 100 mg / ml. A certain amount of insulin analog precursor was weighed and dissolved in 0.1 mol / L sodium carbonate to 10 mg / ml. The pH was controlled at 10.5-11.5. Under stirring at room temperature, the molar ratio of modifier to insulin analog precursor was 4:1-6:1. The reaction was continued for 0.5 hours, and the pH was adjusted to 7.5 with acetic acid. After completion of the reaction, the insulin analog was purified by reverse phase chromatography, freeze-dried, and analyzed by LC-MS to obtain molecular weight information. 18 、C 20 or C 22 For the synthesis method of analogs 1-14, refer to our prior patent CN116655770A (Fatty acid-modified thrombopoietin mimetic peptide homotetramers, preparation methods, and applications thereof). The molecular weights of analogs 1-14 synthesized in this protocol are shown in Table 4, and the mass spectrometry results of analogs 2, 5, 6, 10, and 11 are shown in Figures 1-5.

[0074] Table 4: Molecular weight information of analogs 1-14

[0075] surface

[0076] Example 5: Blood sugar reduction experiment in type 1 diabetes mouse model

[0077] (1) Mouse modeling

[0078] A type Ⅰ diabetes model was established in mice by intraperitoneal injection of streptozotocin (STZ), and the blood glucose level was measured to be above 16.8 mmol / L, thus establishing the model.

[0079] (2) Dosing in mice

[0080] The mice were divided into three groups: the model group, analogue 1, and analogue 6. Normal mice served as blank controls. The normal and model groups were given PBS six times, with 0.2 ml of PBS used each time. Analogue 1 was administered once every 36 hours; analogue 6 was administered once every 36 hours, with a dose of 25 U / kg for both groups. After six consecutive administrations, blood glucose levels were measured in each group of mice at 0, 12, 24, 36, 48, 72, 78, 84, and 96 hours after administration, and the blood glucose levels were measured until the blood glucose levels in the treatment group returned to pre-dose levels. Experimental data are shown in Table 5. With analogue 1, blood glucose levels returned to pre-dose levels at 72 hours. However, with analogue 6, blood glucose levels remained below pre-dose levels at 96 hours after administration, indicating that analogue 6 has a longer duration of action and a better effect.

[0081] Table 5: Blood glucose test results (mmol / L)

[0082] Group 0h12h24h36h48h72h78h84h96h Model group 22.923.724.825.123.124.322.622.622.6 Blank group 8.48.78.39.78.58.38.18.18.1 Analogs 112.02.21.93.217.825.2 --- Analogs 66.99.73.26.24.33.56.79.110.5

[0083] Example 6: Blood sugar reduction experiment in rat type 1 diabetes model

[0084] After one week of acclimatization, the experimental animals were allowed free access to food and water. After passing all tests, the rats were fasted for 12 hours, followed by a tail vein injection of 55 mg / kg of 1% streptozocin (STZ). STZ was dissolved in citrate buffer (pH 4.5) at a concentration of 1%, and the injection was completed within 30 minutes based on the fasting body weight. Model establishment criteria: Fasting blood glucose was measured three days after STZ administration (after a 6-hour fast, with no water restriction) once daily. A fasting blood glucose ≥16.8 mmol / L for three consecutive days was considered a successful type 1 diabetes model.

[0085] The animals were randomly divided into a vehicle control group, a positive control group, and different test drug groups according to the principle of stratification based on fasting blood glucose and body weight, with 9-10 animals in each group. Drugs were administered via subcutaneous injection at a volume of 1 mL / kg, with the day of administration designated as Day 0. The dosing period was 6 weeks.

[0086] Diabetic rats were given multiple subcutaneous injections of peptides at 1.8 mg / kg (analog 10) and 2 mg / kg (analog 6). LC-MS was used to measure plasma drug concentrations at 3, 12, 24, 36, 48, 60, 72, 96, 120, 144, 168, 192, and 216 hours after the final dose. The results of the plasma concentrations of the parent drug at different times are shown in Table 6. The experimental data are expressed as mean ± standard deviation (mean ± SD) and analyzed using GraphPad Prism 8.3 software. The parameters in Table 6 are as follows:

[0087] t 1 / 2 Half-life: refers to the time required for the concentration of a drug in plasma to drop to half of its initial concentration. It is an important indicator for measuring the rate at which a drug is cleared from the body.

[0088] T max Peak time: refers to the time when the drug reaches its maximum concentration in the blood after administration (C max This can be used to assess how quickly a drug is absorbed into the bloodstream. maxPeak concentration: The maximum concentration of a drug in the blood. It reflects the highest exposure level of the drug in the body and is very important for evaluating the safety and efficacy of the drug.

[0089] AUC 0-216h Area under the curve (AUC) from 0 to 216 hours: This value is used to estimate the total exposure to the drug during this period.

[0090] AUC 0-∞ (Area under the curve from zero to infinity): Same as AUC 0-216h Similar to the dapoxetine, but considers the total drug exposure from the time of administration until theoretically complete elimination of the drug. It provides a more comprehensive picture of overall drug exposure.

[0091] MRT 0-216h Average residence time (0 to 216 hours): This refers to the average time a drug molecule remains in the body from the time of administration until 216 hours. It is a measure of how long a drug remains in the body.

[0092] MRT 0-∞ (mean residence time from zero to infinity): similar to MRT 0-216h , but covers the entire timeframe a drug is present until it is completely eliminated from the body. It provides a more complete view of how long a drug remains in the body.

[0093] Table 6: Summary and comparison of pharmacokinetic parameters after multiple administration in diabetic rats

[0094] Parameter Unit Analog 10 (1.8 mg / kg) Analog 6 (2 mg / kg) t 1 / 2 h40±14.932±12.5T max h12±15.218±13.9C max ng / mL15200±669017400±9050AUC 0-216h h·ng / mL1070000±5880001300000±979000AUC 0-∞ h·ng / mL1110000±6220001350000±1050000MRT 0-216h h53.7±14.253.2±14.2MRT 0-∞ h60.1±20.457.7±19.1

[0095] Example 7: Insulin analog affinity experiment

[0096] The affinity of the insulin derivatives of the present invention for the human insulin receptor (IR) and insulin-like growth factor receptor (IGF-1R) was determined using SPR (surface plasmon resonance). This experiment employed SPR to determine sample affinity. The analysis software offers two modes: "Steady state affinity" and "1:1 binding." The specific analysis mode used is determined by the sample's dissociation pattern. The insulin derivatives were immobilized on the chip: the receptor was diluted to 25 μg / mL with acetate (pH 4.0), 180 μL was selected, and amino coupling was performed on channel 2 of a single-channel chip. Kinetic parameters were then set: the receptor (IR / IGF-1R) was adjusted to 250 nM using 1× PBS buffer (pH 6), then diluted to 125, 62.5, 31.25, 15.625, 7.8125, and 3.90625 nM, respectively. The flow rate was set to 20 μL / min, the association time to 60 s, the dissociation time to 90 s, and the reaction temperature to the default of 25°C. The assay was then run on a cytometer. Binding and dissociation analysis was performed using the "Steady state affinity" and / or "1:1 binding" analysis modes, and KD values ​​were calculated. In this example, affinity experiments were conducted for commercially available insulin, analogs 1, 6, and 10. Detailed experimental results are shown in Tables 7 and 8. The experimental results show that the affinity constant for the insulin receptor is 336 nM, and for each insulin analog ranges from 52.3 to 213 nM. The affinity constant between insulin-like growth factor receptor and insulin is 1.43 μM, and that of various insulin analogs is between 19.3-39.1 nM.

[0097] Table 7: Affinity constants of insulin receptor and various insulin analogs

[0098] Peptide KaKdKAKD Insulin / / / 3.36E-07 Analog 16.45E+031.38E-034.67E+062.13E-07 Analog 61.96E+042.63E-037.45E+061.34E-07 Analog 101.58E+048.26E-041.91E+075.23E-08

[0099] Table 8: Affinity constants of insulin-like growth factor receptors and various insulin analogs

[0100] Peptide KaKdKAKD Insulin / / / 1.43E-06 Analog 11.37E+054.98E-032.75E+073.63E-08 Analog 65.79E+041.12E-035.17E+071.93E-08 Analog 103.35E+041.31E-032.56E+073.91E-08

[0101] Example 8: In vitro activity test of insulin analogs

[0102] Stable cell lines and luciferase detection kits are used to detect cell activity in vitro. Early test experiments have shown that after the human insulin receptor (INSR) expressed on the surface of the cell line binds to insulin or its analogs, it will rapidly cause receptor activation and continue to activate other downstream signaling pathway elements, which can cause the transcription and translation of the luciferase reporter gene stably inserted on the chromosome. After lysing the cells, the luciferase detection reagent is added, and a significant chemiluminescent signal can be detected. The detected signal intensity is closely dose-dependent with the biological activity of the added insulin sample. On this cell detection platform, an in vitro pharmacodynamic curve of insulin can be obtained, which enables the comparison of the relative biological activity (potency) of the test sample and the standard.

[0103] HEK293 / Luc / INSR cells were cultured in DMEM + 10% FBS and maintained at 37°C in a humidified incubator with 5% CO2. Cells were passaged by trypsinization. Cell confluence reached 80%-90% and viability was above 90%, ready for plating. Cell density was adjusted to 5 × 10 cells / well using DMEM + 10% FBS. 5 cells / mL, add 20μL / well to a 384-well plate, i.e. 10,000 cells / well. Add 50μL / well PBS solution to the wells around the cells, and then place the cell plate in a 37°C, 5% CO2 incubator for overnight culture. Remove the cell plate from the incubator, add 20μL / well of sample working solution, and set up 3 replicates for each concentration. Preparation of working solution: Take out the aliquoted standard stock solution from the -20°C refrigerator and equilibrate to room temperature. In a 96-well U-bottom plate, dilute the standard stock solution to 44μM with (DMEM+10%FBS) culture medium, and perform 2-fold dilutions continuously to obtain a series of solutions with a total of 10 concentrations. Place the cell plate back into the incubator and continue culturing for 6 hours. Export the raw data in Excel format from PHERAstar, open Prism GraphPad, calculate the logarithm of the standard concentration, use the logarithm as the horizontal axis and RLU as the vertical axis, perform a four-parameter fitting to obtain the EC of the standard. 50 EC values ​​for insulin, analog 6, and analog 1 50The experimental results of the values ​​are detailed in Table 7. Specifically, the EC values ​​of human insulin, analog 6 and analog 1 are 50 The values ​​are 3.047×10 −9 , 1.261e×10 −6 , 1.099×10 −6 M. Human insulin refers to a commercial batch of human insulin (Novolin R, JVGT586).

[0104] Table 9: EC for insulin, analog 6 and analog 1 50 Test results of the value

[0105] surface

[0106] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. An ultra-long-acting insulin analog, characterized in that: Includes A chain: GIVEQCCTSICSLX1QLENYCX2X3X4, and B chain; FVX5QHLCGSHLVEALX6LVCGERGFX7X8X9X 10 X 11 X 12 ; Wherein, X1 is Y or E or D; X2 is G or N or K; X3 is K or does not exist; X4 is A or G or does not exist; X5 is N or D; X6 is Y or H or E; X7 is F or H or I; X8 is K or Y; X9 is T or G or does not exist; 10 P or G or K or not present; X 11 K or P or not present; X 12 is T or does not exist; In X8 or X 10 or X 11 The amino acid residues are modified with fatty acids.

2. The ultra-long-acting insulin analog according to claim 1, characterized in that The amino acid residues of X2 or X3 are also modified with fatty acids.

3. The ultra long-acting insulin analog according to claim 2, wherein The fatty acid includes a structural formula as shown in formula (2): 18 Modifier, structural formula is shown in formula (3) C 20 Modifier; the structural formula is shown in formula (4) C 22 modifiers; Formula (2) Formula (3) Formula (4).

4. The ultra long-acting insulin analog according to claim 3, wherein X1 is E; X2 is N or K; X3 is K or not present; X4 is not present; X5 is N; X6 is H; X7 is H; X8 is Y; X9 is T or G; X 10 P or G; X 11 K; X 12 Does not exist; in X 11 The amino acid residue of X2 or X3 is modified with a fatty acid.

5. The ultra-long-acting insulin analog according to claim 4, characterized in that: 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 analog according to claim 5, wherein Chain A and chain B are linked by disulfide bonds.

7. The method for preparing an ultra-long-acting insulin analog according to any one of claims 1 to 6, characterized in that: The method includes the following steps: S1: Obtain insulin analog A precursor; S2: Fatty acid modification of insulin analogs.

8. The method for preparing an ultra-long-acting insulin analog according to claim 7, wherein: In S1, recombinant expression is performed using Escherichia coli or yeast to obtain the insulin analog A precursor.

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

10. Use of an ultra-long-acting insulin analog according to any one of claims 1 to 6 in the preparation of a drug for treating diabetes or a drug for lowering blood sugar.

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