Long-acting acylated insulin derivative and application thereof

Abstract

The invention relates to the technical field of insulin derivatives, in particular to a long-acting acylated insulin derivative and application thereof. The long-acting acylated insulin derivative is formed by acylating and connecting a fatty acid side chain and an insulin peptide chain, and the fatty acid side chain is acylated and connected with epsilon amino of amino acid K on the insulin peptide chain. The insulin derivative provided by the invention has excellent hypoglycemic ability, the action time is remarkably prolonged, the insulin derivative can be administrated once a week, the compliance and sugar control willingness of a patient can be remarkably improved, and the insulin derivative has a wide market prospect.

Description

[0001] The present application claims priority to the Chinese patent application No. 202410762875.9, filed on June 13, 2024, and entitled "Long-acting acylated insulin derivative and application thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the technical field of insulin derivatives, in particular to a long-acting acylated insulin derivative and application thereof, and more particularly to a long-acting acylated insulin derivative and a preparation method thereof, an injection liquid preparation and a pharmaceutical composition containing the same. BACKGROUND

[0003] Diabetes mellitus is a group of metabolic disorders characterized by high blood sugar, which is caused by absolute or relative deficiency of insulin secretion and / or insulin utilization disorder, and can be caused by genetic and environmental factors. Diabetes mellitus is one of the three major causes of death in human beings, and its mortality rate is only next to cardiovascular and cerebrovascular diseases and cancer. Diabetes mellitus is mainly divided into type 1 diabetes and type 2 diabetes, and most of the patients are type 2 diabetes (accounting for about 90% according to statistics). The characteristics of type 2 diabetes mellitus (T2DM) patients are high blood sugar, relative lack of insulin, insulin resistance, etc. At present, the drugs used in clinical treatment of type 2 diabetes mellitus mainly include biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors and GLP-1 derivatives, etc.

[0004] Insulin is the only hormone in the body that can lower blood sugar, and it also promotes the synthesis of glycogen, fat and protein. Exogenous insulin and insulin derivatives are mainly used to treat diabetes mellitus. Human insulin (Insulin Human) A chain has 11 21 amino acids, and B chain has 15 30 amino acids, a total of 51 amino acids; among them, the sulfhydryl groups in the four cysteines of A7(Cys)-B7(Cys) and A20(Cys)-B19(Cys) form two disulfide bonds to connect the A and B peptide chains, in addition, there is also a disulfide bond between A6(Cys) and A11(Cys) in the A chain. Insulin is secreted by the islet beta cells in the pancreas in response to endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. The biological action of insulin at the cellular level is initiated by binding to specific receptors on the target cell membrane; the insulin receptor is a specific site on the target cell membrane for insulin to act, and can only bind to insulin or proinsulin containing insulin molecules, and has high specificity.

[0005] Although insulin is the most effective means of treating diabetes, the insulin products currently on the market are used once a day at most, so patients need to inject frequently to meet the body's demand for insulin, but frequent injections can bring great pain to patients, so it is hoped that the hypoglycemic effect of insulin can be prolonged to reduce the number of injections, reduce the pain of patients and improve patient compliance.

[0006] Icodec insulin is a long-acting basal insulin derivative under development, which is designed to remove B30 of insulin while introducing several amino acid mutations: A14E, B16H, B25H. And a C20 fatty acid side chain is connected to B29K. Compared with detemir and degludec, Icodec has a longer half-life. The purpose of A14E, B16H, B25H mutations is to reduce enzymatic degradation and weaken the affinity with insulin receptor (IR), reduce IR-mediated clearance, and further prolong the half-life. After injection into the human body, Icodec insulin will be closely but reversibly combined with albumin. This result can continuously, slowly and stably reduce blood glucose for a week. Based on its concentrated formula, the dosage of Icodec insulin injected once a week is equivalent to that of insulin glargine U100 injected once a day, so that once-a-week administration can be achieved.

[0007] However, there is still a need for better modification to obtain better efficacy, prolong the half-life of insulin derivatives, improve their hypoglycemic activity, and provide more long-acting insulin derivatives that can be injected once a week or even more than once a week. SUMMARY

[0008] To solve the above technical problems, the present application provides a new long-acting acylated insulin derivative with significantly prolonged action time and its application, which can achieve at least once-a-week administration.

[0009] The term "insulin derivative" in the present application refers to a chemically modified insulin analogue, in which one or more fatty acid side chains are covalently linked to the insulin peptide chain skeleton.

[0010] The term "amino acid" includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) and non-proteinogenic (or unnatural) amino acids. Proteinogenic amino acids are amino acids that naturally occur in proteins, and proteinogenic amino acids are amino acids encoded by the genetic code. Non-proteinogenic amino acids either do not exist in proteins or are not produced by standard cellular mechanisms (for example, they can have undergone post-translational modification).

[0011] In a first aspect, the present application provides a long-acting acylated insulin derivative, said derivative is acylated by a fatty acid side chain with an epsilon amino group of an amino acid K in an insulin peptide chain;

[0012] The insulin peptide chain is composed of an A chain and a B chain, wherein the sequence of the A chain is as follows:

[0013] GIVX1QCCTSICSLEQLEX2YCN, wherein X1 is E or D, and X2 is N or S;

[0014] The sequence of the B chain is as follows:

[0015] (GQAP) m FVNQHLCGSHLVEALX3LVCGERGFHYTP(GQAP) n K, wherein m is selected from any integer between 0-6, such as 0, 1, 2, 3, 4, 5, 6; n is selected from any integer between 0-6, such as 0, 1, 2, 3, 4, 5, 6; X3 is D or H;

[0016] The fatty acid side chain is HOOC(CH2) a CO-γ-Glu-(AEEA)2, wherein a is selected from any integer between 14-20, such as a can be 14, 15, 16, 17, 18, 19, 20, etc.

[0017] wherein the AEEA is 2-[2-(2-amino-ethoxy)-ethoxy]-acetic acid, and the chemical structure of the γ-Glu-(AEEA)2 is as follows (s and n are both 1):

[0018]

[0019] Preferably, in the B chain, m is selected from any integer between 0-3, such as 0, 1, 2, 3; n is selected from any integer between 0-3, such as 0, 1, 2, 3.

[0020] Preferably, in the B chain, m is selected from 0, and n is selected from 0, 1, 2, or 3; or, in the B chain, n is selected from 0, and m is selected from 0, 1, 2, or 3.

[0021] Preferably, the A chain is selected from:

[0022] GIVEQCCTSICSLEQLENYCN, or

[0023] GIVDQCCTSICSLEQLESYCN;

[0024] and / or, the B chain is selected from:

[0025] GQAPGQAPGQAPFVNQHLCGSHLVEALDLVCGERGFHYTPGQAPGQAPGQAPK, or

[0026] FVNQHLCGSHLVEALHLVCGERGFHYTPK.

[0027] Preferably, the insulin peptide chain of the insulin derivative of the present application is specifically combined with each of the A chain and the B chain, respectively.

[0028] Preferably, the fatty acid side chain is selected from the group consisting of HOOC(CH2) 14 CO-γ-Glu-(AEEA)2, HOOC(CH2) 16 CO-γ-Glu-(AEEA)2, HOOC(CH2) 18 CO-γ-Glu-(AEEA)2or HOOC(CH2) 20 CO-γ-Glu-(AEEA)2.

[0029] Preferably, the fatty acid side chain is selected from the group consisting of HOOC(CH2) 18 CO-γ-Glu-(AEEA)2.

[0030] Preferably, the derivative is acylated by a fatty acid side chain and an insulin peptide chain, the fatty acid side chain is acylated with the epsilon amino group of the amino acid K, the fatty acid side chain is selected from the group consisting of HOOC(CH2) 18 CO-γ-Glu-(AEEA)2, and the insulin peptide chain is composed of an A chain and a B chain, specifically, the insulin peptide chain of the insulin derivative is as follows:

[0031] (1) Insulin derivative HSP002-051

[0032] The A chain is: GIVEQCCTSICSLEQLENYCN (SEQ ID NQ.1),

[0033] The B chain is: GQAPGQAPGQAPFVNQHLCGSHLVEALDLVCGERGFHYTPGQAPGQAPGQAPK (SEQ ID NQ.2);

[0034] (2) Insulin derivative HSP002-080

[0035] The A chain is: GIVDQCCTSICSLEQLESYCN (SEQ ID NQ.3),

[0036] The B chain is: FVNQHLCGSHLVEALHLVCGERGFHYTPK (SEQ ID NQ.4).

[0037] In a second aspect, the present application provides a long-acting acylated insulin derivative injection formulation, which comprises the long-acting acylated insulin derivative of the first aspect and 1.5-12 moles of zinc ions per 6 moles of insulin derivative.

[0038] Preferably, the content of zinc ions is 1.5-8 moles of zinc ions per 6 moles of insulin derivative, 1.5-6 moles of zinc ions per 6 moles of insulin derivative, 1.5-3.5 moles of zinc ions per 6 moles of insulin derivative, 1.5-2.5 moles of zinc ions per 6 moles of insulin derivative, or 3.5-5.5 moles of zinc ions per 6 moles of insulin derivative, or 5.5-7.7 moles of zinc ions per 6 moles of insulin derivative.

[0039] Preferably, the content of insulin derivative in the injection formulation of the present application is 1-9 mM, 1-3 mM, 3-8 mM, 3.5-7 mM or 4-6 mM.

[0040] Further, the injection formulation further comprises glycerol, phenol and / or m-cresol, sodium chloride.

[0041] Preferably, the injection formulation contains 1-2% (w / w) of glycerol, 0-75 mM of phenol, 0-35 mM of m-cresol and 0-75 mM of sodium chloride.

[0042] Preferably, the content of phenol is 0-60 mM or 30-75 mM.

[0043] Preferably, the content of m-cresol is 0-30 mM or 15-35 mM.

[0044] Preferably, the content of sodium chloride is 5-75 mM, 5-30 mM, 10-30 mM, 15-25 mM or 20 mM.

[0045] Further, the injection formulation contains 1-2% (w / w) of glycerol, 0-60 mM or 30-75 mM of phenol, 0-30 mM or 15-35 mM of m-cresol and 0-75 mM of sodium chloride.

[0046] Further, the injection formulation further comprises 1-9 mM of the insulin derivative of the present application, 1-2% (w / w) of glycerol, 15-75 mM of phenol, 0-30 mM of m-cresol, 0-75 mM of sodium chloride and 1.5-12 moles of zinc ions per 6 moles of insulin derivative.

[0047] The content of the insulin derivative can be 3-8 mM, 3.5-7 mM, or 4-6 mM; the content of the phenol can be 0-60 mM or 25-60 mM; the content of the m-cresol can be 0-30 mM or 15-35 mM; the content of the sodium chloride can be 5-50 mM, or 5-30 mM, or 10-30 mM, or 15-25 mM, or 20 mM; and the content of the zinc ion can be 1.5-8 moles of zinc ion per 6 moles of insulin derivative, or 1.5-6 moles of zinc ion per 6 moles of insulin derivative, or 1.5-3.5 moles of zinc ion per 6 moles of insulin derivative, or 1.5-2.5 moles of zinc ion per 6 moles of insulin derivative, or 3.5-5.5 moles of zinc ion per 6 moles of insulin derivative, or 5.5-7.7 moles of zinc ion per 6 moles of insulin derivative.

[0048] Further, the injection liquid preparation further comprises 1-9 mM of the insulin derivative of the present application, 1-2% (weight / weight) of glycerol, 25-60 mM of phenol, 0-20 mM of m-cresol, 0-75 mM of sodium chloride, and 1.5-12 moles of zinc ion per 6 moles of insulin derivative. The content of the insulin derivative can be 3-8 mM, or 3.5-7 mM, or 4-6 mM; the content of the phenol can be 25-60 mM, or 60 mM; the content of the m-cresol can be 0-20 mM, or 0 mM; the content of the sodium chloride can be 5-50 mM, or 5-30 mM, or 10-30 mM, or 15-25 mM, or 20 mM; and the content of the zinc ion can be 1.5-8 moles of zinc ion per 6 moles of insulin derivative, or 1.5-6 moles of zinc ion per 6 moles of insulin derivative, or 1.5-3.5 moles of zinc ion per 6 moles of insulin derivative, or 1.5-2.5 moles of zinc ion per 6 moles of insulin derivative, or 3.5-5.5 moles of zinc ion per 6 moles of insulin derivative, or 5.5-7.7 moles of zinc ion per 6 moles of insulin derivative.

[0049] Preferably, the pH of the injection liquid preparation is 6.6-8.5, preferably 6.8-8.2.

[0050] In a third aspect, the present application provides a recombinant engineering bacterium expressing the insulin peptide chain of the long-acting acylated insulin derivative of the first aspect, wherein the recombinant engineering bacterium is transfected with a recombinant plasmid, and the recombinant plasmid can express a recombinant fusion protein comprising an insulin peptide chain, and the recombinant fusion protein is composed of an inclusion body promoting sequence, a lysine endopeptidase cleavage sequence, a B chain, a C peptide, and an A chain.

[0051] The inclusion body promoting sequence is preferably FKFEFKFE (SEQ ID NQ.5), HQHQHQHQHQ (SEQ ID NQ.6), HQHQHQHQHQHQ (SEQ ID NQ.7), HQHQHQHQHQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK (SEQ ID NQ.8) or HQHQHQHQHQHVEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK (SEQ ID NQ.9); the lysine endonuclease cleavage sequence is K; and the C peptide is preferably GGGPGRK (SEQ ID NQ.10). Taking the inclusion body promoting sequence FKFEFKFE as an example, the structure of the fusion protein is FKFEFKFEK-B chain-GGGPGRK-A chain.

[0052] Preferably, the recombinant engineering bacteria are recombinant E. coli engineering bacteria, and more preferably, the recombinant engineering bacteria are recombinant BL21(DE3) E. coli engineering bacteria.

[0053] Preferably, the recombinant plasmid is a pET-28a(+), pET-30a(+) or pET-32a(+) recombinant plasmid.

[0054] In a fourth aspect, the present application provides a preparation method of the recombinant engineering bacteria of the third aspect, which comprises the following steps:

[0055] (1) constructing a gene expression fragment encoding a fusion protein composed of an inclusion body promoting sequence, a lysine endonuclease cleavage sequence, a B chain, a C peptide and an A chain;

[0056] (2) inserting the gene expression fragment into a prokaryotic expression plasmid to obtain an expression plasmid of the corresponding fusion protein;

[0057] (3) transforming the expression plasmid into E. coli to obtain recombinant engineering bacteria expressing the fusion protein.

[0058] The inclusion body promoting sequence is preferably FKFEFKFE, HQHQHQHQHQ, HQHQHQHQHQHQ, HQHQHQHQHQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK or HQHQHQHQHQHVEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK; the lysine endonuclease cleavage sequence is K; and the C peptide is preferably GGGPGRK.

[0059] Preferably, the recombinant engineering bacteria of the present application are recombinant E. coli engineering bacteria, and more preferably, the recombinant engineering bacteria are recombinant BL21(DE3) E. coli engineering bacteria, and the recombinant plasmid is preferably a pET-28a(+), pET-30a(+) or pET-32a(+) recombinant plasmid.

[0060] In a fifth aspect, the present application provides use of the long-acting acylated insulin derivative of the first aspect in the manufacture of a medicament for the treatment of diabetes.

[0061] Compared with the prior art, the technical scheme provided by the embodiments of the present application has the following advantages:

[0062] The insulin derivative provided by the present application has excellent blood glucose lowering ability, and the action time is significantly prolonged, which can realize once-a-week administration, can significantly improve the compliance and sugar control willingness of patients, and has broad market prospects. BRIEF DESCRIPTION OF DRAWINGS

[0063] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and serve to explain the principles of the present application together with the description.

[0064] In order to more clearly illustrate the technical schemes in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, for those skilled in the art, other drawings can also be obtained from these drawings without any creative labor.

[0065] Figure 1 Biological activity data of insulin receptor in vitro of the insulin derivative HSP002-051 of the embodiment 2 of the present application;

[0066] Figure 2 Biological activity data of insulin receptor in vitro of the insulin derivative HSP002-080 of the embodiment 2 of the present application;

[0067] Figure 3 Hypoglycemic data of the insulin derivatives HSP002-051 and HSP002-080 of the embodiment 3 of the present application;

[0068] Figure 4 Hypoglycemic data of the insulin derivatives HSP002-051 and HSP002-080 of the embodiment 3 of the present application;

[0069] Figure 5 Hypoglycemic data of the insulin derivatives HSP002-051 and HSP002-080 of the embodiment 3 of the present application;

[0070] Figure 6 Blood glucose values of beagles 24-36h after the first clamp administration of the insulin derivatives HSP002-051, HSP002-080 and the positive control Icodec;

[0071] Figure 7Glucose infusion rate results 24-36h after the first clamp dosing for insulin derivative HSP002-051, HSP002-080 and positive control Icodec;

[0072] Figure 8 Beagle dog blood glucose values 48-60h after the second clamp dosing for insulin derivative HSP002-051, HSP002-080 and positive control Icodec.

[0073] Figure 9 Glucose infusion rate results 48-60h after the second clamp dosing for insulin derivative HSP002-051, HSP002-080 and positive control Icodec. DETAILED DESCRIPTION

[0074] In order to enable every intended person to understand the present application more clearly and completely, the schemes of the present application will be further described below. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0075] In the following description, a lot of specific details are set forth in order to give a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein; obviously, the embodiments described in the specification are only a part of the embodiments of the present application, and not all the embodiments.

[0076] Example 1 Preparation of insulin derivative

[0077] The present embodiment provides a preparation method of long-acting acylated insulin derivative, taking insulin derivative HSP002-051 as an example, the preparation method comprises the following steps:

[0078] (1) The peptide chain of the insulin derivative HSP002-051 of the present application is prepared according to the method described in Example 1 of patent CN94193852.2, and the peptide chain comprises a modified insulin A chain (as shown in SEQ ID NQ.1) and a modified insulin B chain (as shown in SEQ ID NQ.2); the prepared peptide chain is reserved after sequencing.

[0079] (2) Fatty acid side chain connection

[0080] The insulin derivative peptide chain prepared in step (1) is formulated at about 6 mg / mL, the pH is adjusted to about 11.0, the fatty acid powder is weighed according to the molar ratio of the peptide chain to eicosanedioic acid mono-tert-butyl ester-glutamic acid (1-tert-butyl ester)-AEEA-AEEA-OSU 1:4 in acetonitrile, the two are mixed, and the mixture is left to stand at room temperature for 1 h. The reaction is terminated by adding an acid solution to adjust the pH to 4.8. Continue to add 2 times the volume of the acid solution, and leave to stand at room temperature for 1 h of deprotection, and then add NaOH dropwise to adjust the pH to 7.5-8.5 to terminate the reaction.

[0081] The reaction solution is diluted 5 times with water, and loaded onto UniPS10-300 (purchased from Suzhou Nanmi Technology Co., Ltd.), eluted with 0-100% eluent (10 mM TFA, 80% acetonitrile), and the purity of the eluted peak is detected by HPLC to be more than 95%, to obtain the insulin derivative HSP002-051, and the eluent is lyophilized and stored at -20°C for standby.

[0082] In the same way, insulin control Icodec and HSP002-080 are prepared.

[0083] Example 2: In vitro insulin receptor biological activity assay experiment

[0084] In order to measure the in vitro insulin receptor biological activity of the insulin derivative provided by the application, a glucose uptake capacity experiment is performed using HEK293-IR-B cells stably expressing human insulin receptors.

[0085] (1) Experimental materials

[0086] HEK293-IR-B is subcultured using Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 100 μg / mL of G418, and the cells are used for glucose uptake capacity experiments when they are in the logarithmic growth phase.

[0087] (2) Experimental method

[0088] The dissociated HEK293-IR-B cells are washed once with DPBS buffer, resuspended in DMEM containing 10% FBS, stained with trypan blue, and counted. The cell density is adjusted to 3×10 5 Each well of a 96-well cell plate is inoculated with 100 μL of the sample diluent (DMEM containing 0.8 mg / mL of glucose), and incubated in a 37°C, 5% CO2 incubator for 24 h.

[0089] The positive control Icodec and the sample solution to be tested were each diluted in a 6-fold gradient from a starting concentration of 120,000 nM, using sample diluent, for a total of 8 concentrations, with 2 replicates per concentration. The cell culture supernatant was discarded from the 96-well plate, and the positive control Icodec and the sample solutions HSP002-051 and HSP002-080, which had been diluted in a gradient, were each added to the cell culture plate in an amount of 50 μL / well, and reacted in a 37°C, 5% CO2incubator for 24 h.

[0090] The remaining glucose in the cell culture medium of the 96-well plate was measured using a glucose assay kit (glucose oxidase method). Glucose oxidase was added to a new 96-well plate in an amount of 200 μL / well, and 20 μL of the culture supernatant was collected per well and added to the plate, which was then reacted at 37°C for 10 min. The OD value at 508 nm was read using a microplate reader. The EC50value was calculated using a four-parameter regression calculation method.

[0091] (3) Experimental results

[0092] The in vitro insulin receptor biological activity data of the insulin derivative HSP002-051 are shown in Table 1 and Figure 1 Table 1.

[0093] Table 1

[0094] Sample EC 50 ]]> Icodec 600.0 HSP002-051 710.6

[0095] As can be seen from Table 1, the insulin derivative HSP002-051 provided by the present application has the ability to bind to the human insulin IR-B receptor in vitro, has insulin receptor agonist activity, and its in vitro insulin receptor agonist activity is close to that of the positive control Icodec.

[0096] The in vitro insulin receptor biological activity data of the insulin derivative HSP002-080 are shown in Table 2 and Figure 2 Table 2.

[0097] Table 2

[0098] Sample EC 50 ]]> Icodec 358.8 HSP002-080 193.8

[0099] As can be seen from Table 2, the insulin derivative HSP002-080 provided by the present application has the ability to bind to the human insulin IR-B receptor in vitro, has insulin receptor agonist activity, and its in vitro insulin receptor agonist activity is superior to that of the positive control Icodec.

[0100] Example 3 Single-dose single administration hypoglycemic test of STZ+HFD-induced hyperglycemic mice

[0101] The hypoglycemic effect of the insulin derivative is proved by the hypoglycemic test of the high blood sugar mice induced by STZ+HFD.

[0102] (1) Experimental materials

[0103] Experimental animals: C57 mice induced by STZ+HFD, 6-8 weeks old, male;

[0104] Experimental drug preparation: glycerol 19.6 mg / mL, phenol 1.5 mg / mL, m-cresol 1.72 mg / mL, zinc acetate dihydrate 110.43 μg / mL, insulin derivative 20.0 nmol / mL.

[0105] (2) Experimental method

[0106] a. Modeling and grouping:

[0107] Modeling: healthy SPF male C57 mice, 6-8 weeks old, weighing 18-20 g, were selected, and after one week of adaptive feeding, the feed was replaced with 60% high-fat feed, and fed for 8-12 weeks. After the weight reached the expected value, the mice were fasted for 16 h, and then injected with STZ (80 mpk) intraperitoneally to induce a high blood sugar model. After 5 days of induction, the blood glucose was detected, and the random blood glucose value was above 16.8 mmol / L, which was considered as successful modeling. If the first STZ induction modeling rate was low, the same method as the first time was used for 2 times of induction one week later. The mice that failed to model were eliminated, and were randomly grouped according to blood glucose and body weight.

[0108] b. Administration method: subcutaneous administration according to Table 3:

[0109] Table 3: Administration method

[0110]

[0111]

[0112] c. Detection index

[0113] Blood glucose value: 0 h blood glucose was measured before administration, and blood glucose was detected at 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h and 48 h after administration, and the blood glucose change curve was drawn.

[0114] (3) Experimental results

[0115] The hypoglycemic data of insulin derivatives HSP002-051 and HSP002-080 are shown in Table 4 and Figure 3

[0116] Table 4

[0117] ​

[0118] As shown in Table 4, the insulin derivative HSP002-051 provided by the present application has a significantly better hypoglycemic effect than Novo Nordisk once-a-week long-acting acylated insulin derivative Icodec within 48 hours of administration, and a better hypoglycemic duration than Icodec.

[0119] The insulin derivative HSP002-080 has a similar hypoglycemic effect and duration to Icodec within 48 hours of administration.

[0120] Example 4: PK test of subcutaneous injection in healthy beagle dogs

[0121] This example demonstrates the half-life of the insulin derivatives HSP002-051 and HSP002-080 through a PK test of subcutaneous injection in healthy beagle dogs.

[0122] (1) Experimental materials

[0123] Experimental animals: beagle dogs, 10-12 months old, male; body weight 8-10 kg;

[0124] Experimental drug preparation: glycerol 19.6 mg / mL, phenol 1.5 mg / mL, m-cresol 1.72 mg / mL, zinc acetate dihydrate 110.43 μg / mL, and insulin derivatives HSP002-051 and HSP002-080 at a concentration of 45.0 nmol / mL.

[0125] (2) Experimental method

[0126] a. Administration method: the insulin derivative HSP002-051 was administered subcutaneously according to Table 5:

[0127] Table 5: Administration method

[0128]

[0129] The insulin derivative HSP002-080 was administered subcutaneously according to Table 6:

[0130] Table 6: Administration method

[0131]

[0132] Due to the difference in molecular weight, the insulin derivatives HSP002-051 and HSP002-080 were administered at an equimolar concentration and equimolar amount compared to the positive control Icodec.

[0133] b. Detection index

[0134] Blood sampling time points: All experimental groups were sampled before administration and at 0h, 1h, 4h, 6h, 8h, 24h, 32h, 48h, 56h, 72h, 80h after administration, and the plasma (EDTA anticoagulation tube, 150 μL of plasma) was stored in a-80℃ refrigerator for testing.

[0135] (3) Experimental results

[0136] The half-life data of the insulin derivative HSP002-051 are shown in Table 7 and Figure 4

[0137] Table 7

[0138] Group T 1 / 2 / h]] Positive control group 56.41 HSP002-051 group 53.91

[0139] As can be seen from Table 7, the half-life of the insulin derivative HSP002-051 provided by the present application is 53.91h, which is basically equivalent to that of the positive control Icodec, which is 56.41h.

[0140] The half-life data of the insulin derivative HSP002-080 are shown in Table 8 and Figure 5

[0141] Table 8

[0142]

[0143]

[0144] As can be seen from Table 8, the half-life of the insulin derivative HSP002-080 of the present application is 55.28h, which is better than that of the positive control Icodec, which is 49.48h.

[0145] Example 5: Investigation of the in vivo pharmacokinetics of subcutaneous injection of healthy beagle dogs by using clamp test

[0146] In this embodiment, healthy beagle dogs were subcutaneously injected with two test samples (HSP002-051 and HSP002-080) and the positive control Icodec, respectively. Through glucose clamp test, the pharmacokinetics of the two test samples and the positive control Icodec in beagle dogs was compared, and the hypoglycemic effect of the insulin derivative of the present application was proved.

[0147] (1) Experimental materials

[0148] Experimental animals: beagle dogs, 8-10 months old, male; body weight 8-12 kg;

[0149] ​​Experimental drug preparation: the concentration of insulin derivatives HSP002-051, HSP002-080 and positive control Icodec is 280.0 nmol / mL;

[0150] Excipients: glycerol 19.6 mg / mL, phenol 1.5 mg / mL, m-cresol 1.72 mg / mL, zinc acetate dihydrate 110.43 μg / mL.

[0151] According to the content of each component, phenol, m-cresol, zinc acetate dihydrate and glycerol are added to each insulin derivative in turn, and the final insulin derivative concentration of the preparation is 280.0 nmol / mL.

[0152] (2) Experimental method

[0153] a. Experimental animal enrollment:

[0154] Animal blood glucose detection:

[0155] Select 3 beagle dogs with uniform baseline blood glucose for enrollment, with a baseline blood glucose range of 3.5-5.5 mmol / L, and conduct the test.

[0156] Surgery:

[0157] The beagle dog is placed on a fixing frame, the hair around the needle placement site on the limbs of the dog is shaved clean with a hair clipper, the skin surface is disinfected, a Y22Gx25-30 tube retention needle is placed in the cephalic vein of the front limb of the dog, and a 20G non-tube retention needle is placed in the saphenous vein of the hind limb. The retention needle is wrapped and fixed firmly with medical tape, and when blood is not taken, heparin sodium injection is injected through the liquid inlet of the retention needle to make the heparin sodium injection fill the retention needle. The health status of the animal during this process is supervised by a veterinarian.

[0158] b. Feeding information

[0159] All animals are adapted for one week, and the dogs are adapted to the training during the adaptive feeding period, 12h / day, at least 4 days. Record the animal state.

[0160] All animals were fed 30g / kg the morning before administration and fasted for 16h overnight; water was freely available.

[0161] All animals were fed 30g / kg after administration and fasted for 16h overnight; water was freely available. The state of the dogs was closely observed after administration.

[0162] All animals were fed 15g / kg after the clamping test, and the food bowl was removed after 0.5h, and water was freely available during this period.

[0163] c. Grouping and administration information:

[0164] Three beagles were used in this experiment, and two test samples and positive control Icodec were administered respectively to detect the clamp in 24-36h and 48-60h time periods.

[0165] The animal numbers were AM01, BM01 and CM01.

[0166] Dosage method: insulin derivatives HSP002-051, HSP002-080 and positive control Icodec were administered subcutaneously according to Table 9:

[0167] Table 9

[0168]

[0169] Due to the difference in molecular weight, the above-mentioned insulin derivatives HSP002-051 and HSP002-080 and positive control Icodec were administered at equimolar concentration and equimolar amount.

[0170] d. Blood glucose detection

[0171] At the designated time point, about 20μL of whole blood was collected from the beagle blood channel, and Roche blood glucose test paper and Roche blood glucose meter were used to detect the blood glucose level.

[0172] The sampling time points were:

[0173] Before administration: 3 dogs, blood was taken every 5min to measure blood glucose, until the CV% of three consecutive detection values was <10%, then the average value of the three blood glucose values was taken as the pre-administration basal blood glucose value BBG;

[0174] The basal blood glucose sampling and detection records are shown in Table 10:

[0175] Table 10

[0176]

[0177]

[0178] First clamp: blood glucose was measured 24h after administration, and the blood glucose results were analyzed by t with the basal blood glucose BBG, if there was a significant difference, the first stage clamp test was started, for a total of 12 hours (24-36h after administration), blood was taken every 5min to measure the blood glucose level (clamp 1).

[0179] Second clamp: blood glucose was measured 48h after administration, and the blood glucose results were analyzed by t with the basal blood glucose BBG, if there was a significant difference, the second stage clamp test was carried out. For a total of 12 hours (48-60h after administration), blood was taken every 5min to measure the blood glucose level (clamp 2).

[0180] e. Detection index

[0181] During the clamp procedure, a continuous infusion of 25% dextrose in normal saline was used to maintain a target blood glucose level with a coefficient of variation of approximately 10%. Blood glucose was measured at each time point according to the protocol, and the glucose infusion rate was adjusted and recorded at each time point, so that the glucose infusion rate (GIR) was quantified by the glucose clamp test, and the GIR-time curve of the drug was obtained, and the hypoglycemic activity of the drug was evaluated.

[0182] (3) Experimental results

[0183] a. First clamp test:

[0184] The blood glucose data of the insulin derivatives HSP002-051, HSP002-080 and the positive control Icodec 24-36h after the first clamp administration are shown in Tables 11, 12 and 13, respectively, Figure 6 The blood glucose values of the insulin derivatives HSP002-051, HSP002-080 and the positive control Icodec 24-36h after the first clamp administration in beagles.

[0185] Table 11

[0186]

[0187]

[0188] Table 12

[0189]

[0190]

[0191] Table 13

[0192]

[0193]

[0194] The glucose infusion rate (GIR) results of the insulin derivatives HSP002-051, HSP002-080 and the positive control Icodec 24-36h after the first clamp administration are shown in Tables 14, 15 and 16, respectively, Figure 7 The glucose infusion rate results of HSP002-051, HSP002-080 and the positive control Icodec 24-36h after the first clamp administration.

[0195] Table 14

[0196]

[0197]

[0198] Table 15

[0199]

[0200]

[0201] Table 16

[0202]

[0203]

[0204] b. Second clamp trial:

[0205] The 48-60h glucose data for insulin derivatives HSP002-051, HSP002-080 and positive control Icodec after second clamp administration are shown in Tables 17, 18 and 19, respectively, Figure 8 The 48-60h glucose values for beagle dogs for insulin derivatives HSP002-051, HSP002-080 and positive control Icodec after second clamp administration.

[0206] Table 17

[0207]

[0208]

[0209] Table 18

[0210]

[0211]

[0212] Table 19

[0213]

[0214]

[0215] The 48-60h glucose infusion rate (GIR) results for insulin derivatives HSP002-051, HSP002-080 and positive control Icodec after second clamp administration are shown in Tables 20, 21 and 22, respectively, Figure 9 The 48-60h glucose infusion rate results for HSP002-051, HSP002-080 and positive control Icodec after second clamp administration.

[0216] Table 20

[0217]

[0218]

[0219] Table 21

[0220]

[0221]

[0222] Table 22

[0223]

[0224] As can be seen from Tables 11-22 and Figures 6-9 It can be seen that the insulin derivative HSP002-051 provided by the present application has a significantly better hypoglycemic effect than the positive control Novo Nordisk once-a-week long-acting acylated insulin derivative Icodec in both the 24-36h and 48-60h clamp-in experiments after administration, and the test results are the same as those of Example 3, fully proving that the insulin derivative HSP002-051 has both a better hypoglycemic effect and a longer duration than the positive control Icodec.

[0225] The insulin derivative HSP002-080 has a better hypoglycemic effect than the positive control Novo Nordisk once-a-week long-acting acylated insulin derivative Icodec (slightly lower than the insulin derivative HSP002-051) in both the 24-36h and 48-60h clamp-in experiments after administration within 48h, fully proving that the insulin derivative HSP002-080 has both a better hypoglycemic effect and a longer duration than the positive control Icodec.

[0226] In summary, the insulin derivative provided by the present application has excellent hypoglycemic ability and a significantly prolonged action time, can achieve once-a-week administration, can significantly improve the compliance and sugar control willingness of patients, and has a broad market prospect.

[0227] It should be noted that, in this document, relational terms such as“first” and“second”, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms“comprises”,“comprising”, or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by“comprises a...” does not, without more constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0228] The foregoing is considered as illustrative only of the principles of the application. Numerous modifications and changes will readily occur to those skilled in the art, and it is intended to embrace all such modifications and changes that fall within the scope of the appended claims to the principles and novel features disclosed herein. Accordingly, the scope of the application should be determined by the scope of the following claims and their equivalents.

Claims

1. A long-acting acylated insulin derivative, said derivative being formed by acylation of a fatty acid side chain with an insulin peptide chain, wherein the fatty acid side chain is acylated to the ε-amino group of amino acid K on the insulin peptide chain. Its features are, The insulin peptide chain consists of an A chain and a B chain, wherein the sequence of the A chain is shown in the following formula: GIVX1QCCTSICSLEQLEX2YCN, where X1 is E or D, and X2 is N or S; The sequence of the B chain is shown in the following formula: (GQAP) m FVNQHLCGSHLVEALX3LVCGERGFHYTP(GQAP) n K, where m and n are selected from any integers between 0 and 6, and X3 is D or H; The fatty acid side chain is HOOC(CH2). a CO-γ-Glu-(AEEA)2, where a is any integer between 14 and 20.

2. The long-acting acylated insulin derivative according to claim 1, characterized in that, In the B-chain, m and n are selected from any integers between 0 and 3; Preferably, in the B chain, m is selected from 0, and n is selected from 0, 1, 2, or 3; or, in the B chain, n is selected from 0, and m is selected from 0, 1, 2, or 3.

3. The long-acting acylated insulin derivative according to claim 1 or 2, characterized in that, The A chain is selected from: GIVEQCCTSICSLEQLENYCN, or GIVDQCCTSICSLEQLESYCN; And / or, the B chain is selected from: GQAPGQAPGQAPFVNQHLCGSHLVEALDLVCGERGFHYTPGQAPGQAPGQAPK, or FVNQHLCGSHLVEALHLVCGERGFHYTPK.

4. The long-acting acylated insulin derivative according to any one of claims 1-3, characterized in that, The fatty acid side chain is selected from HOOC(CH2). 14 CO-γ-Glu-(AEEA)2, HOOC(CH2) 16 CO-γ-Glu-(AEEA)2, HOOC(CH2) 18 CO-γ-Glu-(AEEA)2 or HOOC(CH2) 20 CO-γ-Glu-(AEEA)2, preferably HOOC(CH2) 18 CO-γ-Glu-(AEEA)2.

5. The long-acting acylated insulin derivative according to any one of claims 1-4, characterized in that, The derivative is formed by acylation of a fatty acid side chain with an insulin peptide chain, wherein the fatty acid side chain is acylated with the ε-amino group of amino acid K on the insulin peptide chain, and the fatty acid side chain is selected from HOOC(CH2). 18 CO-γ-Glu-(AEEA)2, the insulin peptide chain is composed of an A chain and a B chain, wherein, Chain A is: GIVEQCCTSICSLEQLENYCN, The B chain is: GQAPGQAPGQAPFVNQHLCGSHLVEALDLVCGERGFHYTPGQAPGQAPGQAPK; And / or, Chain A is: GIVDQCCTSICSLEQLESYCN. The B chain is: FVNQHLCGSHLVEALHLVCGERGFHYTPK.

6. An injectable formulation of a long-acting acylated insulin derivative, characterized in that, The injectable formulation comprises the long-acting acylated insulin derivative as described in any one of claims 1-5 and 1.5-12 moles of zinc ions / 6 moles of insulin derivative; Preferably, the zinc ion content is 1.5-8 moles of zinc ions / 6 moles of insulin derivative, 1.5-6 moles of zinc ions / 6 moles of insulin derivative, 1.5-3.5 moles of zinc ions / 6 moles of insulin derivative, 1.5-2.5 moles of zinc ions / 6 moles of insulin derivative, or 3.5-5.5 moles of zinc ions / 6 moles of insulin derivative, or 5.5-7.7 moles of zinc ions / 6 moles of insulin derivative; Preferably, the content of the insulin derivative is 1-9 mM, 1-3 mM, 3-8 mM, 3.5-7 mM or 4-6 mM.

7. The injectable formulation of the long-acting acylated insulin derivative according to claim 6, characterized in that, The injectable formulation further comprises glycerin, phenol and / or m-cresol, and sodium chloride; Preferably, the injection formulation contains 1-2% (by weight) glycerol, 0-75 mM phenol, 0-35 mM m-cresol, and 0-75 mM sodium chloride. Preferably, the phenol content is 0-60 mM or 30-75 mM; Preferably, the content of m-cresol is 0-30 mM or 15-35 mM; Preferably, the sodium chloride content is 5-75mM, 5-30mM, 10-30mM, 15-25mM, or 20mM.

8. The injectable formulation of the long-acting acylated insulin derivative according to claim 6, characterized in that, The injectable formulation further comprises 1-2% (by weight) glycerol, 0-60 mM or 30-75 mM phenol, 0-30 mM or 15-35 mM m-cresol, and 0-75 mM sodium chloride.

9. The injectable formulation of the long-acting acylated insulin derivative according to any one of claims 6-8, characterized in that, The pH of the injectable formulation is 6.6-8.5, preferably 6.8-8.

2.

10. A recombinant engineered bacterium expressing an insulin peptide chain of a long-acting acylated insulin derivative according to any one of claims 1-5, characterized in that, The recombinant engineered bacteria were transfected with a recombinant plasmid, which was able to express a recombinant fusion protein containing an insulin peptide chain. The recombinant fusion protein was composed of an inclusion body sequence, a lysine endonuclease digestion sequence, a B chain, a C peptide, and an A chain. The inclusion body sequence is preferably FKFEFKFE, HQHQHQHQHQ, HQHQHQHQHQHQ, HQHQHQHQHQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK or HQHQHQHQHQHVEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK; the lysine endonuclease digestion sequence is K; and the C peptide is GGGPGRK. Preferably, the recombinant engineered bacteria is a recombinant Escherichia coli engineered bacteria, more preferably a recombinant BL21(DE3) Escherichia coli engineered bacteria; Preferably, the recombinant plasmid is pET-28a(+), pET-30a(+), or pET-32a(+) recombinant plasmid.

11. The method for preparing the recombinant engineered bacteria according to claim 10, characterized in that, The preparation method includes the following steps: (1) Construct a gene expression fragment encoding a fusion protein consisting of an inclusion body sequence, a lysine endonuclease digestion sequence, a B chain, a C peptide, and an A chain; (2) Insert the gene expression fragment into a prokaryotic expression plasmid to obtain the expression plasmid of the corresponding fusion protein; (3) The expression plasmid is transferred into Escherichia coli to obtain a recombinant engineered bacterium expressing the fusion protein.

12. Use of the long-acting acylated insulin derivative according to any one of claims 1-5 in the preparation of a medicament for treating diabetes.

Images