Long-acting acylated insulin derivatives and uses thereof

Abstract

The present application 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 of the present application is acylated by connecting a fatty acid side chain with an insulin peptide chain, and the fatty acid side chain is acylated with the epsilon amino group of amino acid K on the insulin peptide chain.The insulin derivative provided by the present application has excellent blood glucose lowering ability, and the action time is significantly prolonged, once-a-week administration can be realized, the compliance and sugar control willingness of patients can be significantly improved, and the insulin derivative has broad market prospects.

Description

[0001] This invention claims priority to Chinese Patent Application No. 202410762875.9, filed on June 13, 2024, entitled "A Long-Acting Acylated Insulin Derivative and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of insulin derivative technology, and more particularly to a long-acting acylated insulin derivative and its applications, specifically to a long-acting acylated insulin derivative and its preparation method, injectable formulations containing the derivative, and pharmaceutical compositions thereof. Background Technology

[0003] Diabetes mellitus is a group of metabolic disorders involving carbohydrates, proteins, and fats caused by absolute or relative insulin deficiency and / or impaired insulin utilization. Hyperglycemia is the primary characteristic of diabetes, and it can be caused by a variety of factors, including genetics and environment. Diabetes is one of the three leading causes of death worldwide, second only to cardiovascular disease and cancer. Diabetes is mainly divided into type 1 and type 2 diabetes, with the majority of patients having type 2 diabetes (approximately 90% according to statistics). Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia, relative insulin deficiency, and insulin resistance. Currently, the main drugs used clinically to treat type 2 diabetes include biguanides, sulfonylureas, thiazolidinediones, DPP-4 receptor inhibitors, SGLT-2 receptor inhibitors, and GLP-1 derivatives.

[0004] Insulin is the only hormone in the body that lowers blood sugar. It also promotes the synthesis of glycogen, fat, and protein. Exogenous insulin and its derivatives are primarily used to treat diabetes. Insulin consists of two peptide chains, A and B. Human insulin (Insulin Human) has 21 amino acids in its A chain (11 types) and 30 amino acids in its B chain (15 types), for a total of 51 amino acids. Two disulfide bonds are formed between the sulfhydryl groups of four cysteine ​​residues: A7 (Cys)-B7 (Cys) and A20 (Cys)-B19 (Cys), linking the A and B peptide chains. Additionally, a disulfide bond exists between A6 (Cys) and A11 (Cys) in the A chain. Insulin is secreted by the pancreatic β-cells in the pancreas in response to stimulation from endogenous or exogenous substances such as glucose, lactose, ribose, arginine, and glucagon. The biological effects of insulin at the cellular level are initiated by binding to specific receptors on the target cell membrane; insulin receptors are specific sites on the target cell membrane where insulin acts, and can only bind to insulin or proinsulin containing insulin molecules, exhibiting high specificity.

[0005] Although insulin is the most effective treatment for diabetes, currently available insulin products can only be used once a day at most. Therefore, patients need to inject frequently to meet the body's insulin needs. However, frequent injections can cause great discomfort for patients. Therefore, it is hoped that by prolonging the blood sugar-lowering effect of insulin, the number of injections can be reduced, patient discomfort can be alleviated, and patient compliance can be improved.

[0006] Icodec insulin is an investigational long-acting basal insulin derivative. Its molecular design removes insulin's B30 and introduces several amino acid mutations: A14E, B16H, and B25H. A C20 fatty acid side chain is attached at B29K. Compared to insulin detemir and insulin degludec, Icodec has a longer half-life. The A14E, B16H, and B25H mutations aim to reduce enzymatic degradation and weaken affinity for the insulin receptor (IR), reducing IR-mediated clearance and further prolonging the half-life. After injection, Icodec insulin binds tightly but reversibly to albumin. This results in a continuous, slow, and stable reduction of blood glucose over a week. Due to its concentrated formulation, a weekly injection of Icodec insulin is equivalent to a daily injection of insulin glargine U100, allowing for once-weekly dosing.

[0007] Nevertheless, the demand remains for insulin derivatives that can be modified to achieve better efficacy, prolong their half-life, enhance their hypoglycemic activity, and provide more long-acting insulin derivatives that can be injected once a week or even more. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a novel long-acting acylated insulin derivative with significantly prolonged duration of action and its applications, enabling administration at least once a week.

[0009] In this invention, the term "insulin derivative" refers to a chemically modified insulin analogue in which one or more fatty acid side chains are covalently linked to the insulin peptide backbone.

[0010] The term "amino acid" includes proteinogenic (or naturally occurring) amino acids (of which there are 20 standard amino acids) and non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are those that are naturally present in proteins and are encoded by the genetic code. Non-proteinogenic amino acids are either not present in proteins or are not produced through standard cellular mechanisms (e.g., they may have undergone post-translational modifications).

[0011] In a first aspect, the present invention provides a long-acting acylated insulin derivative, wherein 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.

[0012] 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:

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

[0014] The sequence of the B chain is shown in the following formula:

[0015] (GQAP) m FVNQHLCGSHLVEALX3LVCGERGFHYTP(GQAP) n K, where m is any integer between 0 and 6, for example 0, 1, 2, 3, 4, 5, 6; n is any integer between 0 and 6, for example 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, where a is any integer between 14 and 20, for example, a can be 14, 15, 16, 17, 18, 19, 20, etc.

[0017] Wherein, AEEA is 2-[2-(2-amino-ethoxy)-ethoxy]-acetic acid, and the chemical formula of γ-Glu-(AEEA)2 is shown below (s and n are both 1):

[0018]

[0019] Preferably, in the B chain, m is selected from any integer between 0 and 3, such as 0, 1, 2, 3; n is selected from any integer between 0 and 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 invention comprises a specific combination of each of the above-mentioned A chains and each of the B chains.

[0028] Preferably, 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.

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

[0030] Preferably, 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, wherein the insulin peptide chain is composed of an A chain and a B chain, specifically, the insulin peptide chain of the insulin derivative is shown below:

[0031] (1) Insulin derivative HSP002-051

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

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

[0034] (2) Insulin derivative HSP002-080

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

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

[0037] In a second aspect, the present invention provides an injectable formulation of a long-acting acylated insulin derivative, the injectable formulation comprising the long-acting acylated insulin derivative described in the first aspect and 1.5-12 moles of zinc ions / 6 moles of insulin derivative.

[0038] 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.

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

[0040] Furthermore, the injectable formulation also contains glycerin, phenol and / or m-cresol, and sodium chloride.

[0041] 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.

[0042] Preferably, the phenol content 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 sodium chloride content is 5-75mM, 5-30mM, 10-30mM, 15-25mM, or 20mM.

[0045] Furthermore, the injectable formulation contains 1-2% (by weight) glycerol, 0-60mM or 30-75mM phenol, 0-30mM or 15-35mM m-cresol, and 0-75mM sodium chloride.

[0046] Furthermore, the injectable formulation further comprises 1-9 mM of the insulin derivative of the present invention, 1-2% (w / w) glycerol, 15-75 mM phenol, 0-30 mM m-cresol, 0-75 mM sodium chloride, and 1.5-12 moles of zinc ions / 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, 5-30 mM, 10-30 mM, 15-25 mM, or 20 mM; the content of the zinc ions can be 1.5-8 mol zinc ions / 6 mol insulin derivative, or 1.5-6 mol zinc ions / 6 mol insulin derivative, or 1.5-3.5 mol zinc ions / 6 mol insulin derivative, or 1.5-2.5 mol zinc ions / 6 mol insulin derivative, or 3.5-5.5 mol zinc ions / 6 mol insulin derivative, or 5.5-7.7 mol zinc ions / 6 mol insulin derivative.

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

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

[0050] Thirdly, the present invention provides a recombinant engineered bacterium that expresses an insulin peptide chain of the long-acting acylated insulin derivative described in the first aspect, wherein the recombinant engineered bacterium is transfected with a recombinant plasmid, the recombinant plasmid being able to express a recombinant fusion protein containing an insulin peptide chain, the recombinant fusion protein being composed of an inclusion body sequence, a lysine endonuclease digestion sequence, a B chain, a C peptide, and an A chain;

[0051] The inclusion-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 HQHQHQHQHQHQVEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK (SEQ ID NQ.9); the lysine endonuclease digestion sequence is K; and the C peptide is preferably GGGPGRK (SEQ ID NQ.10). Taking the inclusion-promoting sequence FKFEFKFE as an example, the fusion protein structure is: FKFEFKFEK-B chain-GGGPGRK-A chain.

[0052] Preferably, the recombinant engineered bacteria is a recombinant Escherichia coli engineered bacteria, more preferably a recombinant BL21(DE3) Escherichia coli engineered bacteria.

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

[0054] Fourthly, the present invention provides a method for preparing the recombinant engineered bacteria described in the third aspect, the method comprising the following steps:

[0055] (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;

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

[0057] (3) The expression plasmid is transferred into Escherichia coli to obtain a recombinant engineered bacterium expressing the fusion protein.

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

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

[0060] Fifthly, the present invention provides the use of the long-acting acylated insulin derivative described in the first aspect in the preparation of a medicament for treating diabetes.

[0061] The technical solution provided by the embodiments of the present invention has the following advantages compared with the prior art:

[0062] The insulin derivative provided by this invention has excellent hypoglycemic ability and a significantly prolonged duration of action, enabling once-weekly dosing. This can significantly improve patient compliance and willingness to control blood sugar, and has broad market prospects. Attached Figure Description

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

[0064] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0065] Figure 1 This refers to the in vitro insulin receptor biological activity data of insulin derivative HSP002-051 in Example 2 of the present invention;

[0066] Figure 2 This refers to the in vitro insulin receptor biological activity data of insulin derivative HSP002-080 in Example 2 of the present invention;

[0067] Figure 3 The hypoglycemic data for insulin derivatives HSP002-051 and HSP002-080 in Example 3 of this invention;

[0068] Figure 4 This refers to the half-life data of the subcutaneous injection PK test of insulin derivative HSP002-051 in beagle dogs in Example 4 of this invention;

[0069] Figure 5 This refers to the half-life data of the subcutaneous injection PK test of insulin derivative HSP002-080 in beagle dogs in Example 4 of this invention;

[0070] Figure 6 The values ​​of blood glucose in beagle dogs 24-36 hours after the first clamp administration of insulin derivatives HSP002-051, HSP002-080, and the positive control Icodec.

[0071] Figure 7The results of glucose infusion rates 24-36 h after the first clamp administration of insulin derivatives HSP002-051, HSP002-080, and the positive control Icodec;

[0072] Figure 8 The values ​​of blood glucose in beagle dogs 48-60 hours after the second clamp administration of insulin derivatives HSP002-051, HSP002-080, and the positive control Icodec.

[0073] Figure 9 The results show the glucose infusion rates 48-60 h after the second clamp administration of insulin derivatives HSP002-051, HSP002-080, and the positive control Icodec. Detailed Implementation

[0074] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0075] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0076] Example 1: Preparation of Insulin Derivatives

[0077] This embodiment provides a method for preparing a long-acting acylated insulin derivative. Taking insulin derivative HSP002-051 as an example, the preparation method includes the following steps:

[0078] (1) Prepare the peptide chain of the insulin derivative HSP002-051 of the present invention according to the method described in Example 1 of patent CN94193852.2. The peptide chain includes 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); sequence the prepared peptide chain for later use.

[0079] (2) Fatty acid side chain linkage

[0080] Take the insulin derivative peptide chain prepared in step (1), prepare a solution of approximately 6 mg / mL, and adjust the pH to approximately 11.0. Weigh fatty acid powder into acetonitrile according to a molar ratio of peptide chain to eicosanoic acid monotert-butyl ester-glutamic acid (1-tert-butyl ester)-AEEA-AEEA-OSU of 1:4. Mix the two solutions, let stand at room temperature for 1 hour, and then add acid to adjust the pH to 4.8 to terminate the reaction. Continue to add 2 times the volume of acid solution, let stand at room temperature for 1 hour to remove protection, and then add NaOH dropwise to adjust the pH to 7.5-8.5 to terminate the reaction.

[0081] The reaction solution was diluted 5 times with water, loaded onto UniPS10-300 (purchased from Suzhou Nanomicro Technology Co., Ltd.), and eluted with 0-100% eluent (10mM TFA, 80% acetonitrile). The purity of the elution peak was found to be above 95% by HPLC, and insulin derivative HSP002-051 was obtained. The eluent was lyophilized at -20℃ and stored for later use.

[0082] Insulin controls Icodec and HSP002-080 were prepared using the same method.

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

[0084] To measure the in vitro insulin receptor biological activity of the insulin derivative provided by this invention, a glucose uptake experiment was conducted using HEK293-IR-B cells that stably express the human insulin receptor.

[0085] (1) Experimental materials

[0086] HEK293-IR-B cells were passaged in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 μg / mL G418 until they were in the logarithmic growth phase for glucose uptake experiments.

[0087] (2) Experimental methods

[0088] After decontamination, HEK293-IR-B cells were washed once with DPBS buffer, resuspended in DMEM containing 10% FBS, stained with trypan blue, and counted. The cell density was adjusted to 3 × 10⁶ cells / mL using DMEM containing 10% FBS. 5 100 μL of the sample dilution buffer (0.8 mg / mL DMEM) was seeded into each well of a 96-well plate and incubated at 37°C with 5% CO2 for 24 h. The supernatant was discarded, and 100 μL of the sample dilution buffer (0.8 mg / mL glucose DMEM) was added to each well. The plates were then incubated at 37°C with 5% CO2 for another 24 h.

[0089] Positive control Icodec and test sample solutions were serially diluted 6-fold starting from 120,000 nM using sample diluent, resulting in a total of 8 concentrations, with 2 replicates per concentration. The cell culture supernatant in the 96-well plate was discarded. The serially diluted positive control Icodec and test sample solutions HSP002-051 and HSP002-080 were added to the cell culture plate at 50 μL / well, and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0090] The residual glucose in the cell culture medium of a 96-well plate was measured using a glucose assay kit (glucose oxidase method). 200 μL of glucose oxidase was added to each well of a new 96-well plate, and 20 μL of culture supernatant was collected from each well and added to the plate. The plate was incubated at 37°C for 10 min. The OD value at 508 nm was read using a microplate reader. The EC50 value was calculated using a four-parameter regression method.

[0091] (3) Experimental Results

[0092] In vitro insulin receptor biological activity data of insulin derivative HSP002-051 are shown in Table 1 and Figure 1 As shown:

[0093] Table 1

[0094] Icodec 600.0 HSP002-051 710.6

[0095] As can be seen from Table 1, the insulin derivative HSP002-051 provided by the present invention 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] In vitro insulin receptor biological activity data of insulin derivative HSP002-080 are shown in Table 2 and Figure 2 As shown:

[0097] Table 2

[0098] Icodec 358.8 HSP002-080 193.8

[0099] As can be seen from Table 2, the insulin derivative HSP002-080 provided by the present invention 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 in STZ+HFD-induced hyperglycemic mice

[0101] This embodiment demonstrates the hypoglycemic effect of the insulin derivative of the present invention through a hypoglycemic experiment in hyperglycemic mice induced by STZ+HFD.

[0102] (1) Experimental materials

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

[0104] Experimental drug formulations: 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 methods

[0106] a. Modeling and grouping:

[0107] Model establishment: Healthy SPF-grade male C57 mice aged 6–8 weeks, weighing 18–20 g, were selected. After one week of acclimatization, their diet was replaced with a 60% high-fat diet and fed for 8–12 weeks. After reaching the expected weight, the mice were fasted for 16 hours and then induced with an intraperitoneal injection of STZ (80 mpk) to establish a hyperglycemic model. Blood glucose was measured 5 days after induction. A random blood glucose level above 16.8 mmol / L was considered a successful model. If the initial STZ induction rate was low, a second induction was performed one week later using the same method. Mice that did not successfully establish the model were culled and randomly grouped according to blood glucose and body weight.

[0108] b. Administration method: Subcutaneous administration as per Table 3:

[0109] Table 3: Administration Methods

[0110]

[0111]

[0112] c. Detection indicators

[0113] Blood glucose levels: Blood glucose was measured at 0 h before administration, and blood glucose was measured at 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h and 48 h after administration. Blood glucose change curves were plotted.

[0114] (3) Experimental Results

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

[0116] Table 4

[0117]

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

[0119] HSP002-080 exhibits similar hypoglycemic effects and duration of action to Novo Nordisk's once-weekly long-acting acylated insulin derivative Icodec within 48 hours of administration.

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

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

[0122] (1) Experimental materials

[0123] Laboratory animals: Beagles, 10-12 months old, male; weighing 8-10 kg;

[0124] Experimental drug formulations: 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, both with a concentration of 45.0 nmol / mL.

[0125] (2) Experimental methods

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

[0127] Table 5 Administration methods

[0128]

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

[0130] Table 6 Administration methods

[0131]

[0132] Due to differences in molecular weight, the above-mentioned insulin derivatives HSP002-051 and HSP002-080 were administered at equimolar concentrations and equimolar amounts to the positive control Icodec.

[0133] b. Detection indicators

[0134] Blood collection time points: Blood was collected from all experimental groups before administration and at 0h, 1h, 4h, 6h, 8h, 24h, 32h, 48h, 56h, 72h, and 80h after administration. The plasma was separated (EDTA anticoagulant tube, 150μL plasma) and stored at -80℃ for testing.

[0135] (3) Experimental Results

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

[0137] Table 7

[0138] Positive control group 56.41 Group HSP002-051 53.91

[0139] As shown in Table 7, compared with Icodec, the half-life of the insulin derivative HSP002-051 provided by the present invention is 53.91h, which is basically the same as the 56.41h of the positive control group Icodec.

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

[0141] Table 8

[0142]

[0143]

[0144] As shown in Table 8, compared with Icodec, the half-life of the insulin derivative HSP002-080 of the present invention is 55.28h, which is better than the 49.48h of the positive control group Icodec.

[0145] Example 5: Investigating the in vivo pharmacokinetics of subcutaneous injection in healthy beagle dogs using a clamp test.

[0146] In this embodiment, healthy beagle dogs were administered two test samples (HSP002-051 and HSP002-080) and a positive control Icodec via subcutaneous injection. The pharmacokinetics of the two test samples and the positive control Icodec in beagle dogs were compared using a glucose clamp test to demonstrate the hypoglycemic effect of the insulin derivative of the present invention.

[0147] (1) Experimental materials

[0148] Laboratory animals: Beagles, 8-10 months old, male; weighing 8-12 kg;

[0149] The concentrations of the experimental drug formulations, insulin derivatives HSP002-051 and HSP002-080, and the positive control Icodec, were all 280.0 nmol / mL.

[0150] Excipients: Glycerin 19.6 mg / mL, Phenol 1.5 mg / mL, m-cresol 1.72 mg / mL, Zinc acetate dihydrate 110.43 μg / mL.

[0151] Based on the content of each component, phenol, m-cresol, zinc acetate dihydrate, and glycerol were added to each insulin derivative in sequence to obtain a formulation with a final insulin derivative concentration of 280.0 nmol / mL.

[0152] (2) Experimental methods

[0153] a. Enrollment of laboratory animals:

[0154] Animal blood glucose testing:

[0155] Three beagle dogs with uniform baseline blood glucose levels (3.5–5.5 mmol / L) were selected for the experiment.

[0156] Operation:

[0157] The beagle was placed on a support frame, and the hair on all four limbs where the indwelling needles were inserted was shaved clean. The skin was disinfected. A Y22G×25-30 indwelling needle with a tube was inserted into the cephalic vein of the forelimb; a 20G indwelling needle without a tube was inserted into the saphenous vein of the hindlimb. The indwelling needles were securely wrapped with medical tape. When no blood was being drawn, heparin sodium injection was injected through the inlet of the indwelling needle until the needle was filled with heparin sodium injection. The animal's health was monitored by a veterinarian throughout the process.

[0158] b. Feeding information

[0159] All animals should undergo one week of acclimatization training. During the acclimatization period, dogs should receive acclimatization restraint training for 12 hours per day for at least 4 days. Animal condition should be recorded.

[0160] All animals were fed 30g / kg the morning before administration and fasted overnight for 16 hours; they were allowed free access to water.

[0161] All animals should be administered 30g / kg after administration, and fasted overnight for 16 hours; water should be available freely. The dogs' condition should be closely monitored after administration.

[0162] All animals were fed 15g / kg after the clamp test, and the food bowls were removed 0.5 hours later. They were allowed free access to water during this period.

[0163] c. Grouping and Dosing Information:

[0164] This experiment used three beagle dogs, administering two test samples and a positive control (Icodec) to each dog, and measuring clamping activity at two time points: 24-36 h and 48-60 h.

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

[0166] Administration method: Insulin derivatives HSP002-051, HSP002-080, and the positive control Icodec were administered subcutaneously according to Table 9.

[0167] Table 9

[0168]

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

[0170] d. Blood glucose testing

[0171] At a designated time point, approximately 20 μL of whole blood was collected from the beagle blood collection channel, and blood glucose levels were measured using Roche blood glucose test strips and a Roche blood glucose meter.

[0172] The sampling time points are:

[0173] Before administration: Blood glucose was measured in 3 dogs every 5 minutes until the CV% of three consecutive measurements was less than 10%. The average of these three blood glucose values ​​was taken as the basal blood glucose value (BBG) before administration.

[0174] The baseline blood glucose sampling and testing records are shown in Table 10:

[0175] Table 10

[0176]

[0177]

[0178] First clamp: Blood glucose was measured 24 hours after administration. The blood glucose result was compared with the baseline blood glucose (BBG) using a t-analysis. If there was a significant difference, the first stage of the clamp test was started for a total of 12 hours (24-36 hours after administration). Blood glucose levels were measured every 5 minutes (clamp 1).

[0179] Second clamp test: Blood glucose was measured 48 hours after drug administration. The blood glucose result was compared with the baseline blood glucose (BBG) using a t-analysis. If there was a significant difference, the second clamp test was conducted. Blood glucose levels were measured every 5 minutes for a total of 12 hours (48-60 hours after drug administration) (clamp test 2).

[0180] e. Detection indicators

[0181] During the clamping process, a 25% glucose saline solution was continuously infused using an infusion pump to ensure that the coefficient of variation of the target blood glucose level remained relatively stable. Blood glucose levels were measured at various time points according to the experimental protocol. After measurement, the glucose infusion rate at each time point was adjusted and recorded. This glucose clamping test quantified the glucose infusion rate (GIR), thereby deriving the drug's GIR-time curve and evaluating its hypoglycemic activity.

[0182] (3) Experimental Results

[0183] a. First clamping test:

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

[0185] Table 11

[0186]

[0187]

[0188] Table 12

[0189]

[0190]

[0191] Table 13

[0192]

[0193]

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

[0195] Table 14

[0196]

[0197]

[0198] Table 15

[0199]

[0200]

[0201] Table 16

[0202]

[0203]

[0204] b. Second clamping test:

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

[0206] Table 17

[0207]

[0208]

[0209] Table 18

[0210]

[0211]

[0212] Table 19

[0213]

[0214]

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

[0216] Table 20

[0217]

[0218]

[0219] Table 21

[0220]

[0221]

[0222] Table 22

[0223]

[0224] From Table 11-22 and Figure 6-9 It can be seen that the insulin derivative HSP002-051 provided by the present invention has significantly better hypoglycemic effect than the positive control Novo Nordisk once-weekly long-acting acylated insulin derivative Icodec in two clamp tests at 24-36h and 48-60h after administration, and the results are the same as those in Example 3. This fully demonstrates that the hypoglycemic effect and duration of insulin derivative HSP002-051 are better than those of the positive control Icodec.

[0225] In the clamp tests conducted within 48 hours of administration, as well as at 24-36 hours and 48-60 hours post-administration, the insulin derivative HSP002-080 demonstrated superior hypoglycemic efficacy compared to the positive control, Novo Nordisk's once-weekly long-acting acylated insulin derivative Icodec (though slightly lower than insulin derivative HSP002-051). This fully demonstrates that the hypoglycemic effect and duration of insulin derivative HSP002-080 are superior to those of the positive control Icodec.

[0226] In summary, the insulin derivative provided by this invention has excellent hypoglycemic ability and a significantly prolonged duration of action, enabling once-weekly dosing. This can significantly improve patient compliance and willingness to control blood sugar, and has broad market prospects.

[0227] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0228] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other instances without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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 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: GIVDQCCTSICSLEQLESYCN. The B-chain is: FVNQHLCGSHLVEALHLVCGERGFHYTPK; The AEEA is 2-[2-(2-amino-ethoxy)-ethoxy]-acetic acid.

2. An injectable formulation of a long-acting acylated insulin derivative, characterized in that, The injectable formulation comprises the long-acting acylated insulin derivative of claim 1 and zinc ions, wherein the zinc ion content is 1.5-12 moles of zinc ions / 6 moles of insulin derivative.

3. The injectable formulation of the long-acting acylated insulin derivative according to claim 2, characterized in that, The zinc ion content is 1.5-8 moles of zinc ions / 6 moles of insulin derivative.

4. The injectable formulation of the long-acting acylated insulin derivative according to claim 2, characterized in that, The content of the insulin derivative is 1-9 mM.

5. The injectable formulation of the long-acting acylated insulin derivative according to claim 2, characterized in that, The injectable formulation further comprises glycerol, sodium chloride, and phenol and / or m-cresol.

6. The injectable formulation of the long-acting acylated insulin derivative according to claim 5, characterized in that, The injectable formulation contains 1-2% (by weight) glycerol, 0-75 mM phenol, 15-35 mM m-cresol, and 5-75 mM sodium chloride.

7. The injectable formulation of the long-acting acylated insulin derivative according to claim 6, characterized in that, The phenol content is 0-60 mM.

8. The injectable formulation of the long-acting acylated insulin derivative according to claim 2, characterized in that, The injectable formulation further comprises 1-2% (by weight) glycerol, 30-75 mM phenol, 0-30 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 2-8, characterized in that, The pH of the injectable formulation is 6.6-8.

5.

10. The injectable formulation of the long-acting acylated insulin derivative according to claim 9, characterized in that, The pH of the injectable formulation is 6.8-8.

2.

11. A recombinant engineered bacterium for preparing the insulin peptide chain of the long-acting acylated insulin derivative of claim 1, 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 FKFEFKFE, HQHQHQHQHQ, HQHQHQHQHQHQ, HQHQHQHQHQEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK or HQHQHQHQHQHVEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK; the lysine endonuclease digestion sequence is K; and the C peptide is GGGPGRK.

12. The recombinant engineered bacteria according to claim 11, characterized in that, The recombinant engineered bacteria is a recombinant Escherichia coli engineered bacteria.

13. The recombinant engineered bacteria according to claim 12, characterized in that, The recombinant engineered bacteria is recombinant BL21(DE3) Escherichia coli engineered bacteria.

14. The recombinant engineered bacteria according to claim 11, characterized in that, The recombinant plasmid is pET-28a(+), pET-30a(+), or pET-32a(+).

15. The method for preparing the recombinant engineered bacteria according to any one of claims 11-14, 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.

16. Use of the long-acting acylated insulin derivative of claim 1 in the preparation of a medicament for treating diabetes.

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