Insulin analogues and uses thereof

Abstract

This application provides an insulin analogue and its use, the insulin analogue comprising an A chain and a B chain; the sequence of the A chain includes: GIVEQCCTSICSLX aa14 QLENYCN, the B-chain sequence contains: FVNQHLCGSHLVEALY aa16 LVCGEY aa22 GF Y aa25 YTPY aa29 Y aa30 ; where X aa14 Selected from E, Y, K, or D, Y aa16 Selected from Y or H, Y aa22 Selected from Q, S, T, Y, or N, Y aa25 Selected from F, H, or K, Y aa29 Let R and Y be... aa30 It does not exist; the condition is: X aa14 and Y aa25 There is only one K in the insulin analogue. The insulin derivative obtained by the above-mentioned insulin analogue has significantly better in vitro INSR phosphorylation activity and glucose uptake activity than the control drug Icodec. The efficacy (PD) was evaluated using a type 1 diabetic rat model. It has the potential to be administered once a week or less frequently and has a significant hypoglycemic effect.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an insulin analog and its use. Background Technology

[0002] Diabetes is a common chronic metabolic disease that affects the body's ability to regulate blood sugar. The main types of diabetes include type 1 diabetes, type 2 diabetes, and other specific types. Type 1 diabetes is caused by insufficient or complete insulin production and usually occurs in children and adolescents. The key to treating type 1 diabetes is injecting exogenous insulin to control blood sugar levels. Type 2 diabetes is the most common type of diabetes, accounting for more than 90% of all diabetes cases. It usually occurs in adults, especially in overweight or obese individuals. Type 2 diabetes is associated with insulin resistance and insufficient insulin secretion. Treatment methods for type 2 diabetes include improving dietary habits, increasing physical activity, losing weight, oral medications, or insulin injections. Initially, improving diet and increasing physical activity can help improve insulin efficiency and control blood sugar levels. If these measures fail to control blood sugar, oral medications or insulin injections are introduced into the treatment plan to help control blood sugar. Therefore, insulin plays a crucial role in diabetes treatment.

[0003] Insulin is a protein hormone produced by pancreatic β cells, playing a crucial role in regulating blood glucose levels in the human body. Human insulin consists of two chains, A and B, linked together by two disulfide bonds. The A chain contains 21 amino acids, and the B chain contains 30 amino acids. Additionally, the A chain itself contains one disulfide bond. With the development of genetic engineering technology, a series of insulin analogs have been publicly disclosed, prepared by adding, deleting, or replacing one or more amino acid residues based on the natural human insulin sequence. The insulin B16H mutation reported in the literature (Biochemistry, 1994, 33:7998 8006.) reduces its affinity for its insulin receptor but decreases receptor-mediated insulin clearance and prolongs its half-life. In addition, EP0425482B1 discloses an insulin analog with His or Tyr substitution at the B25 position, which has significantly improved activity compared to human insulin; WO2008034881A1 (CN200780043130) discloses a novel insulin analog resistant to proteases, in which at least two hydrophobic amino acids are replaced by hydrophilic amino acids compared to the parent insulin molecule.

[0004] Insulin preparations are typically administered via subcutaneous injection. However, recombinant human insulin has a short half-life, requiring frequent injections and causing significant discomfort to patients. Therefore, the development of insulin preparations with longer durations of action has been ongoing. Currently, various insulin preparations with different durations of action have been developed, including rapid-acting, intermediate-acting, and long-acting insulin preparations. Currently marketed long-acting insulins include detemir insulin and insulin degludec. WO1995007931A1 discloses the structure of detemir insulin, which has a C14 fatty acid chain linked to the B29 lysine residue of deB30 human insulin, allowing it to act for up to 24 hours. WO2005012347A1 discloses the structure of insulin degludec, which has a C16 fatty acid linked to the B29 lysine residue of deB30 human insulin via a glutamate linker, allowing it to act for up to 42 hours, but still requiring once-daily dosing.

[0005] Icodec insulin is an investigational long-acting insulin derivative. Its structure was disclosed in CN102037008A. The molecular design incorporates amino acid mutations—A14E, B16H, and B25H—in human insulin, removing B30 and attaching a C20 fatty acid side chain to the lysine residue at position B29. Compared to detemir and degludec insulin, Icodec has a longer half-life, allowing for once-weekly dosing. However, compared to human insulin, Icodec has 100-fold reduced activity, requiring a relatively higher dosage (Nishimura, Erica et al. (2021)).

[0006] Therefore, current insulin derivatives have not yet met the needs of diabetic patients, and there is still a need to develop more novel insulin analogs and insulin derivatives with activities close to natural human insulin. Summary of the Invention

[0007] In view of some problems existing in the prior art, this application provides an insulin analog and its use, wherein the insulin analog and derivative have significant hypoglycemic effects.

[0008] The specific technical solution of this application is as follows:

[0009] 1. An insulin analog, wherein the insulin analog comprises an A chain and a B chain;

[0010] Chain A contains: GIVEQCCTSICSLX aa14 QLENYCN

[0011] The B-chain sequence contains: FVNQHLCGSHLVEALY aa16LVCGEY aa22 GF Y aa25 YTPY aa29 Y aa30 ;

[0012] Among them, X aa14 Selected from E, Y, K, or D, Y aa16 Selected from Y or H, Y aa22 Selected from Q, S, T, Y, or N, Y aa25 Selected from F, H, or K, Y aa29 Let R and Y be... aa30 It does not exist;

[0013] The condition is: X aa14 and Y aa25 There is exactly one K among them.

[0014] 2. The insulin analogue according to claim 1, wherein,

[0015] X aa14 For E or K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H or K, Y aa29 Let R and Y be... aa30 It does not exist.

[0016] 3. The insulin analogue according to claim 1 or 2, wherein X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 Does not exist; or

[0017] X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 It does not exist.

[0018] 4. An insulin derivative, wherein the insulin derivative has the structure shown in the following formula:

[0019] AM, wherein A is a fatty acid side chain and M is an insulin analogue according to any one of claims 1 to 3;

[0020] The K residue in the insulin analog sequence is linked to A.

[0021] 5. The insulin derivative according to claim 4, wherein the K residue in the insulin analog sequence is linked to A via an amide bond.

[0022] 6. The insulin derivative according to claim 4 or 5, wherein A has the structure shown in the following formula:

[0023] WXY-; where,

[0024] W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22;

[0025] X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp;

[0026] Y is -(OEG) n - where n is an integer from 1 to 3; and

[0027] Y is connected to M.

[0028] Preferably, W is connected to X and X is connected to Y via amide bonds.

[0029] 7. The insulin derivative according to any one of items 4 to 6, wherein A is selected from one of the following structures: Preferred options are:

[0030] Where m is an integer from 18 to 22, and n is an integer from 1 to 3.

[0031] 8. The insulin derivative according to item 6 or 7, wherein m is 18 or 20; and / or

[0032] n is 2.

[0033] 9. The insulin derivative according to any one of items 4 to 8, wherein,

[0034] X in the insulin analog sequence aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30If not present, A is selected from one of the following: eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-βAsp-OEG, eicosidine-βAsp-2xOEG, eicosidine-βAsp-3xOEG, eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2 ...2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu The following are listed: docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, docosyl-γGlu-OEG, docosyl-γGlu-2xOEG, docosyl-γGlu-3xOEG, docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, and docosyl-βAsp-3xOEG, preferably docosyl-γGlu-2xOEG;

[0035] Wherein, X in the insulin analog sequence aa14 The K residue is linked to A via an amide bond.

[0036] 10. The insulin derivative according to any one of claims 4 to 8, wherein X in the insulin analog sequence aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 If not present, A is selected from one of the following: eicosanodiacyl-γGlu-OEG, eicosanodiacyl-γGlu-2xOEG, eicosanodiacyl-γGlu-3xOEG, eicosanodiacyl-βAsp-OEG, eicosanodiacyl-βAsp-2xOEG, eicosanodiacyl-βAsp-3xOEG, docosanodiacyl-γGlu-OEG, docosanodiacyl-γGlu-2xOEG, docosanodiacyl-γGlu-3xOEG, docosanodiacyl-βAsp-O EG, docosacyl-βAsp-2xOEG, docosacyl-βAsp-3xOEG, tetracosacyl-γGlu-OEG, tetracosacyl-γGlu-2xOEG, tetracosacyl-γGlu-3xOEG, tetracosacyl-βAsp-OEG, tetracosacyl-βAsp-2xOEG, and tetracosacyl-βAsp-3xOEG, preferably eicosacyl-γGlu-2xOEG, wherein the Y in the insulin analog sequence... aa25 The K residue is linked to A via an amide bond.

[0037] 11. A method for preparing an insulin derivative according to any one of items 4 to 10, comprising acylation of a K residue in an insulin analog with a fatty acid side chain.

[0038] 12. A conjugate comprising any one of claims 1 to 3 of the insulin analog and a compound for prolonging the half-life of the insulin analog, wherein optionally, the compound for prolonging the half-life of the insulin analog is selected from one or more of the following: fatty acids, albumin, and Fc domains.

[0039] 13. The conjugate according to item 12, wherein the structure of the compound for prolonging the half-life of the insulin analog is as follows:

[0040] WXY-; where,

[0041] W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22;

[0042] X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, or α-D-Asp; and

[0043] Y is -(OEG) n - where n is an integer from 1 to 3;

[0044] Preferably, W is connected to X and X is connected to Y via amide bonds;

[0045] Preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures:

[0046] Preferred options are:

[0047] Where m is an integer from 18 to 22, and n is an integer from 1 to 3;

[0048] More preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures:

[0049] Eicosyl-γGlu-OEG), Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG, Eicosyl-βAsp-2xOEG, Eicosyl-βAsp-3xOEG, Eicosyl-γGlu-OEG, Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG,

[0050] Docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, tetracosyl-γGlu-OEG, tetracosyl-γGlu-2xOEG, tetracosyl-γGlu-3xOEG, tetracosyl-βAsp-OEG, tetracosyl-βAsp-2xOEG, and tetracosyl-βAsp-3xOEG.

[0051] 14. A pharmaceutical composition comprising any one of claims 1 to 3, any one of claims 4 to 10, or any one of claims 12 to 13, an insulin analogue.

[0052] 15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0053] 16. The pharmaceutical composition according to item 14 or 15, wherein the pharmaceutical composition is an injectable formulation.

[0054] 17. Use of any one of the insulin analogs in items 1 to 3, any one of the insulin derivatives in items 4 to 10, or any one of the conjugates in items 12 to 13 in the preparation of medicaments for the treatment or prevention of type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia.

[0055] 18. A method for treating or preventing type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia, comprising administering to a subject in need an effective amount of any one of claims 1 to 3, any one of claims 4 to 10, or any one of claims 12 to 13, an insulin analogue.

[0056] The effects of the invention

[0057] This application uses the insulin analogue to obtain an insulin derivative, whose in vitro INSR phosphorylation activity and glucose uptake activity are significantly superior to the control drug Icodec. The efficacy (PD) was evaluated using a type 1 diabetic rat model, and it has the potential to be administered once a week or less frequently, and has a significant hypoglycemic effect. Attached Figure Description

[0058] Figure 1 This diagram illustrates the determination of compound purity using reduced polyacrylamide gel electrophoresis (SDS-GAGE). The sample information for each lane is as follows: Lane 1: Marker; Lane 2: Compound 1; Lane 3: Precursor protein of Compound 1; Lane 4: Compound 3; Lane 5: Precursor protein of Compound 3; Lane 6: Compound 2; Lane 7: Precursor protein of Compound 2; Lane 8: Human insulin.

[0059] Figures 2A-2C The diagram shows a schematic of the purity determination of a compound using reversed-phase high-performance liquid chromatography (RP-HPLC), in which... Figure 2A , Figure 2B and Figure 2C These are schematic diagrams showing the purity of compounds 1, 2, and 3, respectively.

[0060] Figure 3 A schematic diagram showing the in vitro INSR phosphorylation activity of a compound detected using a chemiluminescence method (AlphaLISA) is presented.

[0061] Figure 4 This diagram illustrates the detection of a compound's glucose uptake activity using a radioactive assay.

[0062] Figure 5 A schematic diagram showing the hypoglycemic curves of the compound in a type 1 diabetic rat model induced by STZ is presented. Detailed Implementation

[0063] The present application will now be described in detail with reference to the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.

[0064] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0065] As used herein, the term insulin refers to insulin obtained or derived from any species (such as mammalian species, especially humans), wherein the natural form is a heterodimeric peptide having two peptide chains (e.g., chain A and chain B) linked by two disulfide bonds, and wherein chain A further has a single intramolecular disulfide bond, chains A and B containing 21 and 30 amino acid residues respectively and linked by two cysteine ​​disulfide bonds. Human insulin is used in this application, wherein the amino acid sequence of chain A of human insulin is shown as SEQ ID NO:1; and the amino acid sequence of chain B is shown as SEQ ID NO:2.

[0066] The amino acid sequence of SEQ ID NO:1 is as follows:

[0067] GIVEQCCTSICSLYQLENYCN

[0068] The amino acid sequence of SEQ ID NO:2 is as follows:

[0069] FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0070] As used herein, the term amino acid residue includes amino acids from which a hydrogen atom has been removed from an amino group and / or from a carboxyl group and / or from a thiol group.

[0071] As used herein, the term "insulin analogue" refers to a compound, such as a peptide or polypeptide, that can induce one or more effects of natural insulin in insulin resistance (IR), but is altered in some way in terms of its amino acid sequence compared to natural insulin due to one or more additions, deletions, insertions, and / or substitutions. Insulin analogues may also include variants of these compounds that are functionally equivalent to natural insulin but have fragments or complete sequences, but which themselves have further additions, deletions, insertions, and / or substitutions. Unless otherwise noted, the amino acid positions of the insulin analogues described herein are based on the corresponding positions in the A chain of SEQ ID NO:1 or the B chain of SEQ ID NO:2 of natural human insulin. In some cases, the insulin analogues described herein may bind to the insulin receptor (INSR) with higher or lower affinity than natural insulin, especially natural human insulin (SEQ ID NO:1 and 2), but exhibit a longer t1 / 2 in vivo or in vitro.

[0072] As used herein, the term "insulin derivative" refers to acylated insulin formed by attaching an acylated group to the ε-amino group of a lysine residue at a position on either the A or B chain of an insulin analog. This is as defined in AM within this application.

[0073] As used herein, the term mutation includes any change in the amino acid sequence (e.g., substitution and insertion with a codeable amino acid, as well as deletion).

[0074] As used herein, the term des indicates the absence of an amino acid residue; for example, for this application, desB30 indicates the absence of the 30th amino acid in the B chain of human insulin.

[0075] As used in this article, OEG is the simplified symbol for the compound NH2(CH2)2O(CH2)2OCH2CO2H.

[0076] As used in this article, γGlu, αGlu, βAsp, and αAsp refer to the simplified symbols for amino acids with the L configuration, while γ-D-Glu, α-D-Glu, β-D-Asp, or α-D-Asp refer to the simplified symbols for amino acids with the D configuration. For example, γGlu is the simplified symbol for the L-configured amino acid γglutamic acid, and γ-D-Glu is the simplified symbol for the D-configured amino acid γglutamic acid.

[0077] As used in this article, A14E refers to the mutation of amino acid Y at position 14 of the human insulin A chain to E; similarly, B16H refers to the mutation of amino acid Y at position 16 of the human insulin B chain to H.

[0078] For example, the insulin derivative of Example 1 (having the sequence / structure given below) named "A14K(Nεeicosanodiacyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin" is a human insulin in which the amino acid Y at position A14 has been mutated to K and modified by acylation of the ε nitrogen (denoted as Nε) of K with eicosanodiacyl-γGlu-2xOEG, the amino acid Y at position B16 has been mutated to H, the amino acid R at position B22 has been mutated to Q, the amino acid F at position B25 has been mutated to H, the amino acid K at position B29 has been mutated to R, and the amino acid T at position B30 has been deleted.

[0079] As used herein, the term "albumin" refers to the most abundant protein in blood plasma, with a molecular weight of approximately 65-67 kilodaltons in its monomeric form, depending on the species from which it is derived. The terms "albumin" and "serum albumin" are used interchangeably, but this does not imply limitation on the source of albumin that forms conjugates with the insulin-stimulating analogues of this application. Therefore, as used herein, the term "albumin" can refer to albumin purified from natural sources such as blood or plasma, or it can refer to chemically synthesized albumin, or albumin produced through recombinant technology.

[0080] As used herein, the term "Fc domain" has the same meaning as "Fc region" or "Fc fragment," referring to the C-terminal region of an immunoglobulin heavy chain capable of binding mammalian Fc(γ) or Fc(Rn) receptors, such as human Fc(γ) or Fc(Rn) receptors. An Fc receptor (FcR) is a receptor that binds to an Fc fragment or Fc region of an antibody. In some embodiments, the FcR is a naturally occurring human FcR sequence. In some embodiments, the FcR binds to IgG antibodies (γ receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. In some embodiments, the Fc fragment, region, or domain may be a naturally occurring Fc region sequence. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of the human IgG heavy chain is generally defined as extending from an amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The residues in the Fc region are numbered using the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. In some embodiments, the Fc fragment contains or is composed of the Fc region of mammalian IgG (e.g., the CH2 and CH3 domains), such as human IgG. In some embodiments, the Fc fragment contains or is composed of the Fc region of human IgG1 (e.g., the CH2 and CH3 domains). In some embodiments, the Fc fragment comprises or consists of an amino acid sequence that has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99% or more) identity with the Fc region (e.g., the CH2 and CH3 domains) of human IgG1.

[0081] As used herein, the term "half-life" or "t1 / 2" refers to the time it takes for half the amount of a compound (such as the insulin analogue in this application) to be removed from an individual's fluid or other physiological space (such as serum or plasma) by biological processes. Alternatively, t1 / 2 may also refer to the time it takes for a given amount of such a compound to lose half of its pharmacological, physiological, or radiological activity.

[0082] As used in this article, the term "pharmaceutically acceptable" means suitable for normal drug use, i.e., it will not produce serious adverse events in the patient.

[0083] As used herein, the term "treatment" refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom. This term is intended to include: delaying the progression of the disease, obstacle, or symptom; alleviating or mitigating symptoms and complications; and / or curing or eliminating the disease, obstacle, or symptom. Patients to be treated are preferably mammals, and especially humans.

[0084] As used herein, the term “prevention” refers to the management and care of an individual at risk of developing a disease prior to its clinical onset. The purpose of prevention is to counteract the progression of a disease, condition, or disorder, and includes the administration of active compounds to prevent or delay the onset of symptoms or complications, and to prevent or delay the progression of the associated disease, condition, or disorder.

[0085] As used in this article, the term "effective dose" refers to: an adequate dose that effectively treats a patient compared to no treatment.

[0086] As used herein, the term “diabetes” includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas produces insufficient amounts of insulin, or in which the body’s cells do not respond adequately to insulin, thus preventing the cells from absorbing glucose, resulting in glucose accumulating in the blood.

[0087] As used in this article, the term "Type 1 diabetes," also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by the destruction of beta cells and typically results in absolute insulin deficiency. The term "Type 2 diabetes," also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance and consequently relative insulin deficiency and / or major insulin secretion defects with insulin resistance.

[0088] As used herein, the term "hyperglycemia" refers to a blood glucose concentration higher than normal fasting blood glucose, typically 126 mg / dL or higher. In some studies, a hyperglycemic episode is defined as a blood glucose concentration exceeding 280 mg / dL (15.6 mM). Examples include, but are not limited to, type 1 diabetes (or insulin-dependent diabetes), type 2 diabetes (or non-insulin-dependent diabetes), impaired glucose tolerance, insulin resistance, hyperlipidemia, hypercholesterolemia, dyslipidemia, syndrome X, metabolic syndrome, obesity, hypertension, or atherosclerosis. It also includes drug-induced hyperglycemia, such as that caused by long-term use of corticosteroids, Zyprexa, octreotide, etc.; and endocrine disorders of the thyroid, adrenal glands, and pituitary gland, such as Cushing's disease and pancreatitis.

[0089] Insulin analog

[0090] This application provides an insulin analogue, wherein the insulin analogue comprises an A chain and a B chain;

[0091] Chain A contains: GIVEQCCTSICSLX aa14 QLENYCN

[0092] The B-chain sequence contains: FVNQHLCGSHLVEALY aa16 LVCGEY aa22 GF Y aa25 YTPY aa29 Y aa30 ;

[0093] Among them, X aa14 Selected from E, Y, K, or D, Y aa16 Selected from Y or H, Y aa22 Selected from Q, S, T, Y, or N, Y aa25 Selected from F, H, or K, Y aa29 Let R and Y be... aa30 It does not exist;

[0094] The condition is: X aa14 and Y aa25 There is exactly one K among them.

[0095] In this application, the insulin analogue is obtained by mutation based on human primitive insulin. This application does not limit the mutation method in any way; it can be performed using methods conventional in the art. Furthermore, this application counts from the N-terminus, for Xaa in the A chain... 14 This refers to the 14th amino acid, counted from the N-terminus of the A chain of insulin analogs.

[0096] In this application, the A chain and B chain of the insulin derivative are respectively relative to the A chain and B chain of human insulin.

[0097] In some implementations, X aa14 For E or K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H or K, Y aa29 Let R and Y be... aa30 It does not exist. In some implementations, X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 Does not exist; or

[0098] X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 It does not exist.

[0099] Insulin derivative

[0100] This application provides an insulin derivative having the structure shown in the following formula:

[0101] AM, where A is a fatty acid side chain and M is the insulin analogue described above;

[0102] The K residue in the insulin analog sequence is linked to A. In some embodiments, the K residue in the insulin analog sequence is linked to A via an amide bond.

[0103] In some implementations, A has the structure shown in the following formula:

[0104] WXY-;

[0105] W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22;

[0106] X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp;

[0107] Y is -(OEG) n - where n is an integer from 1 to 3; and

[0108] Y is connected to M.

[0109] Preferably, W is connected to X and X is connected to Y via amide bonds.

[0110] m can be, for example, an integer from 18 to 21, an integer from 18 to 20, an integer from 18 to 19, an integer from 19 to 22, an integer from 19 to 21, an integer from 19 to 20, an integer from 20 to 22, an integer from 20 to 21, an integer from 21 to 22, and so on.

[0111] n can be an integer from 1 to 2, an integer from 2 to 3, etc.

[0112] For example, m can be 18, 19, 20, 21, or 22; n can be 1, 2, or 3.

[0113] In some implementations, A is selected from one of the following structures:

[0114]

[0115] Preferred options are:

[0116]

[0117] Where m is an integer from 18 to 22, and n is an integer from 1 to 3. In some implementations, m is 18 or 20; and / or

[0118] n is 2.

[0119] In some embodiments, X in the insulin analog sequence aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 If not present, A is selected from one of the following: eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-βAsp-OEG, eicosidine-βAsp-2xOEG, eicosidine-βAsp-3xOEG, eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2 ...2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu The following are listed: docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, docosyl-γGlu-OEG, docosyl-γGlu-2xOEG, docosyl-γGlu-3xOEG, docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, and docosyl-βAsp-3xOEG, preferably docosyl-γGlu-2xOEG;

[0120] Wherein, X in the insulin analog sequence aa14 The K residue is linked to A via an amide bond. In some embodiments, X in the insulin analog sequence aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30If not present, A is selected from one of the following: eicosanodiacyl-γGlu-OEG, eicosanodiacyl-γGlu-2xOEG, eicosanodiacyl-γGlu-3xOEG, eicosanodiacyl-βAsp-OEG, eicosanodiacyl-βAsp-2xOEG, eicosanodiacyl-βAsp-3xOEG, docosanodiacyl-γGlu-OEG, docosanodiacyl-γGlu-2xOEG, docosanodiacyl-γGlu-3xOEG, docosanodiacyl-βAsp-O EG, docosacyl-βAsp-2xOEG, docosacyl-βAsp-3xOEG, tetracosacyl-γGlu-OEG, tetracosacyl-γGlu-2xOEG, tetracosacyl-γGlu-3xOEG, tetracosacyl-βAsp-OEG, tetracosacyl-βAsp-2xOEG, and tetracosacyl-βAsp-3xOEG, preferably eicosacyl-γGlu-2xOEG, wherein the Y in the insulin analog sequence... aa25 The K residue is linked to A via an amide bond.

[0121] Where X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-γGlu-OEG, and insulin derivatives can be represented as: A14K(Nεeicosanodiacyl-γGlu-OEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0122] Similarly, when X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14K (Nε eicosanodiacyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0123] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30When not present, A is eicosanodiacyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14K (Nε eicosanodiacyl-γGlu-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0124] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-OEG, and insulin derivatives can be represented as: A14K(Nεeicosanodiacyl-βAsp-OEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0125] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14K(Nεeicosanodiacyl-βAsp-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0126] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14K(Nεeicosanodiacyl-βAsp-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0127] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-OEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-γGlu-OEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0128] When Xaa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0129] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-γGlu-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0130] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-βAsp-OEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-βAsp-OEG), B16H, B22Q, B25H, B29R, and DesB30 human insulin.

[0131] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-βAsp-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0132] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30When not present, A is docosacyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14K(Nε docosacyl-βAsp-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0133] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-OEG, and insulin derivatives can be represented as: A14K(Nε tetracosyl-γGlu-OEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0134] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14K(Nε tetracosyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0135] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14K(Nε tetracosyl-γGlu-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin.

[0136] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-βAsp-OEG, and insulin derivatives can be represented as: A14K (Nε tetracosyl-βAsp-OEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0137] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14K(Nε tetracosyl-βAsp-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0138] When X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14K(Nε tetracosyl-βAsp-3xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin;

[0139] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-γGlu-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-γGlu-OEG), DesB30 human insulin;

[0140] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-γGlu-2xOEG), DesB30 human insulin;

[0141] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30When not present, A is eicosanodiacyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-γGlu-3xOEG), DesB30 human insulin;

[0142] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-βAsp-OEG), DesB30 human insulin;

[0143] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-βAsp-2xOEG), DesB30 human insulin;

[0144] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is eicosanodiacyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε eicosanodiacyl-βAsp-3xOEG), DesB30 human insulin;

[0145] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-γGlu-OEG), DesB30 human insulin;

[0146] When Xaa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-γGlu-2xOEG), DesB30 human insulin;

[0147] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-γGlu-3xOEG), DesB30 human insulin;

[0148] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-βAsp-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-βAsp-OEG), DesB30 human insulin;

[0149] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is docosacyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-βAsp-2xOEG), DesB30 human insulin;

[0150] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30When not present, A is docosacyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε docosacyl-βAsp-3xOEG), DesB30 human insulin;

[0151] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosyl-γGlu-OEG), DesB30 human insulin;

[0152] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosyl-γGlu-2xOEG), DesB30 human insulin;

[0153] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-γGlu-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosyl-γGlu-3xOEG), DesB30 human insulin;

[0154] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-βAsp-OEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosyl-βAsp-OEG), DesB30 human insulin;

[0155] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When absent, A is tetracosanodiacyl-βAsp-2xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosanodiacyl-βAsp-2xOEG), DesB30 human insulin and

[0156] When X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 When not present, A is tetracosyl-βAsp-3xOEG, and insulin derivatives can be represented as: A14E, B16H, B22Q, B29R, B25K (Nε tetracosyl-βAsp-3xOEG), DesB30 human insulin;

[0157] Preferred

[0158] A14K (Nε-eicosanodicyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin or A14E, B16H, B22Q, B29R, B25K (Nε-eicosanodicyl-γGlu-2xOEG), DesB30 human insulin.

[0159] This application provides a method for preparing any of the above-mentioned insulin derivatives, comprising reacting an insulin analog with an adipose side chain via acylation.

[0160] In this application, the acylation conditions for preparing insulin derivatives are all conventional acylation conditions in the art, and this application does not impose any restrictions. Those skilled in the art can react insulin analogs with fatty side chains to obtain the insulin derivatives described in this application using conventional acylation conditions.

[0161] Conjugate

[0162] This application provides a conjugate comprising any one of the insulin analogs described above and a compound for extending the half-life of the insulin analog, optionally, the compound for extending the half-life of the insulin analog is selected from one or more of the following: fatty acids, albumin, and Fc domains. In some embodiments, the structure of the compound for extending the half-life of the insulin analog is as follows:

[0163] WXY-; where,

[0164] W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22;

[0165] X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, or α-D-Asp; and

[0166] Y is -(OEG) n - where n is an integer from 1 to 3;

[0167] Preferably, W is connected to X and X is connected to Y via amide bonds;

[0168] Preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures:

[0169] Preferred options are:

[0170] Where m is an integer from 18 to 22, and n is an integer from 1 to 3;

[0171] More preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures:

[0172] Eicosyl-γGlu-OEG), Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG, Eicosyl-βAsp-2xOEG, Eicosyl-βAsp-3xOEG, Eicosyl-γGlu-OEG, Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG,

[0173] Docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, tetracosyl-γGlu-OEG, tetracosyl-γGlu-2xOEG, tetracosyl-γGlu-3xOEG, tetracosyl-βAsp-OEG, tetracosyl-βAsp-2xOEG, and tetracosyl-βAsp-3xOEG.

[0174] Pharmaceutical composition

[0175] This application provides a pharmaceutical composition comprising any of the insulin analogs, insulin derivatives, or conjugates described in any of the preceding claims.

[0176] The pharmaceutical composition can be prepared using methods known in the art, namely by using similar excipients commonly used in human insulin compositions.

[0177] The pharmaceutical composition described herein can be prepared using conventional pharmaceutical industry techniques, which include, where appropriate, dissolving and mixing the components to obtain the desired final product. Thus, according to one technique, the aforementioned insulin derivative is dissolved in a volume of water slightly less than the final volume of the pharmaceutical composition to be prepared, excipients (isotonic agents, preservatives, and buffers) are added as needed, and, if necessary, the pH of the solution is adjusted as needed using an acid such as hydrochloric acid or a base such as an aqueous sodium hydroxide solution. Finally, the volume of the solution is adjusted with water to obtain the desired concentration of the component.

[0178] In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is an injectable formulation.

[0179] The term "excipient" broadly refers to any component other than the active therapeutic ingredient. Excipients can be inert substances, inactive substances, and / or substances without pharmaceutical activity.

[0180] Excipients can be used for a variety of purposes, depending on the pharmaceutical composition itself, such as as carriers, solvents, diluents, tablet excipients, and / or to improve the delivery and / or absorption of the active substance. Examples of excipients include, but are not limited to, diluents, buffers, preservatives, tension modifiers (also known as tension modifiers or isotonic agents), chelating agents, surfactants, protease inhibitors, wetting agents, emulsifiers, antioxidants, fillers, metal ions, oily solvents, proteins and / or zwitterions, and stabilizers.

[0181] Treatment or prevention of disease

[0182] This application provides the use of any of the above-described insulin analogs, insulin derivatives, or conjugates in the preparation of medicaments for the treatment or prevention of type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia.

[0183] This application also provides a method for treating or preventing type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia, comprising administering an effective amount of any of the above-described insulin analogs, insulin derivatives, or conjugates to a subject in need.

[0184] This application also provides the use of any of the above-mentioned insulin analogs, insulin derivatives, or conjugates in the treatment or prevention of type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia.

[0185] In this application, the administration method for insulin derivatives or pharmaceutical compositions can be determined based on the patient's general knowledge and in conjunction with the physician's general knowledge. The administration method is preferably chosen according to the patient's convenience. Therefore, the final administration method depends on both the product's efficacy and the patient's intentions and preferences. This is due to the fact that the effectiveness of any insulin product depends on the individual patient's insulin needs, the patient's sensitivity to the effects of said insulin in a given situation, and ultimately, other preferences. These factors may change over time, both over longer periods (years) and daily. The optimal dose level for any patient will depend on a variety of factors, including the patient's age, weight, physical activity, and diet; combinations with other medications; and the severity of the condition being treated.

[0186] Example

[0187] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0188] Example 1: Preparation of Compound 1

[0189] Compound 1, namely A14K(N ε Eicosanodiacyl-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin, named N-{ε-A14}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(19-carboxynonadenylatedamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[HisB16, GlnB22, HisB25, ArgB29], des-Thr B30-insulin (human), structure:

[0190]

[0191] The preparation method is as follows:

[0192] (1) Preparation of precursor protein of compound 1

[0193] The precursor protein was prepared using well-known techniques in the art, including vector construction, *E. coli* expression, and renaturation. In simple terms, the gene was synthesized based on the protein's amino acid sequence and codon optimization, inserted into an expression vector, transformed into competent *E. coli* cells, and positive transformants were obtained through antibiotic screening. The precursor protein was then induced to express in a fermenter. After cell disruption using a homogenizer, inclusion bodies were harvested. Following denaturation and renaturation of the inclusion bodies, the mature precursor protein was obtained through separation. The amino acid sequence of the precursor protein of compound 1 is shown in SEQ ID NO. 3.

[0194] SEQ ID NO.3:

[0195] FVNQHLCGSHLVEALHLVCGEQGFHYTPRAARGIVEQCCTSICSLKQLENYCN

[0196] (2) Preparation of compound 1

[0197] Take the precursor protein prepared in step (1) of Example 1, prepare a solution with a concentration of 0.7 mg / mL, adjust the pH to 11.4, weigh out the eicosanodiacyl-γGlu-2xOEG-OSu dry powder according to the molar ratio of precursor protein to eicosanodiacyl-γGlu-2xOEG-OSu (manufacturer: Xiamen Sainobang) 1:10, dissolve it in N-methylpyrrolidone (manufacturer: Sinopharm Shanghai Experimental), and then add it to the protein solution. React for 30 min. Then adjust the pH to 8.0, add trypsin (manufacturer: Hangzhou Putai), and digest overnight at room temperature.

[0198] The enzyme digestion buffer was purified using Capto Q (manufacturer: Cytiva), washed with 15% B (solution A: 20mM Tris-HCl pH 8.0; solution B: 20mM Tris-HCl 1M NaCl pH 8.0), and eluted with 20% B. The purity and molecular weight of the eluent were determined by RP-HPLC (manufacturer: Waters) and LC-MS (manufacturer: Waters). The purity reached 90%, and the measured molecular weight was 6375.2, consistent with the theoretical molecular weight of 6375.03, yielding compound 1. The eluent was concentrated, replaced with DPBS, and stored at -20℃.

[0199] The protein amino acid sequence of compound 1 is shown in SEQ ID NO.4 and SEQ ID NO.5, that is, the precursor protein is digested by enzymes to obtain the amino acid sequence as shown in SEQ ID NO.4 and SEQ ID NO.5.

[0200] SEQ ID NO.4:

[0201] GIVEQCCTSICSLKQLENYCN

[0202] SEQ ID NO.5:

[0203] FVNQHLCGSHLVEALHLVCGEQGFHYTPR

[0204] Wherein, SEQ ID NO.4 represents the amino acid sequence of chain A of compound 1, and SEQ ID NO.5 represents the amino acid sequence of chain B of compound 1.

[0205] Example 2 Preparation of Compound 2

[0206] Compound 2, namely A14E, B16H, B22Q, B29R, B25K(N) ε Eicosanoyl-γGlu-2xOEG), DesB30 human insulin, has the following name:

[0207] N-{ε-B25}-[2-[2-[2-[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[GluA14, HisB16, GlnB22, ArgB29],des-Thr B30-insulin (human).

[0208] The structure is as follows:

[0209]

[0210] The preparation method was the same as in Example 1. The purity and molecular weight of the eluent were determined by RP-HPLC (manufacturer: Waters) and LC-MS (manufacturer: Waters). The purity reached 90%, and the measured molecular weight was 6367.1, which is consistent with the theoretical molecular weight of 6367.01. The amino acid sequence of the precursor protein of compound 2 is shown in SEQ ID NO.6.

[0211] SEQ ID NO.6:

[0212] FVNQHLCGSHLVEALHLVCGEQGFKYTPRAARGIVEQCCTSICSLEQLENYCN

[0213] The protein amino acid sequence of compound 2 is shown in SEQ ID NO.7 and SEQ ID NO.8.

[0214] SEQ ID NO.7:

[0215] GIVEQCCTSICSLEQLENYCN

[0216] SEQ ID NO.8:

[0217] FVNQHLCGSHLVEALHLVCGEQGFKYTPR

[0218] Wherein, SEQ ID NO.7 represents the amino acid sequence of chain A of compound 2, and SEQ ID NO.8 represents the amino acid sequence of chain B of compound 2.

[0219] Example 3 Preparation of Compound 3

[0220] Compound 3, namely A14K(N ε Dodecanoic acid-γGlu-2xOEG), B16H, B22Q, B25H, B29R, DesB30 human insulin, named N-{ε-A14}-[2-[2-[2-[2-[2-[2-[(4S)-4-carboxy-4-(21-carboxytetraalkylamino)butyryl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-[HisB16, GlnB22, HisB25, ArgB29], des-Thr B30-insulin (human), structure:

[0221]

[0222] The preparation method was the same as in Example 1. The purity and molecular weight of the eluent were determined by RP-HPLC (manufacturer: Waters) and LC-MS (manufacturer: Waters). The purity reached 90%, and the measured molecular weight was 6403.2, which is consistent with the theoretical molecular weight of 6403.06. The fatty acid side chain was docosanodiacyl-γGlu-2xOEG-OSu.

[0223] Comparative Example 1: Preparation of the control drug Icodec

[0224] The precursor protein of the control drug Icodec (CN102037008A) was prepared according to the preparation method of the precursor protein of compound 1 in Example 1. After obtaining the mature refolded protein, the refolded protein was digested with recombinant lysine endonuclease (manufacturer: Hangzhou Putai) at room temperature, and the precursor protein was obtained by separation. Fatty acid modification was performed according to the preparation method of compound 1 in Example 1. After modification, it was purified by Capto Q (manufacturer: Cytiva), washed with 10% B (solution A: 20mM Tris-HCl pH 8.0; solution B: 20mM Tris-HCl 1M NaCl pH 8.0), and eluted with 15% B. The purity and molecular weight of the eluent were detected by RP-HPLC (manufacturer: Waters) and LC-MS (manufacturer: Waters). The purity reached 90%, and the measured molecular weight was 6376.1, which is consistent with the theoretical molecular weight of 6376.01. The control drug Icodec was obtained. The eluent was concentrated and replaced with DPBS, and then stored at -20℃.

[0225] The amino acid sequence of the precursor protein of Icodec is shown in SEQ ID NO.9.

[0226] SEQ ID NO.9:

[0227] FVNQHLCGSHLVEALHLVCGERGFHYTPKAAKGIVEQCCTSICSLEQLENYCN

[0228] The protein amino acid sequence of Icodec is shown in SEQ ID NO.10 and SEQ ID NO.11.

[0229] SEQ ID NO.10:

[0230] GIVEQCCTSICSLEQLENYCN

[0231] SEQ ID NO.11:

[0232] FVNQHLCGSHLVEALHLVCGERGFHYTPK

[0233] In this sequence, SEQ ID NO.10 represents the amino acid sequence of the A chain of Icodec, and SEQ ID NO.11 represents the amino acid sequence of the B chain of Icodec.

[0234] Example 4: Analysis by reducing polyacrylamide gel electrophoresis (SDS-PAGE)

[0235] Take 80 μl of the samples from Examples 1-3 and human insulin, mix with 20 μl of 5x Loading Buffer, heat at 100°C for 10 min, and centrifuge at 12000 rpm for 3-5 min to obtain the electrophoresis samples.

[0236] Pre-prepared protein gels (4%–20% by GenScript) and Tris-MES-SDS electrophoresis buffer (by GenScript) were used, and electrophoresis was performed at 200V for 30 minutes. After electrophoresis, the protein gels were peeled off, washed with purified water, stained with Coomassie Brilliant Blue rapid staining solution, destained with purified water, and photographed for recording. The results are shown below. Figure 1 As shown, lane 1: Marker; lane 2: Compound 1; lane 3: Precursor protein of Compound 1; lane 4: Compound 3; lane 5: Precursor protein of Compound 3; lane 6: Compound 2; lane 7: Precursor protein of Compound 2; lane 8: Human insulin.

[0237] Example 5: Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis

[0238] (1) Precursor protein RP-HPLC detection method:

[0239] Chromatographic column: Peptide BEH C18, 3.5μm, 4.6×250mm, 1 / pkg;

[0240] Mobile phase A: 100% H2O + 0.2% trifluoroacetic acid (TFA);

[0241] Mobile phase B: 90% acetonitrile (ACN) + 10% isopropanol (IPA) + 0.2% trifluoroacetic acid (TFA);

[0242] Perform gradient elution according to Table 1:

[0243] Table 1

[0244]

[0245]

[0246] Injection temperature: 4℃, column temperature: 40℃;

[0247] Detection wavelengths: 280nm and 214nm.

[0248] (2) Fatty acid modification enzymatic digestion RP-HPLC detection method:

[0249] Chromatographic column: Peptide BEH C18, 3.5μm, 4.6×250mm, 1 / pkg;

[0250] Mobile phase A: 100% H2O + 0.2% trifluoroacetic acid (TFA);

[0251] Mobile phase B: 90% acetonitrile (ACN) + 10% isopropanol (IPA) + 0.2% trifluoroacetic acid (TFA);

[0252] Perform gradient elution according to Table 2:

[0253] Table 2

[0254]

[0255] Injection temperature: 4℃, column temperature: 40℃;

[0256] Detection wavelengths: 280nm and 214nm;

[0257] The RP-HPLC purity results of each compound after fatty acid modification and enzymatic digestion purification are as follows: Figures 2A-2C As shown, where Figure 2A This is a schematic diagram showing the purity of compound 1. Figure 2B This is a schematic diagram showing the purity of compound 2. Figure 2C This is a schematic diagram showing the purity of compound 3.

[0258] Example 6: In vitro insulin receptor (INSR) phosphorylation assay

[0259] The in vitro INSR phosphorylation activity of the samples was detected using a chemiluminescence immunoassay (AlphaLISA).

[0260] First, stably transfected CHO INSR 1284 cells (purchased from ATCC (CRL-3307)) were cultured overnight in 384-well cell culture plates. After removing the culture medium, 25 μl of serially diluted test sample was added, and the cells were incubated at room temperature before removing the sample. 25 μl of lysis buffer (purchased from Promega, catalog number: E194A) was added to lyse the cells and terminate the reaction. The cell lysis buffer was transferred to Optiplate 384-well plates, and 5 μl of recipient microbead solution (purchased from PerkinElmer) was added. After sealing the plates, the plates were rotated at 1000 rpm for 1 minute and incubated at room temperature for 1 hour. 5 μl of donor microbead solution (purchased from PerkinElmer) was added. After sealing the plates, the plates were rotated at 1000 rpm for 1 minute and incubated at room temperature for 1 hour. Fluorescence signals were measured using the Envision follow AlphaScreen model (which is the AlphaScreen mode of the PerkinElmer Envision instrument). The EC50 of each sample was calculated using the GraphPad Prism5 software with the function "Dose-response-Stimulation—log[agonist] vs. response-variable slope". The results are shown in Table 3 and 4 respectively. Figure 3 As shown.

[0261] The in vitro INSR phosphorylation activity assay results for human insulin, compound 1, compound 2, compound 3, and the control drug Icodec are as follows: Figure 3 As shown in Table 3.

[0262] Table 3

[0263] Human insulin lcodec Compound 1 Compound 2 Compound 3 log(agonist) vs. response - Variable slope Best-fit values Bottom ＝0.000 ＝0.000 ＝0.000 ＝0.000 ＝0.000 Top 103.9 107.5 110.5 93.27 108.3 LogEC50 0.7133 2.802 2.136 3.398 2.494 Hill Slope 1.197 1.002 0.7305 0.9379 0.8883 EC50 5.168 633.9 136.9 2502 312.0

[0264] from Figure 3 As shown in Table 3, the in vitro INSR phosphorylation activity of Icodec in this experiment was approximately 100 times lower than that of human insulin, which is consistent with the results reported in the literature (Nishimura, Erica et al. (2021)). Furthermore, the in vitro INSR phosphorylation activities of compounds 1 and 3 of this application are significantly superior to the control drug Icodec, and are closer to natural human insulin, potentially reducing the dosage.

[0265] Example 7: Determination of glucose uptake activity

[0266] The activity of the samples in glucose uptake was detected using a radioactive assay.

[0267] First, 3T3-L1 (ATCC) adipocytes cultured in 96-well plates were replaced with FBS-free DMEM (Thermo Fisher) medium and starved for 2 hours. After washing the 3T3-L1 adipocytes with 200 μL of assay buffer (5 mM Na2HPO4, 20 mM HEPES, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, 0.1% casein, pH 7.4), 50 μL of serially diluted 2× sample was added to each well, and the plates were incubated at 37°C for 10 minutes in a 5% CO2 incubator. Then, 50 μL of 2× [3H]-deoxy-D-glucose (Revvity) solution was added to each well, and the plates were incubated at 37°C for 20 minutes in a 5% CO2 incubator. The solution in the wells was removed, and the plates were washed with cold DPBS to stop the reaction. After lysing adipocytes with 10% NaOH, the lysate was transferred to a scintillation tube (manufacturer: Revvity), and 2 mL of scintillation fluid (manufacturer: Revvity) was added to the tube. Radioactivity was read using a Tri-Carb instrument (manufacturer: PerkinElmer). The EC50 of each sample was calculated using GraphPadPrism 5 software under the function "Dose-response-Stimulation—log[agonist]vs.response--Variableslope". The results are shown in Table 4 and [Table 5 is missing from the original text]. Figure 4 As shown.

[0268] The results of glucose uptake activity assays for human insulin, compound 1, compound 2, compound 3, and the control drug Icodec are as follows: Figure 4 As shown in Table 4.

[0269] Table 4

[0270] lcodec Compound 1 Compound 2 Compound 3 Human insulin log(agonist) vs. response - Variable slope Best-fit values Bottom ＝0.000 ＝0.000 ＝0.000 ＝0.000 ＝0.000 Top 124.3 113.9 107.2 114.0 124.5 LogEC50 1.818 0.4373 2.299 1.020 -0.3371 Hill Slope 0.5308 0.6194 0.6907 0.6211 0.5497 EC50 65.75 2.737 199.3 10.46 0.4601

[0271] from Figure 4 As can be seen from Table 4, compounds 1 and 3 of this application have significantly better glucose uptake activity than the control drug Icodec.

[0272] Example 8: Hypoglycemic effects of different compounds in streptozotocin (STZ)-induced type 1 diabetic rats

[0273] (1) Rat modeling

[0274] Healthy male Sprague-Dawley rats, 6–8 weeks old, 180–220g, were used to establish a rat model of type 1 diabetes by intraperitoneal injection of STZ (Maclean's reagent, 55mg / kg) after a 3-day acclimatization period. The model was considered established when blood glucose levels reached 16.7 mmol / L or higher.

[0275] (2) Administration to rats

[0276] The rats were grouped to provide comparable changes in blood glucose and body weight, and then randomly divided into 7 groups of 5 rats each. The dosage and volume of drugs administered to each group are shown in Table 5. The appropriate solvent (DPBS) or drug was administered subcutaneously (SC) as a single dose. Animals had free access to food and water during the experiment.

[0277] Table 5

[0278] Group Drug Dose nmol / animal Dosing volume μl Dosing method Dosing frequency Model control group DPBS / 300 S.C. 1 Positive control group 1 Icodec 100 300 S.C. 1 Positive control group 2 Icodec 200 300 S.C. 1 Experimental group 1 Compound 1 100 300 S.C. 1 Experimental group 2 Compound 1 200 300 S.C. 1 Experimental group 3 Compound 3 100 300 S.C. 1 Experimental group 4 Compound 3 200 300 S.C. 1

[0279] (3) Detection indicators

[0280] Blood glucose levels were measured at 0, 2, 4, 6, 24, 48, 72 and 96 hours after drug administration.

[0281] (4) Experimental Results

[0282] The blood glucose change curves after administration of each compound are as follows: Figure 5 As shown. From Figure 5 It can be seen that the hypoglycemic effect of Icodec, compound 1, and compound 3 all increased with increasing dosage, showing a clear dose-response relationship. At all dosages, the hypoglycemic nadir of compound 1 was 2–6 hours after administration, while that of Icodec and compound 3 was 24–48 hours after administration. At the same dosage, the hypoglycemic nadir of compound 1 was lower than that of Icodec and compound 3, but the blood glucose recovery was earlier for compound 1 than for Icodec and compound 3, while the blood glucose recovery trend of compound 3 was significantly slower than that of Icodec. In conclusion, compound 3 is superior to the control Icodec in hypoglycemic effect and has the potential for administration once a week or less frequently.

[0283] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. An insulin analogue, wherein, The insulin analogue comprises an A chain and a B chain; Chain A contains: GIVEQCCTSICSLX aa14 QLENYCN The B-chain sequence contains: FVNQHLCGSHLVEALY aa16 LVCGEY aa22 GF Y aa25 YTPY aa29 Y aa30 ; Among them, X aa14 Selected from E, Y, K, or D, Y aa16 Selected from Y or H, Y aa22 Selected from Q, S, T, Y, or N, Y aa25 Selected from F, H, or K, Y aa29 Let R and Y be... aa30 It does not exist; The condition is: X aa14 and Y aa25 There is exactly one K among them.

2. The insulin analogue according to claim 1, wherein, X aa14 For E or K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H or K, Y aa29 Let R and Y be... aa30 It does not exist; Preferably, X aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 Does not exist; or X aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 It does not exist.

3. An insulin derivative, wherein the insulin derivative has the structure shown in the following formula: AM, wherein A is a fatty acid side chain and M is an insulin analogue according to any one of claims 1 to 2; The K residue in the insulin analog sequence is linked to A.

4. The insulin derivative according to claim 3, wherein, The K residue in the insulin analog sequence is linked to A via an amide bond; Preferably, A has the structure shown in the following formula: WXY-; where, W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22; X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp or α-D-Asp; Y is -(OEG) n - where n is an integer from 1 to 3; and Y is connected to M. Preferably, W is connected to X and X is connected to Y via amide bonds. Preferably, A is selected from one of the following structures: Preferred options are: Where m is an integer from 18 to 22, and n is an integer from 1 to 3; Preferably, m is 18 or 20; and / or n is 2.

5. The insulin derivative according to any one of claims 3 to 4, wherein, X in the insulin analog sequence aa14 For K, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For H, Y aa29 Let R and Y be... aa30 If not present, A is selected from one of the following: eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-βAsp-OEG, eicosidine-βAsp-2xOEG, eicosidine-βAsp-3xOEG, eicosidine-γGlu-OEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2 ...2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu-2xOEG, eicosidine-γGlu-3xOEG, eicosidine-γGlu The following are listed: docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, docosyl-γGlu-OEG, docosyl-γGlu-2xOEG, docosyl-γGlu-3xOEG, docosyl-βAsp-OEG, docosyl-βAsp-2xOEG, and docosyl-βAsp-3xOEG, preferably docosyl-γGlu-2xOEG; Wherein, X in the insulin analog sequence aa14 The K residue is linked to A via an amide bond.

6. The insulin derivative according to any one of claims 3 to 4, wherein, X in the insulin analog sequence aa14 For E, Y aa16 For H, Y aa22 Let Q and Y be the values ​​of Q and Y respectively. aa25 For K, Y aa29 Let R and Y be... aa30 If not present, A is selected from one of the following: eicosanodiacyl-γGlu-OEG, eicosanodiacyl-γGlu-2xOEG, eicosanodiacyl-γGlu-3xOEG, eicosanodiacyl-βAsp-OEG, eicosanodiacyl-βAsp-2xOEG, eicosanodiacyl-βAsp-3xOEG, docosanodiacyl-γGlu-OEG, docosanodiacyl-γGlu-2xOEG, docosanodiacyl-γGlu-3xOEG, docosanodiacyl-βAsp-O EG, docosacyl-βAsp-2xOEG, docosacyl-βAsp-3xOEG, tetracosacyl-γGlu-OEG, tetracosacyl-γGlu-2xOEG, tetracosacyl-γGlu-3xOEG, tetracosacyl-βAsp-OEG, tetracosacyl-βAsp-2xOEG, and tetracosacyl-βAsp-3xOEG, preferably eicosacyl-γGlu-2xOEG, wherein the Y in the insulin analog sequence... aa25 The K residue is linked to A via an amide bond.

7. A method for preparing an insulin derivative according to any one of claims 3 to 6, comprising acylation of a K residue in an insulin analog with a fatty acid side chain.

8. A conjugate comprising any one of claims 1 to 2 of an insulin analog and a compound for prolonging the half-life of the insulin analog, wherein, optionally, the compound for prolonging the half-life of the insulin analog is selected from one or more of the following: fatty acids, albumin, and Fc domains; Preferably, the structure of the compound used to prolong the half-life of the insulin analog is as follows: WXY-; where, W stands for -CO(CH2) m COOH, where m is an integer between 18 and 22; X is an amino acid residue, preferably γGlu, αGlu, βAsp, αAsp, γ-D-Glu, α-D-Glu, β-D-Asp, or α-D-Asp; and Y is -(OEG) n - where n is an integer from 1 to 3; Preferably, W is connected to X and X is connected to Y via amide bonds; Preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures: Preferred options are: Where m is an integer from 18 to 22, and n is an integer from 1 to 3; More preferably, the compound used to prolong the half-life of the insulin analog is selected from one of the following structures: Eicosyl-γGlu-OEG), Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG, Eicosyl-βAsp-2xOEG, Eicosyl-βAsp-3xOEG, Eicosyl-γGlu-OEG, Eicosyl-γGlu-2xOEG, Eicosyl-γGlu-3xOEG, Eicosyl-βAsp-OEG, Docosyl-βAsp-2xOEG, docosyl-βAsp-3xOEG, docosyl-γGlu-OEG, docosyl-γGlu-2xOEG, docosyl-γGlu-3xOEG Tetracosyl-βAsp-OEG, Tetracosyl-βAsp-2xOEG, and Tetracosyl-βAsp-3xOEG.

9. A pharmaceutical composition comprising the insulin analogue of any one of claims 1 to 2, the insulin derivative of any one of claims 3 to 6, or the conjugate of claim 8; Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an injectable formulation.

10. Use of the insulin analogue of any one of claims 1 to 2, the insulin derivative of any one of claims 3 to 6, or the conjugate of claim 8 in the preparation of a medicament for the treatment or prevention of type 1 diabetes, type 2 diabetes, obesity, or hyperglycemia.

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