An insulin derivative, pharmaceutical composition thereof, and use thereof

Abstract

The present application belongs to the field of biological medicine, and relates to a novel insulin derivative, a pharmaceutical composition thereof, and medical uses of the insulin derivative and the pharmaceutical composition.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a novel insulin derivative, a pharmaceutical composition thereof, and the pharmaceutical uses of the insulin derivative and the pharmaceutical composition. Background Technology

[0002] Icodec (trade name "Novo Nordisk") is a basal weekly insulin formulation developed by Novo Nordisk. It was approved in China in June 2024 for the treatment of type 2 diabetes in adults. Utilizing an innovative molecular design, it prolongs the duration of action, reducing the frequency of injections from once daily to once weekly, thus reducing the treatment burden. Icodec is based on human insulin, with the removal of threonine at position B30 and three amino acid substitutions: tyrosine at position A14 is replaced with glutamic acid (A14E), tyrosine at position B16 is replaced with histidine (B16H), and phenylalanine at position B25 is replaced with histidine (B25H). Icodec introduces a 20-carbon fatty acid side chain, linked to lysine at position B29 via a linker-spacer (L-γ-glutamic acid-2x oligoethylene glycol). The substitution of amino acids and the introduction of fatty acid side chains enable icoinsulin to bind potently and reversibly to albumin, and slow down insulin receptor-mediated clearance. icoinsulin is then slowly and continuously released from the albumin reservoir, thus exerting a blood glucose-lowering effect for one week. The structure of icoinsulin is shown in the figure below:

[0003]

[0004] However, once-weekly insulin Icosinate for diabetes treatment has a higher incidence of hypoglycemia compared to once-daily insulin Degludec.

[0005] Therefore, there is still a need to develop new insulin derivatives that have better efficacy or efficacy, faster and longer duration of action, lower dosing frequency, or lower dosage than insulin ico or other known insulin derivatives, in order to provide safe and effective new treatments for diabetes and its complications. Summary of the Invention

[0006] The first aspect of this invention provides a novel insulin derivative, more specifically an acylated insulin, comprising an insulin matrix and an acyl moiety.

[0007] The parent insulin is either desB30 human insulin or A14E, B16H, B25H, or desB30 human insulin.

[0008] The acyl group is linked to the ε-amino group of the lysine residue at position B29 of the insulin matrix.

[0009] The acyl moiety is shown as in formula X':

[0010]

[0011] in,

[0012] A is ,

[0013] B is selected from γGlu and Lys.

[0014] G is selected from aliphatic diacids containing 18-22 carbon atoms.

[0015] D is selected from Lys and Gly.

[0016] E is selected from Where z is 0, 1, 2 or 3,

[0017] m is 1, 2 or 3

[0018] n is 0 or 1,

[0019] r is 0 or 1.

[0020] p is 0 or 1,

[0021] a is 0 or 1.

[0022] Among them, p and a have the same value, which is either both 0 or both 1;

[0023] x is an integer from 1 to 5, preferably x is 1, 2, or 3.

[0024] y is an integer from 2 to 8, preferably 2, 3, 4, 5, or 6.

[0025] A, B, G, and D are linked by amide bonds, and E is linked to other groups by CN bonds;

[0026] Furthermore, the acyl moiety does not include the following structures:

[0027] .

[0028] In one implementation, z is 0.

[0029] In some embodiments, the acyl moiety is as shown in Formula X:

[0030]

[0031] in,

[0032] A is ,

[0033] B is selected from γGlu and Lys.

[0034] G is selected from aliphatic diacids containing 18-22 carbon atoms.

[0035] D is selected from Lys and Gly.

[0036] m is 1, 2 or 3

[0037] n is 0 or 1,

[0038] r is 0 or 1.

[0039] p is 0 or 1,

[0040] a is 0 or 1.

[0041] Among them, p and a have the same value, which is either both 0 or both 1;

[0042] x is an integer from 1 to 5, preferably x is 1, 2, or 3.

[0043] y is an integer from 2 to 8, preferably 2, 3, 4, 5, or 6.

[0044] A, B, G, and D are connected by amide bonds;

[0045] Furthermore, the acyl moiety does not include the following structures:

[0046] .

[0047] It should be noted that, in this paper, the positions and orders of groups / parts in formulas X' and X (and any variants thereof such as X-1 and X-2, etc.) can be interchanged, and are not limited to the positions and orders shown in formulas X' and X. As an example, and Individually, these groups can be interchanged in position or order. As an example, any one, two, or three A groups can be interchanged in position or order with a B group. As another example, any one, two, or three A groups can be interchanged in position or order with an E group.

[0048] It should also be noted that in this article, group A ( In ) and The positions and order can also be interchanged, and are not limited to the positions and order shown for group A in this paper. As an example, and Each of them, as a whole, can be interchanged in position or order. As an example, any one, two, or three, where appropriate, can be... In It can be with any one, two, three, four, five, or six Exchange positions or order.

[0049] In some embodiments, the G group in the acyl moiety is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0050] In one embodiment, the acyl moiety is shown as in Formula X-1:

[0051] ,

[0052] Among them, the A, B, and G groups have the meanings described above for formula X.

[0053] In one implementation, r is 0.

[0054] In one embodiment, the acyl moiety is shown as in Formula X-2:

[0055] ,

[0056] Among them, the A, B, and G groups have the meanings described above for formula X.

[0057] In some embodiments, the acyl moiety is as shown in Formula X-2:

[0058] ,

[0059] in,

[0060] A is ,

[0061] B is selected from γGlu and Lys.

[0062] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0063] m is 1, 2 or 3

[0064] When n is 1,

[0065] x is 1, 2, or 3.

[0066] y is 3, 4, 5, or 6.

[0067] A, B, and G are connected by amide bonds.

[0068] In some embodiments, the acyl moiety is as shown in Formula X-2:

[0069] ,

[0070] in,

[0071] A is ,

[0072] B is selected from γGlu and Lys.

[0073] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0074] m is 1, 2 or 3

[0075] When n is 1,

[0076] x is 2 or 3

[0077] y is 3, 4, 5, or 6.

[0078] A, B, and G are connected by amide bonds.

[0079] In some embodiments, the acyl moiety is as shown in Formula X-2:

[0080] ,

[0081] in,

[0082] A is ,

[0083] B is selected from γGlu.

[0084] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0085] m is 1, 2 or 3

[0086] When n is 1,

[0087] x is 2 or 3

[0088] y is 3, 4, 5, or 6.

[0089] A, B, and G are connected by amide bonds.

[0090] In some embodiments, the acyl moiety is as shown in Formula X-2:

[0091] ,

[0092] in,

[0093] A is ,

[0094] B is selected from γGlu and Lys.

[0095] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0096] m is 1, 2 or 3

[0097] When n is 0,

[0098] x is 2 or 3

[0099] y is 3, 4, 5, or 6.

[0100] A, B, and G are connected by amide bonds.

[0101] In some embodiments, the acyl moiety is as shown in Formula X-2:

[0102] ,

[0103] in,

[0104] A is ,

[0105] B is selected from γGlu.

[0106] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0107] m is 2 or 3

[0108] When n is 1,

[0109] x is 2,

[0110] y is 3 or 5.

[0111] A, B, and G are connected by amide bonds.

[0112] In some implementations, both p and a are 1.

[0113] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0114] ,

[0115] The A, B, G, D, and E groups have the meanings described above for formula X'.

[0116] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0117] ,

[0118] in,

[0119] A is ,

[0120] B is selected from γGlu and Lys.

[0121] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0122] D is selected from Lys and Gly.

[0123] E is selected from Where z is 0, 1, 2 or 3,

[0124] m is 1, 2 or 3

[0125] n is 0 or 1,

[0126] r is 0 or 1.

[0127] x is 1, 2, or 3.

[0128] y is 2, 3, 4, 5, or 6.

[0129] A, B, G, and D are linked by amide bonds, and E is linked to other groups by CN bonds.

[0130] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0131] ,

[0132] in,

[0133] A is ,

[0134] B is selected from γGlu and Lys.

[0135] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0136] D is selected from Lys and Gly.

[0137] E is selected from Where z is 0 or 3,

[0138] m is 1, 2 or 3

[0139] When n is 1,

[0140] r is 0 or 1.

[0141] x is 1, 2, or 3.

[0142] y is 2, 3, 4, 5, or 6.

[0143] A, B, G, and D are linked by amide bonds, and E is linked to other groups by CN bonds.

[0144] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0145] ,

[0146] in,

[0147] A is ,

[0148] B is selected from γGlu and Lys.

[0149] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0150] D is selected from Lys and Gly.

[0151] E is selected from Where z is 0 or 3,

[0152] m is 1 or 2.

[0153] When n is 1,

[0154] r is 0 or 1.

[0155] x is 2 or 3

[0156] y is 2, 3, 4, 5, or 6.

[0157] A, B, G, and D are linked by amide bonds, and E is linked to other groups by CN bonds.

[0158] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0159] ,

[0160] in,

[0161] A is ,

[0162] B is selected from γGlu.

[0163] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0164] D is selected from Lys and Gly.

[0165] E is selected from where z is 0,

[0166] m is 1, 2 or 3

[0167] When n is 1,

[0168] r is 0 or 1.

[0169] x is 2 or 3

[0170] y is 2, 3, 4, 5, or 6.

[0171] A, B, G, and D are connected by amide bonds.

[0172] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0173] ,

[0174] in,

[0175] A is ,

[0176] B is selected from γGlu.

[0177] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0178] D is selected from Lys.

[0179] E is selected from where z is 0,

[0180] m is 1 or 2.

[0181] When n is 1,

[0182] r is 0,

[0183] x is 2 or 3

[0184] y is 2, 3, 4, 5, or 6.

[0185] A, B, G, and D are connected by amide bonds.

[0186] In some embodiments, the acyl moiety is as shown in Formula X-3:

[0187] ,

[0188] in,

[0189] A is ,

[0190] B is selected from γGlu.

[0191] G is selected from octadecanoic acid, eicosanoic acid, or docosanoic acid.

[0192] D is selected from Lys.

[0193] E is selected from where z is 0,

[0194] m is 1 or 2.

[0195] When n is 1,

[0196] r is 0,

[0197] x is 2 or 3

[0198] y is 3, 4, or 5.

[0199] A, B, G, and D are connected by amide bonds.

[0200] In some embodiments, B in formula X is γGlu, if present. In some embodiments, B in formula X is Gly, if present. In some embodiments, D in formula X is Lys, if present. In other embodiments, D in formula X is Gly, if present.

[0201] In some embodiments, the insulin derivative of the present invention is selected from:

[0202]

[0203]

[0204]

[0205] A second aspect of the present invention provides a pharmaceutical composition comprising the insulin derivative described in the first aspect of the present invention and one or more pharmaceutically acceptable excipients.

[0206] In some embodiments, pharmaceutically acceptable excipients may include, but are not limited to, lubricants and preservatives. The pharmaceutical composition further comprises glycerol, phenol, m-cresol, NaCl, and / or Na₂HPO₄, zinc acetate, or zinc chloride; preferably, the pharmaceutical composition further comprises glycerol, phenol, m-cresol, NaCl, and zinc acetate.

[0207] A third aspect of the present invention provides the use of the insulin derivative described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a medicament for the treatment or prevention of diabetes, hyperglycemia, and / or impaired glucose tolerance.

[0208] In addition, the present invention provides a novel method for preparing an insulin derivative, which includes the steps of preparing an insulin analog, coupling the insulin analog to a fatty acid side chain, and preparing an insulin derivative after coupling.

[0209] definition

[0210] In this article, the term insulin includes naturally occurring insulin, such as human insulin, and its insulin analogs. Human insulin consists of two polypeptide chains (A chain and B chain), which contain 21 and 30 amino acid residues respectively and are linked by two cystine disulfide bonds.

[0211] In this document, the term insulin analogue includes polypeptides having a molecular structure that is formally derived from the structure of naturally occurring insulin (e.g., human insulin) by the deletion and / or substitution (replacement) of one or more amino acid residues present in natural insulin and / or the addition of one or more amino acid residues. The added and / or substituted amino acid residues may be codeable amino acid residues, other naturally occurring amino acid residues, or purely synthetic amino acid residues. Preferably, the added and / or substituted amino acid residues are codeable amino acid residues.

[0212] In this document, the term "insulin derivative" refers to insulin that has been chemically modified, such modification being, for example, the introduction of a side chain at one or more positions on the insulin backbone (sometimes referred to herein as the "insulin matrix"), the oxidation or reduction of groups on amino acid residues of insulin, or the conversion of a free carboxyl group into an ester group or the acylation of a free amino or hydroxyl group. The insulin derivative of this invention is an acylated insulin. Therefore, the insulin derivative of this invention is sometimes also referred to as "acylated insulin."

[0213] In this document, the term "insulin matrix" refers to insulin without the additional acyl moiety. The insulin matrix may optionally have a mutation relative to human insulin.

[0214] In this article, terms such as desB30 refer to insulin analogs lacking the B30 amino acid residue.

[0215] In this article, the term "acyl moiety" refers to the chemically modified group portion attached to the "insulin matrix," which further includes albumin-binding residues, amino acid residues, hydrophilic linker groups, etc.

[0216] In this document, the term "amino acid residue" includes amino acids in which hydrogen atoms have been removed from the amino group and / or hydroxyl groups have been removed from the carboxyl group and / or hydrogen atoms have been removed from the thiol group. In imprecisely, an amino acid residue may be called an amino acid.

[0217] In this document, the term "albumin-binding residue" refers to a residue that can non-covalently bind to human serum albumin. In this document, "albumin-binding residue" refers to an aliphatic diacid containing 16-24 carbon atoms (more preferably an aliphatic diacid with 18-22 carbon atoms), wherein one of the carboxyl groups of the formally described aliphatic diacid is removed. In this document, the term "aliphatic diacid" includes straight-chain or branched aliphatic dicarboxylic acids having at least two carbon atoms and being saturated or unsaturated. Non-limiting examples of aliphatic diacids include adipic acid, octanoic acid, sebacic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, and tetradecanoic acid.

[0218] In this article, "hydrophilic linker" refers to the linker group that separates the insulin precursor from the albumin-binding residues. In this article, "hydrophilic linker" can also specifically refer to the A group in formulas X and X' and any variants thereof (general formula: Where x is an integer from 1 to 5, and y is an integer from 2 to 8). In the A group... It can also be represented as PEGy, and can also be called "y polyethylene glycol". For example, when y is 2, It can also be represented as PEG2, and can also be called "diethylene glycol".

[0219] In this document, the term "chemical stability" refers to the fact that, chemically, the insulin derivative of the present invention is sufficiently stable in the desired formulation. That is, only a small amount of chemical degradation products are formed that do not impair the shelf life of the final pharmaceutical product. Chemical degradation products include deamidation products, isoaspartate ester formation, dimer formation, racemization products, and products arising from dehydration processes. Chemical stability can be determined by HPLC analysis of aged samples or formulations.

[0220] In this article, "the ability to phosphorylate the insulin receptor" refers to the phosphorylation of the insulin receptor (IR-β) after insulin activates it. The intensity of this phosphorylation can be measured using, for example, homogeneous real-time fluorescence (HTRF) technology. For instance, in HTRF assays, when a solution containing insulin is incubated with cells stably converted to the insulin receptor INSR-B, the interaction between insulin and the insulin receptor leads to a phosphorylation change in the receptor. This change can be detected based on the principle of a double-antibody sandwich assay. The double-antibody sandwich assay uses two different specific antibodies; one antibody specifically binds to the phosphorylation motif of INSR-B and is labeled Eu. 3+ - A cavitation sac (donor) and another antibody, labeled d2 (receptor), recognize the phosphorylated state of the receptor. When the two are close together, a light source excites the donor to trigger fluorescence resonance energy transfer (FRET) to the receptor, which then fluoresces at a specific wavelength (665 nm). The specific signal is positively correlated with phosphorylation-INSR-B. Generally, a higher specific signal indicates a greater ability to phosphorylate the insulin receptor.

[0221] Peptides with affinity for both the insulin receptor and the IGF-1 receptor are peptides that can interact with the insulin receptor and the human IGF-1 receptor in a suitable binding assay. Such receptor assays are well known in the art.

[0222] In this document, "efficacy," "potency," or "potency" refers to the ability of a drug or active compound to produce a certain effect or function (e.g., lowering blood glucose). For example, administration of the same dose of the insulin derivative of the present invention produces a greater effect or function in lowering blood glucose compared to insulin ico or other known insulin derivatives.

[0223] 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 properly to insulin, thus preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.

[0224] Type 1 diabetes, also known as insulin-dependent diabetes mellitus (IDDM) and juvenile diabetes, is caused by the destruction of beta cells and usually results in absolute insulin deficiency. Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with major insulin resistance and thus relative insulin deficiency and / or major insulin secretion defects with insulin resistance.

[0225] In this paper, insulin is named according to the following principles: it is named according to the mutation and modification (acylation) relative to human insulin. For the acyl moiety, it is named according to IUPAC nomenclature and, in other cases, peptide nomenclature. For example, naming the acyl moiety:

[0226]

[0227] It can be named, for example, “N,N-di(octadecanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetaminopropyl)-glycyl”, “N,N-di(octadecanoic acid-γGlu-PEG2-acetyl-PEG2-acetyl-aminopropyl)-glycyl”, “N,N-di(octadecanoyl-gGlu-PEG2-acetyl-PEG2-acetyl-aminopropyl)-glycyl”, or “N,N-di(17-carboxyheptadecanoyl-γGlu-PEG2-acetyl-PEG2-acetyl-aminopropyl)-glycyl”, where γGlu (and gGlu) is the abbreviation for the L-configured amino acid γ-glutamic acid.

[0228] For example, the insulin derivative CL9 of Example 4 (having the sequence / structure given below) is referred to as “A14E,B16H,B25H,B29K-N(ε)-eicosanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl,desB30 human insulin”, to indicate that amino acid Y at position A14 in human insulin has been mutated to E, amino acid Y at position B16 in human insulin has been mutated to H, amino acid F at position B25 in human insulin has been mutated to H, and amino acid K at position B29 in human insulin has been mutated to E. The insulin derivative is modified by acylation of the ε nitrogen (called Nε) of the lysine residue at B29 with the residue "eicosanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl" (also known as "eicosanoic acid-gGlu-PEG3-propionyl-PEG3-propionyl"), and the amino acid T at position B30 in human insulin has been deleted; in addition, this insulin derivative is sometimes referred to as "N(ε)-eicosanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl)-A14E,B16H,B25H,desB30 human insulin".

[0229] For example, the insulin derivative CL14 of Example 6 (having the sequence / structure given below) is referred to as "A14E,B16H,B25H,B29K-N(ε)-docosadiyl-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl,desB30 human insulin", indicating that amino acid Y at position A14 in human insulin has been mutated to E, amino acid Y at position B16 in human insulin has been mutated to H, amino acid F at position B25 in human insulin has been mutated to H, and amino acid K at position B29 in human insulin has been mutated to E. It has been modified by acylation of the ε nitrogen (called Nε) of the lysine residue at B29 with the residue "eicosidine-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl" (also known as "eicosidine-gGlu-PEG5-propionyl--PEG5-propionyl"), and the amino acid T at position B30 in human insulin has been deleted. In addition, this insulin derivative is sometimes referred to as "eicosidine-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl)-A14E,B16H,B25H,desB30 human insulin".

[0230] Human insulin was prepared in microorganisms by expressing fusion proteins in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7th American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, III. pp. 729-739).

[0231] The construction, expression, processing, and purification of insulin analog vectors can be performed using techniques well known to those skilled in the art. For example, insulin analogs can be prepared by expressing a DNA sequence encoding the target insulin analog in a suitable host cell using well-known techniques disclosed in U.S. Patent No. 6,500,645. Insulin analogs can also be prepared, for example, by methods reported in the following literature: Glendorf T, AR, Nishimura E, Pettersson I, & Kjeldsen T: Importance of the Solvent-Exposed Residues of the Insulin B Chain α-Helix for Receptor Binding; Biochemistry 2008 47 4743-4751. In this literature, overlapping extension PCR was used to introduce mutations into the insulin-encoding vector. The insulin analog was expressed as a pre-insulin-like fusion protein with an Ala-Ala-Lys small C-peptide in Saccharomyces cerevisiae strain MT663. The single-chain precursor was enzymatically converted to a double-chain desB30 analog using an Achromobacterium lyticum endonuclease.

[0232] The isolated insulin analogue can be acylated at the desired location using acylation methods known in the art, examples of which have been described in, for example, Chinese patent applications with publication numbers CN1029977C, CN1043719A and CN1148984A.

[0233] Generally, in this article, the term "excipient" refers to any component in a pharmaceutical composition other than the active therapeutic ingredient, and may also be referred to as a "pharmaceutically acceptable carrier or excipient." Excipients can be inert, inactive, and / or pharmaceutically inactive substances. Excipients can be used for a variety of purposes, such as as carriers, vehicles, fillers, binders, diluents, lubricants, flow aids, disintegrants, anti-adhesion agents, flow control agents, crystallization inhibitors, solubilizers, stabilizers, colorants, flavor enhancers, surfactants, emulsifiers, and / or to improve the administration and / or absorption of the active substance, such as absorption enhancers.

[0234] 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.

[0235] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. For example, a description of a range from 1 to 6 should be considered as specifically disclosing subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and specific numerical points within those ranges, such as 1, 2, 3, 4, 5, 6. The above principles apply equally regardless of the breadth of the numerical values. When a range description is used, the range includes the endpoints of the range.

[0236] When referring to measurable values ​​such as various quantities, time, etc., the term "about" means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0237] The inventors unexpectedly discovered that the insulin derivative of this invention, compared to marketed Icosuga (trade name "Novogene") or other insulin derivatives, has comparable binding affinity to the insulin receptor, but exhibits satisfactory chemical stability, unexpectedly significant increased efficacy, and a longer duration of action. Furthermore, the inventors found that, compared to marketed Icosuga (Novogene), the insulin derivative of this invention maintains comparable efficacy even with a 50% or 75% reduction in dosage, suggesting that it can reduce dosage in clinical applications, alleviate the risk of hypoglycemia due to dosage, and reduce the psychological stress of diabetic patients using weekly insulin formulations due to excessive dosage. Attached Figure Description

[0238] Figure 1 This is the result of the activity of insulin derivatives CL5~CL10 on the phosphorylation of insulin receptor INSR-B.

[0239] Figure 2 This is the result of the activity of insulin derivatives CL13 and CL14 in phosphorylating insulin receptor INSR-B.

[0240] Figure 3 This is the blood glucose-time curve after a single dose of insulin derivative CL3 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0241] Figure 4The graphs show the blood glucose-time changes after a single administration of insulin derivatives CL6 and CL8 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0242] Figure 5 The graphs show the blood glucose-time changes after a single administration of insulin derivatives CL5 and CL7 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0243] Figure 6 The graphs show the blood glucose-time changes after a single administration of insulin derivatives CL9 and CL10 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0244] Figure 7 This is the blood glucose-time curve after a single dose of insulin derivative CL11 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0245] Figure 8 The graphs show the blood glucose-time changes after a single administration of insulin derivatives CL1 and CL2 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0246] Figure 9 This is a graph showing the blood glucose-time changes after a single dose of insulin derivative CL9 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0247] Figure 10 The pharmacokinetic curves of insulin derivatives CL1, CL2, and CL9 after a single dose in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model are shown.

[0248] Figure 11 The graphs show the blood glucose-time changes after a single administration (1000 nmol / kg) of insulin derivatives CL13 and CL14 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0249] Figure 12 This is a graph showing the blood glucose-time changes after a single dose of insulin derivative CL13 at different doses in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0250] Figure 13 This is a graph showing the blood glucose-time changes after a single dose of insulin derivative CL14 at different doses in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model.

[0251] Figure 14The figures show the drug-time curves of insulin derivatives CL13 and CL14 after a single dose at different doses in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model. Detailed Implementation

[0252] The following detailed description of specific embodiments further illustrates the above-mentioned content of the present invention, but should not be construed as limiting the scope of protection of the present invention in any way. All technical solutions implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. The present invention provides a general and / or specific description of the materials and experimental methods used in the experiments.

[0253] Example 1: Preparation of A14E, B16H, B25H, desB30 human insulin

[0254] Experimental materials: Nde I, Xho I restriction endonuclease, T4 DNA ligase (purchased from NEB), manganese sulfate monohydrate, copper sulfate pentahydrate, zinc sulfate heptahydrate, ammonium molybdate (purchased from Sigma-Aldrich), sodium chloride, magnesium sulfate (purchased from Tianjin Kemei Chemical Reagent Co., Ltd.), yeast extract and peptone (purchased from OXOID), glucose, IPTG, citric acid monohydrate, EDTA, Triton X-100, tris(hydroxymethyl)aminomethane, urea, dithiothreitol and L-cysteine ​​(purchased from Sangon Biotech (Shanghai) Co., Ltd.), dilute hydrochloric acid and sodium acetate (purchased from Hunan Ercon Pharmaceutical Co., Ltd.), ammonia, hydrochloric acid, glacial acetic acid (purchased from Chengdu Kelong Chemical Co., Ltd.), potassium dihydrogen phosphate, sodium hydroxide (purchased from Xilong Scientific Co., Ltd.), sodium dihydrogen phosphate (purchased from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd.), ammonium sulfate (purchased from Sinopharm Chemical Reagent Co., Ltd.), lysyl endonuclease (purchased from Zhuhai Jibaikang Biotechnology Co., Ltd.).

[0255] Experimental steps: Based on the structure and characteristics of human insulin (A14E, B16H, B25H, desB30), a leader peptide-Ins fusion protein expression sequence was designed, and its gene synthesis was commissioned to GenScript Biotech Co., Ltd. The synthesized fusion protein gene sequence was then... Nde I, Xho Digested with restriction endonucleases (I), ligated with T4 DNA ligase, and constructed into plasmids containing the T7 promoter (pET41a, etc.) to create a recombinant expression vector. The recombinant expression vector was transformed into E. coli QLE2103 host cells that had undergone selection pressure modification to construct an engineered expression bacterium. The engineered bacterium expressed the target protein, which accounted for more than 20% of the total bacterial protein.

[0256] Successfully constructed A14E, B16H, B25H, and desB30 human insulin-expressing engineered bacteria were cultured and expanded on 2YT medium (1.6% peptone, 1% yeast extract, 0.5% sodium chloride). They were then transferred at a 10% inoculum to an inorganic salt medium containing 1%–2% glucose (0.5%–2% ammonium sulfate, 0.1%–0.5% citric acid, 1%–5% potassium dihydrogen phosphate, 0.01%–0.05% ferrous sulfate, 0.5%–2% magnesium sulfate) for high-density fermentation. During fermentation, carbon source depletion and dissolved oxygen rebound were observed. At this point, 60% glucose solution and 6% yeast extract solution were added to control the specific growth rate of the cells, achieving high-density fermentation. The fermentation cell density OD was [not specified]. 600 Once the concentration reaches 80% or higher, IPTG is added to induce the expression of the target protein. Fermentation is terminated after induction for at least 12 hours, and the cells are harvested. The harvested cell pellet is analyzed by SDS-PAGE to determine if the target protein expression level is above 12 g / L. The cell pellet is resuspended in lysis buffer (20 mM Tris, 0.5 mM EDTA, 750 mM NaCl, 0.1% Triton X-100), and then homogenized using a high-pressure homogenizer at 700 bar or higher. Afterward, inclusion body (IB) washing and centrifugation are performed to obtain an inclusion body pellet containing a target protein purity higher than 70%.

[0257] Denaturing agents containing 20 mmol / L tris(hydroxymethyl)aminomethane, 8 mol / L urea, 15 mmol / L L-cysteine, and 20 mmol / L dithiothreitol were added to inclusion bodies. The volume was brought to a final level with water, and the pH of the reaction system was adjusted to 10.0. The mixture was stirred at room temperature for 90 min. The denaturing solution was then slowly added to a dilution buffer (20 mmol / L tris(hydroxymethyl)aminomethane, pH 10.0), maintaining the pH of the reaction system at 10.0 and the reaction temperature at 10–20 °C. The annealing time was at least 15 h. After annealing, the protein concentration in the annealed solution was measured, and the total amount of annealed protein was calculated. The required amount of lysyl endonuclease was calculated according to a specific ratio, and the mixture was stirred thoroughly. The pH of the reaction system was maintained at 10.0, the reaction temperature at least 30 °C, and the digestion time at least 30 h. After digestion, the digestion solution was obtained, and parameters such as the protein concentration were measured. The enzyme digest was purified by ion exchange chromatography, including anion and cation exchange chromatography or hydrophobic and ion complexation mode chromatography, which are well known in the art. The binding-elution mode was used, and a washing step was used to remove a large number of process and product-related impurities. Then, the digest was eluted with elution buffer to collect high-purity A14E, B16H, B25H, and desB30 human insulin protein.

[0258] Experimental resultsDuring the preparation of human insulin analogs (A14E, B16H, B25H, desB30), the fermentation process resulted in a cell OD of 210, a cell wet weight of 260 g / L fermentation broth, a target protein expression level of 13.5 g / L fermentation broth, an inclusion body yield of 120 g / L fermentation broth, and an inclusion body purity of 70%. After purification, a high-purity human insulin analog was obtained.

[0259] Example 2: Preparation of insulin derivative CL3

[0260] Prepare A14E,B16H,B25H,B29K-eicosanoic acid-glutamic acid-diethylene glycol acetyl-diethylene glycol acetyl,desB30 human insulin (abbreviated as CL3, which is the active pharmaceutical ingredient of the marketed Icodec insulin (trade name "Novogene").

[0261]

[0262] Experimental materials:

[0263] The A14E, B16H, B25H, and desB30 human insulins prepared using the method described in Example 1 above consisted of eicosanodiayl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl active ester (purchased from Xiamen Sainuobang), DMSO (purchased from ThermoFisher), sodium hydroxide (purchased from Hunan Ercon), glacial acetic acid (purchased from Merck, Germany), Tris (purchased from Shanghai Sangon Biotech), sodium chloride (purchased from Hebei Huachen), acetonitrile (purchased from Tianjin Kangkede), trifluoroacetic acid (purchased from Chengdu Kelong), and sodium acetate (purchased from Jinan Qiguang).

[0264] Experimental steps:

[0265] Take 3.3g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 1.0g of eicosanoic acid-glutamic acid-diethylene glycol acetyl-diethylene glycol acetate active ester dissolved in DMSO under vigorous stirring, and let it react at room temperature for 60min. Adjust the pH of the reaction solution to 7.88 with acetic acid.

[0266] Purification was performed using anion exchange chromatography on Äkta pure.

[0267] Column: 250 ml (50 mm x 12.5 mm); Buffer A: 20 mM Tris, pH 7.5; Buffer B: 20 ​​mM Tris + 500 mM sodium chloride, pH 7.5; Gradient: 0-100% B, 5-20 CV; Flow rate: 20 mL / min; Collect eluent fractions, analyze purity using liquid chromatography, and combine qualified solutions.

[0268] The anion purification collection solution was further purified using preparative liquid chromatography:

[0269] Column: 500 ml (50 mm × 250 mm); Buffer A: 0.1% trifluoroacetic acid in water; Buffer B: acetonitrile; Gradient: 25-55% B, 0-120 min; Flow rate: 60 ml / min; Collect eluent fractions in segments, analyze purity by liquid chromatography, and combine qualified solutions.

[0270] Column: 500 ml (50 mm × 250 mm); A1 buffer: 50 mM sodium acetate aqueous solution; A2 buffer: purified water; B buffer: acetonitrile; gradient: 90% A1 / A2 + 10% B, 30% A2 + 70% B wash sequentially; flow rate: 60 ml / min; the collected solution was concentrated under reduced pressure in a water bath at less than 40 °C, and most of the acetonitrile was evaporated using a rotary evaporator to obtain an insulin sodium salt aqueous solution, which was then freeze-dried to obtain the insulin derivative product.

[0271] Experimental results: The coupling rate, purity, and mass spectrometry molecular weight of insulin derivative CL3 are shown in Table 1 below.

[0272] Table 1. Coupling rate, purity, and mass spectrometry molecular weight of insulin derivative CL3

[0273]

[0274] Example 3: Preparation of insulin derivatives CL1 and CL2

[0275] Preparation of A14E, B16H, B25H, B29K-Nα, Nε-bis(eicosanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysyl, desB30 Human Insulin (CL1)

[0276] Preparation of A14E,B16H,B25H,B29K-Nα,Nε-bis(octadecanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysyl-,desB30 Human Insulin (CL2)

[0277] Experimental materials:

[0278] The A14E, B16H, B25H, desB30 human insulin, Nα,Nε-bis(eicosanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysine active ester and Nα,Nε-bis(octadecanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysine prepared by the method in Example 1 above were provided by Qilu Pharmaceutical Research Institute. Other experimental materials were the same as those in Example 2.

[0279] Experimental steps:

[0280] Take 1.8g of A14E,B16H,B25H,desB30 human insulin into a reaction vessel, add triethylamine to adjust the pH to 10.8, and under vigorous stirring, add 1.5g of Nα,Nε-bis(eicosanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysine active ester dissolved in DMSO. Allow the reaction to proceed at room temperature for 60min, dilute the reaction solution with water, and adjust the pH of the reaction solution to 9.7 using acetic acid.

[0281] Take 2.3g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, add triethylamine to adjust the pH to 10.8, and under vigorous stirring, add 1.7g of Nα,Nε-bis(octadecanoyl-glutamyl-diethylene glycol acetyl-diethylene glycol acetyl)-lysine active ester dissolved in DMSO. Allow the reaction to proceed at room temperature for 60min, dilute the reaction solution with water, and adjust the pH of the reaction solution to 9.5 using acetic acid.

[0282] Further purification by anion exchange chromatography and preparative liquid chromatography was carried out in the same manner as in Example 2.

[0283] Experimental results: The coupling rate, purity, and mass spectrometry molecular weight of insulin derivatives CL1 and CL2 are shown in Table 2 below.

[0284] Table 2. Coupling rates, purity, and mass spectrometry molecular weights of insulin derivatives CL1 and CL2

[0285]

[0286] Example 4: Preparation of insulin derivatives CL5 to CL10

[0287] Preparation of A14E, B16H, B25H, B29K-N(ε)-eicosanoyl-glutamyl-tetraethylene glycol propionyl, desB30 human insulin (CL5)

[0288] Preparation of A14E, B16H, B25H, B29K-N(ε)-octadecanoyl-glutamyl-tetraethylene glycol propionyl, desB30 human insulin (CL6)

[0289] Preparation of A14E, B16H, B25H, B29K-N(ε)-eicosanoyl-glutamyl-pentaethylene glycol propionyl, desB30 human insulin (CL7)

[0290] Preparation of A14E, B16H, B25H, B29K-N(ε)-octadecanoic acid-glutamyl-pentaethylene glycol propionyl, desB30 human insulin (CL8)

[0291] Preparation of A14E, B16H, B25H, B29K-N(ε)-eicosanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin (CL9)

[0292] Preparation of A14E, B16H, B25H, B29K-N(ε)-octadecanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin (CL10)

[0293] Experimental materials: The A14E, B16H, B25H, and desB30 human insulins prepared using the method described in Example 1 above, along with eicosanoyl-glutamyl-tetraethylene glycol propionyl active ester, octadecanoyl-glutamyl-tetraethylene glycol propionyl active ester, eicosanoyl-glutamyl-pentaethylene glycol propionyl active ester, octadecanoyl-glutamyl-pentaethylene glycol propionyl active ester, eicosanoyl-glutamyl-triethylene glycol propionyl-triethylene glycol propionyl active ester, and octadecanoyl-glutamyl-triethylene glycol propionyl-triethylene glycol propionyl active ester, were provided by Qilu Pharmaceutical Research Institute. Other experimental materials were the same as those used in Example 2.

[0294] Experimental steps:

[0295] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.44g of eicosanoyl-glutamyl-tetraethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.7 with acetic acid.

[0296] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.43g of octadecanoyl-glutamyl-tetraethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.5 with acetic acid.

[0297] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.48g of eicosadecanoyl-glutamyl-pentaethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.5 with acetic acid.

[0298] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.45g of octadecanoyl-glutamyl-pentaethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.6 with acetic acid.

[0299] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.56g of eicosanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.4 with acetic acid.

[0300] Take 2.0g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, adjust the pH to 11.0 with 1M NaOH solution, add 0.53g of octadecanoyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl active ester dissolved in DMSO under vigorous stirring, and allow the reaction to proceed at room temperature for 60min. Adjust the pH of the reaction solution to 7.5 with acetic acid.

[0301] Further purification by anion exchange chromatography and preparative liquid chromatography was carried out in the same manner as in Example 2.

[0302] Experimental results: The coupling rates, purity, and mass spectrometry molecular weights of insulin derivatives CL5 to CL10 are shown in Table 3 below.

[0303] Table 3. Coupling rates, purity, and mass spectrometric molecular weights of insulin derivatives CL5 to CL10.

[0304]

[0305] Example 5: Preparation of insulin derivative CL11

[0306] Preparation of A14E, B16H, B25H, B29K-Nα, Nε-bis(eicosanoyl-tetraethylene glycol propionyl)-lysyl-tetraethylene glycol propionyl, desB30 human insulin (CL11)

[0307] Experimental materials:

[0308] A14E, B16H, B25H, desB30 human insulin was prepared using the method described in Example 1 above. The active ester of Nα,Nε-bis(eicosanoyl-tetraethylene glycol propionyl)-lysyl-tetraethylene glycol propionyl was provided by Qilu Pharmaceutical Research Institute. Other experimental materials were the same as those in Example 2.

[0309] Experimental steps:

[0310] Take 2.4g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, add TEA to adjust the pH to 10.8, and under vigorous stirring, add 1.0g of Nα,Nε-bis(eicosanoyl-tetraethylene glycol propionyl)-lysyl-tetraethylene glycol propionyl active ester dissolved in DMSO. Allow the reaction to proceed at room temperature for 60min, dilute the reaction solution with water, and adjust the pH of the reaction solution to 9.5 using acetic acid.

[0311] Further purification by anion exchange chromatography and preparative liquid chromatography was carried out in the same manner as in Example 2.

[0312] Experimental results: The coupling rate, purity, and mass spectrometry molecular weight of insulin derivative CL11 are shown in Table 4 below.

[0313] Table 4. Coupling rate, purity, and mass spectrometry molecular weight of insulin derivative CL11

[0314]

[0315] Example 6: Preparation of insulin derivatives CL13 and CL14

[0316] Preparation of A14E, B16H, B25H, B29K-N(ε)-docosadiyl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin (CL13)

[0317] Preparation of A14E, B16H, B25H, B29K-N(ε)-docosadiyl-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl, desB30 human insulin (CL14)

[0318] Experimental materials:

[0319] The A14E, B16H, B25H, and desB30 human insulins, docosanoyl-glutamyl-triethylene glycol propionyl-triethylene glycol propionyl active ester, and docosanoyl-glutamyl-pentaethylene glycol propionyl active ester prepared by the method in Example 1 above were provided by the Peptide Drug Research Department of Qilu Pharmaceutical Research Institute. Other experimental materials were the same as those in Example 2.

[0320] Experimental steps:

[0321] Take 5.4g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, add 10% DMSO to a final concentration, adjust the pH to 11.0 with 1M NaOH solution, and add 1.4g of docosanodiayl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl-polyethylene glycol propionyl active ester dissolved in DMSO while stirring vigorously. Allow the reaction to proceed at room temperature for 60min, and adjust the pH of the reaction solution to 7.9 with acetic acid.

[0322] Take 5.5g of A14E, B16H, B25H, desB30 human insulin into a reaction vessel, add 10% DMSO to a final concentration, adjust the pH to 11.0 with 1M NaOH solution, and add 1.4g of docosanodiayl-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl active ester dissolved in DMSO while stirring vigorously. Allow the reaction to proceed at room temperature for 60min, and adjust the pH of the reaction solution to 8.0 with acetic acid.

[0323] Further purification by anion exchange chromatography and preparative liquid chromatography was carried out in the same manner as in Example 2.

[0324] Experimental results: The coupling rates, purity, and mass spectrometry molecular weights of insulin derivatives CL13 and CL14 used in cell and animal experiments are shown in Table 5 below.

[0325] Table 5. Coupling rate, purity, and mass spectrometry molecular weight of insulin derivatives CL13 and CL14

[0326]

[0327] Example 7: Study on the receptor phosphorylation ability of insulin derivatives CL5-CL10

[0328] The ability of the insulin derivatives CL5-CL10 and the control CL3 (the active pharmaceutical ingredient of Icodec (trade name "Novogene")) of the present invention to phosphorylate the insulin receptor (INSR-B) was tested using HTRF technology. CHO-INSR-1284 cells were seeded at a density of 20,000 cells / well and incubated overnight. The old culture medium was discarded, and the cells were washed once with phosphate-buffered saline (PBS). Serum-free culture medium was added and incubated for 4 h to starve the cells. The test samples were diluted with basal medium containing 0.1% BSA (serialized dilutions from 500 μg / mL, for a total of 9 concentration points), and added to the corresponding wells to replace the serum-free culture medium. The cells were incubated for 30 min. Then, pre-prepared lysis buffer was added and the cells were lysed by shaking at room temperature for 40 min. 4 μL of premixed antibody reaction solution (EU antibody and d2 antibody ratio 1:1) was added to each well of a 96-well low-volume plate using an automatic dispensing pipette. According to the plate layout, 16 μL of each group of samples, positive control, and negative control (lysis buffer) were added to each well. The plates were sealed with a membrane and incubated at room temperature for 2 h. The values ​​were then read using a microplate reader.

[0329] Experimental results: The insulin derivatives CL5-CL10 have INSR-B receptor phosphorylation activities that are essentially equivalent to CL3, as detailed in Table 6 below. Figure 1 .

[0330] Table 6. Results of the activity of insulin derivatives CL5-CL10 on INSR-B receptor phosphorylation.

[0331]

[0332] Example 8: Study on the receptor phosphorylation ability of insulin derivatives CL13-CL14

[0333] The ability of insulin derivatives CL13-CL14 and control CL3 to phosphorylate insulin receptor (INSR-B) was tested using a method similar to that in Example 7, with concentrations starting at 500 μg / mL. The results are detailed in Table 7 below. Figure 2 .

[0334] Experimental results: The insulin derivatives CL13 and CL14 have similar INSR-B receptor phosphorylation activities to CL3.

[0335] Table 7. Results of the activity of insulin derivatives CL13 and CL14 on INSR-B receptor phosphorylation.

[0336]

[0337] Example 9: Pharmacodynamic study in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model

[0338] Ten-week-old male C57BL / 6J mice (purchased from Vital River) were housed in an SPF-grade animal facility using IVC (independent ventilation system, constant temperature and humidity) cages at a relative temperature of 20-26℃ and a relative humidity of 30-70%, with free access to standard food and purified water. After a 5-7 day acclimatization period, the mice were fasted for 15 hours and then intraperitoneally injected with streptozotocin (Sigma) solution (STZ, 20 mg / mL, in 50 mM sodium citrate buffer) or citrate buffer (control group n=6) at a dose of 200 mg / kg. Fasting blood glucose was measured 72 hours and 1 week after streptozotocin administration. A fasting blood glucose level >16.7 mmol / L was considered a successful model, and the mice were used for subsequent experiments. On day 8, based on the mice's random blood glucose and body weight, the successfully modeled mice were randomly divided into 5 groups of 6-8 mice each. The animal grouping and drug administration details are shown in Table 8 below.

[0339] Table 8 Grouping and Dosing Regimens of Experimental Animals

[0340]

[0341] Note: The solvent is PBS.

[0342] Animals were administered the appropriate solvent or drug via subcutaneous (SC) administration as a single dose, with free access to food and water throughout the experiment. Blood was collected via tail clipping, and random blood glucose levels were measured before administration and at 1, 2, 6, 24, 48, 72, 96, 120, and 144 hours after administration using a glucometer and matching test strips (Roche). Data are presented as mean ± SEM, and graph analysis was performed using Graphpad Prism 8.0.2 software. t-tests or one-way ANOVA were used for analysis. P <0.05 was used as the criterion for judging significant differences.

[0343] Experimental results: Compared with the model group, within 24 hours after CL3 administration, blood glucose levels significantly decreased at three doses: 200, 500, and 1500 nmol / kg, reaching the lowest level at 24 hours, followed by a slow rise. The blood glucose-lowering effect and effective blood glucose control time of CL3 were dose-dependent; that is, the higher the dose, the stronger the hypoglycemic effect and the longer the blood glucose control time. At a dose of 1500 nmol / kg, the blood glucose control time was approximately 96 hours. Specific data are shown in Table 9 below. Figure 3 As shown.

[0344] Table 9 Blood glucose levels at different time points after drug administration in each group of mice

[0345]

[0346] Example 10: Pharmacodynamic study of a streptozotocin-induced type 1 diabetic C57BL / 6J mouse model

[0347] Eight-week-old male C57BL / 6J mice (purchased from Vital River) were housed in an SPF-grade animal facility using IVC (independent ventilation system, constant temperature and humidity) cages at a relative temperature of 20-26°C and a relative humidity of 30-70%, with free access to standard food and purified water. After a 5-7 day acclimatization period, the mice were fasted for 15 hours and then intraperitoneally injected with streptozotocin (Sigma) solution (STZ, 15 mg / mL, in 50 mM sodium citrate buffer) or citrate buffer (control group n=6) at a dose of 150 mg / kg. Fasting blood glucose was measured 3 and 6 days after streptozotocin administration. A fasting blood glucose level >16.7 mmol / L was considered a successful model, and the mice were used for subsequent experiments. On day 8, based on the random blood glucose and body weight of the mice that day, the successfully modeled mice were randomly divided into 10 groups of 6 mice each. The animal grouping and drug administration details are shown in Table 10 below.

[0348] Table 10 Grouping and Dosing Regimens of Experimental Animals

[0349]

[0350] Note: The solvent for group CL11 was PBS at pH 11, while the other groups used PBS at pH 10; the insulin degludec was the commercially available product Novo Nordisk, 3ml: 300 units / vial.

[0351] Animals were administered the appropriate solvent or drug via subcutaneous (SC) administration as a single dose, with free access to food and water during the experiment. Blood was collected by tail clipping, and random blood glucose levels were measured using a glucometer and matching test strips (Roche) before administration and at 1, 2, 6, 24, 30, 48, 72, 96, 120, 144, 168, and 192 hours after administration. Data are presented as mean ± SEM. GraphpadPrism 8.0.2 software was used for plotting and analysis. t-tests or one-way ANOVA were employed, with P < 0.05 considered statistically significant.

[0352] Experimental results:Compared with the model group, blood glucose levels gradually decreased after administration of CL3 (1000 nmol / kg), reaching their lowest levels at 24h and 48h, gradually rising after 48h, and disappearing after 96h. At the same dose (1000 nmol / kg), CL5 and CL7 had comparable hypoglycemic effects to CL3, while CL9 and CL11 had better hypoglycemic effects than CL3. CL9 and CL11 had a prolonged effective blood glucose control time, maintaining low blood glucose levels in mice for 72h. The effect of CL9 disappeared after 120h, and that of CL11 disappeared after 144h. In this experiment, insulin degludec exerted a significant hypoglycemic effect within 24h. At a dose of 1000 nmol / kg, CL8 had comparable efficacy to insulin degludec, while CL10 was slightly better than insulin degludec. Specific data are shown in Table 11 below. Figures 4-7 .

[0353] Table 11 Blood glucose levels at different time points after drug administration in each group of mice

[0354]

[0355] Note:* P <0.05,** P <0.01, *** P <0.001 vs. model group. CL3 / CL9 / CL11 had a longer duration of action, requiring a detection time of 168 hours, while the remaining groups were detected within 96 hours.

[0356] Example 11: Efficacy and PK / PD Study of Streptozotocin-Induced Type 1 Diabetic C57BL / 6J Mouse Model

[0357] Pharmacodynamic and pharmacokinetic studies of insulin derivatives CL1, CL2, CL9 and control CL3 in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model were conducted using methods similar to those in Example 10. On day 8, mice that successfully modeled diabetes were randomly divided into 7 groups of 8 mice each, based on their random blood glucose and body weight. The grouping and administration details are shown in Table 12 below.

[0358] Table 12 Grouping and Dosing Regimens of Experimental Animals

[0359]

[0360] Note: All solvents were PBS solutions with pH adjusted to 10.0 ± 0.05 using 2.5M NaOH solution.

[0361] Animals were administered the appropriate solvent or drug via subcutaneous (SC) administration as a single dose, with free access to food and water throughout the experiment. Blood was collected via tail clipping, and random blood glucose levels were measured using a glucometer and matching test strips (Roche) before administration and at 1, 2, 6, 24, 30, 48, 72, 96, 120, 144, 168, and 192 hours post-administration. PK blood samples were also collected at 24, 96, and 168 hours. Data are presented as mean ± SEM. Graphpad Prism 8.0.2 software was used for plotting and analysis, employing t-tests or one-way ANOVA. P< 0.05 was used as the criterion for judging significant differences.

[0362] Experimental results:

[0363] Pharmacodynamic results showed that, compared with the model group, blood glucose levels gradually decreased after administration of CL3 (1000 nmol / kg), reaching their lowest levels at 24h and 48h, gradually rising after 48h, and disappearing after 72h. The blood glucose-lowering effect of CL9 (500 nmol / kg) was comparable to that of CL3 (1000 nmol / kg). Compared with CL3 (1000 nmol / kg), at the same dose (1000 nmol / kg), blood glucose levels significantly decreased after administration of CL1, CL2, and CL9, all reaching their lowest levels at 48h with stronger hypoglycemic effects. Blood glucose levels gradually rose after 48h, prolonging the effective hypoglycemic control time, and still showing hypoglycemic effects 192h after administration (P<0.05). (v vs model group); Throughout the experimental period, the blood glucose-lowering effect of CL9 was dose-dependent, meaning that the higher the dose, the stronger the hypoglycemic effect; as the dose increased, the effective blood glucose control time was prolonged. The effective blood glucose control time of low and medium doses of CL9 (250 nmol / kg and 500 nmol / kg) was about 72 hours, while the effective blood glucose control time of high dose of CL9 (1000 nmol / kg) could reach more than 192 hours, as shown in Table 13 below.

[0364] The pharmacokinetic parameters showed that at the same dose (1000 nmol / kg), within 24–168 h after administration, the plasma concentrations of CL1 and CL2 were significantly higher than CL3 (approximately 3–37 times), while CL9 was comparable to CL3 (approximately 1–2 times). CL9 plasma concentrations showed a linear correlation within the dose range of 250–1000 nmol / kg. Detailed data are shown in Table 14 below. Figures 8-10 .

[0365] Table 13 Blood glucose levels at different time points after drug administration in each group of mice

[0366]

[0367] Note:* P <0.05,**P <0.01, *** P <0.001 vs model group.

[0368] Table 14 Plasma exposure at different time points after drug administration (1000 nmol / kg) in each group of mice

[0369]

[0370] Note: Quantitative range: 20ng / ml-20000ng / ml; BLQ is below the lower limit of quantification.

[0371] Example 12: Efficacy and PK / PD Study of Streptozotocin-Induced Type 1 Diabetic C57BL / 6J Mouse Model

[0372] Pharmacodynamic and pharmacokinetic studies of insulin derivatives CL13-CL14 and control CL3 were conducted in a streptozotocin-induced type 1 diabetes C57BL / 6J mouse model using methods similar to those in Examples 10 and 11. On day 8, mice that successfully modeled the disease were randomly divided into 8 groups of 8 mice each, based on their random blood glucose and body weight. The grouping and administration details are shown in Table 15 below.

[0373] Table 15 Grouping and Dosing Regimens of Experimental Animals

[0374]

[0375] Note: All solvents were PBS solutions with pH adjusted to 10.0 ± 0.05 using 2.5M NaOH solution.

[0376] Animals were administered the appropriate solvent or drug via subcutaneous (SC) administration as a single dose, with free access to food and water during the experiment. Blood was collected via tail clipping, and random blood glucose levels were measured before administration and at 1, 2, 6, 24, 48, 72, 96, 120, 144, 168, and 192 hours post-administration using a glucometer and matching test strips (Roche). Simultaneously, pharmacokinetic (PK) blood samples were collected at 24, 96, and 168 hours to determine plasma drug concentrations. Data are presented as mean ± SEM. Graphpad Prism 8.0.2 software was used for graphical analysis, and t-tests or one-way ANOVA were employed. A p-value < 0.05 was considered statistically significant.

[0377] Experimental results:

[0378] Pharmacodynamic results showed that, compared with the model group, blood glucose gradually decreased after administration of CL3 (1000 nmol / kg), reaching the lowest level at 48h, and then gradually rose. The hypoglycemic effects of CL13 (250 nmol / kg), CL14 (250 nmol / kg) and CL3 (1000 nmol / kg) were comparable within 192h, indicating that CL13 and CL14 had more significant hypoglycemic effects than CL3. The efficacy of CL13 and CL14 dose groups (250, 500 and 1000 nmol / kg) was dose-dependent, and at the same dose, the duration of action and blood glucose control effect of the two were comparable.

[0379] Pharmacokinetic results showed that CL13 and CL14 exhibited a linear correlation in plasma concentrations within the dose range of 250–500 nmol / kg; within the dose range of 500–1000 nmol / kg, plasma concentrations increased proportionally to the dose; at the same dose, their plasma concentrations were essentially equivalent; at the same dose of 1000 nmol / kg, within 24–168 hours post-administration, the plasma concentration of CL13 was slightly higher than that of CL3 (approximately 1.8–2.6 times), and that of CL14 was higher than that of CL3 (approximately 2.1–3 times). Detailed data are shown in Tables 16–17 below. Figures 11-14 .

[0380] Table 16 Blood glucose levels at different time points after drug administration in each group of mice

[0381]

[0382] Note:* P <0.05,** P <0.01, *** P <0.001 vs model group.

[0383] Table 17 Plasma exposure at different time points after drug administration in each group of mice

[0384]

[0385] Note: Quantitative range: 20ng / ml-20000ng / ml; BLQ is below the lower limit of quantification.

Claims

1. An insulin derivative, wherein the insulin derivative is selected from the following: A14E,B16H,B25H,B29K-N(ε)-eicosanodiayl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin; A14E,B16H,B25H,B29K-N(ε)-eicosanodiayl-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin; A14E,B16H,B25H,B29K-N(ε)-eicosanodiayl-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl, desB30 human insulin.

2. The insulin derivative according to claim 1, wherein the insulin derivative is selected from the following: A14E,B16H,B25H,B29K-N(ε)-docosadicialog-glutamyl-polyethylene glycol propionyl-polyethylene glycol propionyl, desB30 human insulin; A14E,B16H,B25H,B29K-N(ε)-docosadicialog-glutamyl-pentaethylene glycol propionyl-pentaethylene glycol propionyl, desB30 human insulin.

3. A pharmaceutical composition comprising the insulin derivative of claim 1 or 2 and one or more pharmaceutically acceptable excipients.

4. Use of the insulin derivative of claim 1 or 2 or the pharmaceutical composition of claim 3 in the preparation of a medicament for the treatment or prevention of diabetes or hyperglycemia.

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