阅读说明：本技术 包含胰岛素肽和egf(a)肽的双功能化合物 (Bifunctional compounds comprising an insulin peptide and an EGF (A) peptide ) 是由 M·W·B·蒙策尔 S·霍斯特鲁普 G·K·波夫尔森 M·诺尔曼 T·B·克杰尔德森 C· 于 2019-10-04 设计创作，主要内容包括：本发明涉及包含胰岛素和EGF(A)类似物或其衍生物的新型共价连接的双功能融合蛋白,及其药学用途。此外,本发明涉及包含这类双功能化合物的药物组合物,并且涉及这类化合物在治疗或预防与糖尿病有关的医学状况和与糖尿病相关的血脂异常中的用途。(The present invention relates to a novel covalently linked bifunctional fusion protein comprising insulin and an egf (a) analogue or derivative thereof, and pharmaceutical uses thereof. Furthermore, the present invention relates to pharmaceutical compositions comprising such bifunctional compounds and to the use of such compounds for the treatment or prevention of medical conditions associated with diabetes and dyslipidemia associated with diabetes.)

1. A fusion protein comprising an insulin peptide, an egf (a) peptide, a spacer, and a substituent, wherein:

i. the insulin peptide is human insulin (SEQ ID NO 2 and 3) or an analogue of human insulin,

the EGF (A) peptide is an analogue of the EGF (A) domain of LDL-R (293-332) according to SEQ ID NO:1,

the spacer is a peptide linker comprising a segment of (GAQP) N or (GQAP) N, where N is 1-20, and the N-terminus of the insulin analogue B-chain is linked to the C-terminus of an egf (a) analogue, and

the substituent has formula (I): Acy-AA2_m-AA3_p-, wherein

Acy is a fatty diacid containing from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue, and wherein m is an integer in the range of 1 to 10, and

AA3 is a neutral alkylene glycol-containing amino acid residue, and p is an integer in the range of 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may occur in any order,

or a pharmaceutically acceptable salt, amide or ester thereof.

2. The fusion protein of claim 1, wherein the egf (a) analog comprises 301L.

3. The fusion protein of claim 2, wherein the EGF (A) analog further comprises 309R; [309R, 312E ] or [309R, 312E,321E ].

4. The fusion protein of any one of the preceding claims, wherein the egf (a) analogue sequences are 301L,309R,312E and 321E.

5. The fusion protein of any one of the preceding claims, wherein the insulin peptide is human insulin or an analogue/derivative of human insulin comprising up to 12 mutations.

6. The fusion protein of any one of the preceding claims, wherein the insulin analogue comprises desB 30.

7. The fusion protein of any one of the preceding claims, wherein the spacer comprises (GAQP) n, wherein n is 2-10.

8. The fusion protein of any one of the preceding claims, wherein the substituents are linked via Lys/K amino acid residues in the insulin sequence within the compound.

9. The fusion protein of claim 8, wherein the substituent is linked via Lys/K amino acid residue B29K in the insulin sequence of the compound.

10. The fusion protein of claim 9, wherein the at least one acyl moiety comprises a fatty diacid group selected from the group consisting of 1, 16-hexadecanedioic acid, 1, 18-octadecanedioic acid, and 1, 20-eicosanedioic acid.

11. The fusion protein of any one of the preceding claims, wherein the fusion protein is selected from the compounds of examples 1-24 (SEQ ID nos 17-40):

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (hexadecanediacyl-gGlu-2 xOEG), desB30) (chemical formula 1)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 10-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 2)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 3)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 4)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-OEG), desB30) (chemical formula 5)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (chemical formula 6)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (chemical formula 7)

EGF (A), (301L,309R,312E,321E) - [ GQAP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 8)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 3-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 9)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 3-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 10)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 4-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 11)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 4-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 12)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 13)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 14)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-OEG), desB30) (chemical formula 15)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (formula 16)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 8-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 17)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 12-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 18)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 19)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 20)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30) (chemical formula 21)

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (chemical formula 22)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (chemical formula 23)

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30) (chemical formula 24).

12. The fusion protein according to any one of claims 1-11 for use as a medicament.

13. The fusion protein according to any one of claims 1-11 for use in the treatment of diabetes and diabetes-associated dyslipidemia.

14. A pharmaceutical composition for treating diabetes in a patient in need thereof, comprising a therapeutically effective amount of a fusion protein according to any one of claims 1-11, and a pharmaceutically acceptable excipient.

15. Use of a fusion protein according to any one of claims 1-11 for the preparation of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and diabetic dyslipidemia.

Technical Field

The present invention relates to a novel bifunctional fusion peptide comprising an insulin analogue or a derivative thereof and an EGF (A) analogue, and pharmaceutical uses thereof. Furthermore, the present invention relates to pharmaceutical compositions comprising such bifunctional fusion peptides and to the use of such fusion peptides for the treatment or prevention of diabetes-related medical conditions and diabetes-associated dyslipidemia.

Incorporation by reference of sequence listing

This application is filed with a sequence listing in electronic form. The entire contents of this sequence listing are incorporated herein by reference.

Background

Diabetes is a metabolic disorder in which the ability to utilize glucose is partially or completely lost. Over 5% of the world's population suffers from diabetes, and millions of people are at risk. Insulin therapy for the treatment of diabetes has been used for decades and involves the administration of several insulin injections per day. Such therapy typically involves administering a long-acting basal injection once or twice daily, and a fast-acting insulin at meal time (i.e., prandial insulin). Type 2 diabetic patients often suffer from various metabolic dysfunctions, such as dyslipidemia, obesity and cardiovascular complications, in addition to hyperglycemia, for which current insulin therapy has only limited beneficial effects. Diabetic dyslipidemia, characterized by elevated LDL-c (low density lipoprotein cholesterol), reduced HDL and elevated triglycerides, is a well-established driver of cardiovascular disease (CVD).

Statins have been used for decades in the treatment of dyslipidemia, the administration of which shows a clear and sustained reduction of cardiovascular events with an acceptable safety profile. Despite the availability and widespread use of statins and other lipid lowering drugs, many patients do not reach their target LDL-C levels and are still at high risk for CVD.

PCSK9 (proprotein convertase subtilisin/Kexin type 9) promotes hepatic LDL-R (LDL receptor) degradation, thereby reducing hepatic LDL-R surface expression and, therefore, LDL particle clearance. In contrast, blocking PCSK9 increased clearance of LDL-C and other atherogenic lipoproteins such as medium density lipoprotein and residual particles. This additional clearance may have therapeutic benefits beyond the effects provided by LDL reduction alone.

The EGF (A) (epidermal growth factor-like domain A) sequence (40 amino acids) of LDL-R (LDL-R- (293-332)) is recognized as the site for PCSK9 binding. Isolated wild type EGF (A) peptides have been shown to have IC in the low μ M range₅₀Inhibits the binding of PCSK9 to LDL-R (Biochemical and Biophysical Research Communications 375(2008) 69-73). This poor binding affinity hampers the practical pharmaceutical use of egf (a) peptides.

WO2012177741 and j.mol.biol. (2012)422,685-696 are said to disclose analogues of egf (a) and Fc fusions thereof. WO 2015/127273 is said to disclose the fusion of anti-PCSK 9 and GLP-1.

WO2017121850 purportedly discloses egf (a) analogs having fatty acid substituents.

Two anti-PCSK 9 antibodies, alirocumab (alirocumab) (II) ((III))Sanofi-Aventis) and Evokumab (evolocumab)Amgen Europe BV), recently approved for useFor treatment of high LDL-C levels, administration was by subcutaneous injection every two weeks.

Insulin therapy is well known for regulating blood glucose levels in diabetic patients. It is also well known that patients are at high risk for CVD and are at risk for microvascular complications such as nephropathy, retinopathy and neuropathy. With current therapies, about 50% of diabetics still die from cardiovascular disease. Therefore, there is currently a strong need to provide treatments that can combine the effects of lowering blood glucose with the effects of lowering LDL cholesterol.

Disclosure of Invention

In its broadest aspect, the present invention relates to the combination of insulin with egf (a).

In another aspect, the compounds of the invention comprise an insulin peptide or analog thereof, an egf (a) peptide or analog thereof, a spacer and a substituent.

In another aspect, the compounds of the invention are fusion proteins comprising an insulin peptide or analog thereof, an egf (a) peptide or analog thereof, a spacer and a substituent.

In another aspect, the fusion protein of the invention comprises an insulin peptide, an EGF (A) peptide, a spacer and a substituent, wherein,

i. the insulin peptide is human insulin (SEQ ID NOS: 2 and 3) or an analogue of human insulin

The EGF (A) peptide is an analogue of the EGF (A) domain of LDL-R (293-332) (SEQ ID NO:1)

The spacer is a peptide linker comprising a segment of (GAQP) N or (GQAP) N, where N is 1-20, and linking the N-terminus of the insulin analogue B-chain to the C-terminus of the egf (a) analogue

The substituent has formula (I): Acy-AA2_m-AA3_p-, wherein

Acy is a fatty diacid containing from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue, and wherein m is an integer in the range of 1 to 10, and

AA3 is a neutral alkylene glycol-containing amino acid residue, and p is an integer in the range of 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may occur in any order,

or a pharmaceutically acceptable salt, amide or ester thereof.

Since diabetic patients requiring insulin administration belong to the high risk group for CVD, the inclusion of LDLc reducing properties in the insulin compound provides improved therapy for diabetic patients, particularly cholesterol reduction, treatment of dyslipidemia and reduction of risk of CVD.

In one aspect, the bifunctional fusion proteins of the invention lower blood glucose levels and bind PCSK9, thereby enhancing the expression of functional LDL-R in the liver.

In one aspect, the present invention provides novel bifunctional fusion proteins that are capable of both activating the insulin receptor and binding PCSK9, i.e., combining the effects of lowering blood glucose with the effects of lowering LDL cholesterol.

In another aspect, the bifunctional fusion peptides of the invention lower blood glucose levels and bind PCSK9, thereby enhancing the expression of functional LDL-R in the liver.

In another aspect, the fusion peptide of the invention lowers blood glucose levels.

In another aspect, the fusion peptide of the invention lowers LDL cholesterol.

In another aspect, the invention relates to a pharmaceutical composition comprising a fusion peptide according to the invention.

In another aspect, the invention relates to a fusion peptide according to the invention for use as a medicament.

In another aspect, the present invention relates to a fusion peptide according to the invention for use in the treatment of diabetes and diabetes-associated dyslipidemia.

In another aspect, the invention relates to the medical use of a fusion peptide according to the invention.

The present invention may also address other issues that will be apparent from the disclosure of exemplary embodiments.

Brief Description of Drawings

FIG. 1 shows the hypoglycemic effects of the compounds of examples 4, 9 and 14 of the present invention, all having octadecanedioyl-gGlu-2 xOEG side chains and having GQAP/GAQP spacers of different lengths.

FIG. 2 shows the hypoglycemic effects of the compounds of examples 2, 12, 17, 18 and 19 of the present invention, all having octadecanedioyl-gGlu-2 xOEG side chains and having GQAP/GAQP spacers of different lengths.

Figure 3 shows the hypoglycemic effects of the compounds of examples 4 and 17 of the invention with GQAP/GAQP spacers of different lengths, all with octadecanedioyl-gGlu-2 xeeg side chains and vehicle, relative to the hypoglycemic effects of comparative compounds 1 and 2 with GQEP spacers.

Figure 4 shows the hypoglycemic effects of the compounds of examples 3, 11 and 13 of the present invention with GQAP/GAQP spacers of different lengths, all with octadecanedioyl-gGlu-2 xeeg side chains and vehicle, relative to the hypoglycemic effects of comparative compounds 1 and 2 with GQEP spacers.

Figure 5 shows the hypoglycemic effects of the compounds of examples 4 and 8 of the invention having a 2xGQAP/GAQP spacer, all of which have octadecanedioyl-gGlu-2 xeeg side chains, relative to the hypoglycemic effect of comparative compound 1 having a 2xGQEP spacer.

Figure 6 shows the hypoglycemic effects of the compounds of examples 11 and 12 of the invention having a 4 xgaiqp spacer, all of which have octadecanedioyl-gGlu-2 xeeg side chains, relative to the hypoglycemic effect of comparative compound 4 having a 4xGQEP spacer.

Figure 7 shows the hypoglycemic effects of the compounds of examples 3, 4 and 5 of the invention having a2 xgaiqp spacer, all of which have octadecanedioyl-gGlu-2 xeeg side chains, relative to the hypoglycemic effect of comparative compound 1 having a 2xGQEP spacer.

Figure 8 shows the hypoglycemic effects of the compounds of examples 13, 14 and 15 of the invention with a 6 xgaiqp spacer, all of which have a C18 side chain, relative to the hypoglycemic effect of comparative compound 6 with a 6xGQEP spacer.

Figure 9 shows the hypoglycemic effect of the compound of example 1 of the invention with a2 xgaiqp spacer, all of which have a hexadecanediacyl-gGlu-2 xeeg side chain, relative to the hypoglycemic effects of comparative compounds 5 and 7 with 2x or 8x GQEP spacers.

Figure 10 shows the hypoglycemic effect of the compound of example 16 of the invention having a 6 xgaiqp spacer, both having eicosanedioyl-gGlu-2 xeeg side chains, relative to the hypoglycemic effect of comparative compound 3 having a 6xGQEP spacer.

FIG. 11 shows the hypoglycemic effect of the compound of example 4 of the present invention having 2xGAQP, both having octadecanedioyl-gGlu-2 xOEG side chains, relative to the hypoglycemic effect of the compound 8 compared to "insulin only".

Figure 12 shows the dose response of the compound of example 3 (0, 3, 10, 30 and 100nmol/kg) administered intravenously at t 0min, followed by intravenous administration of vehicle or hPCSK9 at t 15 min. Hepatic LDL-r protein expression following administration of vehicle or compound of example 3 to diabetic mice followed by administration of vehicle or hPCSK 9.

Figure 13 shows the dose response of the compound of example 3 (0, 3, 10, 30 and 100nmol/kg) administered intravenously at t 0min, followed by intravenous administration of vehicle or hPCSK9 at t 15 min. Blood glucose profile following administration of vehicle or compound of example 3 to diabetic mice.

Figure 14 shows administration to diabetic mice, the compounds of examples 3 and 13 (0 and 10nmol/kg) were administered intravenously at t ═ 0min, followed by intravenous administration of vehicle or hPCSK9 at t ═ 15 min. Hepatic LDL-r protein expression following administration of vehicle, the compounds of examples 3 and 13 or egf (a) derivative (comparative compound 10) to diabetic mice followed by hPCSK 9.

Description of the invention

The present invention relates to bifunctional compounds that activate the insulin receptor and bind to PCSK 9.

In one embodiment, the present invention relates to a fusion protein comprising an insulin peptide and an egf (a) peptide.

In one embodiment, the invention relates to a fusion protein comprising an insulin analogue and an EGF (A) analogue, wherein the insulin analogue is an analogue of human insulin (SEQ ID NO:2 and 3) and the EGF (A) analogue is an analogue of the EGF (A) domain of LDL-R (293) -332) (SEQ ID NO: 1).

In another embodiment, the EGF (A) peptide is an analog of the peptide of SEQ ID NO 1.

In one embodiment, the insulin analogue is fused to the C-terminal amino acid of the egf (a) peptide analogue via the N-terminal amino acid residue of the insulin analogue B-chain.

In one embodiment, the present invention relates to a fusion protein comprising an insulin peptide, an EGF (A) peptide, a spacer and a substituent, wherein,

i. the insulin peptide is human insulin (SEQ ID NOS: 2 and 3) or an analogue of human insulin

The EGF (A) peptide is an analogue of the EGF (A) domain of LDL-R (293-332) (SEQ ID NO:1)

The spacer is a peptide linker comprising a segment of (GAQP) N or (GQAP) N, where N is 1-20, and linking the N-terminus of the insulin analogue B-chain to the C-terminus of the egf (a) analogue

The substituent has formula (I): Acy-AA2_m-AA3_p-, wherein

Acy is a fatty diacid containing from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue, and wherein m is an integer in the range of 1 to 10, and

AA3 is a neutral alkylene glycol-containing amino acid residue, and p is an integer in the range of 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may occur in any order,

or a pharmaceutically acceptable salt, amide or ester thereof.

In one embodiment, the insulin analogue is fused to the C-terminal amino acid of the egf (a) peptide analogue via the B1 amino acid residue of the N-terminal B chain of the insulin analogue.

In one embodiment, the insulin analogue is fused via the N-terminal amino acid residue of the insulin analogue B-chain, via the spacer, to the C-terminal amino acid of the egf (a) peptide analogue.

In one embodiment, the insulin analogue is fused to the C-terminal amino acid of an egf (a) peptide analogue via the N-terminal amino acid residue of the insulin analogue B-chain, via a spacer comprising a segment of (GAQP) N or (GQAP) N (where N-2-19).

Since diabetic patients who require insulin administration belong to the high risk group for CVD, the inclusion of LDLc reducing properties in the insulin fusion peptide will provide improved therapy for diabetic patients and reduce their CVD risk.

In one embodiment, the fusion peptide of the invention lowers blood glucose levels.

In another embodiment, the fusion peptide of the present invention exhibits superior blood glucose reduction relative to a comparative fusion peptide comprising (GQEP) n.

In one embodiment, the fusion peptide of the invention combines the effects of lowering blood glucose with the effects of lowering LDL cholesterol.

In another embodiment, the invention relates to a pharmaceutical composition comprising a fusion peptide according to the invention.

In another embodiment, the invention relates to a pharmaceutical composition comprising the fusion peptide of the invention and a pharmaceutically acceptable excipient.

In another embodiment, the invention relates to a fusion peptide according to the invention for use as a medicament.

In another embodiment, the invention relates to a fusion peptide according to the invention for use in the treatment of diabetes and diabetes-associated dyslipidemia.

In another embodiment, the invention relates to the medical use of a fusion peptide according to the invention.

General definitions

The term "compound" is used herein to denote a molecular entity, and thus a "compound" may have different structural elements in addition to the smallest element defined for each compound or group of compounds. Thus, the compound may be a fusion compound/peptide or derivative thereof, as long as the compound comprises the defined structural and/or functional elements. The term "compound" is also intended to encompass pharmaceutically relevant forms thereof, i.e., the invention relates to a compound as defined herein, or a pharmaceutically acceptable salt, amide or ester thereof.

The term "peptide" or "polypeptide" as used, for example, in the context of the present invention, refers to a compound comprising a series of amino acids interconnected by amide (or peptide) bonds. In a particular embodiment, the peptide consists of amino acids linked to each other by peptide bonds.

The term "analog" generally refers to a peptide having one or more amino acid changes in its sequence as compared to a reference amino acid sequence. Analogs that "comprise" certain specified changes can comprise further changes as compared to their reference sequence. In particular embodiments, an analog "has" or "includes" the specified changes. In other particular embodiments, an analog "consists of changes. When the term "consisting of" or "consisting of … …" is used with respect to an analog, e.g., where the analog consists of a set of designated amino acid mutations, it is understood that the designated amino acid mutations are the only amino acid mutations in the analog. In contrast, an analog that "comprises" a specified set of amino acid mutations can have additional mutations. In the context of the present application, the term "analogue" also denotes analogues of the egf (a) human insulin fusion protein.

The term "derivative" generally refers to a compound that can be prepared from a native peptide or an analog thereof by chemical modification, particularly by covalent attachment of one or more substituents. Derivatives may also be referred to as acylated analogs.

The term "amino acid" includes proteinogenic (or natural) amino acids (of which there are 20 standard amino acids) as well as non-proteinogenic (or non-natural) amino acids. Proteinogenic amino acids are amino acids that are naturally incorporated into proteins. The standard amino acid is the amino acid encoded by the genetic code. Non-protein amino acids are either not present in the protein or are not produced by standard cellular mechanisms (e.g., they may have undergone post-translational modification).

Generally, amino acid residues (peptide/protein sequences) as used herein may be represented by their full name, their single letter code, and/or their three letter code. These three approaches are fully equivalent and are used interchangeably. Hereinafter, each amino acid of the peptide of the present invention whose optical isomer is not specified should be understood as meaning L-isomer (unless otherwise specified). Amino acids are molecules containing an amino group and a carboxylic acid group and optionally one or more additional groups commonly referred to as side chains. Herein, the term "amino acid residue" is an amino acid from which a hydroxyl group has been formally removed from a carboxyl group, and/or an amino acid from which a hydrogen atom has been formally removed from an amino group.

The terms "fusion" and "fused" are used for compounds comprising two separately defined peptide/protein sequences, which are linked by a peptide bond or by a peptide spacer (also linked by a peptide bond).

Insulin

The term "human insulin" as used herein means human insulin hormone, the structure and nature of which is well known. Human insulin has two polypeptide chains, designated as the A chain and the B chain. The a chain is a21 amino acid peptide and the B chain is a 30 amino acid peptide, the two chains being connected by a disulfide bridge: a first bridge between the cysteine at position 7 of the a-chain and the cysteine at position 7 of the B-chain, and a second bridge between the cysteine at position 20 of the a-chain and the cysteine at position 19 of the B-chain. The third bridge is present between the cysteines at positions 6 and 11 of the A chain.

The human insulin a chain has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO:2), and the B chain has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 3).

In the human body, the hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a 24 amino acid propeptide followed by 86 amino acid proinsulin, which is configured as: the propeptide-B-Arg-C-Lys Arg-A, wherein C is a 31 amino acid linker peptide. Arg-Arg and Lys-Arg are cleavage sites for the linker peptide from the A and B chains.

"insulin" according to the invention is to be understood here as human insulin or insulin from another species, such as porcine or bovine insulin.

The term "insulin peptide", "insulin compound" or "insulin" as used herein means a peptide that is human insulin or an analog or derivative thereof that has insulin activity (i.e., activates the insulin receptor).

Insulin analogues

The term "insulin analogue" as used herein means a modified human insulin, wherein one or more amino acid residues of the insulin have been substituted by other amino acid residues, and/or wherein one or more amino acid residues have been deleted from the insulin, and/or wherein one or more amino acid residues have been added and/or inserted into the insulin.

The term "mutation" as used herein means a substitution or deletion of an amino acid within the sequence of human insulin. The term mutation does not include addition, elongation or extension to the sequence of human insulin. Mutations in the insulin molecule are indicated by the specification of the chain (a or B) in which the amino acid residue replacing the natural amino acid residue is located, its position and the single or three letter code.

Any mutation of an insulin analogue as used herein refers to a mutation of the insulin peptide alone, without including any spacer peptide linked to the insulin peptide/analogue.

By "connecting peptide" or "C-peptide" is meant the connecting portion "C" of the B-C-A polypeptide sequence of the single-chain proinsulin-molecule. In the human insulin chain, the C-peptide is linked at position 30 of the B chain and at position 1 of the A chain and is 35 amino acid residues in length. In human insulin, the linker peptide includes two terminal dibasic amino acid sequences, such as Arg-Arg and Lys-Arg, which serve as cleavage sites for the linker peptide to cleave from the A and B chains to form a double-stranded insulin molecule.

"desB 30" or "B (1-29)" means the B chain of native insulin or an analog thereof lacking the B30 amino acid, while "A (1-21)" means the A chain of native insulin. Thus, for example, desB30 human insulin is an analog of human insulin in which the amino acid at position 30 in the B chain is deleted.

Herein, terms like "a 1", "a 2" and "A3" denote the amino acids at positions 1,2 and 3, respectively, in the a chain of insulin (counting from the N-terminus). Similarly, terms such as "B1", "B2" and "B3" denote the amino acids at positions 1,2 and 3, respectively, in the B chain of insulin (counting from the N-terminus). Using the one letter code for amino acids, terms such as a21A, a21G, and a21Q denote the amino acid at position a21 as A, G and Q, respectively. Using the three letter code for amino acids, the corresponding designations are a21Ala, a21Gly, and a21Gln, respectively.

In one embodiment, an analogue or derivative of human insulin of the invention has the ability to lower blood glucose levels.

In one embodiment, an analog or derivative of human insulin of the invention activates the insulin receptor.

In one embodiment, an analogue or derivative of human insulin of the invention lowers blood glucose.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin or a derivative thereof.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising up to 12 mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising up to 10 mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising 1-6 mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising 1-3 mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising one mutation.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising two mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising three mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin containing four mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin containing five mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin containing six mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising seven mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin containing eight mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising nine mutations.

In one embodiment, the fusion peptide of the invention comprises an analogue of human insulin comprising 10 mutations.

In one embodiment, the fusion peptide of the invention comprises human insulin.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB 30.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising a 14E.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising B3E.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising a14E, desB 30.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising B3E, desB 30.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 9 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 8 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 7 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 6 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 5 mutations in said insulin analogue.

In one embodiment the fusion peptide of the invention comprises an insulin analogue containing desB30 and further 4 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 3 mutations in said insulin analogue.

In one embodiment, the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further 2 mutations in said insulin analogue.

In one embodiment the fusion peptide of the invention comprises an insulin analogue comprising desB30 and further comprising one further mutation in said insulin analogue.

EGF(A)

The term "EGF (A) compound" or "EGF (A) peptide" is used herein to broadly refer to fusion proteins comprising an EGF (A) peptide encompassing wt-LDL-R (293-332) and analogs thereof as defined in SEQ ID NO: 1. The term EGF (a) compound includes derivatives of the EGF- (a) peptide and analogues thereof, i.e. EGF (a) peptide analogues having an acyl moiety as described herein are typical examples of EGF (a) compounds. The term "EGF (A) analog" herein refers to the modified EGF (A) domain of LDL-R (293-332) (SEQ ID NO: 1).

The terms "EGF (A) domain of LDL-R", "LDL-R (293) -332)", "native LDL-R (293) -332)," EGF (A) (293) -332) "," wild-type EGF (A) "," wt-EGF (A) "or" native EGF (A) "as used herein refer to a peptide consisting of the sequence SEQ ID NO: 1:

Gly-Thr-Asn-Glu-Cys-Leu-Asp-Asn-Asn-Gly-Gly-Cys-Ser-His-Val-Cys-Asn-Asp-Leu-Lys-Ile-Gly-Tyr-Glu-Cys-Leu-Cys-Pro-Asp-Gly-Phe-Gln-Leu-Val-Ala-Gln-Arg-Arg-Cys-Glu。

in this formula, the numbering of the amino acid residues follows that of the EGF (A) domain of LDL-R (LDL-R- (293) -332)), where the first (N-terminal) amino acid residue is numbered 293 or is given position 293, and the subsequent amino acid residues towards the C-terminus are numbered 294, 295, 296, etc., up to the last (C-terminal) amino acid residue, which is Glu numbered 332 in the EGF (A) domain of LDL-R.

The different numbering is done in the sequence listing, where the first amino acid residue (Gly) of SEQ ID NO. 1 is designated as number 1 and the last (Glu) is designated as number 40. The same applies to the other sequences in the sequence listing, i.e.the designated N-terminal amino acid is number 1, regardless of its orientation relative to the 293Gly or 293 replacement amino acid residue of the reference LDL-R (293-332). However, as explained above, the numbering of the amino acid positions herein is with reference to LDL-R (293-332).

EGF (A) analogues

The term "egf (a) analogue" generally refers to a peptide whose sequence has one or more amino acid changes compared to a reference amino acid sequence.

The terms "EGF (A) domain of LDL-R (293-332)," "EGF (A) domain of LDL-R (293-332) analogue of SEQ ID NO: 1", "LDL-R (293-332) analogue", "EGF (A) analogue" or "analogue of SEQ ID NO: 1" as used herein may be referred to as peptides, the sequences of which comprise mutations, i.e.amino acid substitutions or deletions relative to the sequence SEQ ID NO: 1.

Any mutation of an egf (a) analogue as used herein refers to a mutation of the egf (a) peptide alone, and does not include any spacer peptide linked to the egf (a) peptide/analogue.

In one embodiment, the EGF (A) domain of LDL-R (293-332) according to SEQ ID NO:1 or an analog thereof is capable of inhibiting the binding of PCSK9 to the human low-density lipoprotein receptor (LDL-R).

In one embodiment, the EGF (A) domain of LDL-R (293-332) according to SEQ ID NO:1 or an analogue thereof has the ability to inhibit the binding of PCSK9 to LDL-R.

In one embodiment, the EGF (A) domain of LDL-R (293-332) according to SEQ ID NO:1 or an analog thereof has the ability to inhibit PCSK9 binding to LDL-R and to lower LDL levels in the blood.

In one embodiment, the EGF (A) domain of LDL-R (293-332) according to SEQ ID NO:1 or an analog thereof reduces LDL blood levels.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analogue of SEQ ID NO:1, wherein said EGF (A) analogue comprises 1-15 amino acid mutations compared to SEQ ID NO: 1.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analogue of SEQ ID NO:1, wherein said EGF (A) analogue comprises 1-10 amino acid mutations compared to SEQ ID NO: 1.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analogue of SEQ ID NO:1, wherein said EGF (A) analogue comprises 1-8 amino acid mutations compared to SEQ ID NO: 1.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analogue of SEQ ID NO:1, wherein said EGF (A) analogue comprises 1-6 amino acid mutations compared to SEQ ID NO: 1.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein said EGF (A) analog comprises 1-5 mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein said EGF (A) analog comprises a mutation.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein said EGF (A) analog comprises two mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises three mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises four mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises five mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises six mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises seven mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein the EGF (A) analog comprises eight mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein said EGF (A) analog comprises nine mutations.

In one embodiment, the fusion peptide of the invention comprises the EGF (A) domain of the LDL-R (293-332) analog of SEQ ID NO:1, wherein said EGF (A) analog comprises 10 mutations.

In other words, peptide analogs may be described with reference to the native LDL-R (293-332) EGF (A) peptide, i.e., as analogs thereof in which many of the amino acid residues have been altered as compared to native LDL-R (293-332) EGF (A) (SEQ ID NO: 1). These changes may independently represent one or more amino acid mutations.

The EGF (A) analogs incorporated into the fusion peptides of the invention may be referred to as the following LDL-R (293-332) EGF (A) analogs: (301Leu,309Arg,312Glu,321Glu) LDL-R (293) -332) EGF (A) or (Leu301, Arg309, Glu312, Glu321) -LDL-R (293) -332) EGF (A) or (301L,309R,312E,321E) LDL-R (293) -332) or (L301, R309, E312, E321) LDL-R (293) -332). This means that when such analogue is aligned with native LDL-R (293) -332), it i) has Leu in the analogue at a position corresponding to position 301 of native LDL-R (293-332) EGF (A) according to the alignment, ii) has Arg in the analogue at a position corresponding to position 309 of native LDL-R (293-332) EGF (A), iii) has Glu in the analogue at a position corresponding to position 312 of native LDL-R (293-332) EGF (A), iv) has Glu in the analogue at a position corresponding to position 321 of native LDL-R (293-332) EGF (A).

Analogs that "comprise" certain specified changes may comprise further changes as compared to SEQ ID NO 1.

The egf (a) peptide analogue within the fusion peptide of the present invention comprises an amino acid substitution of amino acid residue 301 from Asn to Leu, further described as Asn301Leu, or simply 301L.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analogue of SEQ ID NO:1, wherein said egf (a) analogue comprises 301 Leu.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 309R.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 312E.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 321E.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 301L and 309R.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 301L,309R and 312E.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analog of SEQ ID NO:1, wherein said egf (a) analog comprises 301L,309R,312E and 321E.

In one embodiment, the fusion peptide of the invention comprises an egf (a) analogue of SEQ ID NO:1, wherein the egf (a) peptide analogue comprises amino acid mutations represented by any one of groups i-vii as set forth in:

i.301Leu

ii.301Leu and 309Arg

iii 301Leu and 312Glu

301Leu and 321Glu

v.301Leu、309Arg、312Glu

vi.301Leu, 309Arg and 321Glu

vii.301Leu, 309Arg,312Glu, and 321 Glu.

EGF (A) insulin fusion protein

The compounds or fusion peptides of the invention comprise an insulin peptide, an EGF (A) peptide, and a spacer, wherein,

i. the insulin peptide is human insulin (SEQ ID NO:2 and 3) or an analogue of human insulin,

the EGF (A) peptide is an analogue of the EGF (A) domain of LDL-R (293-332) (SEQ ID NO:1), and

the spacer is a peptide linker linking the N-terminus of the insulin analogue B-chain to the C-terminus of the egf (a) analogue.

The compounds or fusion peptides of the invention further comprise a substituent attached to an amino acid residue of the fusion protein.

The designation of egf (a) insulin fusion protein is given as relative to analogues and derivatives of egf (a) and human insulin protein as detailed above. The designation of a spacer that links two proteins and a substituent attached to the fusion protein is detailed below.

The compounds of the invention may be interchangeably referred to as "compounds of the invention", "bifunctional compounds", "fusion proteins of the invention".

Spacing body

Typically, the fusion peptide/protein comprises a spacer to ensure that any functionality present at the ends of the two peptides/proteins is not disturbed by the proximity of the other peptides/proteins.

In one embodiment, the spacer is a peptide, which is referred to herein as a spacer peptide or peptide spacer or peptide linker. Various spacer peptides are known in the art and may be placed between an insulin analogue and an egf (a) analogue to obtain fusion compounds.

When two peptide segments are to be fused, the order may affect the functionality of the resulting fusion compound as well as derivatives comprising it.

In one embodiment of the invention the order of egf (a) analogue and insulin analogue starting from the N-terminus is egf (a) analogue followed by insulin analogue, optionally separated by a spacer peptide. In one embodiment, the C-terminus of the egf (a) analogue is fused to the N-terminus of the insulin analogue B-chain.

In one embodiment, the spacer is a peptide segment consisting of 4-80 amino acids linked via peptide bonds.

In one embodiment, the spacer comprises one or more of the following amino acid residues: ala (A), Gly (G), Pro (P), Gln (Q).

Surprisingly, the inventors have found that the amino acid composition of the spacer affects the ability of the compound to lower blood glucose levels. Compounds of the invention comprising uncharged spacers (GQAP) n or (GAQP) n show superior blood glucose reduction relative to comparative compounds comprising charged spacers such as (GQEP) n. In addition, the length of the spacer has also been found to affect the ability of the compound to lower blood glucose levels.

TABLE 1 examples of spacers comprised in the inventive Compounds/fusion peptides and spacers comprised in the comparative Compounds

The spacer in the EGF (A) -insulin fusion protein derivative of example 4 was named [ GAQP]2, this means that the spacer linking the C-terminal residue of the EGF (A) peptide to the N-terminal residue of the insulin B chain has the sequence (GAQP)₂It may also be denoted as GAQPGAQP or 2xGAQP or [ GAQP]2 or 2x (gaqp). Amino acid residues may be represented by their full name, their single letter code, and/or their three letter code. These three approaches are fully equivalent and are used interchangeably.

Similarly, the spacer within the EGF (A) -insulin fusion protein derivative of example 2 was named [ GAQP]10, this means that the spacer linking the C-terminal residue of the EGF (A) peptide to the N-terminal residue of the insulin B chain has the sequence (GAQP)₁₀It can also be represented as 10xGAQP, [ GAQP ]]10. 10x (GAQP) or GAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQP.

In one embodiment, the fusion protein of the invention exhibits superior blood glucose reduction relative to a comparative compound comprising (GQEP) n.

In one embodiment, the fusion protein of the invention wherein the spacer comprises (GAQP) n or (GQAP) n exhibits superior blood glucose reduction relative to a comparative compound comprising (GQEP) n.

In another aspect, a fusion protein of the invention wherein the spacer comprises (GAQP) n or (GQAP) n (wherein n is 1-20) exhibits superior blood glucose reduction relative to a comparative compound comprising (GQEP) n.

In another aspect, a fusion protein of the invention wherein the spacer comprises (GAQP) n or (GQAP) n (wherein n is 2-19) exhibits superior blood glucose reduction relative to a comparative compound comprising (GQEP) n.

In another embodiment, the fusion protein of the invention wherein the spacer comprises (GAQP) n (wherein n-2-10) exhibits superior blood glucose reduction relative to a comparative compound comprising (GQEP) n.

In another embodiment, the fusion protein of the invention wherein the spacer comprises (GAQP) n (wherein n-2-10) exhibits superior blood glucose reduction relative to both the comparative compound comprising (GQEP) n and the compound comprising (GAQP) n (n-12-19).

In one embodiment, the fusion protein of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 1-20.

In one embodiment, the fusion protein of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-19.

In one embodiment, the fusion protein of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-12.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-10.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-8.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-6.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 2-4.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 4-6.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-2.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-3.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-4.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-5.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-6.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-7.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-8.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-9.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 10.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-11.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-12.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-13.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-14.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-15.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n ═ 16.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n ═ 17.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n is 18.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n ═ 19.

In one embodiment, the fusion peptide of the invention comprises a spacer comprising (GAQP) n or (GQAP) n, wherein n-20.

In one embodiment, the fusion peptide of the invention comprises a spacer consisting of (GAQP) n or (GQAP) n, wherein n is 2-19, or [ (GAQP) n or (GQAP) n ], wherein n is 2-10.

In one embodiment, the fusion peptide of the invention comprises a spacer consisting of (GAQP) n or (GQAP) n, wherein n is 2-19, or [ (GAQP) n or (GQAP) n ], wherein n is 2-6.

Substituent group

In one embodiment, the substituent/acyl moiety is linked to a fusion protein of the invention (i.e., a bifunctional insulin egf (a) fusion compound or a bifunctional compound).

Ideally, the substituent has no or minimal effect on the functionality of the egf (a) peptide and has the expected effect on the functionality of insulin, i.e. a reduction in the affinity of the insulin receptor, similar to the effect of linking an acyl moiety to insulin without the egf (a) peptide.

In one embodiment, the acyl moiety is linked via a Lys/K amino acid residue within the insulin analogue sequence.

In one embodiment, the substituent attached to the compounds of the present invention has the general formula (I): Acy-AA2_m-AA3_p-, wherein

Acy is a fatty diacid containing from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue, and wherein m is an integer in the range of 1 to 10, and

AA3 is a neutral alkylene glycol-containing amino acid residue, and p is an integer in the range of 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may occur in any order,

or a pharmaceutically acceptable salt, amide or ester thereof.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said Acy comprises a fatty diacid group selected from the group consisting of 1, 16-hexadecanedioic acid, 1, 18-octadecanedioic acid and 1, 20-eicosanedioic acid.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said Acy comprises the fatty diacid group 1, 16-hexadecanedioic acid.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said Acy comprises the fatty diacid group 1, 18-octadecanedioic acid.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said Acy comprises a fatty diacid group 1, 20-eicosanedioic acid.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said AA2_mComprising a gGlu representing a gamma glutamic acid residue represented by the following structure:

in one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, wherein said AA3_pComprising [2- (2-aminoethoxy) ethoxy]Acetyl or amino acid residue 8-amino-3, 6-dioxaoctanoic acid-NH (CH)₂)₂O(CH₂)₂OCH₂CO-, and is represented by the following structure:

in one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_pAnd wherein AA2_m-AA3_p-is independently represented by gGlu-OEG or gGlu-OEG-OEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_pAnd wherein AA2_m-AA3_p-is represented by gGlu-OEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_pAnd wherein AA2_m-AA3_p-is represented by gGlu-OEG-OEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, independently represented by:

i.e. the acid C16-gGlu,

ii.C18 diacid-gGlu-OEG,

c18 diacid-gGlu-2 xOEG or

C20 diacid-gGlu-2 xOEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, represented by C16 diacid-gGlu.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, represented by C18 diacid-gGlu-OEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, represented by the C18 diacid-gGlu-2 xOEG.

In one embodiment, the substituent is of the formula (I) Acy-AA2_m-AA3_p-, represented by the C20 diacid-gGlu-2 xOEG.

In another embodiment, the acyl moiety attached to the fusion peptide of the invention has the general formula Acy-AA2_m-AA3_p- (I) wherein AA2 is selected from L-or D-gGlu, L-or D-Glu, L-or D-Asp, L-or D-homoGlu.

The acidic amino acid residue designated AA2 is an amino acid with a molecular weight of up to about 200Da, which comprises two carboxylic acid groups and one primary or secondary amino group.

The neutral alkylene glycol-containing amino acid residue designated AA3 is an alkylene glycol moiety, optionally an oligomeric or polyalkylene glycol moiety containing a carboxylic acid functional group at one terminus and an amino functional group at the other terminus.

Herein, the term alkylene glycol moiety encompasses mono-alkylene glycol moieties as well as oligo-alkylene glycol moieties. The mono-and oligo-alkylene glycols comprise mono-and oligo-polyethylene glycol-based, mono-and oligo-propylene glycol-based and mono-and oligo-polybutylene glycol-based chains, i.e. based on the repeating unit-CH₂CH₂O-、-CH₂CH₂CH₂O-or-CH₂CH₂CH₂CH₂Chain of O-. The alkylene glycol moiety is monodisperse (with well defined length/molecular weight). The monoalkylene glycol moiety comprising a-OCH containing moiety at each terminus₂CH₂O-、-OCH₂CH₂CH₂O-or-OCH₂CH₂CH₂CH₂Different groups of O-.

As mentioned herein, AA2 and AA3 are in the form of (I) (Acy-AA 2)_m-AA3_p-) the order of occurrence in the acyl moiety may be independently interchanged. Thus, the formula Acy-AA2_m-AA3_pAlso encompassed are moieties such as, for example, the formula Acy-AA2_m-AA3_p-, of the formula Acy-AA2-AA3_n-AA 2-and of the formula Acy-AA3_p-AA2_m-, wherein AcyAA2, AA3, n, m and p are as defined herein.

As mentioned herein, the linkage between the Acy, AA2 and/or AA3 moieties is obtained by removing water from the parent compound which formally constitutes it, to form an amide bond (peptide bond) (-CONH-). This means that in order to obtain a compound of formula (I) (Acy-AA 2)_m-AA3_p-, where Acy, AA2, AA3, m and p are as defined herein), it is necessary to formally employ the compounds given by the terms Acy, AA2 and AA3 and to remove hydrogen and/or hydroxyl groups therefrom and formally attach at the free end the structural units thus obtained.

For the nomenclature of substituents, in some cases, the nomenclature is according to IUPAC nomenclature, while in other cases, the nomenclature is according to peptide nomenclature.

As an illustration, the acyl moiety of the compound of example 2 having the structure:

for example, they may be named "octadecanedioyl-gGlu-2 xOEG", "octadecanedioyl-gGlu- (OEG)₂"," octadecanedioyl-gamma Glu-2xOEG "," octadecanedioyl-gamma Glu- (OEG)₂"," 1, 18-octadecanedioyl-gGlu-2 xOEG "," (C18 diacid) -gGlu-2xOEG "," C18d-gGlu-2xOEG ", etc., wherein gamma Glu (and gGlu) is a shorthand form of the amino acid gamma-glutamic acid in the L-configuration, and" 2x "means that the subsequent residue is repeated 2 times.

Gamma Glu, Gamma Glu and gGlu are amino acids of L-configuration Gamma-glutamic acid H₂N-CH(CO₂H)-CH₂CH₂-CO₂Abbreviated form of H (attached via alpha amino and via gamma (side chain) carboxy).

OEG is the amino acid residue 8-amino-3, 6-dioxa-octanoic acid NH₂(CH₂)₂O(CH₂)₂OCH₂CO₂Abbreviated form of H.

In one embodiment, of the formula Acy-AA2_m-AA3_p-the substituents of (a) are represented by:

in one embodiment, of the formula Acy-AA2_m-AA3_p-the substituents of (a) are represented by:

in one embodiment, of the formula Acy-AA2_m-AA3_p-the substituents of (a) are represented by:

in one embodiment, of the formula Acy-AA2_m-AA3_p-the substituents of (a) are represented by:

formula Acy-AA2_m-AA3_pAny one of the above non-limiting examples of substituents of (E) may be attached to the epsilon amino group of a lysine residue present in any of the compounds of the invention, giving other specific examples of acylated compounds of the invention. The desired formula Acy-AA2 can be introduced by any convenient method_m-AA3_pAnd a number of methods for such reactions are disclosed in the prior art.

The EGF (A) -insulin fusion compound derivative of example 1 was named "EGF (A) (301L,309R,312E,321E) - [ GAQP]2-insulin (B3E, B29K (hexadecanediacyl-gGlu-2 xOEG), desB30) ", which indicates that the EGF (A) peptide contains substitutions 301L,309R,312E,321E relative to native EGF (A), that the insulin peptide contains substitutions B3E and desB30, and that the lysine at position B29 of insulin has been partially derivatized (acylated) with hexadecanediacyl-gGlu-2 xOEG. The spacer linking the C-terminal residue of the EGF (A) peptide to the N-terminal residue of the insulin B chain has the sequence (GAQP)₂It may also be denoted as GAQPGAQP or 2xGAQP or [ GAQP]2。

Similarly, the EGF (A) -insulin fusion protein derivative of example 2 was named "EGF (A) (301L,309R,312E,321E) - [ GAQP]10-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30) ", indicating that the EGF (A) peptide contains the substitutions 301L,309R,312E,321E relative to native EGF (A), that the insulin peptide contains the substitution desB30, and that the lysine at position B29 of the insulin has been partially derivatized (acylated) with octadecanedioyl-gGlu-2 xOEG. The spacer linking the C-terminal residue of the EGF (A) peptide to the N-terminal residue of the insulin B chain has the sequence (GAQP)₁₀It can also be used as a watchShown as 10xGAQP, [ GAQP]10 or GAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQPGAQP.

Throughout this application, the general formulae and names of preferred compounds of the present invention are given.

TABLE 2 examples of compounds of the invention

In one embodiment, the invention relates to a compound selected from the fusion proteins of examples 1 to 24.

In one embodiment, the invention relates to a compound selected from the fusion proteins of example 1.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 2-5, 8-15 and 17-21.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 6,7, 16, 22, 23 and 24.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1-4, 5-14 and 16-24.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1-18.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1 and 3-17.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1 and 3-16.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1 and 3-12.

In one embodiment, the invention relates to a compound independently selected from the fusion proteins of the examples of examples 1 and 3-8.

In one embodiment, the invention relates to the fusion peptide of example 1: egf (a) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (hexadecanediacyl-gGlu-2 xeeg), desB30), and is represented by chemical formula 1.

In one embodiment, the invention relates to the fusion peptide of example 3: egf (a) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xeeg), desB30), and is represented by chemical formula 3.

In one embodiment, the invention relates to the fusion peptide of example 4: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 4.

In one embodiment, the invention relates to the fusion peptide of example 5: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-OEG), desB30), and is represented by chemical formula 5.

In one embodiment, the invention relates to the fusion peptide of example 6: egf (a) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xeeg), desB30), and is represented by chemical formula 6.

In one embodiment, the invention relates to the fusion peptide of example 7: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 7.

In one embodiment, the invention relates to the fusion peptide of example 8: EGF (A) (301L,309R,312E,321E) - [ GQAP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 8.

In one embodiment, the invention relates to the fusion peptide of example 9: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 3-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 9.

In one embodiment, the invention relates to the fusion peptide of example 10: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 3-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 10.

In one embodiment, the invention relates to the fusion peptide of example 11: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 4-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 11.

In one embodiment, the invention relates to the fusion peptide of example 12: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 4-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 12.

In one embodiment, the invention relates to the fusion peptide of example 13: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 13.

In one embodiment, the invention relates to the fusion peptide of example 14: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 6-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 14.

In one embodiment, the invention relates to the fusion peptide of example 15: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-OEG), desB30), and is represented by chemical formula 15.

In one embodiment, the invention relates to the fusion peptide of example 16: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 16.

In one embodiment, the invention relates to the fusion peptide of example 17: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 8-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 17.

In one embodiment, the invention relates to the fusion peptide of example 18: EGF (A) (301L,309R,312E,321E) - [ GAQP ] 12-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 18.

In one embodiment, the invention relates to the fusion peptide of example 19:

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 19.

In one embodiment, the invention relates to the fusion peptide of example 20:

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 20.

In one embodiment, the invention relates to the fusion peptide of example 21:

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 21.

In one embodiment, the invention relates to the fusion peptide of example 22:

EGF (A) (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 22.

In one embodiment, the invention relates to the fusion peptide of example 23:

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 23.

In one embodiment, the invention relates to the fusion peptide of example 24: EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30), and is represented by chemical formula 24.

Intermediate product

The invention further relates to novel backbone-form intermediates to which the substituents of the invention are attached, thereby producing the fusion peptides of the invention.

The invention also relates to intermediates of novel backbone forms of the fusion peptides of the invention selected from:

i. frameworks of examples 1,3, 5 and 6 (SEQ ID NOS: 17 and 2)

The scaffold of example 2 (SEQ ID NOS: 18 and 2)

Frameworks of examples 4 and 7 (SEQ ID NOS: 19 and 2)

The scaffold of example 8 (SEQ ID NOS: 30 and 2)

v. backbone of example 9 (SEQ ID NOS: 20 and 2)

The frameworks of example 10 (SEQ ID NOS: 20 and 29)

The scaffold of example 11 (SEQ ID NOS: 21 and 29)

Skeleton of example 12 (SEQ ID NOS: 21 and 2)

Frameworks of examples 13, 15 and 16 (SEQ ID NOS: 22 and 29)

x. backbone of example 14 (SEQ ID NOS: 22 and 2)

Framework of example 17 (SEQ ID NOS: 23 and 2)

xii. framework of example 18 (SEQ ID NOS: 24 and 2)

The frameworks of examples 19 and 22 (SEQ ID NOS: 27 and 2)

xiv. frameworks of examples 20 and 23 (SEQ ID NOS: 27 and 29)

xv. framework of example 21 (SEQ ID NOS: 28 and 2)

The frameworks of example 24 (SEQ ID NOS: 28 and 29).

Dual functionality

Different functionalities are associated with both insulin analogues and egf (a) analogues. When combining the two analogues in the fusion compound derivative of the invention, it is preferred that the analogues remain functional, i.e. the insulin analogue has the ability to activate the insulin receptor and the egf (a) analogue binds to PCSK 9. The functionality of such compounds can be tested as described below.

Insulin function

The relative binding affinity of insulin analogs to human Insulin Receptor (IR) can be determined by competitive binding in the Scintillation Proximity Assay (SPA) as described in example 25.

In one embodiment, the fusion peptide of the invention has the ability to bind to the insulin receptor.

The lipogenesis assay described in example 26 can be used as a measure of the functional (agonistic) activity of insulin analogues.

In one embodiment, a fusion peptide of the invention comprising an insulin analog has the ability to bind to and activate the insulin receptor.

In one embodiment, the fusion peptide of the invention has the ability to lower blood glucose levels.

The fusion peptides of the invention may be tested for pharmacokinetic parameters and/or insulin-related pharmacodynamic properties as described in examples 29 and 30.

The fusion peptides of the invention can be tested by subcutaneous administration to rats, for example, in accordance with this protocol in comparison to comparative fusion compounds and/or similar B29K acylated insulin analogues.

In one embodiment, the fusion peptide of the invention lowers blood glucose levels.

In another embodiment, the fusion peptide of the invention exhibits comparable blood glucose reduction relative to a similar B29K acylated insulin analog.

The fusion peptides of the present invention comprise a spacer with an uncharged spacer (GQAP) n or (GAQP) n, which has an unexpectedly superior blood glucose reduction relative to a comparative compound comprising a charged spacer (GQEP) n.

The fusion peptide of the present invention comprising a spacer (GQAP) n or (GAQP) n (where n is 2-10) was found to be equivalent in hypoglycemic effect, whereas its effect was less pronounced for spacers where n is higher than 10 (fig. 1-2).

It was found that the fusion peptide of the present invention comprising the spacer (GQAP) n or (GAQP) n (where n is 2-8) showed superior hypoglycemic effects than all the comparative compounds tested comprising (GQEP) n (where n is 2-8) (fig. 3-11).

It was found that the fusion peptide of the present invention comprising the spacer (GQAP) n or (GAQP) n (where n ═ 2) showed a dose-dependent decrease in blood glucose (fig. 13).

EGF (A) function

Egf (a) peptide analogs have the ability to bind to PCSK 9. This binding can be assessed using the assay described in example 27 herein.

In one embodiment, the fusion protein of the invention is a PCSK9 inhibitor.

In one embodiment, the fusion protein of the invention inhibits the binding of PCSK9 to the human low density lipoprotein receptor (LDL-R).

In one embodiment, the invention provides a fusion protein comprising an EGF (A) peptide analog of SEQ ID NO:1, wherein the fusion protein is capable of inhibiting the binding of PCSK9 to a human low-density lipoprotein receptor (LDL-R).

In one embodiment, the fusion protein of the invention has the ability to inhibit the binding of PCSK9 to LDL-R.

In one embodiment, the fusion protein of the invention has the ability to inhibit PCSK9 binding to LDL-R and reduce LDL levels in the blood.

In one embodiment, the fusion protein of the invention lowers LDL blood levels.

In one embodiment, the fusion protein of the invention has an improved ability to bind PCSK9 compared to native LDL-R (293-332) (SEQ ID NO:1, native EGF- (A)). In one embodiment, the ratio of the total relative to LDL-R (293-332) EGF (A) analog: (301Leu,309Arg,312Glu,321Glu) (compare fusion protein 9), the fusion protein of the invention has comparable binding ability to PCSK 9.

In one embodiment, the K of a fusion protein of the invention described herein, as measured in a PCSK9-LDL-R binding competition ELISA assay_iLess than 20nM, such as less than 15nM, or such as less than 10nM, or such as less than 5 nM.

In another embodiment, the K of a fusion protein of the invention described herein, as measured in a PCSK9-LDL-R binding competition ELISA assay_iLess than 5 nM.

The functionality of the egf (a) analogues within the fusion proteins and derivatives thereof of the present invention may be further enhanced by their use as described in example 28 hereinImproved LDL uptakeIs measured.

In one embodiment, the fusion protein of the invention increases LDL uptake in the presence of PCSK 9.

In one embodiment, the fusion protein of the invention is capable of reversing or reducing PCSK 9-mediated reduction in LDL uptake.

In one embodiment, the fusion protein of the invention has an EC50 of less than 1500nM, such as less than 1000nM, such as less than 500nM, or such as less than 200nM, as measured in an LDL uptake assay.

In one embodiment, the fusion protein of the invention has an EC50 of less than 1500nM as measured in an LDL uptake assay.

In one embodiment, the fusion protein of the invention has an EC50 of less than 500nM as measured in an LDL uptake assay.

In one embodiment, the fusion protein of the invention has an EC50 of less than 200nM as measured in an LDL uptake assay.

Administration of hPCSK9 to mice was found to result in almost complete down-regulation of hepatic LDL receptor protein (figure 12). The insulin-egf (a) fusion protein effectively prevented PCSK 9-mediated down-regulation of LDLr protein in a dose-dependent manner. Furthermore, two insulin-egf (a) fusion proteins have been shown to prevent hPCSK 9-mediated down-regulation of LDLr protein, similar to that seen with the egf (a) derivative alone (fig. 14).

Dual functionality

To demonstrate bifunctional or dual activity, selected compounds of the invention were tested in the in vivo model described in example 31. Dual activity means increased LDL receptor expression levels in mouse liver by inhibiting the action of intravenous hPCSK9 with an insulin-egf (a) -based anti-PCSK 9 peptide, as well as the hypoglycemic effect of the insulin part of the molecule.

Selected compounds were found to lower blood glucose and prevent hPCSK9 mediated down-regulation of LDLr protein, similar to that seen with egf (a) derivatives alone.

In one embodiment, the fusion protein of the invention activates the insulin receptor.

In one embodiment, the fusion protein of the invention lowers blood glucose.

In one embodiment, the fusion protein of the invention exhibits superior blood glucose reduction compared to a comparative fusion protein.

In one embodiment, the fusion protein of the invention exhibits improved blood glucose reduction compared to a comparative fusion protein.

In one embodiment, the fusion protein of the invention binds to PCSK 9.

In one embodiment, the fusion protein of the invention inhibits the binding of PCSK9 to LDL-R.

In one embodiment, the fusion protein of the invention exhibits improved ability to bind PCSK9 compared to wild-type egf (a).

In one embodiment, the fusion proteins of the invention have a Ki of less than 20nM when measured in a PCSK9-LDL-R binding competition ELISA assay.

In one embodiment, the fusion proteins of the invention have a Ki of less than 5nM when measured in a PCSK9-LDL-R binding competition ELISA assay.

In one embodiment, the fusion protein of the invention increases LDL uptake.

In one embodiment, the fusion protein of the invention has an EC50 of less than 1000nM when measured in an LDL uptake assay.

In one embodiment, the fusion protein of the invention has an EC50 of less than 500nM when measured in an LDL uptake assay.

In one embodiment, the fusion protein of the invention has an EC50 of less than 200nM when measured in an LDL uptake assay.

As mentioned above, it has been found in an in vitro assay that the bifunctional fusion proteins of the present invention bind to both insulin receptor and PCSK9, leading to the activation of the insulin response and preventing PCSK9 binding, thereby preventing LDLR degradation. Furthermore, the inventors have surprisingly found a combined effect on lowering glucose (insulin action) and enhancing hepatic LDLR expression (PCSK9i action) in vivo.

Furthermore, it was surprisingly found that the fusion proteins of the invention comprising an uncharged spacer show an excellent reduction in blood glucose relative to a comparative fusion protein comprising a charged spacer (GQEP) n, and that the level of further reduction depends on the length of the uncharged spacer.

Pharmaceutical composition

The invention also relates to pharmaceutical compositions comprising a fusion protein of the invention, including, for example, an analog of the invention or a pharmaceutically acceptable salt, amide, or ester thereof, and one or more pharmaceutically acceptable excipients. Such compositions may be prepared as known in the art.

The term "adjuvant" refers broadly to any component other than an active therapeutic ingredient. The adjuvants may be inert substances, inactive substances and/or non-pharmaceutically active substances. Adjuvants may be used for various purposes, for example as carriers, vehicles, diluents, tablet auxiliaries and/or to improve administration and/or absorption of the active substance. Non-limiting examples of adjuvants are: solvents, diluents, buffers, preservatives, tonicity adjusting agents, chelating agents and stabilizers. The formulation of pharmaceutically active ingredients with various excipients is known in The art, see, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21 st edition (2005) and any subsequent editions).

The composition of the present invention may be in the form of a liquid formulation, i.e., an aqueous formulation comprising water. The liquid formulation may be a solution or a suspension. Alternatively, it may be a solid formulation, such as a freeze-dried or spray-dried composition.

The pharmaceutical compositions of the invention may further comprise a second active ingredient, such as a therapeutic agent, which may simplify administration in the case of combination therapy.

The compositions of the invention may be used for parenteral administration, for example by subcutaneous, intramuscular, intraperitoneal or intravenous injection.

Indications of drugs

Diabetes mellitus

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 body cells do not respond appropriately to insulin to prevent the cells from absorbing glucose. As a result, glucose accumulates in the blood.

Type 1 diabetes, also known as Insulin Dependent Diabetes Mellitus (IDDM) and juvenile diabetes, is caused by B cell destruction, often resulting 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 with relative insulin deficiency, and/or with major insulin secretion defects with insulin resistance.

Other indications

In one embodiment, the fusion protein of the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes.

In another embodiment, the fusion protein of the invention is used as a medicament for delaying or preventing disease progression in type 2 diabetes.

In one embodiment of the invention, the fusion protein is used as a medicament for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes.

In a further embodiment, the invention relates to a method of treating or preventing hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, comprising administering to a patient in need of such treatment an effective amount of such treatment with a fusion protein of the invention.

In one embodiment, the fusion protein of the invention or a composition thereof may be inDiabetic patientsIs used for:

(i) improving lipid parameters, such as preventing and/or treating dyslipidemia, reducing total serum lipids; lowering LDL-C, increasing HDL; lowering small, dense LDL; reduction of VLDL; lowering triglycerides; reducing cholesterol; reducing plasma levels of lipoprotein a (lp (a)); in another embodiment, the invention relates toDiabetic patientsA method of treatment of (a), for:

i. improving lipid parameters, such as preventing and/or treating dyslipidemia, reducing total serum lipids; increasing HDL-C; lower LDL-C, lower and smallerDense LDL; reduction of VLDL-C; lowering triglycerides; reducing cholesterol; reducing plasma levels of lipoprotein a (lp (a)); wherein a pharmaceutically active amount of a fusion protein according to the invention, e.g. a peptide analogue or derivative according to the invention, is administeredDiabetic patients。

In one embodiment, the invention relates to the use of a fusion protein as described herein for the preparation of a medicament.

The invention also relates to the fusion protein of the invention or a pharmaceutical composition thereof for use as a medicament or for the preparation of a medicament.

Mode of administration

The term "treating" is intended to include preventing and minimizing the disease, disorder or condition referred to (i.e., "treating" refers to prophylactic and therapeutic administration of a fusion protein of the invention or a composition comprising a fusion protein of the invention, unless otherwise indicated or clearly contradicted by context).

The route of administration may be any route effective to deliver the fusion protein of the invention to a desired or appropriate location in the body, such as parenteral, e.g., subcutaneous, intramuscular, or intravenous routes. Alternatively, the fusion protein of the invention may be administered orally, pulmonary, rectally, transdermally, buccally, sublingually, or nasally.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended embodiments are intended to cover all such modifications and changes as fall within the true scope of the invention.

Detailed description of the preferred embodiments

1. A fusion protein comprising an insulin peptide, an EGF (A) peptide, a spacer and substituents, wherein the insulin peptide is human insulin (SEQ ID NO 2-3) or an analogue of human insulin

The EGF (A) peptide is an analogue of the EGF (A) domain of LDL-R (293-332) (SEQ ID NO:1)

The spacer is a peptide linker comprising a segment of (GAQP) N or (GQAP) N, N-2-19, and linking the N-terminus of the insulin analogue B-chain to the C-terminus of the egf (a) analogue

The substituent has formula (I): Acy-AA2_m-AA3_p-, wherein

Acy is a fatty diacid containing from about 16 to about 20 carbon atoms,

AA2 is an acidic amino acid residue, and wherein m is an integer in the range of 1 to 10, and

AA3 is a neutral alkylene glycol-containing amino acid residue, and p is an integer in the range of 1 to 10, and

wherein the maximum number of AA2 and AA3 residues is 10, and

wherein the AA2 and AA3 residues may occur in any order,

or a pharmaceutically acceptable salt, amide or ester thereof.

2. A fusion protein according to embodiment 1, wherein the egf (a) peptide analogue is fused to the N-terminus of the insulin analogue B-chain via the C-terminal amino acid residue of the egf (a) analogue.

3. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-15 amino acid mutations compared to SEQ ID No. 1.

4. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-13 amino acid mutations compared to SEQ ID No. 1.

5. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-11 amino acid mutations compared to SEQ ID No. 1.

6. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-9 amino acid mutations compared to SEQ ID No. 1.

7. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-7 amino acid mutations compared to SEQ ID No. 1.

8. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-5 amino acid mutations compared to SEQ ID No. 1.

9. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 1-3 amino acid mutations compared to SEQ ID No. 1.

10. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises one or two amino acid mutations compared to SEQ ID No. 1.

11. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises eight amino acid mutations compared to SEQ ID No. 1.

12. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises seven amino acid mutations compared to SEQ ID No. 1.

13. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises six amino acid mutations compared to SEQ ID No. 1.

14. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises five amino acid mutations compared to SEQ ID No. 1.

15. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises four amino acid mutations compared to SEQ ID No. 1.

16. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises three amino acid mutations compared to SEQ ID No. 1.

17. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises two amino acid mutations compared to SEQ ID No. 1.

18. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 301L.

19. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analog comprises 309R.

20. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 312E.

21. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises 321E.

22. The fusion protein according to any one of the preceding embodiments, wherein the egf (a) analogue comprises one of the following combinations:

a.301Leu and 309Arg

301Leu,309Arg and 312Glu

c.301Leu, 309Arg,312Glu and 321Glu

23. The fusion protein according to embodiment 22, wherein the egf (a) analog comprises 301L and 309R.

24. The fusion protein according to embodiment 22, wherein the egf (a) analog comprises 301L,309R, and 312E.

25. The fusion protein according to embodiment 22, wherein the egf (a) analog comprises 301L,309R,312E, and 321E.

26. The fusion protein according to embodiment 22, wherein the egf (a) analogue is 301L,309R,312E, 321E.

27. The fusion protein according to any of the preceding embodiments, wherein the insulin analogue/derivative is an analogue/derivative of human insulin comprising 0-10 mutations.

28. A fusion protein according to embodiment 27, wherein said insulin comprises 1-10 mutations.

29. A fusion protein according to embodiment 28, wherein said insulin comprises 1-8 mutations.

30. A fusion protein according to embodiment 28, wherein said insulin comprises 1-6 mutations.

31. The fusion protein according to embodiment 28, wherein the insulin comprises five mutations.

32. A fusion protein according to embodiment 28, wherein said insulin comprises 1-4 mutations.

33. The fusion protein according to embodiment 28, wherein the insulin comprises four mutations.

34. A fusion protein according to embodiment 28, wherein said insulin comprises 1-3 mutations.

35. A fusion protein according to embodiment 28, wherein said insulin comprises one or two mutations.

36. A fusion protein according to embodiment 28, wherein said insulin comprises a mutation.

37. A fusion protein according to any one of the preceding embodiments, wherein the insulin analogue comprises one of the following combinations:

a.A14E

b.B3E

c.desB30

d.A14E、desB30

e.B3E、desB30

38. a fusion protein according to embodiment 37, wherein the insulin analogue comprises desB 30.

39. A fusion protein according to embodiment 37, wherein the insulin analogue comprises B3E.

40. A fusion protein according to embodiment 37, wherein the insulin analogue comprises a14E, desB 30.

41. A fusion protein according to embodiment 37, wherein the insulin analogue comprises B3E, desB 30.

42. A fusion protein according to embodiment 37, wherein the insulin analogue is a14E, desB 30.

43. A fusion protein according to embodiment 37, wherein the insulin analogue is B3E, desB 30.

44. The fusion protein according to embodiment 37, wherein the insulin analog is insulin desB30 human insulin.

45. A fusion protein according to any one of the preceding embodiments, wherein the fusion protein activates the insulin receptor and binds PCSK 9.

46. A fusion protein according to any one of the preceding embodiments, wherein the fusion protein comprises a spacer linking the egf (a) analogue and the insulin analogue/derivative.

47. The fusion protein according to any one of the preceding embodiments, wherein the spacer comprises an amide bond.

48. The fusion protein according to any one of the preceding embodiments, wherein the spacer comprises 4-80 amino acid residues.

49. The fusion protein according to any one of the preceding embodiments, wherein the spacer comprises one or more of the following amino acid residues: ala (A), Gly (G), Pro (P) and/or Gln (Q).

50. The fusion protein according to any one of the preceding embodiments, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-1-20.

51. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-19.

52. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-12.

53. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-10.

54. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-8.

55. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-6.

56. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2-4.

57. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-2.

58. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-3.

59. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-4.

60. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-5.

61. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-6.

62. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-7.

63. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-8.

64. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-10.

65. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-12.

66. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-14.

67. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-16.

68. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-18.

69. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-19.

70. The fusion protein according to embodiment 50, wherein the spacer comprises (GAQP) n or (GQAP) n, wherein n-20.

71. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein comprises one substituent.

72. A fusion protein according to any one of the preceding embodiments, wherein the substituents are linked via Lys/K amino acid residues.

73. A fusion protein according to any one of the preceding embodiments, wherein the substituent is linked to Lys/K amino acid residue B29K within the insulin sequence of the fusion protein.

74. The fusion protein according to any one of the preceding embodiments, wherein the order of occurrence of AA2 and AA3 in formula (la) are independently interchangeable.

75. The fusion protein according to any one of the preceding embodiments, wherein formula (I) Acy-AA2_m-AA3_pAcy of (a) comprises a fatty diacid group selected from 1, 16-hexadecanedioic acid, 1, 18-octadecanedioic acid and 1, 20-eicosanedioic acid.

76. The fusion protein according to embodiment 75, wherein the Acy comprises a fatty diacid group of 1, 16-hexadecanedioic acid.

77. The fusion protein according to embodiment 75, wherein the Acy comprises a fatty diacid group of 1, 18-octadecanedioic acid.

78. The fusion protein according to embodiment 75, wherein the Acy comprises a fatty diacid group of 1, 20-eicosanedioic acid.

79. The fusion protein according to any one of the preceding embodiments, wherein formula (I) Acy-AA2_m-AA3_pSaid AA2 of (A-A)_mComprising a gGlu representing a gamma glutamic acid residue represented by the following structure:

80. the fusion protein according to any one of the preceding embodiments, wherein formula (I) Acy-AA2_m-AA3_pSaid AA3 of (A-A)_pContaining 1xOEG or [2- (2-aminoethoxy) ethoxy]Acetyl or amino acid residue 8-amino-3, 6-dioxaoctanoic acid-NH (CH)₂)₂O(CH₂)₂OCH₂CO-, and is represented by the following structure:

81. the fusion protein according to any one of the preceding embodiments, wherein formula (I) Acy-AA2_m-AA3_pSaid AA3p of (a) comprises 2 xeog.

82. The fusion protein according to any one of the preceding embodiments, wherein formula (I) Acy-AA2_m-AA3_pAA2 of_m-AA3_p-is independently represented by gGlu-OEG or gGlu-OEG-OEG.

83. The fusion protein according to embodiment 82, wherein formula (I) Acy-AA2_m-AA3_pAA2 of_m-AA3_p-is represented by gGlu-OEG.

84. The fusion protein according to embodiment 82, wherein formula (I) Acy-AA2_m-AA3_pAA2 of_m-AA3_p-is represented by gGlu-OEG-OEG.

85. According to the foregoing embodimentThe fusion protein of any one of embodiments, wherein the substituent Acy-AA2_m-AA3_p-is represented by:

86. the fusion protein according to embodiment 85, wherein the substituent Acy-AA2_m-AA3_p-is selected from the following:

87. the fusion protein according to any one of the preceding embodiments, wherein the substituent Acy-AA2_m-AA3_p-independently represented by:

c16 diacid-gGlu-2 xOEG

C18 diacid-gGlu-OEG

C18 diacid-gGlu-2 xOEG

C20 diacid-gGlu-2 xOEG

88. The fusion protein according to embodiment 87, wherein the substituent Acy-AA2_m-AA3_pIndependently by the C16 diacid-gGlu-2 xOEG.

89. The fusion protein according to embodiment 87, wherein the substituent Acy-AA2_m-AA3_pIndependently by C18 diacid-gGlu-OEG.

90. The fusion protein according to embodiment 87, wherein the substituent Acy-AA2_m-AA3_pIndependently by the C18 diacid-gGlu-2 xOEG.

91. The fusion protein according to embodiment 87, wherein the substituent Acy-AA2_m-AA3_pIndependently by the C20 diacid-gGlu-2 xOEG.

92. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein activates the insulin receptor.

93. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has the ability to reduce blood glucose.

94. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein exhibits superior blood glucose reduction compared to a comparative fusion protein.

95. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein exhibits an improved blood glucose reduction compared to a comparative fusion protein.

96. A fusion protein according to any one of the preceding embodiments, wherein the fusion protein binds to PCSK 9.

97. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein inhibits the binding of PCKS9 to LDL-R.

98. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has an improved ability to bind PCSK9 compared to wild type egf (a).

99. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has a Ki of less than 20nM when measured in a PCSK9-LDL-R binding competition ELISA assay.

100. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has a Ki of less than 5nM when measured in a PCSK9-LDL-R binding competition ELISA assay.

101. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein increases LDL uptake.

102. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has an EC50 of less than 1000nM when measured in an LDL uptake assay.

103. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has an EC50 of less than 500nM when measured in an LDL uptake assay.

104. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein has an EC50 of less than 200nM when measured in an LDL uptake assay.

105. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1-24.

106. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion protein of example 1.

107. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1-24.

108. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 2-5, 8-15, and 17-21.

109. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 6,7, 16, 22, 23 and 24.

110. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1-4, 5-14, and 16-24.

111. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1-18.

112. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1 and 3-17.

113. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1 and 3-16.

114. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1 and 3-12.

115. The fusion protein according to any one of the preceding embodiments, wherein the fusion protein is represented by the fusion proteins of examples 1 and 3-8.

116. A fusion protein according to any one of the preceding embodiments for use as a medicament.

117. The fusion protein according to any one of the preceding embodiments for use in the prevention or treatment of cardiovascular disease in a diabetic patient.

118. The fusion protein according to any one of the preceding embodiments for use in a method of improving lipid parameters in a diabetic patient.

119. An intermediate product of a novel backbone form of the fusion protein of the invention, selected from the group consisting of:

i. frameworks of examples 1,3, 5 and 6 (SEQ ID NOS: 17 and 2)

The scaffold of example 2 (SEQ ID NOS: 18 and 2)

Frameworks of examples 4 and 7 (SEQ ID NOS: 19 and 2)

The scaffold of example 8 (SEQ ID NOS: 30 and 2)

v. backbone of example 9 (SEQ ID NOS: 20 and 2)

The frameworks of example 10 (SEQ ID NOS: 20 and 29)

The scaffold of example 11 (SEQ ID NOS: 21 and 29)

Skeleton of example 12 (SEQ ID NOS: 21 and 2)

Frameworks of examples 13, 15 and 16 (SEQ ID NOS: 22 and 29)

x. backbone of example 14 (SEQ ID NOS: 22 and 2)

Framework of example 17 (SEQ ID NOS: 23 and 2)

xii. framework of example 18 (SEQ ID NOS: 24 and 2)

The frameworks of examples 19 and 22 (SEQ ID NOS: 27 and 2)

xiv. frameworks of examples 20 and 23 (SEQ ID NOS: 27 and 29)

xv. framework of example 21 (SEQ ID NOS: 28 and 2)

The frameworks of example 24 (SEQ ID NOS: 28 and 29).

120. A fusion protein according to any one of embodiments 1-115 for use in the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia and/or dyslipidemia.

121. A fusion protein according to any one of embodiments 1-115 inDiabetic patientsFor improving lipid parameters, such as prevention and/or treatment of dyslipidemia, lowering total serum lipids, increasing HDL-C, lowering LDL-C, lowering small, dense LDL-C, lowering VLDL-C, lowering triglycerides, lowering cholesterol, lowering plasma levels of lipoprotein a (lp (a)), or inhibiting the production of apolipoprotein a (apo (a)).

122. A pharmaceutical composition comprising a fusion protein according to any one of the preceding embodiments 1-118 and a pharmaceutically acceptable excipient.

123. The pharmaceutical composition according to embodiment 122, for subcutaneous administration.

124. A pharmaceutical composition for treating diabetes in a patient in need thereof, comprising a therapeutically effective amount of a fusion protein according to any one of embodiments 1-118, and a pharmaceutically acceptable carrier.

125. The pharmaceutical composition of embodiment 122 for use as a medicament.

126. The pharmaceutical composition of embodiment 122 for use in treating a patient having diabetes and having a high risk of cardiovascular disease.

127. Use of a fusion protein according to any one of embodiments 1-118 for the preparation of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia.

128. A method of improving lipid parameters comprisingDiabetic patientsA step of administering a pharmaceutically active amount of a fusion protein according to any one of the preceding embodiments 1-115.

129. Improvements in or relating toDiabetic patientsThe method of lipid parameters of (a), comprising the step of administering a pharmaceutically active amount of a fusion protein according to any one of the preceding embodiments 1-118, such as preventing and/or treating dyslipidemia, lowering total serum lipids; increasing HDL; lowering LDL-C; lower small, dense LDL-C; reduction of VLDL-C; non-HDL-C; lowering triglycerides; reducing cholesterol; reduce plasma levels of lipoprotein a (lp (a)).

130. An insulin analogue according to any one of the preceding embodiments 1-118 inDiabetic patientsIn a method of treatment of (a): improving lipid parameters, such as preventing and/or treating dyslipidemia, lowering total serum lipids, increasing HDL-C, lowering LDL-C, lowering small and dense LDL-C, lowering VLDL-C, lowering triglycerides,lowering cholesterol, lowering plasma levels of lipoprotein a (lp (a)) or inhibiting the production of apolipoprotein a (apo (a)); and the prevention and/or treatment of cardiovascular diseases.

131. A method for the prevention and/or treatment of cardiovascular diseases in diabetic patients, comprising the step of administering a pharmaceutically active amount of a fusion protein according to any of the preceding embodiments 1-118.

132. A method of treating or preventing diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, the method comprising administering to a subject in need thereof a therapeutically effective amount of a fusion protein according to any one of embodiments 1-118.

Examples

List of abbreviations

AcOH: acetic acid

ADO: 8-amino-3, 6-dioxaoctanoic acid

ALP hydrolysis of Achromobacter (Achromobacter lyticus) protease

API: active pharmaceutical ingredient

Area under the AUC curve;

area under curve normalized by AUC/D dose;

BG: blood sugar

BHK: baby hamster kidney

Boc: tert-butyloxycarbonyl radical

C_maxMaximum plasma concentration;

C_maxdose normalized maximum plasma concentration;

c-peptide linker peptides

Clt: 2-Chlorotriphenylmethyl radical

Collidine (collidine): 2,4, 6-trimethylpyridine

D dose;

DCM: methylene dichloride

DIC: diisopropylcarbodiimide

DIPEA ═ DIEA N, N-diisopropylethylamine

DMAP: 4-dimethylaminopyridine

DMF: n, N-dimethylformamide

DMSO, DMSO: dimethyl sulfoxide

DTT: DL-dithiothreitol

EC 50: half maximal effective concentration

EDC: n- (3-dimethylaminopropyl) -N' -ethylcarbodiimide

EDTA: ethylenediaminetetraacetic acid

EGF: epidermal growth factor-like

EGF (A): epidermal growth factor-like domain A

eps： epsilon

F bioavailability (fraction absorbed);

fmoc: 9-fluorenylmethoxycarbonyl group

Gamma Glu (gGlu) gamma L-glutamyl;

HCl hydrochloric acid

HDL: high density lipoprotein

HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid

HFIP 1,1,1,3,3, 3-hexafluoro-2-propanol or hexafluoroisopropanol

HI: human insulin

hLDL-R: human LDL receptor

hPCSK 9: human PCSK9

HPLC: high performance liquid chromatography

HSA: human serum albumin

IC₅₀: half maximal inhibitory concentration

IGF-1R insulin-like growth factor 1 receptor

IP: intraperitoneal cavity

IR: insulin receptor

i.v. intravenous

LC liquid chromatography

LCMS or LC-MS: liquid chromatography mass spectrometry

LDL-R or LDLr: LDL receptors

LDL: low density lipoprotein

LDL-C: LDL cholesterol

MALDI-TOF matrix assisted laser desorption ionization time of flight

MeCN: acetonitrile

MeOH: methanol

MRT mean residence time;

MS mass spectrometry

Mtt: 4-Methyltriphenylmethyl group

NMP: n-methyl pyrrolidone

OEG [2- (2-aminoethoxy) ethoxy ] ethylcarbonyl;

OSu: o-succinimidyl ester (hydroxysuccinimide ester)

OtBu: tert-butyl ester

OxymaCyano-hydroxyimino-acetic acid ethyl ester

% extrap extrapolation Curve percentage

Pbf: 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl

PBS: phosphate buffered saline

PD pharmacodynamics (e.g. blood/plasma glucose lowering effect)

PK pharmacokinetics (blood/plasma insulin concentration versus time curve)

Pra: l-propargylglycine

rhLDL-R: recombinant human LDL receptor

RP: inverse phase

RP-HPLC: reversed phase high performance liquid chromatography

RT: at room temperature

s.c.: under the skin

SD: standard deviation of

SEM: standard error of mean

SPA: scintillation proximity assay

SPPS: solid phase peptide synthesis

T1/2 terminal half-life;

tBu: tert-butyl radical

T_maxTime to maximum plasma exposure;

TCTU: o- (6-chlorobenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate

TFA: trifluoroacetic acid

THPTA: trihydroxypropyl triazolyl methylamine

TIS or TIPS: tri-isopropyl silane

TRIS: tris (hydroxymethyl) aminomethane or 2-amino-2-hydroxymethyl-propane-1, 3-diol

TBS-T: tris buffered saline

Trt: triphenylmethyl (trityl)

TSTU O- (N-succinimidyl) -1,1,3, 3-tetramethyluronium tetrafluoroborate

And (3) UPLC: ultra-high performance liquid chromatography

wt. wild type

The invention is further illustrated with reference to the following examples, which are not intended to limit the scope of the claimed invention in any way.

The analogue, i.e. the double stranded non-acylated insulin egf (a) fusion protein, may be expressed in e.coli or yeast as described further below.

Recombinant expression and purification of EGF (A) insulin fusion compound analogs in E.coli

EGF (A) insulin fusion protein analogs can be well expressed as Inclusion Bodies (IB) in E.coli.

The cDNA of the insulin-egf (a) fusion protein analogue was subcloned into a pET11 b-derived vector, and the plasmid was then transformed into a BL21(DE3) -derived host strain. The fermentation was carried out in a chemically defined medium via a fed-batch process.

The insulin-EGF (A) fusion protein analog was further purified as follows:

the cells were harvested and lysed in 20mM pH8.0Tris buffer containing 150mM NaCl using a cell disruptor. Insoluble fractions of IB containing the fusion protein were collected and sequentially treated with 20mM pH8.0Tris buffer containing 500mM NaCl and H₂And O washing. Then, IB was dissolved to a concentration of 10mg/mL using 20mM Tris, 6M Urea, 10mM DTT (pH 9.0) at room temperature (20-25 deg.C)And (4) degree. After solubilization, the solution was diluted 10-fold with 20mM glycine, 1mM cysteine, 10mM CaCl2(pH 10.5) for refolding, which was completed within 6-10 hours at room temperature. After clarification (centrifugation or depth filtration), Q Sepharose beads (20mM Tris, 10mM CaCl) were used₂10% EtOH, pH 8.0) capture proteins. Thereafter, the elution pool was treated with ALP at a ratio of 1:1000 for 3-4 hours at room temperature. The cleaved protein was then added to Source 30RPC for isolation. As a final purification step, Source 30Q (20mM Tris, 10mM CaCl) using a shallow NaCl gradient was used₂10% EtOH, pH 8.0). For spacers with longer length (i.e. repetition number)>6), binding should be facilitated using pH 8.5 in both anion exchange steps.

Recombinant expression and purification of EGF (A) insulin fusion Compounds in Yeast

Egf (a) insulin fusion analogues are expressed and cultured by well-known techniques, e.g. as disclosed in WO 2017/032798. More specifically, an insulin-EGF (A) fusion protein was prepared and purified as follows:

capture of precursor on SP Sepharose BB:

yeast supernatant was applied to a column packed with SP Sepharose BB at a flow rate of 10-20 cv/h. Wash with 0.1M citric acid pH 3.5 and 40% EtOH. The analogue was eluted with 0.2M sodium acetate pH 5.5/35% EtOH.

Shuffling of disulfide bonds:

the SP pool was adjusted to pH 9. Adding a shuffling reagent to a final concentration; cysteine 2.5mM, cystine 0.25mM, CaCl 225 mM, then UPLC.

ALP digestion:

the shuffling pool was adjusted to pH 9 and ALP enzyme was added at 1:100 (w/w). The reaction was then subjected to UPLC. The ALP lysate pool was adjusted to pH 2.5 and diluted 2-fold in preparation for RPHPLC purification.

RPHPLC purification:

purification was performed by RPHPLC C18 as follows:

column: 15um C1850 x250mm

Buffer solution:

A：25mM CaCl₂0.2% formic acid, 5% EtOH,

b: 0.2% formic acid, 50% EtOH

Gradient: 20-55% B-buffer.

Gradient: 20CV of

Flow rate 20CV/h

Sample loading g-5 g/l resin

The fractions were analyzed by UPLC, combined, and lyophilized.

General description of the invention

The following examples and general procedures relate to the end products identified in the present specification and synthetic schemes. The preparation of the compounds of the invention is described in detail using the following examples, but the chemical reactions described are disclosed with respect to their general applicability to the preparation of the compounds of the invention.

Occasionally, the reaction may not be as described for every compound included within the scope of the present disclosure. Those skilled in the art will readily recognize compounds where this occurs. In these cases, the reaction can be successfully carried out by conventional modifications known to the person skilled in the art, i.e. by appropriate protection of interfering groups, by exchange with other conventional reagents or by conventional modification of the reaction conditions.

Alternatively, other reactions disclosed herein or conventional would be applicable to the preparation of the corresponding compounds of the present invention. In all preparation methods, all starting materials are known or can be readily prepared from known starting materials. All temperatures are expressed in degrees celsius and, unless otherwise indicated, all parts and percentages are by weight when referring to yield and all parts are by volume when referring to solvents and eluents.

The analysis method comprises the following steps:

LC-MS method 1 (electrospray):

the system comprises the following steps: waters Acquity UPLC SQD 2000

Column: acquisty UPLC BEH 1.7 μ C182.1x50mm

A detector: UV: PDA, SQD 2000

An ionization method comprises the following steps: ES +

Scanning range: 500-2000

Cone voltage: 60V

Scan time 0.5

Linear gradient: 10% to 90% B

Gradient run time: 3min

Total run time: 4min

Flow rate: 0.3ml/min

Column temperature: 40 deg.C

PDA：210-400nm

Solvent A: 99.90% H2O, 0.1% TFA

Solvent B: 99.90% CH3CN, 0.1% TFA

Solvent C: NA

LC-MS method 2 (electrospray):

the system comprises the following steps: waters Acquity UPLC H-Class SQD 22000

Column: acquity UPLC BEH 1,7C 181002, 1x50 mm. Part number: 186002350

A detector: UV: PDA, SQD 2000

An ionization method comprises the following steps: ES +

Scanning range: 500-2000

Cone voltage: 60V

Scan time 0.5

Linear gradient: 10% to 80% B

Gradient run time: 2.50min

Total run time: 4min

Flow rate: 0.3ml/min (0-2.51min) and 0.8ml/min (2.51-4.00min)

Column temperature: 40 deg.C

PDA：210-400nm

Solvent A: 99.90% H2O, 0.1% TFA

Solvent B: 99.90% CH3CN, 0.1% TFA

Solvent C: NA

LC-MS method 3 (TOF):

the system comprises the following steps: agilent 1290 definition series UPLC

Column: eclipse C18+2.1x50 mm 1.8u

A detector: agilent Technologies LC/MSD TOF 6230(G6230A)

An ionization method comprises the following steps: agilent Jet Stream source

Scanning range: m/z minimum 100, m/z maximum 3200

Linear reflector mode

Positive mode

Linear gradient: 5% to 95% B

Gradient run time: 0-4.5min for 6 min, 5-95% of B, 4.5-595% of B, 5-5.595-5% of B, 5.5-65% of B

Flow rate: fixing at 0.40ml/min

Column temperature: 40 deg.C

Solvent A: 99.90% H2O, 0.02% TFA

Solvent B: 99.90% CH3CN, 0.02% TFA

Solvent C: NA

The calculated mass is the average molecular weight of the desired compound.

For the compound of m 3000, the measured masses (averages) are the result of deconvolution using the Masshunter workshop Software Version B.05.00Build 5.0.519.13SP1 (Agilent).

LC-MS method 4 (TOF):

the system comprises the following steps: agilent 1290 definition series UPLC

Column: phenomenex Aeris wire position 3.6 mu C4502.1mm

A detector: agilent Technologies LC/MSD TOF 6230(G6230A)

An ionization method comprises the following steps: agilent Jet Stream source

Scanning range: m/z minimum 100, m/z maximum 3200

Linear reflector mode

Positive mode

Step gradient: 5% to 90% B

Gradient run time: 10 minutes: 0-1min 5-20% B, 1-7min 20-90% B, 7-8min 90% B8-8.5 min 90-5% B8.5-10 min 5% B

Flow rate: fixing at 0.40ml/min

Column temperature: 40 deg.C

Solvent A: 99.90% H2O, 0.02% TFA

Solvent B: 99.90% CH3CN, 0.02% TFA

Solvent C: NA

The calculated mass is the average molecular weight of the desired compound.

For the compound of m 3000, the measured masses (averages) are the result of deconvolution using the Masshunter workshop Software Version B.05.00Build 5.0.519.13SP1 (Agilent).

And (3) synthesis of a substituent:

for example, the substituents may be synthesised in solution or on a solid phase as described in WO 2009/115469.

General procedure for acylation of insulin-EGRA) fusion proteins of the invention:

the insulin-egf (a) fusion protein is dissolved in an aqueous buffer, optionally with the addition of an organic co-solvent (e.g., EtOH, acetonitrile, DMSO, or NMP), and the pH is adjusted to 11.2. While the pH was maintained at about 11.2 by the addition of 1M NaOH, a solution of activated side chains in NMP (100mg/mL) was added dropwise and the progress of the reaction was monitored by LC-MS. After completion of the reaction, TFA, acetic acid or 1M HCl was added to the mixture. After dilution with water, the mixture was purified by preparative HPLC. Pure fractions were combined and lyophilized to give the compounds of the invention.

This general procedure is further illustrated in example 1 below. All other compounds of the invention are prepared analogously.

Example 1

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (hexadecanediacyl-gGlu-2 xOEG), desB30)

Chemical formula 1:

a solution (48mL, 2.44mg/mL, 117mg) of EGF (A) (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, desB30) was dissolved in a buffer consisting of 20mM Tris, pH8.0, 10mM CaCl2, 10% EtOH, 50mM NaCl. The pH was raised to 11.1 with 1M NaOH. While the pH was kept constant at 11.1 using 1M NaOH, a solution of hexadecanediacyl-Glu-2 xOEG-OSu (24mg in 0.5mL NMP) was added dropwise. After the reaction was complete, TFA was added to the mixture to pH 1.6. Acetonitrile (14mL) was added to the mixture followed by water to 100 mL. The mixture was purified by RP-HPLC.

Column: phenomenex Gemini, 5. mu.M 5u C1830x250mm

Flow rate: 20mL/min

And (3) buffer solution A: 0.1% TFA in MilliQ Water

And (3) buffer solution B: 0.1% TFA in acetonitrile

Gradient: 20% B to 50% B, linear

Gradient time: 40min

Fraction size: 6mL

The pure fractions were combined and lyophilized to give 56mg of the title compound.

LC-MS 38 (electrospray): m/z is 1873.28(M + 6)/6. Calculated values: 1873.27.

Rt＝1.67min

example 2

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 10-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 2:

LC-MS 38 (electrospray): m/z is 1597.35(M + 9)/9. Calculated values: 1597.00

Rt＝1.75min

Example 3

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 3:

LC-MS 27 (TOF): 11552.89. Calculated values: 11552.00

Example 4

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 4:

LC-MS method 1 (electrospray): m/z is 1924.39(M + 6)/6. Calculated values: 1923.83

Rt＝2.13min

Example 5

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (octadecanedioyl-gGlu-OEG), desB30)

Chemical formula 5:

LC-MS method 2 (electrospray): m/z is 1902.01(M + 6)/6. Calculated values: 1902.1

Rt＝1.8min

Example 6

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 6:

LC-MS method 2 (electrospray): m/z is 1930.74(M + 6)/6. Calculated values: 1931.02

Rt＝1.89min

Example 7

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 2-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 7:

LC-MS method 1 (electrospray): m/z is 1928.67(M + 6)/6. Calculated values: 1928.51

Rt＝2.27min

Example 8

EGF (A), (301L,309R,312E,321E) - [ GQAP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 8:

LC-MS method 1 (electrospray): m/z is 1924.33(M + 6)/6. Calculated values: 1923.83

Rt＝1.87min

Example 9

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 3-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 9:

LC-MS method 1 (electrospray): m/z is 1983.44(M + 6)/6. Calculated values: 1982.72

Rt＝2.00min

Example 10

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 3-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 10:

LC-MS method 2 (electrospray): m/z is 1977.18(M + 6)/6. Calculated values: 1977.05

Rt＝1.8min

Example 11

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 4-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 11:

LC-MS method 2 (electrospray): m/z is 1527.13(M + 8)/8. Calculated values: 1527-2

Rt＝1.78min

Example 12

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 4-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 12:

LC-MS method 2 (electrospray): m/z is 1749.96(M + 7)/7. Calculated values: 1750.10

Rt＝1.71min

Example 13

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 13:

LC-MS method 2 (electrospray): m/z is 1845.9(M + 7)/7. Calculated values: 1846.2

Rt＝1.74min

Example 14

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 14:

LC-MS method 1 (electrospray): m/z is 1851.01(M + 7)/7. Calculated values: 1851.07

Rt＝2.07min

Example 15

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (octadecanedioyl-gGlu-OEG), desB30)

Chemical formula 15:

LC-MS method 4 (TOF): 12771.82; calculated values: 12771.26

Example 16

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 6-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 16:

LC-MS method 2 (electrospray): m/z is 1850.16(M + 7)/7. Calculated values: 1850.21

Rt＝1.98min

Example 17

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 8-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 17:

LC-MS method 2 (electrospray): m/z is 1708.15(M + 8)/8. Calculated values: 1708.15

Rt＝1.74min

Example 18

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 12-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 18:

LC-MS method 1 (electrospray): m/z is 1884.76(M + 8)/8. Calculated values: 1884.84

Rt＝2.04min

Example 19

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 19:

LC-MS method 3 (TOF): 17545.17; calculated values: 17544.34

Example 20

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 20:

LC-MS method 1 (electrospray): m/z is 1947.0(M + 9)/9. Calculated values: 1946.6

Rt＝1.73min

Example 21

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 21:

LC-MS method 3 (TOF): 17559.93; calculated values: 17559.35

Example 22

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 22:

LC-MS method 3 (TOF): 17573.37; calculated values: 17572.39

Example 23

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (A14E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 23:

LC-MS method 1 (electrospray): m/z is 1462.45(M + 12)/12. Calculated values: 1462.53

Rt＝1.84min

Example 24

EGF (A), (301L,309R,312E,321E) - [ GAQP ] 19-insulin (B3E, B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

Chemical formula 24:

LC-MS method 3 (TOF): 17587.97; calculated values: 17587.40

Comparative Compound 1

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 2-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

LC-MS method 1 (electrospray): m/z is 1943.59(M + 6)/6. Calculated values: 1943.18

Rt＝2.09min

Comparative Compound 2

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 8-insulin (A14E, B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

LC-MS method 1 (electrospray): m/z is 1566.29(M + 9)/9. Calculated values: 1566.27

Rt＝1.79min

Comparative Compound 3

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 4-insulin (B29K (eicosanedioyl-gGlu-2 xOEG), desB30)

LC-MS method 1 (electrospray): m/z is 1787.19(M + 7)/7. Calculated values: 1787.28

Rt＝2.16min

Comparative Compound 4

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 4-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

LC-MS method 3 (TOF): 12473 for m/z; calculated values: 12475

Comparative Compound 5

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 2-insulin (B29K (hexadecanediacyl-gGlu-2 xOEG), desB30)

LC-MS method 3 (TOF): 11625.00; calculated values: 11625.00

Comparative Compound 6

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 6-insulin (B29K (octadecanedioyl-gGlu-2 xOEG), desB30)

LC-MS method 3 (TOF): 13296; calculated values: 13298

Comparative Compound 7

EGF (A), (301L,309R,312E,321E) - [ GQEP ] 8-insulin (A14E, B29K (hexadecanediacyl-gGlu-2 xOEG), desB30)

LC-MS method 1 (electrospray): m/z is 1758.57(M + 8)/8. Calculated values: 1758.43

Rt＝1.67min

Comparative Compound 8

B29K (octadecanedioyl-gGlu-2 xOEG), desB30 human insulin

This is the prior art molecule disclosed in example 1 of WO 2009/022006.

Comparative Compound 9

EGF(A)(301L,309R,312E,321E)

This compound was prepared as described in WO 2017121850.

Comparative Compound 10

EGF (A) (301L,309R,312E,321E,328K (hexadecanediacyl-gGlu-2 xOEG),330K (hexadecanediacyl-gGlu-2 xOEG))

This is the molecule disclosed in example 151 of WO 2017/121850.

Example 25

Insulin receptor affinity of selected insulin derivatives of the invention, as measured at the solubilized receptor

The relative binding affinity of insulin analogues of the present invention for human Insulin Receptor (IR) was determined by competitive binding in the Scintillation Proximity Assay (SPA) according to Glendorf T et al (2008) Biochemistry 4743 and 4751.

Briefly, dilution series of human insulin standards and insulin analogs to be tested were performed in 96-well Optiplates (Perkin-Elmer Life Sciences) followed by addition at 100mM HEPES (pH 7.8), 100mM NaCl, 10mM MgSO₄And 0.025% (v/v) Tween 20 in a binding buffer¹²⁵I-A14Y]Human insulin, anti-IR mouse antibody 83-7, solubilized human IR- A (semi-purified by wheat germ agglutinin chromatography from Baby Hamster Kidney (BHK) cells overexpressing IR- A whole receptor (holoreceptor)) and spA beads (anti-mouse polyvinyltoluene spA beads, GE Healthcare). Plates were incubated at 22 ℃ for 22-24 hours with gentle shaking, centrifuged at 2000rpm for 2 minutes, and counted on TopCount NXT (Perkin-Elmer Life Sciences).

According to a four-parameter logical model (A (1978) Biometrics 34357-365) analyzed the data for SPA and calculated the binding affinity of the analogs relative to the binding affinity of human insulin standards measured in the same plate.

Related assays were also used, where the binding buffer contained 1.5% HSA (w/v) (Sigma a1887) to mimic more physiological conditions.

The insulin receptor affinities and other in vitro data for selected insulin analogs of the invention are listed below in table 3, and the data for comparative compounds are listed below in table 4.

Example 26

Adipogenesis in rat adipocytes

As a measure of the in vitro potency of the insulins of the invention, lipogenesis can be used. Primary rat adipocytes were isolated from epididymal fat pads and incubated with 3H-glucose in a buffer containing, for example, 1% fat-free HSA and a standard of the invention (human insulin, HI) or insulin. Labeled glucose is converted to extractable lipids in a concentration-dependent manner, resulting in a complete concentration response curve. The results are expressed as the relative potency (%) of the insulin of the invention compared to the standard (HI) with a 95% confidence limit.

The data are given in tables 3 and 4 below.

TABLE 3 insulin receptor binding data for selected analogs of the invention in the absence and presence of HSA (0 or 1.5%), and functional adipogenesis data from rat adipocytes

TABLE 4 comparison of IR (isoform A) receptor binding data for compounds in the absence and presence of HSA (0 and/or 1.5%), and functional adipogenesis data from rat adipocytes

It can be seen that the insulin receptor binding (and adipogenic potency) of the compounds of the invention and the comparative compounds are not different and therefore independent of the composition (charge) of the linker. This is in sharp contrast to the observed difference in vivo potency (example 30), where the comparative compound comprising a charged GQEP linker is significantly less potent than the compound of the invention.

It can also be seen that addition of the egf (a) peptide to the N-terminus of insulin via the peptide linker, the insulin receptor affinity was approximately halved in the absence and presence of 1.5% HSA.

Example 27

PCSK9-LDL-R binding competition (ELISA)

The purpose of this assay is to measure the apparent binding affinity of egf (a) compounds to PCSK 9.

Due to their ability to inhibit PCSK9 interaction with LDL-R, the compounds of the invention may also be referred to as PCSK9 inhibitors.

The day before the experiment, recombinant human low density lipoprotein receptor (rhLDL-R; NSO-derived; R)&Dsystems #2148-LD or in-house preparation) was dissolved at 1 μ g/mL in 50mM sodium carbonate (pH 9.6) and then 100 μ L of this solution was added to each well of the assay plate (Maxisorp 96, NUNC #439454) and coated overnight at 4 ℃. On the day of the experiment, eight-point concentration curves of egf (a) compound containing biotinylated PCSK9(0.5ug/mL, BioSite/BPSBioscience catalog No. 71304 or prepared internally) were made in duplicate. A mixture of EGF (A) compound and biotinylated PCSK9 was prepared and incubated at room temperature with 25mM Hepes, pH7.2(15630-056, 100ml, 1M), 150mM NaCl (Emsure 1.06404.1000), 1% HSA (Sigma A1887-25G), 0.05% Tween 20(Calbiochem 655205), 2mM CaCl₂(Sigma 223506-500G) for 1 hour. The coated assay plate was then washed 4 times with 200 μ Ι assay buffer, then 100 μ Ι of a mixture of egf (a) compound and biotinylated PCSK9 was added to the plate and incubated for 2 hours at room temperature. The plate was washed 4 times with 200. mu.L of assay buffer and then incubated with streptavidin-HRP (25 ng/ml; VWR #14-30-00) for 1 hour at room temperature. The reaction was detected by adding 50. mu.L of TMB-on (KEM-EN-TEC) and incubated for 10min in the dark. Then by adding 50. mu.L of 4M H to the mixture₃PO₄The reaction was terminated by electron multiplex addition. Then used within 1 hourSpectramax reads plates at 450 and 620 nm. The 620nm reading was used for background subtraction. IC50 values were calculated by nonlinear regression log (inhibitor) and response variable slope (four parameters) using Graphpad Prism and converted to Ki values using the following formula: Ki-IC 50/(1+ (Biotin-PCSK 9)/(Kd (Biotin-PCSK 9))), with the Kd of Biotin-PCSK9 of 1.096727714 μ g/mL, [ Biotin-PCSK9 ]]＝0.5(μg/mL)。

The results for the compounds of the invention are shown in table 5 below, and the data for the comparative compounds are shown in table 5 below. Higher Ki values reflect lower apparent binding affinity for PCSK9, and vice versa. Typically, a number of the egf (a) compounds tested showed the ability to inhibit PCSK9 binding to hLDL-R in the low nM range.

Table 5: apparent PCSK9 binding affinity (Ki, in nM) and LDL uptake (EC) in HepG2 cells of the compounds of the invention₅₀In nM)

TABLE 6 comparison of apparent PCSK9 binding affinity (Ki, in nM) and LDL uptake (EC) in HepG2 cells for compounds₅₀In nM)

It can be seen that the binding of PCSK9 for the compounds of the invention and the comparative compounds is not different and therefore independent of the composition (charge) of the linker.

It was also observed that the addition of linker and insulin to the C-terminus of the egf (a) peptide did not alter binding to PCSK 9.

Example 28

LDL uptake assay in HepG2 cells

Described below are alternative assays to determine the inhibitory potency of PCSK9 peptides and derivatives thereof for measuring LDL uptake in HepG2 cells.

The test principle is as follows: LDL uptake is primarily mediated by endogenously expressed hLDL-R, and thus LDL uptake capacity is an indirect measure of LDL-R expression. Incubation with exogenous PCSK9 can downregulate hLDL-R in a dose-dependent manner. Therefore, PCSK9 incubation decreased the ability of cells to take up LDL molecules. Downregulation of LDL uptake can then be antagonized by the addition of compounds that neutralize or inhibit PCSK9/LDL-R binding. Accordingly, PCSK9 inhibitors may be characterized based on the ability of PCSK9 inhibitors to increase LDL uptake in the presence of PCSK9 and, for example, counteract PCSK 9-mediated down-regulation of hdlk-R.

Experiments were performed using HepG2 cells (Sigma Aldrich ECACC: accession number 85011430) grown in 10% lipoprotein deficient fetal bovine serum (Sigma Aldrich # S5394) and cells were measured for their ability to take up BODIPY fluorescently labeled LDL particles (Life technologies Europe BV # L3483).

Test protocol: 96-well plates (Perkin Elmer, Viewplate-96 Black #60005182) were coated with poly-D-lysine (10mg/L, Sigma Aldrich # P6407, dissolved in PBSGibco #14190-094) in an incubator for 1 hour at 37 ℃. The plate was then washed 2 times with 100. mu.l of PBS (Gibco # 14190-094). To obtain an 8-point concentration profile for the EGF (A) compounds, test compositions were prepared, all containing PCSK9(10 ug/mL; prepared internally) diluted in assay medium (DMEM (Gibco #31966-021), 10% lipoprotein-deficient fetal bovine serum (Sigma Aldrich # S5394), and 1% Pen Strep (Cambrex # DE17-602E)) and added to the plates in a volume of 50 uL/well.

After 30-60 minutes, 50,000 HepG2 cells (Sigma-Aldrich: ECACC: accession No. 85011430, batch No. 13B023) diluted in assay medium were added in a volume of 50. mu.L/well and the plates were placed in CO₂Permeability plastic bag (Antalis Team, LDPE bag 120/35x300x0.025mm #281604) incubated for 20 hours (5% CO at 37 ℃)₂Below). Plates were emptied thereafter, immediately followed by the addition of 50 μ L FL-LDL (Life technologies Europe BV # L3483) at a concentration of 10 μ g/mL in assay medium to each well, and plates were placed in CO₂Permeable plastic bagIncubated for 2 hours (5% CO at 37 ℃ C.)₂Lower), it was covered with a black lid to protect from light. The plate was emptied and washed 2 times with 100. mu.L of PBS (Gibco # 14190-. Then 100 μ L of PBS (Gibco #14190-094) was added and the plate was read (bottom read) over 15 minutes thereafter on a SpecktraMax M4(Molecular Probes, Invitrogen Detection Technologies) using the following filters Ex (515nm)/Em (520 nm).

Finally, EC50 values were calculated using GraphPad Prism, nonlinear regression curve fitting, sigmoidal dose-response (variable slope).

The results are shown in the table above. Lower EC50 values reflect a higher ability to reverse PCSK 9-mediated down-regulation of LDL uptake, whereas conversely, high EC50 values indicate compounds with a low ability to inhibit PCSK 9-mediated down-regulation of LDL uptake.

As can be seen, the tested compounds exhibited an EC50 of 100-200nM in the LDL uptake assay, indicating that the compounds have a high ability to reverse PCSK 9-mediated down-regulation of LDL uptake.

Example 29

Rat pharmacokinetics, intravenous rat PK:

different doses of insulin analogues were administered intravenously (i.v.) to 300 grams of conscious, non-fasting approximately male Sprague-Dawley rats and plasma concentrations of the compounds used were determined using immunoassay or mass spectrometry at specified time intervals up to 48 hours after administration. Pharmacokinetic parameters were then calculated using WinNonLin Professional (Pharsight inc., Mountain View, CA, USA)).

TABLE 7 PK data (mean residence time) in rats after intravenous administration of the compounds of the invention

TABLE 8 PK data (mean residence time) in rats after intravenous administration of the comparative compounds

In conclusion, the compounds of the invention have similar PK profiles to similar comparative compounds. Thus, the PK data cannot account for the unexpected in vivo potency differences observed (example 30), where the comparative compound comprising a charged GQEP linker was significantly less potent than the compound of the invention.

Example 30

Subcutaneous PK/PD profiles of insulin analogues of the invention and of the prior art in Sprague Dawley rats

The fusion peptide derivatives of the invention can be tested by subcutaneous administration to rats, for example, in accordance with this protocol in comparison to a similar B29K acylated insulin analogue of the prior art. The derivatives may be tested for pharmacokinetic and/or pharmacodynamic parameters.

In vivo protocol

Approximately 350 grams of conscious, non-fasted male Sprague-Dawley rats were used for these experiments. During the study period (up to 30 hours after dosing), rats had free access to water and food. Using NovoPenRats were administered subcutaneously (90 nmol/kg; 600. mu.M insulin derivative preparation) to the neck. Blood samples were drawn at time point 0 (pre-administration) and 15min, 1,2, 4, 5.5, 7, 24, 29/30 hours after administration of the insulin derivative and finally daily up to 30 hours (sublingual vein; 200. mu.l was added200EDTA tubes) and plasma was collected. Plasma concentrations of glucose and the final insulin derivative were quantified using a BIOSEN analyzer and immunoassay/LCMS analysis, respectively.

The following conclusions are drawn from fig. 1 and 2: spacers 2 xgaiqp, 3 xgaiqp, 4 xgaiqp, 6 xgaiqp, 8 xgaiqp and 10 xgaiqp confer equal insulin potency, while longer spacers 12 xgaiqp and 19 xgaiqp impair insulin potency.

Observing fig. 3 leads to the following conclusions: the spacers 2xgaq and 8xgaq confer equal insulin potency, while the spacers 2xGQEP and 8xGQEP unexpectedly and significantly impair insulin potency.

Observing fig. 4 leads to the following conclusions: the hypoglycemic effects of the compounds containing linkers 2 xgaqqp, 4xGAQP and 6xGQAP and insulin replacement a14E or B3E were equivalent and superior to the hypoglycemic effects of comparative compounds 1 and 2 and vehicle containing linkers 2xGQEP and 8xGQEP, respectively.

Observing fig. 5 leads to the following conclusions: the spacers 2xGAQP and 2xGQAP confer equal insulin potency, while the spacer 2xGQEP unexpectedly attenuates insulin potency.

Observing fig. 6 leads to the following conclusions: for spacer 4 xgaiqp, the insulin substitution desB30 and a14E, desB30 conferred equal insulin potency, while spacer 4xGQEP unexpectedly attenuated insulin potency.

Observing fig. 7 leads to the following conclusions: the insulin substitutions desB30 and B3E, desB30 conferred equal insulin potency and the side chain containing one and two OEG moieties conferred equal insulin potency, while the spacer 2xGQEP unexpectedly attenuated insulin potency compared to a similar compound with a2 xgaqqp spacer.

Observing fig. 8 leads to the following conclusions: the insulin substitutions desB30 and a14E, desB30 conferred equal insulin potency and the side chain containing one and two OEG moieties conferred equal insulin potency, while the spacer 6xGQEP unexpectedly attenuated insulin potency compared to a similar compound with a 6 xgaqqp spacer.

Observing fig. 9 leads to the following conclusions: the insulin of example 1 with the side chain hexadecanediacyl-gGlu-2 xOEG, spacer 2xGAQP unexpectedly confers insulin potency much higher than the spacers 2xGQEP and 8 xGQEP.

Observing fig. 10 leads to the following conclusions: insulin with the side chain eicosanedioyl-gGlu-2 xOEG, spacer 6xGAQP surprisingly confers insulin potency much higher than spacer 4 xGQEP.

Observing fig. 11 leads to the following conclusions: the egf (a) moiety of the present invention is attached to insulin via an uncharged linker, resulting in a compound with similar glucose kinetic (glucodynemic) potency compared to that of insulin alone. The fusion proteins of the invention have a slower onset of action, which is advantageous for basal insulin.

Example 31

Acute in vivo proof of concept model: streptozotocin-induced human PCSK9(hPCSK9) challenge model in diabetic mice

The purpose of this model was to demonstrate the dual activity of the insulin-EGF (A) fusion protein. Dual activity means increased LDL receptor expression levels in mouse liver by inhibiting the action of intravenous hPCSK9 with an insulin-egf (a) -based anti-PCSK 9 peptide, as well as the hypoglycemic effect of the insulin part of the molecule.

The method comprises the following steps: healthy male BalBC mice (Charles River, Germany) were made diabetic by a single high dose subcutaneous (s.c.) injection of streptozotocin (230-250 mg/kg). After 5-7 days, diabetic animals were randomized into the indicated treatment groups. On the day of the experiment, animals were injected intravenously (i.v.) with vehicle, egf (a) derivative or insulin-egf (a) fusion protein at t-0 min (first dose on the figure). hPCSK9 or vehicle was injected intravenously at a dose of 0.4mg/kg at t-15 min (2 nd dose on the figure). Blood glucose levels were measured at times 0, 15, 45 and 75 min. 60 minutes after the injection of hPCSK9 (t 75min), animals were anesthetized with isoflurane and euthanized by cervical dislocation. Livers were rapidly excised and frozen in liquid nitrogen. Liver samples were kept at-80 degrees celsius until analysis. LDL-r protein in liver samples was quantified by ELISA.

Mouse LDL-R ELISA: a piece of liver (10mg) was homogenized on a TissueLyser using a steel ball in 500uL PBS at 30Hz for 2.5 min. Then, the tissue was lysed by adding 500uL 2X Lysis Buffer 2(R & D systems catalog No. 895347) and incubated on a shaker (500rpm) for 1 h. The liver lysate was centrifuged at 20000g for 10min at 4 ℃. The clarified supernatant was diluted 50-fold in a calibration dilution and 50uL was used for analysis on mdll-R ELISA (RD Systems MLDLR 0). The LDL-R concentration values in liver lysates were normalized to the protein concentration in the same samples. Lysates were diluted 20-fold in PBS and Protein assays were performed with 25uL, 2 replicates according to Pierce BCA Protein Assay Kit (cat No. 23225).

The dual activity of the insulin-EGF (A) fusion protein in vivo was demonstrated by studying blood glucose changes and hepatic LDL-r protein expression following administration of the compound to streptozotocin-diabetic mice. The insulin-egf (a) fusion protein of example 3 was administered at 0, 3, 10, 30 and 100nmol/kg, with each group of n-5-6 animals. Figure 12 shows that administration of hPCSK9 to mice results in almost complete down-regulation of hepatic LDL receptor protein. The insulin-egf (a) fusion protein effectively prevented PCSK 9-mediated down-regulation of LDLr protein in a dose-dependent manner. In addition, insulin-egf (a) fusion protein dose-dependently reduced blood glucose (fig. 13). Furthermore, two insulin-egf (a) fusion proteins have been shown to prevent hPCSK 9-mediated down-regulation of LDLr protein, similar to that seen with the egf (a) derivative alone (fig. 14).