Acylated insulin compounds with delayed action

Abstract

The compounds described herein are directed to the treatment of Type I and / or Type II diabetes and / or hyperglycemia. More specifically, the described compounds are directed to acylated insulin compounds that have a prolonged effect on lowering blood glucose, pharmaceutical compositions containing such compounds, therapeutic uses of such compounds, and intermediate compounds useful in the preparation of the described acylated insulin compounds.

Description

Technical Field

[0001] This invention belongs to the field of treating diabetes and / or hyperglycemia. Specifically, this invention relates to compounds that lower blood sugar, pharmaceutical compositions containing such compounds, and the therapeutic use of such compounds. Background Technology

[0002] Insulin replacement therapy for patients with diabetes should ideally closely resemble the endogenous insulin secretion pattern in healthy individuals. The physiological need for insulin can be divided into two phases: (a) the nutrient absorption phase, which requires insulin pulses to manage diet-related glucose spikes, also known as “dietary” insulin; and (b) the postabsorption phase, which requires continuous delivery of insulin to regulate hepatic glucose output to maintain optimal fasting blood glucose, also known as “basal” insulin.

[0003] Effective insulin therapy for people with diabetes typically involves the combined use of two types of exogenous insulin preparations: fast-acting mealtime insulin and long-acting basal insulin administered once or twice daily to control blood glucose levels between meals. One or more characteristics of endogenous insulin that may need to be emulated include binding affinity to the human insulin receptor, preferential binding to the human insulin receptor relative to the human IGF-1 receptor, phosphorylation of the human insulin receptor, and lower blood glucose levels.

[0004] Desired exogenous basal insulin should also provide a delayed effect—that is, it should control blood glucose levels preferably for 24 hours or longer, and most preferably for 168 hours or longer, without a significant risk of hypoglycemia. Some basal insulins have a duration of action of 24 hours or longer. Compounds with a delayed effect (during which efficacy does not change significantly) may reduce the risk of nocturnal hypoglycemia and allow for greater variability in the frequency of daily dosing without increasing the patient's risk of hypoglycemia. Therefore, weekly dosing is highly desirable. Potential characteristics of exogenous basal insulin may include weakened receptor binding, which can lead to reduced clearance from the bloodstream, and / or chemical stability at multiple concentrations, which would promote extended shelf-life stability and / or stability in concentrated formulations, which would allow for use in multi-dose devices.

[0005] There is a need for alternative treatments for diabetes and / or hyperglycemia in patients. Some acylated insulin compounds are known, see, for example, U.S. Patent Nos. 7,615,532, 10,400,021, 9,045,560, and 9,018,161, but additional alternative treatments are still required. Summary of the Invention

[0006] This invention provides acylated insulin compounds with a delayed effect for treating diabetes, lowering hemoglobin A1c, and / or lowering blood glucose levels in patients with this need. The compounds of this invention possess any of the following desirable characteristics: slower clearance than known acylated insulins (e.g., insulin degludec), enhanced bioavailability, more stable pharmacokinetic characteristics in humans over time, and / or an increased duration of action in vivo. Furthermore, the compounds of this invention exhibit low chemical degradation rates, indicating increased chemical stability and / or the potential for longer shelf life than known acylated insulins.

[0007] This invention provides compounds of formula I or pharmaceutically acceptable salts thereof:

[0008]

[0009] Where X is selected from the following group: -Lys-Gly-, -Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- and -εLys-Gly-.

[0010] Therefore, the compound of formula I is a modified human insulin or a pharmaceutically acceptable salt thereof, which consists of the following:

[0011] The sequence of chain A in SEQ ID NO:1, wherein the native tyrosine amino acid residue at position A14 is mutated to a glutamic acid amino acid residue; and the sequence of chain B in SEQ ID NO:2, wherein the native tyrosine amino acid residue at position B16 is mutated to a histidine amino acid residue, the native phenylalanine amino acid residue at position B25 is mutated to a histidine amino acid residue, and the native threonine amino acid residue at position B30 is deleted; wherein: a disulfide bond exists between cysteine ​​at position 6 and cysteine ​​at position 11 in SEQ ID NO:1, a disulfide bond exists between cysteine ​​at position 7 and cysteine ​​at position 7 in SEQ ID NO:2, and a disulfide bond exists between cysteine ​​at position 20 and cysteine ​​at position 19 in SEQ ID NO:2; and wherein the lysine amino acid residue at position B29 is bonded to HO2C-(CH2) via the ε-amino group of the lysine side chain. 18 -CO-γGlu-γGlu-γGlu-Lys-Gly-, HO2C-(CH2) 18 -CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- or HO2C-(CH2) 18It is chemically modified by the conjugation of -CO-γGlu-γGlu-γGlu-εLys-Gly-.

[0012] According to a preferred embodiment of the invention, X is -Lys-Gly-; or a pharmaceutically acceptable salt thereof.

[0013] According to another preferred embodiment of the invention, X is -Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-; or a pharmaceutically acceptable salt thereof.

[0014] According to a further preferred embodiment of the invention, X is -εLys-Gly-; or a pharmaceutically acceptable salt thereof.

[0015] In a more preferred embodiment of the invention, X is -Lys-Gly-; or a pharmaceutically acceptable salt thereof.

[0016] A preferred embodiment of the present invention provides the following compound:

[0017]

[0018] It is a compound of formula I consisting of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:3, or a pharmaceutically acceptable salt thereof, wherein the ε-amino group of the lysine side chain at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-Lys-Gly- conjugates are chemically modified.

[0019] Preferably, the present invention provides the following compounds:

[0020]

[0021] It is a compound of formula I consisting of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:4, or a pharmaceutically acceptable salt thereof, wherein the ε-amino group of the lysine side chain at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- is chemically modified by conjugation.

[0022] Preferably, the present invention comprises the following compounds:

[0023]

[0024] It is a compound of formula I consisting of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:5, or a pharmaceutically acceptable salt thereof, wherein the ε-amino group of the lysine side chain at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-εLys-Gly- conjugates are chemically modified.

[0025] According to another aspect of this application, a pharmaceutical composition is provided comprising a compound of formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

[0026] Another aspect of this application provides a method for treating type I and / or type II diabetes in patients, the method comprising administering to a patient in need an effective amount of a compound of formula I according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a compound of formula I.

[0027] Another aspect of this application provides a method for treating hyperglycemia in a patient, the method comprising administering to a patient in need an effective amount of a compound of formula I according to the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a compound of formula I.

[0028] Another aspect of this application also provides compounds of formula I or pharmaceutically acceptable salts thereof for use in therapeutics.

[0029] This application further provides compounds of formula I or pharmaceutically acceptable salts thereof for the treatment of diabetes and / or hyperglycemia.

[0030] The compounds of the present invention were prepared using the novel intermediate compounds described below.

[0031] According to another aspect of this application, compound A having the following formula is provided:

[0032]

[0033] This application also provides compound B having the following formula:

[0034]

[0035] This application also provides compound C having the following formula:

[0036]

[0037] Another aspect of the invention provides a method for preparing a compound of formula I using a compound selected from any one of compounds A, B and C.

[0038] Another aspect of this application provides the use of any of the compounds of Formula I or pharmaceutically acceptable salts thereof in the preparation of a medicament for treating type I and / or type II diabetes and / or hyperglycemia.

[0039] This application further provides the use of any one of compounds A, B or C in the preparation or manufacture of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0040] As used herein, the term "treatment" refers to the management and care of a patient with diabetes or hyperglycemia, or other conditions for which insulin is recommended, with the aim of combating or alleviating the symptoms and complications of those conditions. The patient to be treated is an animal, and preferably a human.

[0041] As used herein, the term "effective amount" refers to the amount or dose of a compound of the present invention or a pharmaceutical composition containing a compound of the present invention that, upon administration of a single or multiple doses to a patient or subject, will elicit a biological or medical response or desired therapeutic effect in a tissue, system, animal, mammal, or human that is being sought by researchers, veterinarians, physicians, or other clinicians. Doses may include a higher initial loading dose followed by lower doses.

[0042] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to animals. Preferably, the terms refer to humans. In some embodiments, the patient, preferably a human, is further characterized by having a disease, disorder, or condition that would benefit from lowering blood glucose levels.

[0043] Pharmaceutical compositions comprising the compounds of the present invention can be administered parenterally to patients requiring such treatment. Parenterical administration can be performed via subcutaneous, intramuscular, or intravenous injection using a syringe, optionally a pen syringe, or a mechanically driven syringe. Alternatively, parenterical administration can be performed using an infusion pump.

[0044] Embodiments of the present invention provide pharmaceutical compositions suitable for administration to patients, preferably weekly, comprising administering to a patient in need a therapeutically effective amount of the compound of the present invention and one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be prepared using conventional excipients well known in the art for pharmaceutical products by any of a variety of techniques (Remington's Pharmaceutical Sciences, 21st edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA (2006)).

[0045] The claimed compound may be used simultaneously, separately, or sequentially in combination with one or more other therapeutic agents that can be used to treat diabetes and / or diabetes-related conditions. Non-limiting examples of other therapeutic agents that may be combined with the claimed compound include: insulin or insulin analogs; biguanides; sulfonylureas; thiazolidinediones; dipeptidyl peptidase-4 (“DPP-4”) inhibitors; sodium-dependent glucose transporter (SGLT2) inhibitors; incretin compounds such as glucagon-like peptide-1 (GLP-1) or GLP-1 analogs, gastric inhibitory peptides (GIPs) or GIP analogs, gastrin or gastrin analogs; or any combination of the foregoing agents. The claimed compound and one or more other therapeutic agents may be administered together via the same route of delivery and device, such as a single pill, capsule, tablet, or injectable formulation; or simultaneously and separately via separate delivery devices or routes; or sequentially.

[0046] In this article, “BHI” refers to biosynthesized human insulin, “TFA” refers to trifluoroacetic acid, “Boc” refers to tert-butyloxycarbonyl, “tBu” refers to tert-butyl, “PyBop” refers to benzotriazol-1-yl-oxytripyrrolidone phosphonium hexafluorophosphate, “DMF” refers to dimethylformamide, “DIEA” refers to diisopropylethylamine, “DCM” refers to dichloromethane, “HFIP” refers to hexafluoroisopropanol, “ACN” refers to acetonitrile, “Fmoc” refers to fluorenylmethoxycarbonyl, “DMSO” refers to dimethyl sulfoxide, “TCI” refers to double-chain insulin, “SCI” refers to single-chain insulin, “TIS” refers to triisopropylsilane, and “TSTU” refers to... (O-(N-succinimide)-N,N,N′,N′-tetramethylureonium tetrafluoroborate, "Su" refers to succinimide, "CpB" refers to carboxypeptidase-B, "RP-HPLC" refers to reversed-phase HPLC, "hr" refers to hours, "min" refers to minutes, "EDTA" refers to ethylenediaminetetraacetic acid, "BSA" refers to bovine serum albumin, "HSA" refers to human serum albumin, "C20-OH" refers to eicosanoic acid, "γGlu" refers to L-glutamic acid linked through its side chain γ-carboxyl group, "Lys" refers to L-lysine, "εLys" refers to L-lysine linked through its side chain ε-amino group, and "Gly" refers to glycine.

[0047] Formula I contains the standard single-letter amino acid codes for the amino acid residues of the insulin A and B chains, except for residue 29 of the B chain, which is lysine, and the structure of this amino acid residue has been expanded. C20-OtBu is CO-(CH2). 18-CO2-tert-butyl, also known as 20-tert-butoxy-20-oxo-eicosanoic acid, γGlu(OtBu) is L-glutamic acid α-tert-butyl ester, and Lys(Boc) is L-lysine ε-amino-tert-butoxycarbonyl. A14E,B16H,B25H,desB30-human double-chain insulin (TCI; A14E,B16H,B25H,desB30-BHI) refers to modified human insulin, in which the tyrosine amino acid residue at position 14 of chain A is mutated to a glutamic acid amino acid residue, the native tyrosine amino acid residue at position 16 of chain B is mutated to a histidine amino acid residue, the native phenylalanine amino acid residue at position 25 of chain B is mutated to a histidine amino acid residue, and the native threonine amino acid residue at position B30 is deleted.

[0048] The structures of 2-[2-(2-aminoethoxy)ethoxy]acetic acid, γGlu and εLys.

[0049] Detailed Implementation

[0050] Example 1 is a compound of Formula I, which can be produced by selective acylation of the ε-amino group of lysine at position 29 of the B chain using a linker-fatty acid intermediate, wherein the linker-fatty acid intermediate is: C20-OtBu-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH, wherein C20-OtBu is 20-tert-butoxy-20-oxo-eicosanoic acid, γGlu(OtBu) is an L-glutamic acid α-tert-butyl ester linked by a γ-carboxyl group on its side chain, Lys(Boc) is an L-lysine ε-aminotert-butoxycarbonyl group, and Gly is glycine.

[0051] Example 2 is a compound of Formula I, which can be produced by selective acylation of the ε-amino group of lysine at position 29 of the B chain using a linker-fatty acid intermediate, wherein the linker-fatty acid intermediate is: C20-OtBu-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-OH, wherein C20-OtBu is 20-tert-butoxy-20-oxo-eicosanoic acid, γGlu(OtBu) is an L-glutamic acid α-tert-butyl ester linked by a γ-carboxyl group on its side chain, and Lys(Boc) is an L-lysine ε-aminotert-butoxycarbonyl group.

[0052] Example 3 is a compound of Formula I, which can be produced by selective acylation of the ε-amino group of lysine at position 29 of the B chain using a linker-fatty acid intermediate: C20-OtBu-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-εLys(Boc)-Gly-OH, wherein C20-OtBu is 20-tert-butoxy-20-oxo-eicosanoic acid, γGlu(OtBu) is an L-glutamic acid α-tert-butyl ester linked through its side chain γ-carboxyl group, εLys(Boc) is an L-lysine α-amino tert-butoxy carbonyl group linked through its side chain ε-amino group, and Gly is glycine, followed by removal of the acid-labile Boc and tBu protecting groups.

[0053] The formation of compounds of Formula I can occur during three main stages: 1) the formation of A14E, B16H, B25H, and desB30-BHI; 2) the synthesis of linker-fatty acid intermediates; and 3) acylation, deprotection, purification, and salt exchange to isolate compounds of Formula I.

[0054] The insulin moiety of the compounds of the present invention can be prepared using various techniques known to those skilled in the art, such as by using recombinant DNA technology to generate precursor protein molecules. The DNA (including cDNA and synthetic DNA) can be double-stranded or single-stranded. The coding sequence encoding the precursor protein molecule as described herein may vary due to redundancy or degeneracy of the genetic code. To generate the precursor protein of the present invention, the DNA can be introduced into a host cell. Suitable host cells are transiently or stably transfected or transformed using an expression system for generating the precursor protein. The host cell can be a bacterial cell such as K12 or B strains of *Escherichia coli*, a fungal cell such as yeast cells, or a mammalian cell such as Chinese hamster ovary (“CHO”) cells.

[0055] Expression vectors, either as episomes or as part of the host's chromosomal DNA, are typically capable of replication in the host organism. Often, expression vectors will contain selection markers, such as tetracycline, neomycin, and dihydrofolate reductase, to allow selection of those cells that are transformed by the desired DNA sequence.

[0056] The compounds of the present invention can be prepared using a variety of procedures known in the art and the methods described below. The specific synthetic steps of each described route can be combined in different ways to prepare the compounds described herein. Examples 2 and 3 were prepared in a manner similar to Example 1.

[0057] Preparation of Formula I Acylated Insulin

[0058] Example 1

[0059]

[0060] Example 1 is a compound of formula I, which consists of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:3, wherein Lys at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-Lys-Gly- conjugates are chemically modified.

[0061] Overview of the synthesis of Example 1: Example 1 was produced by selective acylation of the ε-amino group of lysine at position 29 of the B chain of mature biosynthesized A14E,B16H,B25H,desB30-human double-chain insulin (TCI; A14E,B16H,B25H,desB30-BHI), or by direct acylation of a similar ε-amino group of lysine at position 29 of the B chain of a single-chain insulin (SCI; proinsulin A14E,B16H,B25H,desB30-BHI) construct with a linker-fatty acid intermediate designated as compound A (C20-OtBu-γGlu(OtBu)-γGlu-(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH). The preferred method is the acylation of the proinsulin SCI construct. Because the A1 and B1 amino terms of the insulin molecule are blocked by a C-peptide and a leader sequence, respectively, this method provides higher acylation efficiency for the ε-amino group of lysine at position 29 of the B chain. Enzymatic digestion with CpB and trypsin, followed by acylation and TFA deprotection steps, yielded the fully matured double-stranded product of Example 1. Although acylation of the N-terminus of the SCI construct does occur, resulting in a diacylation byproduct, enzymatic removal of the leader sequence also converts this substance into the desired product.

[0062] The formation of molecules occurs during three main stages:

[0063] 1) Expression and purification of single-chain proinsulin construct (SCI) in E. coli: This can be used for preferred acylation methods, or it can be enzymatically processed into mature double-chain insulin analogs (TCI, A14E, B16H, B25H, desB30-BHI).

[0064] 2) Synthesis of the linker fatty acid intermediate (compound A); and

[0065] 3a) Acylation and deprotection of TCI, or 3b) Acylation, deprotection and enzymatic digestion of SCI, followed by purification and salt exchange to produce Example 1.

[0066] A novel expression vector was developed and used in E. coli fermentation to produce an SCI insulin construct; it consists of the following sequence (SEQ ID NO:7):

[0067]

[0068] Residues 1-34 constitute the leader peptide, residues 35-63 constitute the B-chain (desB30T), residues 64-97 constitute the modified C-peptide (des64K using the conventional numbering of native insulin), and residues 98-118 constitute the A-chain. The disulfide bonds correspond to the disulfide bonds in native insulin. These disulfide bonds are located between C41 and C104; between C53 and C117; and between C103 and C108. Enzymatic digestion with CpB and trypsin yields a fully mature TCI analog.

[0069]

[0070] The disulfide bonds are located between C7 (SEQ ID NO:1) and C7 (SEQ ID NO:8); between C20 (SEQ ID NO:1) and C19 (SEQ ID NO:8); and between C6 (SEQ ID NO:1) and C11 (SEQ ID NO:1). It should be noted that both the SCI and TCI constructs were purified using a standard RP-HPLC column and lyophilized as solid TFA salts prior to acylation. The linker fatty acid molecule was generated using solid-phase synthesis. This molecule can be generated using solution-phase methods alone, or in combination with solid-phase methods.

[0071] Due to the solubility of linker fatty acids based on Boc / tBu protected amino acids, the conjugation of linker fatty acids with TCI or SCI A14E, B16H, B25H, desB30-BHI is carried out in a dry organic solvent (DMSO). However, alternative protecting / deprotecting schemes can be designed to make the linker fatty acids soluble in aqueous solutions to minimize the use of organic solvents. Similarly, acidic chemical transformations using trifluoroacetic acid (TFA) to remove the Boc / tBu protecting groups can be achieved through alternative methods.

[0072] Solid-phase synthesis was used to produce linker fatty acid molecule compound A.

[0073] Compound A (C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH was generated via solid-phase peptide synthesis. Fmoc-Lys(Boc)-OH (769 mg, 1.6 mmol, 1.5 equivalents relative to resin) was mixed with PyBop (849 mg, 1.6 mmol, 1.49 equivalents relative to resin) and DIEA (1520 μL, 8.7 mmol, 8 equivalents) in 10 mL of DMF for 2 minutes, then transferred to a reaction vessel containing H-Gly-2-chlorotriphenylmethylchloro resin (1.1 g, 0.99 mmol / g, 1.1 mmol; Peptides International RHG-1160-PI), which was pre-expanded in DCM and pre-washed with DMF. The slurry was mixed for 1.5 h, filtered, and then the resin was thoroughly washed with DMF (Kaiser test negative). The Fmoc protecting group was removed by treating the resin with 20% piperidine / DMF (10 mL, 30 min). After washing the resin with DMF (40 mL), the Kaiser test was positive, and the final product was H-Lys(Boc)-Gly-2-chlorotriphenylmethyl chloride resin (theoretical value 1.1 mmol).

[0074] Fmoc-Glu-OtBu (701 mg, 1.6 mmol, 1.5 equivalence) was pre-activated with PyBop (845 mg, 1.6 mmol, 1.49 equivalence) for 2 min using DIEA as a base in 10 mL of DMF (1520 μl, 8.7 mmol, 8.0 equivalence) and transferred to resin. The slurry was mixed for 3 h, filtered, and the resin was thoroughly washed with DMF (Kaiser test negative). The Fmoc protecting group was removed by treating the resin with 20% piperidine / DMF (10 mL, 30 min), followed by washing the resin with DMF (40 mL, Kaiser test positive). The second and third Fmoc-Glu-OtBu residues were coupled to the resin by repeating the above conditions with a coupling time of 1.5 h.

[0075] After removing the final Fmoc protecting group, 20-tert-butoxy-20-oxo-eicosanoic acid (660 mg, 1.60 mmol, 1.5 equivalence) was pre-activated (2 min) with PyBop (845 mg, 1.6 mmol, 1.49 equivalence) in 10 mL DMF using DIEA as a base (1520 μl, 8.7 mmol, 8.0 equivalence) and transferred to resin. The slurry was mixed for 3 h, filtered, and the resin was thoroughly washed with DMF (Kaiser test negative). Resin cleavage: Protected linker-fatty acids were cleaved from the resin by mixing with 30% HFIP / DCM (20 mL) for 1 h. The resin was filtered and thoroughly washed with DCM. The combined filtrates were evaporated to oil under vacuum. The residual oil was diluted with ACN (15–20 mL) and evaporated again under vacuum to oil. The sample was redissolved with ACN (15–20 mL) and evaporated under vacuum to form oil. A gentle stream of nitrogen evaporated the residual ACN to produce 2.3 g of crude amorphous solid (theoretical yield = 1.4 g).

[0076] Purification: The crude sample was dissolved in 2 mL DMF and 20 mL ACN (including flask wash). Water was added, which produced 35 mL of turbid solution. An additional 10 mL of ACN produced a clear solution (total volume equal to 45 mL (33% aqueous content)). The sample was then loaded onto a semi-preparative cyano RP-HPLC column (SilaChrom XDB1-CN; 10 μm). Purification was performed on a 2.1 x 25 cm plate. The sample was eluted for 71 min using a 40–75% B gradient at 15 mL / min at 60 °C (A-buffer = 0.15% TFA in water and B-buffer = ACN). Fractions containing the desired product, as determined by analytical RP-HPLC, were combined, freeze-dried, and yielded 696 mg of compound A as a white amorphous solid (52% of theoretical value; 91% purity determined by RP-HPLC; observed MW = 1239.6 Daltons; theoretical MW = 1239.65 Daltons).

[0077] Compound B (C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-OH was prepared essentially as described in the procedure used for the synthesis of compound A, except that the starting resin was changed to H-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-2-chlorotriphenylmethyl chloride resin (1.8 g, 0.62 mmol / g, 1.1 mmol; Peptides International RHX-11074-PI). Compound B was separated as a white amorphous solid by RP-HPLC. The observed MW was 1327.5 Daltons; the theoretical MW was 1327.73 Daltons.

[0078] Compound C (C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-εLys(Boc)-Gly-OH was prepared essentially as described in the procedure for synthesizing compound A, but the Fmoc-Lys(Boc)-OH structural unit was replaced with Boc-L-Lys(Fmoc)-OH. Compound C was separated into a white amorphous solid by RP-HPLC. The observed MW was 1239.5 Daltons; the theoretical MW was 1239.65 Daltons.

[0079] Acylation, deprotection, purification, and salt exchange to separate Example 1

[0080] Acylation: Compound A ((C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH) (60.8 mg; 0.0437 mmol; 1.2 equivalents; synthesized above) and TSTU (12.04 mg 0.040 mmol; 1.1 equivalents) were dissolved in 200 μL of dimethyl sulfoxide (DMSO). Diisopropylethylamine (DIEA, 25.3 μL; 0.145 mmol; 4 equivalents) was added to the solution, and the resulting mixture was incubated at room temperature for 30 minutes to produce compound A-OSu ester in DMSO, which was used directly.

[0081] To a solution of double-chain insulin A14E, B16H, B25H, desB30-BHI (TFA salt; 205 mg; 0.0364 mmol) dissolved in 2 mL of dry dimethyl sulfoxide (DMSO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 81.6 μL 0.546 mmol, 15 equivalents, Sigma-Aldrich catalog 33482) was added, followed immediately by the addition of compound A-OSu ester as described above in dry DMSO. The reaction mixture was stirred at ambient temperature for 12 minutes and then added to a mixture of diethyl ether / DCM / TFA (30:10:0.2 v / v; 40 mL volume). The resulting white precipitate was separated by centrifugation and ground once with diethyl ether.

[0082] Deprotection (removal of Boc and OtBu groups): The white precipitate moistened with diethyl ether was treated with a mixture of TFA / triisopropylsilane (TIS) / water (92.5:5.0:2.5 v / v; 5 mL) for 20 min. Diethyl ether (40 mL) was added, and the resulting precipitate was collected by centrifugation, washed once with diethyl ether, and dried. Reversed-phase (RP)-HPLC analysis showed that the deprotection step was complete (9% unreacted A14E, B16H, B25H, desB30-BHI; 61% B29-acylated product; 13% A1, B29-diacylated product).

[0083] Purification: The crude product was dissolved in 20 mM Tris HCl, pH 8 / ACN (60:40 v / v; 15 mL). The pH reading, measured using test strips, was acidic, between 2 and 3. The solution was adjusted to pH 8 by adding 2 mL of 1 M Tris HCl (pH 8). Anion exchange chromatography was performed as the initial purification step using a GE Source 30Q anion exchange column (2.6 x 10 cm) with buffer A: 20 mM Tris HCl, pH 8 / ACN (60 / 40 v / v) and buffer B: 20 ​​mM Tris HCl, pH 8 / ACN (60 / 40 v / v) containing 0.5 NaCl. The sample was loaded onto the column and washed with buffer A until a stable UV baseline was reached. The sample was eluted with a multi-step gradient: 0–8% B for 1 min, then 8–50% B for 59 min, and finally 90% B for 12 min. The flow rate was set to 10 mL / min, UV monitoring was performed at 225 nm and 280 nm, and the fraction collection time was set to 0.5 min. An analytical RP-HPLC system with a Waters X Select CSH C18, 4.6 x 50 mm column was used to identify fractions containing the desired product. The desired fractions were combined (~80 mL total volume) and diluted to 200 mL with milli-Q water.

[0084] Purification by RP-HPLC: The diluted fractions were loaded onto a Kromasil C18 column (2.1 x 25 cm) and further purified using a standard TFA / water / ACN gradient. Buffer A = 0.15% TFA / water, and buffer B = ACN. The sample was eluted with a multi-gradient gradient: 0–10% B for 1 min, then 10–45% B for 71 min, at a flow rate of 15 mL / min, a fraction collection time of 0.5 min, and a column temperature of 50 °C. UV monitoring was performed at 225 nm and 280 nm. An analytical RP-HPLC system with a Waters XSelect CSH C18, 4.6 x 50 mm column was used to identify fractions containing the desired product. The desired fractions were combined (~113 mL total volume; 99.8%, by RP-HPLC).

[0085] Salt exchange and conversion to HCl salt: The elution buffers used for HCl conversion were A1-buffer: 0.1M ammonium chloride aqueous solution and A2-buffer: 0.01% HCl with buffer B: ACN. The combined fractions were diluted with water to 200 mL and reloaded onto a Kromasil C18 column (2.1 x 25 cm) HPLC column. The column was washed with three column volumes of A1-buffer, followed by three column volumes of A2-buffer. The sample was eluted using a gradient of 0–10% (A2-B) for 1 min, followed by a gradient of 10–70% (A2-B) for 71 min, and UV monitoring was performed at 225 nm and 280 nm. Fractions containing the desired product were identified by analytical RP-HPLC (column: Waters X Select CSH C18, 4.6 x 50 mm), combined, frozen, and lyophilized to produce the compound of Example 1 as a white powder (153 mg, 0.023 mmol; 64% overall yield). Purity was confirmed by analytical RP-HPLC and found to be 98.9%. ESMS deconvolution spectra: observed MW: 6531.2 Daltons; theoretical MW: 6533.4 Daltons.

[0086] An alternative method for acylation of SCI: Compound A ((C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-Gly-OH) (584.6 mg; 0.424 mmol; 3.5 equivalents) and TSTU (125.6 mg; 0.417 mmol; 3.4 equivalents) were dissolved in 1.0 mL of dry DMSO. DIEA (168.3 μL; 0.966 mmol; 8 equivalents) was added, and the resulting mixture was incubated at room temperature for 30 minutes to produce compound AO-(N-succinimide ester) (compound A-OSu ester) in DMSO, which was then used directly.

[0087] To a solution of SCI-A14E,B16H,B25H,desB30-BHI (TFA salt; 1587 mg; 0.121 mmol) dissolved in 16 mL of dry DMSO, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 452 μL 3.02 mmol, 25 equivalents, Sigma-Aldrich catalog 33482) was added, followed by fractions of compound A-OSu ester (1.11 equivalents, 0.4 equivalents, 0.4 equivalents, 0.7 equivalents, 0.4 equivalents) added to DMSO at approximately 10 min intervals. The reaction progress was monitored by analytical RP-HPLC. After stirring the reaction mixture at ambient temperature for a total of 60 min, it was divided into two equal 50 mL fractions and filled into a mixture of diethyl ether / DCM / TFA (30:10:0.2 v / v; 40 mL volume). The resulting white precipitate was separated by centrifugation, washed, and ground twice with 30 mL of diethyl ether.

[0088] Deprotection (removal of Boc and OtBu groups): The white precipitates moistened with diethyl ether were each treated with a mixture of TFA / triisopropylsilane (TIS; Aldrich) / water (92.5:5.0:2.5 v / v; 10 mL) for 30 min. Diethyl ether (40 mL) was added to each mixture, and the resulting precipitates were collected by centrifugation and washed once with diethyl ether. The collected precipitates were thoroughly dried to remove residual diethyl ether and crushed into a fine powder. RP-HPLC analysis indicated the deprotection step was complete (4% unreacted SCI-A14E, B16H, B25H, desB30-BHI; 51% B29-acylated product; 38% N-terminal, B29 diacylated product).

[0089] Enzymatic digestion (removal of leader sequence and C-peptide): The dried, powdered crude product was completely dissolved in 0.1 M TrisHCl pH 8 (600 mL). To this solution, 6.0 mL of 0.1 M CaCl2 solution, 6.363 mL of carboxypeptidase-B stock solution (2.9 mg / mL in water), and 1.587 mL of trypsin stock solution (1.0 mg / mL in water) were added. The solution was incubated at room temperature for 120 min. Analytical RP-HPLC and LC-MS analysis confirmed the complete production of the mature compound (IFA-197) from Example 1. The solution was acidified with 45 mL of glacial acetic acid (pH 2-3 determined using pH paper).

[0090] Separation of digestion products by preparative RP-HPLC: The desired product contained in the acidified digestion solution is separated in a series of preparative RP-HPLC purification runs. Typically, the desired product is eluted from the RP-HPLC column using a linear water / ACN gradient (containing 0.05% TFA), the fractions containing the desired product are combined, frozen, and lyophilized. Anion exchange (AEX) chromatography can be omitted if the desired purity is achieved in this step.

[0091] Initial purification steps (optional) via preparative AEX chromatography: AEX was performed using a GE Source 30Q anion exchange column (2.6 x 11.5 cm) with A-buffer: 20 mM Tris HCl, pH 8 / ACN (60 / 40 v / v) and B-buffer: 20 mM Tris HCl, pH 8 / ACN (60 / 40 v / v) containing 0.5 NaCl to remove any residual C-peptide or leader sequence. Two batches of lyophilized powder were dissolved in 12 mM Tris, pH 8 / 40% ACN (with an additional 1 M Tris HCl, pH 8 buffer to reach pH 8). Each solution was then loaded onto an AEX column equilibrated with 100% A-buffer and washed with A-buffer until a steady-state UV baseline was reached. The sample was eluted as follows: a linear gradient of 0–50% B for 60 min and a hold of 100% B for 12 min. The flow rate was set to 10 mL / min, UV monitoring was performed at 225 nm and 280 nm, and the fraction collection time was set to 0.5 min.

[0092] Fractions containing the desired product were identified using an analytical RP-HPLC system equipped with a Waters XSelect CSH C18, 4.6 x 50 mm column. The desired fractions were combined (225 mL total volume) and stored at 4°C until the desalting / HCl conversion step.

[0093] Salt exchange; conversion to HCl salt: using A1-buffer: 0.1M ammonium chloride aqueous solution and A2-buffer: 0.01% HCl with buffer B: ACN, using a Phenomenex Luna C18(2) column (2.1 x 25cm, 5μm, Desalting and conversion to HCl salt form were simultaneously achieved by preparative RP-HPLC. The combined solution from the AEX purification step was aliquoted into three equal parts (75 mL each). Each part was diluted with 75 mL of milli-Q water and 1.5 mL of glacial acetic acid. Each solution (~151 mL) was loaded onto an RP-HPLC column equilibrated with A1-buffer and washed with 2 column volumes of A1-buffer and 2 column volumes of A2-buffer. The sample was eluted at 55 °C with a linear gradient of 5–10% (A2-B) for 1 min at 15 mL / min, followed by 10–40% (A2-B) for 71 min. Fractions containing the desired product were identified by RP-HPLC (column: Waters XSelect CSH C18, 4.6 x 50 mm), combined, frozen, and lyophilized to produce Example 1-HCl salt (354 mg, 0.054 mmol; 45% overall yield) as a white amorphous powder. Purity was confirmed to be 99% by analytical RP-HPLC. ESMS: Deconvolution spectrum: Observed MW: 6531.2 Daltons; Theoretical MW: 6533.4 Daltons.

[0094] Example 2

[0095]

[0096] Example 2 is a compound of formula I consisting of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:4, wherein the ε-amino group of the Lys side chain at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- is chemically modified by conjugation.

[0097] Example 2 was prepared essentially as described in the procedure of Example 1, except that ((C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-Lys(Boc)-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-OH)(compound B) was used in the acylation step. ESMS: Deconvolution spectrum: Observed MW: 6620.1 Daltons; Theoretical MW: 6621.5 Daltons.

[0098] Example 3

[0099]

[0100] Example 3 is a compound of formula I consisting of chain A of SEQ ID NO:1 and chain B of SEQ ID NO:5, wherein the ε-amino group of the Lys side chain at position 29 of chain B is reacted with HO2C-(CH2). 18 -CO-γGlu-γGlu-γGlu-εLys-Gly- conjugates are chemically modified.

[0101] Example 3 was prepared essentially as described in the procedure of Example 1, except that ((C20-OtBu)-γGlu(OtBu)-γGlu(OtBu)-γGlu(OtBu)-εLys(Boc)-(Gly)-OH)(compound C) was used in the acylation step. ESMS deconvolution spectrum: observed MW: 6532.0 Daltons; theoretical MW: 6533.4 Daltons.

[0102] In vitro receptor affinity

[0103] Examples 1, 2, and 3 and control compounds (biosynthetic human insulin (BHI) and insulin-like growth factor 1 (IGF-1)) were tested in a competitive radioligand binding assay for human insulin receptor (hIR) and human IGF-1 receptor (hIGF-1R) scintillation proximity assay (SPA). The membranes were prepared from stably transfected 293HEK cells overexpressing recombinant hIR-A, hIR-B, or hIGF-1R using a differential centrifugation step.

[0104] Receptor cDNA was subcloned into human embryonic kidney (HEK) 293 cells using the pcDNA3.1 expression plasmid, followed by selection with genimycin to prepare stably transfected cell lines. The resulting cell lines were human IR-A, human IR-B containing a C-terminal C9 tag (TETSQVAPA (SEQ ID NO:6)), human IGF-1R, and rat IR-A containing a C-terminal C9 tag (TETSQVAPA). Cells were grown in a 5% CO2 humidified environment. Typically, cell pellets from passages 6 to 12 were frozen to prepare membranes, depending on the receptor.

[0105] The frozen cell pellet was thawed in ice-cold homogenization / resuspending buffer (50 mM Tris-HCl, pH 7.5), the buffer containing one [cell sample] per 50 mL buffer. Protease inhibitor tablets with EDTA (Roche Diagnostics). Cells were homogenized using a Teflon-glass Potter-Elvehjem homogenizer driven by an overhead motor with 15 to 20 shocks, followed by centrifugation at 1100 x g for 10 min at 4 °C. The supernatant was kept on ice, and the pellet was homogenized as before and centrifuged at 1100 x g for 10 min at 4 °C. All supernatants were combined and then centrifuged at 35,000 x g for 60 min at 4 °C. The pellet was resuspended in a buffer containing protease inhibitor (4 to 5 mL / g starting cell paste) and rapidly frozen in liquid nitrogen before storage at -80 °C. Protein concentrations were determined using a quinolinic acid (BCA) kit (Thermo Scientific) with BSA as a standard.

[0106] Personnel restructuring (3-[ 125 I]-iodotyrosine-A14)-insulin (2200 Ci / mmol) or recombinant human [ 125 [I]-Insulin-like growth factor-1 (1853 Ci / mmol) (both obtained from Perkin Elmer) receptor binding affinity (Kb) was determined from competitive radioligand binding assays. i The assay was performed using SPA beads (PerkinElmer) coupled with polyvinyltoluene (PVT) wheat germ lectin via scintillation proximity assay (SPA). The assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) contained 0.1% (w / v) fatty acid-free BSA, 0.001% (w / v) NP-40 (4-nonylphenyl-polyethylene glycol), or 0.1% (w / v) fatty acid-free HSA and was used for all compound tests and reagent preparation. The Freedom / Evo robot was used. Prepare 10-point concentration response curves in the assay buffer using a 3-fold serial dilution of the test sample or control. Fifty μL of compound dilution was transferred using a TeMO robot. Add the contents to a 96-well white transparent microplate (Corning), followed by the addition of radioligand (50 μL), membrane (50 μL), and SPA beads (50 μL), all using a Multiflo F / X (Biotek) large-volume dispensing instrument. The final concentration of the radioligand was ~40 pM, and the amount of SPA beads added was 0.15 mg / well.

[0107] Based on the equation Ki = IC 50 / (1+L* / Kd) from IC 50The affinity constant (Ki) is calculated, where L* equals the concentration of the radioligand used in the experiment, and Kd equals the equilibrium binding affinity constant of the radioligand for each receptor determined from saturated binding analysis.

[0108] Geometric mean, Ki = 10 ( Log Ki值的算术平均值 )

[0109] The error is calculated using the Delta method, where SEM = geometric mean x ((standard deviation of Log10Ki value) / (square root of n)) x ln10.

[0110] Table 1A, B, C: Binding affinity of human insulin receptor subtypes A and B (hIR-A and hIR-B) and human insulin-like growth factor-1 receptor (hIGF-1R). Ki values ​​are geometric mean, and SEM values ​​are errors calculated using the Delta method.

[0111] Table 1A: 0.1% (w / v) BSA

[0112]

[0113] Table 1B: 0.001% (w / v) NP-40

[0114]

[0115] Table 1C: 0.1% (w / v) HSA

[0116]

[0117] The data in Tables 1A, 1B, and 1C indicate that using 0.1% BSA, 0.001% NP-40, or 0.1% HSA in the assay buffer, Examples 1, 2, and 3, when combined with human IR-A and human IR-B, showed very low binding to hIGF-R.

[0118] Receptor functional activation

[0119] The insulin receptor contains an intracellular tyrosine kinase domain that autophosphorylates its own tyrosine residues upon ligand binding to allow recruitment of adaptor proteins that function to induce the insulin signaling pathway. Functional cellular activity of receptor autophosphorylation in response to tyrosine residue stimulation was determined after ligand treatment of HEK293 cells expressing hIR-A, hIR-B, or hIGF-1R, each possessing a C-terminal C9 epitope (TETSQVAPA, SEQ ID NO: 6).

[0120] HEK293 cells overexpressing hIR-A or hIR-B-C9 were trypsinogenized and rapidly centrifuged at 1000 rpm (spin-down). Cells were resuspended in starvation medium containing DMEM high glucose (without sodium pyruvate) + 0.1% BSA and seeded at a density of 50-60K cells / well in 96-well poly-D-lysine-coated plates. Cells were incubated overnight under standard tissue culture conditions. To assess the effect of albumin binding, cells were stimulated in medium containing DMEM high glucose (without sodium pyruvate) + 0.1% BSA. hIR-A or hIR-B cells were stimulated for 60 minutes at 37°C with ligands at various concentrations ranging from 10 to 0.0000169 μM. Cells were then lysed with lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, and 1% NP40, and freshly added to a complete protease inhibitor mixture (Pierce A32955) + 2 mM vanadate (Sigma S6508) on the day of assay. The level of tyrosine autophosphorylation achieved by the kinase domain of each receptor was determined by applying cell lysates to ELISA plates; activated receptors were captured by antibodies to IR (Ab 83-14) (at 3.5 μg / ml for IR-A) or C9 epitope tags (at 2 μg / ml for IR-B), and the level of tyrosine phosphorylation was subsequently detected at a ratio of 1:5000 using an anti-phosphorylated tyrosine horseradish peroxidase conjugate HRP antibody (Millipore 16-105).

[0121] For IR ELISA, coat 96-well plates with 3.5 μg / mL Ab 83-14 (for IR-A) or 2 μg / mL anti-C9Ab (for IR-B). Dilute the antibody in 20 mM sodium carbonate at pH 9.6 and incubate overnight at 4°C. The next day, wash the plates three times with TBST (1X TBS containing 0.1% Tween 20). Block the plates with 1% BSA in TBST for 1 hour. Remove the blocking buffer and apply cell lysates to the plates. Incubate the plates on a shaker at room temperature for 1 hour. Wash the plates three times with 1X TBST. Apply secondary antibody (anti-phosphorylated tyrosine-HRP (Millipore 16-105), 2 μL in 10 mL (1 / 5000), in TBST containing a mixture of 1X protease and phosphatase inhibitor). Further incubate the plates on a shaker at room temperature for 1 hour. To generate a pIR signal, the plate was washed four times with TBST. TMB substrate (Pierce 34021) was added to the plate at a volume of 100 μL / well and incubated at room temperature for 5 minutes. The reaction was stopped by adding 100 μL / well of 2N H₂SO₄ and the plate was incubated for another 5 minutes. The plate was read at OD₄50 nm.

[0122] Insulin receptor activation, 0.1% casein (BSA-free method)

[0123] To measure the effect of the examples on IRA and IRB phosphorylation in the presence of 0.1% casein, hIRA-HEK293-zeo or hIRB-C9-HEK293-G418 cells were first seeded at 60,000 cells / well in DMEM (Gibco 11965-084) (containing 0.1% BSA) high in glucose and without sodium pyruvate in 96-well poly-D-lysine-coated plates (Biocoat 354461). The cells were then incubated overnight at 37°C in a tissue incubator. 96-well ELISA plates were also coated with 3.5 μg / mL anti-insulin receptor antibody 83-14 (for IRA assay) or 2.0 μg / mL anti-C9 antibody (for IRB assay) in 20 mM sodium carbonate buffer (pH 9.6) and incubated overnight at 4°C.

[0124] The following day, the ELISA plate was washed with 200 μL of wash buffer (containing 0.1%... Wash three times with TBS-T and block with 1% BSA in TBS-T for at least one hour. Prepare the assay medium with 0.1% casein in DMEM containing high glucose and free of sodium pyruvate. Dilute the following compounds in the assay medium to various concentrations: human insulin to 200 nM, examples to 60 μM, hIGF-1 to 20 μM, and AspB10 to 200 nM. Serially dilute the compounds at a 1:3 ratio. Wash the cell plate twice with 50 μL of assay medium, then transfer 50 μL of assay medium and 50 μL of diluted compound to each well of the cell plate. Transfer 200 nM BHI. Incubate the plate at 37°C for 1 hour.

[0125] Add the protease inhibitor (Pierce A32965) and sodium orthovanadate (final concentration 2 mM) to lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, and 1% NP40). After 1 hour of incubation, wash the cell plate with 100 μL of cold DPBS and lyse it in 100 μL of lysis buffer at 4°C for 15 min. Mix the cell lysate plates and transfer 300 μL of BHI-treated cell lysate from the BHI-treated plate to the first column of the dilution plate. Add 200 μL of lysis buffer to the other eleven columns of this dilution plate and serially dilute the lysate eleven times at a 1:3 ratio as follows: transfer 100 μL of lysate to 200 μL of lysis buffer and repeat eleven times. Transfer 10 μL of lysate from the compound-treated cell plate to the dilution plate and add 190 μL of lysis buffer to each well. Wash the ELISA plate as described above, and then transfer 100 μL of diluted lysate to the appropriate ELISA plate. Incubate the plate on a shaker at room temperature for one hour.

[0126] Wash the plates as described above, and add 100 μL of secondary antibody diluted 1:5000 in TBST (containing protease inhibitor and 40 μM sodium orthovanadate) to each plate. Incubate the plates on a shaker at room temperature for one hour. Wash the plates as described above, and add 50 μL of TMB to the plates for color development for approximately 2 minutes. Stop the TMB reaction with 50 μL of 2N sulfuric acid and read the plate at 450 nm.

[0127] Functional efficacy is reported as the concentration that elicits the half-maximal response (EC50) relative to the maximum effective concentration (100 nM) of positive control human insulin (hIR-A and hIR-B phosphorylation assays) or 10 nM of positive control hIGF-1 (hIGF-1R phosphorylation assays). 50 EC was determined from 4-parameter logistic nonlinear regression analysis (NGR Screener 13). 50 Value. If necessary, set the curve top or bottom parameter to 100 or 0 respectively.

[0128] The report will be submitted to the EC 50 The values ​​are displayed as geometric mean, and the standard error of the mean (SEM) is calculated using the Delta method, where the number of independent determinations is indicated by “n” (Table 2).

[0129] Table 2: Geometric Mean of Activation of Human Insulin Receptor Subtypes A and B (hIR-A and hIR-B) (SEM, n)

[0130]

[0131] The data in Table 2 show that Examples 1, 2 and 3 bind to and stimulate human insulin receptors A and B.

[0132] Evaluation of in vivo efficacy in a rat model of type 1 diabetes

[0133] The glucose-lowering effects and pharmacokinetics of Examples 1, 2, and 3 were investigated in a streptozotocin (STZ)-treated rat model of diabetes. Male Sprague-Dawley rats weighing 400–425 g were obtained from Envigo, Indianapolis, India. After approximately one week of acclimatization, the rats were anesthetized with isoflurane and given a single injection of Zanosar (89256, Teva Parenteral Medicines, 40 mg / kg, IV). The rats were used in the studies 3 days after the Zanosar injection; only animals with a non-fasting blood glucose level of 400–550 mg / dL were used in these studies.

[0134] Rats were grouped to provide comparable differences in blood glucose and body weight; the rats were randomized. Blood glucose was measured using an Accu-ChekAviva blood glucose meter (Roche).

[0135] The test substance (peptide solution; 1 mL / kg; subcutaneous; single dose on day 1 of the dosing phase) or the medium (10 mM Tris, 19 mg / mL glycerol, 3.15 mg / mL m-cresol, pH 7.6; 1 mL / kg; subcutaneous; single dose on day 1 of the dosing phase) was administered based on the animal's body weight at 8:00 AM (0800) on day 1 of the dosing phase. Blood samples for glucose measurement were collected by tail bleeding. Animals had free access to food and water throughout the study. Plasma samples from these studies were used for compound level analysis.

[0136] Whole blood was collected at 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108, and 120 hours after administration and glucose was measured using a glucometer. Values ​​were recorded in duplicate unless the difference exceeded 30 mg / dL. If this occurred, values ​​were recorded in triplicate. If a rat's glucose level dropped below 35 mg / dL, the entire group was orally administered 2 mL of 50% dextran, except for rats in the same group with levels >100 mg / dL. The rescued rats were observed, and blood glucose was measured every 2 to 4 hours after the dextran loading. During the observation period, if a rat's glucose level dropped below 35 mg / dL, the entire group was orally administered 2 mL of 50% dextran, except for rats in the same group with levels >100 mg / dL. Observation continued, and blood glucose was measured every 2 to 4 hours after the dextran loading. If rats are orally administered 2 mL of 50% dextran, all subsequently recorded glucose values ​​are excluded from the study calculations. Even if the animals are rescued, studies continue on them to collect PK time points because PK is not affected by large doses (bolus) of dextran.

[0137] Immunoaffinity-LC / MS Rat Plasma Assay: To characterize the pharmacokinetics of the examples, plasma samples (10 μL aliquots) were analyzed by immunoprecipitation followed by LC / MS to obtain complete insulin concentrations. Standards and blank samples were prepared in 100% control rat plasma using the examples, and a stable isotope-labeled antibody (unrelated to the examples) was added to all standards and samples as internal standards. For immunoprecipitation, a biotin-labeled anti-human mouse antibody (Fitzgerald, 10R-I134E) was used as the capture reagent, which was subsequently bound to Dynal M-280 streptavidin-coated magnetic beads (Invitrogen 60210). The magnetically immobilized samples were washed with 0.1% CHAPS, followed by PBS, and the examples were eluted with a solution containing 20% ​​ACN, 2% formic acid aqueous solution, and 20% Invitrosol (Invitrogen, 46-5553). Plasma samples were quantified using a Thermo Q / Exactive Plus mass spectrometer in the range of 0.01 to 5.3 μg / mL (1.6 to 802 nM).

[0138] Non-compartmental pharmacokinetic analyses were performed using Phoenix WinNonLin v8.1. For rat PK analyses, the concentrations used in estimating elimination half-lives were taken as averages of the following: 44 to 120 hours post-administration for the 50 nmol / kg dose group, 32 to 120 hours post-administration for the 100 nmol / kg dose group, 18 to 120 hours post-administration for the 200 nmol / kg dose group, and 18 to 120 hours post-administration for the 400 nmol / kg dose group.

[0139] Tables 3A and 3B: Glucose reduction (3A) and pharmacokinetic effects (3B) in a rat model of diabetes treated with streptozotocin (STZ) in Example 1.

[0140] Table 3A: Following a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg of Example 1, glucose levels decreased in STZ-treated male Sprague-Dawley rats of Example 1.

[0141]

[0142] * No values ​​higher than 601 were measured, as that is a high reading on the blood glucose meter.

[0143] Table 3B: Mean pharmacokinetic parameters of Example 1 in STZ-treated male Sprague-Dawley rats after a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg.

[0144]

[0145] Table 4A: Following a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg, glucose levels in male Sprague-Dawley rats treated with STZ decreased according to Example 2.

[0146]

[0147] * No values ​​higher than 601 were measured, as that is a high reading on the blood glucose meter.

[0148] Table 4B: Mean pharmacokinetic parameters of Example 2 in STZ-treated male Sprague-Dawley rats after a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg.

[0149]

[0150] Table 5A: Following a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg, glucose levels decreased in STZ-treated male Sprague-Dawley rats of Example 3.

[0151]

[0152] * No values ​​higher than 601 were measured, as that is a high reading on the blood glucose meter.

[0153] Table 5B: Mean pharmacokinetic parameters of Example 3 in STZ-treated male Sprague-Dawley rats after a single subcutaneous administration of 50, 100, 200, or 400 nmol / kg.

[0154]

[0155] Abbreviations for Tables 3B, 4B, and 5B: AUC 0-inf =Area under the curve from 0 to infinity, CL / F = clearance / bioavailability, Tmax = time to maximum concentration, Cmax / D = maximum plasma concentration per dose, T 1 / 2 = Half-life [Data is average ± SD (N = 5).

[0156] The data in Tables 3A, 4A, and 5A demonstrate the robust, dose-dependent glucose-lowering effects in vivo for all three embodiments. The data in Tables 3B, 4B, and 5B demonstrate the duration of action in vivo for all three embodiments.

[0157] Evaluation of drug clearance in a pig model of type 1 diabetes

[0158] The aim of this study was to investigate the glucose reduction and pharmacokinetics of the example in diabetic Yucatan miniature pigs following a single subcutaneous dose of 3.6 nmol / kg. Blood samples were collected within 168 hours after administration.

[0159] Castrated, male Yucatan miniature pigs (mean age 23 months, mean weight 44 kg) with alloxan-induced diabetes were housed individually, with free access to fresh water at all times, and fed two pen meals daily. Animals received appropriate maintenance basal and dietary insulin twice daily to manage their diabetes condition when not in the study. Animals were randomly assigned to the treatment group and returned to their pens. The sample was formulated at 400 U / mL in a hexameric formulation (19 mg / mL glycerol, 4 Zn / hex, 3.15 mg / mL m-cresol, 10 mM Tris, pH 7.6).

[0160] The day before the study, pigs were fed half their daily dose and received 0.2 U / kg Humalog Mix 75 / 25 as a morning maintenance administration. Approximately 12 hours before the scheduled test dose, pigs were fed half their daily dose and received only 0.2 U / kg Humalog. All animals remained fasted until 24 hours after sample collection.

[0161] On the morning of the study, all animals were restrained in slings, and their vascular access points (equipped for blood sampling) were connected and patency checked. Animals were randomly assigned to the treatment group (n=6) and returned to their respective enclosures. Two pre-administration samples were collected, and the animals were subcutaneously injected (0 min) into the flank using a U100 insulin syringe at a dose of 0.6 U / kg / 3.6 nmol / kg (based on an insulin concentration of 400 U / ml, with an average volume of 7 units / pig). During the remaining blood collection phase, all study animals were provided with clean fresh water at will.

[0162] A series of blood samples (4.0 mL each) were collected from each animal at the following time points and placed in tubes containing K3EDTA anticoagulant: 0.5, 0, 1.5, 3, 6, 12, 18, 24, 36, 42, 48, 54, 60, 72, 96, 120, 144 and 168 hours after subcutaneous (sc) administration.

[0163] Animals were fed a 300g S-9 diet and administered 0.2U / kg Humalog subcutaneously 24 and 60 hours after sampling. After 72 hours of sampling, animals were resumed their normal maintenance insulin and feeding regimens. All samples were collected before breakfast and maintenance insulin administration.

[0164] Whole blood samples should be kept on wet ice immediately after collection. The plasma should then be separated by centrifugation (at least 15 min at ~4°C to 3000 RPM) and divided into two aliquots, which should be stored frozen at approximately -20°C (for glucose analysis) or -70°C (for PK analysis).

[0165] Blood glucose concentrations were measured using an automated Beckman AU480 Clinical Chemistry Analyzer (Beckman Coulter, Brea, CA) and plotted using a Graphpad Prism 8.

[0166] Immunoaffinity-LC / MS assay of porcine plasma: To characterize the pharmacokinetics (PK) of the test examples, plasma samples (100 μL aliquots) were analyzed by immunoprecipitation followed by LC / MS to obtain their complete example concentrations. Standards and blank samples were prepared in 100% control porcine plasma using the test examples, and a stable isotope-labeled antibody (unrelated to the examples) was added to all standards and samples as an internal standard. For immunoprecipitation, a biotin-labeled anti-human mouse antibody (Fitzgerald, catalog number 10R-I134E) was used as the capture reagent, which was subsequently bound to Dynal M-280 streptavidin-coated magnetic beads (Invitrogen 60210). The magnetically immobilized samples were washed with 0.1% CHAPS, followed by PBS, and the test examples and their internal standards were eluted with a solution containing 20% ​​ACN, 2% formic acid aqueous solution, and 20% Invitrosol (Invitrogen, 46-5553). Plasma samples were quantified using a Thermo Q / Exactive Plus mass spectrometer in the range of 0.1 to 52.3 ng / mL (0.016 to 8.0 nM).

[0167] Pharmacokinetic analysis: Non-compartmental pharmacokinetic analysis was performed using Phoenix WinNonLin v8.1. For PK analysis, the concentrations used in estimating the elimination half-life were taken as the average values ​​from 50 to 160 hours after administration (corresponding to 3.6 nmol / kg subcutaneously).

[0168] Table 6A: Glucose Decrease Data

[0169]

[0170] Table 6B: Mean PK parameters in male diabetes-induced Yucatan pigs after a single subcutaneous dose in Examples 1, 2, and 3

[0171]

[0172] Abbreviation: T 1 / 2 = Half-life, T max = Time to reach maximum concentration, C max =Maximum plasma concentration, AUC 0-inf =Area under the curve from 0 to infinity, CL / F = Scavenging rate / Bioavailability (N = 6, for experimental examples).

[0173] The data in Tables 6A and 6B demonstrate that all three examples exhibited robust and prolonged glucose-lowering effects in a pig model of type 1 diabetes.

[0174] sequence

[0175] SEQ ID NO: 1 Modified insulin A-chain (A14E); A-chain of Formula I

[0176]

[0177] SEQ ID NO: 2 Modified insulin B-chain (B16H, B25H, desB30); B-chain of Formula I

[0178] The Lys at position 29 is linked to HO2C-(CH2) via the ε-amino group of the Lys side chain. 18 The chemical modification is achieved by conjugation of -CO-γGlu-γGlu-γGlu-X-, where X is -Lys-Gly-, -Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- or -εLys-Gly-.

[0179] SEQ ID NO: 3 B-chain of Example 1

[0180]

[0181] The Lys at position 29 is linked to HO2C-(CH2) via the ε-amino group of the Lys side chain. 18 It is chemically modified by the conjugation of -CO-γGlu-γGlu-γGlu-Lys-Gly-.

[0182] SEQ ID NO: 4 B-chain of Example 2

[0183]

[0184] The Lys at position 29 is linked to HO2C-(CH2) via the ε-amino group of the Lys side chain. 18 It is chemically modified by the conjugation of -CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)-.

[0185] SEQ ID NO: 5 B-chain of Example 3

[0186]

[0187] The Lys at position 29 is linked to HO2C-(CH2) via the ε-amino group of the Lys side chain. 18 It is chemically modified by the conjugation of -CO-γGlu-γGlu-γGlu-εLys-Gly-.

[0188] SEQ ID NO: 6C-terminal C9 epitope

[0189]

[0190] SEQ ID NO: 7 Single-chain insulin

[0191]

[0192] SEQ ID NO: 8 Modified insulin B-chain (B16H, B25H, desB30)

[0193] sequence list <110> Eli Lilly and Company <120> Acylated insulin compounds with delayed effects <130> X22664 <150> US 63 / 025,463 <151> 2020-05-15 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> disulfides <221> <222> (6)..(11) <400> 1 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> 2 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> <221> MOD_RES <222> (29)..(29) <223> The ε-amino group of Lys is chemically converted through conjugation with HO2C-(CH2)18-CO-γGlu-γGlu-γGlu-X-. Modifications, wherein X is -Lys-Gly-, -Lys-(2-[2-(2-aminoethoxy)ethoxy]acetic acid)- or -εLys-Gly- <400> 2 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys 20 25 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> <221> MOD_RES <222> (29)..(29) <223> The ε-amino group of Lys is formed through the conjugation of HO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-Gly-. Chemically modified <400> 3 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys 20 25 <210> 4 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> <221> MOD_RES <222> (29)..(29) <223> The ε-amino group of Lys reacts with HO2C-(CH2)18-CO-γGlu-γGlu-γGlu-Lys-(2-[2-(2-aminoethyl)-) Chemically modified by the conjugation of (oxy)ethoxy]acetic acid. <400> 4 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys 20 25 <210> 5 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> <221> MOD_RES <222> (29)..(29) <223> The ε-amino group of Lys is conjugated with HO2C-(CH2)18-CO-γGlu-γGlu-γGlu-εLys-Gly- Chemically modified <400> 5 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys 20 25 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <400> 6 Thr Glu Thr Ser Gln Val Ala Pro Ala 1 5 <210> 7 <211> 118 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <220> <221> disulfides <222> (41)..(104) <220> <221> disulfides <222> (53) (117) <220> <221> disulfides <222> (103)...(108) <400> 7 Met His His His His His Gln Ala Ile Phe Val Leu Gln Gly Ser 1 5 10 15 Leu Asp Gln Asp Pro Glu Phe Glu Asn Leu Tyr Phe Gln Ile Glu Gly 20 25 30 Gly Arg Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala 35 40 45 Leu His Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys Arg 50 55 60 Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly 65 70 75 80 Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln 85 90 95 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln 100 105 110 Leu Glu Asn Tyr Cys Asn 115 <210> 8 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Synthesized constructs <400> 8 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu His 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe His Tyr Thr Pro Lys 20 25

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: ###0001### wherein X is selected from the group consisting of -Lys-Gly-, -Lys-(2-[2-(2- aminoethyloxy)ethyloxy]acetic acid)- and -εLys-Gly-.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -Lys-Gly-.

3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -Lys-(2-[2-(2-aminoethyloxy)ethyloxy]acetic acid)-.

4. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X is -εLys-Gly-.

5. The compound of claim 1 or a pharmaceutically acceptable salt thereof, which is ###0002### 6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, which is ###0003### 7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, which is ###0004### 8. A pharmaceutical composition comprising a compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients.

9. A compound selected from the group consisting of: ###0005### or a pharmaceutically acceptable salt thereof.

10. A method of preparing a compound of formula I or a pharmaceutically acceptable salt thereof, ###0006### wherein X is selected from: -Lys-Gly-, -Lys-(2-[2-(2-aminoethyloxy)ethyloxy]acetic acid)- and -εLys-Gly-; using a compound selected from the group according to claim 9, wherein said compound of formula I is produced by selective acylation of the epsilon-amino group of lysine at position 29 of the B chain of mature biosynthetic A14E, B16H, B25H, desB30-human insulin or direct acylation of a similar epsilon-amino group of lysine at position 29 of the B chain of single chain insulin preproinsulin A14E, B16H, B25H, desB30-BHI construct with a compound selected from the group according to claim 9 as a linker-fatty acid intermediate.

11. Use of a compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of diabetes.

12. Use of a compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of hyperglycemia.

13. Use of a pharmaceutical composition of claim 8 for the preparation of a medicament for the treatment of type I and / or type II diabetes.

14. Use of a pharmaceutical composition of claim 8 for the preparation of a medicament for the treatment of hyperglycemia. ​ ​ ​

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