Insulin conjugates

Abstract

The present invention relates to a compound represented by formula (I) [Formula 1] The present invention relates to an insulin conjugate comprising a human serum albumin binder of formula (I) and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at a point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of the human insulin analogue via an amide bond, and related aspects such as pharmaceutical compositions, uses in medicine, methods for preparing such insulin conjugates.

Description

[Technical field]

[0001] The present invention relates to insulin conjugates comprising a human serum albumin binder of formula (I) and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at the point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached via an amide bond to the epsilon amino group of lysine B29 of the human insulin analogue, and related aspects such as pharmaceutical compositions, uses in medicine, methods for preparing such insulin conjugates. [Background technology]

[0002] Over 400 million people worldwide suffer from type 1 or type 2 diabetes. Type 1 diabetes is treated by insulin replacement. In contrast to type 1 diabetes, there is essentially no insulin deficiency in type 2 diabetes, but in many cases, especially in advanced stages, type 2 diabetes patients are treated with insulin.

[0003] In healthy people, the release of insulin by the pancreas is strictly linked to the concentration of blood glucose. An increase in blood glucose level occurs after a meal and is quickly compensated by a corresponding increase in insulin secretion. In the fasting state, plasma insulin levels fall to a basal value that is sufficient to ensure a continuous supply of insulin-sensitive organs and tissues with glucose and to keep hepatic glucose production low during the night. In many cases, the replacement of endogenous insulin secretion by exogenous, most often subcutaneous, administration of insulin does not achieve the above-mentioned quality of physiological regulation of blood glucose. An upward or downward deviation of blood glucose levels may occur, which in its most severe form can be life-threatening. It may result from this that the improvement of diabetes treatment is mainly aimed at keeping blood glucose as close as possible within the physiological range.

[0004] Human insulin is a polypeptide of 51 amino acids, which is divided into two amino acid chains, the A chain with 21 amino acids and the B chain with 30 amino acids. The chains are linked to each other by two disulfide bridges. The third disulfide bridge is between the cysteines at positions 6 and 11 of the A chain. Some products currently used for the treatment of diabetes are insulin conjugates, i.e. insulin variants whose sequence differs from that of human insulin by one or more amino acid substitution units in the A and / or B chains.

[0005] Like many other peptide hormones, human insulin has a short half-life in vivo, which requires frequent administration, which is associated with patient discomfort. Therefore, insulin conjugates with a prolonged half-life in vivo and therefore a long duration of action are desirable.

[0006] Currently, there are various approaches to extend the half-life of insulin.

[0007] One approach is based on the development of formulations that are soluble at low pH but have reduced solubility at physiological pH compared to native insulin. The isoelectric point of the insulin conjugate is increased by the addition of two arginines to the C-terminus of the B chain. The addition of two arginines in combination with a glycine substitution at A21 (insulin glargine) provides insulin with an extended duration of action. The insulin conjugate precipitates and slowly solubilizes in the presence of zinc immediately after injection into a subcutaneous site, resulting in a sustained presence of insulin glargine.

[0008] WO 2016006963A1 (Jung, et al., HANMI PHARM. CO., LTD.) discloses insulin conjugates that have reduced insulin receptor-mediated clearance rates compared to human insulin.

[0009] WO 2018056764 A1 (Choi, et al., HANMI PHARM. & Sanofi) discloses insulin conjugates that have reduced insulin receptor-mediated clearance rates compared to human insulin.

[0010] WO 2008034881 A1 (Nielsen, et al., NOVO NORDISK A / S) discloses protease stabilized insulin conjugates.

[0011] In another approach, a long-chain fatty acid group is conjugated to the epsilon amino group of LysB29 of insulin. The presence of this group allows insulin to attach to serum albumin by non-covalent reversible binding. As a result, this insulin conjugate has a significantly prolonged time-action profile compared to human insulin (see, for example, Mayer et al., Inc. Biopolymers [Pept Sci] 88:687-713, 2007; or WO2009115469A1 [Madsen, et al., NOVO NORDISK A / S]).

[0012] None of the prior art teaches or provides a solution in this regard that meets the needs of all patients. Summary of the Invention [Means for solving the problem]

[0013] The first aspect of the present invention is a compound represented by formula (I) [ka] and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at the point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of the human insulin analogue via an amide bond.

[0014] The first aspect also relates to certain human insulin analogues.

[0015] A second aspect of the invention relates to a pharmaceutical composition comprising a pharma- ceutically effective amount of an insulin conjugate according to the first aspect.

[0016] A third aspect of the invention relates to an insulin conjugate according to the first aspect for use as a medicament. [Brief description of the drawings]

[0017] [Figure 1] The structure of conjugate 1 is disclosed. The sequences of A-chain (SEQ ID NO: 13) and B-chain (SEQ ID NO: 9) are shown in three letter code. [Diagram 2] The structure of conjugate 2 is disclosed. The sequences of A-chain (SEQ ID NO: 11) and B-chain (SEQ ID NO: 9) are shown in three letter code. [Diagram 3] The structure of conjugate 3 is disclosed. The sequences of A-chain (SEQ ID NO: 16) and B-chain (SEQ ID NO: 9) are shown in three letter code. [Figure 4] The structure of conjugate 4 is disclosed. The sequences of A-chain (SEQ ID NO: 16) and B-chain (SEQ ID NO: 8) are shown in three letter code. [Diagram 5] The structure of conjugate 5 is disclosed. The sequences of A-chain (SEQ ID NO: 17) and B-chain (SEQ ID NO: 3) are shown in three letter code. [Figure 6] The structure of conjugate 6 is disclosed. The sequences of A-chain (SEQ ID NO: 18) and B-chain (SEQ ID NO: 3) are shown in three letter code. [Figure 7]The structure of conjugate 7 is disclosed. The sequences of A-chain (SEQ ID NO: 16) and B-chain (SEQ ID NO: 3) are shown in three letter code. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Theory and Advantages of the Invention Half-life plays a major role in extending the duration of action of a drug. 1 / 2 ) is proportional to the volume of distribution divided by the clearance. For human insulin, clearance is driven primarily by binding to the insulin receptor, internalization and subsequent degradation. However, in clinical steady-state situations, not only elimination of the drug plays a role; the rate of absorption is also important because it affects the absence / occurrence of undesirable side effects such as hypoglycemia by influencing the peak-to-trough ratio during steady state.

[0019] In pharmacokinetics, steady state refers to a situation in which the entire intake of a drug is in fairly dynamic equilibrium with the elimination of the drug. In practice, it is generally believed that the steady state is reached 4-5 times the half-life of the drug after normal administration is started. The peak-to-trough ratio is important, especially for drugs such as insulin, which have a small therapeutic index. This index is defined as a measure of the relative desirability of a drug to achieve a particular medical purpose, which is usually expressed as the ratio of the maximum dose that does not produce toxic symptoms to the minimum dose that routinely produces a cure. The trough level is the lowest concentration of the drug in the patient's body, and the peak level is the highest concentration of the drug in the patient's body during the steady state. Thus, the peak-to-trough ratio should be as close to 1 as possible to exert optimal therapeutic effect with minimal side effects.

[0020] Therefore, to achieve effective glucose lowering combined with a minimized risk of hypoglycemic events, insulin conjugates that not only have a long half-life in vivo but also are slowly absorbed are needed.

[0021] Surprisingly, in the context of the studies underlying the present invention, it can be shown that substitutions, preferably at position A14 of human insulin with aspartic acid or glutamic acid, also preferably at position B16 with glutamic acid, also preferably at position B25 with histidine, preferably also in combination with at least four additional substitutions with basic amino acids such as arginine, result in a slow onset of action with a very long duration of action, which allows a once-weekly administration of the insulin conjugate with minimal risk of hypoglycemia.

[0022] Oral delivery of insulin is hindered due to instability in the gastrointestinal tract and poor mucosal penetration. Mucosal penetration is addressed in the present invention by the introduction of arginine. See Uhl et al. Coating of PLA-nanoparticles with cyclic, arginine-rich cell penetrating peptides enables oral delivery of liraglutide. Nanomedicine: Nanotechnology, Biology, and Medicine 24 (2020) 102132. Surprisingly, preferred insulin conjugates of the present invention with arginine substitutions at positions A5, A15, A18, B27 show advantageous stability in the presence of trypsin.

[0023] Detailed Description of the First Aspect The first aspect of the present invention is a compound represented by formula (I) [ka] and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at the point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of the human insulin analogue via an amide bond.

[0024] The expression "insulin analogue" as used herein refers to a peptide having a molecular structure that can be derived formally from the structure of naturally occurring insulin (also referred to herein as "parent insulin", e.g., human insulin), preferably by deleting and / or substituting at least seven amino acid residues present in naturally occurring insulin. The insulin analogue has a human serum albumin binder of formula (I) attached to the epsilon amino group of lysine B29. The added and / or replaced amino acid residues can be either codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. The insulin conjugates referred to herein can, for example, reduce blood glucose levels in vivo in a human subject.

[0025] In at least one embodiment, the human insulin analogue provided herein comprises two peptide chains, an A chain and a B chain. Typically, the two chains are linked by disulfide bridges between cysteine ​​residues. For example, in at least one embodiment, the human insulin analogue comprises three disulfide bridges: one between the cysteines at positions A6 and A11, one between the cysteine ​​at position A7 of the A chain and the cysteine ​​at position B7 of the B chain, and one between the cysteine ​​at position A20 of the A chain and the cysteine ​​at position B19 of the B chain. In at least one embodiment, the human insulin analogue comprises cysteine ​​residues at positions A6, A7, A11, A20, B7 and B19. The human serum albumin binder of formula (I) is attached to the epsilon amino group of lysine B29 of the human insulin analogue.

[0026] Mutations of human insulin, i.e. mutations of the parent insulin, are indicated herein by reference to the chain of the conjugate, i.e. either the A or B chain, the position of the mutated amino acid residue in the A or B chain (e.g. A14, B16 and B25) and the three letter code of the amino acid which replaces the natural amino acid in the parent insulin. For example, Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-human insulin is an analogue of human insulin in which the amino acid residue at position 5 of the A chain of human insulin (A5) is replaced by arginine, the amino acid residue at position 14 of the A chain of human insulin (A14) is replaced by glutamic acid and the amino acid residue at position 15 of the A chain of human insulin (A15) is replaced by arginine. , the amino acid residue at position 18 (A18) of the A chain of human insulin is substituted with arginine, the amino acid residue at position 21 (A21) of the A chain is substituted with glycine, the amino acid residue at position 16 (B16) of the B chain is substituted with glutamic acid, the amino acid residue at position 25 (B25) of the B chain of human insulin is substituted with histidine, the amino acid residue at position 27 (B27) of the B chain of human insulin is substituted with arginine, and an arginine is added to the C-terminus of the B chain at position 31 (B31). The term "desB30" refers to a conjugate that lacks the B30 amino acid of the parent insulin (i.e., the amino acid residue at position B30 is not present).

[0027] In some embodiments, the human insulin analogue comprises at least one mutation compared to the parent insulin, and the insulin conjugate comprises a mutation at position B16 substituted with a hydrophobic amino acid and / or a mutation at position B25 substituted with a hydrophobic amino acid. The human insulin analogue may optionally comprise further mutations. For example, the amino acid residue at position 14 (A14) of the A chain of the parent insulin (such as human insulin) may be substituted with glutamic acid and the amino acid at position 30 of the B chain may be deleted, i.e. absent (desB30 mutation).

[0028] In another embodiment herein, the insulin conjugate is long acting and for once weekly administration, eg, oral administration.

[0029] In at least one embodiment, the insulin conjugate is in the form of a pharma- ceutically acceptable salt. Pharmaceutically acceptable salts of insulin conjugates can include acid addition salts and base salts. Suitable acid addition salts are formed from acids that form non-toxic salts. In at least one embodiment, pharma- ceutically acceptable salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, iodide ... The base salts are selected from the group consisting of phosphate, lactate, maleate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1,5-naphthalenedisulfonate and xinafoate. Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum salt, arginine salt, benzathine salt, calcium salt, choline salt, diethylamine salt, bis(2-hydroxyethyl)amine (diolamine) salt, glycine salt, lysine salt, magnesium salt, meglumine salt, 2-aminoethanol (olamine) salt, potassium salt, sodium salt, 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris or tromethamine) salt and zinc salt. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0030] Optionally, insulin conjugates and their pharmaceutically acceptable salts may exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term "hydrate" is used.

[0031] Examples of isotopes suitable for inclusion in the conjugates herein include: 2 H and 3 Hydrogen such as H 11 C. 13 C and 14 Carbon such as C, 36 Chlorine such as Cl, 18 Fluorine such as F, 123 I and 125 Iodine, such as I 13 N and 15 Nitrogen such as N 15 O. 17 O and 18 Oxygen such as O 35 Examples include isotopes of sulfur such as S.

[0032] Certain isotopically labeled insulin conjugates, for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and ready means of detection.

[0033] Deuterium, i.e. 2 Substitution with heavier isotopes such as H may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0034] 11 C. 18 F, 15 O and 13Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0035] Isotopically labeled conjugates may generally be prepared by conventional techniques known to those of skill in the art.

[0036] Pharmaceutically acceptable solvates according to the invention include those in which the solvent of crystallization may be isotopically substituted, e.g. D 2 O, d 6 -Acetone, d 6 -DMSO.

[0037] The human insulin analogues provided herein preferably contain at least seven mutations (amino acid substitutions, deletions or additions) compared to the parent insulin. As used herein, the term "at least seven" means seven or more than seven, such as "at least eight", "at least nine", etc. In another embodiment, the human insulin analogues provided herein contain at least two mutations in the B chain and at least four mutations in the A chain. For example, the human insulin analogues may contain substitutions at positions B3, B16, B25 and B27, a deletion at position B30, and substitutions at positions A5, A9, A14, A18 and A21. Alternatively, the human insulin analogues may contain substitutions at positions B16, B25 and B27, and substitutions at positions A5, A14, A15, A21. Additionally, human insulin analogues may include substitutions at positions B16, B25, B27, an additional amino acid at the C-terminus of the B chain (B31) and substitutions at positions A5, A14, A15, A18 and A21.

[0038] The human insulin analogues provided herein may contain mutations in addition to those mentioned above. In some embodiments, the number of mutations does not exceed a certain number. In some embodiments, the insulin conjugate contains fewer than 12 mutations (i.e., deletions, substitutions, additions) compared to the parent insulin. In another embodiment, the human insulin analogue contains fewer than 11 mutations compared to the parent insulin. In another embodiment, the human insulin analogue contains fewer than 10 mutations compared to the parent insulin.

[0039] The expression "parent insulin" as used herein refers to naturally occurring insulin, ie insulin without any mutations, also known as wild-type human insulin.

[0040] The sequence of human insulin is well known in the art and is provided in the Examples section. Human insulin comprises an A chain having the amino acid sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1) and a B chain having the amino acid sequence shown in FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).

[0041] Human insulin contains three disulfide bridges: one between the cysteines at positions A6 and A11, one between the cysteine ​​at position A7 of the A chain and the cysteine ​​at position B7 of the B chain, and one between the cysteine ​​at position A20 of the A chain and the cysteine ​​at position B19 of the B chain.

[0042] The insulin receptor can be any mammalian insulin receptor, such as a bovine, porcine or human insulin receptor, in some embodiments, the insulin receptor is a human insulin receptor, such as human insulin receptor isoform A or human insulin receptor isoform B (which is used in the Examples section).

[0043] Advantageously, the insulin conjugates provided herein are t max The half-life is at least as long as, and longer than, the insulin conjugates provided in WO 14009316 (Pridal, et al., NOVO NORDISK A / S). max is C max is the time point at which the max is the maximum (or peak) serum concentration achieved by the insulin conjugate. In addition, insulin conjugates have a very long half-life, resulting in a favorable peak-to-trough ratio.

[0044] Methods for determining the binding affinity of insulin conjugates to insulin receptor are well known in the art. For example, insulin receptor binding affinity can be determined by scintillation proximity assay, which is based on the evaluation of the competitive binding between [125I]-labeled parent insulin, such as [125I]-labeled human insulin, and (unlabeled) insulin conjugates to insulin receptor. Insulin receptor can be present in the membrane of cells that overexpress recombinant insulin receptor, such as CHO cells. In one embodiment, insulin receptor binding affinity is determined as described in the Examples section.

[0045] Binding of naturally occurring insulin or insulin conjugates to the insulin receptor activates the insulin signaling pathway. The insulin receptor has tyrosine kinase activity. Binding of insulin to its receptor induces a conformational change that stimulates the autophosphorylation of the receptor on tyrosine residues. Autophosphorylation of the insulin receptor stimulates the tyrosine kinase activity of the receptor on intracellular substrates involved in the transmission of signals. Therefore, autophosphorylation of the insulin receptor by insulin conjugates is considered a measure for the signal transduction caused by said conjugates.

[0046] Insulin receptor autophosphorylation in response to the parent insulin can be determined as described in the Examples section.

[0047] Typically, human insulin analogues provided herein exhibit sufficient insulin receptor binding and autophosphorylation activity to account for their pharmacological effects, hi one embodiment, a human insulin analogue exhibits 0.5%-10%, such as 1%-10%, of the insulin receptor binding of the parent insulin, and / or 0.2%-2% of the autophosphorylation activity of the parent insulin (typically human insulin).

[0048] In at least one embodiment, the human insulin analogue comprises at least 7 mutations compared to the parent insulin, hi at least one embodiment, the human insulin analogue comprises at least 7 mutations but fewer than 12 mutations, such as fewer than 11 mutations, compared to the parent insulin.

[0049] In some embodiments, the insulin conjugates provided herein include a human insulin analog having a mutation at position A14 substituted with aspartic acid or glutamic acid, a mutation at position B16 substituted with glutamic acid or histidine, a mutation at position B25 substituted with histidine, and at least four additional substitutions. The insulin conjugate may have additional amino acids attached to the human insulin analog. For example, the human insulin analog may have an additional arginine residue attached to the human insulin analog, such as one or two arginine residues. For example, it may have two arginine residues attached to the C-terminus of the B chain. In one embodiment, an additional arginine residue is added to the C-terminus of the B chain.

[0050] In another embodiment, the insulin conjugates provided herein comprise a human insulin analog having a mutation at position A14 substituted with aspartic acid or glutamic acid, a mutation at position B16 substituted with aspartic acid or glutamic acid, a mutation at position B25 substituted with histidine and at least four additional substitutions, all of which are arginine, histidine or glycine.

[0051] In another embodiment, the insulin conjugates provided herein include a human insulin analog having a mutation at position A14 substituted with aspartic acid or glutamic acid, a mutation at position B16 substituted with aspartic acid or glutamic acid, a mutation at position B25 substituted with histidine, a mutation at position A21 substituted with glycine or arginine, and at least three additional substitutions. In at least one embodiment, all additional substitutions are arginine. In at least one embodiment, the insulin conjugate has an additional amino acid attached to the human insulin analog. For example, the human insulin analog can have an additional arginine residue attached to the human insulin analog, such as one or two arginine residues. For example, it can have two arginine residues attached to the C-terminus of the B chain. In one embodiment, an additional arginine residue is added to the C-terminus of the B chain.

[0052] In another embodiment, the insulin conjugates provided herein include a human insulin analog having a mutation at position A14 substituted with aspartic acid or glutamic acid, a mutation at position B16 substituted with glutamic acid, a mutation at position B25 substituted with histidine, a mutation at position A21 substituted with glycine or arginine, and at least three additional substitutions. In at least one embodiment, all additional substitutions are arginine. In at least one embodiment, the insulin conjugate has an additional amino acid attached to one or more insulin chains of the human insulin analog. For example, the human insulin analog may have an additional arginine residue attached to the human insulin analog, such as one or two arginine residues. For example, it may have two arginine residues attached to the C-terminus of the B chain. In one embodiment, the additional arginine residue is covalently attached to the C-terminus of the B chain.

[0053] At least one embodiment relates to an insulin conjugate comprising a human insulin analog having at least seven mutations relative to the parent insulin, optionally wherein the mutations are selected from the group consisting of substitutions, deletions and additions of amino acid residues.

[0054] At least one embodiment relates to an insulin conjugate, the insulin conjugate comprising: A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, a mutation at position B16 that results in a substitution with histidine or glutamic acid; A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, the insulin conjugate has an additional amino acid residue attached to the human insulin analog.

[0055] At least one embodiment relates to an insulin conjugate, the insulin conjugate comprising: A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine, glycine, or a combination thereof; Optionally, the insulin conjugate has an additional amino acid residue attached to the human insulin analog.

[0056] At least one embodiment relates to an insulin conjugate, the insulin conjugate comprising: A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the insulin conjugate has an additional amino acid residue attached to the human insulin analog.

[0057] At least one embodiment relates to an insulin conjugate, the insulin conjugate comprising: A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the human insulin analog has an additional arginine residue attached to the human insulin analog.

[0058] In one embodiment the additional amino acid is an additional arginine residue attached to the human insulin analogue, such as one or two additional arginine residues, in one embodiment the additional arginine residue is attached, i.e. covalently attached, to the C-terminus of the B chain.

[0059] In one embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVRQHLCGSHLVEALELVCGERGFHYTPK (SEQ ID NO:3).

[0060] In another embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVRQHLCGSHLVEALELVCGERGFHYRPK (SEQ ID NO: 4).

[0061] In another embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALELVCGERGFHYRPK (SEQ ID NO:5).

[0062] In another embodiment, the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALELVCGERGFHYTPK (SEQ ID NO: 6).

[0063] In another embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYTPK (SEQ ID NO: 7).

[0064] In another embodiment, the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALELVCGERGFHYTPKTR (SEQ ID NO:8).

[0065] In another embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALELVCGERGFHYRPKTR (SEQ ID NO: 9).

[0066] In another embodiment the B-chain of the human insulin analog comprises or consists of the amino acid sequence FVNQHLCGSHLVEALHLVCGERGFHYRPKTR (SEQ ID NO: 10).

[0067] In one embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLERLERYCG (SEQ ID NO:11).

[0068] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVEQCCTSICSLERLERYCR (SEQ ID NO: 12).

[0069] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLEQLERYCG (SEQ ID NO: 13).

[0070] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLEQLERYCR (SEQ ID NO: 14).

[0071] In another embodiment, the A-chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLERLENYCR (SEQ ID NO: 15).

[0072] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLERLERYCR (SEQ ID NO: 16).

[0073] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLERLERYCR (SEQ ID NO: 17).

[0074] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLERLERYCG (SEQ ID NO: 18).

[0075] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVEQCCTRICSLERLERYCR (SEQ ID NO: 19).

[0076] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLEQLERYCR (SEQ ID NO: 20).

[0077] In another embodiment the A-chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLERLENYCR (SEQ ID NO:21).

[0078] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDRLERYCG (SEQ ID NO: 22).

[0079] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDRLERYCG (SEQ ID NO: 23).

[0080] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDQLERYCG (SEQ ID NO:24).

[0081] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVEQCCTSICSLDRLERYCR (SEQ ID NO:25).

[0082] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDQLERYCR (SEQ ID NO:26).

[0083] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDRLENYCR (SEQ ID NO:27).

[0084] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTSICSLDRLERYCR (SEQ ID NO:28).

[0085] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLDRLERYCR (SEQ ID NO: 29).

[0086] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLDRLERYCG (SEQ ID NO: 30).

[0087] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVEQCCTRICSLDRLERYCR (SEQ ID NO:31).

[0088] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLDQLERYCR (SEQ ID NO: 32).

[0089] In another embodiment the A chain of the human insulin analog comprises or consists of the amino acid sequence GIVERCCTRICSLDRLENYCR (SEQ ID NO: 33).

[0090] Derivatives of the aforementioned amino acids are known in the art.

[0091] In at least one embodiment, the insulin conjugate comprises a human insulin analog comprising the A and B chains shown above.

[0092] In at least one embodiment, the human insulin analog is Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B31)-insulin (human insulin) is selected from the group consisting of:

[0093] In another embodiment, the human insulin analog is Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-Insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin) is selected from the group consisting of:

[0094] In at least one embodiment, the human insulin analog is Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-Insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin) is selected from the group consisting of:

[0095] In one embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:13 and a B chain having the amino acid sequence set forth in SEQ ID NO:9.

[0096] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:11 and a B chain having the amino acid sequence set forth in SEQ ID NO:9.

[0097] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:16 and a B chain having the amino acid sequence set forth in SEQ ID NO:9.

[0098] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:16 and a B chain having the amino acid sequence set forth in SEQ ID NO:8.

[0099] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:17 and a B chain having the amino acid sequence set forth in SEQ ID NO:3.

[0100] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:18 and a B chain having the amino acid sequence set forth in SEQ ID NO:3.

[0101] In another embodiment, the human insulin analog comprises an A chain having the amino acid sequence set forth in SEQ ID NO:16 and a B chain having the amino acid sequence set forth in SEQ ID NO:3.

[0102] In at least one embodiment, the insulin conjugate is conjugate 1 as depicted in Figure 1 or conjugate 2 as depicted in Figure 2 or conjugate 3 as depicted in Figure 3 or conjugate 4 as depicted in Figure 4. For clarity, [ka] It is.

[0103] In at least one embodiment, the insulin conjugate is conjugate 5 depicted in FIG. 5 or conjugate 6 depicted in FIG.

[0104] In at least one embodiment, the insulin conjugate is conjugate 7 as depicted in FIG.

[0105] The first aspect further relates to a human insulin analogue as defined in relation to the insulin conjugates of the invention, e.g. as defined in any one of items 3-10, e.g. items 5-10, e.g. items 7-10. The items can be found in the section preceding the Examples section. The definitions and explanations for the insulin analogue given in the conjugates typically apply mutatis mutandis. In one embodiment, the insulin analogue is covalently attached to a moiety capable of binding to serum albumin, preferably human serum albumin. A serum albumin binding moiety (also referred to herein as "albumin binder" or "binder") is a moiety that, when attached to a peptide such as an insulin analogue provided herein, typically results in improved pharmacodynamic and / or pharmacokinetic properties of the peptide, e.g. an extended pharmacokinetic half-life in blood and / or plasma and / or an extended profile of action, i.e. an extended reduction in blood glucose levels. Typically, the albumin binder is covalently attached to the insulin analogue via the epsilon amino group of lysine B29.

[0106] Detailed Description of the Second Aspect The second aspect relates to a pharmaceutical composition comprising a pharma- ceutical effective amount of an insulin conjugate according to the first aspect. The second aspect further relates to a pharmaceutical composition comprising a pharma- ceutical effective amount of a human insulin analogue according to the first aspect. In at least one embodiment, the pharmaceutical composition comprises a pharma- ceutical acceptable excipient.

[0107] In at least one embodiment, the pharmaceutical composition comprises zinc ions. In at least one embodiment, the pharmaceutical composition comprises a buffer. In at least one embodiment, the pharmaceutical composition comprises a surfactant. In at least one embodiment, the pharmaceutical composition comprises glycerol. In at least one embodiment, the pharmaceutical composition comprises water.

[0108] In at least one embodiment, the pharmaceutical composition has a pH of pH 3 to pH 8, preferably pH 3.5 to pH 6.

[0109] Examples of pharmaceutical compositions: 1 ml of the pharmaceutical composition consists of 4.2 mmol of conjugate 1, zinc chloride, metacresol (Ph.Eur.), glycerol, hydrochloric acid (adjusting the pH value to pH 4), polysorbate 20, sodium hydroxide (adjusting the pH value to pH 4), water and an injection agent.

[0110] Other insulin preparations known in the art may be used herein as pharmaceutical compositions in accordance with the present invention.

[0111] In at least one embodiment, the pharmaceutical composition comprises the insulin conjugate in the form of a pharma- ceutically acceptable salt. Pharmaceutically acceptable salts of the insulin conjugate can include acid addition salts and base salts. Suitable acid addition salts are formed from acids that form non-toxic salts. In at least one embodiment, pharma- ceutically acceptable salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, iodide ... The base salts are selected from the group consisting of phosphate, lactate, maleate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1,5-naphthalenedisulfonate and xinafoate. Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum salt, arginine salt, benzathine salt, calcium salt, choline salt, diethylamine salt, bis(2-hydroxyethyl)amine (diolamine) salt, glycine salt, lysine salt, magnesium salt, meglumine salt, 2-aminoethanol (olamine) salt, potassium salt, sodium salt, 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris or tromethamine) salt and zinc salt. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0112] Detailed Description of the Third Aspect The third aspect relates to an insulin conjugate according to the first aspect for use as a medicament. Furthermore, the third aspect relates to a human insulin analogue according to the first aspect for use as a medicament.

[0113] One embodiment relates to an insulin conjugate according to the first aspect for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes and hyperglycemia and / or for lowering blood glucose levels.

[0114] One embodiment relates to a pharmaceutical composition according to the second aspect for use as a medicament.

[0115] One embodiment relates to a pharmaceutical composition according to the second aspect for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes and hyperglycemia and / or for lowering blood glucose levels.

[0116] Detailed Description of the Fourth Aspect The fourth aspect relates to a method of treating a patient comprising administering to the patient an insulin conjugate of the first aspect or a pharmaceutical composition of the second aspect.Furthermore, the fourth aspect relates to a method of treating a patient comprising administering to the patient a human insulin analogue of the first aspect or a pharmaceutical composition of the second aspect.

[0117] In at least one embodiment, a patient is dosed, for example, by oral administration, once a week.

[0118] Provided herein are methods of treating a patient having a disease, comprising administering to the patient a pharma- tically effective amount of one or more insulin conjugates provided herein or pharmaceutical compositions thereof.

[0119] In some embodiments, the disease is diabetes, such as type II diabetes. In at least one embodiment, the disease is selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes, and hyperglycemia, and / or for reducing blood glucose levels.

[0120] Detailed Description of the Fifth Aspect A fifth aspect relates to a proinsulin comprising an insulin A chain as disclosed in the first aspect and / or an insulin B chain as disclosed in the first aspect.

[0121] Also provided herein is a proinsulin comprising the insulin A chain and / or insulin B chain of the insulin conjugates provided herein. The B chain can be any B chain as defined herein above for the insulin conjugates provided herein. The insulin B chain can include further mutations as described herein above for the B chain.

[0122] The A-chain comprised by the proinsulins provided herein can be any A-chain defined herein above for the insulin conjugates provided herein.

[0123] In addition to the insulin A chain and / or insulin B chain, the proinsulins provided herein can include additional elements such as a leader sequence or a C peptide. In some embodiments, the proinsulins can further include a C peptide located between the insulin B chain and the insulin A chain. The C peptide can be 0-15 amino acids in length. In at least one embodiment, the proinsulin does not include a C peptide. The configuration can be as follows (from N-terminus to C-terminus): B chain, C peptide, A chain.

[0124] Detailed Description of the Sixth Aspect A sixth aspect of the invention relates to a method for preparing an insulin conjugate of the first aspect.

[0125] In at least one embodiment, the human serum albumin binder of formula (I) is covalently attached to the human insulin analog by forming an amide bond between the terminal carboxy group "a" of the human serum albumin binder of formula (I) and the epsilon amino group of lysine B29 of the human insulin analog.

[0126] The insulin conjugates can be prepared by any method deemed appropriate, for example, they can be prepared by recombinant methods or solid phase synthesis.

[0127] The above definitions and explanations apply mutatis mutandis to the sixth aspect.

[0128] Provided herein is a human insulin B chain, i.e. a human insulin B chain peptide, as defined herein above in relation to the B chain of an insulin conjugate. Thus, provided herein is an insulin B chain that comprises at least two mutations compared to the insulin B chain of the parent insulin. The insulin B chain may comprise further mutations as described herein above, such as a Des(B30) deletion.

[0129] Provided herein are polynucleotides encoding human insulin analog B chain and proinsulin provided herein. The polynucleotide may be operably linked to a promoter that allows expression of the polynucleotide. In some embodiments, the promoter is heterologous to the polynucleotide. In some embodiments, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter.

[0130] Further provided herein is a vector comprising a polynucleotide encoding an insulin conjugate provided herein, hi some embodiments, the vector is an expression vector.

[0131] Provided herein are host cells comprising nucleic acids, polynucleotides and / or vectors provided herein encoding insulin conjugates, insulin B chain and proinsulin. In some embodiments, the host cell is a bacterial cell, such as a cell belonging to the genus Escherichia, e.g., an E. coli cell. In other embodiments, the host cell is a yeast cell, such as a Komagataella phaffii (sometimes referred to as "Pichia pastoris") cell or a Klyveromyces lactis cell.

[0132] Another aspect of the invention relates to a polynucleotide encoding a human insulin analogue according to the first aspect.

[0133] Another aspect of the invention relates to an expression vector comprising a polynucleotide encoding a human insulin analogue according to the first aspect.

[0134] Another aspect of the invention relates to a host cell comprising a human insulin analogue according to the first aspect, a polynucleotide encoding a human insulin analogue according to the first aspect and / or an expression vector comprising a polynucleotide encoding a human insulin analogue according to the first aspect.

[0135] Detailed Description of the Seventh Aspect The seventh aspect is a compound of formula (I) [ka] This invention relates to stable derivatives of human albumin binders.

[0136] In at least one embodiment, the stable derivative of the human albumin binder of formula (I) is an ester, preferably, "a" is an ester bond.

[0137] In at least one embodiment, the stable derivative of the human albumin binder of formula (I) is an O-succinimide ester.

[0138] Detailed Description of the Eighth Aspect An eighth aspect relates to the non-therapeutic use of an insulin conjugate or an insulin analogue according to the first aspect.

[0139] Detailed description of specific embodiments (provisions and items) The following provides an overview of certain embodiments. The above provisions typically apply mutatis mutandis to the following clauses and items.

[0140] Terms 1. Formula (I) [ka] and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at a point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of the human insulin analogue via an amide bond. 2. The insulin conjugate according to clause 1, comprising a human insulin analogue having at least seven mutations relative to the parent insulin, optionally wherein the mutations are selected from the group consisting of substitutions, deletions and additions of amino acid residues. 3. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, a mutation at position B16 that results in a substitution with histidine or glutamic acid; A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, an insulin conjugate according to any of the preceding clauses, having an additional amino acid residue attached to the human insulin analogue. 4. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine, glycine, or a combination thereof; Optionally, the insulin conjugate according to any of the preceding clauses, wherein the insulin conjugate has an additional amino acid residue attached to the human insulin analogue. 5. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the insulin conjugate according to any of the preceding clauses, wherein the insulin conjugate has an additional amino acid residue attached to the human insulin analogue. 6. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the insulin conjugate according to any of the preceding clauses, wherein the insulin conjugate has an additional arginine residue attached to the human insulin analogue. 7.Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B31)-insulin (human insulin) An insulin conjugate according to any of the preceding clauses, comprising a human insulin analogue selected from the group consisting of: 8.Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-Insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin) An insulin conjugate according to any of the preceding clauses, comprising a human insulin analogue selected from the group consisting of: 9. An insulin conjugate according to any of the preceding clauses, which is conjugate 1, 2, 3, 4, 5, 6 or 7 (see Figures 1 to 7). 10. A human insulin analogue as defined in any one of clauses 3 to 10, such as clauses 5 to 8. 11. A human insulin analogue as defined in clause 7 or 8. 12. A pharmaceutical composition comprising a pharma- ceutically effective amount of an insulin conjugate according to any of clauses 1 to 9 or an insulin analogue of clause 10 or 11. 13. An insulin conjugate according to any of clauses 1 to 9 or an insulin analogue of clause 10 or 11 for use as a medicament. 14. An insulin conjugate according to any of clauses 1 to 9 or an insulin analogue of clause 10 or 11 for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes and hyperglycemia and / or for lowering blood glucose levels. 15. An insulin conjugate according to any of clauses 1 to 9 or an insulin analogue of clause 10 or 11 for use as a medicament for the treatment of type 2 diabetes.

[0141] item 1. Formula (I) [ka] and a human insulin analogue, wherein the human serum albumin binder of formula (I) is covalently attached to the human insulin analogue at a point where the terminal carboxy group "a" of the human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of the human insulin analogue via an amide bond. 2. The insulin conjugate according to item 1, comprising a human insulin analogue having at least seven mutations relative to the parent insulin, optionally the mutations being selected from the group consisting of substitutions, deletions and additions of amino acid residues. 3. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, a mutation at position B16 that results in a substitution with histidine or glutamic acid; A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, an insulin conjugate according to any of the preceding paragraphs, having an additional amino acid residue attached to the human insulin analogue. 4. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine, glycine, or a combination thereof; Optionally, the insulin conjugate according to any of the preceding paragraphs has an additional amino acid residue attached to the human insulin analog. 5. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the insulin conjugate according to any of the preceding paragraphs has an additional amino acid residue attached to the human insulin analog. 6. A mutation at position A14 resulting in a substitution with aspartic acid or glutamic acid, A mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all of the additional substitutions are to arginine; Optionally, the insulin conjugate according to any of the preceding paragraphs has an additional arginine residue attached to the human insulin analog. 7.Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B31)-insulin (human insulin) An insulin conjugate according to any of the preceding items, comprising a human insulin analog selected from the group consisting of: 8.Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-Insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-Insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin) An insulin conjugate according to any of the preceding items, comprising a human insulin analog selected from the group consisting of: 9. Conjugate 1 (A strand sequence: SEQ ID NO: 13; B strand sequence: SEQ ID NO: 9): [ka] Or conjugate 2 (A chain sequence: SEQ ID NO:11; B chain sequence: SEQ ID NO:9): [ka] Or conjugate 3 (A chain sequence: SEQ ID NO: 16; B chain sequence: SEQ ID NO: 9): [ka] Or conjugate 4 (A chain sequence: SEQ ID NO: 16; B chain sequence: SEQ ID NO: 8): [ka] An insulin conjugate according to any of the preceding items, wherein 10. Conjugate 5 (A strand sequence: SEQ ID NO: 17; B strand sequence: SEQ ID NO: 3): [ka] Or conjugate 6 (A chain sequence: SEQ ID NO: 18; B chain sequence: SEQ ID NO: 3): [ka] An insulin conjugate according to any of the preceding items, wherein 11. A pharmaceutical composition comprising a pharma- ceutical effective amount of an insulin conjugate according to any of items 1 to 10. 12. An insulin conjugate according to any of items 1 to 10 for use as a medicament. 13. An insulin conjugate according to any of items 1 to 10 for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes and hyperglycemia and / or for lowering blood glucose levels. EXAMPLES

[0142] Synthesis Example Synthetic Section 1: Synthetic Human Serum Albumin Binder. Details: Synthesis of 2-(nonadec-2-yn-1-yloxy)tetrahydro-2H-pyran [ka] BuLi (2.5 M in hexanes, 30 mL, 75 mmol) was added dropwise to a stirred solution of C (10 g, 72 mmol) in THF (100 mL) at -78 °C and the resulting mixture was stirred at this temperature for 30 min. DMPU (10 g, 78 mmol) was then added and stirring was continued for an additional 10 min. A (10 g, 78 mmol) was then added dropwise and the resulting mixture was allowed to warm to room temperature and then heated at 55 °C for 20 h. The mixture was cooled to room temperature and saturated NH 4 Aqueous Cl (200 mL) was added. The layers were separated and the aqueous layer was extracted with EA (300 mL). The combined organic layers were washed with water and brine and diluted with anhydrous Na 2 SO 4 The crude product B was used directly without further purification (9.6 g, 80%).

[0143] Synthesis of nonadec-2-yn-1-ol [ka] 4-Methylbenzenesulfonic acid (2 g, 0.4 mmol) was added to B (9.6 g, 26.3 mmol) in MeOH (100 mL) and the reaction mixture was stirred for 12 h. After hydrolysis with HO (300 mL) and extraction with EA (2×300 mL), the solvent was removed and the ethyl acetate solution was added to Na 2 SO 4 It was dried over and concentrated under vacuum. The crude compound was purified by silica gel chromatography (eluted with 5% EA in PE) to give the desired product D. (6.2 g, 85%) LCMS no

[0144] Synthesis of nonadec-18-yn-1-ol [ka] To NaH (60% in mineral oil, 5.7 g, 142.8 mmol, oil-free washed three times with hexane) was added 1,3-diaminopropane (80 mL). The mixture was stirred in a thermostatic oil bath at 70° C. After 10 min, gas evolution was recorded and after 1 h, a clear solution of D (4 g, 14.28 mmol) in 1,3-diaminopropane (40 mL) was added. The red-brown mixture was stirred overnight at 55° C., then cooled, water was added and the organic products were extracted four times with ether. The combined ether phases were washed successively with water, dilute HCl and NaCl solutions, then diluted with NaCl. 2 SO 4 It was dried over and concentrated under vacuum. The crude compound was purified by silica gel chromatography (eluted with 10% EA in PE) to give the desired product E. (2.8 g, 70%)

[0145] Synthesis of nonadec-18-ynoic acid [ka] E (2.8 g, 10 mmol) and TEMPO (782 mg, 5 mmol), CH 3 CN (20 mL), THF (20 mL) and pH 4 buffer solution (20 mL) with NaClO 2 (5 g, 55 mmol) and a 10% solution of NaOCl (372 mg, 5 mmol) were added simultaneously. The reaction mixture was stirred at RT overnight, diluted with EA (150 mL), washed with water (100 mL) and brine, and diluted with Na 2 SO 4 It was dried over water and concentrated under reduced pressure. The crude product F was used directly without further purification. (2.5 g, 86%) 1 H NMR (400 MHz, CDCl 3 )δ 2.40-2.31(m,2H),2.18(td,J=7.0,2.4Hz,2H),1.94(t,J=2.5Hz,1H),1.69-1.58(m,2H),1.51(dd,J=14.8,7.2Hz,2H),1.37-1.18(m,24H).

[0146] Synthesis of tert-butyl nonadec-18-ynoate [ka] F (1.3 g, 4.42 mmol), TFAA (2.8 g, 13.2 mmol) were added to THF (10 mL), and the mixture was reacted at room temperature for 1 h. Then, t-BuOH (10 mL) was added to the mixture, and the mixture was stirred at room temperature for 16 h. The pH of the reaction mixture was adjusted with NaHCO 3 The pH was adjusted to 8 with EA (200 mL x 3), and the solution was extracted with Na 2 SO 4 After drying and concentration, the target compound G was obtained (1.2 g, 80%).

[0147] Preparation of compound 2 [ka] General procedure: A solution of compound 1 (4.62 g, 12.0 mmol) in anhydrous DCM (60 mL) was added to 2-Cl-trt-resin (5.0 g, 6.0 mmol), followed by DIEA (1.55 g, 12.0 mmol). The mixture was vortexed at 5-10 °C for 20 h. The mixture was filtered and washed with NMP (60 mL x 3), DCM (60 mL x 3) and NMP (60 mL x 3) to give compound 2 (10 g, crude compound) as a yellow solid, which was used directly in the next step. LCMS:WH01668-109-2A m / z 386.2[M+1] +

[0148] Preparation of compound 3 [ka] General procedure: 20% piperidine / NMP (60 mL) was added to compound 2 (10 g, crude compound, theoretical 6.0 mmol). The mixture was vortexed at 5-10 °C for 30 min, filtered, and the residue was treated twice with the same procedure. The residue was washed with NMP (60 mL x 3), DCM (60 mL x 3) and NMP (60 mL x 3) to give compound 3 (7.0 g, crude compound) as a yellow solid, which was used directly in the next step. LCMS showed no compound 2 remained, but compound 3 was not detected. A red resin was obtained with a positive TNBS test.

[0149] Preparation of compound 4 [ka] General procedure: To a solution of compound 1 (4.63 g, 12.0 mmol) in NMP (30 mL) and DCM (30 mL) was added N-hydroxysuccinimide (1.66 g, 14.4 mmol) and diisopropylcarbodiimide (1.82 g, 14.4 mmol). The mixture was stirred at 5-10 °C for 4 h. TLC showed the reaction was complete. The reaction mixture containing compound 4 was used directly in the next step.

[0150] Preparation of compound 5 [ka] General procedure: The reaction mixture containing compound 4 (12.0 mmol theoretical) was added to compound 3 (7.0 g, crude compound, 6.0 mmol theoretical), followed by DIEA (1.86 g, 14.4 mmol). The mixture was vortexed at 5-10 °C for 16 h. The mixture was filtered and washed with NMP (60 mL x 3), DCM (60 mL x 3) and NMP (60 mL x 3) to give compound 5 (12.0 g, crude compound) as a yellow solid, which was used directly in the next step. A colorless resin was obtained with a positive TNBS test. LCMS:WH01668-112-3A m / z 531.3[M+1] +

[0151] Preparation of compound 6 [ka] General procedure: 20% piperidine / NMP (60 mL) was added to compound 5 (10 g, crude compound, theoretical 6.0 mmol). The mixture was vortexed at 5-10 °C for 30 min, filtered, and the residue was treated twice with the same procedure. The residue was washed with NMP (60 mL x 3), DCM (60 mL x 3) and NMP (60 mL x 3) to give compound 6 (7.0 g, crude compound) as a yellow solid, which was used directly in the next step. LCMS showed that no compound 5 remained, but compound 6 was not detected. A positive TNBS test gave a red resin.

[0152] Synthesis of tert-butyl 2-(2-benzyloxy-2-oxo-ethoxy)-5-iodo-benzoate [ka] General procedure: 2 CO 3 (0.86 g, 6 mmol) was added to a solution of H (1 g, 3 mmol) in DMF (10 mL) and the mixture was stirred at room temperature for 5 min. Benzyl bromoacetate (4.5 mmol, 1.05 g) was then added to the mixture, the temperature was raised to 45 °C and the reaction was stirred at room temperature for 2 h. The reaction was then diluted with 50 mL of EtOAc and washed with brine (3 x 50 mL). The organic phase was removed using MgSO 4 The crude compound was purified by stirring the mixture with pentane (5 mL) after drying on a kettle, filtration, and removing the solvent under reduced pressure. The solid product was collected on a filter to give I (0.98 g, 70%) as a tan solid. 1 H NMR:(400Mhz,DMSO)δ 7.8(d,1H),7,7(dd,1H),7,38(s,5H),6.9(d,1H),5.21(s,2H),4.92(s,2H),1.5(s,9H)

[0153] Synthesis of 19-[4-(2-benzyloxy-2-oxo-ethoxy)-3-tert-butoxycarbonyl-phenyl]nonadeca-18-ynoic acid [ka] General procedure: I (0.98 g, 2.1 mmol) and triethylamine (2 mL) were added to a suspension of trans-dichloro-bis-(triphenylphosphine)palladium (0.03 g, 0.043 mmol) and copper(I) iodide (0.017 g, 0.086 mmol) in THF (10 mL), then G (0.8 g, 23 mmol) was added to the mixture and stirred at room temperature for 24 h. The mixture was concentrated in vacuo and filtered through a plug of Celite and silica with diethyl ether (3×25 mL). The organic solution was concentrated in vacuo and purified by flash chromatography (10% dichloromethane in petroleum ether 40-60) to give product K (1.3 g, 90%).

[0154] Synthesis of 2-[2-tert-butoxycarbonyl-4-(19-tert-butoxy-19-oxo-nonadecyl)phenoxy]acetic acid [ka] General procedure: A mixture of K (1.2 g, 1.73 mmol) and Pd / C (0.1 g) in ethyl acetate (10 mL) was stirred under a hydrogen atmosphere for 16 h. The mixture was filtered and concentrated under reduced pressure to give L (1 g, 1.65 mmol).

[0155] Preparation of compound 7 [ka] General procedure: To a solution of compound L (1 g, 1.65 mmol) in NMP (5 mL) and dichloromethane (5 mL) was added HOBt (0.243 g, 1.8 mmol) and diisopropylcarbodiimide (0.227 g, 1.8 mmol). The mixture was stirred at 5-10 °C for 1 h, then compound 6 (0.825 g, crude compound, theoretical 0.55 mmol) was added along with DIEA (0.232 g, 1.8 mmol). The mixture was vortexed at 5-10 °C for 16 h. The mixture was filtered and washed with NMP (3 x 10 mL), DCM (3 x 10 mL) and NMP (3 x 10 mL) to give compound 7 (1.3 g, crude compound) as a yellow solid, which was used directly in the next step. A colorless resin was obtained with a positive TNBS test.

[0156] Synthesis of 2-[2-[2-[[2-[2-[[2-[2-[2-[2-[2-[2-tert-butoxycarbonyl-4-(19-tert-butoxy-19-oxo-nonadecyl)phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid [ka] General procedure: 5% TFA / DCM (10 mL) was added to compound 7 (1.3 g, crude compound, theoretical 0.55 mmol). The mixture was vortexed at 5-10 °C for 20 min, filtered, and the residue was treated twice with the same procedure. The residue was washed with DCM (3 x 60 mL). The combined organic phase was cooled to 0 °C, diluted with water (200 mL), neutralized to pH = 3 with saturated sodium bicarbonate solution, the organic phase was separated, and the aqueous phase was extracted with DCM (3 x 100 mL). The organic phase was dried over sodium sulfate, filtered, concentrated, and the residue was purified by preparative HPLC (TFA) to give formula (I) (0.180 g, yield: 38%) as a white solid.

[0157] Synthesis Section 2: Conjugates Provided herein is, inter alia, a compound of formula (I) [ka] and a human serum albumin binder of the formula:

[0158] The human serum albumin binder of formula (I) is covalently attached to the insulin conjugate at the point where the terminal carboxy group "a" of the compound of formula (I) is covalently attached to the epsilon amino group of lysine B29.

[0159] Pharmaceutically acceptable salts of insulin conjugates include acid addition salts and base salts. Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, Salts include maleate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, 1,5-naphthalenedisulfonate and xinafoate. Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, bis(2-hydroxyethyl)amine (diolamine) salts, glycine salts, lysine salts, magnesium salts, meglumine salts, 2-aminoethanol (olamine) salts, potassium salts, sodium salts, 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris or tromethamine) salts and zinc salts. Hemi-salts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0160] The conjugate and its pharmaceutically acceptable salts can exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex that comprises a compound of formula I or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules, such as ethanol. When the solvent is water, the term "hydrate" is used.

[0161] Examples of isotopes suitable for inclusion in the conjugate include: 2 H and 3 Hydrogen such as H 11 C. 13 C and 14 Carbon such as C, 36 Chlorine such as Cl, 18 Fluorine such as F, 123 I and 125 Iodine, such as I 13 N and 15 Nitrogen such as N 15 O. 17 O and 18 Oxygen such as O 35 Examples include isotopes of sulfur such as S.

[0162] Certain isotopically labeled conjugates, for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and ready means of detection.

[0163] Deuterium, i.e. 2 Substitution with heavier isotopes such as H may offer certain therapeutic advantages due to greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements.

[0164] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.

[0165] Isotopically labeled conjugates may generally be prepared by conventional techniques known to those of skill in the art.

[0166] Pharmaceutically acceptable solvates according to the invention include those in which the solvent of crystallization may be isotopically substituted, e.g. D 2 O, d 6 -Acetone, d 6 -DMSO.

[0167] In some embodiments, the insulin conjugate is an insulin conjugate according to the first aspect above. The definitions and explanations above apply accordingly.

[0168] Synthesis Section 3: Human Insulin Analogues and Conjugates Synthesis 3.1 Human insulin The amino acid sequences of the A and B chains of human insulin are: A chain: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) B chain: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2) It is.

[0169] An intrachain disulfide bridge exists between Cys(A6) and Cys(A11), and two interchain disulfide bridges exist between Cys(A7) and Cys(B7) and between Cys(A20) and Cys(B19).

[0170] 3.2 Conjugate 1 Conjugate 1 is based on human insulin with mutations at positions A5, A14, A18, A21, B16, B25, B27 and an addition of an amino acid at position B31: Arg(A5): The amino acid at position 5 of the A chain of human insulin (Q, glutamine, Gln) is replaced by arginine (R, Arg); Glu(A14): The amino acid at position 14 of the A chain of human insulin (Y, tyrosine, Tyr) is replaced by glutamic acid (E, Glu); Arg(A18): The amino acid at position 18 of the A chain of human insulin (N, asparagine, Asn) is replaced by arginine (R, Arg); Gly(A21): the amino acid at position 21 of the A chain of human insulin (N, asparagine, Asn) is replaced by arginine (G, Gly); Glu(B16): The amino acid at position 16 of the B chain of human insulin (Y, tyrosine, Tyr) is replaced by glutamic acid (E, Glu); His(B25): The amino acid at position 25 of the B chain of human insulin (F, phenylalanine, Phe) is replaced by histidine (H, His); Arg(B27): The amino acid at position 25 of the B chain of human insulin (T, threonine, Thr) is replaced by Arg (R, Arg); Arg(B31): The 31st amino acid in the B chain of human insulin is arginine.

[0171] The complete amino acid sequence of conjugate 1, taking into account the A and B chains, is: A chain: GIVERCCTSICSLEQLERYCG (SEQ ID NO: 13) B chain: FVNQHLCGSHLVEALELVCGERGFHYRPKTR (SEQ ID NO: 9) It is.

[0172] One intrachain and two interchain disulfide bridges follow human insulin.

[0173] 3.3 Conjugate with insulin analogues 1 / Synthesis of Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)2-[2-[2-[2-[2-[2-[2-[2-(carbonylmethoxy)ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]-5-(18-carboxyoctadecyl)benzoateLys(B29)Arg(B31)-insulin. The conjugate was prepared from Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin and tert-butyl 5-(19-tert-butoxy-19-oxo-nonadecyl)-2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]benzoate according to 3.2.

[0174] Synthesis of tert-butyl 5-(19-tert-butoxy-19-oxo-nonadecyl)-2-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]benzoate: [ka] To a solution of 296 mg of 2-[2-[2-[[2-[2-[2-[[2-[2-[2-[2-tert-butoxycarbonyl-4-(19-tert-butoxy-19-oxo-nonadecyl)phenoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid in 9 ml DMF was added 92.7 μl of triethylamine, 106 mg of TSTU and a trace of DMAP. The solution was stirred for 1 h. The product was used without further purification. The conversion to the O-succinimide ester was 65%.

[0175] A solution of 500 mg of insulin analog was suspended in 25 ml of water, followed by the addition of 0.45 ml of triethylamine. To the clear solution was added 25 ml of MeCN, followed by 0.9 ml (45.89 mM in DMF) of tert-butyl 5-(19-tert-butoxy-19-oxo-nonadecyl)-2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxoethoxy]benzoate. The solution was stirred at room temperature for 1 hour. The reaction was analyzed by waters UPLC H-class at 214 nm in sodium chloride phosphate buffer. Waters BEH300 10 cm. The product was purified by HPLC on an Aecta avant 25. Kinetex 5μm C18 100 A 250×21.2mm. Column volume (CV) 88ml. Column volume (CV): 88 ml. Solvent A: 0.5% acetic acid in water Solvent B: 0.5 acetic acid in water / MeCN 2:8 Gradient: 95%A 5%B to 40%A 60%B in 14CV

[0176] The reaction was analyzed by waters UPLC H-class at 214 nm in sodium chloride phosphate buffer. Waters BEH300 10 cm. The solution was lyophilized to give the desired product. 98 mg, 34% yield. Mass Spec: 6859 g / mol.

[0177] After lyophilization of the product, the powder was dissolved in 2 ml of trifluoroacetic acid. After 1 h, the solution was neutralized to pH 4 with dilute sodium bicarbonate. The product was purified by HPLC on an Aecta avant 25. Kinetex 5 μm C18 100 A 250×21.2 mm. Column volume (CV) 88 ml. Solvent A: 0.5% acetic acid in water Solvent B: 0.5% acetic acid in water / MeCN 4:6 Gradient: 70%A 30%B~30%A 70%B in 8CV

[0178] The reaction mixture was analyzed by waters UPLC H-class at 214 nm in sodium chloride phosphate buffer. Waters BEH300 10cm.

[0179] The solution was lyophilized to give the desired product. 60mg, 62% yield. Mass spectrometry: 6746.8g / mol.

[0180] 4.Analysis Data 4.1 Liquid Chromatography Mass Spectrometry (LCMS)

[0181] [Table 1]

[0182] [Table 2]

[0183] 5. Insulin Receptor Binding Affinity Insulin receptor binding affinity for insulin and conjugate 1 was determined as described by Hartmann et al. (Effect of the long-acting insulin conjugates glargine and degludec on cardiomyocyte cell signaling and function. Cardiovasc Diabetol. 2016;15:96). Isolation of plasma membrane embedded insulin receptor (M-IR) and competitive binding experiments were performed as previously described (Sommerfeld et al., PLoS One. 2010;5(3):e9540). Briefly, CHO cells overexpressing IR were harvested and incubated with ice-cold 2.25 STM buffer (2.25 M sucrose, 5 mM Tris-HCl pH 7.4, 5 mM MgCl 2The homogenates were resuspended in 0.8 STM buffer (0.8 M sucrose, 5 mM Tris-HCl pH 7.4, 5 mM MgCl, complete protease inhibitors), disrupted using a Dounce homogenizer, and then sonicated. 2 , Complete Protease Inhibitors) and ultracentrifuged at 100,000 g for 90 min. The plasma membrane at the interface was collected and washed twice with phosphate-buffered saline (PBS). The final pellet was diluted with dilution buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl 2 The cells were resuspended in 0.1% Tris-HCl, 150 mM NaCl, 0.1% BSA, complete protease inhibitors, and homogenized again with a Dounce homogenizer. Competitive binding experiments were performed in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, complete protease inhibitors, adjusted to pH 7.8) in 96-well microplates. In each well, 2 μg of isolated membranes were incubated with 0.25 mg of wheat germ agglutinin polyvinyltoluene polyethyleneimine scintillation proximity assay (SPA) beads. A constant concentration of [125I]-labeled human Ins (100 pM) and various concentrations of the respective unlabeled Ins (0.001–1000 nM) were added for 12 h at room temperature (23 °C). Radioactivity was measured at equilibrium by a microplate scintillation counter (Wallac Microbeta, Freiburg, Germany).

[0184] The insulin receptor binding affinity to human insulin for conjugate 1 is shown in Table 2.

[0185] Table 2 Insulin receptor B binding affinity for human insulin

[0186] [Table 3]

[0187] Example 1: Production of human insulin and insulin conjugates Human insulin and insulin conjugates were produced recombinantly. Polynucleotides encoding pre-proinsulin were ordered from Geneart®. The designed polynucleotides were optimized for expression in yeast. They were inserted into expression vectors by classical restriction cloning allowing functional expression and secretion in Klyveromyces lactis K. As a secretion leader, the genes were C-terminally fused to a DNA sequence encoding the alpha mating factor signal of Saccharomyces cerevisiae. Recombinant gene expression was controlled by the lactose-inducible K. lactis promoter.

[0188] Human insulin and insulin conjugates were produced as pre-proinsulin. A genetically fused N-terminal presequence was used to improve expression and secretion yields and to stabilize the peptide in the culture broth. A variety of sequences could be used for this purpose and were tested for efficiency. Proinsulin itself consists of a B chain fused to a C-peptide followed by a C-terminal A chain. Various amino acid combinations have been described as C-peptides. It has been shown that short peptides of 1-10 amino acids function well as C-sequences. For the subsequent processing of insulin, recognition sites for specific proteases adjacent to the C-peptide to allow its excision are important.

[0189] K. lactis cells were made competent by chemical means. The cells were then transformed with expression plasmids encoding the respective pre-proinsulins. After insertion of the plasmids, the cells were plated on selective agar plates containing geneticin. The grown colonies were isolated and tested for recombinant gene expression. The cells were grown to a sufficiently high cell density in yeast peptone dextrose medium supplemented with geneticin. After the initial growth phase, salt-buffered yeast extract medium with geneticin supplemented with lactose was added to the culture to induce recombinant gene expression. The cultures were grown for several days and the supernatant was collected by centrifugation.

[0190] The purification of functional insulin or insulin conjugates was initiated by a filtration step. An initial chromatographic capture step was performed by ion exchange resin. Cleavage of pre-proinsulin to insulin was performed by the highly specific protease trypsin or endoproteinase Lys-C. Depletion of host cell proteins, presequence and product associated products was performed by a cascade of two additional chromatographic steps. Hydrophobic interaction chromatography followed by another ion exchange step was used to achieve this goal. Final polishing was performed by reversed phase chromatography. Filtration, precipitation and lyophilization were used to finish the production process of the insulin molecule.

[0191] After the coupling reaction with the activated carboxylic acid derivative, the solution containing the conjugated insulin molecule was filtered. Final purification was performed by reverse phase chromatography. Filtration, precipitation and lyophilization were used to complete the synthesis of the target molecule.

[0192] For example, mutations of conjugate 1 (human insulin analogue moiety) compared to human insulin were generated at positions B16, B25 and / or A14. Table 1 shows an overview of the insulins generated.

[0193] [Table 4]

[0194] Example 2: Insulin receptor binding affinity assay / insulin receptor autophosphorylation assay Insulin binding and signaling of the various insulin conjugates produced were determined by binding and receptor autophosphorylation assays.

[0195] A) Insulin receptor binding affinity assay Insulin receptor binding affinity for the conjugates listed in Table 4 was determined as described by Hartmann et al. (Effect of the long-acting insulin conjugates glargine and degludec on cardiomyocyte cell signaling and function. Cardiovasc Diabetol. 2016;15:96): Isolation of plasma membrane embedded insulin receptor (M-IR) and competitive binding experiments were performed as previously described (Sommerfeld et al., PLoS One. 2010;5(3):e9540). Briefly, CHO cells overexpressing IR were harvested and resuspended in ice-cold 2.25 STM buffer (2.25M sucrose, 5mM Tris-HCl pH7.4, 5mM MgCl2, Complete protease inhibitors), disrupted using a Dounce homogenizer, and then sonicated. The homogenate was covered with 0.8 STM buffer (0.8 M sucrose, 5 mM Tris-HCl pH 7.4, 5 mM MgCl2, Complete protease inhibitors) and ultracentrifuged at 100,000 g for 90 min. The plasma membranes at the interface were collected and washed twice with phosphate-buffered saline (PBS). The final pellet was resuspended in dilution buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, Complete protease inhibitors) and homogenized again with a Dounce homogenizer. Competitive binding experiments were performed in binding buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% BSA, Complete protease inhibitors, adjusted to pH 7.8) in 96-well microplates. In each well, 2 μg of isolated membranes were incubated with 0.25 mg of wheat germ agglutinin polyvinyltoluene polyethyleneimine scintillation proximity assay (SPA) beads. A constant concentration of [125I]-labeled human Ins (100 pM) and various concentrations of each unlabeled Ins (0.001–1000 nM) were added for 12 h at room temperature (23°C). Radioactivity was measured at equilibrium by a microplate scintillation counter (Wallac Microbeta, Freiburg, Germany).

[0196] The results of the insulin receptor binding affinity assay for the tested conjugates to human insulin are shown in Table 5.

[0197] B) Insulin receptor autophosphorylation assay (as a measure of signal transduction) To determine the signaling effect of insulin conjugate binding to insulin receptor B, autophosphorylation was measured in vitro.

[0198] CHO cells expressing human insulin receptor isoform B (IR-B) were used for IR autophosphorylation assays using the In-Cell Western technique as previously described (Sommerfeld et al., PLoS One. 2010;5(3):e9540). For analysis of IGF1R autophosphorylation, the receptor was overexpressed in mouse embryonic fibroblast 3T3 Tet off cell line (BD Bioscience, Heidelberg, Germany) stably transfected with an IGF1R tetracycline-regulated expression plasmid. To determine receptor tyrosine phosphorylation levels, cells were seeded in 96-well plates and grown for 44 h. Cells were serum-starved for 2 h with serum-free medium Ham's F12 medium (Life Technologies, Darmstadt, Germany). Afterwards, cells were treated with increasing concentrations of either human insulin or insulin conjugates for 20 min at 37°C. After incubation, the medium was discarded and the cells were fixed in 3.75% freshly prepared paraformaldehyde for 20 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Blocking was performed with Odyssey blocking buffer (LICOR, Bad Homburg, Germany) for 1 h at room temperature. Anti-pTyr 4G10 (Millipore, Schwalbach, Germany) was incubated for 2 h at room temperature. After primary antibody incubation, cells were washed with PBS + 0.1% Tween 20 (Sigma-Aldrich, St Louis, MO, USA). Secondary anti-mouse IgG-800-CW antibody (LICOR, Bad Homburg, Germany) was incubated for 1 h. Results were normalized by quantification of DNA with TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained as relative units (RU).

[0199] The results of the insulin receptor autophosphorylation assay for the tested insulin conjugates to human insulin are shown in Table 5.

[0200] [Table 5]

[0201] C) Conclusion Conjugate 1 still exhibits sufficient receptor binding and autophosphorylation activity to demonstrate a pharmacological effect.

Claims

1. Formula (I) 【Chemistry 1】 and a human insulin analogue, wherein said human serum albumin binder of formula (I) is covalently attached to said human insulin analogue at a point where the terminal carboxy group "a" of said human serum albumin binder of formula (I) is covalently attached to the epsilon amino group of lysine B29 of said human insulin analogue via an amide bond.

2. 2. The insulin conjugate of claim 1, comprising a human insulin analogue having at least seven mutations relative to the parent insulin, optionally wherein the mutations are selected from the group consisting of substitutions, deletions and additions of amino acid residues.

3. a mutation at position A14 substituted with aspartic acid or glutamic acid, a mutation at position B16 substituted with histidine or glutamic acid, A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula:

2. The insulin conjugate of claim 1, optionally having an additional amino acid residue attached to the human insulin analog.

4. a mutation at position A14 that results in a substitution with aspartic acid or glutamic acid; a mutation at position B16 resulting in a substitution with glutamic acid, A mutation at position B25 that results in a substitution with histidine, and At least four additional substitutions and a human insulin analog having the formula: Optionally, all additional substitutions are to arginine, glycine, or a combination thereof; 2. The insulin conjugate of claim 1, optionally having an additional amino acid residue attached to the human insulin analog.

5. a mutation at position A14 that results in a substitution with aspartic acid or glutamic acid; a mutation at position B16 resulting in a substitution with glutamic acid, a mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all additional substitutions are to arginine; 2. The insulin conjugate of claim 1, optionally having an additional amino acid residue attached to the human insulin analog.

6. a mutation at position A14 that results in a substitution with aspartic acid or glutamic acid; a mutation at position B16 resulting in a substitution with glutamic acid, a mutation at position B25 that results in a substitution with histidine, A mutation at position A21 that is substituted with glycine or arginine, and At least three additional substitutions and a human insulin analog having the formula: Optionally, all additional substitutions are to arginine; 2. The insulin conjugate of claim 1, optionally having an additional arginine residue attached to the human insulin analog.

7. Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A21)Glu(B16)His(B25)Arg(B27)Arg(B31)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Arg(B31)-insulin (human insulin) 2. The insulin conjugate of claim 1, comprising a human insulin analog selected from the group consisting of:

8. Arg (A5) Arg (A9) Glu (A14) Arg (A15) Arg (A18) A rg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Glu(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Gly(A21)Arg(B3)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Glu(B16)His(B25)Arg(B27)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Gly(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A18)Arg(A21)Arg(B3)Glu(B16)His(B25)Des(B30)-insulin (human insulin); Arg(A5)Arg(A9)Asp(A14)Arg(A15)Arg(A18)Arg(A21)Glu(B16)His(B25)Des(B30)-insulin (human insulin) 2. The insulin conjugate of claim 1, comprising a human insulin analog selected from the group consisting of:

9. Conjugate 1 (A chain sequence: SEQ ID NO: 13; B chain sequence: SEQ ID NO: 9): 【Chemistry 2】 or conjugate 2 (A chain sequence: SEQ ID NO: 11; B chain sequence: SEQ ID NO: 9): 【Transformation 3】 or conjugate 3 (A chain sequence: SEQ ID NO: 16; B chain sequence: SEQ ID NO: 9): 【Chemistry 4】 or conjugate 4 (A chain sequence: SEQ ID NO: 16; B chain sequence: SEQ ID NO: 8): 【Transformation 5】 2. The insulin conjugate of claim 1, wherein:

10. Conjugate 5 (A chain sequence: SEQ ID NO: 17; B chain sequence: SEQ ID NO: 3): 【Transformation 6】 or conjugate 6 (A chain sequence: SEQ ID NO: 18; B chain sequence: SEQ ID NO: 3): 【Transformation 7】 or conjugate 7 (A chain sequence: SEQ ID NO: 16; B chain sequence: SEQ ID NO: 3): 【Transformation 8】 2. The insulin conjugate of claim 1, wherein:

11. 4. The human insulin analogue of claim 3.

12. A pharmaceutical composition comprising a pharmaceutically effective amount of an insulin conjugate according to any one of claims 1 to 10 or an insulin analogue according to claim 11.

13. A pharmaceutical composition comprising an insulin conjugate according to any one of claims 1 to 10 or an insulin analogue according to claim 11 for use as a medicament.

14. 12. A pharmaceutical composition comprising an insulin conjugate according to any one of claims 1 to 10 or an insulin analogue according to claim 11 for use as a medicament for the treatment of a disease selected from the group consisting of gestational diabetes, type 1 diabetes, type 2 diabetes and hyperglycemia and / or for lowering blood glucose levels.

15. A pharmaceutical composition comprising an insulin conjugate according to any one of claims 1 to 10 for use as a medicament for the treatment of type 2 diabetes.

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