JP7634756B2 - Glucose-sensitive insulin derivatives - Google Patents

Description

The present invention relates to novel insulin derivatives and their pharmaceutical uses. Furthermore, the present invention relates to pharmaceutical compositions comprising such insulin derivatives and to the use of such compounds for the treatment or prevention of medical conditions associated with diabetes.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING SEQUENCE LISTING This application is submitted with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference.

Insulin is the most effective drug for treating hyperglycemia, but insulin dosage is a delicate balance between too much and too little due to the narrow physiological glucose range. Healthy people have glucose levels of approximately 5 mM in the fasting state, and diabetics attempt to obtain approximately 5 mM by administering both prandial and basal insulin preparations. However, blood glucose levels below about 3 mM (hypoglycemia) often occur during insulin treatment, and hypoglycemia can lead to discomfort, loss of consciousness, brain damage, or death. Thus, diabetics are hesitant to aggressively treat high or moderately high blood glucose levels for fear of hypoglycemia. The development of an insulin drug that is only active at higher blood glucose levels or released from a depot and is inactive or weakly active at lower blood glucose levels could aid in diabetes treatment.

U.S. Patent No. 5,399,433 discloses glucose-sensitive insulin derivatives having a diboron-containing moiety that exhibits glucose-sensitive albumin binding. These insulin derivatives therefore exhibit insulin activity that is dependent on glucose concentration. There is a need for glucose-sensitive insulin derivatives with longer half-lives to allow for less frequent administration.

International Publication No. 2020/201041

In its broadest aspect, the present invention relates to insulin derivatives that exhibit insulin activity that is dependent on glucose concentration and thus function as glucose-sensitive insulin derivatives.

The insulin derivatives of the present invention bind both glucose and albumin (human serum albumin, HSA), and the HSA affinity is glucose sensitive. Thus, the apparent human insulin receptor (HIR) affinity in the presence of HSA also becomes glucose sensitive. The fraction of HSA-bound insulin is shielded from binding to the HIR, but glucose-facilitated release from HSA increases the free fraction of insulin, and thus glucose increases the apparent affinity of the HIR. Thus, the insulin derivatives of the present invention exhibit higher apparent insulin receptor affinity in the presence of glucose and albumin than in the absence of glucose.

The insulin derivative of the present invention exhibits high glucose sensitivity and has a long half-life.

In one embodiment, the insulin derivative of the invention comprises an insulin peptide containing B29K, a peptide extension at the N-terminus of the insulin peptide B chain, and three modifying groups, one of which is linked to a Lys residue at position 29 of the insulin peptide B chain, and the other two modifying groups are linked to Lys residues in the peptide extension.

In one aspect the peptide extension is
Z-Lys-Y-Lys-aa1-aa2-(Gly) ₃ -Ser-((Gly) ₄ -Ser) _p -#
wherein Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
In the formula, aa1 is absent or Pro;
In the formula, aa2 is Glu or Gly;
In the formula, p is 1, 2, or 3;
where # indicates the point of attachment to the N-terminus of the B-chain of human insulin or a human insulin analogue.

In one aspect, the modifying group has formula A:
wherein one of R1, R2, or R3 is an electron-withdrawing group and the other two are hydrogen;
wherein m is 0, 1, 2, or 3, n is 0, 1, 2, or 3, and * indicates a connection point.

In one aspect, the present invention relates to a pharmaceutical composition comprising an insulin derivative according to the present invention. In one aspect, the present invention relates to an insulin derivative according to the present invention for use as a medicament. In one aspect, the present invention relates to an insulin derivative according to the present invention for use in the treatment of diabetes. In one aspect, the present invention relates to a medical use of an insulin derivative according to the present invention.

In one aspect, the insulin derivatives of the present invention exhibit high glucose sensitivity. In one aspect, the insulin derivatives of the present invention have a long half-life. In one aspect, the insulin derivatives of the present invention exhibit high glucose sensitivity and have a long half-life.

The present invention may also solve further problems that will become apparent from the disclosure of the exemplary embodiments.

FIG. 1 shows the concentration-response curves of insulin receptor phosphorylation in human primary hepatocytes for the insulin derivative of Example 4 and human insulin at 3 mM and 20 mM D-glucose, respectively (see Example 13). FIG. 2 shows representative concentration-response curves from a lipogenesis assay (rFFC) in adipocytes from Sprague Dawley rats for the insulin derivative of Example 4 at 3 mM and 20 mM L-glucose, respectively (see Example 14). FIG. 3 shows the concentration-response curves of glycogen accumulation in rat primary hepatocytes for the insulin derivative of Example 4 and human insulin at 20 mM D-glucose (see Example 15).

In one embodiment, the insulin derivative of the invention comprises an insulin peptide, a peptide extension at the N-terminus of the insulin peptide B chain, and three modifying groups.

Insulin Derivatives As used herein, the term "insulin derivative" refers to modified insulin peptides, the modification being in the form of the attachment of chemical moieties and/or the presence of peptide extensions.

In one aspect, the modification is in the form of a covalent attachment of a modifying group of formula A. In one aspect, the modification is in the form of the presence of a peptide extension at the N-terminus of the insulin peptide B chain. In one aspect, the modification is in the form of a covalent attachment of a modifying group of formula A and the presence of a peptide extension at the N-terminus of the insulin peptide B chain.

Insulin Peptides As used herein, the term "insulin peptide" refers to a peptide that is either human insulin or a human insulin analog. In one embodiment, the term "insulin peptide" as used herein refers to a peptide that is either human insulin or an analog thereof that has insulin activity, i.e., activates the insulin receptor.

Human insulin As used herein, the term "human insulin" refers to the human insulin hormone, whose structure and properties are well known. Human insulin has two polypeptide chains, designated the A and B chains. The A chain is a peptide of 21 amino acids, and the B chain is a peptide of 30 amino acids, and the two chains are connected by disulfide bridges, the first bridge being between the cysteine at position 7 of the A chain and the cysteine at position 7 of the B chain, and the second bridge being between the cysteine at position 20 of the A chain and the cysteine at position 19 of the B chain. The third bridge is between the cysteine at position 6 of the A chain and the cysteine at position 11 of the A chain. Human insulin A-chain has the sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO:1), while human insulin B-chain has the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).

Insulin Analogs As used herein, the term "insulin analog" refers to modified human insulin in which one or more amino acid residues of insulin have been replaced by other amino acid residues and/or one or more amino acid residues have been deleted from insulin and/or one or more amino acid residues have been added and/or inserted into insulin. As used herein, the term "insulin analog" refers to an insulin analog that exhibits insulin activity, i.e., binds to and activates the insulin receptor.

The human insulin analog contains fewer than 10 amino acid modifications (substitutions, deletions, additions (i.e., lengthening), insertions, and any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin. In one aspect, the human insulin analog has fewer than 10 amino acid modifications (substitutions, deletions, additions (i.e., lengthening), insertions, and any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin.

Modifications in the insulin molecule are represented by stating the chain (A or B), the position, and the one-letter or three-letter code of the amino acid residue replacing the natural amino acid residue. As used herein, terms such as "A1", "A2", and "A3" represent the amino acids at positions 1, 2, and 3, etc., respectively, of the A chain of insulin (counting from the N-terminus). Similarly, terms such as B1, B2, and B3 represent the amino acids at positions 1, 2, and 3, etc., respectively, of the B chain of insulin (counting from the N-terminus). Using the one-letter code of amino acids, the term B29K specifies that the amino acid at position B29 is K. Using the three-letter code of amino acids, the corresponding expression is B29Lys. "desB30" refers to an insulin analog lacking the B30 amino acid.

In certain embodiments, an analog "has" or "contains" a particular change. In other particular embodiments, an analog "consists of" a change. When the terms "consisting of" or "consisting of" are used in reference to an analog, e.g., an analog consists of or consists of a particular group of amino acid mutations, it will be understood that the particular amino acid mutations are the only amino acid mutations in the analog. In contrast, an analog that "contains" a particular group of amino acid mutations may have additional mutations.

Examples of insulin analogues include:
desB30 human insulin (A chain of SEQ ID NO:1 and B chain of SEQ ID NO:3).

As mentioned above, the insulin analogues of the invention contain fewer than 10 amino acid modifications (substitutions, deletions, additions (i.e., extensions), insertions, and any combination thereof) relative to human insulin, or fewer than 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification relative to human insulin. In addition to these up to 9 modifications, the insulin peptides of the invention have a peptide extension at the N-terminus of the B-chain of the insulin peptide.

A variety of peptide extensions or spacer peptides are known in the art and may be used in the insulin derivatives of the present invention. In one embodiment, the peptide extension is a peptide segment of 28-58 amino acids connected via peptide bonds. The peptide extension of the present invention comprises one or more of the following amino acid residues: Gly (G), Glu (E), Ala (A), Ser (S), Thr (T), Pro (P), Gln (Q), and Lys (K).

The peptide extension in the insulin derivatives of the present invention contains two Lys (K) residues to allow for the attachment of two modifying groups. The exact location of the attachment of the modifying groups may vary, and some variation in the exact peptide sequence is permitted.

The peptide extension used in the insulin derivatives of the invention has the following peptide sequence:
Z-Lys-Y-Lys-aa1-aa2-(Gly) ₃ -Ser-((Gly) ₄ -Ser) _p -#
wherein Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
In the formula, aa1 is absent or Pro;
In the formula, aa2 is Glu or Gly;
In the formula, p is 1, 2, or 3;
where # indicates the point of attachment to the N-terminus of the B-chain of human insulin or a human insulin analogue.

In one embodiment, the peptide extension is kept generally polar, for example by having at least five glutamic acid (Glu) amino acid residues in the peptide extension.

When insulin is produced recombinantly, it is expressed as a single-chain insulin precursor, which is subsequently matured to two-chain insulin using a protease such as Achromobacter lyticus lysyl-specific endoprotease (ALP) or trypsin. ALP specifically and exclusively cleaves lysyl bonds, including lysyl-proline bonds. However, because the peptide extension used in the insulin derivatives of the present invention contains two lysine (Lys) residues, cleavage by ALP can result in undesirable overcleavage products. Surprisingly, it has been found that by having Pro-Glu after Lys, the rate of cleavage by ALP is greatly reduced, thus reducing overcleavage and increasing the overall yield.

In one embodiment the peptide extension used in the insulin derivative of the invention has the following peptide sequence:
Z-Lys-Y-Lys-aa1-aa2-(Gly) ₃ -Ser-((Gly) ₄ -Ser) _p -#
wherein Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
wherein Y consists of Pro-Gly followed by 13 to 28 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
In the formula, aa1 is Pro;
In the formula, aa2 is Glu;
In the formula, p is 1, 2, or 3;
where # indicates the point of attachment to the N-terminus of the B-chain of human insulin or a human insulin analogue.

More specifically, the peptide extension is linked to the insulin peptide via the carboxylic acid of the C-terminal serine residue of the peptide extension and the amino group of the N-terminal amino acid residue of the B chain of the insulin peptide, forming an amide bond between the two amino acid residues.

An example of a peptide extension at the N-terminus of the B-chain of insulin peptide is GKPE(GEQP) ₄ GEQGGKPEGGGS(G ₄ S) ₂ (SEQ ID NO: 4).

Modification Group A
An insulin derivative of the invention comprises three modifying groups A. In one embodiment an insulin derivative of the invention has exactly three modifying groups of formula A. The modifying groups may be the same or different. In one embodiment the modifying groups are the same. The modifying groups are covalently linked to the epsilon amino groups of lysine residues in the insulin peptide and in the peptide extension.

The modifying group of the insulin derivatives of the present invention has the formula A:
(wherein one of R1, R2, or R3 is an electron-withdrawing group and the other two are hydrogen;
In the formula, m is 0, 1, 2, or 3;
wherein n is 0, 1, 2, or 3.

Throughout the formulas of this application, B represents a boron atom, H represents a hydrogen atom, N represents a nitrogen atom, and O represents an oxygen atom.

In other words, R1, R2, and R3 are independently selected from the group of electron-withdrawing groups and hydrogen, provided that when R1 is an electron-withdrawing group, R2 and R3 are hydrogen, when R2 is an electron-withdrawing group, R1 and R3 are hydrogen, and when R3 is an electron-withdrawing group, R1 and R2 are hydrogen.

In one embodiment, R1 is an electron-withdrawing group and R2 and R3 are hydrogen.

Electron-withdrawing groups are attached to adjust the pKa of boronic acids and boroxoles to facilitate glucose binding at neutral pH.

An electron withdrawing group (EWG) is an atom or group that attracts electron density from adjacent atoms toward itself. In one embodiment, the electron withdrawing group is selected from the group of _CF3 , F, _NO2 , CN, COX, _SO2X , and _POX2 , where X is OR or _NR2 , and R is independently selected from H, alkyl, and aryl. In one embodiment, the electron withdrawing group is selected from the group of _CF3 , F, _NO2 , CN, COX, and _SO2X , where X is OR or _NR2 , and R is independently selected from H, alkyl _, and aryl. In one embodiment, the electron withdrawing group is _CF3 , F, _SO2NR2 , or CN, where R is alkyl. In one embodiment, the electron withdrawing group is _CF3 . In one embodiment, R1 is _CF3 , and R2 and R3 are hydrogen.

In the insulin derivative of the present invention, one modifying group is linked to the lysine (Lys) residue at position 29 of the B chain of the insulin peptide, and the other two modifying groups are linked to each of the lysine (Lys) residues in the peptide extension. More specifically, each modifying group is linked to the epsilon amino group of a lysine forming an amide bond.

Formula A is covalently linked to Lys via the attachment point indicated by *.

The modifying group of formula A has two chiral centers. Each of the chiral atoms in the modifying group of formula A may be independently in the (R) or (S) form. In one embodiment, both chiral atoms are in the (S) form. In yet another embodiment, the chiral center is of formula A1:
As shown in.
R1, R2, R3, m and n are as defined above for Formula A.

In certain embodiments, the modifying group is of formula A, where R1 is _CF3 , R2 and R3 are hydrogen, m is 2, and n is 0.

In certain embodiments, the modifying group is of formula A1, where R1 is _CF3 , R2 and R3 are hydrogen, m is 2, and n is 0.

Function of the Insulin Derivatives of the Invention Surprisingly, it was found that the insulin derivatives of the invention exhibit both a long half-life and high glucose sensitivity in the presence of HSA.

The relative binding affinities of insulin analogs and insulin derivatives to the human insulin receptor (HIR) can be determined by competitive binding in a scintillation proximity assay (SPA) as described in Example 12.

In one embodiment, the insulin derivative of the present invention has the ability to bind to the insulin receptor. In one embodiment, the insulin derivative of the present invention has a higher apparent insulin receptor affinity in the presence of HSA and 20 mM glucose than in the absence of glucose. The increase in the apparent relative affinity (HIR glucose coefficient) from 0 to 20 mM glucose in the presence of HSA reflects the glucose sensitivity of the insulin derivative. If the relative insulin receptor affinity is higher in the presence of 20 mM glucose compared to the absence of glucose, the glucose coefficient is greater than 1. In one embodiment, the insulin derivative of the present invention has a glucose coefficient of at least 20, 30, or 40 in the presence of 1.5% HSA.

As can be seen from the data presented in Example 12, the peptide extension at the N-terminus of the B chain of the insulin backbone results in a decrease in glucose sensitivity in the presence of HSA, as measured by the fold change in affinity (glucose coefficient) from 0 to 20 mM glucose. Surprisingly, the insulin derivatives of the present invention were found to exhibit high glucose sensitivity in the presence of HSA.

The insulin receptor phosphorylation assay described in Example 13, the lipogenesis assay described in Example 14, and the glycogen accumulation assay described in Example 15 can be used as measures of functional (agonist) activity of the insulin derivatives. As can be seen from the data presented in Examples 13, 14, and 15, the insulin derivatives of the present invention activate the insulin receptor. The half-life (T1/2) upon subcutaneous administration to LYD pigs can be determined using the method described in Example 16. As can be seen from the pharmacokinetic (PK) study in LYD pigs in Example 16, the compounds of the present invention exhibit extended half-life (T1/2).

Intermediate Products The present invention further provides intermediate products in the form of insulin analogues having a peptide extension at the N-terminus of the insulin peptide.

Examples of insulin analogues having a peptide extension at the N-terminus of the B-chain of the insulin peptide include:
and GKPE(GEQP) ₄ GEQGGKPEGGGS(G ₄ S) ₂ -B1 desB30 human insulin (A chain of SEQ ID NO:1 and B chain of SEQ ID NO:5).

GKPE(GEQP) ₄ GEQGGKPEGGGS(G ₄ S) ₂ -B1 desB30 human insulin means desB30 human insulin extended from B1 with GKPE(GEQP) ₄ GEQGGKPEGGGS(G ₄ S) _2. The C-terminal serine (S) is connected to the phenylalanine (F) in position B1 of desB30 human insulin.

General Definitions The term "compound" is used herein to refer to a molecular entity, and therefore a "compound" may have different structural elements other than the minimal elements defined for each compound or group of compounds. The term "compound" is also meant to encompass pharma- ceutically relevant forms of the compounds herein, i.e., the present invention relates to the compounds defined herein, or their pharma- ceutically acceptable salts, amides, or esters.

The term "peptide" or "polypeptide", for example, as used in the context of the present invention, refers to a compound that includes a series of amino acids interconnected by amide (or peptide) bonds. In certain embodiments, a peptide consists of amino acids interconnected by peptide bonds.

The term "amino acid" includes proteinogenic (or naturally occurring) amino acids (among which the 20 standard amino acids) as well as non-proteinogenic (or unnatural) amino acids. Proteinogenic amino acids are those that are naturally incorporated into proteins. Standard amino acids are those that are encoded by the genetic code. Non-proteinogenic amino acids are either not found in proteins or are not produced by standard cellular mechanisms (e.g., they may have been subject to post-translational modifications).

In general, amino acid residues (peptide/protein sequences) can be identified by their full name, their one-letter code, and/or their three-letter code. These three methods are fully equivalent. Throughout this application, each amino acid of the insulin peptides and peptide extensions of the invention for which no optical isomer is described should be understood to mean the L-isomer (unless otherwise specified). Amino acids are molecules containing an amino group and a carboxylic acid group and, optionally, one or more additional groups, often referred to as side chains.

As used herein, the term "amino acid residue" refers to an amino acid that has formally had a hydroxy group removed from its carboxy group and/or a hydrogen atom removed from its amino group.

As used herein, the term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms. Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and tetradecyl.

The term "aryl" means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl.

The terms "comprise" or "comprising" certain features are to be interpreted as meaning that the subject matter in question includes those particular features, but does not exclude the presence of other features.

Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising an insulin derivative of the present invention, or a pharma- ceutically acceptable salt, amide, or ester thereof, and one or more pharma- ceutically acceptable excipients. Such compositions may be prepared as known in the art.

The term "excipient" broadly refers to any ingredient other than the active therapeutic ingredient. An excipient may be an inactive, non-active, and/or non-pharmaceutical active substance. Excipients may serve various purposes, for example, as carriers, vehicles, diluents, and/or function to improve administration and/or absorption of the active ingredient. Non-limiting examples of excipients are solvents, diluents, buffers, preservatives, tonicity adjusting agents, chelating agents, and stabilizers. Formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005) and any subsequent editions).

The compositions of the invention may be in the form of a liquid formulation, i.e. an aqueous formulation comprising water. The liquid formulation may be a solution or a suspension. The compositions of the invention may be for parenteral administration, for example, by subcutaneous, intramuscular, intraperitoneal or intravenous injection.

Aryl boron compounds may have low stability in aqueous solutions at near-neutral pH. The insulin derivatives of the present invention exhibit improved stability in aqueous solutions. Stability can be assessed, for example, by measuring the purity of the insulin derivative after standing in aqueous solution at neutral pH at 25° C. or 37° C. for an extended period of time, e.g., one week.

Pharmaceutical Indications Diabetes The term "diabetes" or "diabetes mellitus" includes type 1 diabetes, type 2 diabetes, gestational diabetes (during pregnancy), and other conditions that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas produces insufficient amounts of insulin or the body's cells fail to respond properly to insulin, thus preventing the cells from absorbing glucose. As a result, glucose accumulates in the blood.

Type 1 diabetes, also called insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes, is usually caused by beta cell destruction resulting in absolute insulin deficiency.

Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) and adult-onset diabetes, is associated with primary insulin resistance and therefore primary insulin secretion failure with relative insulin deficiency and/or insulin resistance.

In one embodiment, the insulin derivative according to the invention is used as a medicine for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes such as metabolic syndrome X or insulin resistance syndrome. In one embodiment, the insulin derivative according to the invention is used as a medicine for the treatment or prevention of hyperglycemia, including stress-induced hyperglycemia, type 2 diabetes, impaired glucose tolerance, or type 1 diabetes. In another embodiment, the insulin derivative according to the invention is used as a medicine for delaying or preventing disease progression in type 2 diabetes. The term "treatment" is meant to include both prevention and minimization of the disease, disorder, or condition referred to (i.e., "treatment" refers to both prophylactic and therapeutic administration of the insulin derivative of the invention or a composition comprising the insulin derivative of the invention, unless otherwise indicated by the context or clearly contradicted).

Methods of Administration The route of administration may be any route that effectively delivers the insulin derivative of this invention to a desired or suitable location in the body, for example parenterally (e.g., subcutaneously, intramuscularly, or intravenously).

For parenteral administration, the insulin derivatives of the present invention are formulated similarly to the formulation of known insulins. Furthermore, for parenteral administration, the insulin derivatives of the present invention may be administered similarly to the administration of known insulins, and physicians are familiar with this procedure.

The amount of insulin derivative of the present invention to be administered, the determination of the frequency with which the insulin derivative of the present invention is administered, and the choice of which compound of the present invention to administer, optionally together with another antidiabetic compound, are determined in consultation with a specialist familiar with the treatment of diabetes.

NON-LIMITING EMBODIMENTS The present invention is further illustrated by the following non-limiting embodiments of the invention.
1. An insulin derivative comprising:
A. An insulin peptide comprising B29K;
B. The peptide extension Z-Lys-Y-Lys-aa1-aa2-(Gly) ₃ -Ser-((Gly) ₄ -Ser) _p -#,
wherein Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln;
In the formula, aa1 is absent or Pro;
In the formula, aa2 is Glu or Gly;
wherein p is 1, 2, or 3;
where # indicates the attachment point to the N-terminus of the B chain of human insulin or a human insulin analogue.
C. three modifying groups of formula A;
wherein one of R1, R2, or R3 is an electron-withdrawing group and the other two are hydrogen;
wherein m is 0, 1, 2, or 3;
wherein n is 0, 1, 2, or 3;
In the formula, * indicates a connection point,
An insulin derivative in which one modifying group is linked to the Lys residue at position 29 of the B-chain of the insulin peptide and two other modifying groups are linked to each of the Lys residues in the peptide extension.
2. An insulin derivative according to the preceding embodiment, wherein Z consists of 1 to 4 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
3. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 3 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
4. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 2 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gin.
5. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of one amino acid residue independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gin.
6. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 4 Gly amino acid residues.
7. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 3 Gly amino acid residues.
8. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 2 Gly amino acid residues.
9. An insulin derivative according to any one of the preceding embodiments, wherein Z is GIy.
10. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
11. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
12. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
13. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
14. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
15. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 15 to 30 amino acid residues independently selected from Gly, Ala, Pro, and Gln.
16. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 20 to 25 amino acid residues independently selected from Gly, Ala, Pro, and Gln.
17. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of 23 amino acid residues independently selected from Gly, Ala, Pro, and Gln.
18. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 18 to 23 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
19. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 20 to 22 amino acid residues independently selected from Gly, Glu, Ala, Ser, Thr, Pro, and Gln.
20. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 21 amino acid residues independently selected from Gly, Ala, Ser, Thr, Pro, and Gln.
21. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 18 to 23 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
22. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 20 to 22 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
23. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Pro-Gly followed by 21 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
24. An insulin derivative according to any one of the preceding embodiments, wherein Y is PE(GEQP) ₄ GEQGG (SEQ ID NO: 13).
25. An insulin derivative according to any one of the preceding embodiments, wherein aa1 is Pro.
26. An insulin derivative according to any one of the preceding embodiments, wherein aa2 is GIu.
27. An insulin derivative according to any one of the preceding embodiments, wherein p is 2.
28. An insulin derivative according to any one of the preceding embodiments, wherein the peptide extension is GKPE(GEQP) ₄ GEQGGKPEGGGS(G ₄ S) ₂ (SEQ ID NO: 4).
29. The modifying group has the formula A1:
wherein one of R1, R2, or R3 is an electron-withdrawing group and the other two are hydrogen;
wherein m is 0, 1, 2, or 3;
wherein n is 0, 1, 2, or 3;
Insulin derivative according to any one of the preceding embodiments, wherein:
30. An insulin derivative according to any one of the previous embodiments, wherein the electron withdrawing group is selected from the group: _CF3 , F, _NO2 , CN, COX, _SO2X , and _POX2 , where X is OR or _NR2 and R is H, alkyl or aryl.
31. An insulin derivative according to any one of the previous embodiments, wherein the electron withdrawing group is selected from the group of _CF3 , F, _NO2 , CN, COX, and _SO2X , where X is OR or _NR2 and R is H, alkyl or aryl.
32. An insulin derivative according to any one of the preceding embodiments, wherein X is OR.
33. An insulin derivative according to any one of the preceding embodiments, wherein X is _NR2 .
34. An insulin derivative according to any one of the preceding embodiments, wherein R is H.
35. An insulin derivative according to any one of the preceding embodiments, wherein R is alkyl.
36. An insulin derivative according to any one of the preceding embodiments, wherein alkyl is selected from the group of methyl, ethyl, n-propyl, and isopropyl.
37. An insulin derivative according to any one of the preceding embodiments, wherein alkyl is selected from the group of methyl and ethyl.
38. An insulin derivative according to any one of the preceding embodiments, wherein alkyl is methyl.
39. An insulin derivative according to any one of the preceding embodiments, wherein R is aryl.
40. An insulin derivative according to any one of the preceding embodiments, wherein aryl is phenyl.
41. An insulin derivative according to any one of the previous embodiments, wherein the electron withdrawing group is selected from the group CF ₃ , F, SO ₂ NR ₂ , or CN, where R is alkyl.
42. An insulin derivative according to any one of the preceding embodiments, wherein the electron withdrawing group is selected from the group of _CF3 and F.
43. An insulin derivative according to any one of the preceding embodiments, wherein the electron withdrawing group is _CF3 .
44. An insulin derivative according to any one of the preceding embodiments, wherein R1 is an electron-withdrawing group, and R2 and R3 are hydrogen.
45. An insulin derivative according to any one of the preceding embodiments, wherein R1 is _CF3 and R2 and R3 are hydrogen.
46. An insulin derivative according to any one of the preceding embodiments, wherein m is 0, 1, 2, or 3, and n is 0.
47. An insulin derivative according to any one of the preceding embodiments, wherein m is 1, 2, or 3 and n is 0.
48. An insulin derivative according to any one of the preceding embodiments, wherein m is 3 and n is 0.
49. An insulin derivative according to any one of the preceding embodiments, wherein m is 2 and n is 0.
50. An insulin derivative according to any one of the preceding embodiments, wherein m is 1 and n is 0.
51. An insulin derivative according to any one of the preceding embodiments, wherein m is 0 and n is 3.
52. An insulin derivative according to any one of the preceding embodiments, wherein m is 0 and n is 2.
53. An insulin derivative according to any one of the preceding embodiments, wherein m is 0 and n is 1.
54. An insulin derivative according to any one of the preceding embodiments, wherein both chiral atoms in formula A are in the (S) form.
55. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is human insulin.
56. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue.
57. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than 10 amino acid modifications relative to human insulin.
58. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than 9 amino acid modifications relative to human insulin.
59. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than eight amino acid modifications relative to human insulin.
60. An insulin derivative according to any one of the previous embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than seven amino acid modifications relative to human insulin.
61. An insulin derivative according to any one of the previous embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than six amino acid modifications relative to human insulin.
62. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than five amino acid modifications relative to human insulin.
63. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than four amino acid modifications relative to human insulin.
64. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than three amino acid modifications relative to human insulin.
65. An insulin derivative according to any one of the previous embodiments, wherein the insulin peptide is a human insulin analogue containing fewer than two amino acid modifications relative to human insulin.
66. An insulin derivative according to any one of the previous embodiments, wherein the insulin peptide is a human insulin analogue comprising desB30.
67. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide is desB30 human insulin.
68. The insulin peptide is desB30 human insulin;
The peptide extension is Z-Lys-Y-Lys-Pro-Glu-(Gly) ₃ -Ser-((Gly) ₄ -Ser) _p -#;
wherein Z consists of 1 to 5 amino acid residues independently selected from Gly, Glu, Pro, and Gln;
wherein Y consists of Lys-Pro followed by 13 to 23 amino acid residues independently selected from Gly, Glu, Pro, and Gln;
wherein p is 1, 2, or 3;
where # indicates the attachment point to the N-terminus of the B chain of human insulin or a human insulin analogue.
An insulin derivative according to any one of the preceding embodiments, wherein R1 is _CF3 , R2 and R3 are hydrogen, m is 2 and n is 0.
69. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of 1 to 3 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
70. An insulin derivative according to any one of the preceding embodiments, wherein Z consists of Gly optionally followed by 1 to 2 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
71. An insulin derivative according to any one of the preceding embodiments, wherein Z is GIy.
72. An insulin derivative according to any one of the preceding embodiments, wherein Y consists of Lys-Pro followed by 20 to 25 amino acid residues independently selected from Gly, Glu, Pro, and Gln.
73. An insulin derivative according to any one of the preceding embodiments, wherein p is 2.
74. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide has the ability to bind to the insulin receptor.
75. An insulin derivative according to any one of the preceding embodiments, wherein the insulin peptide has the ability to bind to and activate the insulin receptor.
76. An insulin derivative according to any one of the preceding embodiments, wherein the insulin derivative has the ability to bind to an insulin receptor.
77. An insulin derivative according to any one of the preceding embodiments, wherein the insulin derivative has the ability to activate the insulin receptor.
78. An insulin derivative according to any one of the preceding embodiments, wherein the insulin derivative has a higher apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) than in the absence of glucose.
79. An insulin derivative according to any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is at least 20-fold higher than in the absence of glucose.
80. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is at least 30-fold higher than in the absence of glucose.
81. An insulin derivative according to any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is at least 40-fold higher than in the absence of glucose.
82. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is 20- to 80-fold higher than in the absence of glucose.
83. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is 30- to 60-fold higher than in the absence of glucose.
84. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is 40- to 60-fold higher than in the absence of glucose.
85. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has an apparent insulin receptor affinity in the presence of 20 mM glucose and human serum albumin (HSA) that is 50- to 60-fold higher than in the absence of glucose.
86. An insulin derivative according to any one of the preceding embodiments, wherein the insulin receptor affinity is determined by the assay described in Example 12.
87. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of at least 15 hours when administered subcutaneously to LYD pigs.
88. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of at least 16 hours when administered subcutaneously to LYD pigs.
89. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of at least 17 hours when administered subcutaneously to LYD pigs.
90. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of at least 18 hours when administered subcutaneously to LYD pigs.
91. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of at least 19 hours when administered subcutaneously to LYD pigs.
92. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of 15-25 hours when administered subcutaneously to LYD pigs.
93. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of 17-23 hours when administered subcutaneously to LYD pigs.
94. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative has a T1/2 of 18-23 hours when administered subcutaneously to LYD pigs.
95. The insulin derivative of any one of the preceding embodiments, wherein T1/2 is determined by the method described in Example 16.
96. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is the insulin derivative of Example 4.
97. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is:
98. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is:
99. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is:
100. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is:
101. The insulin derivative of any one of the preceding embodiments, wherein the insulin derivative is:
102. An insulin derivative, which is the insulin derivative of Example 4.
103. An insulin derivative, wherein the insulin derivative is:
104. A composition comprising an insulin derivative according to any one of embodiments 1 to 103.
105. An insulin derivative according to any one of embodiments 1-103 for use as a medicament.
106. An insulin derivative according to any one of embodiments 1 to 103 for use in the prevention or treatment of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes, such as metabolic syndrome X or insulin resistance syndrome.
107. An insulin derivative according to any one of embodiments 1-103 for use in the prevention or treatment of diabetes, including type 1 diabetes and type 2 diabetes.
108. An insulin derivative according to any one of embodiments 1-103 for use in the prevention or treatment of diabetes.
109. An insulin derivative according to any one of embodiments 1-103 for use in the treatment of diabetes.
110. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 for the manufacture of a medicament for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes, such as metabolic syndrome X or insulin resistance syndrome.
111. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 for the manufacture of a medicament for the treatment or prevention of diabetes, including type 1 diabetes and type 2 diabetes.
112. Use of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104 for the manufacture of a medicament for the treatment or prevention of diabetes.
113. A method for the treatment or prevention of diabetes, type 1 diabetes, type 2 diabetes, impaired glucose tolerance, hyperglycemia, and metabolic syndromes, such as metabolic syndrome X or insulin resistance syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.
114. A method for the treatment or prevention of diabetes, including type 1 diabetes and type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.
115. A method for the treatment or prevention of diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of an insulin derivative according to any one of embodiments 1 to 103 or a composition according to embodiment 104.

The insulin conjugates in the examples are depicted using standard single letter abbreviations for amino acids. The sulfur atoms of cysteine residues are specifically depicted to illustrate disulfide bridges. Residues that are modified by conjugation are depicted to show the exact location where the modification was made on the relevant amino acid. As is standard in peptide chemistry, the N-terminus of insulin is indicated with small letters H- and the C-terminus with small letters -OH. H- and -OH are not used when the terminal residue is modified by conjugation, in which case the residue is depicted expanded as above.
Materials and Methods Abbreviations List

Preparation of insulin mutants [Example 1]
[Expression of insulin mutants in yeast and transformation with ALP, etc.]
Insulin analogs were expressed in yeast using well-known techniques, for example as disclosed in WO 2017/032798. More specifically, insulin analogs were expressed as single-chain precursors that were isolated by ion exchange capture and cleaved into two-chain insulin analogs by treatment with ALP as described below.

Capture of precursors on SP Sepharose BB:
Yeast supernatant was loaded onto a column packed with SP Sepharose BB at a flow rate of 10-20 CV/hr. Washes with 0.1 M citric acid, pH 3.5, and 40% EtOH were performed. Analogs were eluted with 0.2 M sodium acetate pH 5.5/35% EtOH.

ALP Digestion:
The solution of single-chain precursor was adjusted to pH 9 and ALP enzyme was added at 1:100 (w/w). The reaction was followed by UHPLC. In preparation for RP-HPLC purification, the ALP cleavage pool was adjusted to pH 2.5 and diluted 2-fold.

RP-HPLC purification:
Purification was carried out by RP-HPLC C18 as follows:
Column: 15um C18 50x250mm 200Å
Buffer solutions:
A: 0.2% formic acid, 5% EtOH,
B: 0.2% formic acid, 50% EtOH
Gradient: 20-55% B buffer.
Gradient: 20CV
Flow rate 20 CV/hr Loading g approx. 5 g/L Resin fractions were analyzed by UHPLC, pooled and lyophilized.

The insulin analogues prepared and used in the examples are:
GKPE-(GEQP) _4- GEQGGKPEGGGSGGGGSGGGGGS-B1 desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 5)
(GQEP) ₄ -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 6)
(GQEP) -GQEGGKPGGGGSGGGGSGGGGS- _B1 desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 7)
(GEQP) ₄ -GEQGGKPEGGGSGGGGSGGGGGS-B1 desB30 human insulin (SEQ ID NO: 1 and SEQ ID NO: 8).
GKPE-(GEQP) ₄ -GEQG-GKPEGGGSGGGGSGGGGGS-B1 desB30 human insulin means desB30 human insulin extended from B1 with GKPE-GEQPGEQPGEQPGEQP-GEQG-GKPEGGGSGGGGSGGGGGS (C-terminal S connected to B1F) and similar for the other backbones.

Preparation of building blocks [Example 2]
[(S)-4-((S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid]
NBS (34.0 g, 191 mmol) was added to a solution of 3-trifluoromethyl-4-methylbenzoic acid (39.0 g, 191 mmol) in concentrated sulfuric acid (400 mL) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was then poured into ice water (2 L). The resulting precipitate was filtered off, washed with water (500 mL), dissolved in ethyl acetate (400 mL), dried over anhydrous sodium sulfate, filtered and evaporated to give 3-bromo-4-methyl-5-trifluoromethylbenzoic acid as a white solid. Yield: 53.4 g (98%). ¹ H NMR spectrum (300 MHz, DMSO-d6, δH): 13.71 (bs, 1H); 8.35 (d, J=0.4 Hz, 1H); 8.15 (d, J=0.9 Hz, 1H); 2.56 (s, 3H).

Concentrated sulfuric acid (24 mL) was added to a solution of 3-bromo-4-methyl-5-trifluoromethylbenzoic acid (35.0 g, 124 mmol) in methanol (500 mL) and the reaction mixture was stirred at reflux for 4 h and at room temperature for 16 h. The reaction mixture was then evaporated under reduced pressure, dissolved in diethyl ether (250 mL) and washed with water (2×100 mL) and a mixture of a saturated solution of potassium carbonate (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate as a white solid. Yield: 35.3 g (96%). ^1H NMR spectrum (300MHz, DMSO-d6, δH): 8.36 (d, J=1.1 Hz, 1H); 8.13 (d, J=1.1 Hz, 1H); 3.90 (s, 3H); 2.55 (d, J=1.3 Hz, 3H).

A suspension of NBS (31.7 g, 178 mmol) and methyl 3-bromo-4-methyl-5-trifluoromethylbenzoate (35.3 g, 119 mmol) in water (300 mL) was stirred under a 100 W bulb at 80° C. for 6 h. The reaction mixture was extracted with diethyl ether (2×200 mL). The organic layer was washed with brine (150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and evaporated to give methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate as a yellow solid. Yield: 44.0 g (98%). ¹ H NMR spectrum (300 MHz, CDCl ₃ , δH): 8.47 (d, J=1.5 Hz, 1H); 8.31 (d, J=1.3 Hz, 1H); 4.75 (s, 2H); 3.98 (s, 3H).

A solution of methyl 3-bromo-4-bromomethyl-5-trifluoromethylbenzoate (44.0 g, 117 mmol) and potassium acetate (22.9 g, 234 mmol) in MeCN (0.5 L) was stirred at 75° C. overnight. The suspension was filtered through filter paper and evaporated. The crude product was dissolved in dichloromethane and filtered again. Evaporation gave methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate as a white solid. Yield: 37.9 g (91%). ¹ H NMR spectrum (300 MHz, CDCl ₃ , δH): 8.49 (d, J=1.3 Hz, 1H); 8.34 (d, J=1.3 Hz, 1H); 5.37 (s, 2H); 3.99 (s, 3H); 2.11 (s, 3H).

A solution of methyl 4-(acetoxymethyl)-3-bromo-5-(trifluoromethyl)benzoate (37.9 g, 107 mmol), bis(pinacolato)diboron (29.8 g, 117 mmol), potassium acetate (31.4 g, 294 mmol), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.57 g, 1.92 mmol) in anhydrous tetrahydrofuran (500 mL) was stirred under an argon atmosphere for 13 days at 75° C. The reaction mixture was then cooled to room temperature, filtered, and evaporated. The crude product was filtered through a silica gel column (Silicagel, 0.063-0.200 mm, eluent: cyclohexane/ethyl acetate 8:1) to give methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)benzoate. Yield: 31.1 g (72%). RF (SiO ₂ , cyclohexane/ethyl acetate 8:1): 0.40. ¹ H NMR spectrum (300 MHz, CDCl ₃ , δH): 8.65 (s, 1H); 8.43 (s, 1H); 5.48 (s, 2H); 3.97 (s, 3H); 2.05 (s, 3H); 1.36 (s, 12H).

A solution of methyl 4-(acetoxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)benzoate (31.0 g, 77.1 mmol) and sodium hydroxide (15.4 g, 386 mmol) in water (300 mL) was stirred at room temperature for 3 h. Then a solution of hydrochloric acid (35 mL) in water (100 mL) was added to lower the pH to 1. The reaction mixture was stirred overnight. The precipitate was filtered off and dried to give 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid as a white solid. Yield: 16.6 g (86%).
^1H NMR spectrum (300 MHz, DMSO-d6, δH): 13.47 (bs, 1H); 9.66 (s, 1H); 8.62 (s, 1H); 8.24 (s, 1H); 5.22 (s, 2H).

A solution of pentafluorophenol (7.48 g, 40.7 mmol), 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylic acid (10.0 mg, 40.7 mmol), and N,N'-dicyclohexylcarbodiimide (DCC, 8.37 mg, 40.7 mmol) in MeCN (0.5 L) was stirred at room temperature overnight. The reaction mixture was filtered, evaporated, dissolved in MeCN, refiltered, and evaporated to give pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate as a white solid.
Yield: 16.7 g (100%). ¹ H NMR spectrum (300 MHz, DMSO-d6, δH): 9.79 (s, 1H); 8.86 (s, 1H); 8.46 (s, 1H); 5.30 (s, 2H).

2-Chlorotrityl chloride resin 100-200 mesh 1.5 mmol/g (3, 4.47 g, 6.71 mmol) was left to swell in anhydrous dichloromethane (30 mL) for 30 minutes. A solution of (2S)-5-(tert-butoxy)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-5-oxopentanoic acid (Fmoc-Glu-OtBu, 1.90 g, 4.47 mmol) and N,N-diisopropylethylamine (2.96 mL, 17.0 mmol) in anhydrous dichloromethane (30 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and treated with a solution of N,N-diisopropylethylamine (1.56 mL, 8.95 mmol) in a methanol/dichloromethane mixture (4:1, 2 x 5 min, 2 x 40 mL). The resin was then washed with DMF (2 x 30 mL), dichloromethane (2 x 40 mL) and DMF (3 x 40 mL). The Fmoc group was removed by treatment with 20% piperidine in DMF (1 x 5 min, 1 x 20 min, 2 x 40 mL). The resin was washed with DMF (3 x 40 mL), 2-propanol (2 x 40 mL) and dichloromethane (3 x 40 mL). A solution of (S)-2,3-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (Fmoc-Dap(Fmoc)-OH, 3.68 g, 6.71 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 2.55 g, 6.71 mmol), and 2,4,6-trimethylpyridine (1.60 mL, 12.1 mmol) in DMF (40 mL) was added to the resin and the mixture was shaken for 2 h. The resin was filtered and washed with DMF (2×40 mL), dichloromethane (2×40 mL), and DMF (2×40 mL). The Fmoc group was removed by treatment with 20% piperidine in DMF (1×5 min, 1×30 min, 2×40 mL). The resin was washed with DMF (3×40 mL), 2-propanol (2×40 mL) and dichloromethane (3×40 mL). A solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (5.53 g, 13.4 mmol) and triethylamine (4.99 mL, 35.8 mmol) in DMF (40 mL) was added to the resin and the mixture was shaken overnight. The resin was filtered and washed with DMF (6×40 mL) and dichloromethane (10×50 mL). The product was cleaved from the resin by treatment with 2,2,2-trifluoroethanol (60 mL) for 16 h. The resin was filtered off and washed with dichloromethane (4 x 50 mL). The crude product (4) was dried in vacuum and extracted with ethyl acetate (2 x 70 mL) and 1 M aqueous potassium hydrogen sulfate (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered and the solvent was evaporated. The crude product was then triturated in diethyl ether (20 mL) to give (S)-4-((S)-2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamido)-5-(tert-butoxy)-5-oxopentanoic acid as a beige solid. Yield: 1.89 g (57%). ^1H NMR spectrum (300MHz, AcOD-d4, δH): 8.50 (s, 1H); 8.46 (s, 1H); 8.29 (s, 1H); 8.26 (s, 1H); 5.28 (d, J = 2.6 Hz, 4H); 5.20 (t, J = 5.9 Hz, 1H); 4.55 (dd, J = 8.5 and 5.2 Hz, 1H); 4.08 (dd, J = 6.0 and 2.1 Hz, 2H); 2.57-2.42 (m, 2H); 2.34-2.16 (m, 1H); 2.17-2.08 (m, 1H); 1.47 (s, 9H). LC-MS: 746.3 (M+H)+.

[Example 3]
[(S)-3-(2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamide)propanoic acid = N ^alpha ,N ^beta -bis-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)-L-diaminopropionant]-beta-alanine]
A solution of L-diaminopropanoic acid hydrochloride ((S)-2,3-diaminopropanoic acid hydrochloride) (15.0 g, 107 mmol), di-tert-butyl dicarbonate (46.6 g, 214 mmol), and potassium bicarbonate (32.0 g, 320 mmol) in a mixture of MeCN (400 mL) and water (400 mL) was stirred overnight. The solvent was removed under reduced pressure and the residue was acidified with a saturated aqueous solution of potassium hydrogen sulfate until pH=1 was achieved. The reaction mixture was extracted with ethyl acetate (3×200 mL) and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give (S)-2,3-bis((tert-butoxycarbonyl)amino)propanoic acid as an off-white solid. Yield: 28.2 g (87%). ^1H NMR spectrum (300 MHz, _CDCl3 , δH): 5.85 (bs, 1H); 5.17 (bs, 1H); 4.31 (bs, 1H); 3.64-3.46 (m, 2H); 1.46 (s, 18H).

A solution of (S)-2,3-bis((tert-butoxycarbonyl)amino)propanoic acid (27.9 g, 91.7 mmol), tert-butyl 3-aminopropanoate (16.7 g, 91.7 mmol), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl, 21.1 g, 110 mmol), 1-hydroxy-7-azabenzotriazole (HOAt, 15.0 g, 110 mmol), and N,N-diisopropylethylamine (64.0 mL, 367 mmol) in dichloromethane (300 mL) was stirred overnight. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (600 mL), washed with 1 M aqueous hydrochloric acid (4×300 mL) and saturated aqueous sodium bicarbonate (4×300 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give tert-butyl (S)-3-(2,3-bis((tert-butoxy-carbonyl)amino)propanamido)propanoate as an off-white solid. Yield: 36.1 g (91%).
^1H NMR spectrum (300MHz, _CDCl3 , δH): 7.01 (bs, 1H); 5.75 (bs, 1H); 5.14 (bs, 1H); 4.15 (bs, 1H); 3.57-3.39 (m, 4H); 2.43 (t, J=6.0 Hz, 2H); 1.45 (s, 27H).

To a solution of tert-butyl (S)-3-(2,3-bis((tert-butoxycarbonyl)amino)propanamido)propanoate (36.1 g, 83.7 mmol) in dichloromethane (50 mL) was added 95% aqueous trifluoroacetic acid (300 mL) and the solution was stirred for 3 h. The solvent was removed under reduced pressure and the residue was coevaporated with MeCN (3×300 mL) and treated with 1 M hydrogen chloride solution in dry diethyl ether (300 mL). The precipitate was filtered off and triturated with MeCN (2×600 mL) to give (S)-3-((2-carboxyethyl)amino)-3-oxopropane-1,2-diaminium chloride as a white powder.
Yield: 22.2 g (100%). ¹ H NMR spectrum (300 MHz, DO, δH): 4.35 (t, J=5.8 Hz, 1H); 3.63-3.46 (m, 4H); 2.67 (t, J=6.6 Hz, 2H).

To a solution of (S)-3-((2-carboxyethyl)amino)-3-oxopropane-1,2-diaminium chloride (6.41 g, 24.3 mmol) and triethylamine (33.8 mmol, 243 mmol) in water (50 mL) was added a solution of pentafluorophenyl 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxylate (from Example 2, 20.0 g, 48.6 mmol) in 1,4-dioxane (100 mL) and the solution was stirred overnight. The reaction mixture was partitioned between ethyl acetate (300 mL) and 1 M aqueous potassium hydrogen sulfate solution (1500 mL). The organic layer was washed with 1 M aqueous potassium hydrogen sulfate solution (1×300 mL) and the solvent was removed under reduced pressure. The residue was triturated with diethyl ether (2×150 mL) and filtered. The solid was dissolved in 70% aqueous MeCN (600 mL) and lyophilized to give (S)-3-(2,3-bis(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxaborole-6-carboxamido)propanamide)propanoic acid as a white powder. Yield: 12.1 g (80%).
¹ H NMR spectrum (300MHz, AcOD-d4, δH): 8.51 (s, 1H); 8.47 (s, 1H); 8.29 (s, 1H); 8.27 (s, 1H); 5.28 (s , 4H); 5.15 (t, J = 6.1Hz, 1H); 4.15-3.99 (m, 2H); 3.61 (t, J = 6.4Hz, 2H); 2.67 (t, J = 6.3Hz, 2H). LC-MS: 632.0 (M+H)+.

Preparation of insulin derivatives The building blocks of Example 2 were activated with NHS/DIC in tetrahydrofuran and the tBu protecting group was removed by treatment with TFA, which was then evaporated prior to conjugation with insulin.

The building blocks of Example 3 were activated using NHS/DIC in MeCN prior to conjugation with insulin.

LCMS analysis was performed using a C18 column and 0.1% TFA in water as buffer A and 0.1% TFA in MeCN as buffer B.

LCMS of boron-insulin derivatives generally show various dehydrated species as major peaks, for example [M+nH-1×m _Mwater ] ⁿ⁺ for ionization state "n" with up to "m" number of boroxoles.

[Example 4]
GKPE-(GEQP) ₄ -GEQGGKPEGGGSGGGGSGGGGGS-B1 desB30 human insulin (536 mg, 0.057 mmol) was dissolved in 0.1 M Na ₂ HPO ₄ (5.6 mL) and DMSO (2.4 mL) and the pH was adjusted to 10.8 with 1.0 M aqueous NaOH. Example 2 building block (147 mg, 0.19 mmol) was NHS-activated and tBu-deprotected as described above, then dissolved in DMF (0.5 mL) and added dropwise to the given insulin solution over 10 min while the pH was kept near 10.8 by dropwise addition of 0.1 M NaOH. LCMS indicates the formation of the desired product. The mixture was diluted with MeCN and water and the pH was adjusted to 3.8 by dropwise addition of 1 M HCl. The product was purified by reversed-phase HPLC (RP-HPLC) on a C18 column using 0.1% TFA in water as buffer A and 0.1% TFA in MeCN as buffer B. The product was isolated by lyophilization. LCMS found 1894.1 [M+6H-5×water] ⁶⁺ , calculated 1894.1; 1623.6 [M+7H-5×water] ⁷⁺ , calculated 1623.6; 1420.8 [M+8H-5×water] ⁸⁺ , calculated 1420.8; and 1263.0 [M+9H-5×water] ⁹⁺ , calculated 1263.0 for C ₄₈₀ H ₆₆₆ B ₆ F ₁₈ N ₁₂₂ O ₁₆₉ S ₆ .

[Example 5]
[(Comparative Compound)]
The insulin derivative of example 5 was prepared similarly to the insulin derivative of example 4 from (GQEP) ₄ -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and the building blocks of example 2. LCMS of the product found 2049.0 [M+5H-3×water] ⁵⁺ , calculated 2049.0.

[Example 6]
[(Comparative Compound)]
The insulin derivative of example 6 was prepared similarly to the insulin derivative of example 4 from (GQEP) ₂₄ -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and the building blocks of example 2. LCMS of the product found 1847.8 [M+10H-3×water] ¹⁰⁺ , calculated 1847.8.

[Example 7]
[(Comparative Compound)]
The insulin derivative of example 7 was prepared similarly to the insulin derivative of example 4 from (GQEP) ₄ -GQEGGKPGGGGSGGGGSGGGGS-B1 desB30 human insulin and the building blocks of example 3. LCMS of the product found 2029.2 [M+5H-2×water] ⁵⁺ , calculated 2029.4.

[Example 8]
[(Comparative Compound)]
The insulin derivative of example 8 was prepared similarly to the insulin derivative of example 4 from (GEQP) ₄ -GEQGGKPEGGGSGGGGSGGGGGS-B1 desB30 human insulin and the building blocks of example 3. LCMS of the product found 2036.3 [M+5H-4×water] ⁵⁺ , calculated 2036.6.

[Example 9]
[(Prior art compound)]
The insulin derivative shown here is the compound of Example 324 in WO 2020/201041. Although this is a prior art compound, the structure is shown here for ease of reference.

[Example 10]
[Prior art compound)]
The insulin derivative shown here is the compound of Example 280 in WO 2020/201041. Although this is a prior art compound, the structure is shown here for ease of reference.

[Example 11]
[ALP cleavage speed]
When insulin is produced recombinantly, it is expressed as a single-chain precursor, which is subsequently matured to the two-chain insulin using a protease such as Achromobacter lyticus lysyl-specific endoprotease EC 3.4.21.50 (ALP) or trypsin. ALP specifically and exclusively cleaves lysyl bonds, including lysyl-proline bonds (Norioka & Sakiyama (1993) Lysine-specific serine protease from Achromobacter lyticus: Its substrate specificity and comparison with trypsin. Methods in Protein Sequence Analysis, pp. 101-106; Masaki et al. (1978) A New Proteolytic Enzyme from Achromobacter lyticus M497-I. Agricultural and Biological Chemistry, pp. 1443-1445).

The amino acid residues (AA) at the peripheral positions P _n -P ₁ to the left of the cleavage site and P ₁ '-P _n ^' to the right of the cleavage site can be varied over a very wide range without affecting the cleavage ability of the enzyme (Sakiyama & Masaki (1994) Lysyl Endopeptidase of Achromobacter lyticus. Methods in Enzymology, Vol 244, pp. 126-137).

However, the cleavage rate is affected by specific adjacent amino acids. For example, cleavage proceeds slowly when a proline is found at the P1' position or when one or two basic amino acids precede the lysine residue (Tsusanawa et al. (1987), Amino Acid Sequence if Thermostable Direct Hemolysin Produced by Vibrio parahaemolyticus, Journal of Biochemistry, vol. 101, pp. 111-121; Sakiyama & Masaki (1994), Lysyl Endopeptidase of Achromobacter lyticus. Methods in Enzymology, Vol 244, pp. 126-137).

In this example, the effect on ALP cleavage rate of having a glutamic acid (E) at the second position (P2') after a lysine (K) was determined. The reference insulin precursor contained a glycine (G) at P2'. In both cases, the amino acid at the first position after the cleavage site (P1') was a proline (P).

To determine the ALP cleavage rates, the two insulin precursors in Table 1 were cleaved with ALP and the formation of the cleavage products was followed over time by UPLC-MS (results are shown in Tables 2 and 3).

Insulin precursors were produced in Saccharomyces cerevisiae. Briefly, DNA fragments encoding the relevant insulin precursors were cloned into vectors designed to direct recombinant proteins into the secretory pathway of S. cerevisiae. The constructs were transformed into S. cerevisiae strains and subsequently transformed into the secretory pathway of S. cerevisiae as described in Verduyn et al. (1992). Effect of Benzoic Acid on Metabolic Fluxes in Yeasts: A Continuous-Culture on the Regulation of Respiration and Alcoholic Fermentation. Yeast, vol. 8, pp. 2171-2175. The cells were cultured in defined glucose medium prepared as described by Friedrichsson, et al., 2002, pp. 501-517, but supplemented with 6% glucose. The supernatant containing the insulin precursor was adjusted to pH 8.5 using 1 M NaHCO ₃ (pH 9.5) and diluted with water to a final concentration of 150 mg precursor/L. ALP was added to the pH-adjusted supernatant to a final concentration of 0.1 mg/ml, and the reaction was followed over time by removing samples at 0, 30, 60, 120, 240, 360, and 1440 min. To stop the ALP reaction, the supernatant was diluted 1:1 in 4 M CH ₃ COOH. The cleavage products in the ALP cleavage samples were identified and quantified using UPLC-MS. The results are shown in Tables 2 and 3.

The desired cleavage product, designated "open insulin precursor", is cleaved only at the Lys residue corresponding to the Lys at position B29 of human insulin (Lys residue at position 77 (also designated A77) in SEQ ID NO:20 and SEQ ID NO:21, respectively). Thus, for open insulin precursor 1, the sequence of the A chain is SDDMRGIVEQCCTSICSLYQLENYCN (SEQ ID NO:22) and the sequence of the B chain is EEAEPRGKPEGEQPGEQPGEQPGEQPGEQGGKPEGGGSGGGGSGGGGSFVNQHLCGSHLVEALYLVCGERGFFYTPK (SEQ ID NO:23). The term "overcleaved insulin precursor" is used to indicate cleavage products that, in addition to cleavage at AA77, are further cleaved at the Lys residues at positions 8 (also designated AA8) and/or 32 (also designated AA32) of SEQ ID NO:20 and SEQ ID NO:21, respectively.

The open insulin precursor can be purified and chemically modified with lysine residues and then fully matured by trypsin cleavage of the N-terminal extensions on the B and A chains, respectively (this concept is described in WO 2005/047508 A1). For the particular open insulin precursor 1 of this example, covalent conjugation at the three lysine residues with appropriate modifying groups, followed by cleavage of the N-terminal extensions EEAEPR and SDDMR by trypsin, would result in the insulin derivative of Example 4.

The ALP cleavage rate was found to be greatly influenced by the amino acid at the P2' position. When P2' was glycine (G), cleavage proceeded rapidly at all three lysine residues, with overcleavage already observed after 60 min, long before AA77 was completely cleaved (Table 3). Conversely, when the AA at the P2' position was glutamic acid (E), cleavage proceeded very slowly. At 60 and 120 min, AA77 was almost 100% cleaved, while cleavage at residues AA8 and AA32 was undetectable (Table 2). Cleavage at AA8 and AA32 was observed only after extended incubation times longer than 240 min, and only to a limited extent in all cases.

In conclusion, precursor loss due to ALP overcleavage at lysine residues AA8 and AA32 can be significantly reduced by having PE at the P1'-P2' positions compared to having PG at the same positions.

Functional data of insulin derivatives [Example 12]
Assay to determine affinity for the human insulin receptor (HIR-A) in the absence or presence of glucose.
Preparation of insulin receptor: Baby hamster kidney (BHK) cells overexpressing human insulin receptor A (HIR-A) were lysed in 50 mM Hepes pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 10% glycerol. The cleared cell lysate was batch absorbed with Wheat Germ Agglutinin (WGA)-agarose (lectin from Triticum vulgaris-Agarose, L1394, Sigma-Aldrich Steinheim, Germany) for 90 min. The receptor was washed with 20 volumes of 50 mM Hepes pH 8.0, 150 mM NaCl, and 0.1% Triton X-100, and then eluted with 50 mM Hepes pH 8.0, 150 mM NaCl, 0.1% Triton X-100, 0.5 M n-acetylglucosamine, and 10% glycerol. All buffers contained the protease cocktail Complete (Roche Diagnostic GmbH, Mannheim, Germany) as described in Andersen et al. 2017 PLos One 12.

Insulin Receptor Scintillation Proximity Assay (SPA) Binding Assay SPA PVT anti-mouse beads (Perkin Elmer) were diluted in SPA binding buffer consisting of 100 mM Hepes pH 7.4, 100 mM NaCl, 10 mM MgSO ₄ , 0.025% (v/v) Tween-20. SPA beads were incubated with IR-specific antibody 83-7 (Soos et al. 1986 Biochem J. 235, 199-208) and solubilized semi-purified HIR-A. Receptor concentration was adjusted to achieve 10% binding of 5000 cpm of ¹²⁵ I-(Tyr31)-insulin (Novo Nordisk A/S). A dilution series of cold ligand was added to a 96-well Optiplate, followed by the tracer ( ¹²⁵ I-insulin, 5000 cpm/well) and finally the receptor/SPA mix. To test glucose sensitivity, binding experiments were set up in the absence or presence of 20 mM glucose. Plates were gently rocked for 22.5 h at 22° C., centrifuged at 1000 rpm for 5 min and counted in a TopCounter (Perkin Elmer). Data points were fitted to a four-parameter logistic model, which determined the relative affinity of the insulin derivative compared to human insulin (in the same plate). The relative affinity of the insulin derivative compared to human insulin was determined and the increase in apparent relative affinity from 0 to 20 mM glucose reflects the glucose sensitivity of the insulin derivative in each experiment. Experiments were performed in the absence and presence of 1.5% HSA (w/w) to mimic physiological conditions. The data are shown in Table 4a (no HSA) and Table 4b (1.5% HSA).

The insulin derivatives of the present invention bind to HSA, and therefore when HIR affinity is measured in the presence of HSA, a certain percentage of the insulin derivative is bound to HSA and cannot bind to the human insulin receptor. Therefore, the term "apparent affinity" is used to describe the affinity measured in the presence of 1.5% HSA.

HIR (human insulin receptor) glucose coefficients were determined in individual experiments as the apparent relative HIR affinity at 20 mM glucose divided by the relative affinity in the absence of glucose. Average glucose coefficients from several experiments are provided in Tables 4a and 4b. The glucose coefficients provided in Tables 4a and 4b are average glucose coefficients determined in individual experiments and therefore may differ slightly from values obtained by dividing the average HIR affinity in the presence of glucose by the average HIR affinity in the absence of glucose.

Insulin derivative of the present invention The data in Table 4a and Table 4b show albumin-dependent glucose sensitivity.The apparent insulin receptor affinity of the insulin derivative of Example 4 is decreased when measured in the presence of HSA compared to the absence of HSA (compare the data in Table 4a with the data in Table 4b).Glucose can move the binding of the insulin derivative of Example 4 to HSA, thereby increasing the apparent HIR in the presence of 20 mM glucose and 1.5% HSA compared to the absence of glucose (see the data in Table 4b).This can be easily seen from the glucose coefficient that exceeds 1, where the relative insulin receptor affinity is higher in the presence of 20 mM glucose compared to the absence of glucose.Therefore, the insulin derivative binds to the insulin receptor in a glucose-sensitive manner in the presence of HSA, and therefore the insulin derivative has the potential for glucose-sensitive treatment of diabetes, and the insulin derivative is inactive or has low activity at low blood glucose levels, and binds to and activates the insulin receptor at higher blood glucose levels. As can be seen from Table 4b, the insulin derivative of Example 4 has a high glucose coefficient of 51.9 in the presence of 1.5% HSA.

Insulin derivatives included for comparison In Table 5, data are shown for three insulin derivatives that are identical except for the peptide extension at the N-terminus of the insulin B chain. The structures of the insulin derivatives are provided in Examples 5, 6, and 9, respectively, and the sequences of the peptide extensions are shown in Table 5 for ease of reference. The modifying groups of these insulin derivatives are identical to those of the insulin derivative of Example 4. However, compared to the insulin derivative of Example 4 of the present invention, these insulin derivatives have only two modifying groups. The prior art compound of Example 324 from WO 2020/201041 (structure shown in Example 9 for ease of reference) has a peptide extension GKP(G ₄ S) ₃ at the B1 position of the insulin backbone. It can be seen that by further lengthening the peptide extension by 20 amino acid residues (as in the comparative insulin derivative of Example 5), the glucose coefficient is reduced from 46.5 to 26.3. If the peptide extension is even longer, as in the comparative insulin derivative of Example 6, by 100 amino acid residues, the glucose coefficient is further reduced by 18.6.

Thus, surprisingly, the insulin derivative of Example 4 according to the invention shows a glucose coefficient of 51.9, despite having a peptide extension of similar length to the insulin derivative of Example 5.

In Table 6, data are presented for three insulin derivatives that are identical except for the peptide extension at the N-terminus of the insulin B chain. The modification groups of these insulin derivatives are different from those of the insulin derivatives in Table 5. The structures of the insulin derivatives are provided in Examples 7, 8, and 10, respectively, and the sequences of the peptide extensions are provided in Table 6 for ease of reference. The prior art compound of Example 280 from WO 2020/201041 (structure shown in Example 10 for ease of reference) has a peptide extension GKP(G ₄ S) ₃ at the B1 position of the insulin backbone. It can be seen that in the comparative insulin derivatives of Examples 7 and 8, further lengthening the peptide extension by 20 amino acid residues reduces the glucose coefficient from 18.0 to 12.6 and 12.0, respectively. This further supports the observation that lengthening the peptide extension in prior art compounds such as Examples 324 and 280 from WO 2020/201041 leads to a reduction in the glucose coefficient.

[Example 13]
Assay for determining glucose-sensitive insulin receptor phosphorylation in human primary hepatocytes.
When insulin binds to insulin receptor, it induces the activation of insulin receptor, which can be measured as tyrosine phosphorylation.This induces downstream signal transduction pathway, resulting in mitogenic and metabolic response.The insulin derivative of the present invention can bind to albumin and be transported by glucose.The glucose sensitivity of the insulin derivative of Example 4 is measured at low and high glucose concentrations, thereby detecting the glucose-dependent cellular response of the insulin derivative of Example 4.

Donor human cryo-plateable hepatocytes were incubated with increasing concentrations of human insulin (HI) or the insulin derivative of Example 4 for 15 minutes in the presence of 1.5% human serum albumin (HSA). Hepatocytes were then washed and lysed, and phosphorylation of the insulin receptor (p-Tyr1158) was revealed using an ELISA with the specific insulin receptor antibody D2 developed by Novo Nordisk (Orstrup et al. 2019, Journal of Immunological Methods, Volume 465, February 2019, Pages 20-26) and the IR p-Tyr1158 antibody (ThermoFischer, Cat. #44-802G).

Insulin Receptor Phosphorylation Methods:
Human hepatocytes obtained from BioIVT (Liverpool cryo-plateable hepatocytes, ref. #X008001-P lot #ACR; single donor adherent hepatocytes #M00995P) were thawed and cultured in Invitrogen supplemented with Torpedo antibiotic mixture (Z99000) at 0.1 mL/well according to the manufacturer's instructions. Cells were seeded at a density of 50.000 cells/well in CP medium. After 4 hours, the medium is changed to M199 medium supplemented with 5.5 mM glucose, 100 units/ml penicillin, and 100 mg/ml streptomycin, 4 mg/ml dexamethasone, 0.1% fetal calf serum (FCS), and 1 nM human insulin (HI) for overnight culture at 37° C. The day after seeding, human hepatocytes were cultured in basal culture medium (M199 w.o. phenol red, tween 100 mM phenol red, 1 ... 80, and adenosine-5-triphosphate) for 15 min at 37°C. After stimulation, hepatocytes were washed twice in ice-cold PBS, and then cells were solubilized by addition of lysis buffer for 30 min at 4°C. Phosphorylation of insulin receptor (p-Tyr1158) was subsequently quantified using an immunoassay ELISA with IR-specific antibody IR (D2 (Orstrup et al. (2019) J. Immunological Methods 465.20-26), batch 0268-0000-0945-1B), and detection pIR:pTyr1158 antibody (ThermoFischer, Cat.#44-802G). SpectraMax_1_190, Molecular The absorbance was read at 450 nm using a 350 nm chromatograph.

The insulin derivative of Example 4 stimulated phosphorylation of IR (pIR) in a concentration-dependent manner in human primary hepatocytes, to the same maximum level as HI, albeit with a higher EC _50. The EC ₅₀ of the insulin derivative of Example 4 was found to be lower at 20 mM (30.8 nM) than at 3 mM (58.1 nM) D-glucose concentration, as revealed by a left shift (n=6) of the concentration-response curve of pIR stimulation (shown in FIG. 1). The ratio of the EC ₅₀ of the insulin derivative of Example 4 at 3 mM D-glucose to the EC ₅₀ at 20 mM D-glucose mM (IR pTyr human hepatocytes 1.5% HSA 3 vs. 20 mM D-glucose coefficient) was then calculated and found to be 1.89 (P value=0.019) (Table 7). This indicates that the insulin derivative of Example 4 has higher activity at higher levels of glucose than at lower levels of glucose and therefore exhibits glucose-sensitive insulin activity.

Example 14: Assay for determining carbohydrate-sensitive glucose uptake in cells (rat lipogenesis assay)
Binding of insulin to the insulin receptor triggers activation of downstream signaling pathways. One metabolic endpoint of insulin signaling is lipid metabolism, and in the presence of insulin, ^3H -glucose uptake by cells is stimulated and incorporated into lipids, so a lipogenesis assay was used to measure the endpoint readout.

The insulin derivative of Example 4 can bind to albumin and be displaced by glucose, conferring glucose sensitivity. Although both L-glucose and D-glucose can displace the derivative from albumin, L-glucose is metabolically inactive and is used to vary the glucose concentration in the medium without affecting metabolism. The fold change in potency (relative to human insulin) of the insulin derivative of Example 4 at L-glucose concentrations of 20 mM and 3 mM was determined.

Rat Adipogenesis Assay (rFFC)
Epididymal fat pads from Sprague Dawley rats were digested with collagenase in Hepes Krebs Ringer buffer at 36.5°C for 1-1.5 hours under vigorous shaking. The suspension was filtered through two layers of gauze. The phases were separated by standing at room temperature for 5 minutes, allowing the adipocytes to collect in the upper phase. The lower phase was removed with a syringe. The adipocytes were washed twice with 20 ml of Hepes Krebs Ringer buffer. The cells were transferred to a 96-well plate in Hepes Krebs Ringer buffer containing 1.5% HSA, 0.5 mM glucose, 0.1 μCi/well glucose (D-[3- ³ H]glucose (20.0 Ci/mmol) Perkin Elmer), and 3 mM or 20 mM L-glucose. Increasing amounts of human insulin or insulin derivatives of the invention to generate concentration response curves were added and incubated for 2 hours at 36.5°C. The reaction was stopped by adding 100 μL of Microscient E (cat# 6013661 Perkin Elmer). Plates were allowed to rest for 3 hours before counting in a Top counter. The ratio of the insulin derivatives' _EC50 at 3 mM L-glucose to the _EC50 at 20 mM L-glucose was determined (see Table 8).

Representative concentration response curves (n=1) are shown in FIG. 2. The EC ₅₀ of the insulin derivative of Example 4 was found to be lower at 20 mM than at 3 mM L-glucose concentration, as revealed by the left shift of the concentration response curve of lipogenesis. The data in Table 8 show that the insulin derivative of Example 4 leads to higher levels of lipogenesis (i.e., more glucose transport) in the presence of higher levels of L-glucose (20 mM) compared to lower levels of L-glucose (3 mM). This indicates that the insulin derivative of Example 4 has higher activity at higher levels of glucose than at lower levels of glucose, and therefore, the insulin derivative of Example 4 exhibits glucose-sensitive insulin activity.

Example 15: Assay to determine glycogen accumulation in primary rat hepatocytes.
When insulin binds to insulin receptor, it induces the activation of downstream signaling pathways, leading to the activation of metabolic processes in insulin-sensitive tissues. Among other functions, insulin induces glycogen accumulation in the liver. Therefore, estimation of glycogen levels was used as an endpoint in response to increasing dose concentrations of human insulin or insulin derivative of Example 4 in primary rat hepatocytes in the presence of 20 mM glucose and 0.1% human serum albumin (HSA) for 24 hours. Quantification of cellular glycogen content was carried out using a quantitative colorimetric assay based on the principle of Periodic Acid Schiff Reagent (PAS) assay with absorbance reading at 550 nm.

Glycogen Accumulation (PAS Assay) Method:
Rat hepatocytes (Lonza, RSCP01) are thawed and seeded in collagen-coated 96-well plates at a density of 50.000 cells/well in basal M199 culture medium (5.5 mM glucose, 100 units/ml penicillin, and 100 mg/ml streptomycin, and 4 mg/ml Decadron) supplemented with 4% fetal calf serum (FCS) and 1 nM human insulin (HI) at 37° C. After approximately 4 hours, the medium was replaced with basal M199 culture medium supplemented with 0.1% FCS and 1 nM HI for overnight culture. The day after isolation, rat hepatocytes were incubated for 24 hours at 37°C in assay medium corresponding to basal M199 medium supplemented with 100u/ml penicillin, 100mg/ml streptomycin, 4mg/ml dexamethasone, 0.1% human serum albumin (HSA), 14.5mM glucose, and increasing concentrations of either HI or the derivative of Example 4. After stimulation, hepatocytes were washed three times in ice-cold PBS, flash frozen in liquid nitrogen, and stored at -80°C for later analysis. To measure glycogen levels, plates were then thawed and cells were solubilized in 1% Triton X100. Subsequently, 100μl of periodic acid, prepared as a solution of 0.1% periodic acid in 7% acetic acid, was added to the plates for 1.5 hours at 37°C, protected from light, to oxidize the hydroxyl groups in glycogen. Then, 85 μl of Schiff's reagent was added for 5 min to react with the oxidized moieties, and the plate was incubated for another 10 min at room temperature to allow color development. The absorbance was then read at 550 nm (spectraMax_1_190, Molecular Devices).

The data show that the insulin derivative of Example 4 induced glycogen accumulation in rat primary hepatocytes in a concentration-dependent manner with a full dose-response curve at a glucose concentration of 20 mM (Figure 3). The maximum response was slightly reduced compared to human insulin (Emax = 86%, n = 5). The in vitro EC50 was calculated to be 20.4 nM for the insulin derivative of Example 4 and 2.8 nM for human insulin at 20 mM glucose (n = 5), suggesting reduced potency relative to human insulin (Table 9).

Example 16: Pharmacokinetic studies in LYD pigs Pharmacokinetic studies in LYD pigs Insulin derivatives were administered intravenously (iv) or subcutaneously (sc) to female Landrace Yorkshire Duroc (LYD) pigs weighing approximately 70-110 kg. The iv dose was 0.3 nmol/kg and the sc dose was either 1 or 2 nmol/kg. Blood was collected at selected time points up to 72 hours and plasma was prepared and analyzed for insulin derivative concentrations (see below). Pigs were fasted overnight and fed 8 hours after administration of the insulin derivative.

Plasma concentration-time profiles from individual animals were analyzed by noncompartmental pharmacokinetic analysis (NCA) using WinNonlin Professional (Certara, CA, USA) or a custom Rshiny application (Rstudio.com, Boston, MA, US) and validated against WinNonlin.

Quantification of insulin analogue concentrations in plasma samples Plasma samples were analyzed for levels of administered insulin derivatives using a luminescence oxygen channeling immunoassay (LOCI), also known by the trademark AlphaLISA (Perkin Elmer, Waltham, Massachusetts, USA). The assay principle is briefly as follows: an antibody specific for the analyte of interest is conjugated to an acceptor bead. A second antibody, also specific for the analyte, is biotinylated. The two antibody conjugates are then incubated with the plasma sample containing the analyte to form an immune complex. Streptavidin donor beads are then added and bind to the biotinylated antibody. Illumination of the donor beads at 680 nm excites ambient oxygen to form singlet oxygen. When the acceptor beads are in close proximity, energy transfer occurs from the singlet oxygen to the acceptor beads, which then emit light at 615 nm. The emitted light signal intensity is proportional to the concentration of the analyte. Plasma samples spiked with known concentrations of the analyte are used to generate a calibration curve. Unknown sample concentrations are then calculated from the calibration curve using a five-parameter logistic regression. All insulin derivatives were tested with Novo Nordisk's in-house antibodies. The results are shown in Table 10.

The PK data in Table 10 show that the insulin derivative of Example 4 of the present invention has a longer half-life than the prior art compounds of Examples 324 and 280 of WO 2020/201041.

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

Claims (4)

An insulin derivative comprising:
An insulin derivative. A composition comprising the insulin derivative of claim 1 . A medicine comprising the insulin derivative according to claim 1 . 4. The medicament according to claim 3 for use in the prevention or treatment of diabetes, including type 1 diabetes and type 2 diabetes.