Provided herein are conjugates comprising a sulfonamide of formula (I) and an active pharmaceutical ingredient such as an insulin analog comprising at least one mutation relative to the parent insulin, wherein the insulin analog comprises a mutation at position B16 which is substituted with a hydrophobic amino acid and/or a mutation at position B25 which is substituted with a hydrophobic amino acid. Further provided herein are sulfonamides of formula (A) and insulin analogs comprising at least one mutation relative to the parent insulin, wherein the insulin analogs comprise a mutation at position B16 which is substituted with a hydrophobic amino acid and/or a mutation at position B25 which is substituted with a hydrophobic amino acid.

BACKGROUND

Worldwide, more than 400 million people suffer from type 1 or type 2 diabetes mellitus. Type 1 diabetes is treated with insulin substitution. In contrast to type 1 diabetes, there is basically no deficiency of insulin in type 2 diabetes, but in a large number of cases, especially in the advanced stage, type 2 diabetes patients are treated with insulin.

In a healthy person, the release of insulin by the pancreas is strictly coupled to the concentration of the blood glucose. Elevated blood glucose levels, such as occur after meals, and are rapidly compensated by a corresponding increase in insulin secretion. In the fasting state, the plasma insulin level falls to a basal value which is adequate to guarantee a continuous supply of insulin-sensitive organs and tissue with glucose and to keep hepatic glucose production low in the night. Often, the replacement of the endogenous insulin secretion by exogenous, mostly subcutaneous administration of insulin does not achieve the quality of the physiological regulation of the blood glucose described above. Deviations of the blood glucose upward or downward can occur, which in their severest forms can be life-threatening. It is to be derived from this that an improved therapy of diabetes is primarily to be aimed at keeping the blood glucose as closely as possible in the physiological range.

Human insulin is a polypeptide of 51 amino acids, which are divided into 2 amino acid chains: the A chain having 21 amino acids and the B chain having 30 amino acids. The chains are connected to one another by means of 2 disulfide bridges. A third disulfide bridge exists between the cysteines at position 6 and 1 1 of the A chain. Some products in current use for the treatment of diabetes mellitus contain are insulin analogs, i.e. insulin variants whose sequence differs from that of human insulin by one or more amino acid substitutions in the A chain and/or in the B chain.

Like many other peptide hormones, human insulin has a short half-life in vivo. Thus, it is administered frequently which is associated with discomfort for the patient. Therefore, insulin analogs are desired which have an increased half-life in vivo and, thus, a prolonged duration of action.

There are currently different approaches for extending the half-life of insulins.

One approach is based on the development of a soluble formulation at low pH, but of reduced solubility relative to native insulin at physiologic pH. The isoelectric point of the insulin analog is increased through 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 an insulin with extended duration of action. The insulin analog precipitates in the presence of zinc upon injection in subcutaneous sites and slowly solubilizes, resulting a sustained presence of insulin glargine.

WO 2016/006963 discloses insulin analogs having a reduced insulin receptor-mediated clearance rate, compared to human insulin.

WO 2018/056764 discloses insulin analogs having a reduced insulin receptor-mediated clearance rate, compared to human insulin.

WO 2008/034881 discloses protease stabilized insulin analogs.

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 the attachment of the insulin to serum albumin by noncovalent, reversible binding. As a consequence, this insulin analog has a significantly prolonged time-action profile relative to human insulin (see e.g. Mayer et al. , Inc. Biopolymers (Pept Sci) 88: 687-713, 2007; or WO 2009/1 15469).

SUMMARY

Provided herein are long-acting insulin analogs. The provided long-acting insulin analogs have a very low binding affinity (hence a lower clearance rate) whilst still maintaining high signal transduction. The insulin analogs are described in section A below.

Provided herein are serum albumin binding moieties (herein also referred to as “albumin binders” or“binders”), which when coupled to a peptide such as an insulin analog provided above lead to improved pharmacodynamics and/or pharmacokinetic properties of the peptide for example, an extended pharmacokinetic half life in blood and/or blood plasma and/or a prolonged profile of action, i.e. a prolonged reduction of blood glucose level. The provided albumin binders are sulfonamides of formula (A)

The serum albumin binding moieties are described in section B below.

Also provided herein are conjugates comprising an active pharmaceutical ingredient, such as an insulin analog defined in section A, and an insulin binder, such as a sulfonamide of formula (A) defined in section B. The conjugates are described in section C below.

DETAILED DESCRIPTION

Section A: Insulin analogs

In order to increase the duration of action of a drug, half-life plays a major role. Half-life (t-1/2) is proportional to the volume of distribution divided by clearance. In the case of human insulin, clearance is mainly driven by binding to the insulin receptor, internalization and subsequent degradation.

Accordingly, there is a need for insulin analogs which have a reduced insulin receptor binding activity, and thus a reduced receptor-mediated clearance rate, but which have a signal transduction activity which allow for sufficiently lowering the blood glucose level in vivo.

Surprisingly, it was shown in the context of the studies underlying the present invention that a substitution at position B16 and/or B25 of human insulin with a hydrophobic amino acid (such as leucine, isoleucine, valine, alanine and tryptophan) resulted in a decrease of insulin receptor binding activity (as compared to the insulin receptor binding activity of the parent insulin, see Examples). The strongest effects on insulin receptor binding activity were observed for substitutions with branched-chain amino acids (leucine, isoleucine and valine). Interestingly, insulin analogs with such substitutions at these positions (such as at position B25) showed up to 6-fold enhancement in signal transduction than expected based on their insulin receptor isoform B (IR-B) binding affinities (see Examples). Further, some tested insulin analogs showed improved proteolytic stability against a-chymotrypsin, cathepsin D and insulin degrading enzyme (see Examples).

Accordingly, provided herein are insulin analogs comprising at least one mutation relative to the parent insulin, wherein the insulin analogs comprise a mutation at position B16 which is substituted with a hydrophobic amino acid, and/or a mutation at position B25 which is substituted with a hydrophobic amino acid.

The expression “insulin analog” as used herein refers to a peptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin (herein also referred to as“parent insulin”, e.g. human insulin) by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or adding at least one amino acid residue. The added and/or exchanged amino acid residue can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues. The analog as referred to herein is capable of lowering blood glucose levels in vivo, such as in a human subject.

In some embodiments, the insulin analog provided herein comprises two peptide chains, an A-chain and a B-chain. Typically, the two chains are connected by disulfide bridges between cysteine residues. For example, in some embodiments, insulin analogs provided herein comprise three disulfide bridges: one disulfide bridge between the cysteines at position A6 and A1 1 , one disulfide bridge 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. Accordingly, insulin analogs provided herein may comprise cysteine residues at positions A6, A7, A1 1 , A20, B7 and B19.

In some embodiments provided herein, the insulin analog is a single-chain insulin. A single-chain insulin is a single polypeptide chains in which the insulin B-chain is linked contiguously with the insulin A-chain via an uncleaved connecting peptide.

Mutations of insulin, i.e. mutations of a parent insulin, are indicated herein by referring to the chain, i.e. either the A-chain or the B-chain of the analog, the position of the mutated amino acid residue in the A- or B-chain (such as A14, B16 and B25), and the three letter code for the amino acid substituting the native amino acid in the parent insulin. The term“desB30” refers to an analog lacking the B30 amino acid from the parent insulin (i.e. the amino acid at position B30 is absent). For example, Glu(A14)lle(B16)desB30 human insulin, is an analog of human insulin in which the amino acid residue at position 14 of the A-chain (A14) of human insulin is substituted with glutamic acid, the amino acid residue at position 16 of the B-chain (B16) is substituted with isoleucine, and the amino acid at position 30 of the B chain is deleted (i.e. is absent).

Insulin analogs provided herein comprise at least one mutation (substitution, deletion, or addition of an amino acid) relative to parent insulin. The term“at least one”, as used herein means one, or more than one, such as“at least two”,“at least three”,“at least four”,”at least five”, etc. In some embodiments, the insulin analogs provided herein comprise at least one mutation in the B-chain and at least one mutation in the A-chain. In a further embodiment, the insulin analogs provided herein comprise at least two mutations in the B-chain and at least one mutation in the A-chain. For example, the insulin analog may comprise a substitution at position B16, a deletion at position B30 and a substitution at position A14. Alternatively, the insulin analog may comprise a substitution at position B25, a deletion at position B30 and a substitution at position A14. Further, the insulin analog may comprise a substitution at position B16, a substitution at position B25, a deletion at position B30 and a substitution at position A14.

The insulin analogs provided herein may comprise mutations in addition to the mutations above. In some embodiments, the number of mutations does not exceed a certain number. In some embodiments, the insulin analogs comprise less than twelve mutations (i.e. deletions, substitution, additions) relative to the parent insulin. In another embodiment, the analog comprises less than ten mutations relative to the parent insulin. In another embodiment, the analog comprises less than eight mutations relative to the parent insulin. In another embodiment, the analog comprises less than seven mutations relative to the parent insulin. In another embodiment, the analog comprises less than six mutations relative to the parent insulin. In another embodiment, the analog comprises less than five mutations relative to the parent insulin. In another embodiment, the analog comprises less than four mutations relative to the parent insulin. In another embodiment, the analog comprises less than three mutations relative to the parent insulin.

The expression“parent insulin” as used herein refers to naturally occurring insulin, i.e. to an unmutated, wild-type insulin. In some embodiments, the parent insulin is animal insulin, such as mammalian insulin. For example, the parent insulin may be human insulin, porcine insulin, or bovine insulin.

In some embodiments, the parent insulin is human insulin. The sequence of human insulin is well known in the art and shown in Table 4 in the Example section. Fluman insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO:

1 (GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 2 (FVNQHLCGSHLVEALYLVCGERGFFYTPKT).

In another embodiment, the parent insulin is bovine insulin. The sequence of bovine insulin is well known in the art. Bovine insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 81 (GIVEQCCASVCSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 82

(FVNQHLCGSHLVEALYLVC-GERGFFYTPKA).

In another embodiment, the parent insulin is porcine insulin. The sequence of porcine insulin is well known in the art. Porcine insulin comprises an A chain having an amino acid sequence as shown in SEQ ID NO: 83 (GIVEQCCTSICSLYQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 84

(FVNQHLCGSHLVEALYLVC GERGFFYTPKA).

Human, bovine, and porcine insulin comprises three disulfide bridges: one disulfide bridge between the cysteines at position A6 and A1 1 , one disulfide bridge 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.

The insulin analogs provided herein have an insulin receptor binding affinity which is reduced as compared to the insulin receptor binding affinity of the corresponding parent insulin, e.g. of human insulin.

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, e.g. human insulin receptor isoform A or human insulin receptor isoform B (which was used in the Examples section).

Advantageously, the human insulin analogs provided herein have a significantly reduced binding affinity to the human insulin receptor as compared to the binding affinity of human insulin to the human insulin receptor (see Examples). Thus, the insulin analogs have a very low clearance rate, i.e. a very low insulin-receptor-mediated clearance rate.

In some embodiments, the insulin analogs have, i.e. exhibit, less than 20 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 10 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin. In another embodiment, the insulin analogs provided herein have less than 5 % of the binding affinity to the corresponding insulin receptor compared to its parent insulin, such as less than 3 % of the binding affinity compared to its parent insulin. For example, the insulin analogs provided herein may have between 0.1 % to 10 %, such as between 0.3 % to 5 % of the of the binding affinity to the corresponding insulin receptor compared to its parent insulin. Also, the insulin analogs provided herein may have between 0.5% to 3 %, such as between 0.5 % to 2 % of the of the binding affinity to the corresponding insulin receptor compared to its parent insulin.

Methods for determining the binding affinity of an insulin analog to an insulin receptor are well known in the art. For example, the insulin receptor binding affinity can be determined by a scintillation proximity assay which is based on the assessment of competitive binding between [125l]-labelled parent insulin, such as [125l]-labelled human insulin, and the (unlabeled) insulin analog to the insulin receptor. The insulin receptor can be present in a membrane of a cell, e.g. of CFIO (Chinese Flamster Ovary) cell, which overexpresses a recombinant insulin receptor. In an embodiment, the insulin receptor binding affinity is determined as described in the Examples section.

Binding of a naturally occurring insulin or an insulin analog 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. The autophosphorylation of the insulin receptor stimulates the receptor’s tyrosine kinase activity toward intracellular substrates involved in the transduction of the signal. The autophosphorylation of the insulin receptor by an insulin analog is therefore considered as a measure for signal transduction caused by said analog.

The insulin analogs in Table 4 of the Examples section were subjected to autophosphorylation assays. Interestingly, insulin analogs with aliphatic substitutions at positions B16 and B25 caused higher than expected insulin receptor autophosphorylation based on their insulin receptor binding affinities. Thus, the insulin analogs provided herein have a low binding activity, and consequently a lower receptor-mediated clearance rate, but are nevertheless capable of causing a relatively high signal transduction. Therefore, the insulin analogs provided herein could be used as long-acting insulins. In some embodiments, the insulin analog provided herein are capable of inducing 1 to 10 %, such as 2 to 8 %, insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). Further, in some embodiments, the insulin analogs provided herein are capable of inducing 3 to 7 %, such as 5 to 7% insulin receptor autophosphorylation relative to the parent insulin (such as human insulin). The insulin receptor autophosphorylation relative to a parent insulin can be determined as described in the Examples section.

Insulin analogs provided herein were subjected to protease stability assays. As shown in Table 6, insulin analogs provided herein had higher stability towards at least some of the tested proteases as compared to human insulin. Improved proteolytic stability was observed against a-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE). Accordingly, insulin analogs provided herein are, typically, proteolytically stable insulin analogs. Thus, they are slower degraded by proteases relative to the parent insulin. In some embodiments, the insulin analog provided herein are stabilized against degradation by a-chymotrypsin, cathepsin D and insulin degrading enzyme (IDE) compared to parent insulin.

As set forth above, the insulin analog comprises at least one mutation as compared to the parent insulin.

In some embodiments insulin analogs provided herein comprise a mutation at position B16 which is substituted with a hydrophobic amino acid. Thus, the amino acid at position B16 (tyrosine in human, bovine and porcine insulin) is replaced with a hydrophobic amino acid.

In another embodiment, insulin analogs provided herein comprise a mutation at position B25 which is substituted with a hydrophobic amino acid. Thus, the amino acid at position B25 (phenylalanine in human, bovine and porcine insulin) is replaced with a hydrophobic amino acid.

In another embodiment, insulin analogs provided herein comprise a mutation at position B16 which is substituted with a hydrophobic amino acid and a mutation at position B25 which is substituted with a hydrophobic amino acid.

The hydrophobic amino acid may be any hydrophobic amino acid. For example, the hydrophobic amino acid may be an aliphatic amino acid such as a branched-chain amino acid.

In some embodiments of the insulin analogs provided herein, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, phenylalanine or tyrosine (or with a derivative of the aforementioned amino acids).

Several parent insulins such as human, bovine and porcine insulin comprise tyrosine at position B16 and phenylalanine at position B25. Thus, the amino acid at position B16 of the parent insulin may be substituted with isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine or phenylalanine (or with a derivative of the aforementioned amino acids). Further, the amino acid at position B25 of the parent insulin may be substituted with isoleucine, valine, leucine, alanine, tryptophan, methionine, proline, glycine, or tyrosine (or with a derivative of the aforementioned amino acids).

Derivatives of the aforementioned amino acids are known in the art.

Derivatives of leucine include, but are not limited to, homo-leucine and terMeucine. Thus, the amino acid at position B16 and/or B25 may be substituted with homo-leucine or tert-leucine.

A derivative of valine is, e.g., 3-ethyl norvaline. Thus, the amino acid at position B16 and/or B25 may be substituted with 3-ethyl norvaline.

Derivatives of glycine include, but are not limited to cyclohexyl-glycine cyclopropylglycine, and trifluorethylglycine.

Derivatives of alanine include, but are not limited to, beta-t-butylalanine, cyclobutyl-alanine, cyclopropyl-alanine and homo-cyclohexylalanine.

In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, leucine, alanine, or tryptophan.

In some embodiments, the aliphatic amino acid is not alanine. Accordingly, the hydrophobic amino acid used for the substitution at position B16 and/or B25 may be isoleucine, valine, leucine, or tryptophan.

In some embodiments, the hydrophobic amino acid used for the substitution at position B16 and/or B25 is isoleucine, valine, or leucine.

Claims

1. A conjugate comprising an insulin analog and a sulfonamide of formula (I)

wherein:

A is selected from the group consisting of oxygen atom, -CH2CH2- group, - OCH2- group and -CH2O- group;

E represents a -C6H3R- group with R being a hydrogen atom or a halogen atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom;

X represents a nitrogen atom or a -CH- group;

m is an integer in the range from 5 to 17;

n is zero or an integer in the range from 1 to 3;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

s is zero or 1 ;

t is zero or 1 ;

R1 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group; R2 represents at least one residue selected from the group of hydrogen atom, halogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group, wherein the sulfonamide of formula (I) is covalently bound to the insulin analog in that terminal carboxy group“a” of the sulfonamide of formula (I) is covalently bound to an amino group of the insulin analog.

2. The conjugate according to claim 1 , wherein the sulfonamide has the formula (1-1 )

wherein:

E represents a -C6H3R- group with R being a hydrogen atom or a halogen atom, wherein the halogen atom is selected from the group consisting of fluorine, chlorine, bromine and iodine atom;

X represents a nitrogen atom or a -CH- group;

p is zero or 1 ;

q is zero or 1 ;

r is an integer in the range from 1 to 6;

R1 represents at least one residue selected from the group of hydrogen atom and halogen atom;

R2 represents at least one residue selected from the group of hydrogen atom, C1 to C3 alkyl group and halogenated C1 to C3 alkyl group; with m being an integer in the range from 5 to 15 if p is zero, or m being an integer in the range from 7 to 15 if p is 1.

3. The conjugate according to claim 1 or 2, wherein the sulfonamide has the formula

wherein X is a nitrogen atom or a -CH- group; m is an integer in the range from 7 to 15; r is an integer in the range from 1 to 6; q is zero or 1 ; Hal is a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine atom; and the H00C-(CH2)m-C6H3Hal-0- group is situated in meta or para position on phenyl ring Ph with respect to the -S(0)2- group.

4. The conjugate according to any of claims 1 to 3, wherein the sulfonamide has the formula (1-1 -1 a)

5. The conjugate according to claim 1 or 2, wherein the sulfonamide has the formula

wherein X is a nitrogen atom or a -CH- group; m is an integer in the range from 5 to 15; r is an integer in the range from 1 to 6; q is zero or 1 ; and the HOOC-(CH2)m- O- group is situated in meta or para position on phenyl ring Ph with respect to the -S(0)2- group.

6. The conjugate according to any of claims 1 to 2 or 5, wherein the sulfonamide has the formula (1-1 -2a)

or the formula (1-1 -2c)

7. The conjugate according to any one of claims 1 to 6, wherein the insulin analog comprises at least one mutation relative to the parent insulin, wherein the insulin analog comprises a mutation at position B16 which is substituted with a

hydrophobic amino acid and/or a mutation at position B25 which is substituted with a hydrophobic amino acid, and optionally, wherein said insulin analog further comprises a mutation at position A14 which is substituted with an amino acid selected from the group consisting of glutamic acid (Glu), aspartic acid (Asp) and histidine (His) and/or a mutation at position B30.

8. The conjugate according to claim 7, wherein the parent insulin is human insulin, porcine insulin, or bovine insulin, and/or wherein the hydrophobic amino acid is a branched-chain amino acid, such as a branched-chain amino acid selected from the group consisting of valine (Val), isoleucine (lie), and leucine (Leu).

9. The conjugate according to any one of claims 1 to 8, wherein the insulin analog is selected from

Leu(B16)-human insulin,

Val(B16)-human insulin,

lle(B16)-human insulin,

Leu(B16)Des(B30)-human insulin,

Val(B16)Des(B30)-human insulin,

lle(B16)Des(B30)-human insulin,

Leu(B25)-human insulin,

Val(B25)-human insulin,

lle(B25)-human insulin,

Leu(B25)Des(B30)-human insulin,

Val(B25)Des(B30)-human insulin,

lle(B25)Des(B30)-human insulin,

Glu(A14)Leu(B16)Des(B30)-human insulin,

Glu(A14)lle(B16)Des(B30)-human insulin,

Glu(A14)Val(B16)Des(B30)-human insulin,

Glu(A14)Leu(B16)-human insulin,

Glu(A14)lle(B16)-human insulin,

Glu(A14)Val(B16)-human insulin,

Glu(A14)Leu(B25)Des(B30)-human insulin,

Glu(A14)lle(B25)Des(B30)-human insulin,

Glu(A14)Val(B25)Des(B30)-human insulin,

Glu(A14)Leu(B25)-human insulin,

Glu(A14)lle(B25)-human insulin,

Glu(A14)Val(B25)-human insulin,

Glu(A14)Gly(A21 )Glu(B3)Val(B25)Des(B30)-human insulin,

Glu(A14)lle(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)Glu(B3)lle(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)lle(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21 )Glu(B3)lle(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Val(B16)lle(B25)Des(B30)-human insulin,

Glu(A14)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21 )Glu(B3)Val(B16)Val(B25)Des(B30)-human insulin,

Glu(A14)Gly(A21 )Glu(B3)Val(B25)-human insulin,

Glu(A14)lle(B16)lle(B25)-human insulin,

Glu(A14)Glu(B3)lle(B16)lle(B25)-human insulin,

Glu(A14)lle(B16)Val(B25)-human insulin,

Glu(A14)Gly(A21 )Glu(B3)lle(B16)Val(B25)-human insulin,

Glu(A14)Val(B16)lle(B25)-human insulin,

Glu(A14)Val(B16)Val(B25)-human insulin,

Glu(A14)Glu(B3)Val(B16)Val(B25)-human insulin, and

Glu(A14)Gly(A21 )Glu(B3)Val(B16)Val(B25)-human insulin.

10. The conjugate according to any one of claims 1 to 9, wherein the insulin analog comprises

(a) an A chain having an amino acid sequence as shown in SEQ ID NO: 43

(GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 44

(FVNQHLCGSHLVEALYLVCGERGFIYTPK),

(b) an A chain having an amino acid sequence as shown in SEQ ID NO: 47

(GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid sequence as shown in SEQ ID NO: 48

(FVNQHLCGSHLVEALYLVCGERGFVYTPK), or

(c) an A chain having an amino acid sequence as shown in SEQ ID NO: 77 (GIVEQCCTSICSLEQLENYCN) and a B chain having an amino acid

sequence as shown in SEQ ID NO: 78

(FVEQHLCGSHLVEALVLVCGERGFVYTPK).

11. The conjugate of any one of claims 1 to 10, wherein the amino group of the insulin analog, to which the sulfonamide of formula (I) is covalently bound, is an epsilon amino group of a lysine present in the insulin analog or is the N-terminal amino group of the B chain of the insulin or insulin analog, e.g. wherein the amino group is the epsilon amino group of lysine present at position B29 of the B chain.

12. The conjugate of any one of claims 1 to 11 , wherein the conjugate is conjugate 1 (A chain sequence: SEQ ID NO: 47; B chain sequence: SEQ ID NO: 48):

conjugate 3 (A chain sequence: SEQ ID NO: 77; B chain sequence: SEQ ID NO: 78):

or

conjugate 4 (A chain sequence: SEQ ID NO: 43; B chain sequence: SEQ ID NO: 44):

13. Pharmaceutical composition comprising in a pharmaceutically effective amount the conjugate comprising a sulfonamide of formula (I) and an insulin analog according to any of claims 1 to 12.

14. The conjugate comprising a sulfonamide of formula (I) and an insulin analog according to any of claims 1 to 12 for use as a medicament.

15. The conjugate comprising a sulfonamide of formula (I) and an insulin analog according to any of claims 1 to 12 for use as a medicament for treatment of a disease selected from the group consisting of gestational diabetes, diabetes mellitus type 1 , diabetes mellitus type 2, and hyperglycemia and/or for lowering blood glucose levels.