INSULIN PRECURSORS FOR DIABETES TREATMENT

FIELD OF THE INVENTION

The invention is related to insulin precursor molecules suitable for diabetes treatment, pharmaceutical formulations comprising such and methods for the treatment or pre- vention of hyperglycemia using such.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a metabolic disorder in which the ability to utilize glucose is partly or completely lost. About 5% of all people suffer from diabetes and the disorder approaches epidemic proportions. Human insulin consists of two polypeptide chains, the A and B chains which contain 21 and 30 amino acid residues, respectively. The A and B chains are interconnected by two disul- phide bridges.

Insulin may be produced in e.g. yeast as a single chain precursor molecule, where the A and B chains are connected by a C-peptide, and the B-chain is extended by a leader peptide for optimal processing in yeast. The C-peptide has to be cleaved to yield active insulin. This is done using either Achromobacter lyticus protease (ALP) or trypsin.

Normally, insulin formulations are administered by subcutaneous injection. However, administration by the oral route would be advantageous due to patient compliance, safety and convenience. Oral administration of protein drugs such as insulin has to pass several barriers before reaching the blodstream. These barriers such as enzymatic degradation in the gastrointestinal (Gl) tract, drug efflux pumps and insufficient and variable absorption from the intestinal mucosa, lead to a low bioavailability. Human insulin is subject to degradation by various digestive enzymes found in the stomach (pepsin), in the intestinal lumen (chymotrypsin, tryp- sin, elastase, carboxypeptidases, etc.) and in mucosal surfaces of the Gl tract (aminopepti- dases, carboxypeptidases, enteropeptidases, dipeptidyl peptidases, endopeptidases, etc.). The digestive machinery of the Gl tract thus both represents a severe challenge to protein/peptide drugs but also a machinery that can mature insulin precursors into active forms of insulin. It would thus be advantageous to provide insulin single chain precursor molecules which can be cleaved in the Gl tract by endogenous enzymes such as trypsin into an active form of insulin and thus can be used in oral administration instead of fully matured two chain insulin. This mode of administration would introduce new possibilities to engineer the stability, duration of action and bioavailability of the molecule.

The physical stability of single chain insulin has been found superior to that of two chain insulin (see e.g. WO20050504291 ). The high physical stability leads to long shelf-life and may facilitate use of highly concentrated formulations.

It is thus the object of the invention to obtain insulin precursor molecules suitable for diabetes treatment.

SUMMARY OF THE INVENTION

The present invention is related to insulin precursor molecules.

In one aspect an insulin precursor of the general structure D-B-C-A-E is obtained, wherein A is the human insulin A chain or an analogue thereof, B is the human insulin B chain or an analogue thereof , C is a peptide chain of 0 -15 amino acid residues connecting the C-terminal amino acid residue in the B-chain with the N-terminal amino acid residue in the A-chain, D is a N-terminal exten-sion peptide on the B-chain of 0-15 amino acid residues and E is a C-terminal extension pep-tide on the A-chain of 0-15 amino acid residues.

The insulin precursor may be proteolytically stable. In one aspect of the invention, the insulin precursor comprises the amino acid sequence C which comprises one or more acidic amino acids, one or more branched amino acids and/or one or more bulky amino acids.

In another aspect of the invention, the insulin precursor comprises the amino acid sequence C which comprises the amino acid sequence XZ(K/R)Y, wherein X and Y are each an amino acid which is selected from the group consisting of: Ala, Asp, GIu, VaI, Ser, Thr, lie, Leu, Trp, Tyr and Phe, or are missing;

(K/R) is Lys or Arg; and

Z is a peptide consisting of 1-13 amino acids.

The insulin precursor of the invention may comprise one or more substitutions in the A-chain and/or the B-chain relative to human insulin.

Also described is a pharmaceutical composition comprising an insulin precursor according to the invention and a method for the treatment or prevention of hyperglycemia.

DESCRIPTION OF THE DRAWINGS Fig. 1 : Blood glucose concentration shown as a function of time after injection into the duodenum of anaesthetized Sprague-Dawley rats of B25H, desB30-AAK-A14E human insulin precursor, A14E, B25H, desB30 human insulin and the vehicle.

Fig. 2: Blood glucose concentration shown as a function of time after placing a solution of the B25H, desB30-AAK-A14E human insulin precursor and the A14E, B25H, desB30 human insulin analogue 20 cm beyond s. pylorus, (duodenum/jejunum) in anaesthetized male mini pigs. Fig. 3: Cleavage of the B25H, desB30-AAK-A14E human insulin precursor to the active A14E, B25H, desB30 human insulin analogue after incubation with luminal content of duodenum from SPDR rats is observed using RP-HPLC by retention time shift from 6.4 min to 6.8 min (top trace). Stock solutions of the A14E, B25H, desB30 human insulin analogue (middle trace) and B25H, desB30-AAK-A14E human insulin precursor (bottom trace) are also shown for comparison.

DESCRIPTION OF THE INVENTION

The present invention is related to insulin precursors which can be cleaved in a mammal such as a human in the Gl tract. In one aspect the insulin precursors are cleaved in the intestine. In another aspect the insulin precursors are cleaved in the duodenum, jejunum and/or ileum. In yet another aspect the insulin precursors are cleaved in the duodenum. In yet another aspect the insulin precursors are cleaved in the jejunum.

In one aspect of the invention the insulin precursors are cleaved in the intestine, intestinal lumen and/or at the mocusal surfaces by the endogenous trypsin.

Insulin is a polypeptide hormone secreted by β-cells of the pancreas. Insulin con- sists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge. The term "human insulin" as used herein means the human hormone whose structure and properties are well-known. Human and other mammal insulins have two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B- chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 1 1 of the A-chain, the second between the cysteine in position 7 of the A- chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain. The hormone is in humans synthesized within the beta cells (β-cells) of the islets of

Langerhans in the pancreas as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acids followed by proinsulin containing 86 amino acids in the configuration: prepeptide-B-Arg-Arg-C-Lys-Arg-A, in which C is a connecting peptide of 31

amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.

When insulin is recombinantly produced it is produced as a single-chain or precursor molecule of the configuration D-B-C-A in which C is a connecting peptide and D is an N- terminal extension on the B-chain used for improved expression in the recombinant host. To obtain a mature insulin molecule without the C and D peptides, the insulin precursor is normally cleaved in vitro by either trypsin or ALP. The inventors have surprisingly discovered that a single chain insulin precursor analogue, such as B25H, desB30-AAK-A14E human insulin precursor analogue, may be used in the oral treatment of diseases such as hypergly- cemia, type 1 and 2 diabetes, impaired glucose tolerance and obesity giving rise to a similar BG (blood glucose) lowering effect as the corresponding two chain matured human insulin analogue, i.e. A14E, B25H, desB30 human insulin.

By an "insulin precursor" as used herein is meant a single chain polypeptide sequence of the general structure D-B-C-A-E , wherein B is the human insulin B chain or an analogue thereof, A is the human insulin A chain or an analogue, C is a peptide chain of 0 - 15 amino acid residues connecting the C- terminal amino acid residue in the B-chain (normally B30) with A1 , D is a N-terminal extension peptide on the B-chain of 0-15 amino acid residues and E is a C-terminal extension peptide on the A-chain of 0-15 amino acid residues. If for example the B chain is a desB30 analogue, the connecting peptide will connect B29 with A1 in the single-chain precursor. The C, D and E peptides are thus optional. The insulin precursor will contain correctly positioned disulphide bridges (three) as in human insulin that is between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulfide bridge between CysA6 and CysA1 1.

In one aspect the insulin precursors of the invention may be used for oral admini- stration. Use of insulin precursors for oral administration may be advantageous compared to the use of mature insulins. The pepsin resistance of the insulin precursor may be considerably higher than that of the corresponding insulin analogue. The higher stability towards pepsin degradation may lead to less degradation in the stomach, and therefore formulation of the precursor insulin analogue is facilitated compared to that of the analogue. In one aspect a precursor insulin according to the invention has equal or higher chemical stability compared to the corresponding two chain insulin. For example, the chemical stability of spray dried B25H, desB30-AAK-A14E human insulin precursor at pH 7.4 has been examined in comparison with A14E, B25H, desB30 human insulin and B28D human insulin. The precursor molecule showed only minor differences in chemical stability compared to the two other insulin molecules.

In one aspect of the invention the insulin precursors are activated in-vivo, i.e. in the patient, upon cleavage of the C-peptide resulting in two chain insulin molecules consisting of an A-chain and a B-chain.

Upon cleavage of the insulin precursor all or some of the amino acids from the C- peptide may still be attached to the A- and/or the B-chain of the resulting mature insulin molecule while insulin activity is still obtained. In one aspect all or some of the amino acids from the C-peptide are attached to the A-chain of the insulin molecule. In another aspect all or some of the amino acids from the C-peptide are attached to the B-chain of the insulin molecule. In yet another aspect none of the amino acids from the C-peptide are attached to the A-chain or the B-chain of the insulin molecule.

The insulin precursors according to the invention may be engineered so as to have e.g. a prolonged or immediate BG lowering profile, improved proteolytic stability and/or chemical moieties attached for targeting towards the epithelium of the intestine compared to the mature insulin molecules. In one aspect of the invention, the C-peptide and/or the D- and/or E-peptide comprises one or more mutations to engineer the molecule, e.g. towards improved proteolytic stability, targeting towards the epithelium of the intestine, optimization of release profiles of active insulin, etc.

In one aspect of the invention the rate of cleavage of the C-peptide may be used as a measure of the onset of action of the insulin precursor. The rate of cleavage of the C- peptide depends e.g. on the sequence of the precursor, where in the Gl compartment the precursor is processed and on the pH of the pharmaceutical composition.

In one aspect a slow rate of cleavage of the C-peptide is obtained by substituting one or more amino acids in the A- and/or B-chain of the precursor molecule of human insulin or an analogue thereof. In another aspect one or more amino acids in the A-chain are substituted. In yet another aspect one or more amino acids in the B-chain are substituted. In a further aspect one or more amino acids are substituted, wherein the substitutions are one or more substitutions selected from the list consisting of: B27E, B27D, B27P, B28E, B28D, B28P, A(O)E, A(O)D, A(O)P, A1 E, A1 D, A1 P, A2E, A2D and A2P. In one aspect a precursor molecule according to the invention comprises less than 8 modifications (substitutions, deletions, additions) in the A- and/or B-chain relative to the parent insulin. In one aspect an insulin analogue comprises less than 7 modifications (substitutions, deletions, additions) in the A- and/or B-chain relative to the parent insulin. In one aspect an insulin analogue comprises less than 6 modifications (substitutions, deletions, addi- tions) in the A- and/or B-chain relative to the parent insulin. In another aspect an insulin ana-

logue comprises less than 5 modifications (substitutions, deletions, additions) in the A- and/or B-chain relative to the parent insulin. In another aspect an insulin analogue comprises less than 4 modifications (substitutions, deletions, additions) in the A- and/or B-chain relative to the parent insulin. In another aspect an insulin analogue comprises less than 3 modifica- tions (substitutions, deletions, additions) in the A- and/or B-chain relative to the parent insulin. In another aspect an insulin analogue comprises 1 modification (substitution, deletion or additions) in the A- or B-chain relative to the parent insulin.

In one aspect of the invention a slow rate of cleavage of the C-peptide is obtained by providing an insulin precursor without C-peptide, i.e. where the A- and the B-chain are di- rectly linked.

In one aspect a slow processing rate is obtained by providing a pharmaceutical formulation comprising a solution comprising water and a precursor, wherein the pH is below 7.5. In one aspect a slow processing rate is obtained by providing an aqueous solution comprising a precursor wherein the pH is above 5.0. In one aspect the pH is between 5.0 and 7.5. In another aspect the pH is between 5.5 and 7.5. In yet another aspect the pH is between 6.0 and 7.5. In still another aspect the pH is between 6.0 and 7.0.

In one aspect a slow processing rate is obtained by providing a solid dosage form such as a microemulsion comprising a precursor according to the invention.

In one aspect an insulin precursor of the invention has a C-peptide which is chosen so as to obtain a molecule with no or only little affinity to the insulin receptor. Thus insulin activity is obtained only upon in vivo cleavage, i.e. cleavage in the patient, of said insulin precursor by an enzyme such as trypsin processing in the Gl tract. In a further aspect the insulin precursor of the invention has a C-peptide which has an amino acid sequence wherein the N-terminal amino acid is Asp, GIu or Pro and/or the C-terminal amino acid is Lys or Arg. In a further aspect the N-terminal amino acid of the C-peptide is Asp, GIu or Pro and the C- terminal amino acid of the C-peptide is Lys or Arg. In a still further aspect of the invention the C-peptide consists of between 3 and 6 amino acids such as 3 amino acids, 4 amino acids, 5 amino acids or 6 amino acids. In a yet further aspect an insulin precursor of the invention has a C-peptide which is selected from the group consisting of: AAK, DGK, EGK, DPK,

EPK,

MWK,

SDDAK,

SEEAK, SEDAK,

SDEAK,

DDHLGK,

EEHLGK,

DEHLGK, EDHLGK,

DGKD,

EGKD,

EGKE,

DGKE, DPDK,

EPDK,

EPEK,

DPEK,

AAR, DGR,

EGR,

DPR,

EPR,

MWR, SDDAR,

SEEAR,

SDEAR,

SEDAR,

DDHLGR, EEHLGR,

DEHLGR,

EDHLGR,

DGRD,

EGRE, DGRE,

EGRD, DPDR, EPER, DPER, EPDR and missing.

In a yet further aspect an insulin precursor of the invention has a C-peptide which is selected from the group consisting of: AAK,

AAR,

DPDK,

DPDR,

DGK, DGR,

SDDAK,

SDDAR,

DPK,

DPR, DGKD,

DGKR,

DDHLGK,

DDHLGR and

Missing.

In a yet further aspect an insulin precursor of the invention has a C-peptide which is selected from the group consisting of:

AAK,

AAR, SDDAK and

SDDAR.

In a yet further aspect an insulin precursor of the invention has a C-peptide which is selected from the group consisting of: DPDK,

DPDR,

DGKD,

DGRD,

DDHLGK,

DDHLGR and

Missing.

In one aspect an insulin precursor of the invention has a C-peptide which is chosen so as to obtain a molecule with a protracted or immediate action. A protracted action could be obtained by selecting a C-peptide that is slowly processed by Gl enzymes into the active insulin. In one aspect of the invention the C-peptide according to the invention comprises acidic, branched and/or bulky amino acids. In another aspect of the invention the C-peptide contains only acidic, branched and/or bulky amino acids. For further reduction of processing speed these C-peptides can be combined with substitutions in the N-terminus of insulin A- chain using acidic, branched or bulky amino acids.

In one aspect the C-peptide of the invention comprises the amino acid sequence XZ(K/R)Y, wherein X and Y are each an amino acid, which is selected from the group consisting of: Ala, Asp, GIu, VaI, Ser, Thr, lie, Leu, Trp, Tyr and Phe, or are missing, (K/R) is Lys or Arg and Z is a peptide consisting of 1-13 amino acids. In another aspect the C-peptide comprises the amino acid sequence XZKY. In yet another aspect of the invention the C- peptide comprises the amino acids: DGK. In still another aspect of the invention the C- peptide consists of the amino acids: DGK.

In one aspect the C-peptide of the invention is for obtaining an insulin precursor with immediate action. In another aspect of the invention the C-peptide comprises the amino acid sequence AAK or AAR. In yet another aspect of the invention the C-peptide consists of the amino acid sequence AAK or AAR.

By "protracted action" of an insulin precursor of the invention is herein meant an insulin precursor which has a time-action of more than 8 hours in standard models of diabetes e.g. pharmacokinetic disappearance and/or appearance in pigs. In one embodiment, the insulin precursor has a time-action of more than about 12 hours. In another embodiment, the insulin precursor has a time-action in the range from about 12 hours to about 168 hours. In another embodiment, the insulin precursor has a time- action in the range from about 24 hours to about 168 hours. In yet another embodiment, the insulin precursor has a time-action in the range from about 48 hours to about 168 hours. In one embodiment, the insulin precursor has a time-action similar to that observed for com-

mercial pharmaceutical compositions of Levemir ^® and Lantus ^® . The term about in relation to the time-action of insulins means + or - 30 minutes.

By "immediate action" of an insulin precursor of the invention is herein meant an insulin precursor which has a time-action of less than 8 hours in standard models of diabetes e.g. pharmacokinetic disappearance and/or appearance in pigs.

In one embodiment, the insulin precursor has a time-action of less than about 5 hours. In another embodiment, the insulin precursor has a time-action in the range from 0 hours to about 4 hours. In one embodiment, the insulin precursor has a time-action similar to that observed for commercial pharmaceutical compositions of Actrapid ^® , Novolog ^® , Huma- log ^® and Apidra ^® . The term about in relation to the time-action of insulins means + or - 30 minutes.

In one aspect an insulin precursor of the invention is of the general structure D-B-C- A-E, wherein D and E are independently selected so as to obtain higher expression yields, resistance to proteolytic degradation and/or improved bioavailability. In one aspect an insulin precursor of the invention is of the general structure D-B-C-A, wherein D is a peptide chain of 1-15 amino acid residues. In another aspect an insulin precursor of the invention is of the general structure B-C-A-E, wherein E is a peptide chain of 1 -15 amino acid residues. In yet another aspect an insulin precursor of the invention is of the general structure B-C-A, i.e. wherein neither D nor E is present. In still another aspect an insulin precursor of the invention is of the general structure B-A, i.e. wherein neither the C nor D nor E is present.

In a further aspect of the invention D is selected from the group consisting of EEAEAEAPK, EEAEPK, EEGEPK,

EEAEAEAPR, EEAEPR and EEGEPR

In a further aspect of the invention E is selected from the group consisting of: (GGG)x,

(PPG)x, (GGE)x and (GGK)x wherein x is a numerical number between 1-10.

By "mature insulin molecule" is herein meant human insulin or an analogue thereof with the same structural conformation as the natural human insulin molecule, i.e. having a two-chain conformation and disulfide bridges between Cys ^A7 and Cys ^B7 and between Cys ^A20 and Cys ^B19 and an internal disulfide bridge between Cys ^A6 and Cys ^A11 , and with insulin activ- ity. Thus, a mature insulin molecule according to the present invention would be e.g. human insulin, B28D human insulin, desB30 human insulin or A14E, B25H,desB30 human insulin. By "insulin analogue" as used herein is meant a polypeptide derived from the primary structure of a naturally occurring insulin, for example that of human insulin, by mutation. One or more mutations are made by deleting and/or substituting at least one amino acid residue occurring in the naturally occurring insulin and/or by adding at least one amino acid residue. The added and/or substituted amino acid residues can either be codable amino acid residues or other naturally occurring amino acid residues.

Mutations in the insulin precursor are denoted stating the chain (A or B), the position, and the three letter code for the amino acid substituting the native amino acid. With "desB30" or "B(1-29)" is meant a natural insulin B chain or analogue thereof lacking the B30 amino acid residue and "A(1-21 )" means the natural insulin A chain.

Herein terms like A1 , A2, A3 etc. indicates the position 1 , 2 and 3, respectively, in the A chain of insulin (counted from the N-terminal end). Similarly, terms like B1 , B2, B3 etc. indicates the position 1 , 2 and 3, respectively, in the B chain of insulin (counted from the N- terminal end). Using the one letter codes for amino acids, terms like A21A, A21 G and A21Q designates that the amino acid in the A21 position is A, G and Q, respectively. Using the three letter codes for amino acids, the corresponding expressions are A21Ala, A21 Gly and A21Gln, respectively. Thus, A14E, B25H, desB30 human insulin is an analogue of human insulin where position 14 in the A chain is mutated to glutamic acid, position 25 in the B chain is mutated to histidine, and the position 30 in the B chain is deleted.

Herein the terms A(O) or B(O) indicate the positions of the amino acids N-terminally to A1 or B1 , respectively. The terms A(-1) or B(-1) indicate the positions of the first amino acids N-terminally to A(O) or B(O), respectively. Thus A(-2) and B(-2) indicate positions of the amino acids N-terminally to A(-1) and B(-1), respectively, A(-3) and B(-3) indicate positions of the amino acids N-terminally to A(-2) and B(-2), respectively, and so forth.

The insulin precursors are primarily named according to the following rule described by the overall sequence D-B-C-A-E: The sequence starts with the D-peptide, continues with the B-chain, continues with the C-peptide, continues with the A-chain and ends with the E- peptide. The amino acid residues are named after their respective counterparts in human insulin and mutations are explicitly described whereas unaltered amino acid residues in the

A- and B-chains are not mentioned. For example, an insulin precursor having the following mutations as compared to human insulin A14E, B25H and desB30 and comprising the C- peptide AAK connecting the C-terminal B-chain and the N-terminal A-chain, and comprising the D-peptide extension EEAEAEAPK on the B-chain N-terminus is named EEAEAEAPK- B25H, desB30-AAK-A14E human insulin precursor.

An insulin precursor according to the invention may be the precursor of an insulin molecule which comprises one or more mutations.

In one aspect an insulin precursor according to the invention is a molecule described by the overall sequence D-B-C-A-E, wherein C, D and E are optional, and wherein the A- and the B-chain contain the amino acids of human insulin.

In one aspect an insulin precursor according to the invention is a molecule described by the overall sequence D-B-C-A-E, wherein C, D and E are optional, and wherein the A- and/or the B-chain comprise one or more mutations selected from the group consist- ing of:

• the amino acid in position AO is GIu, Asp, Pro, VaI, lie, Thr or is absent

• the amino acid in position A1 is GIu, Asp, Pro, VaI, lie, Thr

• the amino acid in position A2 is GIu, Asp, Pro, VaI, lie, Thr

• the amino acid in position A12 is GIu or Asp • the amino acid in position A13 is His, Asn, GIu or Asp

• the amino acid in position A14 is Asn, GIn, GIu, Arg, Asp, GIy or His

• the amino acid in position A15 is GIu or Asp • the amino acid in position A22 is Lys

• the amino acid in position B24 is His or GIy • the amino acid in position B25 is His

• the amino acid in position B26 is His, GIy, Asp, GIu or Thr

• the amino acid in position B27 is His, GIu, Lys, GIy or Arg

• the amino acid in position B28 is His, GIy, GIu or Asp; and

• the amino acid residue in position B28 is Pro, Asp, Lys, Leu, VaI or Ala and the amino acid residue in position B29 is Lys or Pro and optionally the amino acid residue in position B30 is deleted;

• the amino acids in positions B26, B27, B28, B29 and B30 are deleted or replaced by GIy

• the amino acids in positions B27, B28, B29 and B30 are deleted or replaced by GIy

• the amino acids in positions B28, B29 and B30 are deleted or replaced by GIy

• the amino acids in positions B29 and B30 are deleted or replaced by GIy

• the amino acid in position B27 is deleted

• the amino acid in position B30 is deleted • the amino acid residue in position B3 is Lys and the amino acid residue in position

B29 is GIu or Asp; and

• the amino acid residue in position A21 is GIy, and wherein the C-peptide comprises two Arg residues which are retained after in vivo cleavage.

The term "diabetes" includes type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

The term "treatment" of a disease includes treatment, prevention or alleviation of the disease.

In one embodiment of the invention the insulin precursor is particularly suitable for oral administration.

"An insulin" according to the invention is herein to be understood as human insulin, an insulin analogue or an insulin derivative.

The term "parent insulin" as used herein is intended to mean an insulin before any mutations according to the invention have been applied thereto. Non-limiting examples of parent insulins are e.g. a wild-type insulin such as human insulin or porcine insulin, an analogue of human insulin or a derivative of human insulin or an insulin analogue such as human insulin or an insulin analogue which has been PEGylated or acylated.

In one embodiment a parent insulin according to the invention is human insulin. A "protease" or a "protease enzyme" is a digestive enzyme which degrades proteins and peptides and which is found in various tissues of the human body such as e.g. the stomach (pepsin), the intestinal lumen (chymotrypsin, trypsin, elastase, carboxypeptidases, etc.) or mucosal surfaces of the Gl tract (aminopeptidases, carboxypeptidases, enteropeptidases, dipeptidyl peptidases, endopeptidases, etc.), the liver (Insulin degrading enzyme, cathepsin D etc), and in other tissues. An insulin precursor according to the invention may be a proteolytically stable insulin precursor.

A proteolytically stable insulin precursor is herein to be understood as an insulin precursor, which is subjected to slower degradation by one or more proteases relative to human insulin. In one embodiment a proteolytically stable insulin precursor according to the invention is subjected to slower degradation by one or more proteases relative to the parent

insulin. In a further embodiment of the invention an insulin precursor according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: pepsin (such as e.g. the isoforms pepsin A, pepsin B, pepsin C and/or pepsin F), chymotrypsin (such as e.g. the isoforms chymotrypsin A, chymotrypsin B and/or chymotryp- sin C), trypsin, Insulin-Degrading Enzyme (IDE), elastase (such as e.g. the isoforms pancreatic elastase I and/or II), carboxypeptidase (e.g. the isoforms carboxypeptidase A, car- boxypeptidase A2 and/or carboxypeptidase B), aminopeptidase, cathepsin D and other enzymes present in intestinal extracts derived from rat, pig or human.

In one embodiment an insulin precursor according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: chymotrypsin, trypsin, Insulin-Degrading Enzyme (IDE), elastase, carboxypeptidases, aminopep- tidases and cathepsin D. In a further embodiment an insulin precursor according to the invention is stabilized against degradation by one or more enzymes selected from the group consisting of: chymotrypsin, carboxypeptidases and IDE. In a yet further embodiment an in- sulin precursor according to the invention is stabilized against degradation by one or more enzymes selected from: chymotrypsin and carboxypeptidases.

In one aspect an insulin precursor according to the invention is proteolytically stable. T/4 may be determined by measuring the amount of intact precursor by RP-HPLC as a function of time and is used as a measure of the proteolytical stability of an insulin precursor ac- cording to the invention towards protease enzymes such as chymotrypsin, pepsin and/or carboxypeptidase A. In one embodiment of the invention T/4 is increased relative to human insulin. In a further embodiment T/4 is increased relative to the parent insulin. In a yet further embodiment T/4 is increased at least 2-fold relative to the parent insulin. In a yet further embodiment T/4 is increased at least 3-fold relative to the parent insulin. In a yet further em- bodiment T/4 is increased at least 4-fold relative to the parent insulin. In a yet further embodiment T/4 is increased at least 5-fold relative to the parent insulin. In a yet further embodiment T/4 is increased at least 10-fold relative to the parent insulin.

An insulin precursor according to the invention may have increased potency and/or bioavalability relative to the parent insulin when compared upon measurement. Standard assays for measuring insulin potency or bioavailability are known to the person skilled in the art and include inter alia (1) insulin radioreceptorassays, in which the relative potency of an insulin is defined as the ratio of insulin to insulin precursor required to displace 50% of ¹²⁵ l-insulin specifically bound to insulin receptors present on cell membranes, e.g. a rat liver plasma membrane fraction; (2) lipogenesis assays, performed e.g. with rat adipocytes, in which relative insulin potency is defined as the ratio of insulin to insulin

precursor required to achieve 50% of the maximum conversion of [3- ³ H] glucose into organic- extractable material (i.e. lipids); (3) glucose oxidation assays in isolated fat cells in which the relative potency of the insulin precursor is defined as the ratio of insulin to insulin precursor to achieve 50% of the maximum conversion of glucose-1 -[ ¹⁴ C] into [ ¹⁴ CO ₂ ]; (4) insulin radio- immunoassays which can determine the immunogenicity of insulin precursors by measuring the effectiveness by which insulin or an insulin precursor competes with ¹²⁵ l-insulin in binding to specific anti-insulin antibodies; and (5) other assays which measure the binding of insulin or an insulin precursor to antibodies in animal blood plasma samples, such as ELISA assays possessing specific insulin antibodies. The production of polypeptides is well known in the art. Insulin precursors according to the invention may for instance be produced by classical peptide synthesis, e.g. solid phase peptide synthesis using t-Boc or Fmoc chemistry or other well established techniques, see e.g. Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999. The insulin precursors may also be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the analogue and capable of expressing the analogue in a suitable nutrient medium under conditions permitting the expression of the analogue. For insulin precursors comprising non-natural amino acid residues, the recombinant cell should be modified such that the non-natural amino acids are incorporated into the analogue, for instance by use of tRNA mutants. The present invention is also related to nucleic acid sequences which code for the claimed insulin precursors. In a further embodiment the present invention is related to vectors containing such nucleic acid sequences and host cells containing such nucleic acid sequences or vectors.

In still a further embodiment, the invention relates to a process for producing an in- sulin precursor comprising (i) culturing a host cell comprising a nucleic acid sequence encoding an insulin precursor; (ii) isolating the insulin precursor from the culture medium.

In still a further embodiment, the invention relates to a process for producing an insulin precursor comprising (i) culturing a host cell comprising a nucleic acid sequence encoding an insulin precursor; (ii) isolating the insulin precursor from the culture medium. In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae.

PHARMACEUTICAL COMPOSITIONS

Another object of the present invention is to provide a pharmaceutical formulation comprising an insulin precursor according to the present invention which is present in a concentration from 0.1 mg/ml to 500 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0. The formulation may further comprise protease inhibitor(s), a buffer system, preservative^), tonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment of the invention the pharmaceutical formulation is an aqueous formulation, i.e. formulation comprising water. Such formulation is typically a solution or a suspension. In a further embodiment of the invention the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50 %w/w water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50 %w/w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50 %w/w water.

In another embodiment the pharmaceutical formulation is a freeze-dried formulation, whereto the physician or the patient adds solvents and/or diluents prior to use.

In another embodiment the pharmaceutical formulation is a dried formulation (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In a further embodiment the invention relates to a pharmaceutical formulation comprising an aqueous solution of an insulin precursor of the present invention, and a buffer, wherein said insulin precursor is present in a concentration from 0.1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.

Pharmaceutical compositions containing an insulin precursor according to the present invention may be orally administered to a patient in need of such treatment.

Compositions of the current invention may be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multiple emulsion, ointments, tablets, coated tablets, capsules, for example, hard gelatine capsules and soft gelatine capsules, drops, gels, sprays, powder, aerosols micro- and nano-suspension, effervescent tablets, sublingual tablets, buccal tablets, granules, in situ transforming solutions, for example in situ gelling, in situ setting, in situ precipitating, in situ crystallization, stomach floating formulation such as floating suspension, floating tablet or the like.

Compositions of the invention may further be compounded in, or attached to, for example through covalent, hydrophobic and electrostatic interactions, a drug carrier, drug delivery system and advanced drug delivery system in order to further enhance stability of the insulin precursor compound, increase bioavailability, increase solubility, decrease adverse

effects, achieve chronotherapy well known to those skilled in the art, and increase patient compliance or any combination thereof.

Compositions of the current invention may be useful in the formulation of controlled, sustained, protracting, retarded, and slow release drug delivery systems. More specifically, but not limited to, compositions may be useful in formulation of controlled release and sustained release systems (both systems leading to a many-fold reduction in number of administrations), well known to those skilled in the art. Without limiting the scope of the invention, examples of useful controlled release system and compositions are hydrogels, oleaginous gels, liquid crystals, polymeric micelles, microspheres, nanoparticles. Methods to produce controlled release systems useful for compositions of the current invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high pressure homogenisation, encapsulation, spray drying, microencapsulating, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Formulations intended for oral use may be prepared according to any known method, and such formulations may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents, and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient in a mixture with non-toxic pharmaceutically-acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as mannitol, maltodextrin, kaolin, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch; binding agents, for example, starch, gelatine, polymers or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration or release of the therapeutically active polypeptide. The orally administerable formulations of the present invention may be prepared and administered according to methods well known in pharmaceutical chemistry, see Remington's Pharmaceutical Sciences, 17 ^th ed. (A. Osol ed., 1985).

In one embodiment of the invention, the pharmaceutical compositions of the present invention may be administered by means of solid dosage forms such as but not limited to

tablets and capsules. The tablets may be prepared by e.g. wet granulation, by dry granulation, by direct compression or melt granulation.

Tablets for this invention may be prepared utilizing conventional tabletting techniques. A general method of manufacture involves blending of an insulin precursor, a water-soluble diluent, hydrophilic binder and optionally a portion of a disintegrant. This blend is then granulated with an aqueous solution of the hydrophilic binder or an aqueous solution of the hydrophilic binder and surfactant and milled, if necessary. The granules are dried and reduced to a suitable size. Any other ingredients, such as lubricants, (e.g. magnesium stearate) and additional disintegrants, are added to the granules and mixed. This mixture is then compressed into a suitable size and shape using conventional tabletting machines such as a rotary tablet press. The tablets may be film coated by techniques well known in the art.

Formulations for oral use may also be presented as hard or soft gelatine capsules where the active ingredient is mixed with an inert solid diluent, for example, such as mannitol, maltodextrin, calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate or sodium phosphate, or a soft gelatine capsule wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Capsules for this invention may be prepared utilizing conventional methods. A general method of manufacture involves blending a therapeutically active peptide, alginate, a water- soluble diluent, a hydrophilic binder, and optionally a portion of a disintegrant. This blend is then granulated with an aqueous solution of the hydrophilic binder or an aqueous solution of the hydrophilic binder and surfactant in water, and milled, if necessary. The granules are dried and reduced to a suitable size. Any other ingredients, such as a lubricant, are added to the granules and mixed. The resulting mixture is then filled into a suitable size hard-shell gelatin capsule using conventional capsule-filling machines. In a further embodiment of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the formulation further comprises a pharmaceutically acceptable preservative. The preservative is present in an amount sufficient to obtain a preserving effect. The amount of preservative in a pharmaceutical formulation is the well-known to the skilled person and may be determined from e.g. literature in the field and/or the known amount(s) of preservative in e.g. commercial products. Each

one of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995. In a further embodiment of the invention the formulation further comprises a chelating agent. The use of a chelating agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995.

In a further embodiment of the invention the formulation further comprises a stabi- lizer. The term "stabiliser" as used herein refers to chemicals added to polypeptide containing pharmaceutical formulations in order to stabilize the peptide, i.e. to increase the shelf life and/or in-use time of such formulations. The use of a stabilizer in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995. In a further embodiment of the invention the formulation further comprises a surfactant. The term "surfactant" as used herein refers to any molecules or ions that are comprised of a water-soluble (hydrophilic) part, the head, and a fat-soluble (lipophilic) segment. Surfactants accumulate preferably at interfaces, which the hydrophilic part is orientated towards the water (hydrophilic phase) and the lipophilic part towards the oil- or hydrophobic phase (i.e. glass, air, oil etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. Furthermore, surfactants lower the surface tension of a liquid. Surfactants are also known as amphipathic compounds. The term "Detergent" is a synonym used for surfactants in general. The use of a surfactant in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19 ^th edition, 1995.

In a further embodiment of the invention the formulation further comprises protease inhibitors.

It is possible that other ingredients may be present in the insulin precursor pharmaceutical formulation of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, tonicity modifiers, chelating agents, metal ions, oleaginous vehicles, proteins (e.g., human serum albumin, gelatine or proteins) and a zwitterion (e.g., an amino acid such as betaine, taurine, arginine, glycine, lysine and his- tidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

The term "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability.

The term "physical stability" of the protein formulation as used herein refers to the tendency of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein formulations is evaluated by means of visual inspection and/or turbidity measurements after exposing the formulation filled in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different tempera- tures for various time periods. Visual inspection of the formulations is performed in a sharp focused light with a dark background. The turbidity of the formulation is characterized by a visual score ranking the degree of turbidity for instance on a scale from 0 to 3 (a formulation showing no turbidity corresponds to a visual score 0, and a formulation showing visual turbidity in daylight corresponds to visual score 3). A formulation is classified physical unstable with respect to protein aggregation, when it shows visual turbidity in daylight. Alternatively, the turbidity of the formulation can be evaluated by simple turbidity measurements well- known to the skilled person. Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule that preferentially binds to a non-native con- former of the protein. One example of a small molecular spectroscopic probe of protein structure is Thioflavin T. Thioflavin T is a fluorescent dye that has been widely used for the detection of amyloid fibrils. In the presence of fibrils, and perhaps other protein configurations as well, Thioflavin T gives rise to a new excitation maximum at about 450 nm and enhanced emission at about 482 nm when bound to a fibril protein form. Unbound Thioflavin T is essen- tially non-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in protein structure from native to non-native states. For instance the "hydrophobic patch" probes that bind preferentially to exposed hydrophobic patches of a protein. The hydrophobic patches are generally buried within the tertiary structure of a protein in its native state, but become exposed as a protein begins to unfold or denature. Examples of these small molecular, spectroscopic probes are aromatic, hydrophobic dyes, such as antrhacene, acridine, phenanthroline or the like. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids, such as phenylalanine, leucine, isoleucine, methionine, and valine, or the like.

The term "chemical stability" of the protein formulation as used herein refers to chemical covalent changes in the protein structure leading to formation of chemical degradation products with potential less biological potency and/or potential increased immunogenic properties compared to the native protein structure. Various chemical degradation products can be formed depending on the type and nature of the native protein and the environment to which the protein is exposed. Elimination of chemical degradation can most probably not be completely avoided and increasing amounts of chemical degradation products is often seen during storage and use of the protein formulation as well-known by the person skilled in the art. Most proteins are prone to deamidation, a process in which the side chain amide group in glutaminyl or asparaginyl residues is hydrolysed to form a free carboxylic acid. Other degradations pathways involves formation of high molecular weight transformation products where two or more protein molecules are covalently bound to each other through transami- dation and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation products (Stability of Protein Pharmaceuticals, Ahern. T.J. & Man- ning M. C, Plenum Press, New York 1992). Oxidation (of for instance methionine residues) can be mentioned as another variant of chemical degradation. The chemical stability of the protein formulation can be evaluated by measuring the amount of the chemical degradation products at various time-points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by for instance increasing tempera- ture). The amount of each individual degradation product is often determined by separation of the degradation products depending on molecule size and/or charge using various chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a "stabilized formulation" refers to a formulation with increased physical stability, increased chemical stability or increased physical and chemical stability. In general, a formulation must be stable during use and storage (in compliance with recommended use and storage conditions) until the expiration date is reached.

In one embodiment of the invention the pharmaceutical formulation comprising the insulin precursor compound is stable for more than 6 weeks of usage and for more than 3 years of storage. In another embodiment of the invention the pharmaceutical formulation comprising the insulin precursor compound is stable for more than 4 weeks of usage and for more than 3 years of storage.

In a further embodiment of the invention the pharmaceutical formulation comprising the insulin precursor compound is stable for more than 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention the pharmaceutical formulation comprising the insulin precursor compound is stable for more than 2 weeks of usage and for more than two years of storage.

Aqueous suspensions may contain the active compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These formulations may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavouring, and colouring agents may also be present. The pharmaceutical formulations comprising a compound for use according to the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, preservative and flavouring and colouring agent.

In a further embodiment of the invention, the formulation further comprises a per- meation enhancer. Bile salts and fatty acids are most often considered to increase the oral permeability of the lipid bi-layer membranes of the epithelial cell lining of the Gl tract. In general, permeation enhancers increase paracellular and trancellular transport of macromole- cules by reversible altering the membrane integrity. The bile salt is selected from the group consisting of cholate, deoxycholate, taurocholate, glycocholate, taurodeoxycholate, ursode- oxycholate, tauroursodeoxycholate, and chenodeoxycholate. The fatty acid is selected from

the group of short, medium and long chain fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, plamitic acid, stearic acid etc. Others enhancers could be surfactants such as monoglycerides, polyoxyethylene esters, sorbitan surfactants (noninic) and sulphates (anionic). In a further embodiment of the invention, the formulation further comprises a muco- adhesive polymer. An intimate contact of the drug delivery system to the mucosa of the gastrointestinal tract can be obtained by use of such a mucoadhesive polymer. An intimate contact of the dosage form to the membrane seems advantageous as an enzymatic degradation of the therapeutically active polypeptide on the way between the delivery system and the ab- sorption membrane can be avoided. Moreover, a step concentration gradient on the absorption membrane representing the driving force for passive drug uptake can be provided.

In a further embodiment of the invention, the formulation further comprises an inhibitor of a proteolytic enzyme(s) to further circumvent the enzymatic barrier and achieving the delivery of the therapeutically active polypeptide such as aminopeptidase inhibitor, amas- tatin, bestatin, boroleucine and puromycin. Examples of protease inhibitors are sodium glyco- late, camostat mesilate, bacitracin, soybean trypsin inhibitor and aprotinin.

Entrapment and encapsulation is a technique used in drug delivery systems for therapeutically active polypeptides to optimize delivery properties including protection against enzymatic degradation. Entrapment or encapsulation could be in the form of polymeric drug delivery systems such as hydrogels and nanocapsules/microspheres, and lipid drug delivery systems such as liposomes and micro emulsions.

In another embodiment, the present invention relates to an insulin precursor according to the invention for use as a medicament.

In one embodiment, an insulin precursor according to the invention is used for the preparation of a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetes,.

In another embodiment, an insulin precursor according to the invention is used as a medicament for delaying or preventing disease progression in type 2 diabetes.

In one embodiment of the invention, the derivative according to the invention is for use as a medicament for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetesor for delaying or preventing disease progression in type 2 diabetes , is provided.

In a further embodiment of the invention, a method for the treatment or prevention of hyperglycemia, type 2 diabetes, impaired glucose tolerance and type 1 diabetes or for delay- ing or preventing disease progression in type 2 diabetes, the method comprising administer-

ing to a patient in need of such treatment an effective amount for such treatment of an insulin precursor according to the invention, is provided.

The treatment with an insulin precursor according to the present invention may also be combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, antiobesity agents, appetite regulating agents, antihypertensive agents, agents for the treatment and/or prevention of complications resulting from or associated with diabetes and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity. Examples of these pharmacologically active substances are: GLP-1 and GLP-1 derivatives and analogues, GLP-2 and GLP-2 derivatives and analogues, Exendin-4 and Exendin-4 derivatives and analogues, amylin and amylin derivatives and analogues, sulphonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, glucose uptake modulators, compounds modifying the lipid metabolism such as antihyperlipidemic agents as HMG CoA inhibitors (statins), compounds lowering food intake, RXR agonists and agents acting on the ATP-dependent potassium channel of the β-cells; Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, neteglinide, re- paglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and α- blockers such as doxazosin, urapidil, prazosin and terazosin; CART (cocaine amphetamine regulated transcript) agonists, NPY (neuropeptide Y) antagonists, MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosis factor) agonists, CRF (corticotropin releas- ing factor) agonists, CRF BP (corticotropin releasing factor binding protein) antagonists, uro- cortin agonists, β3 agonists, MSH (melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentrating hormone) antagonists, CCK (cholecystokinin) agonists, serotonin re-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors, mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists, bombesin agonists, galanin antago- nists, growth hormone, growth hormone releasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP 2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DA agonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR (retinoid X receptor) modulators, TR β agonists; histamine H3 antagonists, gastrin and gastrin analogues and derivatives.

It should be understood that any suitable combination of the derivatives according to the invention with one or more of the above-mentioned compounds and optionally one or more further pharmacologically active substances are considered to be within the scope of the present invention.

The following is non-limiting list of aspects, which are further described elsewhere herein:

1. An insulin precursor of the general structure D-B-C-A-E, wherein A is the human insulin A chain or an analogue thereof, B is the human insulin B chain or an analogue thereof , C is a peptide chain of 0 -15 amino acid residues connecting the C-terminal amino acid residue in the B-chain with the N-terminal amino acid residue in the A-chain, D is a N- terminal extension peptide on the B-chain of 0-15 amino acid residues and E is a C-terminal extension pep-tide on the A-chain of 0-15 amino acid residues.

2. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 , which is proteolytically stable. 3. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects, wherein C comprises one or more acidic amino acids, one or more branched amino acids and/or one or more bulky amino acids.

4. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects, wherein C has an amino acid sequence wherein the N-terminal amino acid is Asp, GIu or Pro and/or the C-terminal amino acid is Lys or Arg.

5. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects, wherein C consists of between 3 and 6 amino acids such as 3 amino acids, 4 amino acids, 5 amino acids or 6 amino acids.

6. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 , wherein C is selected from the group consisting of:

AAK,

DGK,

EGK,

DPK, EPK,

MWK,

SDDAK,

SEEAK,

SEDAK, SDEAK,

DDHLGK,

EEHLGK,

DEHLGK,

EDHLGK, DGKD,

EGKD,

EGKE,

DGKE,

DPDK, EPDK,

EPEK,

DPEK,

AAR,

DGR, EGR,

DPR,

EPR,

MWR,

SDDAR, SEEAR,

SDEAR,

SEDAR,

DDHLGR,

EEHLGR, DEHLGR,

EDHLGR,

DGRD,

EGRE,

DGRE, EGRD,

DPDR,

EPER,

DPER,

EPDR, and Missing.

7. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 , wherein C comprises the amino acid sequence XZ(K/Y)Y, wherein X and Y are each an amino acid which is selected from the group consisting of: Ala, Asp, GIu, VaI, Ser, Thr, lie, Leu, Trp, Tyr and Phe, or are missing;

(K/R) is Lys or Arg; and Z is a peptide consisting of 1-13 amino acids.

8. An insulin precursor of the general structure D-B-C-A-E according to aspect 7, wherein C comprises the amino acid sequence DGK. 9. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 , wherein C comprises the amino acid sequence AAK or AAR.

10. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 or 2, wherein C is absent.

11. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects, wherein D is selected from the group consisting of

EEAEAEAPK, EEAEPK, EEGEPK, EEAEAEAPR, EEAEPR, and EEGEPR.

12. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects, wherein E is selected from the group consisting of:

(GGG)x, (PPG)x,

(GGE)x, and

(GGK)x wherein x is a numerical number between 1 -10.

13. An insulin precursor of the general structure D-B-C-A-E according to any one of the pre-ceding aspects which comprises one or more substitutions in the A-chain and/or one or more substitutions in the B-chain relative to human insulin.

14. An insulin precursor of the general structure D-B-C-A-E according aspect 13, wherein the substitution(s) in A and/or B are one or more substitutions selected from the list consisting of: B27E, B27D, B27P, B28E, B28D, B28P, A(O)E, A(O)D, A(O)P, A1 E, A1 D, A1 P, A2E, A2D and A2P.

15. An insulin precursor of the general structure D-B-C-A-E according to any one of aspects 1-13, wherein C, D and E are optional, and wherein A and/or B comprise one or more mutations selected from the group consisting of: the amino acid in position AO is GIu, Asp, Pro, VaI, lie, Thr or is absent; the amino acid in position A1 is GIu, Asp, Pro, VaI, lie, Thr; the amino acid in position A2 is GIu, Asp, Pro, VaI, lie, Thr; the amino acid in position A12 is GIu or Asp; the amino acid in position A13 is His, Asn, GIu or Asp; the amino acid in position A14 is Asn, GIn, GIu, Arg, Asp, GIy or His; the amino acid in position A15 is GIu or Asp; the amino acid in position A22 is Lys; the amino acid in position B24 is His or GIy; the amino acid in position B25 is His; the amino acid in position B26 is His, GIy, Asp, GIu or Thr; the amino acid in position B27 is His, GIu, Lys, GIy or Arg; the amino acid in position B28 is His, GIy, GIu or Asp; the amino acid residue in position B28 is Pro, Asp, Lys, Leu, VaI or Ala and the amino acid residue in position B29 is Lys or Pro and optionally the amino acid resi-due in position B30 is deleted; the amino acids in positions B26, B27, B28, B29 and B30 are deleted or replaced by

GIy; the amino acids in positions B27, B28, B29 and B30 are deleted or replaced by GIy; the amino acids in positions B28, B29 and B30 are deleted or replaced by GIy; the amino acids in positions B29 and B30 are deleted or replaced by GIy; the amino acid in position B27 is deleted; the amino acid in position B30 is deleted; the amino acid residue in position B3 is Lys and the amino acid residue in position B29 is GIu or Asp; and the amino acid residue in position A21 is GIy, wherein the C-peptide comprises two Arg residues which are retained after in vivo cleavage.

16. An insulin precursor of the general structure D-B-C-A-E according to aspect 1 or 2, which is selected from the group consisting of:

EEAEAEAPK-B25H-desB30-AAK-A14E human insulin precursor, EEAEAEAPK-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor, B25H-desB30-AAK-A14E human insulin precursor,

EEAEPK-B25H-desB30-DGK-A14E human insulin precursor,

EEAEAEAPK-B1 E-B25H-desB30-AAK-A14E human insulin precursor,

EEAEAEAPK-B25H-B27E-desB30-AAK-A8H-A14E human insulin precursor,

EEAEAEAPK-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor, EEAEAEAPK-B1 E-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor,

B22K, B25H, B29R, desB30-AAR-A14E human insulin precursor,

B25H, B29R, desB30-AAR-A14E, A22K human insulin precursor,

B25H, 29R, desB30-A14E human insulin precursor,

B25H, desB30-DGK-A14E human insulin precursor, B25H, desB30-SDDAK-A14E human insulin precursor,

B25H, desB30-A14E human insulin precursor,

B25H, desB30-DPK-A14E human insulin precursor,

B25H, desB30-DGKD-A14E human insulin precursor,

B25H, desB30-DDHLGK-A14E human insulin precursor, B22A,B25H desB30-AAK-A14E human insulin precursor,

B22E, B25H desB30-AAK-A14E human insulin precursor,

B3K, B25H, B29R, desB30-AAR-A14E human insulin precursor,

B25H desB30-DPDK-A14E human insulin precursor, and

B25H, desB30-AAR-A14E human insulin precursor

17. A pharmaceutical composition comprising an insulin precursor of the general structure D-B-C-A-E according to any one of the preceding aspects.

18. A pharmaceutical composition comprising an insulin precursor of the general structure D-B-C-A-E according to aspect 17, which is in the form of a solution which further comprises water, wherein pH is below 7.5.

19. A pharmaceutical composition comprising an insulin precursor of the general structure D-B-C-A-E according to aspect 17 or 18, which is in the form of a solution which further corn-prises water, wherein pH is above 5.0.

20. An insulin precursor of the general structure D-B-C-A-E according to any one of aspects 1 -16 for use as a medicament for the treatment or prevention of hyperglycemia including stress induced hyperglycemia and hyperglycemia in acute critical illness, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds, and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease, and other cardio-vascular disorders, and treatment of critically ill diabetic and non-diabetic patients and polyneuropathy.

21. An insulin precursor of the general structure D-B-C-A-E according to any one of aspects 1-16 for use as a medicament for delaying or preventing disease progression in type 2 diabetes.

22. A method for the treatment or prevention of hyperglycemia including stress in- duced hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, and burns, operation wounds, and other diseases or injuries where an anabolic effect is needed in the treatment, myocardial infarction, stroke, coronary heart disease, and other cardiovascular disor-ders, stroketreatment of critically ill diabetic and non-diabetic patients and polyneuropathy, the method comprising administering to a patient in need of such treatment an effective amount for such treatment of An insulin precursor of the general structure D-B-C-A- E according to any one of aspects 1 -16.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each refer- ence were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

EXAMPLES

Generel procedures

Plasmids and DNA

All expressions plasmids were of the C-POT type, similar to those described in EP 171 142 A1 , which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cere- visiae. The plasmids furthermore contained the S. cerevisiae triose phosphate isomerase terminator. These sequences were similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as were all sequences except the sequence of the EcoRI-Xbal fragment encoding the fusion of the leader and insulin precursor.

In order to express different fusion proteins, the EcoRI-Xbal fragment of pKFN1003 or a similar C-POT type expression plasmid was simply replaced by an EcoRI-Xbal fragment encoding the leader insulin precursor of interest. Such EcoRI-Xbal fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques.

Yeast strain and transformation S. cerevisiae strain MT663 {MATa/α pep4-3/pep4-3 HIS4/his4 tpi1::LEU2/tpi1::LEU2

Cir+) was used as host cell for transformation. Transformation of strain MT663 was conducted as described in for examples in patents WO 97/22706 or WO 98/01535 or WO 2004/085472 or WO 09/021955, respectively. S. cerevisiae strain MT663 transformed with expression plasmids were grown in YEPD (1 % Yeast Extract, 2 % Peptone, 2 % glucose) for 72 hours and 30 ^" C). A number of expressed and secreted insulin analogue precursors were produced as described above and isolated from the culture medium and purified by conventional means for further testing

Example 1 Construction of an expression vector encoding the EEAEAEAPK - B25H,desB30 - AAK - A14E human insulin precursor

A yeast expression plasmid was constructed by ligation of three DNA fragments of different origin. The vector part originated from pAK1214, directly derived from pAK729.6 described in WO 00/04172. Fragment 1 : pAK1214 was digested with restriction endonucleases Xbal-Eagl and the 9671 bp fragment was isolated using the MinElute Gel extraction kit (Qiagen catno: 28606). Fragment 2: pAK1214 was digested with restriction endonucleases Eagl-EcoRI and the1082 bp fragment was isolated using the MinElute Gel extraction kit (Qiagen cat 28606). Fragment 3: A synthetic gene encoding the EEAEAEAPK-B25H, desB30 - AAK - A14E human insulin precursor was obtained by PCR using synthetic oligonucleotides and standard techniques. The PCR fragment was digested with restriction endonucleases EcoRI-Xbal and

the 522 bp fragment was isolated using the MinElute Gel extraction kit (Qiagen cat 28606). This fragment encoded the MFalphal -signal and MFalpha1*-leader peptide sequence including the dibasic Kex2p recognition motif (Lys-Arg) followed by the extension EEAEAEAPK connected N-terminally to B1 of the B-chain (B1-B29) followed by the connecting peptide AAK between B29 and A1 of the A-chain (A1 -A21). The MFalphai-signal and MFalphal*- leaders peptide sequences were removed within the yeast secretory pathway resulting in secretion into the culture medium of the EEAEAEAPK - B25H, desB30 - AAK - A14E human insulin precursor, which was subsequently purified by conventional means

Example 2 Pepsin resistance of oral insulin precursors

The half-life, Vλ, for pepsin degradation at 37°C was measured to 0.67 min for human insulin, 16.7 min for the A14E, B25H, desB30 human insulin analogue, and 239 min for the B25H-desB30-AAK-A14E human insulin precursor. The methods used to determine the pepsin resistance are described in WO 08/034881 Example 1.

Example 3 Physical stability of oral insulin precursors

The physical stability of the two precursor analogues EEAEAEAPK-B25H-desB30-AAK- A14E human insulin precursor and B25H-desB30-AAK-A14E human insulin precursor was measured as described previously in WO 04/056347 (Example 1010). The physical stabilities of the precursor-analogues EEAEAEAPK-B25H-desB30-AAK-A14E human insulin precursor and B25H-desB30-AAK-A14E human insulin precursor were compared to their two chain molecule, A14E, B25H, desB30 human insulin, and to B28D human insulin in standard formulations (0.6 mM insulin-/ precursor analogue, 10 mM phosphate alone or in combination with 0.2 mM zinc acetate or 0.2 mM zinc acetate and 30 mM phenol). The precursor ana- logues EEAEAEAPK-B25H-desB30-AAK-A14E human insulin precursor and B25H-desB30- AAK-A14E human insulin precursor did not show any signs of fibrillation during the 45 h duration of the assay in any of the formulations, with the exception of EEAEAEAPK-B25H- desB30-AAK-A14E human insulin precursor formulated in buffer which showed signs of beginning fibrillation after 40 h incubation. B28D human insulin formulated in buffer initiated fib- rillation after 0.3 h, and A14E, B25H, desB30 human insulin formulated in buffer initiated fibrillation after 1.3 h (all values determined by visual inspection of graphs).

Example 4 Chemical stability of oral insulin precursors

The chemical stability of spray dried powder at pH 7.4 of the two precursor analogues EEAEAEAPK-B25H-desB30-AAK-A14E human insulin precursor and B25H-desB30-AAK-

A14E human insulin precursor was measured essentially as described previsously in WO 2004/056347, Example 1012. The percentage deamidation per year was found to be lower for B25H-desB30-AAK-A14E human insulin precursor compared to A14E, B25H, desB30 human insulin and B28D human insulin. The percentage of hydrophobic products was slightly higher in the precursor molecule and the percentage of high molecular weight products formed were in the same range as in the two chain insulins (Table 1).

Table 1

Insulin % deamid % Other related hyHMWP

(hphil+hphobi) drophobic impurities

(hphob2) per year per year per year

B25H-desB30-AAK-A14E 0 (5°C) 1.3 (25°C) 1.3 (5°C) 4.29 25°C) 0 (5 ⁰ C) 1.95 (25 ⁰ C human insulin precursor

A14E, B25H, desB30 hu2. 6 (5°C) 3.9 (25°C) 0 (5°C) 2.08 (25°C) 0 (5 ⁰ C) 1.56 (25 ⁰ C man insulin

B28D human insulin 3. 12 (5°C) 3.12 (25°C) 0 (5°C) 2.47 (25°C) 0 (5 ⁰ C) 2.47 (25 ⁰ C

Example 5 Cleavage of oral insulin precursors

In vitro studies were conducted to verify that enzymes in the intestinal lumen are able to cleave oral insulin precursors to active forms of insulin. In figure 3 cleavage of the precursor EEAEAEAPK-B25H, desB30-AAK-A14E human insulin precursor in the intestinal lumen is shown. Different insulin precursors have been investigated in rat duodenal extracts (see below). The insulin precursors were activated upon cleavage of the C-peptide resulting in two chain insulin molecules. Complete removal of the C-peptide or the leader peptide was not necessary for insulin activity. Insulin receptor binding of A14E B25H desB30 human insulin and A14E B25H desB30 human insulin with a B-chain C-terminal extension of AAK was similar approximately 25% of human insulin. The rate of cleavage of the C-peptide depended

on the sequence of the precursor and on pH. Slow rates of cleavage of the C-peptide were observed for B27E substitutions (4 times slower in comparison to B27T substitutions), and for analogues without C-peptide (>10 times slower in comparison to AAK). Slow processing rates were also observed for pH-values below 5.5 (~65 times more intact precursor was found after 1 h incubation at 37°C at pH=5 relative to pH=7).

Tested insulin precursors:

EEAEAEAPK-B25H-desB30-AAK-A14E human insulin precursor EEAEAEAPK-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor B25H-desB30-AAK-A14E human insulin precursor

EEAEPK-B25H-desB30-DGK-A14E human insulin precursor EEAEAEAPK-B1 E-B25H-desB30-AAK-A14E human insulin precursor EEAEAEAPK-B25H-B27E-desB30-AAK-A8H-A14E human insulin precursor EEAEAEAPK-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor EEAEAEAPK-B1 E-B16E-B25H-desB30-AAK-A8H-A14E human insulin precursor

Example 6 Blood glucose lowering in rats

The same extent of blood glucose reduction was observed after injection of the B25H, desB30-AAK-A14E human insulin precursor analogue and the A14E, B25H, desB30 human insulin analogue, A14E, B25H, desB30 human insulin, at the same concentration into duodenum (6-7 cm from the ventricle) of anaesthetized Sprague-Dawley rats. The duration of action of the precursor-analogue was shorter (Fig. 1).

Example 7 Blood glucose lowering in minipigs The same extent of blood glucose reduction was observed as after dosing of the B25H, desB30-AAK-A14E human insulin precursor analogue and the A14E, B25H, desB30 human insulin analogue, A14E, B25H, desB30 human insulin, at the same concentration to duodenum of male minipigs. The duration of action of the precursor-analogue was shorter (Fig. 2).

Example 8 Stability of insulin precursors in extracts from rat duodenum

Degradation of insulin precursors was measured using duodenum lumen enzymes (prepared by filtration of duodenum lumen content) from SPD rats. The assay was performed by a robot in a 96 well plate (2ml) with 16 wells available for insulin precursors and standards. Insulin

precursors -15 μM were incubated with duodenum enzymes in 100 mM Hepes, pH=7.4 at 37°C, samples were taken after 1 , 15, 30, 60, 120 and 240 min and the reaction was quenched by addition of TFA. Intact insulin precursors at each point were determined by RP- HPLC. Degradation half-life was determined by exponential fitting of the data and normalized to half-life determined for the reference insulin precursors in each assay. The result is given as the degradation half-life for the insulin analogue precursor in rat duodenum divided by the degradation half-life of the reference insulin (i.e. the corresponding B25H, desB30-AAK- A14E human insulin analogue precursor) from the same experiment (relative degradation rate).

Table 2