WO2014161835A1

WO2014161835A1 - Modified blood glucose regulating proteins with altered pharmacological activity profile and preparation thereof

Info

Publication number: WO2014161835A1
Application number: PCT/EP2014/056496
Authority: WO
Inventors: Thomas Wendrich; Ulrich Werner; Norbert Tennagels; Marcus Hermann Korn; Gerhard Seipke; Thomas Stillger; Paul Habermann; Ronald Schmidt
Original assignee: Sanofi
Priority date: 2013-04-03
Filing date: 2014-04-01
Publication date: 2014-10-09
Also published as: AR095986A1

Abstract

The present invention relates to modified insulin analogs with improved activity profiles, methods of preparing these molecules, pharmaceutical formulations thereof, as well as their use in therapy. Further the invention relates to methods for inhibiting protease degradation in serum and/or increasing serum stability of said modified insulin analogs, preferably by attaching one or more D-amino acids, in particular D-Arg, to the C-terminus of said insulin analogs.

Description

Modified blood glucose regulating proteins with altered pharmacological activity profile and preparation thereof

Description

Insulin-deficiency disorders such as diabetes mellitus are severe disorders many patients are suffering from worldwide. There are two main types of diabetes mellitus, classified as type I and type II.

Type I is characterized by a failure of endocrine insulin secretion. Type II is characterized by an insulin resistance, i.e., a failure of the cells to use insulin properly. Both types rely on a therapy with blood glucose regulating proteins. The therapy for type I diabetes is the substitution of insulin, e.g. by insulin or insulin analogs. For type II diabetes several therapeutic possibilities are available among them is the treatment with GLP-1 agonists, e.g. lixisenatide an exendin analog. Although type II is not basically caused by the body's failure to produce insulin, in some cases, particularly in more advanced cases, a therapy with insulin is also advisable for type II diabetes.

An effective insulin therapy uses so-called basal insulins which are formulations of insulin with a depot effect. Such formulations allow for a basal insulin concentration in the body for a prolonged time which is associated with physiological advantages for the patient.

The recombinant insulin analog insulin glargine, which is human insulin with Gly at amino acid position A21 and Arg at the positions B31 and B32 (SEQ ID NOs: 1 1 and 12) is characterized in that it has to be administered only once in 24 hours in order to reach a basal effect. The„once-per day" - administration improves the quality of life and physiology of the patient with lower or no long-term effects of insulin deficiency disorders and an increased life span of the patient.

However, a problem that still remains is the limited serum stability of blood glucose regulating proteins, e.g. of insulin analogs. It would be desirable to improve the pharmaceutical efficacy of such compounds by increasing their serum stability.

The present invention solves this problem by providing modified insulin analogs and other blood glucose regulating proteins, such as GLP-1 (receptor) agonists. The inventive compounds exhibit an increased serum stability by decreasing their metabolism in blood serum by serum proteases and an improved activity profile compared to the unmodified protein. Further the inventive molecules show a reduced hypoglycemia risk since peaks in the blood glucose lowering kinetics are avoided. Preferred examples of blood glucose regulating proteins to be modified are insulin, insulin analogs such as insulin glargine (SEQ ID NOs: 1 1 and 12) and A0(Arg)-insulin glargine (SEQ ID NOs: 13 and 14), a GLP-1 agonist, particularly exendin, e.g., exendin-4 (SEQ ID NO: 4), liraglutide (SEQ ID NO: 10) or lixisenatide (SEQ ID NO: 3); a GLP-1 receptor agonist, particularly GLP-1 (SEQ ID NO: 7), GLP-1 (7-36) (SEQ ID NO: 8), GLP-2 (SEQ ID NO: 9), oxyntomodulin (SEQ ID NO: 5), or glucagon (SEQ ID NO: 6).

In one aspect, the invention provides modified insulin analogs or salts of the formula I

wherein

(a) AO is absent or Arg,

(b) (A1 -A5) are amino acid residues in the positions A1 to A5 in the A chain of human (SEQ ID NO:1 ),

(c) (A8-A10) are amino acid residues in the positions A8 to A10 in the A chain of human (SEQ ID NO:1 ),

(d) (A12-A19) are amino acid residues in the positions A12 to A19 in the A chain of human (SEQ ID NO:1 ),

(e) A21 is Asn or Gly,

(f) B1 is Phe or His, (g) B3 is Asn or His,

(h) B2 and (B4-B6) are amino acid residues in the positions B2 and B4 to B6 in the B chain of human (SEQ ID NO:2),

(i) (B8-B18) are amino acid residues in the positions B8 to B18 in the B chain of human (SEQ ID NO:2),

(j) (B20-B27) are amino acid residues in the positions B20 to B27 in the B chain of human (SEQ ID NO:2),

(k) B28 is Pro or Lys,

(I) B29 is Lys or absent,

(m) B30 is Thr or absent,

(n) B31 is absent, Arg or a basic D-amino acid, particularly D-Arg, D-His, D-

Lys, D-Hyl, D-Ornithine, or D-Citrulline.

(o) B32 is a basic D-amino acid, particularly D-Arg,

with the proviso that if B31 is Arg, then A21 is Gly, or if B31 is absent, then AO is Arg; and with the proviso that if B28 is Lys and B29, B30 and B31 are absent, then AO is Arg.

The term "analog", e.g., an insulin analog as used herein refers to an analog of naturally occurring insulin such as human (SEQ ID NOs: 1 and 2), which differs from the naturally occurring insulin by substitution of at least one naturally occurring amino acid by another amino acid in the A and/or B chain and/or the addition/deletion of at least one amino acid in the A and/or B chain of the respective naturally occurring insulin. Examples of insulin analogs are insulin glargine, (A21 (Gly), B31 (Arg) and B32(Arg)-insulin; SEQ ID NOs: 1 1 and 12) or A0(Arg)-insulin glargine (AO(Arg), A21 (Gly), B31 (Arg) and B32(Arg)-insulin; SEQ ID NOs: 13 and 14).

The letters A and B as used herein refer to the A chain of insulin (SEQ ID NO:1 ) or the B chain of insulin (SEQ ID NO:2). The numbers following the letter, e.g. A, refer to the amino acid position within the A chain optionally together the respective amino acid in brackets which is located at said amino acid position. For example AO(Arg) defines that the amino acid Arg is located at position 0 of the A chain of insulin. The terminology, in which the amino acid position is indicated before the letter equals the above name convention, e.g. 0^A-Arg equals AO(Arg). The term "di" as inter alia used herein indicated that two amino acids are affected. For example, (31 -32)^B-di-D-Arg means that both amino acid positions B31 and B32 carry a D-Arg. The term "des" as inter alia used herein indicates that an amino acid position is omitted. For example, (30-32)B-des-Thr-insulin glargine means that the amino acids positions B30, B31 , and B32 are omitted in the given molecule, e.g. insulin glargine.

Amino acids that are described herein by their one letter code, three letter code or full name are L-amino acids.

The position AO of the amino acid sequence of the A chain of the (modified) insulin analogs as defined in the description corresponds to the amino acid position 1 in the corresponding amino acid sequences of the sequence listing, e.g., in SEQ ID NO:14, SEQ ID NO: 20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26. In addition, in some insulin analog amino acid sequences as defined in the description, one or more amino acids are omitted in the B chain compared to the native insulin B chain sequence (SEQ ID NO:1 ). In the sequence listing those B chain amino acid sequences are presented as consecutive sequences, i.e., without gaps. As a consequence, the amino acid position of the C-terminal amino acids B29-B32 may adopt other amino acid positions in the amino acid sequences listed in the sequence listing. For example, in the B chain of insulin analog of formula I with B28 is Pro, B29 is Lys, B30 is Thr, and B31 is absent, and B32 is D-Arg, the position of B32 (e.g. D-Arg) goes to amino acid position B31 in the corresponding sequence of the sequence listing (SEQ ID NO:23).

The term "modified" as used herein, refers to a protein or an analog which are diastereomers of the corresponding unmodified proteins or analogs.

According to the present invention, a modified insulin analog is provided which comprises one or two basic D-amino acids at the C-terminus of the insulin B chain. The term "basic amino acid" refers to amino acids having a basic side chain, e.g. a side chain comprising an amino group or a guanidine group. Examples of basic D-analogs of amino acids are proteinogenic amino acids such as D-Arg, D-Lys, D-His, or D-Hyl (hydroxy-lysine) or D-analogs of non-proteinogenic amino acids such as D-Ornithine or D-Citrulline.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B30 is Thr.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein A21 is Gly.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein AO is absent.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B31 is D-Arg, B32 is D-Arg or B31 and B32 are D-Arg.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B29 is Lys.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B28 is Pro.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein AO is Arg. Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B28 is Lys.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B29 is absent.

Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein A21 is Asn. Preferred insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B31 is L-Arg.

Preferred modified insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B31 is absent.

Preferred modified insulin analogs of the present invention are modified insulin analogs or a salt of formula I described herein wherein B30 is absent. Particularly preferred modified insulin analogs are modified insulin analogs or a salt of formula I described herein wherein AO is absent, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is Arg, and B32 is D-Arg (SEQ ID NOs: 15 and 16), AO is absent, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is D-Arg, and B32 is D-Arg (SEQ ID NOs: 17 and 18), AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is Arg, and B32 is D-Arg (SEQ ID NOs: 19 and 20), AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is D-Arg, and B32 is D-Arg (SEQ ID NOs: 21 and 22), AO is Arg, A21 is Asn, B28 is Pro, B29 is Lys, B30 is Thr, and B31 is absent, and B32 is D-Arg (SEQ ID NOs: 23 and 24), AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is absent, and B32 is D-Arg (SEQ ID NOs: 25 and 26), AO is absent, A21 is Asn, B28 is Lys, B29 is absent, B30 is Thr, B31 is D-Arg, and B32 is D-Arg (SEQ ID NOs: 29 and 30), or AO is absent, A21 is Gly, B28 is Lys, B29 is absent, B30 is Thr, B31 is D- Arg, and B32 is D-Arg (SEQ ID NOs: 33 and 34).

A particularly preferred modified blood glucose regulating protein is an insulin analog such as insulin glargine or A0(Arg)-insulin glargine comprising at its C-terminus two or more L-amino acids which are replaced by their corresponding D-amino acid(s), particularly D-Arg and/or D-Lys.

A further aspect of the invention is a modified blood regulating protein comprising at its C-terminus at least one, e.g. one, two, three, four or five basic D-amino acids as described above. Preferably, one or more basic L-amino acids are replaced by their basic D-amino acid(s), particularly D-Arg and/or D-Lys.

The term "blood glucose regulating protein" as used herein may be any protein that is directly or indirectly involved in regulating the level of blood glucose in a human or animal. For example a blood regulating protein to be modified is a GLP-1 agonist, such as an exendin, e.g. exendin-4 (SEQ ID NO: 4), liraglutide (SEQ ID NO: 10) or lixisenatide (AVE0010, SEQ ID NO: 3) or GLP-1 receptor agonist like GLP-1 (SEQ ID NO: 7), GLP-1 (7-36) (SEQ ID NO: 8) or -2 (SEQ ID NO: 9), oxyntomodulin (SEQ ID NO: 5), glucagon SEQ ID NO: 6) or peptides which bind and activate both the glucagon and the GLP-1 receptor (Hjort et al., Journal of Biological Chemistry, 269, 30121 - 30124, 1994; Day JW et al., Nature Chem. Biol. 5:749-757, 2009) and suppress body weight gain and reduce food intake which are described in patent applications WO 2008/071972, WO 2008/101017, WO 2009/155258, WO 2010/096052, WO 2010/096142, WO 201 1/075393, WO 2008/152403, WO 2010/070251 , WO 2010/070252, WO 2010/070253, WO 2010/070255, WO 201 1/160630, WO 201 1/006497, WO 201 1/152181 , WO 201 1/152182, WO 201 1/1 17415, WO 201 1/1 17416, the contents of which are herein incorporated by reference, or GIP and peptides which bind and activate both the GIP and the GLP-1 receptor and optionally the glucagon receptor, and improve glycemic control, suppress body weight gain and reduce food intake as described in patent applications WO 201 1/1 19657, WO 2012/138941 , WO 2010/01 1439, WO 2010/148089, WO 201 1/094337, and WO 2012/0881 16, the contents of which are herein incorporated by reference. Further examples of blood glucose regulating proteins are insulins and insulin analogues or DPP-4 inhibitors.

Preferred examples of blood glucose regulating proteins to be modified are GLP-1 agonists, particularly exendin, e.g., exendin-4 (SEQ ID NO: 4), liraglutide (SEQ ID NO: 10) or lixisenatide (SEQ ID NO: 3); a GLP-1 receptor agonist, particularly GLP-1 (SEQ ID NO: 7), GLP-1 (7-36) (SEQ ID NO: 8), GLP-2 (SEQ ID NO: 9), oxyntomodulin (SEQ ID NO: 5), or glucagon (SEQ ID NO: 6).

Another particularly preferred blood glucose regulating protein to be modified is lixisenatide. Lixisenatide (SEQ ID NO: 3) is an exendin-4 analog comprising amino acids 1 to 39 of exendin-4 (SEQ ID NO:4) with a deletion of Pro at amino acid position 36 (des Pro36). Lixisenatide comprises six C-terminal lysine residues.

Another particularly preferred modified GLP-1 agonist is a GLP-1 agonist or salt of formula II:

(C1 -C38)-C39-C40-C41 -C42-C43-C44 wherein

(a) C1 -C38 are amino acid residues in the positions C1 to C38 of des Pro36 Exendin-4(1 -39) (SEQ ID NO:3),

(b) C39-C43 is Lys or a basic D-amino acid, preferably D-Lys,

(c) C44 is a basic D-amino acid, preferably D-Lys.

A particularly preferred modified GLP-1 agonist or salt of formula II is one wherein C39- C42 is Lys, C43 and C44 are D-Lys (SEQ ID NO: 35).

A particularly preferred modified GLP-1 agonist or salt of formula II is one wherein C39- C43 is Lys and C44 is D-Lys (SEQ ID NO: 36).

A particularly preferred modified GLP-1 agonist or salt of formula II is one wherein C39 is Lys and C40-C44 is D-Lys (SEQ ID NO: 37). A particularly preferred modified GLP-1 agonist or salt of formula II is an agonist wherein C39 and C40 is Lys and C41 -C44 is D-Lys (SEQ ID NO:38).

A preferred salt of the compounds of the present invention is a pharmaceutically acceptable salt. A preferred pharmaceutically acceptable salt is acetate.

Another embodiment of the invention relates to a conjugate of the modified insulin analog or the modified blood regulating protein both as described herein and an immunoglobulin Fc region linked via a non-peptidyl linker selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combinations thereof.

The term "non-peptidyl linker" as used herein preferably refers to a biocompatible polymer including two or more repeating units linked to each other via a covalent bond excluding a peptide bond.

Preferably, the conjugate comprises a modified insulin analog as described herein and the non-peptidyl linker is attached to the amino terminus of the B chain of the insulin analog.

Preferably, the immunoglobulin Fc region is composed of one to four domains selected from the group consisting of CH1 , CH2, CH3, and CH4 domains, particularly derived from IgG, IgA, IgD, IgE, or IgM.

Another embodiment of the invention refers to a pharmaceutical formulation comprising the modified insulin analog as described herein and/or the modified blood glucose regulating protein described herein, preferably modified lixisentatide and a pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutical formulation is a long-acting formulation. Such a formulation is characterised by maintaining an improved in vivo duration, titer and stability, preferably the serum stability, of the inventive modified molecules compared to the formulation comprising the unmodified molecule.

The pharmaceutical formulation may be suitable for parenteral administration, such as intraperitoneally, intravenously, intramuscularly, subcutaneaously, or intradermally, preferably for subcutaneous administration, preferably injection. The pharmaceutical formulation may be prepared as single-dose or multi-dose formulation.

Suitable injection devices, for instance so-called "pens", comprising a cartridge with the active agent as described herein, and an injection needle, are known in the art. The pharmaceutical formulation may be a liquid formulation. Liquid pharmaceutical formulations of insulin analogs or blood glucose proteins, preferably lixisenatide are known in the prior art. For example, such liquid formulations may comprise pharmaceutically acceptable carriers such as a buffering agent, e.g., a phosphate, citrate and/or acetate; a suitable preservative such as phenol, m-cresol, and benyzl alcohol; a tonicity agent such as glycerol, lactose, sorbitol, mannitol, glucose, sucrose, NaCI, calcium or magnesium containing compounds such as CaCI₂; an analgestic; a solubiliser and/or a stabiliser.

The formulation may further comprise an antioxidant such as methionine or ascorbate.

The invention further provides the modified insulin analog or the modified blood glucose regulating protein both as described herein for use in therapy.

In particular, the modified insulin analog or the modified blood glucose regulating protein as described herein may be for use in the prevention, alleviation, or treatment of an insulin-deficiency disorder, insulin resistance, obesity, hypoglycemia, and/or a neurodegenerative disorder.

The neurodegenerative disorder may involve oxidative stress, loss of neurite integrity, apoptosis, neuronal loss and/or inflammatory response. The neurodegenerative disorder may also be associated with cognitive impairment.

A preferred insulin-deficiency disorder is diabetes, particularly type I or type II diabetes.

A preferred neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson's disease, dementia, epilepsy, stroke, Huntington's Chorea, cerebral hypoxia, multiple sclerosis, and peripheral neuropathy. A particularly preferred neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, and stroke, particularly early- stage Alzheimer's disease or early-stage Parkinson's disease.

The invention further provides a method for inhibiting protease degradation in serum, preferably by a carboxyprotease or trypsin, and/or increasing serum stability of a protein by attaching one or more D-amino acids into said protein.

The percent inhibition of protease degradation is determined by inhibition = 1 - [(cleavage of modified protein in % / cleavage of unmodified protein in %)] x 100 % measured under identical conditions. Preferably, the inhibition is 50 %, 55 %, 60 %, 65 %, 70 %, 72 %, 75 %, 80 %, 90 %, 95 %, 96 %, 97 %, 98 %, or 99 %. The increase in serum stability is determined by comparing the duration of presence of biological active modified protein to biological unmodified protein in the blood measured under identical conditions.

The D-amino acid(s) may be attached to the N-terminus or the C-terminus of said protein. Preferably the D-amino acid(s) is/are attached to the C-terminus of said protein.

The protein modified by above mentioned method may in principle be any protein that is sensitive to protease degradation in serum. In one embodiment the protein is a blood glucose regulating protein as defined herein, preferably insulin (SEQ ID NOs: 1 and 2), or insulin analog such as insulin glargine (SEQ ID NOs: 1 1 and 12) or A0(Arg)-insulin glargine (SEQ ID NOs: 13 and 14), GLP-1 agonist, particularly exendin, e.g., exendin-4 (SEQ ID NO: 4), liraglutide (SEQ ID NO: 10) or lixisenatide (SEQ ID NO: 3) or a GLP-1 receptor agonist, particularly GLP-1 (SEQ ID NO: 7), GLP-1 (7-36) (SEQ ID NO: 8), GLP-2 (SEQ ID NO: 9), oxyntomodulin (SEQ ID NO: 5), or glucagon (SEQ ID NO: 6). The D-amino acid(s) may be introduced by the methods as described herein, particularly by the methods used in the Examples. In one embodiment the D-amino acid(s) are introduced by:

(a) providing a protein, e.g. a recombinantly produced protein, which optionally has been cleaved with an endopeptidase, preferably a lysyl-endopeptidase or trypsin, and

(b) attaching a peptide to the protein wherein the peptide comprises one or more C- terminal D-amino acids and optionally one or more L-amino acids, wherein the D- amino acids replace the corresponding L-amino acids in said protein or wherein the D-amino acids are added to the C-terminus. The peptide has preferably a length of 2-10, more preferably 2, 3, 4, or 5 amino acids.

Methods of recombinantly producing proteins are known in the art. Likewise methods of attaching a peptide are known the art. Preferably, the peptide to be attached is pre- synthesised, e.g. by chemical synthesis. Preferably, the peptide is added via trypsin catalyzed transpeptidation.

In one embodiment the method described herein comprises providing e.g. recombinantly producing said protein which is a fragment of insulin or the insulin analog such as insulin glargine or A0(Arg)-insulin glargine comprising the A chain of said insulin or insulin analog and the B chain terminating at amino acid position B29 (Lys) prior to step (b). The term "fragment" as used herein refers to a protein, e.g. an insulin analog or glucose regulating protein as described herein, which lacks 1 , 2, 3, 4 or 5 amino acids at its C-terminus.

For example, the protein is insulin glargine which lacks amino acid Thr at position B30.

In one embodiment, the protein may be an insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine and the method comprises cleaving the protein with an endopeptidase, preferably a lysyl-endopeptidase or trypsin prior to step (b). In another embodiment of the method describe herein the added D-amino acids may replace L-amino acids of the protein that have been cleaved by the endopeptidase, preferably the lysyl endopeptidase or trypsin.

The method may comprise introducing a D-amino acid, particularly a basic D-amino acid that is an analog of a proteinogenic amino acid such as D-Arg, D-His, D-Hyl, or D- Lys. It may further comprise introducing a D-amino acid, particularly a basic D-amino acid which is an analog of a non-proteinogenic amino acid such as D-ornithine or D- citrulline. The protein may be an insulin analog such as insulin glargine or A0(Arg)-insulin glargine and the introduced D-amino acid is D-Arg at position B31 or B32, preferably at positions B31 and B32. The protein may be produced by cleavage with a protease such as lysylendopeptidase or trypsin.

For example, the peptide is L-Thr-L-Arg- D-Arg or L-Thr- D-Arg -D-Arg or Lys-Arg -D-Arg or Lys- D-Arg -D-Arg.

One embodiment of the invention refers to a method for inhibiting protease degradation of a protein by attaching one or more D-amino acids to said protein, wherein the protein is an insulin analog such as insulin glargine or A0(Arg)-insulin glargine and the introduced amino acid is D-Arg at amino acid positions B31 and B32 and the protease is trypsin. Another aspect of the invention refers to a method of preparing a modified insulin analog or a modified blood glucose regulating protein comprising:

(b) attaching a peptide to the protein wherein the peptide comprises one or more C- terminal D-amino acids and optionally one or more L-amino acids, wherein the D- amino acids replace the corresponding L-amino acids in said protein or wherein the D-amino acids are added to the C-terminus. In the methods described herein, the D-amino acid that is added may be a basic D- amino acid, such as D-Arg, D-His, D-Hly, D-Lys, D-ornithine, and/or D-citrulline, preferably D-Arg and/or D-Lys.

In a preferred embodiment, the method refers to the preparation of a modified insulin analog or a modified blood glucose regulating protein both as described herein, preferably those of formula I or formula II.

The method is suitable for providing an insulin or insulin analog which is a fragment of insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine in step (a), e.g. by recombinant production, comprising the A chain of said insulin or insulin analog and a B chain terminating at amino acid position B29 (Lys) prior to step (b), which may be cleaved with an endopeptidase, preferably a lysyl-endopeptidase or trypsin prior to step (b).

In one embodiment of the method as described herein the added D-amino acids may replace L-amino acids of the insulin, insulin analog or protein that have been cleaved by the endopeptidase, preferably the lysyl-endopeptidase or trypsin. For example, the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the peptide is L-Thr-L-Arg-D-Arg or L-Thr-D-Arg-D-Arg.

A further embodiment of the invention is a pharmaceutical formulation, preferably a long-acting formulation, comprising as an active agent a modified insulin analog as described above or a conjugate as described above and a pharmaceutically acceptable carrier.

A further embodiment of the invention is a modified insulin analog as described above or a conjugate as described above for use in therapy.

A further embodiment of the invention is a modified insulin analog as described above or a conjugate as described above for use in the prevention, alleviation, or treatment of an insulin-deficiency disorder, insulin resistance, obesity, hypoglycemia, and/or a neurodegenerative disorder, particularly a neurodegenerative disorder which involves or is associated with oxidative stress, loss of neurite integrity, apoptosis, neuronal loss, cognitive impairment and/or inflammatory response.

A further embodiment of the invention is a modified insulin analog or a conjugate for use as described above wherein the insulin-deficiency disorder is diabetes, particularly type I or type II diabetes.

A further embodiment of the invention is a modified insulin analog or a conjugate for use as described above wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson's disease, dementia, epilepsy, stroke, Huntington's Chorea, cerebral hypoxia, multiple sclerosis, and peripheral neuropathy.

A further embodiment of the invention is a method for inhibiting protease degradation in serum and/or increasing serum stability of an insulin analog by attaching one or more D-amino acids to said insulin analog resulting in a modified insulin analog as described above, preferably wherein the D-amino acid(s) is/are attached to the C-terminus of said insulin analog and furthermore preferred wherein said protease is a carboxyprotease.

A further embodiment of the invention is a method as described above wherein the insulin analog is preferably insulin (SEQ ID NOs: 1 and 2), or insulin glargine (SEQ ID NOs: 1 1 and 12) or A0(Arg)-insulin glargine (SEQ ID NOs: 13 and 14).

A further embodiment of the invention is a method as described above wherein the D- amino acid(s) is/are introduced by:

(a) providing an insulin analog, e.g. a recombinantly produced insulin analog, which optionally has been cleaved with an endopeptidase, preferably a lysyl- endopeptidase or trypsin, and

(b) attaching a peptide to the insulin analog wherein the peptide comprises one or more C-terminal D-amino acids and optionally one or more L-amino acids, wherein the D-amino acids replace the corresponding L-amino acids in said insulin analog or wherein the D-amino acids are added to the C-terminus.

A further embodiment of the invention is a method as described above wherein the insulin analog is a recombinantly produced fragment of insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine comprising the A chain of said insulin or insulin analog and the B chain terminating at amino acid position B29 (Lys) prior to step (b).

A further embodiment of the invention is a method as described above wherein the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the method comprises cleaving the protein with an endopeptidase, preferably a lysyl-endopeptidase or trypsin. A further embodiment of the invention is a method as described above wherein the added D-amino acids replace L-amino acids of the insulin analog that have been cleaved by the endopeptidase, preferably wherein the D-amino acid is a basic D-amino acid, more preferably D-Arg or D-Lys.

A further embodiment of the invention is a method as described above wherein the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the peptide is L-Thr-L- Arg-D-Arg or L-Thr- D-Arg -D-Arg. A further embodiment of the invention is a method as described above wherein the peptide is added via trypsin catalysed transpeptidation.

A further embodiment of the invention is a method of preparing a modified insulin analog comprising:

A further embodiment of the invention is a method as described above wherein the D- amino acid is a basic D-amino acid, preferably D-Arg and/or D-Lys.

A further embodiment of the invention is a method as described above for producing a modified insulin analog as described above or a conjugate as described above. A further embodiment of the invention is a method as described above comprising the recombinant production of said insulin or insulin analog which is a fragment of insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine in step (a) comprising the A chain of said insulin or insulin analog and a B chain terminating at amino acid position B29 (Lys) prior to step (b). A further embodiment of the invention is a method as described above wherein the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the peptide is L-Thr-L- Arg-D-Arg or L-Thr-D-Arg-D-Arg

Brief Description of the Figures

Figure 1

Blood glucose lowering effect of 12 nmol/kg s.c. 32^B-D-Arg-insulin glargine (SEQ ID NOs:15 and 16), (31 -32)^B-di-D-Arg-insulin glargine (SEQ ID NOs: 17 and 18), 0^A-Arg- 32^B-D-Arg-insulin glargine (SEQ ID NOs: 19 and 20), and 0^A-Arg-(31 -32)^B-di-D-Arg- insulin glargine (SEQ ID NOs: 21 and 22) in a rat

Figure 2

Blood glucose lowering effect of 1 lU/kg s.c. (31 -32)^B-di-D-Arg-insulin glargine (SEQ ID NOs: 17 and 18) and insulin glargine in a rat Figure 3

Pharmacokinetics and metabolism profile of (31 -32)^B-di-D-Arg-insulin glargine (SEQ ID NOs: 17 and 18) and insulin glargine in the rat one hour after subcutanous injection of 1 lU/kg Figure 4

Structure of insulin glargine

Figure 5

(30-32)^B-des-Thr-insulin glargine

Figure 6

32^B-D-Arg-insulin glargine (SEQ ID NOs: 15 and 16), D-Arg outlined in blue

Figure 7 (31 -32) -di-D-Arg-insulin glargine, (SEQ ID NOs: 17 and 18)

Figure 8

0^A(Arg)-30^B-des-Thr-insulin glargine

Figure 9

0^A-Arg-32^B-D-Arg-insulin glargine (SEQ ID NOs: 19 and 20), D-Arg outlined in blue Figure 10

0^A(Arg)-(31 -32)^B-di-D-Arg-insulin glargine (SEQ ID NOs: 21 and 22) Figure 1 1

Flow chart for the preparation of insulin diastereomers

Examples

The following examples are set forth to illustrate the present invention. Preparation and characterisation of C-terminal D-Arg variants of insulin glargine (SEQ ID NOs: 11 and 12)

List of Tables:

Table 1 : Name designation of D-Arg variants of insulin glargine

Table 2: MALDI-MS data for proof of the molecular structure of educts and products

Table 3: Results of the chiral analysis of amino acids

Table 4: Cleavage inhibition of CpB by introduction of D-Arg 1. Concept for the preparation of modified insulin analogs 1.1 C-terminally D-Arg variants of insulin glargine

Insulin glargine encompasses a single Lysin in 29^B, which provides the possibility of preparing D-Arg variants with a semi-synthetic approach. Based on the recombinant insulin glargine, the (30-32)^B-des-Thr-insulin glargine resulting from the Lys-C cleavage is used as educt for the trypsin-catalysed transpeptidisation/coupling with two different tripeptides: The coupling with L-Thr-L-Arg-D-Arg results in 32^B-D-Arg-insulin glargine (SEQ ID NOs: 15 and 16), whereas the coupling with L-Thr-D-Arg-D-Arg results in (31 -32)^B-di-D- Arg-insulin glargine (SEQ ID NOs: 17 and 18).

1.2 C-terminally D-Arg variants of 0^A(Arg)-insulin glargine

The concept can be transferred to other insulins, provided that they can be prepared as B30-des-Thr-insulin. In the present case, a cleavage by-product has been used, which can also be described as 0^A(Arg)-30^B-des-Thr-insulin glargine. A trypsin-catalysed coupling with L-Thr-L-Arg-D-Arg then results in 0^A(Arg)-32^B-D-Arg-insulin glargine (SEQ ID NOs:19 and 20), a coupling with L-Thr- D-Arg -D-Arg results in 0^A(Arg)-(31 - 32)^B-di-D-Arg-insulin glargine (SEQ ID NOs:21 and 22).

2. Flow chart for the preparation of insulin diastereomers

See Figure 1 1

3. Naming convention of D-Arg variants of insulin glargine

Table 1 Name designation of D-Arg variants of insulin glargine

Name Alias SEQ ID NOs

Gly(A21 )Arg(B31 )D-Arg(B32) insulin 32^B-D-Arg-insulin glargine 15/16

Gly(A21 )D-Arg(B31 )D-Arg(B32) (31-32)^B-di-D-Arg-insulin glargine 17/18

insulin

Arg(A0)Gly(A21 )Arg(B31 )D-Arg(B32) 0^A-Arg-32^B-D-Arg-insulin glargine 19/20

insulin

Arg(A0)Gly(A21 )D-Arg(B31 )D- 0^A-Arg-(31-32)^B-di-D-Arg-insulin 21/22

Arg(B32) insulin glargine

4. Characterisation of modified insulin analogs

4.1 Mass spectrometry analyses

4.1.1 Total mass and masses of A- and B-chains by means of MALDI-MS measurements Both the total mass and the masses of A- and B-chains of the 30^B-des-Thr-insulins and of the products after coupling have been measured by MALDI-MS after reduction of the disulfide bonds. The masses that had been expected were found for each substance, as demonstrated in Table 2.

Table 2 MALDI-MS Data for proof of molecular structure of educts and products

N.B.: (30-32) -des-tripeptide-... equals 30 -des-Thr-...

4.2 Chiral analysis of amino acids

In order to prove the amount of D-Arg in insulin, samples had been sent to CAT GmbH & Co KG, Tubingen where a chiral analysis of the amino acids was conducted. The experimental amount of L-Arg and D-Arg in the respective samples corresponded very well with the theoretical amount to be expected (Table 3). Thus, the structure of every insulin diastereomer has been unambiguously determined.

Table 3 Results of chiral analysis of amino acids

Sam Name Number L- Number % L-Arg % L-Arg

pie Arg D-Arg theor. exp.

no

1 Gly(A21 )Arg(B31 )Arg(B32)- 3 0 100.0% 99.9%

insulin

2 Gly(A21 )Arg(B31 )D- 2 1 66.7% 69.1 %

Arg(B32)-insulin

3 Gly(A21 )D-Arg(B31 )D- 1 2 33.3% 31.9%

Arg(B32)-insulin

4 Arg(A0)Gly(A21 )Arg(B31 ) 4 0 100.0% 99.9%

Arg (B32)-insulin

5 Arg(A0)Gly(A21 )Arg 3 1 75.0% 78.1 %

(B31 )D-Arg(B32)-insulin

6 Arg(A0)Gly(A21 )D-Arg 2 2 50.0% 50.8%

(B31 )D-Arg(B32)-insulin

4. Inhibition of carboxypeptidase activity in vitro

In HPLC-MS tests, it was found out to what extend the carboxypeptidase B (CpB) cleaves insulin glargine in vitro. Table 4 shows that 32^B-D-Arg strongly inhibits the cleavage kinetics. However, (31 -32)^B-di-D-Arg completely inhibits the cleavage in vitro.

Table. 4 CpB cleavage inhibition by D-Arg introduction

Sample Time Rate Det. Possible correlation Theor.

name mol mol

masses masses

Da Da

(av/mo) (av/mo)

Insulin 0 h* 77% 5750.26 (31-32)^B-des-di-Arg-insulin glargine 5750.52

glargine

23% 6062.67 Insulin glargine 6062.90

1 h 100% 5750.29 (31-32)^B-des-di-Arg-insulin glargine 5750.52 after 1 h 2325.25 (1-21 )^A-insulin glargine 2324.98

reduction

3429.80 (1-30)^B-insulin glargine 3429.92

32^B-D-Arg- 0 h* 100% 6062.71 insulin glargine 6062.90

insulin

glargine 1 h 7% 5649.33 (1-29)^B-des-tri-peptid-insulin glargine 5649.42

45% 5750.27 (31-32)^B-des-di-Arg-insulin glargine 5750.52

48% 6062.75 insulin glargine 6062.90

after 1 h 2323.25 (1-21 )^A-insulin glargine 2324.98

reduction

3328.27 (1-29)^B-insulin glargine 3328.82

3429.77 (1-30)^B-insulin glargine 3429.92

3742.04 (1-32)^B-insulin glargine 3742.29

(31-32)^B-di- 0 h* 100% 6062.70 insulin glargine 6062.90

D-Arg- insulin 1 h 100% 6062.66 insulin glargine 6062.90

glargine

22 h 100% 6062.80 insulin glargine 6062.90 after 1 h 2323.26 (1-21 )^A-insulin glargine 2324.98

reduction

3742.26 (1-32)^B-insulin glargine 3742.29

0^A-Arg- 0 h* 21 % 5906.59 0^A-Arg-(31-32)^B-des-di-Arg-insulin glargine 5906.71

insulin

79% 6218.97 0^A-Arg-insulin-glargine 6219.08

glargine

1 h 15% 5805.49 0^A-Arg-(30-32)^B-des-tri-peptid-insulin glargine 5805.61

85% 5906.52 0^A-Arg-(31-32)^B-des-di-Arg-insulin 5906.71

glargine

after 1 h 2479.39 (0-21 )^A-insulin glargine 2481.08

reduction

3328.04 (1-29)^B-insulin glargine 3328.82 3430.04 (1-30)^B-insulin glargine 3429.92

0^A-Arg-32^B- 0 h* 100% 6218.94 0^A-Arg-insulin glargine 6219.08

D-Arg- insulin

glargine 1 h 5% 5805.38 0^A-Arg-(1-29)^B-des-tri-peptid-insulin glargine 5805.61

24% 5906.53 0^A-Arg-(31-32)^B-des-di-Arg-insulin glargine 5906.71

71 % 6218.81 0^A-Arg-insulin glargine 6219.08

after 1 h 2479.41 (0-21 )^A-insulin glargine 2481.08

reduction

3328.22 (1-29)^B-insulin glargine 3328.82

3429.26 (1-30)^B-insulin glargine 3429.92

3742.23 (1-32)^B-insulin glargine 3742.29

0^A-Arg-(31- 0 h* 100% 6218.87 0^A-Arg-insulin glargine 6219.08

32)^B-di-D- Arg-insulin 1 h 100% 6218.94 0^A-Arg-insulin glargine 6219.08

glargine

22 h 100% 6218.88 0^A-Arg-insulin glargine 6219.08 after 1 h 2480.41 (0-21 )^A-insulin glargine 2481.08

reduction

3741.95 (1-32)^B-insulin glargine 3742.29

Mass deviation ± 0.01 %

* the 0 h-value was analysed after addition of the enzyme

The primary and secondary structures were examined by means of mass spectrometry and the amount of the D-amino acids in the modified insulin analogs was examined by means of chiral analysis of the amino acids. A resistance to cleavage of 100 % towards carboxypeptidase B (CpB) in vitro in the di-D-Arg variants (31 -32)^B-di-D-Arg-insulin glargine and 0^A-Arg-(31 -32)^B-di-D-Arg-insulin glargine was observed (no formation of metabolit M1 ((31 -32)^B-des-di-Arg-insulin glargine = 21 -Gly-insulin)), however, not in 32^B-D-Arg variants 32^B-D-Arg-insulin glargine and 0^A-Arg-32^B-D-Arg-insulin glargine which showed at least a significantly lower CpB- cleavage rate compared to insulin glargine. The formation of 30^B-des-Thr was observed in the in vitro tests with these modified insulin analogs (metabolit M2 (21^A-Gly-30^B-des-Thr-insulin) which indicates a trypsin activity. Such degradation was not observed with (31 -32)^B-di-D-Arg-insulin glargine and 0^A-Arg-(31 -32)^B-di-D-Arg-insulin glargine. The data might indicate that 31 ^B-D-Arg inhibits the trypsin induced M2 formation. 6. Pharmacology The pharmacological testing of modified insulin analogs was performed as follows. The blood glucose lowering effect of the test substances has been investigated in male, normoglycemic Wistar rats. The animals had free access to food and water during the night. In the morning, test substances were administered via subcutaneous injection after fasting the rats for two hours. During the experiment, the animals remained without food but had free access to water.

The effect of the test substances on the blood glucose of the animals was determined after sequential blood collection from the tail tip. A time/efficacy - profile of the blood glucose had been established. A blood glucose lowering effect was found for all 4 test substances (cf. Fig. 1 ). It was shown exemplarily with (31 -32)^B-di-D-Arg-insulin glargine that the introduced modification leads to a prolonged duration of action in direct comparison with insulin glargine and to a flattening of the of blood glucose lowering curve. The latter additionally points to a reduced hypoglycemia risk as a consequence of the introduced modification (cf. Fig. 2).

7. LC-MS/MS analysis of C-terminal D-Arg variants of insulin glargine from rat plasma samples

List of abbreviations

<LLOQ Below lower limit of quantification

>ULOQ Above upper limit of quantification

°C Degree centigrade

g Microgram

μΙ_ Microliter

μιτι Micrometer

BA Bioanalytical

Cone. Concentration

Cs Calibration sample

g Gravity (also used as g for speed of centrifuge, where "g"

means gravity)

h Hour(s)

HPLC High performance liquid chromatography

ID Inner diameter

ISTD Internal Standard

L Liter

LC-MS/MS Liquid chromatography coupled to tandem mass

spectrometry LLOQ Lower limit of quantification

M Molar

mg Milligram

min Minute(s)

mL Milliliter

mM Millimolar

MS/MS Tandem mass spectrometry

n Number of individual values

ng Nanogram

nm Nanometer

PBS Phosphate Buffered Saline (One tablet dissolved in 200 mL of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25 °C.)

PH Negative common logarithm of the proton concentration

QC Quality control

RT Retention time

sec Second(s)

ULOQ Upper limit of quantification

[U-15N]- Abbreviation used as the prefix for the labeled internal standard in

combination with the compound name (e.g: [U15N]-insulin glargine)

VAL Validation Samples

v/v Volume/volume

7.1 Test system (Matrix)

The test system used is rat plasma. Potassium EDTA (K₂-EDTA) is used as anticoagulant. 7.2 Materials

7.2.1 Equipment

Reaction tubes, 1 .5 mL (polypropylene)

Multi pipettes

Vortex Mixer (Heidolph) Centrifuge: Rotanta 46 RSC / RP (Hettich)

Centrifuge: Mikro 22 R / RP (Hettich)

Vivacon 500 Filter VN01 H02

Autosampler Vials (polypropylene)

Immunoaffinity columns (filled with 100 μί of 10 mg/mL Zentech^® anti-insulin mouse antibody coupled to CNBr-activated Sepharose 4 Fast Flow)

7.2.2 Reagents and solvents

All chemicals used in this study were reagent grade or higher.

Rat plasma: from different animals (K₂-EDTA as anticoagulant)

Acetonitnle: Merck Lichrosolv, # 1 .00030 (or equivalent)

Water for LC-MS/MS: Roth Rotisolv, # AE72.1 (or equivalent)

Water: Millipore purified (or equivalent)

Formic acid: Merck, #1 .00253.1000 (or equivalent)

Brij35: Sigma-Aldrich, # P1254 (or equivalent)

PBS (Phosphate Buffered Saline): Sigma-Aldrich, # P4417-100Tab (or equivalent) ProClin 300: Supelco, # 48912-U (or equivalent)

Sodium hydroxide: Riedel-de-Haen, # 30620 (or equivalent)

Insulin glargine: Certified batch¹

Insulin glargine-M1 : Certified batch

Insulin glargine-M2: Certified batch

[U-15N]-insulin glargine: Certificate with purity information available²

[U-15N]-insulin glargine-M1 : Certificate with purity information available

[U-15N]-insulin glargine-M2: Certificate with purity information available

7.3 Preparation of required solutions and mixtures

7.3.1 Mixture of acetonitrile/water/formic acid (20:80:0.1 , v/v/v)

20 mL of acetonitrile are placed in a 100 mL container and mixed with 80 mL of water. 100 }L of formic acid is added. 7.3.2 Mixture of acetonitrile/Brij35 (100:0.1 , v/w)

100 mL of acetonitrile are placed in a 100 mL container and mixed with 0.1 g of Brij35. 7.3.3 Mixture of acetonitrile/water/formic acid/Brij^'35 (20:80:0.1 :0.001 , v/v/v/w)

20 mL of acetonitrile are placed in a 100 mL container and mixed with 80 mL of water and 100 L of acetonitrile/Brij35 (100:0.1 , v/w). 100 μί of formic acid is added.

7.3.4 Mixture of acetonitrile/formic acid (100:0.5, v/v)

1000 mL of acetonitrile are placed in a 1 L container and mixed with 5 mL of formic acid.

7.3.5 Mixture of water/formic acid (100:0.5, v/v)

1000 mL of water are placed in a 1 L container and mixed with 5 mL of formic acid.

7.3.6 Dilution and Washing Buffer - Mixture of 0.01 M PBS/Brij35 (100:0.001 v/w) pH 7.8

1000 mL of water are placed in a 1 L container and mixed with 5 tablets of PBS. The solution is adjusted to pH 7.8 with 0.1 M sodium hydroxide and 1 mL of acetonitrile/Brij35 (100:0.1 , v/w).

7.3.7 Elution Buffer - Mixture of water/formic acid/acetonitril/Brij35 (90:1 :10:0.001 , v/v/v/w)

1000 mL of water are placed in a 1 L container and mixed with 10 mL of formic acid and 1 mL of acetonithle/Brij^'35 (100:0.1 , v/w).

7.3.8 Storage Buffer - Mixture of 0.01 M PBS/Brij35/ProClin 300 (100:0.001 :0.04, v/w/v) pH 7.8

200 mL of water are placed in a 200 mL container and mixed with 1 tablet of PBS, 200μί of acetonitrile/Brij35 (100:0.1 , v/w) and 80μί ProClin 300. The solution is adjusted to pH 7.8 with 0.1 M sodium hydroxide.

7.3.9 0.1 M Sodium hydroxide

0.4 g of sodium hydroxide are placed in a 100 mL container and mixed with 100 mL of water.

7.3.10 Stock solution insulin glargine

About 1 mg of insulin glargine is filled up to 1 mL with acetonitrile/water/formic acid (20:80:0.1 , v/v/v). This solution needs to be prepared twice (for calibration standards and for quality controls). Other weightings and volumes may be used as far as the resulting concentrations of the stock solutions are about 1 mg/mL.

7.3.11 Stock solution of [U-15N]-insulin glargine

22.3 μΙ_ of [U-15N]-insulin glargine stock solution [448 000 ng/mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v)] are filled up to 10 mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v). Depending on the concentration of the available [U-15N]-insulin glargine stock solution, other weightings and volumes may be used as far as the resulting concentration of the stock solution is 1000 ng/mL. All dilutions are stored in a freezer at about -80°C. 7.3.12 Stock solution of insulin glargine-M1

About 1 mg of insulin glargine-M1 is filled up to 1 mL with acetonitrile/water/formic acid (20:80:0.1 , v/v/v). This solution needs to be prepared twice (for calibration standards and for quality controls). Other weightings and volumes may be used as far as the resulting concentrations of the stock solutions are about 1 mg/mL. 7.3.13 Stock solution of [U-15N]-insulin glargine-M1

20.9 μί of [U-15N]-insulin glargine-M1 stock solution [478 000 ng/mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v)] are filled up to 10 mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v). Depending on the concentration of the available [U-15N]-insulin glargine-M1 stock solution, other weightings and volumes may be used as far as the resulting concentration of the stock solution is 1000 ng/mL. All dilutions are stored in a freezer at about -80°C.

7.3.14 Stock solution of insulin glargine-M2

About 1 mg of insulin glargine-M2 is filled up to 1 mL with acetonitrile/water/formic acid (20:80:0.1 , v/v/v). This solution needs to be prepared twice (for calibration standards and for quality controls). Other weightings and volumes may be used as far as the resulting concentrations of the stock solutions are about 1 mg/mL.

7.3.15 Stock solution of [U-15N]-insulin glargine-M2

22.9 μί of [U-15N]-insulin glargine-M2 stock solution [437 000 ng/mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v)] are filled up to 1 mL in acetonitrile/water/formic acid (20:80:0.1 , v/v/v). Depending on the concentration of the available [U-15N]-insulin glargine-M2 stock solution, other weightings and volumes may be used as far as the resulting concentration of the stock solution is 1000 ng/mL. All dilutions are stored in a freezer at about -80°C.

7.4Standard, Quality Control and Internal Standard Preparation 7.4.1 Preparation of Calibration Standards

The stock solutions of insulin glargine, insulin glargine-M1 and insulin glargine-M2 are stored at -80°C. Stock solutions can be used over the proven stability period.

Calibration standards are prepared as follows:

The first stock solution of insulin glargine, insulin glargine-M1 and insulin glargine-M2 is diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 100 000 ng/mL and subsequently diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 1000 ng/mL. This solution (1000 ng/mL) is used for preparation of calibration standards 6, calibration standard 5 and calibration standard 4. Calibration standard 6 is used for preparation of calibration standard 3, calibration standard 5 is used for preparation of calibration standard 2 and calibration standard 3 is used for preparation of calibration standard 1 . The calibration samples are prepared daily and kept in a refrigerator at about 4°C. Additionally, the calibration samples might be used over any proven stability period. The following mixtures will be used for preparation of calibration standards, though the volumes may be modified to adapt the volume of the mixtures to the respective validation test as far as the resulting target concentrations are equal to those in the following table:

Target

Concentrati Resulting

Initial Cone, on of final of insulin insulin Concentrati

glargine, will be made up with glargine, on of insulin

acetonitrile/water/formi insulin glargine,

Standard insulin Volume

c acid/Brij35 glargine- insulin

No. glargine-Ml [jiL]

and (20:80:0.1 :0.001 , Ml and glargine-M1

insulin v/v/v/w) to insulin and glargine-M2 glargine- insulin [ng/mL] M2 glargine-M2

[ng/mL] [ng/mL]

in plasma

in solvent

Std. 6 1000 100 1000 μΙ_ 100 10

Std. 5 1000 60 1000 μΙ_ 60 6 Std. 4 1000 30 1000 μΙ_ 30 3

Std. 3 100 100 1000 μΙ_ 10 1

Std. 2 60 100 1000 μΙ_ 6 0.6

Std. 1 10 0 1000 μΙ_ 2 0.2

7.4.2 Preparation of Quality Control/Validation Samples

Quality control samples/Validation samples are prepared as follows:

The first stock solutions of insulin glargine, insulin glargine-M1 and insulin glargine-M2 are diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 100 000 ng/mL and subsequently diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 1000 ng/mL. This solution (1000 ng/mL) is used for preparation of quality control 3 and quality control 2. The solution of quality control 3 is used for preparation of quality control 1 and the solution of quality control 2 is used for preparation of the LLOQ level. The dilutions are prepared daily and are kept in a refrigerator at about 4°C. Additionally, the dilutions might be used over any proven stability period. The following mixtures will be used for preparation of quality control samples/validation samples, though the volumes may be modified to adapt the volume of the mixtures to the respective validation test as far as the resulting target concentrations are equal to those in the following table:

Resulting

Target final

Initial Cone. will be made Concentration Concentrati

Of insulin up with of insulin on of glargine, acetonitrile/ glargine, insulin insulin insulin

Quality Volum water/formic glargine,

glargine-Ml glargine-M1 insulin Control No. e [nL] acid/Brij35

and and glargine-M1

insulin (20:80:0.1 :0. insulin and glargine-M2 001 , v/v/v/w) glargine-M2 insulin

[ng/mL] to [ng/mL] glargine-M2

in solvent ⁴ [ng/mL]

in plasma

VAL 4 / QC 1000 160 2000 μί

3 80 8

VAL 3 / QC 100 2000 μί

2 1000 50 5

VAL 2 / QC 150 2000 μί

1 80 6 0.6

VAL 1 /

50 80 2000 μί 2 0.2 LLOQ 7.4.3 Preparation of Validation Samples (spiked with the reference compound)

Validation samples for stability tests are prepared as spiked K₂-EDTA plasma samples. For this purpose, K₂-EDTA plasma is spiked with a solution of insulin glargine, insulin glargine-M1 and insulin glargine-M2 to obtain HIGH QC-Level (8 ng/mL) and further diluted with K₂-EDTA plasma to obtain LOW QC-Level (0.6 ng/mL). The spiked plasma samples might be used over any proven stability period.

The following mixtures will be used as quality control samples/validation samples prepared directly in K₂-EDTA plasma, though the volumes may be modified to adapt the volume of the mixtures to the respective validation test as far as the resulting target concentrations are equal to those in the following table:

7.4.4 Internal Standard Preparation

The stock solution of [U-15N]-insulin glargine (1000 ng/mL) is diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 50 ng/mL. This solution is prepared daily and is kept in a refrigerator at about 4°C. The stock solution of [U-15N]-insulin glargine-M1 (1000 ng/mL) is diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 25 ng/mL. This solution is prepared daily and is kept in a refrigerator at about 4°C. The stock solution of [U-15N]-insulin glargine-M2 (1000 ng/mL) is diluted with acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) to a nominal concentration of 25 ng/mL. This solution is prepared daily and is kept in a refrigerator at about 4°C. 7.5 Assay procedure

If study samples have to be thawed prior LC-MS/MS analysis store them on ice or in a refrigerator at 4°C to prevent degradation of insulin glargine, insulin glargine-M1 and insulin glargine-M2 in K₂-EDTA plasma. The immunoaffinity columns (lAC) can be reused. To reuse the lAC columns follow Step C, if more samples will be running over the respective column on that particular day, or follow Step D, if the lAC columns won't be used on that particular day again.

Step A - Sample Preparation: · Place 300 μΙ_ of K₂-EDTA plasma into a 1 .5 ml_ reaction tube.

• Add 50 μΙ_ of the internal standard working solution [50 ng/mL of [U-15N]-insulin glargine, 25 ng/mL [U-15N]-insulin glargine-M1 , 25 ng/mL [U-15N]-insulin glargine-M2 in acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w)]. In case of double blank samples, pure acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) is added.

• Add 30 μί working solution of insulin glargine, insulin glargine-M1 and insulin glargine-M2. In case of calibration standards or QC-samples, this will be the respective working solution in acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) · Add 300 μί of 0.01 M PBS/Brij35 (100:0.001 , v/w) pH 7.8

• Seal tubes and mix.

Step B - Immunoaffinity Purification:

• Equilibrate the lAC columns with 2 mL (2 x 1 mL) of washing buffer - 0.01 M PBS/Brij35 (100:0.001 , v/w) pH 7.8

• Apply the centrifuged sample from Step A onto the lAC columns

• Wash the lAC columns with 2 mL (2 x 1 mL) of washing buffer - 0.01 M PBS/Brij35 (100:0.001 , v/w) pH 7.8 • Pipet 80 μΙ_ of elution buffer - water/formic acid/ (100:1 , v/v/)

• Place the IAC columns over the Vivacon 500 Filter VN01 H02

• Pipet 300 μΙ_ of the elution buffer - water/formic acid/acetonitrile/Brij35 (90:1 :10:0.001 , v/v/v/w) · Seal tubes and centrifuge the samples for 15 minutes at ~ 21 500 g

• Pipet the eluate in Matrix ScreenMates tubes

• Close the Matrix ScreenMates tubes and use the eluate for LC-MS/MS sample analysis (injection volume typically 60 μΙ_) Step C - Wash of IAC columns for a following immunoaffinity purification:

• Wash the IAC columns with 4 mL (4 x 1 mL) of washing buffer - water/formic acid/ (100:1 :, v/v)

• Wash the IAC columns with 2 mL (2 x 1 mL) of washing buffer - 0.01 M PBS/Brij35 (100:0.001 , v/w) pH 7.8. The IAC columns can now be used for the next extraction

Step D - Procedure for storage of IAC columns:

• Wash the IAC columns with 4 mL (4 x 1 mL) of elution buffer - water/formic acid/ (100:1 :, v/v) · Wash the IAC columns with 0.5 mL of storage buffer - PBS (0.01 M in water)/Brij35/ProClin 300 (100:0.001 :0.04, v/w/v) pH 7.8

• Plug the IAC column and pipet 0.5 mL of storage buffer - PBS (0.01 M in water)/Brij35/Proclin 300 (100:0.001 :0.04, v/w/v) pH 7.8

• Seal the columns with parafilm and store the columns in an upright position at 4°C. 7.6 Sample Analysis

7.6.1 Chromatography and Mass Spectrometry Configuration 7.6.1.1 Analytical Equipment

7.6.1.1.1 Liquid chromatography system

Dionex Ultimate 3000 series liquid chromatography system or equivalent HPLC system:

Degasser, type Ultimate 3000

LC-Pump, type Ultimate 3000

Autosampler, type Ultimate 3000

Column oven, type Ultimate 3000

7.6.1.1.2 Tandem Mass spectrometer

API 5000 from Applied Biosystems MDS Sciex or equivalent mass spectrometer

7.6.1.1.3 VIGI Valco Valve or equivalent Valve

Valco Instruments Co Inc.

Two position actuator control module

Chromatographic Conditions

Gradient Profile:

Time A B

[min] [%] [%]

0.00 95 5

2.80 35 65

5.50 35 65

7.50 95 5

8.25 95 5

Valco Valve:

Time Postion

[min]

0.00 Waste

1 .50 LCMS

3.50 Waste

7.6.3 Mass Spectrometric Conditions

Actual Precursor- and Product Ion masses may vary due to different PPG mass calibration settings from one mass spectrometer to the other or on the same instrument by up to ± 0.5 mass units. Collision Energy may vary from one mass spectrometer to the other or on the same instrument. Dwell time for both mass transitions may vary from 5 ms to 500 ms. References ¹ (31 -32)^B-di-D-Arg-insulin glargine has the same amino acid sequence compared to insulin glargine, but with two D-Arg on position 31 and 32 instead of L-Arg. Since the mass spectrometric detection does not differentiate between D- and L-amino acids, insulin glargine can be used for preparation of calibration standards and quality control samples.

² (31 -32)^B-di-D-Arg-insulin glargine has the same amino acid sequence compared to insulin glargine, but with two D-Arg on position 31 and 32 instead of L-Arg. Since the mass spectrometric detection does not differentiate between D- and L-amino acids, [U15-N]-insulin glargine can be used as internal standard for (31 -32)^B-di-D-Arg-insulin glargine.

³ The resulting working solutions in acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) (2 to 100 ng/mL) are used to prepare the calibration samples in plasma: 30 μί of these working solutions are added to 300 μί of plasma and correspond to a dilution factor of 10. The resulting final concentrations in plasma can be found in the last column of the table.

⁴ The resulting working solutions in acetonitrile/water/formic acid/Brij35 (20:80:0.1 :0.001 , v/v/v/w) (2 to 100 ng/mL) are used to prepare the quality control samples in plasma: 30 μί of these working solutions are added to 300 μί of plasma and correspond to a dilution factor of 10. The resulting final concentrations in plasma can be found in the last column of the table.

Claims

Claims:

1 . A modified insulin analog or a salt thereof of the formula I

wherein

(a) AO is absent or Arg,

(b) (A1 -A5) are amino acid residues in the positions A1 to A5 in the A chain of human (SEQ ID NO:1 ) or animal insulin,

(c) (A8-A10) are amino acid residues in the positions A8 to A10 in the A chain of human (SEQ ID NO:1 ) or animal insulin,

(d) (A12-A19) are amino acid residues in the positions A12 to A19 in the A chain of human (SEQ ID NO:1 ) or animal insulin,

(e) A21 is Asn or Gly,

(f) B1 is Phe or His,

(g) B3 is Asn or His,

(h) B2 and (B4-B6) are amino acid residues in the positions B2 and B4 to B6 in the B chain of human (SEQ ID NO:2) or animal insulin,

(i) (B8-B18) are amino acid residues in the positions B8 to B18 in the B chain of human (SEQ ID NO:2) or animal insulin,

(j) (B20-B27) are amino acid residues in the positions B20 to B27 in the B chain of human (SEQ ID NO:2) or animal insulin,

(k) B28 is Pro or Lys,

(I) B29 is Lys or absent,

(m) B30 is Thr or absent,

(n) B31 is absent, Arg or a basic D-amino acid, particularly D-Arg, D-His, D- Lys, D-Hyl, D-Ornithine, or D-Citrulline. (o) B32 is a basic D-amino acid, particularly D-Arg,

with the proviso that if B31 is Arg, then A21 is Gly, or if B31 is absent, then AO is Arg,

and with the proviso that if B28 is Lys and B29, B30 and B31 are absent, then AO is Arg.

2. The modified insulin analog of claim 1 wherein B30 is Thr.

3. The modified insulin analog of claims 1 or 2 wherein A21 is Gly.

4. The modified insulin analog of any one of claims 1 to 3 wherein AO is absent.

5. The modified insulin analog of any one of claims 1 to 4 wherein at least one of B31 and B32 is D-Arg.

6. The modified insulin analog of any one of claims 1 to 5 wherein B29 is Lys.

7. The modified insulin analog of any one of claims 1 to 6 wherein B28 is Pro.

8. The modified insulin analog of any one of claims 1 to 3 or 5 to 7 wherein AO is Arg.

9. The modified insulin analog of any one of claims 1 to 6 or 8 wherein B28 is Lys.

10. The modified insulin analog of any one of claims 1 to 5 or 7 to 9 wherein B29 is absent.

1 1 . The modified insulin analog of any one of claims 1 to 2 or 4 to 10 wherein A21 is Asn.

12. The modified insulin analog of any one of claims 1 to 4 or 6 to 1 1 wherein B31 is L-Arg.

The modified insulin analog of any one of claims 1 to 4 or 6 to 1 1 wherein B31 is absent.

The modified insulin analog of any one of claims 1 or 3 to 13 wherein B30 is absent.

The modified insulin analog of any one of claims 1 to 14, wherein

(a) AO is absent, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is Arg, and B32 is D-Arg (SEQ ID NOs: 15 and 16),

(b) AO is absent, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is D-Arg, and B32 is D-Arg (SEQ ID NOs: 17 and 18),

(c) AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is Arg, and B32 is D-Arg (SEQ ID NOs: 19 and 20),

(d) AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is D-Arg, and B32 is D-Arg (SEQ ID NOs: 21 and 22),

(e) AO is Arg, A21 is Asn, B28 is Pro, B29 is Lys, B30 is Thr, B31 is absent, and B32 is D-Arg (SEQ ID NOs: 23 and 24),

(f) AO is Arg, A21 is Gly, B28 is Pro, B29 is Lys, B30 is Thr, B31 is absent, and B32 is D-Arg (SEQ ID NOs: 25 and 26),

(g) AO is absent, A21 is Asn, B28 is Lys, B29 is absent, B30 is Thr, B31 is D- Arg, and B32 is D-Arg (SEQ ID NOs: 29 and 30), or

(h) AO is absent, A21 is Gly, B28 is Lys, B29 is absent, B30 is Thr, B31 is D- Arg, and B32 is D-Arg (SEQ ID NOs: 33 and 34).

A conjugate of a modified insulin analog of any one of claims 1 to 15 linked via a non-peptidyl polymer selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combinations thereof.

17. The conjugate of claim 16 wherein said insulin analog is defined according to any one of claims 1 to 15 and said non-peptidyl polymer is linked to the amino terminus of the B chain of said insulin analog.

18. A pharmaceutical formulation, preferably a long-acting formulation, comprising as an active agent a modified insulin analog according to any one of claims 1 -15 or a conjugate according to claims 16 to 17 and a pharmaceutically acceptable carrier.

19. A modified insulin analog according to any one of claims 1 -15 or a conjugate according to claims 16 to 17 for use in therapy.

20. A modified insulin analog according to any one of claims 1 -15 or a conjugate according to claims 16 to 17 for use in the prevention, alleviation, or treatment of an insulin-deficiency disorder, insulin resistance, obesity, hypoglycemia, and/or a neurodegenerative disorder, particularly a neurodegenerative disorder which involves or is associated with oxidative stress, loss of neurite integrity, apoptosis, neuronal loss, cognitive impairment and/or inflammatory response.

21 . A modified insulin analog or a conjugate for use according to claim 20 wherein the insulin-deficiency disorder is diabetes, particularly type I or type II diabetes.

22. A modified insulin analog or a conjugate for use according to claim 20 wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson's disease, dementia, epilepsy, stroke, Huntington's Chorea, cerebral hypoxia, multiple sclerosis, and peripheral neuropathy.

23. A method for inhibiting protease degradation in serum and/or increasing serum stability of an insulin analog by attaching one or more D-amino acids to said insulin analog resulting in a modified insulin analog according to any one of claims 1 -15.

24. The method of claim 23 wherein the D-amino acid(s) is/are attached to the C- terminus of said insulin analog.

25. The method of claim 23 or 24 wherein said protease is a carboxyprotease.

26. The method of any one of claims 23 to 25 wherein the insulin analog is preferably insulin (SEQ ID NOs: 1 and 2), or insulin glargine (SEQ ID NOs: 1 1 and 12) or A0(Arg)-insulin glargine (SEQ ID NOs: 13 and 14).

27. The method of any one of claims 23 to 25 wherein the D-amino acid(s) is/are introduced by:

28. The method of claim 27 wherein the insulin analog is a recombinantly produced fragment of insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine comprising the A chain of said insulin or insulin analog and the B chain terminating at amino acid position B29 (Lys) prior to step (b).

29. The method of claim 27 wherein the insulin analog is insulin glargine or AO(Arg)- insulin glargine and the method comprises cleaving the protein with an endopeptidase, preferably a lysyl-endopeptidase or trypsin.

30. The method of claim 27 wherein the added D-amino acids replace L-amino acids of the insulin analog that have been cleaved by the endopeptidase.

31 . The method of any one of claims 27 to 30 wherein the D-amino acid is a basic D- amino acid, preferably D-Arg or D-Lys.

32. The method of any one of claims 23 to 31 wherein the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the peptide is L-Thr-L-Arg- D-Arg or L-Thr- D-Arg- D-Arg.

33. The method of any one of claims 27 to 32 wherein the peptide is added via trypsin catalysed transpeptidation.

34. A method of preparing a modified insulin analog comprising:

35. The method of claim 34 wherein the D-amino acid is a basic D-amino acid, preferably D-Arg and/or D-Lys.

36. The method of claim 34 or 35 for producing a modified insulin analog according to any one of claims 1 -15 or a conjugate according to claims 16 to 17.

37. The method of any one of claims 34 to 36 comprising the recombinant production of said insulin or insulin analog which is a fragment of insulin or an insulin analog such as insulin glargine or A0(Arg)-insulin glargine in step (a) comprising the A chain of said insulin or insulin analog and a B chain terminating at amino acid position B29 (Lys) prior to step (b).

38. The method of any one of claims 34 to 36 wherein the insulin analog is insulin glargine or A0(Arg)-insulin glargine and the peptide is L-Thr-L-Arg- D-Arg or L-Thr- D-Arg- D-Arg.