ES2353649T3 - INSULIN DERIVATIVES - Google Patents

Abstract

An insulin derivative comprising a progenitor insulin and a substituent, wherein the substituent is well linked to a ε-amino group of a Lys residue present in the A chain of the progenitor insulin at position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or a ε-amino group of a Lys residue in the B chain of the progenitor insulin at position B1, B2, B3, B4; B20; B21 or B22 provided that when B3 is Lys, then B29 is not Glu, where the substituent is of general formula: -WXY-ZdondeW is: - an α-amino acid residue with a carboxylic acid group in the side chain whose residue it forms, with one of its carboxylic acid groups, an amide group with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin; or - a chain composed of two, three or four α-amino acid residues linked together by means of amide bonds, this chain, by means of an amide bond, is attached to the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin, the amino acid residues of W being selected from the group of amino acid residues with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least an amino acid residue having a carboxylic acid group in the side chain; or - a covalent bond of X to the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin; X is: - -CO; --CH (COOH) CO-; - -CON (CH2COOH) CH2CO-; - -CON (CH2COOH) CH2CON (CH2 COOH) CH2CoO - -CON (CH2CH2COOH) CH2CH2O; - -CON (CH2CH2COOH) CH2CH2CON (CH2 CH2 COOH) CH2CH2CO; - -CONHCH (COOH) (CH2) 4NHCO-; - -CON (CH2CH2COOH) CH2CO-; or - -CON (CH2COOH) CH2 CH2CO On condition that a) when W is an amino acid residue or a chain of amino acid residues, the carbonyl carbon underlined in X forms an amide bond with an amino group in W, b) when W is a covalent bond, the carbonyl carbon underlined in X forms an amide bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin; Y is: - - (CH2) m- where m is an integer in the range of 6 to 32; - a bivalent hydrocarbon chain comprising 1, 2 or 3 groups -CH = CH- and a number of groups -CH2- sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; - a bivalent hydrocarbon chain of the formula - (CH2) vC6H4 (CH2) w - where v and w are whole numbers or one of these is zero so that the sum of v and w is in the range of 6 to 30 and z is: - - COOH; - -CO-Asp; - -CO-Glu; - -CO-Gly; - -CO-Sar; - -CH (COOH) 2; - -N (CH2 COOH) 2; - -SO3 H; or - -PO3 H; and any Zn2 + complex thereof.

Description

Insulin derivatives.

Field of the Invention

The present invention relates to derivatives of new human insulin that are soluble at physiological pH values and have a prolonged action profile. The invention is also refers to the pharmaceutical compositions containing them, to a method of treating diabetes and hyperglycemia using the insulin derivatives of the invention and the use of such derivatives insulin in the treatment of diabetes and hyperglycemia

Background of the invention

Usually, the treatment of diabetes, both of type 1 diabetes and type 2 diabetes, it is based increasingly in the so-called intensive insulin treatment. Under this regimen, patients are treated with multiple daily insulin injections comprising one or two injections Daily long-acting insulin to meet the requirement of basal insulin supplemented by bolus injections of an insulin Fast-acting to meet the related insulin requirement with meals.

Long acting insulin compositions are known in the art. Thus, a main type of compositions of long-acting insulin comprises aqueous injectable suspensions of insulin crystals or amorphous insulin. In these compositions, insulin compounds typically used are insulin protamine, cynical insulin or cynical protamine insulin.

Certain drawbacks are associated with the use of insulin suspensions. So, to ensure a precise dosage, insulin particles must be suspended homogeneously by gentle agitation before a Defined volume of the suspension is removed from a bottle or eject from a cartridge. Also, for storage of insulin suspensions, the temperature must be maintained within narrower limits than for insulin solutions to avoid the formation of nodules or coagulation.

Other type of long insulin compositions performance are solutions with a pH value below pH physiological where insulin will precipitate due to the increase in pH value when the solution is injected. An inconvenience of these solutions is that the particle size distribution of the precipitate formed in the tissue with the injection, and therefore the medication release profile, it depends somewhat unpredictable blood flow at the injection site and others parameters Another drawback is that the solid particles of the insulin can act as a local irritant that causes inflammation of the tissue at the injection site.

Human insulin has three amino groups primary: the N-terminal group of chain A and of the B chain and the ε-amino group of LysB29. Different insulin derivatives that are substituted in one or More of these groups are known in the prior art.

WO 95/07931 (Novo Nordisk A / S) discloses derivatives of human insulin where the group ε-amino of LysB29 has a substituent lipophilic These insulin derivatives have a prolonged profile of action and are soluble at physiological pH values.

WO international patent application 96/29344 concerns an insulin derivative, where a chain lipophilic side is fixed to the N-terminal amino group of chain B or a residue Lys in position B26-B29 of the progenitor insulin molecule.

WO 97/31022 discloses insulin derivatives where the α-amino group of the group N-terminal of chain B and / or group ε-amino of Lys at position B28; B29 or B30 has a substituent of the formula CO-W-COOH where W is a group long chain hydrocarbon having 12 to 22 carbon atoms.  These insulin derivatives have a prolonged action profile and They are soluble at physiological pH values.

Another insulin derivative is described in the international patent application. Patent application WO 2005 /
012347 describes an insulin derivative with a side chain attached to either the α-amino group of the N-terminal amino acid residue of the B chain or the ε-amino acid of an Lys residue present in the B chain of the progenitor insulin.

Unfortunately, many diabetics are reluctant to take intensive care due to the associated discomfort to the many injections required to maintain control thorough glucose levels. This type of therapy can be both psychologically and physically painful. Behind the oral administration, insulin is rapidly degraded in the gastrointestinal tract and is not absorbed into the bloodstream. Therefore, many researchers have studied alternative pathways for the administration of insulin, such as oral routes, rectal, transdermal, and nasal. So far, however, you are routes of administration have not resulted in absorption of effective insulin

The effective pulmonary supply of a protein depends on the ability to supply the protein to the epithelium alveolar deep lung. The proteins that are deposited in the Upper airway epithelium is not absorbed at a significant extension This is due to the overlying mucus that is approximately 30-40 µm thick and acts as a barrier to absorption. In addition, the proteins deposited in this epithelium is clarified by mucociliary transport on the airways and then removed by the tract gastrointestinal. This mechanism also contributes substantially at low absorption of some protein particles. The measure in which proteins are not absorbed and instead are eliminated by these pathways depend on their solubility, their size, as well as others less understood features.

There is still a need for insulins with a longer action profile than derivatives insulins known so far and at the same time be soluble at physiological pH values and having a potency comparable to that of human insulin. In addition, there is the need for more insulin formulations that are well suited for pulmonary application.

The present invention is directed and relieves prior art problems.

Summary of the Invention

The present invention is based on the recognition that insulin acylation can be performed in a Lys residue present in the A chain or in a Lys residue in the B chain of a progenitor insulin.

According to the invention a derivative is provided insulin comprising a progenitor insulin and a substituent, where the substituent is well linked to a group ε-amino of a Lys residue present in the A chain of the progenitor insulin at position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or a group ε-amino of a Lys residue in the B chain of the progenitor insulin at position B1, B2, B3, B4; B20; B21 or B22 provided that when B3 is Lys, then B29 does not is Glu, where the substituent is of general formula:

-W X Y Z

where

W is:

\ sqbullet

a residue of α-amino acid with an acid group carboxylic in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or

\ sqbullet

a chain composed of two, three or four α-amino acid residues linked together through amide bonds, whose chain, by through an amide link, it is linked to the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin, the residues of W amino acids being selected from the residue group of amino acids with a neutral side chain and residues of amino acids with a carboxylic acid group in the side chain so that W has at least one amino acid residue that has a carboxylic acid group in the side chain; or

\ sqbullet

a covalent bond of X to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin;

X is:

\ sqbullet

-CO-;

\ sqbullet

-CH (COOH) C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CONHCH (COOH) (CH 2) 4 NH C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 C O-; or

\ sqbullet

-CON (CH 2 COOH) CH 2 CH 2 C O-

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On condition that

a) when W is an amino acid residue or a amino acid residue chain, carbonyl carbon underlined in X forms an amide bond with an amino group in W, or

b) when W is a covalent bond, carbon of the carbonyl underlined in X forms an amide bond with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin;

And it is:

\ sqbullet

- (CH 2) m - where m is an integer in the range of 6 to 32;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of groups -CH_ {2} - enough to give a total number of atoms of carbon in the chain in the range of 10 to 32;

\ sqbullet

a hydrocarbon chain bivalent of the formula - (CH2) v C 8 H 4 (CH 2) w - where v and w are integers or one of these is zero so that the sum of v and w It is in the range of 6 to 30 and Z is:

\ sqbullet

COOH;

\ sqbullet

CO-Asp;

\ sqbullet

CO-Glu;

\ sqbullet

CO-Gly;

\ sqbullet

CO-Sar;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH 2 COOH) 2;

\ sqbullet

-SO 3 H; or

\ sqbullet

-PO 3 H;

and any Zn 2+ complexes of the same.

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The substituent fixed to the group ε-amino of the Lys residue can comprise a lipophilic group, a fatty acid or a fatty diacid, an aromatic group or an amino acid residue containing optionally a group that can be negatively charged. He fatty diacid will typically comprise from 4 to 22, from 6 to 22, from 8 to 20, from 8 to 18, from 4 to 18, from 6 to 18, from 8 to 16, from 8 to 22, from 8 to 17 or 8 to 15 carbon atoms in the carbon chain.

Non-limiting examples of the fraction of fatty diacids are diacid with the formula HOOC- (CH2) r1 -COOH, where r1 is 4 to 22. Examples of fatty diacids are succinic acid, acid hexanedioic acid, octanedioic acid, decanedioic acid, acid dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid or octadecenedioic acid.

Definitions

With " insulin desB30 ", " human insulin desB30 " means a natural insulin or an analogous derivative lacking the amino acid residue B30. Similarly, " human insulin desB29desB30 " or " insulin desB29desB30 " means a natural insulin or an analogous derivative that lacks amino acid residues B29 and B30.

With " B (1-29) " and " chain B " is meant a natural insulin B chain or an analogous derivative lacking the amino acid residue B30. " A (1-21) " and " A chain " means the natural insulin A chain or an analog thereof. "A (1-24)" means a modified A chain, where the A chain has been extended at the C-terminal with 3 encodable amino acids.

With, " B1 ", " A1 " etc. the amino acid residue in position 1 in the insulin B chain (counted from the N-terminal end) and the amino acid residue in position 1 in the insulin chain A (counted from the N-terminal end) is understood, respectively. The amino acid residue at a specific position may also be referred to as, for example, PheB1, which means that the amino acid residue at position B1 is a phenylalanine residue.

With " insulin " as used in this case, human insulin with disulfide bridges between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulfide bridge between CysA6 and CysA11, porcine insulin and bovine insulin is understood.

" POT " is the triosis phosphate isomerase gene from Schizosaccharomyces pombe .

A "leader" means a sequence of amino acids consisting of a prepeptide (the signal peptide) and a propeptide

The term "signal peptide" is understood to mean a prepeptide that is present as an N-terminal sequence in the precursor form of a protein. The function of the signal peptide is to allow the heterologous protein to facilitate translocation in the endoplasmic reticulum. The signal peptide is normally cut during this process. The signal peptide can be heterologous or homologous to the yeast organism that produces the protein. Several signal peptides that can be used with the DNA construct of the invention including the aspartic protease 3 signal peptide (YAP3) or any functional analog (Egel-Mitani et al . (1990) Yeast 6: 127-137 and US 5,726,038 ) and the α signal factor of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathern et al ., eds., pp. 143-180, Cold Spring Harbor Laboratory, NY and US 4,870 , 00.

The term " propeptide " means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and also to a secretory vesicle for secretion in the culture medium (ie, export of the polypeptide through from the cell wall or at least through the cell membrane in the periplasmic space of the yeast cell). The propeptide may be the yeast factor α propeptide, see US 4,546,082 and 4,870,008. Alternatively, the propeptide can be a synthetic propeptide, that is to say a propeptide not found in nature. Suitable synthetic propeptides are those described in US 5,395,922, 5,795,746, 5,162,498 and WO 98/32867. The propeptide may contain an endopeptidase processing site at the C-terminal end, such as a Lys-Arg sequence or any functional analog thereof.

By " insulin precursor " as used herein, a single chain polypeptide is understood that after cleavage with an appropriate protease, for example trypsin, produces a double-chain insulin, insulin analogue, or insulin derivative. An example of such an insulin precursor is "B'A", which is a single chain insulin precursor where the C-terminal amino acid residue of the B chain is directly linked to the amino acid residue A1 in the A chain. An example Specific to such B'A insulin precursor is LysA9 ArgB29 desB30 B'A, where ArgB29 is directly connected to GlyA1.

By " insulin analog " as used in this case is meant a polypeptide having a molecular structure that can be formally derived from the structure of a naturally occurring insulin, for example human insulin, removing and / or replacing at least one amino acid residue that originates from natural insulin and / or by adding at least one amino acid residue. The substituted and / or added amino acid residues may either be encodable amino acid residues or other naturally occurring amino acid residues or purely synthetic amino acid residues.

Examples of insulin analogs are analogues of human insulin desB30; insulin analogs where one or both of B1 and B2 have been deleted; insulin analogs where the chain A and / or the B chain have an N-terminal extension and insulin analogs where chain A and / or chain B have a C-terminal extension. So one or two Arg can be add to position B1. Also one or more of B26-B30 may have been deleted.

By " progenitor insulin " is meant an insulin analog containing only a residue of Lys in chain A and / or chain B, whose residue of Lys is not present in position B29.

Specific examples of progenitor insulins are human insulin LysA9 ArgB29 desB30 and human insulin LysB22 ArgB29 desB30.

By " insulin derivative ", as used herein is meant a naturally occurring insulin or an insulin analogue that has been chemically modified, for example by introducing a substituent at one or more positions of the insulin skeleton or by oxidation or reduction of the groups of amino acid residues in insulin or by conversion of a free carboxylic group to an ester group or by acylation of a free amino group or a hydroxy group.

The insulin derivatives of the invention are named according to the following rule: the sequence begins with the chemical modification, continue with chain A, and end with the chain B. The amino acid residues are named after their respective equivalents in human insulin and mutations and acylations are explicitly described while waste of unchanged amino acids in chains A and B are not mentioned. For example, an insulin that has the following mutations in comparison with human insulin LysA9; ArgB29; desB30 and acylates with myristyl in the N ^ {\} of LysA9 it is called human insulin LysA9 ArgB29 desB30 N ^ {\ varepsilon A9} -myristyl.

When an insulin derivative according to the invention it is declared "soluble at physiological pH values" this means that the insulin derivative can be used to prepare insulin compositions that are completely dissolved at values of physiological pH. Such favorable solubility may well be due to the inherent properties of the insulin derivative alone or a result of a favorable interaction between the insulin derivative and one or more ingredients contained in the vehicle.

Abbreviations for amino acids

one

The expression " an amino acid residue with a carboxylic acid group in the side chain " designates amino acid residues such as Asp, Glu and hGlu. The amino acids can be either in L or D configuration. If nothing is specified it is understood that the amino acid residue is in the L configuration.

The expression " an amino acid residue with a neutral side chain " designates amino acid residues such as Gly, Ala, Val, Leu, Ile, Phe, Pro, Ser, Thr, Cys, Met, Tyr, Asn and Gln.

Abbreviations used in the examples

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2

hGlu is acidic homoglutamic.

TFA: trifluoroacetic acid

DMF: N, N-dimethylformamide

EtOAc: ethyl acetate

THF: tetrahydrofuran

TSTU: O- (N-succinimidyl) -1,1,3,3-tetramethyluronium tetrafluoroborate

DIPEA: diisopropylethylamine

α-Asp is the L-form of -HNCH (CO-) CH2COOH.

β-Asp is the L-form of -HNCH (COOH) CH2CO-.

α-Glu is the L-shape of -HNCH (CO-) CH2CH2COOH.

γ-Glu is the L-shape of -HNCH (COOH) CH2CH2CO-.

α-hGlu is the L-shape of -HNCH (CO-) CH 2 CH 2 CH 2 COOH.

δ-hGlu is the L-shape of -HNCH (COOH) CH2CH2CH2CO-.

β-Ala is -NH-CH 2 -CH 2 COOH.

Sar is (N-methylglycine) sarcosine

Detailed description of the invention

The present invention concerns a derivative insulin comprising a progenitor insulin and a substituent, where the substituent is well linked to a group ε-amino of a Lys residue present in the A chain of the progenitor insulin at position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or a group ε-amino of a Lys residue in the B chain of the progenitor insulin at position B1, B2; B3, 84; B20; B21 or B22 provided that when B3 is Lys, then B29 is not Glu, where the substituent is of general formula:

W X Y Z

where

W is:

\ sqbullet

a residue of α-amino acid with an acid group carboxylic in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or

\ sqbullet

a chain composed of two, three or four α-amino acid residues attached each other by means of amide bonds, whose chain, by means of a amide bond, binds to the ε-amino group of a residue of Lys present in the A or B chain of insulin parent, the amino acid residues of W being selected of the amino acid residue group with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least one amino acid residue having a carboxylic acid group in the side chain; or

\ sqbullet

a covalent bond of X to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin;

X is:

\ sqbullet

-CO-;

\ sqbullet

-CH (COOH) C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CONHCH (COOH) (CH 2) 4 NH C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 C O-; or

\ sqbullet

-CON (CH 2 COOH) CH 2 CH 2 C O-

On condition that

a) when W is an amino acid residue or a amino acid residue chain, carbonyl carbon underlined in X forms an amide bond with an amino group in W, or

b) when W is a covalent bond, carbon of the carbonyl underlined in X forms an amide bond with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin;

And it is:

\ sqbullet

- (CH 2) m - where m is an integer in the range of 6 to 32;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of -CH_ {2} groups - sufficient to give a total number of atoms of carbon in the chain in the range of 10 to 32;

\ sqbullet

a hydrocarbon chain formula bivalent - (CH 2) v C 6 H 4 (CH 2) w - where v and w are integers or one of these is zero so that The sum of v and w is in the range of 6 to 30 and Z is:

\ sqbullet

-COOH;

\ sqbullet

-CO-Asp;

\ sqbullet

-CO-Glu;

\ sqbullet

-CO-Gly;

\ sqbullet

-CO-Sar;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH 2 COOH) 2;

\ sqbullet

-SO 3 H; or

\ sqbullet

-PO 3 H;

And any complex of Zn 2+ of the same.

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In one aspect the substituent of the derivative insulin according to the invention is fixed to the group ε-amino of the Lys residue present in the A8 position in the A chain of the progenitor insulin.

In one aspect the substituent of the derivative insulin is fixed to the ε-amino group of the Lys residue present at position A9 in chain A of the progenitor insulin.

In one aspect the substituent of the derivative insulin is fixed to the ε-amino group of the Lys residue present at position A14 in chain A of the progenitor insulin.

In one aspect the substituent of the derivative insulin is fixed to the ε-amino group of the Lys residue present at position A18 in chain A of the progenitor insulin.

In one aspect the substituent of the derivative insulin is fixed to the ε-amino group of the Lys residue present at position A21 in chain A of the progenitor insulin.

In one aspect the substituent of the derivative insulin is fixed to the ε-amino group of the Lys residue present at position A22 in chain A of the progenitor insulin.

In one aspect the substituent of the derivative insulin according to the invention is fixed to the group ε-amino of the Lys residue present in position A23 in the A chain of the progenitor insulin.

In one aspect the substituent of the derivative insulin according to the invention is fixed to the group ε-amino of the Lys residue present in the A24 position in the A chain of the progenitor insulin.

In one aspect the acyl group is connected to a Lysine residue using an amino acid linker. According to this aspect the acyl group is advantageously connected to a residue of lysine by means of a γ-linker α-glutamyl, or by means of a linker β- or α-aspartyl or by means of a linker α-amido- γ-glutamyl, or through a linker α-amido- β-aspartyl.

In one aspect of the invention the substituent The insulin derivative has a general formula:

-W X Y Z

where

W is:

\ sqbullet

a residue of α-amino acid with an acid group carboxylic in the side chain, this residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or

\ sqbullet

a chain composed of two, three or four α-amino acid residues attached each other by means of amide bonds, whose chain, by means of a amide bond, binds to the ε-amino group of a residue of Lys present in the A or B chain of insulin parent, the amino acid residues of W being selected of the amino acid residue group with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least one amino acid residue having a carboxylic acid group in the side chain; or

\ sqbullet

a covalent bond of X to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin;

X is:

\ sqbullet

-CO-;

\ sqbullet

-CH (COOH) C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CONHCH (COOH) (CH 2) 4 NH C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 C O-; or

\ sqbullet

-CON (CH 2 COOH) CH 2 CH 2 C O-

On condition that

a) when W is an amino acid residue or a amino acid residue chain, carbonyl carbon underlined in X forms an amide bond with an amino group in W, or

b) when W is a covalent bond, carbon of the carbonyl underlined in X forms an amide bond with the group - ε of a Lys residue present in the A or B chain of the progenitor insulin;

And it is:

\ sqbullet

- (CH 2) m - where m is an integer in the range of 6 to 32;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of -CH_ {2} groups - sufficient to give a total number of atoms of carbon in the chain in the range of 10 to 32;

\ sqbullet

a hydrocarbon chain formula bivalent - (CH 2) v C 6 H 4 (CH 2) w - where v and w are integers or one of these is zero so that The sum of v and w is in the range of 6 to 30 and Z is:

\ sqbullet

-COOH;

\ sqbullet

-CO-Asp;

\ sqbullet

-CO-Glu;

\ sqbullet

-CO-Gly;

\ sqbullet

-Sew;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH2) COOH) 2;

\ sqbullet

-SO 3 H; or

\ sqbullet

-PO 3 H;

And any complex of Zn 2+ of the same.

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In one aspect of the invention, the side chain -W-X-Y-Z is fixed to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin. In a more specific aspect of this aspect, the side chain -W-X-Y-Z is fixed to ε-amino group of a Lys residue present in position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23; A24 or B1, B2, B3, B4; B20; B21 or B22.

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The substructure W of the substituent -W-X-Y-Z can be a covalent bond Alternatively, W may be a residue of a amino acid with a carboxylic acid group in the side chain, this residue forms, with one of its carboxylic acid groups, a amide group with the ε-amino group of a residue of Lys present in the A or B chain of insulin parent and where said residue comprises a total of 4 to 10 carbon atoms Specifically, W can, for example, be a linker

In one aspect W can be connected to the group ε-amino of the Lys residue in the chain A or B of the progenitor insulin by means of a linker urea.

In one aspect the linker comprises 1-4 amino acid residues linked together by means of amide bonds of which at least one has a group of free carboxylic acid or a group that is negatively charged at pH neutral.

In one aspect the linker is a residue of amino acid, a peptide chain of 2-4 residues of amino acids or has the motive is α-Asp, β-Asp, α-Glu, γ-Glu, α-hGlu and δ-hGlu; -N (CH 2 COOH) CH 2 CO-; -N (CH 2 CH 2 COOH) CH 2 CH 2 CO-; -N (CH 2 COOH) CH 2 CH 2 CO- or -N (CH 2 CH 2 COOH) CH 2 CO-

The bonds are typically a residue of amino acid or an amino acid residue chain comprising Up to four amino acids Specifically, the linker can be selected from the group consisting of α-Asp;  β-Asp; α-Glu; γ-Glu; α-hGlu; δ-hGlu; -N (CH 2 COOH) CH 2 CO-, -N (CH 2 CH 2 COOH) CH 2 CH 2 CO-; -N (CH 2 COOH) CH 2 CH 2 CO- or -N (CH 2 CH 2 COOH) CH 2 CO-.

In another aspect the linker can be a chain composed of two amino acid residues one of which it has 4 to 10 carbon atoms and a carboxylic acid group in the side chain while the other has 2 to 11 atoms of carbon but no free carboxylic acid group. The residue amino acid without free carboxylic acid group may be a neutral α-amino acid residue. Examples of such links are:? -Asp-Gly; Gly-? -Asp; β-Asp-Gly; Gly-? -Asp; α-Glu-Gly; Gly-?-Glu; γ-Glu-Gly; Gly- γ-Glu; α-hGlu-Gly; Gly-? -HGlu; δ-hGlu-Gly; Y Gly- \ delta-hGlu.

In another aspect the linker is a string composed of two amino acid residues, regardless of they have 4 to 10 carbon atoms, and both with an acid group carboxylic on the side chain. One of this waste from amino acid or both can be a residue of α-amino acid. Examples of such linkers They are: α-Asp-a-Asp; α-Asp-a-Glu; α-Asp-? -hGlu; α-Asp-? -Asp; α-Asp-?-Glu; α-Asp- δ-hGlu; β-Asp-? -Asp; β-Asp-?-Glu; β-Asp-? -hGlu; β-Asp-? -Asp; β-Asp-?-Glu; β-Asp- δ-hGlu; α-Glu-? -Asp; α-Glu-?-Glu; α-Glu-? -hGlu; α-Glu-? -Asp; α-Glu-γ-Glu; α-Glu- δ-hGlu; γ-Glu-? -Asp; γ-Glu-?-Glu; γ-Glu-? -hGlu; γ-Glu-? -Asp; γ-Glu- γ-Glu; γ-Glu- δ-hGlu; α-hGlu-? -Asp; α-hGlu-?-Glu; α-hGlu-? -hGlu; α-hGlu-? -Asp; α-hGlu-γ-Glu; α-hGlu-δ-hGlu; δ-hGlu-? -Asp; δ-hGlu-?-Glu; δ-hGlu-? -hGlu; δ-hGlu-? -Asp; δ-hGlu-γ-Glu; Y δ-hGlu- δ-hGlu.

In another aspect the linker is a string composed of three amino acid residues, having independently of 4 to 10 carbon atoms, the residues of chain amino acid that are selected from the residue group with a neutral side chain and residues with an acid group carboxylic on the side chain so that the chain has the minus a residue having a carboxylic acid group in the side chain In one aspect, amino acid residues are α-amino acid residues.

In another aspect, the linker is a string composed of four amino acid residues, having independently of 4 to 10 carbon atoms, the residues of chain amino acids that are selected from the group with a Neutral side chain and residues with a carboxylic acid group in the side chain so that the chain has at least one residue having a carboxylic acid group in the chain side. In one aspect, amino acid residues are residues of α-amino acids.

The substructure X of the side chain -WXYZ can be a group of the formula -C O- which, by means of a carbon bond of the carbonyl underlined in X, forms an amide bond with an amino group in W or, when W is a covalent bond forms a bond with the α-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the substructure X of the side chain can be a group of the formula -COCH (COOH) C O- which, by means of a carbon bond of the carbonyl underlined in X, forms an amide bond with an amino group in W or, when W is a covalent bond, it forms a bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the X infrastructure side chain can be a group of formula -CON (CM_ {2} COOH) CH {2} C O- that, via a carbon of the carbonyl stressed X forms an amide bond with an amino group in W or, when W is a covalent bond, forms a bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the substructure X of the side chain may be a group of the formula -CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O - which, by means of a carbon bond of the carbonyl underlined in X, forms an amide bond with an amino group in W or, when W is a covalent bond, forms a bond with the ε-amino group of a residue of Lys present in the A chain or B of the progenitor insulin.

In another aspect, the substructure X of the side chain may be a group of the formula -CON (CH 2 CH 2 COOH) CH 2 CH 2 C O - which, by means of a link carbonyl carbon underlined in X, forms an amide bond with an amino group in W or, when W is a covalent bond, forms a bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the substructure X of the side chain may be a group of the formula -CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH ) CH 2 CH 2 C O- which, by means of a carbon bond of the carbonyl underlined in X, forms an amide bond with an amino group in W or, when W is a covalent bond, forms a bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the X infrastructure of the side chain can be a group of the formula -CON (CH2CH2COOH) CH2CO- which, by means of a carbonyl carbon bond underlined in X, forms a amide bond with an amino group in W or, when W is a bond covalent, form a link with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin.

In another aspect, the substructure X of the side chain can be a group of the formula -CON (CH 2} {COOH) CH 2} {2} C {CH O-, through a carbon bond carbonyl underlined at X, it forms an amide bond with an amino group in W or, when W is a covalent bond, it forms a bond with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

The substructure Y of the side chain -W-X-Y-Z can be a group of the formula - (CH 2) m - where m is a integer in the range of 6 to 32, 8 to 20, or 12 to 20, or m It is 11, 12, 13, 14, 15 or 16.

In another aspect, Y is a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of groups -CH_ {2} - enough to give a total number of atoms of carbon in the chain in the range of 6 to 32, 10 to 32, or 12 to twenty.

In one aspect, Y is a hydrocarbon chain formula bivalent - (CH 2) v C 6 H 4 (CH 2) w -  where v and w are integers or one of these is zero so that the sum of v and w is in the range of 6 to 30 or 10 to 20.

In one aspect, the substructure Z of the chain side -W-X-Y-Z is COOH provided that when W is a covalent bond and X is -CO-, then Z is different from COOH.

In one aspect, Z is co-Asp.

In one aspect, Z is co-Glu.

In one aspect, Z is co-Gly.

In one aspect, Z is Co-Ser.

In one aspect, Z is -CH (COOH) 2.

In one aspect, Z is -N (CH 2 COOH) 2.

In one aspect, Z is -SO 3 H.

In one aspect, Z is -PO_ {H}.

In one aspect of the invention the derivative insulin has a formula:

       \ vskip1.000000 \ baselineskip
    

3

       \ vskip1.000000 \ baselineskip
    

where Ins is a fraction of progenitor insulin and -C (O) -X_ {1} -W- [CH 2] m -X-Y-Q-Z is a substituent and where the SNI is fixed to the substituent by of an amide link between a group ε-amino of a Lys residue present in the A or B chain of Ins and a group CO- linked to X_ {1}, W, [CH 2] m, Y or Q in the substituent;

X_ {1} is

\ sqbullet

- (CH 2) n where N is 1, 2, 3, 4, 5 or 6;

\ sqbullet

NR, where R is hydrogen or - (CH 2) p COOH; - (CH 2) p -SO 3 H; - (CH 2) p -PO 3 H 2 - (CH 2) p -O-SO 3 H 2;  - (CH 2) p -O-PO 3 H 2; a aryl group substituted with 1 or 2 groups - (CH 2) p O-COOH; - (CH2) p -tetrazol-5-yl, where p is an integer in the range of 1 to 6;

\ sqbullet

- (CR 1 R 2) q -NR-CO-, where R1 and R2 can be H, COOH, or OH, Q is 1-6 and R is defined as above;

\ sqbullet

- ((CR 3 R 4) q1 -NR-CO) 2-4 -, where R 3 and R 4 can be H, COOH, or OH, q1 is 1-6 and R is defined as above; or

\ sqbullet

a link

W is arylene or heteroarylene, which can be replace with one or two groups selected from the group consisting in COOH, -SO 3 H, and -PO 3 H 2 and tetrazol-5-yl, or W is a bond;

m is 0, 1, 2, 3, 4, 5 or 6;

X is

\ sqbullet

-OR-;

\ sqbullet

4

where R is defined as above; or

\ sqbullet

a link;

And it is

\ sqbullet

- (CR 1 R 2) q -NR-CO-, where R1 and R2 can be H, -COOH, a bond or OH, which is 1-6 and R is defined as above;

\ sqbullet

NR where R is defined as higher;

\ sqbullet

- ((CR 3 R 4) q1 -NR-CO) 2-4 -, where R 3 and R 4 can be H, -COOH, or OH, q 1 is 1-6 and R is defined as above; or

\ sqbullet

a link;

What is it

\ sqbullet

- (CH 2) r - where r is an integer from 4 to 22;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of -CH_ {2} groups - sufficient to give a total number of atoms of carbon in the chain in the range of 4 to 22 o

\ sqbullet

a bivalent chain of the formula

- (CH 2) s -Q 1 - (C 6 H 4) v 1 -Q 2 - (CH 2) w -Q_ { 3} - (C 6 H 4) v2 -Q 4 - (CH 2) t -Q 5 - (C 6 H 4) v 3 -Q 6 - (CH 2) _ { z} -

where Q_ {1} -Q_ {6} one independently of the other they can be O; S or a link; where s, w, t and z one independently of the other they are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range of 4 to 22, and v_ {1}, v_ {2}, and v_ {3} one independently of the other can be zero or 1, with the proviso that Q_ {1}, Q_ {2}, Q_ {3}, Q_ {4}, Q_ {5} and Q_ {6} may not form links to each other and if s, w, t and z are zero or 1, so no -CH_ {2} - can join 2 of the following atoms: O, S; Y

Z is:

\ sqbullet

-COOH;

\ sqbullet

-CO-Asp;

\ sqbullet

-CO-Glu;

\ sqbullet

-CO-Gly;

\ sqbullet

-Sew;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH 2 COOH) 2;

\ sqbullet

-SO 3 H

\ sqbullet

-PO 3 H 2;

\ sqbullet

O-SO_ {3} H;

\ sqbullet

O-PO_ {3} H2;

\ sqbullet

-tetrazol-5-il or

\ sqbullet

-O-W_ {1},

where W1 is arylene or heteroarylene substituted with one or two groups selected from COOH, -SO3H, and -PO_ {H} {2} and tetrazol-5-yl;

and any Zn 2+ complex of same.

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In one aspect of the invention X_ {1} may be - (CH 2) n where n is 1, 2, 3, 4, 5 or 6.

In one aspect of the invention X, it can be NR, where R is hydrogen or - (CH 2) p COOH; - (CH 2) p -SO 3 H;
- (CH 2) p -PO 3 H 2 - (CH 2) p -O-SO 3 H 2; - (CH 2) p -O-PO 3 H 2; an aryl group substituted with 1 or 2 groups - (CH 2) p O-COOH; - (CH2) p -tetrazol-5-yl, where p is an integer in the range of 1 to 6.

In one aspect X 1 may be - (CR 1 R 2) q -NR-CO-. For example, X1 can be - (CH2) - (CHCOOH) -NH-CO- or
- (CH 2) 2 - (CHCOOH) -NH-CO-.

In an aspect X_ {1} it can be - ((CR 3 R 4) q1 -NR-CO) 2-4 - or X_ {1} can be a link.

In one aspect W can be phenylene or W can be a 5-7 membered heterocyclic ring system comprising nitrogen, oxygen or sulfur. When W is a ring 5-membered heterocyclic it may comprise at least one oxygen.

In one aspect W is arylene, which can be replace with -COOH. In one aspect W is a link.

In an aspect X is O,

5

where R can be hydrogen or - (CH 2) p COOH. In one aspect p is 1 or 2.

       \ vskip1.000000 \ baselineskip
    

In an aspect X is a link.

In one aspect Y is - (CR 1 R 2) q -NR-CO-, where q is 1 and R1 and R2 are hydrogen. For example And you can be -CH 2 -NH-CO- or - (CH 2) 3 - (CHCOOH) -NH-CO-.

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In one aspect Y is (CR 3 R 4) q1 NR-CO where R 3 and R 4 can be H, -COOH, or OH, q 1 is 1-6 and R is defined as above.

In one aspect Y is - ((CR 3 R 4) q1 -NR-CO) 2-4 -, where R 3 and R 4 can be H, -COOH, or OH, q 1 is 1-6 and R is defined as above. In an aspect Y It is a link.

In one aspect q is - (CH 2) r - where R is an integer from 4 to 22, from 8 to 20 or from 10 to 18. By Example R can be 12, 13, 14, 15, 16, 17 or 18.

In a Q aspect it is a bivalent chain comprising 1, 2 or 3 groups -CH = CH- and a number of groups -CH_ {2} -. Enough to give a total number of carbon atoms in the chain in the range of 4 to 22.

In one aspect Q is a bivalent chain of the formula:

- (CH 2) s -Q 1 - (C 6 H 4) v 1 -Q 2 - (CH 2) w -Q_ { 3} - (C 6 H 4) v 2 -Q 4 - (CH 2) t -Q 5 - (C 6 H 4) _ {v3} -Q_ {6} - (CH2) z -

where Q_ {1} -Q_ {6} one independently of the other they can be O; S or a link; where s, w, t and z one independently of the other they are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range of 4 to 22, and v_ {1}, v_ {2}, and v_ {3} one independently of the other can be zero or one.

       \ vskip1.000000 \ baselineskip
    

In one aspect s is 1 or 2, 9, 10 or 11. In one aspect v_ {1} is 1 or 2. In an aspect Q_ {1}, Q_ {2}, Q_ {5} and Q_ {6} are all a link. In one aspect Q_ {1} is S or O. In a aspect W is 2. In one v_ {2} is 1 and t is 1.

In one aspect Z is COOH, -CO-Asp, -CO-Glu, -CO-Gly, -CO-Ser, -CH (COOH) 2, -N (CH 2 COOH) 2 , -SO 3 H,
-PO 3 H 2, -O-SO 3 H, -O-PO H 2 or -tetrazol-5-yl.

In one aspect Z is -O-W_ {1}, where W1 is arylene or heteroarylene substituted with one or two groups selected from -COOH, -SO 3 H, and -PO 3 H 2 and tetrazol-5-yl.

The insulin fraction - in this text also referred to as the derivative progenitor insulin insulin according to the invention may be an insulin analog, which it may contain only a lysine residue, which is not present in position B29 of the B chain of the insulin analog. This residue lysine may be in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 in chain A of the progenitor insulin or a group ε-amino of a Lys residue in the chain B in position B1, B2, B3, B4; B20; B21 or B22 in the chain B of the progenitor insulin.

The progenitor insulin can for example be human insulin or porcine insulin, where the residue of Lys in position B29 is replaced and a residue of Lys has been inserted into a position in chain A or chain B, whose position is not B29 of the chain B.

In one aspect the amino acid residue in one of positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 of the progenitor insulin chain A is a residue of Lys.

In one aspect the amino acid residue in one of positions B1, B2, B3, B4; B20; B21 or B22 of the B chain of the Progenitor insulin is a residue of Lys.

In a group of progenitor insulin analogs, The amino acid residue at position B29 is Arg, Pro or Thr.

In a group of progenitor insulin analogs, the amino acid residue at position B1 and / or B30 has been Deleted

In a group of insulin analogues parent the amino acid residue at position B29 is Arg, Pro or Thr and the amino acid residue in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23; A24, B1, B2, B3, B4; B20; B21 or B22 of the A or B chain is Lys. A specific example of This group is the human insulin LysA17 ArgB29.

In a group of insulin analogues parent, amino acid residues in position B30 have been deleted and the amino acid residue in position B29 it can be any encodable amino acid except Lys and the residue of amino acid in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21 A22; A23; A24, B1, B2, B3, B4; B20; B21 or B22 of the string A or B is Lys, provided that when Lys is in position B3, so B29 is not Glu. A specific example of this group of progenitor insulin analogs is human insulin LysA12 HisB29 desB30.

In a group of progenitor insulin analogs, amino acid residues at position B29 and B30 have been deleted and the amino acid residue in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23; A24, B1, B2, B3, B4; B20; B21 or B22 of the A or B chain is Lys. A specific example of this group of progenitor insulin analogs is human insulin LysA8 desB29 desB30.

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In a group of progenitor insulin analogs, the amino acid residue at position B26; B27; B28; B29 and B30 it can be any encodable amino acid except Lys or a deletion and the amino acid in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23; A24, B1, B2, B3, B4; B20; B21 or B22 of the string A or B is Lys, provided that when Lys is in position B3, then B29 is not Glu. A specific example of this group of progenitor insulin analogs is human insulin LysB4 desB26 desB27 desB28 desB29 desB30.

In a group of progenitor insulin analogs, chain A has been extended in the C-term with one, two or three amino acid residues, the positions of the extended amino acid residues being in position A22; A23 or A24. Lys substitutes one of the amino acids at position A22; A23 or A24 of chain A. Either one or two positions of amino acid remaining in the extension can be any residue of encodable amino acid except Lys. A specific example of this group of progenitor insulin analogs is human insulin LysA22 ArgB29 desB30 or human insulin GlyA22 LysA23 ArgB29 desB30.

In one aspect of the invention the residue of amino acid at position A21 of the progenitor insulin is Gly or Asn. The amino acid residue in insulin position A21 parent should be Gly, Ala or Gln when the residue of amino acid at position A23 or A24 of the progenitor insulin is Lys.

In one aspect the amino acid residue in the B3 position of the progenitor insulin is Lys or the residue of amino acid at position B28 of the progenitor insulin is Asp.

Examples of progenitor insulin analogs are human insulin ArgB29 or human insulin ArgB29desB30.

In another aspect, the insulin derivative is selected from the group consisting of human insulin N ^ {A8} -miristil LysA8 ArgB29 desB30, human insulin N ^ {A9} -miristil LysA9 ArgB29 desB30, human insulin N ^ {\ varepsilon A10} -miristil LysA10 ArgB29 desB30, human insulin  N ^ {A12} -miristil LysA12 ArgB29 desB30, human insulin N ^? A14} -miristil LysA14 ArgB29 desB30, insulin human N ^ {\ varepsilon A15} -miristil LysA15 ArgB29 desB30, human insulin N ^? A17} -miristil LysA17 ArgB29 desB30, insulin human N ^ {A18} -miristil LysA18 ArgB29 desB30, human insulin N ^? A21} -miristil LysA21 ArgB29 desB30, human insulin N ^ {A22} -miristil LysA22 ArgB29 desB30, human insulin N ^? B1} -myristil LysB1 ArgB29 desB30, human insulin N ^ {\ varepsilon B2} -miristil LysB2 ArgB29 desB30, human insulin N <3> -myristil LysB3 ArgB29 desB30, human insulin N ^ {\ varepsilon B4} -miristil LysB4 ArgB29 desB30, human insulin N ^ {\ vrepsilon B20} -miristil LysB20 ArgB29 desB30, human insulin N ^? B21} -miristil LysB21 ArgB29 desB30 and insulin human N ^ {\ varepsilon B22} -miristil LysB22 ArgB29 desB30.

In another aspect, the insulin derivative is selected from the group consisting of human insulin N ^ {\ varepsilon A8} -? -Carboxypentadecanoyl-? -Glu LysA8 ArgB29 desB30, human insulin N ^? A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30, human insulin N ^? A10} -? -Carboxypentadecanoyl-? -Glu  LysA10 ArgB29 desB30, human insulin N ^? A12} -? -Carboxypentadecanoyl-?-Glu LysA12 ArgB29 desB30, human insulin N ^? A14} -? -Carboxypentadecanoyl-? -Glu LysA14 ArgB29 desB30, human insulin N ^? A15} -? -Carboxypentadecanoyl-?-Glu LysA15 ArgB29 desB30, human insulin N ^? A17} -? -Carboxypentadecanoyl-?-Glu LysA17 ArgB29 desB30, human insulin N ^? A18} -? -Carboxypentadecanoyl-?-Glu LysA18 ArgB29 desB30, human insulin N ^? A21} -? -Carboxypentadecanoyl-? -Glu LysA21 ArgB29 desB30, human insulin N ^? A22} -? -Carboxypentadecanoyl-? -Glu  LysA22 ArgB29 desB30, human insulin N ^ {\ varepsilon B1} -? -Carboxypentadecanoyl-? -Glu LysB1 ArgB29 desB30, human insulin N ^? B2} -? -Carboxypentadecanoyl-? -Glu LysB2 ArgB29 desB30, human insulin N ^? B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30, human insulin N ^? B4} -? -Carboxypentadecanoyl-? -Glu LysB4 ArgB29 desB30, human insulin N ^? B20} -? -Carboxypentadecanoyl-? -Glu LysB20 ArgB29 desB30, human insulin N ^? B21} -? -Carboxypentadecanoyl-? -Glu LysB21 ArgB29 desB30 and human insulin N ^ {\ varepsilon B22} -? -Carboxypentadecanoyl-? -Glu  LysB22 ArgB29 desB30.

In another aspect the insulin derivative is selected from the group consisting of human insulin N ^ {\ Arepsilon A22} - \ omega-carboxypentadecanoyl- \ gamma-Glu LysA22 GlyA21 ArgB29 desB30, human insulin N ^ {\ arepsilon A22} - \ omega- Carboxypentadecanoyl- γ-Glu LysA22 AlaA21 ArgB29 desB30, human insulin N ^ {\ varepsilon A22} - \ omega-carboxypentadecanoyl- \ gamma-Glu LysA22 GlnA21 ArgB29 desB30, human insulin N ^ \ \ ε-carboxy-ε-carboxy γ-Glu LysA23 GlyA21 GlyA22 ArgB29 desB30, human insulin N ^ {\ re A23} - \ omega-carboxypentadecanoyl- γ-Glu LysA23 AlaA21 GlyA22 ArgB29 desB30, human insulin N ^ \ ε-carbox-ε-carboxy γ-Glu LysA23 GlnA21 GlyA22 ArgB29 desB30, human insulin N ^ {\ re A24} - \ omega-carboxypentadecanoyl- γ-Glu LysA24 GlyA21 GlyA22 GlyA23 ArgB29 desB30, human insulin N ^ {\ ε-omega-carbox -24 - γ-Glu LysA24 AlaA21 GlyA22 GlyA23 ArgB29 desB30, human insulin N 24 A24 - \ omega-carboxypentadecanoyl- \ gamma-Glu LysA24 GlnA21 GlyA22 GlyA23 ArgB29 desB30, human insulin N ^ {\ varepsilon A22} - \ omega-carboxiheptadecanoyl- \ gamma-Glu LysA22 ArgB29 desB30, human insulin N23 \ a - omega -23 -carboxyheptadecanoyl- γ-Glu LysA23 GlyA22 ArgB29 desB30, human insulin N ^ {\ varepsilon A23} - \ omega-carboxyheptadecanoyl- \ gamma-Glu LysA23 GlyA21 GlyA22 ArgB29 desB30, human insulin N23 carboxyheptadecanoyl- γ-Glu LysA23 AlaA21 GlyA22 ArgB29 desB30, human insulin N ^ {\ re A23} - ome omega-carboxyheptadecanoyl- γ-Glu LysA23 GlnA21 GlyA22 ArgB29 desB30, human insulin N ^ {a - ga-24 - 24 carboxyheptadecanoyl- \ gamma-Glu LysA24 GlyA21 GlyA22 GlyA23 ArgB29 desB30, human insulin N ^ {\ varepsilon A24} - \ omega-carboxiheptadecanoyl- \ gamma-Glu LysA24 AlaA21 GIyA22 GlyA23 ArgB29
desB30 and human insulin N ^? A24 -? - carboxyheptadecanoyl-? - Glu LysA24 GlnA21 GlyA22 GlyA23ArgB29 desB30.

Insulin derivatives according to the invention are they can provide in the form of essentially free compounds of zinc or in the form of zinc complexes. When complexes are provided zinc of an insulin derivative according to the invention, approximately two zinc ions, approximately three zinc ions or approximately four zinc ions or even up to 12 ions of zinc can be attached to 6 derivative molecules (hexamer) of insulin. Solutions of zinc complexes of derivatives insulin will contain mixtures of such species.

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The zinc content in a composition Pharmaceutical can be up to about 12 zinc ions for 6 molecules of insulin derivative. The upper limit for the zinc content is the zinc content that would cause the precipitation of insulin and that turns the solution into a suspension.

In one aspect of the invention the composition pharmaceutical comprises between approximately 4.3 and approximately 12 zinc ions per 6 insulin derivative molecules or between about 4.5 and about 12 zinc ions by 6 insulin derivative molecules. In another aspect of the invention the pharmaceutical composition comprises between about 5 and about 11.4 zinc ions per 6 insulin derivative molecules or between about 5.5 and about 10 zinc ions by 6 insulin derivative molecules.

The invention further comprises a method for the production of a pharmaceutical composition comprising a insulin derivative where more than about 4 zinc ions per 6 molecules of insulin derivative are added to the composition.

In one aspect of the invention the method comprises the addition of up to about 12 zinc ions per 6 molecules of insulin derivative to the composition.

In one aspect of the invention the method comprises the addition of between about 4.3 and about 12 zinc ions per 6 insulin derivative molecules to the composition.

In another aspect of the invention between about 4.5 and about 12 zinc ions by 6 insulin derivative molecules are added to the composition, by example about 5 and about 11.4 zinc ions per 6 insulin derivative molecules are added to the composition or between about 5.5 and about 10 zinc ions by 6 insulin derivative molecules are added to the composition.

An aspect of the invention concerns a method for the production of a pharmaceutical composition. The method comprises the addition of zinc to the composition before the addition of a preservative In one aspect of the invention between approximately 4.5 and approximately 12 zinc ions per 6 derivative molecules insulin are added to the composition before the addition of a preservative or for example about 5 and about 11.4 Zinc ions per 6 molecules of insulin derivative are added to the composition before the addition of a preservative or for example between about 5.5 and about 10 zinc ions by 6 insulin derivative molecules are added to the composition before of the addition of a preservative.

In one aspect of the invention the method comprises the addition of up to about 12 zinc ions to the composition after the addition of a preservative.

In one aspect of the invention at least 0.5 ions of zinc per 6 molecules of insulin derivative is added to the composition after the addition of a preservative or at least 1 ion of zinc per 6 molecules of insulin derivative is added to the composition after the addition of a preservative.

In another aspect of the invention more than approximately 2, 3, 4, 5, or 6 zinc ions per 6 molecules of insulin derivative are added to the composition after the addition of a preservative

In another aspect of the invention between about 4.5 and about 12 zinc ions by 6 insulin derivative molecules are added to the composition after of the addition of a preservative or between approximately 5.5 and approximately 10 zinc ions per 6 derivative molecules insulin are added to the composition after the addition of a preservative

In one aspect of the invention the method comprises the addition of part of the zinc before the addition of a preservative and part of the zinc after the addition of a preservative

In one aspect the method comprises the addition of at least 1 zinc ion per 6 insulin derivative molecules before of the addition of a preservative and the addition of at least 1 ion of zinc for 6 molecules of insulin derivative after addition of a preservative

In another aspect of the invention the method comprises the addition of at least 1, 2, 3, 4, 5 or 6 zinc ions for 6 molecules of insulin derivative before the addition of a preservative and the addition of at least 2, 3, 4, 5 or 6 zinc ions per 6 insulin derivative molecules after the addition of a preservative

In one aspect of the invention the number of ions zinc added before the addition of a preservative is at least 3 zinc ions per 6 insulin derivative molecules and the number of zinc ions added after the addition of a preservative they are at least 3 zinc ions per 6 derivative molecules insulin

In one aspect of the invention the preservative added is phenol and / or m-cresol.

In another aspect the invention relates to a pharmaceutical composition comprising a therapeutically amount  Effective of an insulin derivative or a zinc complex of the derivative insulin according to the invention together with a carrier Pharmaceutically acceptable can be provided for the treatment of type 1 diabetes, type 2 diabetes and others states that cause hyperglycemia in patients in need of such treatment.

       \ global \ parskip1.000000 \ baselineskip
    

An insulin derivative or a composition pharmaceutical comprising the insulin derivative or a complex of zinc of the insulin derivative according to the invention can be used for the production of a pharmaceutical composition for use in the treatment of type 1 diabetes, type 2 diabetes and others states that cause hyperglycemia.

In another aspect of the invention, it is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other conditions that cause hyperglycemia in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention in mixture with an insulin or an insulin analogue that has a rapid onset of action, along with acceptable carriers Pharmaceutically and additives.

In one aspect the invention provides a pharmaceutical composition being a mixture of a derivative insulin or a zinc complex of the insulin derivative according to the invention and human insulin or a group selected from the analog of rapid-acting insulin consisting of human insulin AspB28; LysB28ProB29 human insulin and LysB3GluB29 human insulin.

In another aspect the invention relates to a pulmonary request to treat type 1 diabetes, type diabetes 2 and other states that cause hyperglycemia in a patient in need for such treatment, comprising an amount therapeutically effective of an insulin derivative or a complex of zinc of the insulin derivative according to the invention optionally in mixture with an insulin or an insulin analogue that has a rapid onset of action, along with carriers and additives pharmaceutically acceptable.

The insulin derivative according to the invention and the fast-acting insulin analog can be mixed in a proportion of approximately 90/10%; approximately 70/30% or approximately 50/50%.

In another aspect of the invention, it is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to the invention together with a pharmaceutically acceptable carrier and Acceptable pharmaceutical additives.

In another aspect of the invention, it is provided a method for the production of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia, the composition comprising the insulin derivative according to the invention together with a pharmaceutically acceptable carrier and pharmaceutical additives acceptable.

In another aspect of the invention, it is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia in a patient in need of such treatment, including administration to the patient of a therapeutically effective amount of an insulin derivative according to the invention in admixture with an insulin or an insulin analog which has a rapid onset of action, along with a carrier Pharmaceutically acceptable and acceptable pharmaceutical additives.

In another aspect of the invention, it is provided a method for the production of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia, the composition comprising an insulin derivative according to the invention in admixture with an insulin or an insulin analogue that has an appearance fast action, along with pharmaceutical additives and carriers Acceptable pharmacists

In another aspect, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention that is soluble at physiological pH values.

In another aspect, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention that is soluble at pH values in the range of about 6.5 to about 8.5.

In another aspect, the invention relates to a pharmaceutical composition with a prolonged action profile that It comprises an insulin derivative according to the invention.

In another aspect, the invention relates to a pharmaceutical composition which is a solution containing about 120 nmol / ml to about 2400 nmol / ml, of about 400 nmol / ml to about 2400 nmol / ml, of approximately 400 nmol / ml to approximately 1200 nmol / ml, of about 600 nmol / ml to about 2400 nmol / ml, or of approximately 600 nmol / ml to approximately 1200 nmol / ml of a insulin derivative according to the invention or a mixture of the derivative insulin according to the invention with an insulin analogue of action fast

The initial product for acylation, the insulin progenitor or insulin analog or a precursor of they can be produced either by well known peptide synthesis or by well-known recombinant production in microorganisms suitable transformed. Thus the insulin start product is can produce by a method comprising the culture a cell host containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a nutrient medium suitable under conditions allowing peptide expression, after which the resulting peptide is recovered from the culture.

The medium used to grow the cells can be any conventional means suitable for growing host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to recipes published (eg in catalogs of the American Collection of Type crops). The peptide produced by the cells can then be recovered from the culture medium by procedures conventional including the separation of host cells of the medium by centrifugation or filtration, precipitation of the protein components of the supernatant or filtered by a salt, for example ammonium sulfate, purification by a variety of chromatographic procedures, for example chromatography of ion exchange, gel filtration chromatography, affinity chromatography, or similar, depending on the type of peptide in question.

The DNA sequence encoding the progenitor insulin may suitably be of genomic or cDNA origin, for example obtained by preparing a genomic or cDNA library and selecting DNA sequences encoding all or part of the polypeptide by hybridization using synthetic oligonucleotide probes according to techniques. standard (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the progenitor insulin can also be synthetically prepared by established standard methods, for example the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al. ., EMBO Journal 3 (1984), 801-805. The DNA sequence can also be prepared by polymerase chain reaction using specific primers, for example as described in US 4,683,202 or Saiki et al ., Science 239 (1988), 487-491.

The DNA sequence can be inserted in any vector that can conveniently be subjected to recombinant DNA procedures, and vector choice frequently it will depend on the host cell in which it should be inserted. Thus, the vector can be a replication vector. autonomous, that is a vector that exists as an entity extrachromosomal, whose replication is independent of the chromosomal replication, for example a plasmid. Alternatively, the vector can be one that, when introduced into a cell host, integrates into the genome of the host cell and replicates with the chromosome (s) in which it has been (n) integrated (s).

The vector is for example an expression vector in which the DNA sequence encoding the progenitor insulin is operatively linked to additional segments required for DNA transcription, such as a promoter. The promoter can be any DNA sequence that shows transcriptional activity in the host cell of choice and can be derived from genes that encode either heterologous or homologous proteins to the host cell. Examples of promoters suitable for directing the transcription of DNA encoding progenitor insulin in a variety of host cells are well known in the art, see for example Sambrook et al ., Supra .

The DNA sequence encoding insulin parent may also, if necessary, be operatively connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and sequences translational enhancers. The recombinant vector of the invention may also comprise a DNA sequence that allows the vector replicates in the host cell in question.

The vector may also comprise a marker selectable, for example a gene whose product complements a defect in the host cell or one that confers resistance to a medicine, for example ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide of the present invention in the secretory pathway of host cells, a signal sequence secretory (also known as a leader sequence, sequence prepro or pre sequence) can be provided in the vector recombinant The secretory signal sequence joins the sequence of DNA encoding the peptide in the correct reading frame. Secretory signal sequences are commonly located at 5 'to the DNA sequence encoding the peptide. Signal sequence secretory can be that normally associated with the peptide or can be from a gene that encodes another secreted protein.

The methods used to link the DNA sequences encoding the progenitor insulin, the promoter and optionally the terminator and / or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to people. skilled in the art (cf., for example, Sambrook et al ., supra ).

The host cell in which the DNA sequence or the recombinant vector is introduced can be any cell that is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli , Saccharomyces cerevisiae , or mammalian BHK or CHO cell lines.

The progenitor insulin molecule is then converted into the insulin derivatives of the invention by introduction of the relevant side chain in either position B1 or in the position of Lys chosen in chain B. The side chain It can be introduced by any convenient method and many methods are described in the prior art for acylation of a amino group. More details will appear from the following examples.

Pharmaceutical compositions

The insulin derivatives of this invention of the claimed formula can, for example, be administered subcutaneously, orally, or pulmonary.

For subcutaneous administration, the compounds of the formula are formulated analogously with the formulation  of known insulins. In addition, for subcutaneous administration, the compounds of the formula are administered analogously with the administration of known insulins and, generally, doctors They are familiar with this procedure.

The insulin derivatives of this invention are can be administered by inhalation in an effective dose manner to increase circulating insulin levels and / or to lower circulating glucose levels Such administration can be effective. to treat disorders such as diabetes or hyperglycemia. The achievement of effective doses of insulin requires the administration of a inhaled dose of insulin derivative of this invention greater than about 0.5 µg / kg to about 50 µg / kg. A therapeutically effective amount can be determined by a expert professional, which will take into account factors including the insulin level, blood glucose levels, the condition patient's physics, the patient's lung status, or Similar.

According to the invention, the insulin derivative of this invention can be delivered by inhalation to achieve a rapid absorption of it. Administration by inhalation may result in pharmacokinetics comparable to administration subcutaneous insulins. Inhalation of an insulin derivative of this invention leads to a rapid rise in insulin level circulating followed by a rapid drop in glucose levels in blood. Different inhalation devices typically provide similar pharmacokinetics when particle sizes similar and similar levels of lung deposition are compared.

According to the invention, an insulin derivative of This invention can be delivered by any variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These Devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Derivative Insulin of this invention is delivered by a powder inhaler Dry or a sprayer. There are different desirable characteristics of an inhalation device to administer a derivative insulin of this invention. For example, delivery by inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver particles small, for example, less than about 10 µm, per example about 1-5 µm, for good breathability Some specific examples of devices commercially available inhalation suitable for the practice of This invention is Turbohaler? (Astra); Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros ™ inhaler (Dura), devices marketed by Inhale Therapeutics, AERx? (Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), the Ventolin® metered dose inhaler (Glaxo), Spinhaler® powder inhaler (Fisons), or the like.

As those skilled in the art will recognize, the insulin derivative formulation of this invention, the amount of the formulation delivered, and the duration of administration of a Single dose will depend on the type of inhalation device employee. For some aerosol delivery systems, such as nebulizers, frequency of administration and duration of time for which the system is activated will depend mainly on the concentration of insulin conjugate in the aerosol. For example, shorter administration periods can be used at higher concentrations of insulin conjugate in the solution of nebulizer Devices such as metered dose inhalers they can produce higher aerosol concentrations, and they can act for shorter periods to deliver the amount Desired conjugate insulin. Devices such as inhalers of powder deliver active agent until a given load of agent It is ejected from the device. In this type of inhaler, the amount of insulin derivative of this invention in an amount given of the powder determines the dose delivered in a single administration.

The particle size of the insulin derivative of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to do so in the lungs, and in the lower airways or alveoli. The insulin derivative of this invention can be formulated so that at least about 10% of the insulin conjugate delivered is deposited in the lung, for example from about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for humans who breathe through the mouth is obtained with particle sizes of about 2 µm to about 3 µm. When particle sizes are above about 5 µm the lung deposition is substantially reduced. Particle sizes below about 1 µm cause lung deposition to be reduced, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, the insulin derivative particles delivered by inhalation have a particle size of less than about 10 µm, for example in the range of about 1 µm to about 5 µm. The insulin derivative formulation is selected to produce the desired particle size in the inhalation device
chosen one.

Advantageously for the administration as a dry powder, an insulin derivative of this invention is prepared in a grainy form with a particle size less than about 10 µm, for example about 1 a about 5 µm. The particle size is effective for delivery to the alveoli of the patient's lung. The dry powder is largely composed of particles produced so that a Most particles have a size in the desired range. Advantageously, at least about 50% of the dry powder is made of particles with a diameter less than about 10 \ mum. Such formulations can be achieved by drying by atomization, crushing, or condensation of critical point of a solution containing conjugated insulin and other ingredients desired Other methods also suitable for generating particles useful in the present invention are known in the art.

The particles are normally separated from a dry powder formulation in a container and then transported in a patient's lung by means of an air current carrier Typically, in today's dry powder inhalers, the force to disperse the solid is provided only by the patient inhalation In another type of inhaler, the flow of air generated by the patient's inhalation activates a motor propellant that deagglomerates particles.

The insulin derivative formulations of this invention for administration by a powder inhaler dry typically include a finely divided dry powder containing the derivative, but the powder may also include a filler, carrier, excipient, other additive, or the like. Be may include additives in a dry powder formulation of conjugated insulin, for example, to dilute the powder as necessary for delivery from the particular powder inhaler, to facilitate formulation processing, to provide advantageous powder properties to the formulation, for facilitate the dispersion of dust from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide flavor to the formulation, or the like. Advantageously, the additive does not adversely affect the pathways Respiratory of the patient. The insulin derivative can be mixed with an additive at a molecular level or the solid formulation can include particles of conjugated insulin mixed with or coated in particles of the additive. Typical additives include mono-, di-, and polysaccharides; polyols and other polyols, such as, by example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, Trehalose, sucrose, mannitol, starch, or combinations of themselves; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or similar. Typically an additive, such as an agent of stabilization, is present in an amount effective for a purpose previously described, often at about 50% until approximately 90% by weight of the formulation. Additional agents known in the art for the formulation of a protein such as  an insulin-like protein may also be included in the formulation.

A sprayer including derivatives Insulin of this invention can be produced by forcing a suspension or conjugate insulin solution through a nozzle under pressure. The nozzle size and configuration, the pressure applied, and the liquid feed rate can be chosen to achieve the desired yield and particle size. A electrospray can be produced, for example, by a field electrical in connection with a capillary or nozzle feed. Advantageously, insulin conjugate particles delivered by a sprayer has a particle size less than about 10 µm, for example in the range of about 1 µm to about 5 µm.

Formulations of insulin derivatives of this invention suitable for use with a sprayer typically includes the insulin derivative in an aqueous solution at a concentration of about 1 mg to about 20 mg of insulin conjugate per ml of solution. The formulation can include agents such as an excipient, a buffer, an agent of isotonicity, a preservative, a surfactant, and, for example zinc. The formulation may also include an excipient or agent for stabilization of the insulin derivative, such as a buffer, a reducing agent, a mass protein, or a carbohydrate. Protein Bulk useful in the formulation of insulin conjugates include albumin, protamine, or the like. Typical carbohydrates useful in The formulation of insulin conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like. The derived formulation Insulin may also include a surfactant, which can reduce  or prevent induced aggregation on the conjugate surface of insulin caused by atomization of the solution in the formation of a spray Several conventional surfactants can be employees, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. The amounts will generally be between approximately 0.001 and approximately 4% by weight of the formulation.

Pharmaceutical compositions containing a insulin derivative according to the present invention may also be administered parenterally to patients in need of such treatment. Parenteral administration can be performed by intravenous, subcutaneous or intramuscular injection using a syringe, optionally a pen syringe. Alternatively, the parenteral administration can be performed using a pump infusion. Other options are to administer insulin via pulmonary or nasal, for example in compositions, powders or liquids, specifically designed for the purpose.

Injectable derivatives compositions Insulin of the invention can be prepared using the conventional techniques of the pharmaceutical industry that involve dissolve and mix the ingredients as appropriate to give the desired end product. Thus, according to a procedure, a derivative insulin according to the invention is dissolved in an amount of water which is somewhat lower than the final volume of the composition that It must be prepared. An isotonic agent, a preservative and a buffer is added as necessary and the pH value of the solution is adjusted - if necessary - using an acid, for example acid hydrochloric, or a base, for example aqueous sodium hydroxide according to is needed Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

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In another aspect of the invention the buffer is selected from the group consisting of sodium acetate, carbonate sodium, citrate, glycyl glycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, hydrogen phosphate of disodium, sodium phosphate, and tris (hydroxymethyl) -aminomethane, bicin, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative aspect of the invention.

In another aspect of the invention the formulation also comprises a pharmaceutically acceptable preservative that can be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate; 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, acid benzoic, imidurea, chlorhexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium, chlorphenesin chloride, 3- (4-chlorophenoxy) propane-1,2-diol or mixtures thereof. In another aspect of the invention the preservative is present in a concentration of 0.1 mg / ml at 20 mg / ml In another aspect of the invention the preservative is present at a concentration of 0.1 mg / ml to 5 mg / ml. In another aspect of the invention the preservative is present in a concentration of 5 mg / ml to 10 mg / ml. In another aspect of the invention the preservative It is present in a concentration of 10 mg / ml to 20 mg / ml. Each of these specific preservatives constitutes an alternative aspect of the invention. The use of a preservative in compositions Pharmaceuticals is well known to the person skilled in the art. By convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In another aspect of the invention the formulation It also comprises an isotonic agent that can be selected from the group consisting of a salt (eg sodium chloride), a sugar or sugar alcohol, an amino acid (eg L-glycine, L-histidine, arginine, lysine, isoleucine, acid aspartic, tryptophan, threonine), an alditol (eg glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol) polyethylene glycol (eg PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or glucans water soluble, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pululane, dextrin, cyclodextrin, soluble starch, hydroxyethyl Starch and sodium carboxymethyl cellulose can be used. In a The sugar additive aspect is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon that has the less an OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one aspect The sugar alcohol additive is mannitol. Sugars or polyols mentioned above can be used individually or in combination. There is no fixed limit for the amount used, since sugar or sugar alcohol is soluble in the liquid preparation and does not adversely affect the effects stabilizers achieved using the methods of the invention. In One aspect, the concentration of sugar or sugar alcohol is between about 1 mg / ml and about 150 mg / ml. In other aspect of the invention the isotonic agent is present in a concentration of 1 mg / ml to 50 mg / ml. In another aspect of the invention the isotonic agent is present in a concentration of 1 mg / ml to 7 mg / ml. In another aspect of the invention the agent Isotonic is present in a concentration of 8 mg / ml to 24 mg / ml. In another aspect of the invention the isotonic agent is present in a concentration of 25 mg / ml to 50 mg / ml. Each of these agents specific isotonics constitutes an alternative aspect of the invention. The use of an isotonic agent in compositions Pharmaceuticals is well known to those skilled in the art. By convenience refers to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

Typical isotonic agents are sodium chloride, Mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are acetate sodium, glycyl glycine, HEPES (acid from 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid) and sodium phosphate.

A composition for nasal administration of a insulin derivative according to the present invention may, for example, be prepared as described in European patent no. 272097 (of Novo Nordisk A / S).

Compositions containing insulin derivatives of this invention can be used in the treatment of conditions that They are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycemia due to example as sometimes seen in seriously injured people and people who have undergone major surgery. The optimal dose level for any patient it will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body mass, physical activity, and diet of the patient, in a possible combination with other drugs, and the severity of the state that must be treated. It is recommended that the dosage daily insulin derivative of this invention be determined for each individual patient by experts in the art in one way similar as for known insulin compositions.

When appropriate, insulin derivatives of this invention can be used in admixture with other types of insulin, for example insulin analogues with one more occurrence quick action. Examples of such insulin analogs are described for example in European patent applications that have publication numbers EP 214826 (Novo Nordisk A / S), EP 375437 (Novo Nordisk A / S) and EP 383472 (Eli Lilly & Co.).

The invention will be summarized in the following paragraphs:

1. An insulin derivative comprising a progenitor insulin and a substituent, where the substituent is well bound to a ε-amino group of a Lys residue present in the progenitor insulin chain A in position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or a ε-amino group of a residue of Lys in the B chain of the progenitor insulin in position B1, B2, B3, B4; B20; B21 or B22 provided that when B3 is Lys, then B29 is not Glu, where the substituent is of formula general:

-W X Y Z

where

W is:

\ sqbullet

a residue of α-amino acid with an acid group carboxylic in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or

\ sqbullet

a chain composed of two, three or four α-amino acid residues linked together through amide bonds, this chain, through of an amide bond, joins the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin, the residues of W amino acids that are selected from the residue group of amino acids with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least one amino acid residue that has a group of carboxylic acid in the side chain; or

\ sqbullet

a covalent bond of X to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin;

X is:

\ sqbullet

-CO-;

\ sqbullet

-CH (COOH) C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 C O-

\ sqbullet

-CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

CONHCH (COOH) (CH 2) 4 NH C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 C O- or

\ sqbullet

-CON (CH 2 COOH) CH 2 CH 2 C O-.

On condition that

a) when W is an amino acid residue or a amino acid residue chain, carbonyl carbon underlined in X forms an amide bond with an amino group in W, or

b) when W is a covalent bond, carbon of the carbonyl underlined in X forms an amide bond with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin;

And it is:

\ sqbullet

- (CH 2) m where m is an integer in the range of 6 to 32;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of -CH_ {2} groups - sufficient to give a total number of atoms of carbon in the chain in the range of 10 to 32;

\ sqbullet

a hydrocarbon chain formula bivalent - (CH 2) v C 6 H 4 (CH 2) w - where v and w are integers or one of them is zero so that the sum of v and w is in the range of 6 to 30 and

Z is:

\ sqbullet

-COOH;

\ sqbullet

-CO-Asp;

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\ sqbullet

-CO-Glu;

\ sqbullet

-CO-Gly;

\ sqbullet

-CO-Sar;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH 2 COOH) 2;

\ sqbullet

-SO 3 H; or

\ sqbullet

-PO 3 H;

and any complex of Zn 2+ of the same.

2. Insulin derivative according to paragraph 1, where W is an α-amino acid residue with a group of carboxylic acid in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin.

3. An insulin derivative according to any of paragraphs 1-2, where W is a residue of α-amino acid having 4 to 10 atoms of carbon.

4. An insulin derivative according to the paragraphs 1-3, where W is selected from the group consisting in α-Asp, β-Asp, α-Glu, γ-Glu, α-hGlu and δ-hGlu.

5. An insulin derivative according to paragraph 1, where W is a chain composed of two residues of α-amino acid of which one has 4 to 10 carbon atoms and a free carboxylic acid group while others have 2 to 11 carbon atoms but no acid group free carboxylic.

6. An insulin derivative according to paragraphs 1 and 5 where W is selected from the group consisting of α-Asp-Gly; Gly-? -Asp; β-Asp-Gly; Gly-? -Asp; α-Glu-Gly; Gly-?-Glu; γ-Glu-Gly; Gly- γ-Glu; α-hGlu-Gly; Gly-? -HGlu; δ-hGlu-Gly; Y Gly- \ delta-hGlu.

7. An insulin derivative according to paragraph 1, where W is a chain composed of two residues of α-amino acids, which has independently from 4 to 10 carbon atoms, and both with an acid group free carboxylic.

8. An insulin derivative according to paragraph 1 and 7, where W is selected from the group consisting of α-Asp-a-Asp; α-Asp-a-Glu; α-Asp-a-hGlu; α-Asp-? -Asp; α-Asp-?-Glu; α-Asp- δ-hGlu; β-Asp-? -Asp; β-Asp-?-Glu; β-Asp-? -hGlu; β-Asp-? -Asp; β-Asp-?-Glu; β-Asp- δ-hGlu; α-Glu-? -Asp; α-Glu-?-Glu; α-Glu-? -hGlu; α-Glu-? -Asp; α-Glu-γ-Glu; α-Glu- δ-hGlu; γ-Glu-? -Asp; γ-Glu-?-Glu; γ-Glu-? -hGlu; γ-Glu-? -Asp; γ-Glu- γ-Glu; γ-Glu- δ-hGlu; α-hGlu-? -Asp; α-hGlu-?-Glu; α-hGlu-? -hGlu; α-hGlu-? -Asp; α-hGlu-γ-Glu; α-hGlu-δ-hGlu; δ-hGlu-? -Asp; δ-hGlu-?-Glu; δ-hGlu-? -hGlu; δ-hGlu-? -Asp; δ-hGlu-γ-Glu;  Y δ-hGlu- δ-hGlu.

9. An insulin derivative according to paragraph 1, where W is a covalent bond.

10. An insulin derivative according to any of paragraphs 1-9, where X is -CO- or -COCH (COOH) CO-.

11. An insulin derivative according to the paragraphs 1-9, where X is

\ sqbullet

-CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-

\ sqbullet

-CON (CH 2 CH 2 COOH) CH 2 C O- or

\ sqbullet

-CON (CH 2 COOH) CH 2 CH 2 C O-.

12. An insulin derivative according to any of paragraphs 1-11, where Y is - (CH 2) m - where m is an integer in the range of from 6 to 32.

13. An insulin derivative according to paragraph 12 where m is in the 8-20 range.

14. An insulin derivative according to the paragraphs 12-13, where m is 11, 12, 13, 14, 15 or 16.

15. Insulin derivative according to any of the paragraphs 1-14, where Z is -COOH.

       \ global \ parskip1.000000 \ baselineskip
    

16. Insulin derivative according to any of the paragraphs 1-14, where Z is -CO-Asp.

17. Insulin derivative according to any of the paragraphs 1-14, where Z is -CO-Glu.

18. Insulin derivative according to any of the paragraphs 1-14, where Z is -CO-Gly.

19. Insulin derivative according to any of the paragraphs 1-14, where Z is -CO-Sar.

20. Insulin derivative according to any of the paragraphs 1-14, where Z is -CH (COOH) 2.

21. Insulin derivative according to any of the paragraphs 1-14, where Z is -N (CH 2 COOH) 2.

22. Insulin derivative according to any of the paragraphs 1-14, where Z is -SO_ {3} H.

23. Insulin derivative according to any of the paragraphs 1-14, where Z is -PO_ {3} H.

24. Insulin derivative according to any of the paragraphs 1-23, where the progenitor insulin is a insulin analogue, which does not have a residue of Lys in position B29 of chain B.

25. Insulin derivative according to any of the paragraphs 1-24, where the progenitor insulin is human insulin or porcine insulin, where the residue of Lys in the position B29 is replaced and a residue of Lys is inserted into a position in chain A or chain B except in position B29.

26. Insulin derivative according to paragraphs 1 and 24-25, where the amino acid residue in one of the positions A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 of the progenitor insulin chain A is a residue of Lys.

27. Insulin derivative according to paragraphs 1 and 24-25, where the amino acid residue in one of the positions B1, B2, B3, B4; B20; B21 or B22 of the B chain of the Progenitor insulin is a residue of Lys.

28. Insulin derivative according to paragraph 24-27, where the amino acid residue in position B30 of the progenitor insulin has been deleted.

29. Insulin derivative according to paragraph 24-28, where the amino acid residue in position B1 of the progenitor insulin has been deleted.

30. Insulin derivative according to paragraph 24-29, where the amino acid residue in position A21 of the progenitor insulin is Gly or Asn.

31. Insulin derivative according to paragraph 24-30, where the amino acid residue in position B3 of the progenitor insulin is Lys.

32. Insulin derivative according to paragraph 24-31, where the amino acid residue in position B28 of the progenitor insulin is Asp.

33. Insulin derivative according to any of the paragraphs 24-32, where the amino acid residue in the B29 position of the progenitor insulin is Pro or Thr.

34. Zinc complex of an insulin derivative according to any of the preceding paragraphs where two ions of zinc, three zinc ions, four zinc ions, five zinc ions, six zinc ions, seven zinc ions, eight zinc ions, nine zinc ions, ten zinc ions, eleven zinc ions or twelve ions of zinc are bound by six molecules of insulin derivative.

35. Pharmaceutical composition for treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of a derivative insulin according to any of the preceding paragraphs optionally together with a pharmaceutically acceptable carrier.

36. Pharmaceutical composition for treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of a derivative insulin according to any of the preceding paragraphs in mixture with an insulin or an insulin analogue that has an appearance quick action optionally together with a carrier pharmaceutically acceptable.

37. Method for the production of a composition pharmaceutical according to paragraphs 35-36 or a complex zinc of an insulin derivative according to paragraph 34, where up to approximately 12 zinc ions per 6 derivative molecules insulin are added to the pharmaceutical composition.

38. Method according to paragraph 37, where approximately 12 zinc ions per 6 derivative molecules insulin are added to the pharmaceutical composition after the addition of a preservative

39. Diabetes treatment method in a patient in need of such treatment, comprising the administration to the patient of a therapeutically effective amount of an insulin derivative according to any of the paragraphs 1-33 optionally together with a carrier pharmaceutically acceptable.

40. Diabetes treatment method in a patient in need of such treatment, comprising the administration to the patient of a therapeutically effective amount of an insulin derivative according to any of the paragraphs 1-33 in mixture with an insulin or an analogue of insulin that has a rapid onset of action optionally together with a pharmaceutically acceptable carrier.

41. Method according to paragraphs 39-40 for the pulmonary treatment of diabetes.

42. Use of an insulin derivative according to any of paragraphs 1-33 for production of a pharmaceutical composition for use in the treatment of type 1 diabetes, type 2 diabetes and other conditions that cause hyperglycemia

43. Use of an insulin derivative according to any of paragraphs 1-33 in admixture with a insulin or an insulin analogue that has a rapid onset of action for the production of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycemia.

44. Mixing an insulin derivative according to any of paragraphs 1-33 and an analogue of rapid-acting insulin selected from the group consisting of human insulin AspB28; LysB28ProB29 human insulin and insulin human LysB3GluB29.

45. Insulin derivative according to paragraph 1, where the insulin derivative is selected from the group consisting in

human insulin N ^ ε A9} -myristyl LysA9 ArgB29 desB30,

human insulin N ^ ε B3} -miristil LysB3 ArgB29 desB30,

human insulin N ^ ε B22} -miristil LysB22 ArgB29 desB30,

human insulin N ^ ε A15} -miristil LysA15 ArgB29 desB30,

human insulin N ^ ε A18} -miristil LysA18 ArgB29 desB30,

human insulin N ^ ε A22} -miristil LysA22 ArgB29 desB30,

human insulin N ^ ε A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30,

human insulin N ^ ε B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30,

human insulin N ^ ε B22} -? -Carboxypentadecanoyl-? -Gtu LysB22 ArgB29 desB30,

human insulin N ^ ε A15} -? -Carboxypentadecanoyl-?-Glu LysA15 ArgB29 desB30,

human insulin N ^ ε A18} -? -Carboxypentadecanoyl-?-Glu LysA18 ArgB29 desB30,

human insulin N ^ ε A22} -? -Carboxypentadecanoyl-? -Glu LysA22 ArgB29 desB30.

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The invention will be further summarized in the following paragraphs:

1st. Insulin derivatives with a side chain well bonded to a ε-amino group of a Lys residue present in chain A at position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or to an amino group of a residue of Lys in chain B at position B1, B2, B3, B4; B20; B21 or B22 of the progenitor insulin

On condition that when B3 is Lys, then B29 other than Glu when the side chain is of the general formula:

-W X Y Z

where

W is:

\ sqbullet

a residue of α-amino acid with an acid group carboxylic in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or

       \ global \ parskip0.930000 \ baselineskip
    

\ sqbullet

a chain composed of two, three or four α-amino acid residues linked together by means of amide bonds, whose chain -by middle of an amide bond- joins the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin, the residues of W amino acids being selected from the residue group of amino acids with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least one amino acid residue that has an acid group carboxylic on the side chain; or

\ sqbullet

a covalent bond of X to ε-amino group of a Lys residue present in the A or B chain of the progenitor insulin;

X is:

\ sqbullet

- C O-;

\ sqbullet

-CH (COOH) C O-;

\ sqbullet

-N (CH 2 COOH) CH 2 C O-;

\ sqbullet

-N (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C O-;

\ sqbullet

-N (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-N (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O-;

\ sqbullet

-NHCH (COOH) (CH 2) 4 NH C O-;

\ sqbullet

-N (CH 2 CH 2 COOH) CH 2 C O- or

\ sqbullet

-N (CH 2 COOH) CH 2 CH 2 C O-.

that

a) when W is an amino acid residue or a chain of amino acid residues, by means of a link underlined carbonyl carbon forms an amide bond with a group amino in W, or

b) when W is a covalent bond, by means of an underlined carbonyl carbon bond forms an amide bond with the ε-amino group of a residue of Lys present in the A or B chain of the progenitor insulin;

And it is:

\ sqbullet

- (CH 2) m where m is an integer in the range of 6 to 32;

\ sqbullet

a hydrocarbon chain bivalent comprising 1, 2 or 3 groups -CH = CH- and a number of -CH_ {2} groups - sufficient to give a total number of atoms of carbon in the chain in the range of 10 to 32;

\ sqbullet

a hydrocarbon chain formula bivalent - (CH 2) v C 6 H 4 (CH 2) w - where v and w are integers or one of these is zero so that the sum of v and w is in the range of 6 to 30 and

Z is:

\ sqbullet

-COOH;

\ sqbullet

-CO-Asp;

\ sqbullet

CO-Glu;

\ sqbullet

-CO-Gly;

\ sqbullet

-CO-Sar;

\ sqbullet

-CH (COOH) 2;

\ sqbullet

-N (CH 2 COOH) 2;

\ sqbullet

-SO 3 H; or

\ sqbullet

-PO 3 H;

and any complex of Zn 2+ of the same.

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2nd. An insulin derivative according to the paragraphs 1a, in which the acyl group is linked to the lysine residue using An amino acid as a linker.

3rd. An insulin derivative according to paragraph 2a, in which the amino acid bond is α-glutamyl or linked to γ-glutamyl or linked to β- or α-aspartyl.

4th. An insulin derivative according to paragraph 2a, in which the amino acid bond is linked to γ-aminobutanoyl, bound to β-alanil, linked to α-amido- γ-glutamyl, or linked to α-amido- γ-aspartyl,  or linked to α-amido- β-aspartyl.

5th. Insulin derivative according to paragraph 1a, where the side chain comprises a fatty acid or a diacid fatty.

6a. Insulin derivative according to paragraph 1a, where the lipophilic group can be negatively charged.

7a. Insulin derivative according to paragraph 6a, where the side chain comprises an amino acid residue lipophilic

8a. Insulin derivative according to paragraph 6a, where the side chain comprises an amino acid residue.

9a. Insulin derivative according to any of the preceding paragraphs, where the progenitor insulin is insulin human desB30, human insulin ArgB29 or human insulin ArgB29desB30.

10th A zinc complex of a derivative insulin according to any of the preceding paragraphs where up to 12 zinc atoms are joined by 6 insulin derivatives.

11a. Pharmaceutical composition for treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any of the paragraphs 1-9a or a zinc complex of the insulin derivative according to paragraph 10a together with a pharmaceutically carrier acceptable.

12th Pharmaceutical composition for treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any of paragraphs 1 a-9a or a zinc complex of the insulin derivative according to paragraph 10a in admixture with an insulin or an analogue of insulin that has a rapid onset of action, along with a pharmaceutically acceptable carrier.

13a. Pharmaceutical composition according to paragraph 11th or 12th intended for pulmonary administration.

14 to. Pharmaceutical composition according to the paragraphs 11th-13th comprising up to about 12 ions of zinc per 6 molecules of insulin derivative.

15th Method for the production of a pharmaceutical composition according to paragraphs 11a-13a, where up to about 12 zinc ions per 6 molecules of insulin derivative are added to the pharmaceutical composition after of the addition of a preservative.

16th Method of treating diabetes in a patient in need of such treatment, comprising the administration to the patient of a therapeutically effective amount of an insulin derivative according to paragraphs 1-9 or a zinc complex of the insulin derivative according to paragraph 10a together with a pharmaceutically acceptable carrier and additives Acceptable pharmacists

17th Method of treating diabetes in a patient in need of such treatment, comprising the administration to the patient of a therapeutically effective amount of an insulin derivative according to paragraphs 1-9 or a zinc complex of the insulin derivative according to paragraph 10a in admixture with an insulin or an insulin analogue that has a rapid onset of action, along with a pharmaceutically carrier Acceptable and acceptable pharmaceutical additives.

Examples General procedures Construction of expression vectors, transformation of yeast cells, and expression of insulin precursors of the invention

All expression plasmids are of the C-POT type, similar to those described in EP 171142, which are characterized in that they contain the Schyzosacaromices pombe triose phosphate (POT) gene for the purpose of the selection and stabilization of the S plasmid cerevisiae Plasmids also contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/10075) as are all sequences except the sequence of the Eco RI- XbaI fragment encoding the leader's fusion protein and insulin product. To express different fusion proteins, the Eco RI-X baI fragment of pKFN1003 is simply replaced by an Eco RI-X baI fragment encoding the leader fusion and the insulin of interest. Such Eco RI-X baI fragments can be synthesized using synthetic oligonucleotides and PCR according to standard techniques.

Yeast transformants were prepared by transforming the host strain of the S. cerevisiae strain MT663 ( MATa / MAT? Pep4-3 / pep4-3 HIS4 / his4 tpi :: LEU2 / tpi :: LEU2 Cir +) . The strain MT663 of yeast was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with the application WO 92/11378 and was given the deposit number DSM 6278.

MT663 was grown in YPGaL (1% bacto-yeast extract, 2% Bacto-peptone, 2% galactose, 1% lactate) to an OD at 600 nm of 0.6. 100 ml of culture were harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na 2 EDTA pH = 8.0 and 6.7 mg / ml dithiothreitol. The suspension was incubated at 30 ° C for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na 2 EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg of Novozym®234. The suspension was incubated at 30 ° C for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCl 2, 10 mM tris HCl ( pH = 7.5) and resuspended in 2 ml of CAS For transformation, 1 ml of suspended CAS cells was mixed with approximately 0.1 mg of plasmid DNA and left at room temperature for 15 minutes 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl2, 10 mM Tris HCl, pH = 7.5) was added and the mixture was left for another 30 minutes at room temperature.The mixture was centrifuged and the granulate was resuspended in 0.1 ml of SOS ( 1.2 M sorbitol, 33% v / v YPD, 6.7 mM CaCl 2) and incubated at 30 ° C. for 2 hours The suspension was then centrifuged and the granulate resuspended in 0.5 ml of 1.2 M sorbitol. ml of upper agar (SC medium from Sherman et al . (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbi tol plus 2.5% agar) at 52 ° C was added and the suspension poured onto plates containing the same solidified agar medium, containing sorbitol. The S. cerevisiae strain MT663 transformed with expression plasmids was grown in YPD for 72 h at 30 ° C.

Production, purification and characterization of derivatives insulin of the invention

Several insulin precursors were produced as described above and isolated from the culture medium and purified. The insulin precursors were processed and acylates as described in the examples below to produce the final insulin derivatives. These insulin derivatives were evaluated for biological insulin activity as measured by binding affinity to the human insulin receptor relative to that of human insulin as described below.

The following examples refer to compounds intermediates and final products identified in the specification and in the examples. The preparation of insulin derivatives of the The present invention is described in detail using the examples. following, but chemical reactions and purification schemes described are described as to their general applicability to the Preparation of the insulin derivatives of the invention. Occasionally, the reaction may not be applicable as described. for each compound included in the scope of the invention described. The compounds for which this occurs will be easily recognized by those skilled in the art. In these cases the reactions can be successfully performed by modifications conventional techniques known to those skilled in the art, that is, for appropriate protection of interfering groups, changing to other conventional reagents, or by routine modification of reaction conditions Alternatively, other reactions described here or conventional otherwise will be applicable to the preparation of the corresponding compounds of the invention. In all preparatory methods, all raw materials are known or can easily be obtained from materials known premiums. All temperatures are set in degrees Celsius and unless otherwise indicated, all parties and percentages are by weight when referring to yields and All parts are by volume when reference is made to solvents and eluents.

The insulin derivatives of the invention can be purified using one or more of the following procedures that are typical within the art. These procedures can - if necessary - be modified with regarding gradients, pH, salts, concentrations, flow, columns, etc. Depending on factors such as impurity profile, solubility of the insulins in question and so on, these modifications can be recognized quickly and made by a skilled in the art.

Example 1 Synthesis of human insulin N ^? A9} -miristil LysA9 ArgB29 desB30

6

Phase one

Synthesis of N-hydroxysuccinimide ester of myristic acid

Synthesis of the myristic acid N-hydroxysuccinimide ester of acylation reagent was performed as described in B. Faroux-Corlay et al ., J. Med. Chem. 2001, 44, 2188-2203.

Phase 2

Preparation and purification of the insulin precursor LysA9 ArgB29 desB30 B'A

The insulin precursor LysA9 ArgB29 desB30 B'A It was purified as described in phases A to C of purification below.

Phase A of Purification

Capture

In phase A 10.75 liters of culture media rinses were diluted by adding 4.5 liters of 99% of ethanol, to give a total volume of 15.25 liters containing 30 vol% ethanol (conductivity 2.7 mS / cm, pH = 3.4). A column of SP Big Beads Sepharose 300 ml (100-300 µm, Amersham Biosciences) was equilibrated with 1 liter of 0.1 M acid citrus pH 3.5 (flow approx. 20 ml / min), before loading 15.25 liters of culture media prepared overnight (flow approx. 10 ml / min). After loading, the column went again washed with 1 liter of 0.1 M citric acid pH 3.5 followed by 1 liter of 40 vol% ethanol (flow approx. 20 ml / min). The forerunner of insulin LysA9 ArgB29 desB30 B'A bound was then eluted with 1.5 0.2 M liters of sodium acetate, 35 vol% ethanol, pH 5.75 (flow: 1.5 ml / min, eluted precursor volume: 400 ml, amount of precursor: 220 mg).

Phase B of purification

Reverse phase HPLC

In phase B the eluate was evaporated to dryness and the granulate was redissolved in 0.25 M acetic acid. PH was further lowered to 1.5 immediately before purification by reverse phase HPLC on a C18 column (ODDMS C18, 20x250 mM, 200 A, 10 µm, FeF Chemicals A / S). Before application to the column the precursor solution was filtered by sterile filter (22 µm, Low Protein Binding Durapore® (PVDF), Millipore). A gradient from 15% of B to 50% of B exceeded the column, where the Buffer A: 0.2 M of (NH 4) 2 SO 4, 0.04 M of acid ortho-phosphoric, 10 vol% ethanol, pH 2.5 and buffer B: 70 vol% ethanol. The gradient exceeded 120 min with a flow of 5 ml / min, column temperature at 40 ° C. The insulin precursor LysA9 ArgB29 desB30 B'A was eluted at approximately 35% of B and grouped (total volume 75 ml).

Phase C of purification

Gel filtration desalination

In phase C the ethanol content in the eluate of reverse phase HPLC was lowered to less than 5 vol% using a rotary evaporator (new volume: 50 ml). A G25 column 1000 ml Sephadex (5x55 cm, Amersham Biosciences) was washed in 0.5 M acetic acid and insulin precursor LysA9 ArgB29 desB30 B'A was then applied to the column and thus desalted by gel filtration in 0.5 M acetic acid. The insulin precursor was followed by UV detection at 280 nm, while the salt was followed by conductivity measurement. Immediately after desalination the insulin precursor was lyophilized (160 mg in weight, 5759.87 m / z).

Phase 3

Synthesis of precursor N ^? A9} -miristil LysA9 ArgB29 desB30 B'A

30 mg lyophilized insulin precursor LysA9 ArgB29 desB30 B'A was dissolved in 5 ml of 50 mM boric acid pH 2.6. The pH was then increased to 10.2 with a few drops of 1 M of NaOH 4 mg acylation reagent N-hydroxysuccinimide myristic acid ester was dissolved in 3.4 ml of CH3CN under careful heating at approximately 50 ° C. 3.2 ml acylation reagent solution was then added to the insulin precursor solution while stirring (hereby the acylation reagent was 1.75 times in excess). After incubation with shaking for 60 minutes at room temperature, the reaction was stopped by addition  2.1 ml of 0.2 M ethanolamine pH 9.0. After 10 minutes of incubation at room temperature the pH was adjusted to 6.4, so which precipitated the acylated insulin precursor N ^? A9} -miristil LysA9 ArgB29 desB30 B'A. He precipitate was stored at 4 ° C overnight (amount of precipitate: 23 mg).

Phase 4

Production of N ^ {A9} -miristil LysA9 ArgB29 desB30 human insulin

The 23 mg precipitate of N? A9} -miristil LysA9 ArgB29 desB30 B'A was dissolved in 4.2 ml of 50 mM glycine, 20 vol% ethanol pH 10.0. 3.6 mg of lyophilized porcine trypsin (Novo Nordisk A / S) was also dissolved in 3.5 ml of 50 mM glycine, 20 vol% ethanol pH 10.0. This 0.5 ml trypsin solution were then added to the solution insulin precursor (hereby the insulin precursor It was 200 times in excess). The mixture was then incubated at temperature. environment for 15 minutes, after which the activity of Trypsin was stopped by reduction of pH <3 (pH = 2.08 with 0.5 M acetic acid).

The human insulin acylated insulin analogue N? A9 -myristyl LysA9 ArgB29 desB30 was then purified (removing trypsin and any non-acylated, doubly acylated molecule etc. or undivided insulin molecules) by reverse phase HPLC. 40% B to 100% gradient B was applied to a C4 column (Jupiter C4, 5 µm, 300 Å 10x250 mm, Phenomenex), where buffer A: 10 mM Tris, 15 mM (NH_ {4} HCO3, 10 vol% ethanol, pH 8.5 and buffer B: 70 vol% ethanol. The gradient exceeded 120 min with a flow of 2 ml / min, column temperature at 40 ° C, and column size 1x25 cm. The human insulin N9? -Myristil LysA9 ArgB29 desB30 was eluted at 70% B (eluate analog volume: 15 ml). The analog was precipitated by reduction of the pH to approximately 6, redissolved in 2 ml of 0.5 M acetic acid and lyophilized (amount of analog by weight: 9.3 mg by weight,
LCMS: 5988.04 m / z).

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Example 2 Synthesis of human insulin N ^? A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30

Phase one

Preparation of ? -carboxypentadecanoyl-L-Glu (OSu) -OH

Acylating reagent ? -carboxypentadecanoyl-L-Glu (OSu) -OH it was prepared essentially as described for octadecandioyl-L-Glu (OSu) -OH (ome-carboxyheptadecanoyl-L-Glu (OSu) -OH) in WO-2005012347-A2, starting from hexadecanedioic acid instead of octadecanedioic acid.

7

Phase 2

Preparation and purification of the insulin precursor LysA9 ArgB29 desB30 B'A

The insulin precursor was purified as described under phase 2 in example 1.

Phase 3

Synthesis of precursor N ^? A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30 B'A

40 mg lyophilized insulin precursor LysA9 ArgB29 desB30 B'A was dissolved in 2 ml of 100 mM Na2CO3 pH 10.8. 13 mg acylation reagent ? -carcarboxypentadecanoyl-L-Glu (OSu) -OH It was suspended in 500 µL of CH 3 CN. The reagent of Acylation was then dissolved by adding 70 µL of NMP. The 570 µl of acylation reagent solution were then slowly added to the insulin precursor solution while stirring (hereby the acylation reagent was 2.5 times in excess). After incubation with shaking for 60 minutes at room temperature, the reaction was stopped by adjusting the pH at about 5, whereby the insulin precursor N ^ {\ varepsilon acylated A9} -? -Carboxypentadecanoyl-? -Glu  LysA9 ArgB29 desB30 B'A precipitated. The precipitate was stored at 4 ° C overnight (LC-MS: 6154.93 m / Z).

Phase 4

Production of human insulin N ^? A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30

The precipitate of N? A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30 B'A was dissolved in 3 ml of 50 mM glycine, 20 vol% ethanol pH 10.0. 1 mg lyophilized porcine trypsin (Novo Nordisk A / S) was also dissolved in 1.5 ml of 50 mM glycine, 20 vol% ethanol pH 10.0, and was then added to the solution of insulin precursor (hereby the insulin precursor was 300 times in excess). The reaction was then incubated at temperature. environment for 15 minutes, after which the activity of Trypsin was stopped by reduction of pH <3.

The human insulin acylated insulin analog N ^ {\ varepsilon A9} -? -Carboxypentadecanoyl-? -Glu  LysA9 ArgB29 desB30 was then purified (removing trypsin and any molecule not acylated, doubly acylated etc. or molecule of undivided insulin) by reverse phase HPLC. A gradient of 25% B to 55% B was applied to a Fuji C4 column (15 µm, 200 {{A}, 10x250 mm, FeF Chemicals A / S), where buffer A: 10 mM of Tris, 15 mM of (NH4) HCO3, 10 vol% ethanol, pH 8.5 and buffer B: 70 vol% ethanol. The gradient exceeded 100 min with a flow 3 ml / min at room temperature. Human insulin N ^ {\ varepsilon A9} -? -Carboxypentadecanoyl-? -Glu  LysA9 ArgB29 desB30 was eluted at approximately 48% B (17 mg of derivative). The analog was precipitated by pH reduction by below 5, redissolved in 2 ml of 0.5 M acetic acid and lyophilisate (LC-MS: 6173 m / z).

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Example 3 Synthesis of human insulin N ^? B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30

8

Phase one

Preparation of ? -carboxypentadecanoyl-L-Glu (OSu) -OH

The acylation reagent preparation of ? -carboxypentadecanoyl-L-Glu (OSu) -OH It was performed as described under Example 2.

Phase 2

Preparation and purification of the insulin precursor LysB3 AraB29 desB30 B'A

The insulin precursor was purified essentially as described under phase 2 in example 1, it is say with a capture phase, followed by reverse phase HPLC, and A final desalination. Immediately after desalination the insulin precursor was lyophilized (LC-MS: 5731.34 m / z).

Phase 3

Synthesis of precursor N ^? B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30 B'A

The synthesis of the insulin precursor N ^ {\ varepsilon B3} -? -Carboxypenta-decanoyl-? -Glu  LysB3 ArgB29 desB30 B'A was performed essentially as it describes in phase 3 of example 2 (LC-MS: 6128.2 m / z).

Phase 4

Production of human insulin N ^? B3} -? -Carboxypentadecanoyl-? -Glu LysB3 AraB29 desB30

The N ^ {\ varepsilon B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30 B'A was essentially trypsin divided as described for N ^ {\ varepsilon A9} -? -Carboxypentadecanoyl-? -Glu  LysA9 ArgB29 desB30 B'A, phase 4 in example 2.

The human insulin acylated insulin analog N ^ {\ varepsilon B3} -? -Carboxypentadecanoyl-? -Glu  LysB3 ArgB29 desB30 was then purified by reverse phase HPLC. First the trypsin cleavage reaction mixture was diluted adding water to 10% ethanol (4.5 ml) and 175 µl of PMSF (1 mg / ml) was added to inhibit trypsin. The pH was adjusted to 8 to dissolve human insulin N ^? B3} -? -Carboxypentadecanoyl-? -Glu  LysB3 ArgB29 desB30. A gradient of 20% B to 50% B was applied to a Fuji C4 column (15 µm, 200 Å, 10x250 mm, FeF Chemicals A / S), where buffer A: 10 mM Tris, 15 mM (NH4) HCO3, 10 vol% ethanol, pH 8.5 and buffer B: 70 vol% ethanol. The gradient exceeded 160 min with a flow of 3 ml / min at room temperature. The human insulin N ^ \ ε B3} -ctrcarboxypentadecanoyl- γ-Glu LysB3 ArgB29 desB30 was eluted at approx. 30% B.

The derivative was further purified by HPLC of reverse phase using a C4 Jupiter analytical column (5, 300 ring {A}, 4.6x150 mm, Phenomenex). A gradient from 20% B to 50% B was applied to the column, where buffer A: 12.5 mM of Tris, 20 mM (NH4) SO4, 20 vol% CH3CN, pH 7 and buffer B: 80 vol% CH 3 CN. The sample of the previous preparatory HPLC was divided into approximately 10 subsamples, and each subsample was purified on the analytical HPLC scale. Total volume after Purification: 20 ml, 91% purity.

The analog was precipitated by pH reduction at 5.12, and the granulate was lyophilized (1 mg, LC-MS:  6148.4 m / z).

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Example 4 Synthesis of human insulin N ^? B3 -miristil LysB3 ArgB29 desB30

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9

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Preparation and purification of the LysB3 precursor ArgB29 desB30 B'A is described in Example 3. The synthesis of precursor N ^ {B3} -miristil LysB3 ArgB29 desB30 B'A and the following trypsin cleavage to produce the final product, human insulin N ^? B3} -miristil LysB3 ArgB29 desB30, was performed essentially as described in Example 1. LC-MS: Intact derivative: 5959.0 m / Z, chain B of reduced derivative: 3581.3 m / Z.

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Example 5 Synthesis of human insulin N ^? B22} -miristil LysB22 ArgB29 desB30

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10

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Preparation and purification of the precursor LysB22 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N? B22} -miristil LysB22 ArgB29 desB30 B'A and the following trypsin cleavage to produce the final product, N ^ {\ varepsilon B22} -miristil LysB22 ArgB29 desB30 human insulin, was performed essentially as described in the Example 1. LC-MS: Intact derivative: 5917.2 m / z, reduced derivative: 2382.60 m / z (A-chain), 3537.70 m / Z (chain B).

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Example 6 Synthesis of human insulin N ^? B22} -? -Carboxypentadecanoyl-? -Glu LysB22 ArgB29 desB30

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eleven

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Preparation and purification of the precursor LysB22 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^ {\ varepsilon B22} -? -Carboxypentadecanoyl-? -Glu  LysB22 ArgB29 desB30 B'A and the following trypsin cleavage for produce the final product, human insulin N ^? B22} -? -Carboxypentadecanoyl-? -Glu LysB22 ArgB29 desB30, was essentially performed as described in Example 2 and 3. MS calculated: 6104.06 m / Z.

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Example 7 Synthesis of human insulin N ^? A15} -miristil LysA15 ArgB29 desB30

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12

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Preparation and purification of the precursor LysA15 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A15} -miristil LysA15 ArgB29 desB30 B'A and the following trypsin cleavage to produce the final product, N ^ {\ Are} -miristil LysA15 ArgB29 desB30 human insulin, was performed essentially as described in the Example 1. MALDI-MS: Intact derivative: 5946.14 m / Z, reduced derivative B chain: 3356.23 m / Z.

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Example 8 Synthesis of human insulin N ^? B15} -? -Carboxypentadecanoyl-? -Glu LysA15 ArgB29 desB30

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13

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Preparation and purification of the precursor LysA15 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A15} -? -Carboxypentadecanoyl-?-Glu  LysA15 ArgB29 desB30 B'A and the following trypsin cleavage for produce the final product, human insulin N ^? A15} -? -Carboxypentadecanoyl-?-Glu LysA15 ArgB29 desB30, was performed essentially as described in example 2 and 3. MS calculated: 6132.12 m / z.

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Example 9 Synthesis of human insulin N ^? A18} -miristil LysA18 ArgB29 desB30

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14

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Preparation and purification of the precursor LysA18 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A18} -miristil LysA18 ArgB29 desB30 B'A and the following trypsin cleavage to produce the final product, N ^ {A18} -miristil LysA18 ArgB29 desB30 human insulin, was performed essentially as described in the Example 1. LC-MS: Intact derivative: 5959.5 m / Z, reduced derivative B chain: 3355.50 m / z.

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Example 10 Synthesis of human insulin N ^? A18} -? -Carboxypentadecanoyl-?-Glu LysA18 ArgB29 desB30

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fifteen

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Preparation and purification of the precursor LysA18 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A18} -? -Carboxypentadecanoyl-?-Glu  LysA18 ArgB29 desB30 B'A and the following trypsin cleavage for produce the final product, human insulin N ^? A18} -? -Carboxypentadecanoyl-?-Glu LysA18 ArgB29 desB30, was performed essentially as described in Example 2 and 3. MALDI-MS: 6146.96 m / z.

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Example 11 Synthesis of human insulin N <2> -22ististil LysA22 ArgB29 desB30

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16

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Preparation and purification of the precursor LysA22 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A22} -miristil LysA22 ArgB29 desB30 B'A and the following trypsin cleavage to produce the final product, human insulin N22 A22-myristyl LysA22 ArgB29 desB30, was essentially performed as described in Example 1. LC-MS: 6072.3 m / z.

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Example 12 Synthesis of human insulin N ^? A22} -? -Carboxypentadecanoyl-? -Glu LysA22 ArgB29 desB30

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17

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Preparation and purification of the precursor LysA22 ArgB29 desB30 B'A was made essentially as described in the Example 1 and 3. Synthesis of the precursor N ^? A22} -? -Carboxypentadecanoyl-? -Glu  LysA22 ArgB29 desB30 B'A and the following trypsin cleavage for produce the final product, human insulin N ^? A22} -carboxypentadecanoyl-? -Glu LysA22 ArgB29 desB30, was essentially performed as described in Example 2 and 3. LC-MS: 6260.2 m / Z.

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Example 13 Synthesis of human insulin N ^? A22} -carboxy-5-hexadecandioylaminobenzoyl LysA22 ArgB29 desB30

18

A human insulin LysA22 ArgB29 desB30 (100 mg) dissolved in 0.1 M Na2CO3 (2.25 ml) 1.2 are added 3-tert-butyl ester equivalents 1- (2,5-dioxopyrrolidin-1-yl) acid ester 5- (15-tert-butoxycarbonylpentadecanoylamino) isophthalic (prepared as described in WO 2006082204) dissolved in acetonitrile (1.13 ml). The insulin derivative is isolated by isoelectric precipitation The dried product is treated with TFA for 30 min. TFA is removed by evaporation. The compound of title is isolated after purification by RP-HPLC, which can be performed on a Gilson system 215 using a C4 SP 250/21 Nucleosil 300-7 column and a water / acetonitrile gradient of 10-80% containing 0.1% of TFA.

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Example 14 Synthesis of human insulin N ^? A22} -3-carboxy-5-octadecandioylaminobenzoyl LysA22 ArgB29 desB30

19

3-tert-butyl ester of 1- (2,5-dioxo-pyrrolidin-1-yl) acid ester 4- (17-tert-butoxycarbonyl-heptadecanoylamino) isophthalic  reacts with human insulin LysA22 ArgB29 desB30 followed by TFA treatment and purification in a similar way as previously described.

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Example 15 Synthesis of human insulin N ^? A22} -10- (3,5-dicarboxyphenoxydecanoyl-? -Glu LysA22 ArgB29 desB30

twenty

Di-tert-butyl acid ester 5- {9 - [(S) -1-tert-butoxycarbonyl-3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) propylcarbamoyl] nonyloxy} isophthalic (prepared as described below) reacts with human insulin LysA22 ArgB29 desB30 followed by treatment with TFA and purification similarly as described above.

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Example 16 Synthesis of human insulin N ^? A22} -4- [10- (3,5-dicarboxyphenoxy) decanoylamino] -butyryl LysA22 ArgB29 desB30

twenty-one

Di-tert-butyl ester of 5- {9- [3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -propylcarbamoyl] -nonyloxy} -isophthalic acid
(prepared as described below) it is reacted with human insulin LysA22 ArgB29 desB30 followed by treatment with TFA and purification in a similar manner as described above.

Synthesis of di-tert-butyl ester of acid 5- {9 - [(S) -1-tert-butoxycarbonyl-3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -propylcarbamoyl] -nonyloxy} -isophthalic

Phase one

Di-tert-butyl acid ester 3-hydroxy-isophthalic

To an acid suspension 5-hydroxy-isophthalic (1.06 g, 5.8 mmol) in dry toluene (20 ml) at 60 ° C was added N, N-dimethylformamide di-tert-butyl acetal (7.0 ml, 29. 2 mmol) and the temperature was increased to 100 ° C. Dry DMF (5 ml) was added. The mixture was stirred under nitrogen. After 2h more N, N-dimethylformamide di-tert butyl Acetal (7.0 ml, 29. 2 mmol) was added. After 3 h the mixture Reactive was cooled to room temperature and filtered. Solid was purified by flash chromatography using heptane / EtOAc / acid acetic 40: 20: 3. The title compound was isolated, contaminated. by the tri-tert-butyl ester.

LC-MS: 239.13 (M-tBu)

Phase 2

Di-tert-butyl acid ester 5- (9-Methoxycarbonyl-nonyloxy) -isophthalic

Di-tert-butyl 3-hydroxy-isophthalic acid ester (90 mg, 0.3 mmol) was dissolved in dry acetone (5 ml), K 2 CO 3 (90 mg, 0.65 mmol) and methyl 10-Bromodecanoate (0.075 ml, 0.3 mmol) was added. The mixture was heated to reflux. The warming caused the dissolution of K2CO3. After a while, a precipitate was formed. Reflux (50 ° C) was continued during night. More K 2 CO 3 (30 mg, 0.2 mmol) and methyl 10-bromodecanoate (0.025 ml, 0.1 mmol) was added. The mixture was refluxed at 50 ° C overnight. To the mix dry reagent, EtOAc and water were added. After extraction, the organic layer was washed with water and saline, dried (Na 2 SO 4) and concentrated to give the title compound Raw as a clear oil. LC-MS: m / z: 423.34 (M-tBu).

Phase 3

Di-tert-butyl acid ester 5- (9-carboxy-nonyloxy) -isophthalic

To a solution of di-tert-butyl acid ester 5- (9-methoxycarbonyl-nonyloxy) -isophthalic (162 mg, 0.34 mmol) in THF (2 ml) 1 N NaOH (0.35 was added ml) The mixture was refluxed for 7 h. The mixture was left at room temperature overnight before concentration. To the residue, EtOAc and 0.1N HCl were added. The aqueous layer was extracted twice with EtOAc. The combined organic layers were washed with saline solution, dried (Na2SO4) and concentrated to give a clear syrup.

LC-MS: m / z: 465.3 (M + 1).

Phase 4

Di-tert-butyl acid ester 5- [9- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -nonyloxy] -isophthalic

To a solution of di-tert-butyl acid ester 5- (9-carboxy-nonyloxy) -isophthalic (156 mg, 0.34 mmol) in dry THF (2 ml) TSTU (125 mg) was added and DIPEA (0.08 ml). The mixture was stirred under nitrogen during the night. The mixture was concentrated. The residue was redissolved in EtOAc and filtered. The filtrate was washed with 0.1N HCl (2x), and solution saline (1x), dried (Na2SO4) and concentrated to give a clear syrup

LC-MS: m / z: 562.3 (M + 1).

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Phase 5

Di-tert-butyl acid ester 5- [9 - ((S) -1-tert-butoxycarbonyl-3-carboxy-propylcarbamoyl) -nonyloxy] -isophthalic

To a solution of di-tert-butyl acid ester 5- [9- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -nonyloxy] -isophthalic  (62 mg, 0.11 mmol) in dry DMF (1 ml), was added H-Glu-OtBu (0.027 g, 0.13 mmol) and DIPEA (0.05 ml). The mixture was stirred overnight under nitrogen for 4 h. The mixture was concentrated. The residue was redissolved in EtOAc and washed with 0.1 N HCl (2x), saline (1x), dried (Na2SO4) and concentrated to give a syrup.

LC-MS: m / Z: 650.44 (M + 1).

Phase 6

Di-tert-butyl acid ester 5- {9 - [(S) -1-tert-butoxycarbonyl-3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -propylcarbamoyl] -nonyloxy} -isophthalic

Di-tert-butyl acid ester 5- [9 - ((S) -1-tert-butoxycarbonyl-3-carboxy-propylcarbamoyl) -nonyloxy] -isophthalic  (70 mg, 0.11 mmol) was dissolved in dry THF (1 ml). TSTU added (40 mg, 0.13 mmol) and DIPEA (0.05 ml). The mixture was stirred at room temperature under nitrogen during the weekend. The mixture was concentrated. The residue was redissolved in EtOAc and filtered out. The filtrate was washed with 0.1 N HCl (2x), washed with saline solution (1x), dried (Na2SO4) and concentrated to Give a clear syrup. There was still raw material present and the TSTU treatment was repeated followed by intensification to Give the title compound as a clear syrup.

LC-MS: m / z: 747 (M + 1).

Synthesis of di-tert-butyl ester of acid 5- {9- [3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -propylcarbamoyl] -nonyloxy} -isof- tantalum

Phase one

Di-tert-butyl acid ester 5- [9- (3-carboxy-propylcarbamoyl) -nonyloxy] -isophthalic

Di-tert-butyl acid ester 5- [9- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -nonyloxy] -isophthalic (100 mg, 0.18 mmol) was dissolved in dry DMF (2 ml). Was added 4-aminobutyric acid (GABA, 23 mg, 0.2 mmol) and DIPEA (0.05 ml). The mixture was stirred under nitrogen for 2 days. The mixture was concentrated. The residue was dissolved in EtOAc and washed with 0.1 N HCl (2x) and saline (1x), dried (Na 2 SO 4) and concentrated to give a clear syrup.

LC-MS: m / Z: 550.33 (M + 1).

Phase 2

Di-tert-butyl acid ester 5- {9- [3- (2,5-dioxo-pyrrolidin-1-yloxycarbonyl) -propylcarbamoyl] -nonyloxy} -isophthalic

To a solution of di-tert-butyl acid ester 5- [9- (3-carboxy-propylcarbamoyl) -nonyloxy] -isophthalic  (62 mg, 0.11 mmol) in dry THF (2 ml) DIPEA (0.05 ml) was added and TSTU (41 mg, 0.14 mmol). The mixture was stirred at temperature. atmosphere under nitrogen overnight. The mixture was concentrated. and EtOAc was added to the residue. The precipitate was removed by filtration. The filtrate was washed with 0.1 N HCl (2x) and solution saline (1x), dried (Na2SO4) and concentrated to give a clear syrup

LC-MS: m / Z: 647.29.

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Example 17 Hydrophobicity of the insulin derivatives of the invention

The hydrophobicity of an insulin derivative is found by reverse phase HPLC performed under conditions isocratic The elution time of the insulin derivative is compared to that of HI or another derivative with a hydrophobicity known under the same conditions. Hydrophobicity, k'rel, is calculated as: k'reld_ {eriv} = ((t_ {deriv} -t_ {0}) / (t_ {ref} -t_ {0})) * k'rel_ {ref}. Using HI as a reference: k'rel_ {ref} = k'rel_ {HI} = 1. HPLC system vacuum time, t_ {0}, It is determined by injecting 5 µL of 0.1 mM NaNO 3.

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Performance Conditions

22

After executing an initial gradient, the isocratic level to execute the derivative and reference (for example HI) is chosen, and the elution times of the derivative and reference under isocratic conditions in the previous equation is use to calculate k'rel_ {deriv}.

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Example 18 Insulin receptor binding of insulin derivatives of the invention

The affinity of insulin derivatives of the invention for the human insulin receptor is determined by a microtiter plate antibody capture assay of a SPA test (scintillation proximity test). Union beads to SPA-PVT antibody, reagent anti-mouse (Amersham Bioscienses, Cat No. PRNQ0017) mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8, 100 mM of sodium chloride, 10 mM of MgSO4, 0.025% Tween-20). Reactive mixture for a single Packard Optiplate (Packard No. 6005190) is composed of 2.4 µl of a 1: 5000 recombinant purified diluted human insulin receptor (either with or without exon 11), a quantity of a stock solution of human insulin A14Tyr [125 I] corresponding to 5000 cpm per 100 µl of reactive mixture, 12 µl of a 1: 1000 dilution of F12 antibody, 3 ml of SPA beads and a binding buffer total of 12 ml. A total of 100 µl of reactive mixture is then added to each well in the Packard Optiplate and a series of dilution of the insulin derivative is made in the Optiplate of appropriate samples. The samples are then incubated for 16 hours while stirring gently. The phases are then separated by centrifugation for 1 min and the plates counted in a Topcounter Binding data were adjusted using the algorithm. Nonlinear regression in GraphPad Prism 2.01 (GraphPad Software,  San Diego, CA).

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Example 19 Human serum albumin affinity test of derivatives insulin of the invention

The affinity for human serum albumin of insulin derivatives of the invention are given as the constant of relative binding of derivative A14Tyr [125 I] to albumin of human serum (HSA) immobilized in Minileak particles and measured at 23 ° C compared to insulin Detemir (equal to 1 in saline buffer): HSA_ {aff} = k_ {D} (Detemir) / k_ {D} (derivative). HSA is immobilized in the Minileak material (KemEnTec 1011 F) by incubation overnight at room temperature with HSA (Sigma A-1887) and PEG (Fluka 95172) in 0.3 M of NaHCO3 pH 8.8. After incubation the reaction is stopped by addition of 1 M ethanolamine pH 9.0, and the material is washed several times with 0.1 M of Tris (HCl) pH 7.4, with 0.1 M of NaHCO 3 pH 9.0, and with 0.1 M phosphate pH 3.0. The material of HSA-Minileak is stored in 0.1 M of Tris (HCl) pH 7.4, 0.02% azide. The amount of HSA immobilized in the material is evaluated by incubating HSA-Minileak with 3 H-myristic acid (PerkinElmen NET-830) and various amounts of HSA free in 0.1 M Tris (HCl) pH 7.4, 0.025% Triton-X-100 for two hours at room temperature. After centrifugation of the samples the supernatant is analyzed in a scintillation counter, and the amount of immobilized HSA is calculated.

In determining the affinity of derivatives insulins for HSA, various amounts of HSA-Minileak are incubated with the derivative A14Tyr [125 I] for two hours at room temperature in 0.1 M Tris (HCl) pH 7.4, 0.025% Triton-X-100 After incubation the samples are centrifuged and half of the supernatant is removed at new tubes Both half of the supernatant (1/2 S) and the rest of the most granulated supernatant (1/2 S + P) is analyzed in a gamma counter, and this data is used for the calculation of k_ {d} for the derivative of A14Tyr [125] I].

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Example 20 Analysis of self-associative properties of insulin derivatives of the invention

The ability of insulin derivatives of the invention to self-associate in large but soluble complexes is analyzed using SEC (size exclusion chromatography):

2. 3

For this analysis the insulin derivatives of the invention are in a solution consisting of 0.6 mM of derivative, 2.1 Zn2 + / hexamer, 16 mM phenol, 7 mM phosphate pH 7.8. The retention time of the derivative is then compared with the retention times of the following standard molecules:

24

The following equation is used to determine the K_ {av} for the derivative:

K_ {av} = (t-t_ {0}) / (V_ {t} / (f + t_ {d} -t_ {0}))

where t is the retention time for a given maximum value, t_ {0} is the retention time for blue dextran, V_ {t} is the total column volume (here 2.4 ml), f is the flow (here 0.04 ml / min), and t_ {d} is the time of retention for dextran blue without the column in the system.

The value K_ {av} indicates the degree of self-association of a derivative, that is a great K_ {av} similar to K_ {av} for the hexamer Co (III) and the monomer X2 shows little or no propensity of the derivative for form large, self-associated complexes while K_ {av} very small near zero or even negative shows large propensity of the derivative to self-association in large soluble complexes.

26

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Example 21 Effect of decreased blood glucose after injection i.v. bolus in rat of insulin derivatives of the invention

Male Wistar rats, 200-300 g, fasted for 18 h, they were anesthetized using well Hypnorm-Dormicum s.c. (1.25 mg / ml of Dormicum, 2.5 mg / ml fluanisone, 0.079 mg / ml fentanyl citrate) 2 ml / kg as a priming dose (to the time point of -30 min before evaluate the dosage of the substance) and an additional 1 ml / kg each 20 minutes.

Animals are dosed with an injection intravenous (caudal vein), 1 ml / kg, control and compounds test (usual dose range 0.125-20 nmol / kg). Blood samples for concentration determination of blood glucose were collected in heparinized glass tubes 10 µl per puncture of the capillary vessels at the tip of the tail in time -20 min and 0 min (before dosing), and in the Times 10, 20, 30, 40, 60, 80, 120, and 180 min after the dosage. Blood glucose concentrations were Measures after dilution in analysis buffer by method of immobilized glucose oxidase using an EBIO Plus autoanalyzer (Eppendorf, Germany). The mean concentration transitions of blood glucose (mean ± SEM) has been made for each dose and each compound

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Example 22 Determination in pigs of T 50% of insulin analogues of the invention

T_ {50%} is the time when 50% of a injected amount of derivative labeled A14 Tyr [125 I] of an insulin to be evaluated has disappeared from the site of injection as measured with a γ-counter external.

The care principles of laboratory animals LYYD without specific pathogen, sows not diabetics, crossing of Danish Landrace, Yorkshire and Duroc, were used (Holmenlund, Haarloev, Denmark) for studies pharmacodynamics and pharmacokinetics. The pigs were aware, 4-5 months of age and weight of 70-95 kg The animals were fasted throughout the night for 8 h before the experiment.

Formulated preparations of derivatives insulins labeled in TyrA14 with125I were injected  sc. in pigs as described above (Ribel, U., Jørgensen, K, Brange, J, and Henriksen, U. The pig as a model for absorption of subcutaneous insulin in man. Serrano-Rios, M and Lefèbvre, P. J. 891-896. 1985. Amsterdam; NY; Oxford, Elsevier Science Publishers. 1985 (Conference Proceeding)).

At the beginning of the experiments a dose of 60 nmol of the insulin derivative according to the invention (composed of test) and a dose of 60 nmol insulin detemir (both marked 125 I in Tyr A14) were injected into two separate sites on the neck of each pig.

The disappearance of the radioactive marker of the injection site sc. was monitored using a modification of the traditional external gamma counting method (Ribel, U. Subcutaneous absorption of insulin analogues. Berger, M. and Gries, F. A. 70-77 (1993). Stuttgart; New York, Georg Time Verlag (Conference Proceeding)). With this modified method it was possible to continuously measure the disappearance of radioactivity from a subcutaneous deposit for several days using device portable wireless (Scancys Laboratorieteknik, Vaerløse, DK-3500, Denmark). The measurements were performed at 1-min intervals, and the values counted were corrected for background activity.

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Example 23 Potency of the relative insulin derivatives of the invention to human insulin

Male Dawley Sprague Rats 238-383 g of weight on the day of the experiment were used for the impingement experiment. The rats had free access to food under controlled environment conditions and were fasted overnight (from 3 pm) before the experiment of grip

Experimental protocol

The rats were set in the animal facilities for at least 1 week before surgical procedure. About 1 week before Tygon catheter clamping experiment were inserted under halothane anesthesia in the jugular vein (for infusion) and carotid artery (for blood sample) and fixed and externalized in the neck. The rats were given Streptocilin vet (Boehringen Ingelheim; 0.15 ml / rat, i.m.) post-surgically and were placed in a unit of animal care (25ºC) during the recovery period. For Get analgesia, Anorphine (0.06 mg / rat, s.c.) was administered During anesthesia and Rimadyl (1.5 mg / kg, s.c.) was administered after complete recovery from anesthesia (2-3 h) and again once a day for 2 days.

The grip technique used was adapted from (1). At 7 am on the day of the experiment, the rats fasted overnight (from 3 pm the day before) were weighed and connected to the sampling syringes and infusion (Harvard 22 Basic, Harvard, and glass syringe pumps Perfectum Hipodermic, Aldrich) and then placed in cages of individual grip where they remained for approx. 45 min before the start of the experiment. The rats were able to move freely in your usual bed throughout the experiment and They had free access to drinking water. After a baseline period of 30 min during which blood glucose levels were measured at 10 min intervals, the insulin derivative that should be evaluated and human insulin (a dose level per rat, n = 6-7 per dose level) were infused (i.v.) to a constant index for 300 min. Glucose levels in blood were measured at 10 min intervals everywhere and the 20% aqueous glucose infusion was adjusted accordingly to maintain euglicemia Samples of resuspended erythrocytes were grouped from each rat and returned in approximately 1/2 ml volumes by means of the carotid catheter.

On each day of the experiment, samples of the solutions of the individual insulin derivatives that should evaluated and the human insulin solution were taken before and at end of the impingement experiments and concentrations of The peptides were confirmed by HPLC. Plasma concentrations of rat insulin and C-peptide as well as the derivative insulin to be evaluated and human insulin were measured in relevant time points before and at the end of the studies. The rats were killed at the end of the experiment using a dose excessive pentobarbital

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Example 24 Pulmonary supply of insulin derivatives to rats

The test substance will be dosed pulmonary by the method of drip instillation. In summary, male Wistar rats (approx. 250 g) are anesthetized in approx. 60 ml of fentanyl / dehydrodenzperidol / - dormicum given as a dose preparatory sc of 6.6 ml / kg and followed by 3 maintenance doses sc of 3.3 ml / kg with an interval of 30 min. Ten minutes after Anesthesia induction, basal samples are obtained from the vein flow rate (t = -20 min) followed by a baseline sample immediately before dosing test substance (t = 0). At t = 0, the Test substance is dosed intratracheally in a lung. A special cannula with a rounded tip is installed in a syringe containing 200 ul of air and test substance (1 ml / kg). By middle of the hole, the cannula is inserted into the trachea and advances in one of the main bronchi - just past the fork. During insertion, the neck is palpated from the outside to ensure intratracheal positioning. The content of the Syringe is injected followed by 2 sec pause. Then the cannula is slowly extracted. Rats are kept anesthetized for the test (blood samples up to 4 or 8 hrs) and are euthanized after the experiment

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References cited in the description This list of references cited by the applicant has been collected exclusively for reader information. Do not It is part of the European patent document. It has been made with the greatest diligence; the EPO however does not assume any responsibility for possible errors or omissions Patent documents cited in the description

WO 9507931 A [0007]

WO 9629344 A [0008]

WO 9731022 A [0009]

WO 2005012347 A [0010]

US 5726038 A [0025]

US 487000 A [0025]

US 4546082 A [0026]

US 4870008 A [0026]

US 5395922 A [0026]

US 5795746 A [0026]

US 5162498 A [0026]

WO 9832867 A [0026]

US 4683202 A [0148]

EP 272097 A [0176]

EP 214826 A [0178]

EP 375437 A [0178]

EP 383472 A [0178]

EP 171142 A [0181]

WO 9010075 A [0181]

WO 9211378 A [0182]

WO 2005012347 A2 [0196]

WO 2006082204 A [0228]

Non-patent bibliography cited in the description

Egel-Mitani et al . YEAST , 1990 , vol. 6, 127-137 [0025]

Thorner et al . The Molecular Biology of the Yeast Saccharomyces cerevisae. Cold Spring Harbor Laboratory , 1981 . 143-180 [0025]

Sambrook , J; Fritsch , EF; Maniatis , T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press 1989 . [0148]

Beaucage ; Caruthers Tetrahedron Letters , 1981 , vol. 22, 1859-1869 [0148]

Matt Matthes et al ., EMBO Journal , 1984 , vol. 3, 801-805 [0148]

Saiki et al. Science , 1988 , vol. 239, 487-491 [0148]

Remington: The Science and Practice of Pharmacy 1995 . [0172] [0173]

B. Faroux-Corlay et al . J. Med. Chem ., 2001 , vol. 44, 2188-2203 [0188]

The pig as a model for subcutaneous insulin absorption in man. Ribel , U .; Jørgensen , K; Brange , J; Henriksen , U. Conference Proceeding. Elsevier Science Publishers , 1985 . 891-896 [0253]

Berger , M .; Gries , FA Conference Proceeding. Georg Thime Verlag , 1993 , 70-77 [0255]

Claims (16)

1. An insulin derivative comprising a progenitor insulin and a substituent, where the substituent is well bound to a ε-amino group of a Lys residue present in the progenitor insulin chain A in position A8, A9; A10; A12; A14; A15; A17; A18; A21; A22; A23 or A24 or a ε-amino group of a residue of Lys in the B chain of the progenitor insulin in position B1, B2, B3, B4; B20; B21 or B22 provided that when B3 is Lys, then B29 is not Glu, where the substituent is of formula general: -W X Y Z where W is: a residue of α-amino acid with an acid group carboxylic in the side chain whose residue forms, with one of its carboxylic acid groups, an amide group with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; or • a chain consisting of two, three or four α-amino acid residues linked together yes through amide bonds, this chain, through a amide bond, binds to the ε-amino group of a residue of Lys present in the A or B chain of insulin parent, the amino acid residues of W being selected from the group of amino acid residues with a neutral side chain and amino acid residues with a carboxylic acid group in the side chain so that W has at least one amino acid residue  having a carboxylic acid group in the side chain; or ? a covalent bond of X to the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; X is: • C O; • CH (COOH) C O-; • -CON (CH 2 COOH) CH 2 C O-; • -CON (CH 2 COOH) CH 2 CON (CH 2 COOH) CH 2 C oO ? -CON (CH 2 CH 2 COOH) CH 2 CH 2 O; • -CON (CH 2 CH 2 COOH) CH 2 CH 2 CON (CH 2 CH 2 COOH) CH 2 CH 2 C O; • -CONHCH (COOH) (CH 2) 4 NH C O-; • -CON (CH 2 CH 2 COOH) CH 2 C O-; or • -CON (CH 2 COOH) CH 2 CH 2 C O On condition that a) when W is an amino acid residue or a amino acid residue chain, carbonyl carbon underlined in X forms an amide bond with an amino group in W, or b) when W is a covalent bond, carbon of the carbonyl underlined in X forms an amide bond with the group ε-amino of a Lys residue present in the A or B chain of the progenitor insulin; And it is: • - (CH 2) m - where m is an integer in the range of 6 to 32; a bivalent hydrocarbon chain comprising 1, 2 or 3 groups -CH = CH- and a number of groups -CH_ {2} - sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; a bivalent hydrocarbon chain of the formula - (CH 2) v C 6 H 4 (CH 2) w  - where v and w are integers or one of these is zero so that the sum of v and w is in the range of 6 to 30 and z is:

?

-COOH;

?

-CO-Asp;

?

-CO-Glu;

?

-CO-Gly;

?

-CO-Sar;

?

-CH (COOH) 2;

?

-N (CH2) COOH) 2;

?

-SO 3 H; or

?

-PO 3 H;

and any complex of Zn 2+ of the same.

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2. An insulin derivative according to claim 1, wherein W is a residue of α-amino acid having 4 to 10 atoms of carbon. 3. An insulin derivative according to claim 1, wherein W is a chain consisting of two amino-residues
acids of which one has 4 to 10 carbon atoms and a free carboxylic acid group while the others have 2 to 11 carbon atoms but no free carboxylic acid group. 4. An insulin derivative according to claim 1, wherein W is a covalent bond. 5. An insulin derivative according to any of claims 1-4, wherein X is -CO- or -COCH (COOH) CO-. 6. An insulin derivative according to any of claims 1-5, where Y is - (CH 2) m where m is an integer in the range of 6 to 32. 7. Insulin derivative according to claims 1-6, wherein Z is -COOH. 8. An insulin derivative according to any of claims 1-7, wherein the insulin Progenitor is an insulin analog. 9. Insulin derivative according to claim 8, where the progenitor insulin is human insulin ArgB29 or human insulin ArgB29desB30. 10. A zinc complex of a derivative insulin according to any of the preceding claims where two zinc ions, three zinc ions, four zinc ions, five zinc ions, six zinc ions, seven zinc ions, eight zinc ions, nine zinc ions, ten zinc ions, eleven ions of zinc or twelve zinc ions are joined by six derivative molecules insulin 11. A pharmaceutical composition for treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any of the claims optionally precedents together with a pharmaceutically carrier acceptable. 12. A method for the production of a pharmaceutical composition according to the claims 1-9 or a zinc complex of an insulin derivative according to claim 10, wherein up to about 12 ions of zinc per 6 insulin derivative molecules are added to the pharmaceutical composition 13. Insulin derivative according to any of the claims 1-9 for use as a medicament optionally together with a pharmaceutically carrier acceptable. 14. Insulin derivative according to claim 13 for pulmonary use. 15. A mixture of an insulin derivative according to any of claims 1-9 and an analog of fast-acting insulin selected from the group consisting of human insulin AspB28; LysB28ProB29 human insulin and insulin human LysB3GluB29. 16. Insulin derivative according to claim 1, where the insulin derivative is selected from the group that consists in human insulin N ^ ε A9} -miristil LysA9 ArgB29 desB30, human insulin N ^ ε B3} -miristil LysB3 ArgB29 desB30, human insulin N ^ ε B22} -miristil LysB22 ArgB29 desB30, human insulin N ^ ε A15} -miristil LysA15 ArgB29 desB30, human insulin N ^ ε A18} -miristil LysA18 ArgB29 desB30, human insulin N ^ ε A22} -miristil LysA22 ArgB29 desB30, human insulin N ^ ε A9} -? -Carboxypentadecanoyl-? -Glu LysA9 ArgB29 desB30, human insulin N ^ ε B3} -? -Carboxypentadecanoyl-? -Glu LysB3 ArgB29 desB30, human insulin N ^ ε B22} -? -Carboxypentadecanoyl-? -Glu LysB22 ArgB29 desB30, human insulin N ^ ε A15} -? -Carboxypentadecanoyl-?-Glu LysA15 ArgB29 desB30, human insulin N ^ ε A1B} -? -Carboxypentadecanoyl-? -Glu LysA18 ArgB29 desB30, human insulin N ^ ε A22} -? -Carboxypentadecanoyl-? -Glu LysA22 ArgB29 desB30.