Method for preparation of insulin compounds

FIELD: medicine.

SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.

EFFECT: preparation of insulin compound from its precursor by efficient improved method.

24 cl, 5 ex

 

The present invention relates to an improved method of making a precursor of insulin in insulin, not necessarily through the ether of insulin.

PREREQUISITES TO the CREATION of INVENTIONS

Insulin is a pancreatic hormone involved in the regulation of glucose concentration in blood. For example, insulin-dependent patients with diabetes to monitor glucose concentration in blood, a dose of insulin human, pig, bull, insulin analogs and premixed insulin.

Insulin pig or bull usually get from the pancreas. Human insulin can be obtained of semisynthetic insulin pigs. Alternatively, human insulin, as well as many insulin analogs can be obtained using genetic engineering. By genetic engineering, which can be, for example, carried out by bacteria or yeast, the resulting precursor of insulin subsequently converted into the desired product. This transformation can be done in different ways.

One option is the so-called transpeptidase, in which cleavage of the peptide and the connection of the peptide occurs sequentially in the same reaction mixture, under the same reaction conditions, see, for example, U.S. patent No. 4343898 (Novo Industri).

Another option is a first article is Hai cleavage of the precursor of insulin, see, e.g., Hoppe-Seyler's Z. Physiol. Chem. 359 (1978), 799, then the selection of the intermediate product, and then carrying out the desired interaction with the reaction mixture, different from the one used in the first stage, see, e.g., Nature 280 (1979), 412.

In accordance with EP 87238, the reaction transpeptidase carried out in a solvent system containing from about 75% to 97% (V/V, at least one non-aqueous miscible with the reaction mixture, the solvent comprising at least about 50% (V/V) butane-1,4-diol.

In accordance with US 4579820 process transpeptidase performed using the enzyme L-specific serine carboxypeptidase, such as carboxypeptidase Y.

In accordance with US 4601979 (Nordisk Insulinlaboratorium), transpeptidase or only peptide interaction is conducted in an aqueous reaction medium essentially free from organic solvent.

In accordance with WO 83/00504 (Nordisk Insulinlaboratorium), pork product was treated with carboxypeptidase A, the resulting des-alanyl-B30 insulin product suspended in the lower alcohol and the suspension was mixed with a solution of the ester of L-threonine and trypsin. The individual examples were allocated des-alanyl-B30 insulin product or by freeze-drying or by precipitation.

The purpose of this invention is to eliminate or reduce, at the ore, some of the shortcomings of the prior art. Therefore, not all described in more detail the deficiencies listed below, can be completely eliminated or reduced.

DEFINITION

Used herein, the term "amino acid" refers to amino acids that can be encoded by nucleotide sequences. Similarly, this applies to the expression of the amino acid residue constituting the amino acid in which the carboxyl group has been removed hydroxy-group and/or amino group has been removed hydrogen.

Similarly, expression of the peptide and peptide residue composed of amino acid residues. Preferably, the peptide contains no more than 10 amino acid residues.

Used herein, the expression amide amino acid refers to an amino having optionally substituted C-terminal carboxamide group.

Used herein, the expression amide peptide refers to a peptide having optionally substituted C-terminal carboxamide group.

Used herein, the term "precursor of insulin" refers to the polypeptide consisting of two peptide chains (corresponding to a and b chains of insulin, and is further marked by a and b chains) which, like insulin, are connected to each other by two disulfide bridges (one cysteine residue (Cys) to each the mu cysteine residue) between the two peptide chains, and where, as in the insulin, there is a disulfide bridge between one cysteine residue in the chain And another cysteine residue in chain A. the precursor of insulin has at least one residue of lysine or arginine In the chain. Optionally, the precursor of the insulin chains a and b are connected to each other through a third peptide chain (corresponding to the connecting peptide in insulin) between the end of the chain and the N-end And chain. In the case when a and b chains connected to each other through the third peptide chain, With the end of the third peptide is lysine. Optionally, the precursor of insulin with N-end circuit can be connected to the fourth peptide chain. In the case when this fourth peptide chain is linked to the N-end In the chain, With the end of the fourth peptide chains is lysine. Moreover, this precursor of insulin identity with amino acid residues of the human insulin is at least 80%, preferably at least 85%, more preferably at least 90% and even more preferably at least 95%, provided that the third and fourth peptide chains are not taken into account for these calculations. In the human insulin has disulfide bridges between CysA6and CysA11between CysA7and CysB7and between CysA20and CysB19and Lisi is in position B29.

Used herein, the expression "ether amino acids" refers to amino acid bearing a C-terminal protected carboxyl group and, optionally protected hydroxyl group.

Used herein, the expression "ether peptide" refers to a peptide in which at least the C-terminal carboxyl group is protected carboxyl group. Optional, any hydroxyl group is protected and, optionally, the ε-amino group of any lysine residue modified, preferably a hydrophobic group, such as acyl group having at least 10 carbon atoms. Preferably, the ester of the peptide contains no more than 10 amino acid residues.

Used herein, the term "nucleophilic compound" refers to ether, amino, amide amino acid, peptide, ether peptide and amide peptide. In any of these esters of amino acids, amides of amino acids, peptides, esters of peptides and peptide amides, amino group, any group of lysine optionally modified, preferably a hydrophobic group, such as acyl group having at least 10 carbon atoms.

Used herein, the terms "insulin compound" refers to insulin any species, such as porcine insulin, bovine insulin, human insulin, and their salts, such as zinc salts and Protamine salt. Moreover, using the my here the expression of the insulin compound" refers to, that brief could be called "insulin analogues". Insulin analogs used here, refers to insulin compounds in which one or more amino acid residues have been substituted by other amino acid residues and/or one or more amino acid residues have been deleted, and/or to which one or more amino acid residues have been added, provided that the insulin analogue has sufficient insulin activity. Examples of insulin analogues are described in the following patents and equivalents thereto: US 5618913; EP 254516; EP 280534; US 5750497; and US 6011007. Examples of individual insulin analogues are insulin-aspart (i.e. [AspB28] human insulin), insulin-lispro (i.e. [LysB28ProB29] human insulin) and insulin glargine (i.e. [GlyA21,ArgB31,ArgB32] human insulin). Used herein, the term "insulin analog" also includes what could be called the derivative of insulin, i.e. compounds that are specialist in this field as a whole may be considered as derivatives of insulin, see General guidelines, such as insulin, containing Deputy, missing in the original molecule of insulin. Examples of derivatives of insulin is insulin or insulin analogs having optionally substituted carboxamido group. Also compounds that can be considered and ka is derived insulin, and as insulin analogs, here covered by the term "insulin analog". Examples of such compounds are described in the following patents and equivalents thereto: US 5750497 and US 6011007. Therefore, an additional example of the insulin analog is insulin-detemir (i.e. des-ThrB30the human insulin γ LysB29the deletion). Insulin compounds obtained by the present invention possess antidiabetic activity, high enough for use in the treatment of diabetes. Antidiabetic activity can be determined using the so-called analysis of free fatty cells.

Used herein, the term "pH" refers to a value measured with a pH meter by dipping calomel combined glass electrode connected to a pH meter directly into the solution, which measure the pH value. pH Meter calibrated water standard buffer.

SEQUENCE LISTING

SEQ ID NO.: 1 represents a part of the peptide molecule of Glu-{Glu-Ala)3-Pro-Lys; SEQ ID NO.: 2 represents a part of the peptide molecule Glu-Glu-Gly-Glu-Pro-Lys-; and SEQ ID NO.: 3 represents a part of the peptide molecule Gly-Phe-Phe-Tyr-Thr-Lys-Pro-Thr.

BRIEF description of the INVENTION

The present invention relates to a method for producing insulin compounds. These joint insulin may be used as medicines. In a preferred embodiment of the present invention receive connections insulin with threonine (Thr) at the s-end In a chain.

Any expert in this field, for example a doctor can determine the dose of insulin compounds be administered to a patient with diabetes and when.

The starting material of the method according to this invention is the precursor of insulin, which is subjected to as peptide cleavage and peptide binding, good conditions for both reactions, but where no allocation of the intermediate product. In other words, the precursor of insulin is subjected to peptide cleavage, and the resulting product, i.e. the intermediate product is subjected to peptide binding. Conditions conducive to cleavage of peptide bonds identical conditions favorable for the peptide linkage. Therefore, in the first stage of the present invention, i.e. the stage of splitting or cleavage reaction conditions for the reaction in the reaction mixture is chosen so that they are conducive to cleavage of peptide bonds, and in the second stage of the present invention, i.e. the stage of joining, or the reactions of addition, the conditions for the reaction in the reaction mixture changed so that they are favorable for the formation of ties.

In one preferred embodiment of the present invention the precursor of insulin in the first stage is dissolved in a predominantly aqueous medium and add the enzyme used for digestion. This reaction mixture may contain no or essentially does not contain an organic solvent. Alternatively, the reaction mixture may contain a certain amount of organic solvent, which can ensure adequate solubility of the precursor of insulin. However, it is undesirable to use an organic solvent in amounts that may undesirable effects on enzymatic cleavage. In the first stage of the method according to this invention the reaction parameters, such as pH value, temperature and time chosen so that they are conducive to cleavage at residue(s) of lysine or residue(s) of arginine.

When the cleavage reaction was to a certain, desired extent, the reaction mixture is mixed nucleophilic compound and an organic solvent (without isolation of intermediate product), so that there was an interaction of nucleophilic substances with the residue of lysine or arginine of the desired intermediate product. At this stage, the parameters of the reaction set so that they who went to the reactions proceed. In a preferred embodiment of the invention the nucleophilic substance is an ester of amino acids such as threonine ester or ether peptide.

Subsequently, the protective group(s), if required, can be removed from the resulting substance.

In comparison with the known reaction transpeptidase advantages obtained when using the method according to this invention, consist in a shorter total time of reaction with the same amount of enzyme and similar or higher yield. Compared to the reaction carried out in two reactors with splitting in the aquatic environment, the selection of the intermediate product and conduct of joining in a mixture of organic solvent and water, the benefits obtained by the method according to this invention, consist in a shorter total time of reaction, the use of smaller amounts of enzyme and easier the flow of the process.

More precisely, this invention relates to the following options for implementation.

DETAILED description of the INVENTION

As can be seen from paragraph 1, first, a cleavage of peptide bonds, and then the reaction occurs join.

Briefly, the cleavage reaction (i.e. enzymatic cleavage) are as follows.

Enzymatic cleavage of the previous the nick of insulin (i.e. peptide cleavage) occurs in the reaction mixture containing at least about 55%, preferably at least about 60%, more preferably at least 70% water (wt./wt.).

In a preferred embodiment of the present invention, the concentration of the precursor of insulin in the reaction mixture, where the enzymatic cleavage is at least 2%, preferably in the range of about 5 to 10% (weight/volume).

The cleavage reaction is carried out in a neutral or alkaline medium, preferably having a pH value in the range of from about 6 to 11, more preferably in the range of from about 8 to 10.

In the preferred embodiment of this invention the amount of enzyme compared to the number of precursor of insulin is in the range from about 0.05 to about 5% (wt./wt.), preferably from about 0.1 to about 2%.

No matter what type trypsinogen enzyme used in this invention. Trypsin is a well characterized enzyme available in high purity, especially bovine or porcine origin. From a microbial source can be obtained protease I Acromobacter lyticus (hereinafter designated ALP). Moreover, the enzyme form, whether it is native enzyme or active is th immobilized enzyme or derivative of an enzyme, is not a matter for the implementation of the present invention. If you want cleavage on the carboxyl group of arginine may be used trypsin, if you want cleavage on the carboxyl group of lysine, can be used with either trypsin or ALP. For cleavage on the carboxyl group of lysine is preferred ALP.

As examples of active derivatives of the enzyme can be mentioned acetylated trypsin, succinylcholine trypsin, trypsin, treated with glutaraldehyde, and immobilized trypsin or derivatives ALP.

When using immobilized trypsin or ALP it can be suspended in the reaction mixture or they may be filled in the column.

To a large extent the activity of the enzyme is controlled by the ratio of the content of water and solvent, pH and reaction temperature. Increasing the concentration of organic solvent in the reaction mixture and reducing the pH to approximately neutral shifts the normal enzymatic reaction from splitting in the direction of the interaction. Lowering the temperature reduces the reaction rate, but also can reduce the formation of by-product and denaturation of the enzyme.

In a preferred embodiment of the present invention the precursor of insulin is dissolved in water, and eUSA the concentration of acetate ions in the range from about 5 mm to 500 mm, preferably in the range from about 20 mm to 200 mm. For example, there can be used sodium acetate, potassium, ammonium or acetate of triethylamine.

In accordance with one embodiment of the present invention the precursor of insulin (which is a peptide) may be represented by the following General formula I:

where Zn and Zm is independently from each other represent two peptide part of the molecule, each containing n and m amino acid residues, respectively, R1represents a peptide residue, which optionally contains a residue of lysine or arginine, R2represents an amino acid residue or a peptide residue, R3represents a peptide residue, which optionally contains a residue of lysine or arginine, R4represents the residue of lysine or arginine, or a residue of the peptide, which contains a residue of lysine or arginine, or R1and R4together represent a peptide residue containing the residue of lysine or arginine, the two vertical lines show the disulfide bond between two cysteine residues and, in addition, there is a disulfide bond between two cysteine residues located in R1and Zn.

Preferably, amino acid residues in the precursor of the insulin of the formula I, are those to whom that can be encoded by nucleotide sequences.

In accordance with a preferred embodiment of the invention using the precursor of insulin, in which the number of amino acid residues in

R1and R4together is in the range of from about 8 to 50. In another preferred embodiment of the present invention Zn contains a 12 amino acid residues. In another preferred embodiment of the present invention Zm contains 11 amino acid residues. In another preferred embodiment of this invention R2contains 1 amino acid residue. For example, Asn or Gly. In another preferred embodiment of this invention R3contains 6 amino acid residues.

In a preferred embodiment of the present invention the precursor of insulin is a predecessor with a single chain, i.e. the compound of formula (I), where R1and R4together represent a peptide residue containing the residue is lysine or arginine. Therefore, preferably, the precursor of insulin is an insulin other than insulin mammals, such as porcine insulin, rabbit insulin, insulin dog or insulin whale.

In accordance with another embodiment of the present invention the precursor of insulin of the formula I contains the same amino acid is basic residues in positions A1 through A21 and in positions B1 through B29, which are present in human insulin in the same positions.

In accordance with another embodiment of the invention, the precursor of the insulin of the formula I contains the same amino acid residues in positions A1 through A21 and provisions with B1 on V provided that the amino acid residue in position B28 is an Asp.

In accordance with another embodiment of the invention, the precursor of the insulin of the formula I contains the same amino acid residues at positions A1 through A21 and in positions B1 through B29, which are present in human insulin in the same positions, provided that the amino acid residue in position B28 is a Lys and the amino acid residue in position B29 is a Pro.

In accordance with another embodiment of the invention, the precursor of the insulin of the formula I contains the same amino acid residues at positions A1 through A21 and in positions B1 through B29, which are present in human insulin in the same positions, provided that the amino acid residue in position A21 is a Gly and the amino acid residues in positions B31 and B32 both are Arg.

Examples of individual precursors of insulin, which can be used in the method according to the present invention are human proinsulin; proinsulin monkeys; [Ala31,Lys32 ]-des{33-63) proinsulin pigs; proinsulin pigs; [Asp28]-des(30-65) human proinsulin, elongated N-end Glu-(Gtu-Ala)3-Pro-Lys- (SEQ ID NO.:1); and [Asp28,Met30,Trp31,Lys32]-des(33-65) human proinsulin, elongated at the N-end-Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID No. 2).

The precursors of insulin of the formula I can be obtained as described or similar to that described in the international applications published under the numbers WO 01/49742, WO 01/49870, WO 01/079250, and WO 02/079254, the contents of which are incorporated herein by reference.

The desired intermediate product (i.e. a target cleavage product) corresponds to the precursor of insulin, where at least one lysine residue or an arginine was split with the formation of litelnogo or orginalnego radical, respectively. In addition, in the desired intermediate product chains a and b, connected to each other via two disulfide bridge, not connected to each other through peptide chain between the end of the chain and the N-end And chain. In a preferred embodiment of the present invention, the number of amino acid residues present in the desired intermediate product, is in the range of from about 48 to 52, preferably in the range of from about 49 to 51, even more preferably 50. In another preferred embodiment of the present invention in the desired promezhutochnaya is not more than 4, preferably not more than 3, more preferably not more than 2 and even more preferably no more than 1 amino acid residues that are missing in the relevant provisions of human insulin.

In accordance with one embodiment of the present invention the desired intermediate product (the desired product splitting) can be represented by the General formula II

where Zn and Zm is independently from each other represent two peptide part of the molecule, each containing n and m amino acid residues, respectively, R'1represents a peptide residue, R'2represents an amino acid residue or a peptide residue, R'3represents a peptide residue, R'4represents the residue of lysine or arginine, or a peptide containing a lysine residue or an arginine at the C-end, two vertical lines indicate disulfide bonds between two cysteine residues and, in addition, there is a disulfide bond between two cysteine residues present in R'1and Zn.

In a preferred embodiment of the present invention R'1represents amino acid residues A1 to A6 in human insulin in that order, where optionally one or two amino acid residue can be replaced with another amino acid residue or where one or D. the and amino acid residue no. In another preferred embodiment of this invention R'2represents-Asn or Gly. In another preferred embodiment of this invention R'3represents amino acid residues from B1 through B6 in human insulin in that order, where optionally one or two amino acid residue can be replaced with another amino acid residue, or one or two amino acid residue no. In another preferred embodiment of this invention R'4amino acid residues with on B20 B29 in human insulin in order, provided that the residue in position B28 is Asp, and in the position B is Lys and the amino acid residues with in20 on B28 in human insulin in order provided that the residue in position B28 is Lys, each of which optionally one or two amino acid residue can be replaced with another amino acid residue, or one or two amino acid residue is absent, or where the part of any of these peptide residue does not contain one or more contiguous C-terminal amino acid residues. In another preferred embodiment of the present invention Zn represents amino acid residues with A8 through A19 in human insulin in that order, where optionally one or two amino acid residue is replaced on whim amino acid residue, or one or two amino acid residues not present. In another preferred embodiment of the invention Zm represents amino acid residues with B8 on B18 human insulin in that order, where optionally one or two amino acid residues are replaced with another amino acid residue, or one or two amino acid residue is not present.

For the cleavage reaction, and the reactions of addition, the reaction temperature is in the range from the freezing point of the reaction mixture to about 50°C. the Preferred temperature is in the range from about 0°to about 25°C.

Briefly, the reaction of the interaction is as follows.

The splitting of at least about 25%, preferably at least 50%, more preferably at least 75%, preferably at least 85%, more preferably at least 95%, the precursor of insulin to the desired intermediate product of nucleophilic substance, on the one hand, and an organic solvent, on the other hand, is mixed with the reaction mixture, which was splitting, thus, to obtain the reaction conditions that are suitable or favourable stage of the interaction. The percentage of cleavage (conversion) based on the equilibrium potential in the reaction mixture used for splitting. Usually, since the beginning of the reaction f is rotating cleavage and before the expiration of a certain period of time, the yield of the desired intermediate product, i.e. the desired product splitting increases and reaches a maximum concentration. In consequence, the concentration of the desired cleavage product may decrease.

In a preferred embodiment of the present invention from the reaction mixture does not remove the components resulting from the cleavage reaction before reaction occurs accession. A simple way to implement this is to add after the cleavage reaction of nucleophilic substances and a sufficient amount of an organic solvent. In this way, for example, the enzyme is used at the stage of cleavage, is also used at the stage of connection.

The method according to this invention also covers the reactions proceed in the reaction mixture, which in addition to the desired intermediate product contains a small amount of partially cleaved precursor of insulin and/or unreacted precursor of insulin.

In another preferred embodiment of the present invention the nucleophilic substance is an amide amino acid or amide peptide, where carboxamidine group is unsubstituted or mono - or disubstituted by alkyl group with not more than 16 carbon atoms, this alkyl group(s), together with the adjacent atom is m nitrogen, may form a ring or carboxamide group is mono - or disubstituted aryl group. Preferred are aliphatic substituents. Examples of substituted carboxamide groups are N,N-dimethylcarbamate, N,N-diethylcarbamoyl and N-hexylcaine.

In a preferred embodiment of the present invention the nucleophilic compound is an ester of the amino acid where the carboxyl group is protected and any hydroxy-group is not necessarily protected. In another preferred variant of the invention, the nucleophilic compound is an ester of threonine, where the carboxyl group is protected, and optionally protected hydroxyl group. Therefore, the ester of L-threonine can be represented by the following General formula IIIa:

where R6represents a protective group of carboxyl and R5represents hydrogen or a protective group of hydroxyl. For clarity, the ester of threonine can be illustrated by the General formula CH3-C(OR5)-CH(NH2)COOR6where R5and R6defined above.

There are several nucleophilic compounds and other nucleophilic compounds can be obtained by analogy with getting known compounds or by analogy with known methods.

Nuclei the global connections can be used in the form of free bases or their soluble salts, such as hydrochloride, acetates, propionate and butyrate.

When the reaction begins attaching, preferably in solution interacting reaction mixture was attended by a significant excess of nucleophilic compounds with a molar ratio of nucleophilic compound and the desired intermediate product, preferably greater than 5:1. At the beginning of the reaction joining the concentration of the nucleophilic compound in the reaction mixture preferably should be less than 0.1 M, the highest concentration that allows solubility.

To obtain a 60% yield, proposed here as an important aspect for the practical application of this invention, the reaction temperature, water content and pH value are interrelated in the described intervals.

Organic solvents suitable for use in this invention are polar solvents that are mixed with water and preferably those which can contain high concentrations of the desired intermediate product (for example of the formula (II) and nucleophilic compounds. Examples of suitable organic solvents are aprotic solvents such as N,N-dimethylformamide, N,N-dimethylacetamide, N-organic-2 and dimethylsulfoxide, and proton solvents, such as acetic acid, ethanol, is milovy alcohol, 2-propanol, 1-propanol, butanol and 1,4-butanediol. Can also be used dioxane, acetone, tetrahydrofuran, formamide and acetonitrile, and even the air of amino acids used as the nucleophilic compound may wholly or partially be used as organic solvent. The nature of the solvent affects the system as a whole, and the relationships that are appropriate for a single solvent, producing high outputs may not be used with other solvents. The best yields were obtained with aprotic solvents, and aprotic solvents are preferred for the practical implementation of this invention.

Obviously, when calculating or determining the water content in the reaction mixture nucleophilic compound is considered as an organic solvent.

Adding acid, such as hydrochloric acid, formic acid, acetic acid, propionic acid or butyric acid, or bases, such as pyridine, TRIS, N-methylmorpholine, triethylamine or N-ethylmorpholine, is optional. They are included in the reaction mixture to achieve a suitable pH. Although in the practical application of the present invention can be used mineral acids or bases, preferred are organic acids and bases, often the spine of the above. The most preferred organic acid.

At the beginning of the reaction merger mass ratio of trypsin or ALP (based on crystalline trypsin or ALP or corresponding to this number is derived trypsin or ALP) and the desired intermediate product in the reaction mixture preferably is in the range of from about 1:1000 to about 1:10, more preferably in the range of from about 1:200 to about 1:50.

In some cases, the enzyme is added at the stage of cleavage, enough for the reaction of accession, and in this case it is not necessary to add additional amounts of the enzyme during the addition step. In other cases, at the stage of accession may want to add additional quantities of the enzyme.

Because of the high concentration of the desired intermediate product and nucleophilic compounds in solution contribute to the high rate of transformation, the choice of solvent biased focus on those solvents in which the reactants are soluble to a high degree. Especially important solubility of nucleophilic compounds, since this reagent should be present in high concentrations. At the beginning of the reaction accession molar ratio of nucleophilic compounds to the desired intermediate product preferably has revisit 5:1, preferably exceed 50:1. At the beginning of the reaction joining the concentration of the nucleophilic compound in the reaction mixture is preferably at least 0,1 M

In a preferred embodiment of the present invention use a nucleophilic compound having a protective group(s) on the carboxyl group that can be removed from the resulting insulin compound under conditions that do not cause significant irreversible changes in the insulin molecule. As examples of such protective groups carboxyl can be mentioned lower alkyl, for example methyl, ethyl and tert-butyl, substituted benzyl groups such as p-methoxybenzyl, diphenylmethyl and 2,4,6-trimethylbenzyl, and a group of General formula-CH2-CH2-SO2R7where R7represents lower alkyl, such as methyl, ethyl, propyl and n-butyl.

Suitable protective groups of hydroxyl are those that can be removed under conditions that do not cause significant irreversible changes in the insulin molecule. As an example of such a group may be mentioned tert-butyl.

In addition, commonly used protective groups described in Wunch: Metoden der Organischen Chemie (Houben-Weyl), Vol. XV/1, editor: Eugen Muller, Georg Thieme Verlag, Stuttgart 1974.

In accordance with one embodiment of the present invention, in the implementation of the Oia method according to this invention produces compound of General formula IV:

where Zn and Zm is independently from each other represent two peptide part of the molecule, each containing n and m amino acid residues, respectively, R'1represents a peptide residue, R'2represents an amino acid residue or a peptide residue, R'3represents a peptide residue, R'4mentioned above, and R'6is an amino acid that carries carboxyl protective group or a peptide residue, optionally bearing a protective group carboxyl.

Any protective group carboxyl (for example, R6and any protective group of hydroxyl (for example, R5), which is present in insulin compounds may be removed by known methods or by methods known in essence. In that case, when the protective group is a methyl, ethyl or a group of General formula-CH2-CH2-SO2R7where R7identified above mentioned protective group can be removed in alkaline conditions in an aqueous medium, preferably at pH values in the range of from about 8 to about 12, for example at about a 9.5. As the base can be used strong bases, for example tertiary amines, such as triethylamine, hydroxides of alkaline earth metals such as calcium hydroxide or magnesium. When the carboxyl C the protective group is a tert-butyl, substituted benzyl such as p-methoxybenzyl or 2,4,6-trimethylbenzyl or diphenylmethyl, this group can be removed by acidolysis, preferably using triperoxonane acid. Triperoxonane acid may be anhydrous or may contain some water, or it may be diluted with an organic solvent, such as dichloromethane. When the hydroxyl protective group (for example, R5) is a tert-butyl specified group can be removed using acidolysis, see above.

Preferably, the received insulin compounds have a protective group of hydroxyl.

In a preferred embodiment of the invention the method according to the present invention the precursor of insulin (e.g., formula I) is transformed into insulin connection (e.g., formula IV)having a protective group carboxyl on the C-end amino acid residue In the chain, which can then be removed with the formation of the insulin compound having no protective group carboxyl.

When selecting the reaction conditions in accordance with the above explanations and taking into account the results obtained in the following examples, it is possible to obtain an output of insulin connection above 60%, and even above 80% and under certain preferred conditions above 90%.

The method according to N. the present invention can be obtained insulin compounds of acceptable purity and if you want, additionally purified for therapeutic purposes.

More specifically, insulin-aspart may, for example, be obtained by enzymatic cleavage using ALP precursor of insulin, such as [Asp28]-des(30-65) human proinsulin, elongated at the N end of the Glu-(Giu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), and interaction with nucleophilic compound, such as methyl ester of L-threonine with subsequent hydrolysis.

Insulin-lispro may be, for example, obtained by enzymatic cleavage of a precursor, such as porcine insulin, using trypsin and interaction with nucleophilic compound, such as Gly-Phe-Phe-Tyr-Thr-Lys-Pro-Thr (SEQ ID NO.: 3).

Insulin-glargine, for example, can be obtained by enzymatic cleavage of the precursor of insulin, such as [Gly86]-des(30-65) human proinsulin using ALP and interaction with nucleophilic compound, such as Thr-Arg-Arg-OMe, followed by hydrolysis.

Abbreviations used here correspond to the rules adopted by the (1974) IUPAC-IUB Commission on biochemical nomenclature, see Collected Tentative Rules & Recommendations of the Commission on Biochemical Nomenclature IUPAC-IUB, 2ndedition, Maryland 1975.

The mention here of links is not recognized components of the prior art.

Here the word "hold" has a wide meaning "include", "contain" or "containing the in-itself" (see guide EPO With 4.13).

The following examples are offered to illustrate, not limit.

Example 1

200 mg [Ala31, Lys32]-des(33-63) of proinsulin pigs suspended 1.35 ml of water and the pH was brought to 9 with 10 μl of triethylamine. At low stirring was added a mixture of 375 μl of N,N-dimethylacetamide and 460 μl of water and the resulting solution was added 315 μl of 5.4 mg/ml aqueous solution of Achromobacter lyticus lysyl-specific protease (EC 3.4.21.50) (denoted here ALP). The pH value was brought to 9.8 using 20 μl of triethylamine and the reaction solution containing 78% of water, left for 1 hour at 23°C. the Reaction solution was acidified by addition of 70 μl of 4 n hydrochloric acid and cooled in an ice bath. Solution was added 300 mg of methyl ester of L-threonine is 4.85 ml of N,N-dimethylacetamide and the pH was brought to 6.5 by addition of 450 μl of 4 n hydrochloric acid. The reaction solution containing 32% of water, left for 4 hours at 23°C, after which the reaction was stopped by adding hydrochloric acid to pH<3. Using HPLC analysis with reversed-phase column (4 mm × 250 mm 5 μm C18 with silicon dioxide with eluent ethanol-water containing 0.125 M ammonium sulfate, brought to pH 4, after a total reaction time of 5 hours was installed 86% yield conversion into methyl ester of human insulin.

For comparison, spent one the landmark transformation:

100 mg [Ala31, Lys32]-des(33-63) of proinsulin pigs suspended in a mixture 887 μl of water and 175 μl of N,N-dimethylacetamide. 150 mg of methyl ester of L-threonine was dissolved in 2,265 ml of N,N-dimethylacetamide and slowly added to ice mixture. The pH was brought to 6.5 with 340 μl of acetic acid was added 158 μl of 5.4 mg/ml aqueous solution of ALP. The reaction conversion was controlled by HPLC-analysis of the acidified samples. After 5 hours it was found 53% conversion to methyl ester of human insulin, and after 24 hours, the conversion reached a maximum of 87%.

Dedicated methyl ester of human insulin was converted into human insulin by dissolving in water at a pH value of 10 with a concentration of 10 mg/ml the Reaction was stopped after 24 hours, bringing the pH to 5.2 1N hydrochloric acid, and precipitated the human insulin were isolated by centrifugation and purified by high-performance liquid chromatography with reversed phase.

During the same period of time, i.e. for 5 hours, the method according to this invention in comparison with known essentially the way, was increased by 62%. Two ways could almost equal the outputs, if the reaction time is essentially the well-known single-stage method of conversion was increased almost 5 times compared to the reaction time for the method according to this invention.

<> Example 2

200 mg of insulin pigs suspended at 1.37 ml of water and prelabor stirring was added a mixture of 294 μl of N-methyl-2-pyrrolidone and 326 μl of water. The pH value was brought to 9.0 with 10 μl of 2n sodium hydroxide and the resulting solution was added 315 μl of 5.4 mg/ml aqueous solution of ALP. The pH value was brought to 9.8 using 12 μl of 2n sodium hydroxide and the reaction solution containing 80% water, was left for 4 hours at 23°C. the Reaction solution was acidified by addition of 70 μl of 4 n hydrochloric acid and cooled in an ice bath. A solution of 300 mg of methyl ester of L-threonine 4.4 ml of N-methyl-2-pyrrolidone was acidified with 500 μl of 4 n hydrochloric acid. Was slowly added a solution of insulin and the pH was brought to 6.5 with 50 μl of 2n hydrochloric acid. The reaction solution containing 34% of the water was left for 4 hours at 23°C, after which the reaction was stopped by adding hydrochloric acid to pH<3. Using HPLC analysis with reversed-phase column (4 mm × 250 mm 5 μm C18 with silicon dioxide with eluent ethanol-water containing 0.125 M ammonium sulfate, brought to pH 4, after a total reaction time of 8 hours was a degree of conversion to methyl ester of human insulin 86%.

For comparison conducted one-step transformation:

100 mg of insulin pigs suspended in a mixture 848 ál water and 147 μl of N-methyl-2-pyrrolidone. 150 mg metrov the th ether of L-threonine was dissolved in 2.2 ml of N-methyl-2-pyrrolidone and slowly added to ice mixture. The pH was brought to 6.5 300 μl of acetic acid was added 158 μl of 5.4 mg/ml aqueous solution of ALP. The reaction solution was left at 23°C. the Reaction conversion was controlled by HPLC-analysis OF the acidified samples. After 8 hours set the transformation to 54% and 48 hours was achieved to a maximum of 86% conversion to methyl ester of human insulin.

Dedicated methyl ester of human insulin can be converted into human insulin by using alkaline hydrolysis.

During the same period of time, i.e. for 8 hours, the method of the present invention was increased by 59%, compared with the known substantive way. Two methods turned out identical outputs, if the reaction time is known essentially a single-stage method of conversion was increased 6-fold compared to the reaction time for the method according to this invention.

Example 3

200 mg [Asp28]-des(30-65) human proinsulin, elongated at the N-end peptide Gin-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), suspended 1.35 ml of water. Under mild stirring was added a mixture of 350 μl of N,N-dimethylformamide and 425 μl of water and the pH was brought to 9 with 45 μl of triethylamine. To the resulting solution were added 200 μl of 8.5 mg/ml aqueous solution of ALP and the pH value was brought to 9.8 20 μl of triethylamine. The reaction solution containing 76% of the water was left for 1 hour at 23°C. auktsionnyi the solution was acidified by addition of 70 μl of 4 n hydrochloric acid and cooled in an ice bath. Solution was added 300 mg of methyl ester of L-threonine of 4.95 ml of N,N-dimethylformamide and the pH was brought to 6.5 by adding 470 μl of 4 n hydrochloric acid. The reaction solution containing 30% of water, left for 4 hours at 23°C, after which the reaction was stopped by adding hydrochloric acid to pH<3. Using HPLC analysis with reversed-phase column (4 mm × 250 mm 5 μm C18 with silicon dioxide with eluent ethanol-water containing 0.125 M ammonium sulfate, brought to pH 4, after a total reaction time of 5 hours was a degree of conversion of 87% [AspB28]-methyl ester of human insulin.

For comparison conducted one-step transformation:

90 mg [Asp28]-des(30-65) human proinsulin, elongated at the N-end of the peptide Glu-(Glu-Ala)3-Pro-Lys- (SEQ ID NO.: 1), suspended in a mixture 887 μl of water and 175 μl of N,N-dimethylformamide. 150 mg of methyl ester of L-threonine was dissolved 2.13 ml of N,N-dimethylformamide and slowly added to ice mixture. The pH was brought to 6.5 250 µl of acetic acid was added 118 ál 8.5 mg/ml aqueous solution of ALP. The reaction conversion was controlled by HPLC-analysis OF the acidified samples. After 5 hours it was determined transformation to 47% and after 24 hours the transformation in [AspB28]-methyl ester of human insulin reached a maximum of 81%.

Dedicated methyl ether insulin human is the AC can be turned into [Asp B28]-human insulin using alkaline hydrolysis.

For the same reaction time that is 5 hours, the method of the present invention was almost doubled compared with the method known by the creature. Comparable outputs were obtained in two ways, if the reaction time is known for being one-step conversion was increased approximately 5-fold compared with the response time for the method according to this invention.

Example 4

1.5 g of the precursor of the insulin-aspart

[Asp28, Met30, Trp31, Lys32]-des(33-65)human proinsulin, elongated at the N-end of the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) suspended in 3.5 g of water. Easy stirring and at ambient temperature dissolved the predecessor gradual addition of 4 M sodium hydroxide to pH 10,67. Added 3.7 g of 45% (mass/mass) solution of ethanol in water. Was added 1.5 ml of 5.8 mg/ml aqueous solution of ALP and a mixture containing 69% water, left to interact for 2 hours. The pH value was brought to 4.7 by addition of 4 n hydrochloric acid. 2,025 g of ethyl ester of L-threonine was dissolved in 16.2 ml of ethanol and this solution was added at a maximum temperature of 15°C. the pH was brought to 6.5 with 4 n hydrochloric acid. The temperature was brought to ambient temperature and the reaction mixture containing 33% of water, left for 20 hours at this temperature. By means of analysis HPLC with reversed-phase column (4 mm × 250 mm 5 μm C18 with silicon dioxide with eluent acetonitrile-water, containing 200 mm sodium sulfate, pH value, increased to 3.6, after 1 hour, the reaction time was set the degree of transformation of 89.1% in ethyl ether insulin-aspart and after 20 hours the reaction time was set the degree of transformation 90,5%.

Dedicated ethyl ester insulin-aspart can be turned into insulin-aspart using alkaline hydrolysis.

Example 5

of 10.9 g of the precursor of the insulin-aspart

[Asp28, Met30, Trp31, Lys32]-des(33-65)human proinsulin, elongated at the N-end of the peptide Glu-Glu-Gly-Glu-Pro-Lys- (SEQ ID NO.: 2) suspended in an average of 49.3 g of water. Under mild stirring and at ambient temperature dissolved the predecessor gradual addition of 37.6 g of a mixture containing 0.36 M sodium hydroxide, 0.27 M sodium acetate and 36% of N-methyl-2-pyrrolidone. The pH value was brought to 9.7 with the help of 9.2 ml of 0.5 M sodium hydroxide. Added to 7.1 ml of 7.1 mg/ml aqueous solution of ALP and the mixture containing 79% water, left to interact for 5 hours. During the reaction the pH value was kept constant while the 9.7 by adding additional 0.5 M sodium hydroxide. The reaction mixture was cooled to 5°C. and the pH value was brought to 5.7 by adding 2,73 g of 4 n hydrochloric acid. Added 14,02 g of ethyl ester of L-threonine and the pH value was brought to 6.0 with 4 n hydrochloric acid. Added 344 g of chilled (4°C) N-methyl-2-Pierre is lidón. The temperature is brought up to 22°C and the pH was brought to 6.5 with hydrochloric acid. The reaction mixture containing 25% of water, left for 9 hours at this temperature. By analysis of HPLC with reversed-phase column (4 mm × 250 mm 5 μm C18 with silicon dioxide with eluent acetonitrile-water containing 200 mm sodium sulfate, pH value, increased to 3.6, was established the degree of transformation of 87.5% in ethyl ether insulin-aspart after a total reaction time of 14 hours.

Dedicated ethyl ester insulin-aspart can be turned into insulin-aspart using alkaline hydrolysis.

1. The method of obtaining insulin from different species and its derivatives, where (a) in a solvent containing from 55 to 70% water (wt./wt.), the precursor or insulin precursor-derived insulin is subjected to enzymatic cleavage under alkaline pH values, where the enzyme used for enzymatic degradation, is a trypsin or litinspector protease, preferably Achromobacter lyticus protease I, then, without isolating the intermediate product from the reaction mixture (b) to the specified intermediate product of the enzymatic nucleophilic attach the connection that represents the ether amino acids, amide amino acid, peptide, ester peptide or amide of the peptide in the reaction mixture is within the water content in the range from 10 to 50% water (wt./wt.), at acidic pH values close to neutral pH value, and (C)optionally removing the protective(Oh) group(s).

2. The method according to claim 1, where prior to the beginning of the reaction accession 25%, 50%, 75%, 85% or 95% of the precursor of insulin is split up to the intermediate product.

3. The method according to claim 1, where the nucleophilic compound is an ester of amino acids.

4. The method according to claim 3, where the ester of the amino acid is an ester of threonine.

5. The method according to claim 1, where the nucleophilic compound is an amide of amino acids.

6. The method according to claim 1, where the nucleophilic compound is a peptide.

7. The method according to claim 1, where the nucleophilic compound is an ester of the peptide.

8. The method according to claim 1, where the nucleophilic compound is an amide of the peptide.

9. The method according to claim 1, providing a stage of removal of the protective(Oh) group(s) of insulin from various species and its derivatives.

10. The method according to claim 1 where the insulin of various types and its derivatives have a threonine in position B30.

11. The method according to claim 1, where the derived insulin is an insulin-aspart, insulin-lispro, insulin glargine or insulin detemir.

12. The method of obtaining insulin from different species and its derivatives, where (a) in a solvent containing from 55 to 70% water (wt./wt.), the precursor or insulin precursor derived insulin will versaut enzymatic cleavage under alkaline pH values, where the enzyme used for enzymatic degradation, is a trypsin or litinspector protease, preferably Achromobacter lyticus protease I, then, b) the intermediate product of the enzymatic nucleophilic attach the connection that represents the ether amino acids, amide amino acid, peptide, ester peptide or amide of the peptide in the reaction mixture used for the reaction of enzymatic degradation, provided that the composition of the reaction mixture change so that the water content in the reaction mixture is in the range from 10 to 50% water (wt./wt.), at acidic pH values close to neutral pH value, and (C)optionally removing the protective(Oh) group(s).

13. The method according to item 12, where between stages of splitting and joining not conduct the selection of the intermediate product.

14. The method according to item 12, where the enzyme is used at the stage of cleavage, is also present at the stage of connection.

15. The method according to item 12, where before reaction accession 25%, 50%, 75%, 85% or 95% of the precursor of insulin is split up to the intermediate product.

16. The method according to item 12, where the nucleophilic compound is an ester of amino acids.

17. The method according to clause 16, where the ester of the amino acid is an ester of threonine.

18. The method according to item 12, where the nucleophilic compound is an amide amino is islote.

19. The method according to item 12, where the nucleophilic compound is a peptide.

20. The method according to item 12, where the nucleophilic compound is an ester of the peptide.

21. The method according to item 12, where the nucleophilic compound is an amide of the peptide.

22. The method according to item 12, providing a stage of removal of the protective(Oh) group(s) of insulin from various species and its derivatives.

23. The method according to item 12, which received insulin from different species and its derivatives have a threonine in position B30.

24. The method according to item 12, where the derived insulin is an insulin-aspart, insulin-lispro, insulin glargine or insulin detemir.



 

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Insulin derivatives // 2352581

FIELD: medicine.

SUBSTANCE: invention concerns derivatives of a misinformation (B30) human insulin which have lateral a chain, attached to ε-amino group of the rest of the lysine which is present at the B-chain of initial insulin where this lateral the chain has the general formula: -W-X-Y-Z where W, X, Y and Z are such as it is defined in the description.

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15 cl, 4 tbl, 37 ex

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17 cl, 2 tbl, 8 ex

FIELD: biotechnology, in particular medical and biological industry.

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9 cl, 10 dwg, 6 tbl, 5 ex

FIELD: biotechnology, in particular for biochemical, immunological and biological investigation and for production of insulin pharmaceuticals.

SUBSTANCE: semisynthetic human insulin is prepared by transpeptidation of swine insulin in presence of trypsin in mass ratio trypsin/swine insulin of 1:300-1000 and molar excess threonyn di-tert-butyl ester in relation to swine insulin, in aqueous-organic medium. Reaction is inhibited by pH reducing and reaction mixture dilution with water by 2-3 times. Obtained human insulin ester is purified by HPLC followed by providing of human crude insulin crystals and repeated HPLC-purifying. Method of present invention makes it possible to increase human insulin ester yield up to 98 % and decrease by-product contamination in finished product up to 0.9 %.

EFFECT: method of improved yield; product of improved purity.

6 ex

The invention relates to biotechnology and can be used to obtain a properly curled, containing the precursor of insulin chimeric protein

The invention relates to water-soluble unit derived insulin, which remains In 24-B30 of the b-chain derived insulin is sequence Phe-x-X-x-X-x-X, where each X independently represents any amino acid or a deletion of at least one X is Nsubstituted by a lysine residue in which the Deputy is a 5--lithocholic acid or 5--lithocholic acid, connected through the-glutamyl,-glutamyl oror-aspartyl as a linker, where the unit size is defined in the gel-filtration system, more than aldolase, and the Assembly includes at least 2 zinc ions per 6 molecules derived insulin

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FIELD: biotechnology, in particular for biochemical, immunological and biological investigation and for production of insulin pharmaceuticals.

SUBSTANCE: semisynthetic human insulin is prepared by transpeptidation of swine insulin in presence of trypsin in mass ratio trypsin/swine insulin of 1:300-1000 and molar excess threonyn di-tert-butyl ester in relation to swine insulin, in aqueous-organic medium. Reaction is inhibited by pH reducing and reaction mixture dilution with water by 2-3 times. Obtained human insulin ester is purified by HPLC followed by providing of human crude insulin crystals and repeated HPLC-purifying. Method of present invention makes it possible to increase human insulin ester yield up to 98 % and decrease by-product contamination in finished product up to 0.9 %.

EFFECT: method of improved yield; product of improved purity.

6 ex

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