MXPA98004945A - New insulin derivatives with rapid initiation of efe - Google Patents

Abstract

The present invention relates to insulin derivatives that have an initiation of the effect that has been accelerated compared to human insulin, to a process for its preparation and to its use especially in pharmaceutical preparations for the treatment of diabetes mellitus. In particular, the present invention concerns insulin derivatives or physiologically compatible salts thereof, in which asparagine (Asn) in position B3 of the B chain has been changed by a basic amino acid residue that appears in nature and by at least one amino acid residue at positions B27, B28óB29 of chain B has been changed by another amino acid residue that appears in nature, asparagine (Asn) at position 21 of chain A can optionally be changed by Asp, Gly, Ser, ThróAla, and missing phenylalanine (Phe) in position B1 of chain B and the amino acid residue located in position B30 of the chain

Description

New insulin derivatives with rapid onset of effect The present invention concerns insulin derivatives, which have an initiation of the effect that has been accelerated in comparison with human insulin (H-insulin), a process for its preparation and its use, especially in pharmaceutical preparations for the treatment of diabetes mellitus. Around the world, approximately 120 million human beings suffer from diabetes mellitus. Among them, around 12 million are type I diabetics, for whom the application of insulin is the only therapy currently possible. Those affected can not do without insulin injections throughout their lives, as a rule several times a day. Even though type II diabetes, of which approximately 100 million human beings suffer, is not fundamentally accompanied by an insulin defect, nevertheless in a large number of cases, it is considered that the treatment with insulin is the form of therapy more favorable or the only possible. With a progressive duration of the disease, a large number of patients suffer from the so-called late diabetic complications. In the case of these, it is essentially micro- and macrovascular damages, which, depending on their type and extension, result in renal failure, blindness, loss of extremities or an increased risk of cardio / circulatory diseases. As an original cause, the chronically increased levels of blood glucose are responsible for this, since even in the case of a careful adjustment of insulin therapy a normal blood glucose profile is not reached, such as would correspond to physiological regulation (Ward., JD (1989) Briishish Bulletin 45, 111-126; Drury, PL et al. (1989) Briishish Bulletin 45, 127-147; Kohner, EM (1989) British Medical Bulletin 45, 148-173).

In a healthy person, insulin secretion relies heavily on the concentration of glucose in the blood. Increased glucose levels, as they appear after meals, are quickly compensated for by an increased release of insulin. In the fasting state, the plasma insulin level decreases to a baseline, which is sufficient to ensure a continuous supply of glucose to insulin-sensitive organs and tissues. An optimization of therapy, the so-called intensified therapy with insulin, aims today mainly to keep as small as possible the fluctuations of blood glucose concentration, especially the deviations towards higher values (6. Bolli, GB (1989 Diabetes Res. Clin.Practice 6, S3-S16; Berger, M. (1989) Diabetes Res. Clin.Pract. 6, S25-S32). This leads to a significant decrease in the onset and progression of late diabetic damage (The Diabetes Control and Complications Trial Research Group (1993) N. Engl. J. Med. 329, 977-986). From the physiology of insulin secretion it can be deduced that to achieve an intensified and improved therapy with insulin, using two subcutaneous (s.c) preparations, two insulin preparations with different pharmacodynamics are needed. To compensate for the increase in blood glucose after meals, the insulin must flow quickly and can only act for a few hours. For the basal supply, especially during the night, a preparation should be available, acting for a long time, not presenting any prolonged maximum and flowing only very slowly. The preparations based on human and animal insulins, however, only fulfill the requirements of an intensified therapy with insulin. Rapid-acting insulins (Al t-insulins) come, after a subcutaneous application, too slowly to the blood and the site of their action and have a too long overall duration of effect. The consequence of this is that the postprandial glucose levels are set too high and, several hours after the meal, the glucose level the blood glucose level decreases too much 5 (Kang, S. et al., (1991) Diabetes Care 14, 142-148; Home, P. J. et al., (1989) Briishish Medical Bulletin 45, 92-110; Bolli, G. B. (1989) Diabetes Res. Clin. Pract 6, S3 -S16). In turn, the available basal insulins, especially NPH-insulins, present a too short duration of the action and have a maximum too intensely pronounced. Together with the possibility of influencing the action profile through galenic principles, the alternative of using genetic technology today is offered to conceive insulin derivatives that achieve certain properties, such as initiation and duration of the effect, only by their structural properties. Through the use of appropriate insulin derivatives, therefore, an adjustment of the glucose in blood that is more closely accommodated to natural relationships and is essentially better. Insulin derivatives with accelerated initiation of effect are described in European Patent Documents EP-0.214.826, EP-0.375.437 and EP-0.678.522. EP-25 0.214.826 relates, among other particularities, to substitutions of B27 and B28, but not in conjunction with the substitution of B3. EP-0 678 522 discloses insulin derivatives having in position B29 different amino acids, preferably proline, but not glutamic acid. He EP-0,375,437 comprises insulin derivatives with lysine or arginine in B28, which optionally can be further modified in B3 and / or A21. EP-0,419,504 discloses insulin derivatives that are protected against chemical modifications, with asparagine being changed to B3 and at least one other amino acid at positions A5, A15, A18 or A21. However, combinations with modifications at positions B27, B28 or B29 are not described. No indication is given that these compounds possess a modified pharmacodynamics, with the consequence of a more rapid initiation of the effect. In the PCT patent document WO 92/00321 insulin derivatives are described, in which at least one amino acid of the positions B1-B6 is replaced by lysine or arginine. Such insulins have, according to WO 92/00321, a prolonged effect. However, combinations with modifications of positions B27, 28, 29 are not disclosed. It is the object of the present invention to develop insulin derivatives, which after an application, especially after subcutaneous application, have an initiation of effect that has been accelerated compared to human insulin. The insulin derivatives are derivatives of insulins that appear in nature, namely human insulin (see SEQ ID NO 1 = human insulin A chain, see SEQ ID NO 2 = human insulin B chain, sequence protocol) or animal insulins , which differ from the corresponding insulin that appears in nature, otherwise the same, by the substitution of at least one amino acid residue that appears in nature and / or by the addition of at least one amino acid residue and / or an organic rest. It is also the object of the present invention to develop a method for the preparation of the insulin derivatives with said property, the corresponding intermediates as well as their precursors. The problem posed by this mission is solved by an insulin derivative, or a physiologically compatible salt thereof, in which asparagine (Asn) has been changed at position B3 of the B chain by a basic amino acid residue that appears in the nature and at least one amino acid residue at positions B27, B28 or B29 of the B chain has been changed to another amino acid residue that appears in nature, asparagine (Asn) can optionally be changed at position 21 of chain A by Asp, Gly, Ser, Thr or Ala, and missing phenylalanine (Phe) in position Bl of chain B and the amino acid residue located in position B30 of chain B. Preferably, the insulin derivative, or one of its physiologically compatible salts, is characterized by Formula I B1-Val-B3-Glu-His-Leu-Cys- (B8-B18) -Cys- (B20-B26) -B27-B28-B29-B30 where they mean (A1-A5) the amino acid residues in the Al up to A5 positions of the human insulin A chain (cf. SEQ ID NO 1) or animal insulin, (A12-A19) the amino acid residues at positions Al2 to A19 of the human insulin A chain (cf. SEQ ID NO 1) or animal insulin, (B8-B18) the amino acid residues at positions B8 to B18 of the B chain of human insulin (compare SEQ ID NO 2) or animal insulin, (B20-B26) the amino acid residues at positions B20 to B26 of the B chain of human insulin (compare SEQ ID NO 2) or animal insulin, A8, A9, A10 the amino acid residues at positions A8, A9 and A10 of the human insulin A chain (compare SEQ ID NO 1) or animal insulin, A21 Asn, Asp, Gly, Ser, Thr or Ala, B30 -OH or the amino acid residue at position B30 of the B chain of human insulin (compare SEQ ID NO 2) or animal insulin, 10 -.-..'.- •; Bl a phenylalanine residue (Phe) or an atom of '•' • '' •• hydrogen, B3 a basic amino acid residue that appears in nature, B27, B28 «> - «- •« »And ß29 the amino acid residues at positions B27, fsÉg? * - '*'! B28 and B29 of the B chain of human insulin 20 (compare SEQ ID NO 2) or of animal insulin, or in each case another amino acid residue that appears in nature, having been changed at least one of the amino acid residues in the -'':. positions B27, B28 and B29 of the B chain by another different amino acid residue that appears in nature.

Of the twenty amino acids that appear in nature, which are genetically codifiable, the amino acids glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (lie), serine are designated here as neutral amino acids. (Ser), threonine (Thr), cysteine (Cys), methionine (Met), asparagine (Asn), glutamine (Gln), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and proline 35 (Pro), as basic amino acids the amino acids arginine (Arg), usina (Lys) and histidine (His), and as amino acids, the amino acids aspartic acid (Asp) and glutamic acid (Glu). Preferably, the insulin derivative, or its physiologically compatible salt, according to the present invention is a derivative of bovine insulin, porcine insulin or human insulin, namely an insulin derivative, or a physiologically compatible salt thereof, having the formula I, which is characterized because they mean A8 alanine (Ala), A9 serine (Ser), A10 valine (Val) and B30 alanine (Ala) (amino acid residues A8 to A10 and B30 of bovine insulin), 15 or A8 threonine (Thr), A9 serine (Ser) and 20 A10 isoleucine (lie) (amino acid residues A8 to A10 of human or porcine insulins), meaning B30 alanine (Ala) (amino acid residue B30 of porcine insulin) or B30 threonine (Thr) (amino acid residue B30 of human insulin, compare SEQ ID NO 2).

Especially preferred is an insulin derivative, or a physiologically compatible salt thereof, having Formula I with amino acid residues A8 through A10 and B30 of human insulin, which is further distinguished by the fact that it means (A1-A5) the amino acid residues in the Al up to A5 positions of the human insulin A chain (compare SEQ ID NO 1), (A12-A19) the amino acid residues at positions A12 to Al9 of the human insulin A chain (compare SEQ ID NO 1), (B8-B18) the amino acid residues at positions B8 to B18 of the B chain of human insulin (compare SEQ ID NO 2) and (B20-B26) the amino acid residues at positions B20 to B26 of the B chain of human insulin (compare SEQ ID NO 2).

Other preferred refinements of the present invention are an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue in position Bl of the B chain is a phenylalanine residue (Phe), or an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue at position B3 of the B chain is a histidine (His), lysine (Lys) or arginine (Arg) moiety . Other preferred refinements of the present invention are an insulin derivative, or a physiologically compatible salt thereof, having Formula I, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain has been replaced by an amino acid residue that appears in nature, which is selected from the group of neutral amino acids or acidic amino acids, an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is an amino acid residue that appears in nature, which is selected from the group consisting of isoleucine (lie), aspartic acid (Asp) and glutamic acid (Glu), preferably characterized by at least one of the amino acid residues at positions B27, B28 of the chain B has been replaced by an amino acid residue that appears in nature, which is selected from the group of neutral amino acids or, especially preferred, because at least one of the amino acid residues at positions B27, B28 and B29 of chain B is a residue of isoleucine (lie), or an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is an amino acid residue which appears in nature, which is selected from the group of acidic amino acids, preferably characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is a residue of aspartic acid (Asp) , further preferably characterized in that the amino acid residue in position B27 or B28 of the B chain is a residue of aspartic acid (Asp), or characterized by at least one of the amino acid residues at positions B27, B28 and B29 of the Chain B is a glutamic acid residue (Glu). A preferred refinement of the present invention is also an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue at position B29 of the B chain is a residue of aspartic acid (Asp). Other preferred refinements are an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue at position B27 of the B chain is a glutamic acid (Glu) residue, an insulin derivative , or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue at position B28 of the B chain is a glutamic acid residue (Glu), or an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue at position B29 of the B chain is a glutamic acid residue (Glu). Especially preferred is an insulin derivative, or a physiologically compatible salt thereof, which is distinguished by the fact that the B chain has the sequence Phe Vat Lys Gln His Leu Cys Giy Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr §ifiBv5; 20 (SEQU ID NO 3), for example Lys (B3), Glu (B29) -human insulin, or an insulin derivative, or a physiologically compatible salt thereof, which is distinguished by the fact that the amino acid residue at position B27 of the B chain is an isoleucine residue (lie), preferably an insulin derivative, or a physiologically compatible salt thereof, which is distinguished by the fact that the B chain presents the sequence 30 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr lie Pro Lys Thr (SEQU ID NO 5), for example Lys (B3), lie (B27) -human insulin, or an insulin derivative, or a physiologically compatible salt thereof, having the Formula I, characterized in that the amino acid residue in position B28 of the chain B is an isoleucine residue (lie), preferably an insulin derivative, or a physiologically compatible salt thereof, which is distinguished by the fact that the B chain has the sequence Phe Val Lys Gln Hts Leu Cys Gly Ser His Leu Val Giu Wing Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr ile Lys Thr (SEQU ID NO 4), for example Lys (B3), He (B28) -human insulin. Especially preferred is also an insulin derivative, or a physiologically compatible salt thereof, having Formula I, which is distinguished by the fact that the amino acid residue in position 28 of the Bes chain is an isoleucine residue (lie) and the amino acid residue at position A21 is an asparagine residue (Asp), preferably because the A chain presents the sequence Gly Lie Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu Tyr Gln Leu Glu Asp Tyr Cys Asp (SEQ ID NO .: 9) and the string B presents the sequence Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr lie Lys Thr (SEQ ID NO .: 10) (Lys (B3), He (B28), Asp (A21) -human insulin).

The insulin derivatives of Formula I can preferably be prepared by genetic technology.

The problem posed by the initially established mission is thus further solved by a process for the preparation of an insulin derivative, or of a physiologically compatible salt thereof, having the Formula I, which comprises the construction of an expression vehicle. replicable, which contains a DNA sequence, which encodes a precursor of the insulin derivative, in which the amino acid residue in the Al position of the A chain is linked to the B30 amino acid residue of the B chain through a peptide chain that has the 10 Formula II Wherein R ^ n is a peptide chain with n amino acid residues and n signifies an integer from 0 to 34, and chain B is extended adjacent to position Bl with a peptide chain of Formula III IS Kg 20 Met- R2m- (Arg) p- wherein R2m is a peptide chain with m amino acid residues, m means an integer from 0 to 40, preferably from 0 to 9, and p means 0, 1 or 2, preferably terminating the chain peptide R2m with Lys for p = 0, expression in a host cell and release of the insulin derivative from its precursor with chemical and / or enzymatic methods. Preferably, the method is characterized in that the host cell is a bacterium, and is especially preferred because the bacterium is E. coli (Escherichia coli). Preferably, the process is characterized in that the host cell is a yeast and is especially preferably characterized in that the yeast is Saccharomyc cerevisiae.

For the preparation of an insulin derivative with the amino acid sequences SEQ. ID. DO NOT. : 9 (string A) and SEQ. ID. DO NOT. : 10 (chain B) the precursor of this insulin derivative preferably has the sequence Met Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Glp His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr lie Lys Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ata Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly lie Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asp (SEQ ID NO .: 11), one Lys (B3), Ile (B28), Asp (A21) -preproinsulin.

For the preparation of an insulin derivative with the amino acid sequence SEQU ID NO 3, the precursor of this insulin derivative preferably has the sequence Met Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Wing Gly Ser Leu Gln Pro Leu Wing Leu Glu Gly Ser Leu Gin Lys Arg Gly He Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (Lys ( B3), Glu (B29) -preproinsulin) (SEQU ID NO 6).

For the preparation of an insulin derivative with the amino acid sequence SEQU ID NO 5, the precursor of this insulin derivative preferably has the sequence Met Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr He Pro Lys Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gin Val Glu Leu Gly Gly Giy Pro Gly Wing Gly Ser Leu Gin Pro Leu Ala Leu Glu Giy Ser Leu Gln Lys Arg Gly He Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (Lys ( B3), lle (B27) -prepro? Nsulin) (SEQ ID NO 8).

For the preparation of an insulin derivative with the amino acid sequence SEQU ID NO 4, the precursor of this insulin derivative preferably has the sequence Met Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Gtn His Leu Cys Gly Ser Hís Leu Val Glu Wing Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr He Lys Thr Arg Arg Glu Wing Glu Asp Pro GIn Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Wing Gly Ser Leu Gln Pro Leu Wing Leu Glu Gly Being Leu Gln Lys Arg Gly Me Vai Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr GIn Leu Glu Asn Tyr Cys Asn (Lys ( B3), l! E (B28) -preproinsulin) (SEQU ID NO 7).

The present invention therefore also concerns the aforementioned precursors of the preferred insulin derivatives, namely the peptides with the sequence numbers SEQ ID NO 11, SEQ ID NO 6, SEQ ID NO 7 and SEQU ID NO 8, the sequences of DNA encoding said precursors, the expression vehicles, which contain these DNA sequences, as well as the host cells that are transformed with these expression vehicles. The preparation of the insulin derivatives of Formula I is carried out mainly via genetic technology by site-directed mutagenesis according to classical methods. For this, a genetic structure is constructed that encodes the desired insulin derivative of Formula I and is brought into expression in a host cell - preferably in a bacterium, such as E. coli, or in a yeast, especially S aechar omy ees cerevisiae - and - in the event that the gene structure encodes a fusion protein - the insulin derivative of Formula I is released from the fusion protein; analogous methods have been described, for example, in EP-A-0,211,299, EP-A-0,227,938, EP-A-0,229,998, EP-A-0,285,956 and in the German patent application. DE P 3821159. The separation of the fusion protein portion can be carried out, after the cell has been disintegrated, chemically by means of a halo cyanogen (cf. EP-A-0,180,920). In the case of preparation by a preproinsulin precursor having a portion of fusion protein (presequence) according to U.S. Pat. No. 5,358,857, the separation of the fusion protein portion is effected in a subsequent step, together with the separation of the C-peptide. The insulin precursor is then subjected to sulfitolysis p. ex. by oxidation according to the method described by R.C. Marshall and A.S. Inglis in "Practical Protein Chemistry-A Handbook" (compiler A. Darbre) 1986, pages 49 -53, and then renatured in the presence of a thiol with formation of the correct disulfide bridges, eg according to the method described by GH Dixon and A.C. Wardlo in Nature (1960), pages 721-724. The insulin precursors can, however, also be folded directly (EP-A-0,600,372; EP-A-0,668,292). The C-peptide is removed by tryptic cleavage - e.g. according to the method of Kemmler et al., J. B. C. (1971), pages 6. 786-6. 791 and the insulin derivative of Formula I is purified by known techniques such as chromatography - see eg EP-A-0 305 760 - and crystallization. In the case that n in Formula II is 0, the tryptic dissociation serves for the separation of the peptide bond between the A and B chains. In this procedure, chain B ends at its C end with arginine or two arginine residues. These can be removed enzymatically via carboxy peptidase B. The insulin derivatives according to the invention have a full biological activity. This was demonstrated by intravenous application to rabbits and the resultant decrease in blood glucose (Examples 5 and 6). The fastest onset of the effect after subcutaneous application was demonstrated with the euglycemic Clamp technique in a fasted dog (Example 7). 0.3 Ul / kg was administered. The reference preparation was human insulin. In the case of the Clamp technique, after the insulin injection the blood glucose value is measured in short time intervals and exactly as much glucose is infused as is needed to compensate for the decrease. This has the advantage that no antagonistic regulation appears in the animals, as would be the case when a large decrease in blood glucose occurs after the administration of insulin. The quantity and evolution over time of the infused glucose characterize the effect of insulin. The Lys (B3), Glu (B29) - (SEQ ID NO 3) and Lys (B3), Ile (B28) - (SEQ ID NO 4) -insulins 5 present a clearly faster onset of effect than human insulin. The maximum effect (glucose infusion rate) is achieved with human insulin after 100 minutes, with Lys (B3), Glu (B29) -insulin (SEQ ID NO 3) on the contrary after 80 minutes, and with 10 Lys (B3), He (B28) -insulin (SEQ ID NO 4) already after 60 minutes. Therefore, these analogous compounds, when injected shortly before a meal, should compensate for the postprandial increase in blood glucose better than human insulin. The described insulin derivatives are suitable both for the therapy of type I diabetes mellitus and also for type II diabetes mellitus, preferably in conjunction with a basal insulin. The present invention therefore also concerns the use of the insulin derivative and / or one of its physiologically compatible salts having Formula I, for the production of a pharmaceutical preparation, which exhibits an insulin activity with a rapid initiation of the effect. As a physiologically harmless vehicle medium compatible with the insulin derivative, a sterile aqueous solution is made, which makes the blood isotonic in the usual way, for example by means of glycerol, sodium chloride or glucose, and in addition to this one of the usual preservatives, for example phenol, m-cresol or esters of p-hydroxybenzoic acid. The vehicle medium may additionally contain a buffer substance, for example sodium acetate, sodium citrate or sodium phosphate. For adjusting the pH, diluted acids (typically HC1) or diluted liquors (typically NaOH) are used. The preparation can also contain zinc ions. The insulin derivatives can also be used in the pharmaceutical preparations in the form of their physiologically compatible salts, such as alkali metal salts or ammonium salts. An arbitrary proportion of one or more insulin derivatives of Formula I or an insulin derivative of Formula I can be present in a mixture of other insulin derivatives, independently of each other, in each case in dissolved, amorphous form. and / or crystalline. Sometimes, it is advantageous to add to the preparation 10 according to the invention an appropriate amount of an appropriate stabilizer, which prevents the precipitation of protein in the case of a thermal-mechanical load upon contact with different materials. Such stabilizers are known, for example, from EP-A-18,609, DE-A 15 3240177 or WO 83/00288. The present invention also concerns a pharmaceutical preparation, which is characterized in that it contains at least one insulin derivative, and / or a physiological saltPF &f * compatible mind thereof, having the Formula I, preferably in dissolved, amorphous and / or crystalline form. The insulin derivatives according to the invention are characterized by a rapid initiation of the effect. In practical therapy with insulin, it is usual in certain circumstances to mix insulins that exhibit rapid action with preparations containing a depot adjuvant substance [= delayed release] (eg NPH-insulin). This results, depending on the composition, of preparations whose activity profiles correspond to the individual profiles superimposed, provided that the individual components in the mixture are stable and do not influence each other. When mixing an insulin derivative with NPH-human insulin, however, it is expected that, especially in the case of prolonged storage, an exchange occurs between the dissolved derivative and the NPH-crystalline insulin. In this way, both the pharmacodynamics of the depot insulin and that of the rapidly acting insulin that is dissolved are altered in an unpredictable manner. In order to avoid this, it is convenient to prepare the derivative with rapid action through the use of a coadjuvant substance, for example, as NPH-insulin. This form of deposit of the insulin derivative can then be arbitrarily mixed with the fast acting form that is dissolved, without the composition of one or the other of the forms being altered in the course of storage, by exchange. Even though the invention concerns rapid-acting insulin derivatives in its nucleus, it also includes, however, also the possibility of preparing such derivatives as a depot form in order to achieve miscibility, the reservoir adjuvant preferably being protamine sulphate. and the insulin derivative and / or its physiologically compatible salt being presented together with the protamine sulfate in a co-crystallized material. The present invention also concerns an injectable solution, which contains in dissolved form the pharmaceutical preparations described above.Examples Example 1: Construction of Lys (B3) -proinsulin as a starting point for the plasmids that are relevant to the invention, corresponding to Examples 2-4 US Pat. No. 5,358,857 describes the pINT 90d vector and the PCR (primer) primers Tir and Insu 11.

These components serve as starting material for the construction of a pINT 125d plasmid, which encodes the desired Lys (B3) -proinsulin. Additionally, the primers are synthesized Insu 35 having the sequence 5 'TTT GTG AAG CAG CAC CTG 3' and Insu 36 having the sequence 5 5 'CAG GTG CTG CTT CAC AAA 3' A first PCR reaction (10-polymerase chain reaction) is carried out with the Tir and Insu 36 primers and a second PCR reaction is carried out with the primers Insu 11 and Insu 35. As a template (template), it serves for this purpose. the DNA of pINT 90d. The products of both PCR reactions are partially complementary, so when they are put together in a third PCR reaction with the Tir and Insu 11 primers, they provide a fragment encoding a variant proinsulin containing lysine in position 3 of the f $ chain B. This PCR fragment is, for its purification, precipitated in ethanol, dried and then digested with the restriction enzymes Neo 1"and Sal 1 according to the manufacturer's data. gel electrophoresis and the desired Neo I / Sal 1 fragment is isolated. In the cited application a plasmid pINT 91d is described, which encodes a mini-proinsulin.If the sequence encoding mini-proinsulin is separated by dissociation by Neo 1 and Sal 1, and the DNA is isolated from the remaining plasmid, this DNA from the remaining plasmid can be reacted with the Neo l / Sal 1 PCR fragment represented, in a reaction with T 4 ligase. , to form the plasmid pINT 125d. This is transformed according to E. coli K12, there it is multiplied and re-isolated. After the plasmid structure has been verified by DNA sequence analysis and by restriction, pINT 125d DNA serves as template DNA for the introduction of other mutations in this variant of proinsulin.

Example 2: Construction of Lys (B3), Glu (B29) -proinsulin For the preparation of the mutein, the 5 primers 329a are synthesized with the sequence 'TTC TAC ACÁ CCC GAG ACC CGC GGC ATC G - 3' and 329b with the sequence 'GCC GCG GGT CTC GGG TGT GTA GAA GAA GC 3' As a template, DNA of the plasmids pINT 125d and pINT 9Id are used. The primer 329a is reacted with the primer Insu '"*' *. * - ~., 11 on the pINT 91d template and the primer 329b is reacted with the Tir primer (see the previous Example) on the pINT 125d template , in a PCR reaction Since both PCR products are partially complementary, they can be pooled in a direct PCR reaction and reacted again with the Tir and Insu 11 primers resulting in a DNA fragment, which encodes the desired mutein. The fragment is digested twice with the restriction enzymes Neo 1 and Sal 1, and the resulting Neo 1 / Sal 1 fragment is inserted into the DNA of the remaining plasmid pINT 91d by a reaction with T 4 ligase. that after amplification in E. coli K12 is verified by DNA sequence analysis and by restriction with respect to the desired structure The proinsulin derivative encoded by the plasmid is characterized by the two intercals mbios of amino acids 35 and a binding member in C, which consists of the amino acid arginine.

Example 3: Construction of Lys (B3), He (B27) -proinsulin The construction is carried out according to the preceding Example with the following pairs of primers: KB3 JB 27A 'TTC TAC ATC CCC AAG ACC CGC CG 3' and Insu 11 as well as K B3 J 27B 'CTT GGG GAT GTA GAA GAA GCC TCG 3' and Tir.

The DNA of the plasmid pINT 125d is used as a template in both PCR reactions. The PCR products of both reactions are combined in a third reaction, as described in Example 1, and the product is cloned correspondingly to the Example. The plasmid pINT 332 results.

Example 4: Construction of Lys (B3), He (B28) -proinsulin The construction is carried out according to Example 3 with the following pairs of primers: KB3 JB 28A 5 'TAC ACÁ ATC AAG ACC CGC CGG GAG - 3' and Insu 1 1 as well as KB J B28B 5 'GGT CTT GAT TGT GTA GAA GAA GCC TCG - 3' and Tir.

The plasmid pINT 333 results.

Expression of the constructed insulin variants Plasmid pINT 329, 332 and 333 are transformed by way of example in each case according to E. coli K12 W3110. The recombinant bacteria containing plasmids, which encode the respective variants, are then fermented according to Example 4 of US Pat. with the number 5,227,293 and in this way the desired raw material for the production of the respective insulin variant is generated. $ z 20 Example 5: Construction of Lys (B3), He (B28), Asp (A21) -proinsulin The construction is carried out according to Example 3. Instead of pINT 125d, however, the plasmid pINT 333, which was constructed in Example 4, serves as the template for the PCR reaction. In this case, the following pair of primers is used: P-pint365 30 5'-TTTTTTGTCGACTATTAGTCGCAGTAGTTCTACCAGCTG-3 ' and Tir.

The plasmid pINT 365 results.

Example 6: Biological activity of Lys (B3), Glu (B29) -insulin after intravenous administration to rabbits 8 rabbits received intravenous g & amp; indicated (at a rate of 0.2 IU / kg). In the course of the following four hours, the blood glucose concentration was determined at the indicated times and calculated as 20% of the initial value at time 0. Mean values do not reveal any significant difference in biological activity between the Human insulin and Lys (B3), Glu (B29) -insulin.

Example 7: Biological activity of Lys (B3), He (B27) - and Lys (B3), He (B28) -insulins after intravenous administration to rabbits 6 rabbits received intravenously the indicated insulins (at a rate of 0.2 IU / kg). In the course of the next four hours, the blood glucose concentration was determined at the indicated times and calculated as% of the initial value at time 0. The mean values do not reveal any significant difference of the biological activity between the human insulin and the Lys (B3), He (B27) - and Lys (B3), He (B28) - insulins.

Example 8: Pharmacodynamics of Lys (B3), Glu (29) -insulin and 15 Lys (B3), He (B28) -insulin after subcutaneous application to Kg dogs! "*" In each case four dogs received subcutaneous injections of the indicated insulins (at a rate of 0.3 20 IU / kg). Blood glucose was maintained at 3.7 to 4 mmol / 1 by continuous infusion of glucose. The mean glucose infusion rate ± SEM (typical error of the mean) from the time of injection (t = 0) over 240 minutes is shown. 26 GLUCOSE CLAMP IN A FISHING DOG WITH ACCIÓ INSULIN DERIVATIVES Characteristic characteristics of the glucose infusion profile Dose: 1 x 0.3 Ul / kg s.c. in the tg (n = 4) GLUCOSE CLAMP IN A FISHING DOG WITH FAST-ACTING INSULIN DERIVATIVES Dose: 1 x 0.3 IU / kg s.c. in the tn (mean ± etm, n = 4) GLUCOSE CLAMP IN A FISHING DOG WITH FAST-ACTING INSULIN DERIVATIVES Dose: 1 x 0.3 IU / kg s.c. in the tQ (mean ± etm, n = 4) 60 90 120 150 180 210 240 minutes minutes

Claims (59)

1. CLAIMS 1. - Insulin derivative, or a physiologically compatible salt thereof, in which asparagine (Asn) has been changed at position B3 of the B chain by a basic amino acid residue that appears in nature and at least one has been changed amino acid residue at positions B27, B28 or B29 of the B chain by another amino acid residue or neutral acid that appears in nature, asparagine (Asn) can be optionally changed at position 21 of the A chain by Asp , Gly, Ser, Thr or Ala, and missing phenylalanine (Phe) in position Bl of chain B and the amino acid residue located in position B30 of chain B. 2.- Derived from insulin, or a physiologically salt compatible thereof, according to claim 1, characterized by Formula I (A1-A5) -Cys-Cys-A8-A9-A10-Cys- (A12-A19) -Cys-A21 B1-Val-B3-GIu-His-Leu-Cys- (B8-B18) -Cys- ( B20-B26) -B27-B28-B29-B30 where they mean (A1-A5) the amino acid residues in the Al up to A5 positions of the A chain of human insulin or animal insulin, (A12-A19) the amino acid residues in positions A12 to A19 of the A chain of human insulin or animal insulin, A21 Asn, Asp, Gly, Ser, Thr or Ala, 5 (B8-B18) the amino acid residues at positions B8 to B18 of the B chain of human insulin or animal insulin, (B20-B26) the amino acid residues at positions B20 10 to B26 of the B chain of human insulin or animal insulin, A8, A9, AlO the amino acid residues at positions A8, A9 and AlO of the human insulin A chain or animal insulin, B30 -OH or the amino acid residue in the B30 position -IíKre ddee ccadena B of human insulin or animal insulin, 20 Bl a phenylalanine residue (Phe) or a hydrogen atom, B3 a basic amino acid residue that appears in nature, B27, B28 and B29 the amino acid residues in positions B27, B28 and B29 of the B chain of human insulin or animal insulin, or in each case another amino acid residue that appears in nature, having been changed at least one of the amino acid residues at positions B27, B28 and B29 of the B chain by another different amino acid residue that appears in nature, which is selected from the group of neutral or acidic amino acids. 3. - Derivative of insulin, or a physiologically compatible salt thereof, according to claim 2, characterized in that they mean 5 A8 alanine (Ala), A9 serine (Ser), AlO valine (Val) and B30 alanine (Ala). 4. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 2, characterized in that they mean A8 threonine (Thr), A9 serine (Ser) and AlO isoleucine (lie). 5. Derived from insulin, or a physiologically compatible salt thereof, according to claim 4, characterized in that it means B30 alanine (Ala). 6.- Derived from insulin, or a salt physiologically Compatible thereof, according to claim 4, characterized in that it means B30 threonine (Thr). 7. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 6, characterized in that they mean (A1-A5) the amino acid residues at the Al up to A5 positions of the human insulin A chain, 30 (A12-A19) the amino acid residues at positions A12 to Al9 of the human insulin A chain, (B8-B18) the amino acid residues at positions B8 to B18 of the B chain of human insulin, 35 (B20-B26) the amino acid residues at positions B20 to B26 of the B chain of human insulin. 8. - Derivative of insulin, or a physiologically compatible salt thereof, according to one or more of claims 1 to 7, characterized in that the amino acid residue in position Bl of the B chain is a phenylalanine residue (Phe). 9. Derivative of insulin, or a physiologically compatible salt thereof, according to one or more of claims 1 to 8, characterized in that the amino acid residue in position B3 of the B chain is a residue of histidine (His), lysine (Lys) or arginine (Arg). 10. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 9, characterized in that the amino acid residue in position B3 of the B chain is a histidine residue (His). 11. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 9, characterized in that the amino acid residue in position B3 of the B chain is an arginine residue (Arg). 12. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 9, characterized in that the amino acid residue in position B3 of the B chain is a lysine residue (Lys). 13. Derivative of insulin, or a physiologically compatible salt thereof, according to one or more of claims 1 to 12, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is an amino acid residue that appears in nature, which is selected from the group consisting of isoleucine (lie), aspartic acid (Asp) and glutamic acid (Glu). 14. Derivative of insulin, or a physiologically compatible salt thereof, according to one or more of claims 1 to 13, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is an amino acid residue that appears in nature, which is selected from the group consisting of acidic amino acids. 15. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 14, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is an aspartic acid residue (Asp). 16. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 14, characterized in that at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is a glutamic acid residue (Glu) 17. Derivative of insulin, or a physiologically compatible salt thereof, according to one or more of claims 1 to 13, characterized in that at least one of the 15 amino acid residues at positions B27, B28 of the B chain has been changed by an amino acid residue that appears in nature, which is selected from the group consisting of neutral amino acids. ** Synthesis 18. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 17, characterized by at least one of the amino acid residues at positions B27, B28 and B29 of the B chain is a remainder of isoleucine (lie). 19. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 15, characterized in that the amino acid residue in position B27 of the B chain is a residue of aspartic acid (Asp). 20. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 15, characterized in that the amino acid residue in position B28 of the B chain is a residue of aspartic acid (Asp). 21. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 15, characterized in that the amino acid residue in position B29 of the B chain is a residue of aspartic acid (Asp). 22. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 16, characterized in that the amino acid residue in position B27 of the B chain is a glutamic acid residue (Glu). 23. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 16, characterized in that the amino acid residue in position B28 of the B chain is a glutamic acid residue (Glu). 24. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 16, characterized in that the amino acid residue in position B29 of the B chain is a glutamic acid residue (Glu). 25.- Insulin derivative, or a physiologically compatible salt thereof, according to claim 18, characterized in that the amino acid residue in position B28 of the B chain is an isoleucine residue (lie). 26.- Insulin derivative, or a physiologically compatible salt thereof, according to one or more of claims 1 to 25, characterized in that the amino acid residue at position A21 of the A chain is an asparagine residue (Asp). 27. Derived from insulin, or a physiologically compatible salt thereof, according to claim 26, characterized in that the A chain presents the sequence Gly lie Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asp (SEQ ID NO .: 9) and the string B presents the sequence Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr lie Lys Thr (SEQ ID NO .: 10) 28 - Derivative of insulin, or a physiologically compatible salt thereof, according to claim 24, characterized in that the A chain presents the sequence Phe Val Lys Gín His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Giu Arg Gly Phe Phe Tyr Thr Pro Glu Thr 5 (SEQ ID NO 3). 29. - Derivative of insulin, or a physiologically compatible salt thereof, according to claim 18, characterized in that the amino acid residue in position B27 of the B chain is an isoleucine residue (lie). 30. Derivative of insulin, or a physiologically compatible salt thereof, according to claim 29, characterized in that the B chain presents the sequence 15 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Giu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr lie Pro Lys Thr $ g. (SEQ ID NO 5). 20 31. - Derivative of insulin, or a physiologically compatible salt thereof, according to claim 25, characterized in that the B chain presents the sequence 25 Phe Val Lys Gln His Leu Cys Giy Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr lie Lys Thr (SEQU ID NO 4). 32. Process for the preparation of an insulin derivative, or a physiologically compatible salt thereof, according to one or more of claims 1 to 30, comprising the construction of a replicable expression vehicle, which contains a sequence of DNA, which encodes a precursor of the insulin derivative, - in which the amino acid residue in the Al position of the A chain is linked to the amino acid residue B30 of the B chain through a peptide chain having the Formula II -P -Arg-II in which R n is a peptide chain with n amino acid residues and n means an integer from 0 to 34, and chain B is extended next to position Bl with a peptide chain having Formula III et-Rm- (Arg) p- III wherein R m is a peptide chain with m amino acid residues, m means an integer from 0 to 40, and p means 0, 1 or 2, expression in a host cell and release of the insulin derivative from its precursor with chemical and / or enzymatic methods. 33. Method according to claim 32, characterized in that the host cell is a bacterium. 34. - Method according to claim 33, characterized in that the bacterium is E. coli. 35.- Method according to claim 32, characterized in that the host cell is a yeast. 36.- Method according to claim 33, characterized in that the yeast is Saccharo yces cerevi -siae. 37.- Method according to one of claims 32 to 36 for the preparation of an insulin derivative according to claim 26, characterized in that the insulin precursor has the sequence Met Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Gfu Arg Gly Phe Phe Tyr Thr lie Lys Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gln Glu Le Val Glu Leu Gly Gly Pro Gly Gfy Wing Be Leu Gln Pro Leu Wing Leu Glu Gly Ser Leu Glp Lys Arg Gly Lie Val Glu Gln Cys Cys Thr Ser lie Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asp (SEQ ID NO: 1 1). 38 - Process according to one of claims 32 to 36 for the preparation of an insulin derivative according to claim 28, characterized in that the insulin precursor has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg 15 Phe Val Lys Gln His Leu Cys Giy Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Glu Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gln Val Glu Leu Gly $ Hg? Gly Gly Pro Gly Wing Gly Be Leu Glp Pro Leu Wing Leu Glu Gly 20 Be Leu Gln Lys Arg Gly ile Val Giu Gln Cys Cys Thr Ser ile Cys Ser Leu Tyr Glp Leu Glu Asp Tyr Cys Asn 25 (SEQU ID NO 6). 39 - Process according to one of claims 32 to 36 for the preparation of an insulin derivative according to claim 30, characterized in that the insulin precursor has the sequence Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg 35 Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr He Prs Lys Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Val Glu Glu Leu Gly Gly Gly Pro Gly Gly Wing Leu Gln Pro Leu Wing Leu Glu Gly Leu Gln Lys Arg Gly Lie Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO 8). 40 - Process according to one of claims 32 to 36 for the preparation of an insulin derivative according to claim 31, characterized in that the insulin precursor has the sequence Meí Wing Thr Thr Ser Thr Gly Asn Wing Arg Phe Val Lys Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr lie Lys Thr Arg Arg Glu Wing Glu Asp Pro Gln Val Gly Gln Glu Le Val Glu Gly Gly Gly Gly Wing Gly Wing Le Gn Gl Le Glu Le Glu Le Glu Le Glu Lys Arg Gly He Val Glu Gl Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn (SEQ ID NO 7). 41. - Insulin derivative precursor according to claim 37. 42.- Insulin derivative precursor according to claim 38. 43.- Insulin derivative precursor according to claim 39. 44.- Precursor of the insulin derivative according to claim 40. 45.- DNA sequence, which encodes a precursor of the insulin derivative according to claim 41. 46.- DNA sequence, which encodes a precursor of the insulin derivative according to claim 42. 47.- DNA sequence, which encodes a The insulin derivative precursor according to claim 43. 48. DNA sequence, which encodes a precursor of the insulin derivative according to claim 44. 49. Expression vehicle, which contains a DNA sequence according to claim 45. 50. - Expression vehicle, which contains a DNA sequence according to claim 46. 51.- Expression vehicle, which contains a DNA sequence according to claim 47. 52.- Vehicle of expression, which contains a DNA sequence according to claim 48. 53.- Host cell, which has been transformed with an expression vehicle according to one of claims 49 to 52. 54.- Pharmaceutical preparation, characterized in that it contains at least an insulin derivative, and / or a physiologically compatible salt thereof, according to one or more of claims 1 to 30. 55.- Pharmaceutical preparation according to claim 54, characterized in that it contains the insulin derivative, and / or the physiological salt. compatible thereof, in dissolved, amorphous and / or crystalline form. 56.- Pharmaceutical preparation according to claim 54, characterized in that it also contains a substance adjuvant deposit (delayed release). 57.- Pharmaceutical preparation according to the claim 50, characterized in that the depot adjuvant substance is protamine sulfate, the insulin derivative and / or the physiologically compatible salt thereof being presented with the protamine sulfate in a co-crystallized material. 58. - Injectable solution with insulin activity, which contains the pharmaceutical preparation according to one of claims 54 to 56 in dissolved form. 59.- Use of the insulin derivative and / or one of its physiologically compatible salts according to one or more of claims 1 to 31 for the production of a pharmaceutical preparation having an insulin activity with rapid initiation of the effect. S. * 3¡