Insulin derivatives having a rapid onset of action

The present invention relates to insulin derivatives which in comparison to human insulin, have an accelerated onset of action, to a process for their preparation and to their use, in particular in pharmaceutical preparations for the treatment of diabetes mellitus. In particular, the present invention relates to insulin derivatives or physiologically tolerable salts thereof in which asparagine (Asn) in position B3 of the B chain is replaced by a naturally occurring basic amino acid residue and at least one amino acid residue in the positions B27, B28 or B29 of the B chain is replaced by another naturally occurring amino acid residue, it optionally being possible for asparagine (Asn) in position 21 of the A chain to be replaced by Asp, Gly, Ser, Thr or Ala and for phenylalanine (Phe) in position B1 of the B chain and the amino acid residue in position B30 of the B chain to be absent.

The present invention relates to insulin derivatives which, in comparison
 to human insulin, have an accelerated onset of action, to a process for
 their preparation and to their use, in particular in pharmaceutical
 preparations for the treatment of diabetes mellitus.
 Approximately 120 million people worldwide suffer from diabetes mellitus.
 Among these are approximately 12 million type I diabetics, for whom the
 administration of insulin is the only therapy possible at present. The
 affected people are assigned insulin injections, as a rule several times
 daily, for life. Although type II diabetes, from which approximately 100
 million people suffer, is not fundamentally accompanied by an insulin
 deficiency, in a large number of cases, however, treatment with insulin is
 regarded as the most favorable or only possible form of therapy.
 With increasing length of the disease, a large number of the patients
 suffer from so-called diabetic late complications. These are essentially
 micro- and macrovascular damage, which depending on the type and extent,
 result in kidney failure, blindness, loss of extremities or an increased
 risk of heart/circulation disorders.
 As a cause, chronically increased blood glucose levels are primarily held
 responsible, since even with careful adjustment of the insulin therapy a
 normal blood glucose profile, such as would correspond to physiological
 regulation, is not achieved (Ward, J. D. (1989) British Medical Bulletin
 45, 111-126; Drury, P. L. et al. (1989) British Medical Bulletin 45,
 127-147; Kohner, E. M. (1989) British Medical Bulletin 45, 148-173)
 In healthy people, insulin secretion is closely dependent on the glucose
 concentration of the blood. Increased glucose levels, such as occur after
 meals, are rapidly compensated by an increased release of insulin. In the
 fasting state, the plasma insulin level falls to a basal value, which is
 sufficient to guarantee a continuous supply of insulin-sensitive organs
 and tissue with glucose. An optimization of the therapy, the so-called
 intensified insulin therapy, is today primarily aimed at keeping
 variations in the blood glucose concentration, especially deviations
 upward, as low as possible (Bolli, G. B. (1989) Diabetes Res. Clin. Pract.
 6, p. 3-p. 16; Berger, M. (1989) Diabetes Res. Clin. Pract. 6, p. 25-p.
 32). This leads to a significant decrease in the occurrence and the
 progression of diabetic late 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 for an
 improved, intensified insulin therapy using subcutaneously administered
 preparations, two insulin preparations having different pharmacodynamics
 are needed. To compensate the blood glucose rise after meals, the insulin
 must flow in rapidly and must only act for a few hours. For the basal
 supply, in particular in the night, a preparation should be available
 which acts for a long time, has no pronounced maximum and only infuses
 very slowly.
 The preparations based on human and animal insulins only fulfill the
 demands of an intensified insulin therapy, however, in a restricted
 manner. After subcutaneous administration, rapidly acting insulins
 (unmodified insulins) pass too slowly into the blood and to the site of
 action and have too long an overall duration of action. The result is that
 the postprandial glucose levels are too high and the blood glucose begins
 to fall severely several hours after the meal (Kang, S. et al. (1991)
 Diabetes Care 14, 142-148; Home, P. J. et al. (1989) British Medical
 Bulletin 45, 92-110; Bolli, G. B. (1989) Diabetes Res. Clin. Pract. 6, p.
 3-p. 16). The available basal insulins in turn, especially NPH insulins,
 have too short a duration of action and have a too severely pronounced
 maximum.
 Beside the possibility of affecting the profile of action by means of
 pharmaceutical principles, the alternative presents itself today of
 designing insulin derivatives, with the aid of genetic engineering, which
 achieve specific properties such as onset and duration of action solely by
 means of their structural properties. By the use of suitable insulin
 derivatives, a significantly better adjustment of the blood glucose more
 closely adapted to the natural conditions could therefore be achieved.
 Insulin derivatives having an accelerated onset of action are described in
 EP 0 214 826, EP 0 375 437 and EP 0 678 522. EP 0 214 826 relates, inter
 alia, to substitutions of B27 and B28, but not in combination with the
 substitution of B3. EP 0 678 522 describes insulin derivatives which have
 various amino acids, preferably proline, but not glutamic acid, in the
 position B29. EP 0 375 437 encompasses insulin derivatives having lysine
 or arginine in B28, which can optionally be additionally modified in B3
 and/or A21.
 EP 0 419 504 discloses insulin derivatives which are protected against
 chemical modifications by changing asparagine in B3 and at least one
 further amino acid in the positions A5, A15, A18 or A21. Combinations with
 modifications in positions B27, B28 or B29 are, however, not described. An
 indication that these compounds have modified pharmacodynamics resulting
 in a more rapid onset of action is not given.
 WO 92/00321 describes insulin derivatives in which at least one amino acid
 of the positions B1-B6 is replaced by lysine or arginine. According to WO
 92/00321, insulins of this type have a prolonged action. Combinations with
 modifications of the positions B27, 28, 29, however, are not disclosed.

The object of the present invention is to prepare insulin derivatives which
 after administration, in particular after subcutaneous administration,
 have an onset of action which is accelerated in comparison with human
 insulin.
 Insulin derivatives are derivatives of naturally occurring insulins, namely
 human insulin (see SEQ ID NO 1=A chain of human insulin; see SEQ ID NO 2=B
 chain of human insulin, sequence listing) or animal insulins which differ
 from the corresponding, otherwise identical naturally occurring insulin by
 substitution of at least one naturally occurring amino acid residue and/or
 addition of at least one amino acid residue and/or organic residue.
 It is further an object of the present invention to provide a process for
 the preparation of the insulin derivatives having the property mentioned,
 the corresponding intermediates and their precursors.
 The object is achieved by an insulin derivative or a physiologically
 tolerable salt thereof in which asparagine (Asn) in position B3 of the B
 chain is replaced by a naturally occurring basic amino acid residue and at
 least one amino acid residue in the positions B27, B28 or B29 of the B
 chain is replaced by another naturally occurring amino acid residue, it
 optionally being possible for asparagine (Asn) in position 21 of the A
 chain to be replaced by Asp, Gly, Ser, Thr or Ala and for phenylalanine
 (Phe) in position B1 of the B chain and the amino acid residue in position
 B30 of the B chain to be absent.
 Preferably, the insulin derivative or its physiologically tolerable salt is
 of formula I
 ##STR1##
 in which
 (A1-A5) are the amino acid residues in the positions A1 to A5 of the A
 chain of human insulin (cf. SEQ ID NO 1) or animal insulin,
 (A12-A19) are the amino acid residues in the positions A12 to A19 of the A
 chain of human insulin (cf. SEQ ID NO 1) or animal insulin,
 (B8-B18) are the amino acid residues in the positions B8 to B18 of the B
 chain of human insulin (cf. SEQ ID NO 2) or animal insulin,
 (B20-B26) are the amino acid residues in the positions B20 to B26 of the B
 chain of human insulin (cf. SEQ ID NO 2) or animal insulin,
 A8, A9, A10 are the amino acid residues in the positions A8, A9 and A10 of
 the A chain of human insulin (cf. SEQ ID NO 1) or animal insulin,
 A21 is Asn, Asp, Gly, Ser, Thr or Ala,
 B30 is --OH or the amino acid residue in position B30 of the B chain of
 human insulin (cf. SEQ ID NO 2) or animal insulin,
 B1 is a phenylalanine residue (Phe) or a hydrogen atom,
 B3 is a naturally occurring basic amino acid residue,
 B27, B28 and B29 are the amino acid residues in the positions B27, B28 and
 B29 of the B chain of human insulin (cf. SEQ ID NO 2) or animal insulin or
 in each case are another naturally occurring amino acid residue, where at
 least one of the amino acid residues in the positions B27, B28 and B29 of
 the B chain is replaced by another naturally occurring amino acid residue.
 Of the twenty naturally occurring amino acids which are genetically
 encodable, the amino acids glycine (Gly), alanine (Ala), valine (Val),
 leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), cysteine
 (Cys), methionine (Met), asparagine (Asn), glutamine (Gln), phenylalanine
 (Phe), tyrosine (Tyr), tryptophan (Trp) and proline (Pro) are designated
 here as neutral amino acids, the amino acids arginine (Arg), lysine (Lys)
 and histidine (His) are designated as basic amino acids and the amino
 acids aspartic acid (Asp) and glutamic acid (Glu) are designated as acidic
 amino acids.
 Preferably, the insulin derivative or its physiologically tolerable salt
 according to the present invention is a derivative of bovine insulin,
 porcine insulin or human insulin, namely an insulin derivative or a
 physiologically tolerable salt thereof of the formula 1, which is
 distinguished in that
 A8 is alanine (Ala),
 A9 is serine (Ser),
 A10 is valine (Val) and
 B30 is alanine (Ala) (amino acid residues A8 to A10 and B30 of bovine
 insulin),
 A8 is threonine (Thr),
 A9 is serine (Ser) and
 A10 is isoleucine (Ile) (amino acid residues A8 to A10 of the insulins of
 man or pigs), where
 B30 is alanine (Ala) (amino acid residue B30 of porcine insulin) or
 B30 is threonine (Thr) (amino acid residue B30 of human insulin, cf. SEQ ID
 NO 2).
 Particularly preferably, an insulin derivative or a physiologically
 tolerable salt thereof of the formula I with the amino acid residues A8 to
 A10 and B30 of human insulin is one which is furthermore distinguished in
 that
 (A1-A5) are the amino acid residues in the positions A1 to A5 of the A
 chain of human insulin (cf. SEQ ID NO 1),
 (A12-A19) are the amino acid residues in the positions A12 to A19 of the A
 chain of human insulin (cf. SEQ ID NO 1),
 (B8-B18) are the amino acid residues in the positions B8 to B18 of the B
 chain of human insulin (cf. SEQ ID NO 2) and
 (B20-B26) are the amino acid residues in the positions B20 to B26 of the B
 chain of human insulin (cf. SEQ ID NO 2).
 Further preferred embodiments of the present invention are an insulin
 derivative or a physiologically tolerable salt thereof of the formula 1,
 wherein the amino acid residue in position B1 of the B chain is a
 phenylalanine residue (Phe) or
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula 1, wherein the amino acid residue in position B3 of the B chain is
 a histidine (His), lysine (Lys) or arginine residue (Arg).
 Further preferred embodiments of the present invention are an insulin
 derivative or a physiologically tolerable salt thereof of the formula 1,
 wherein at least one of the amino acid residues in the positions B27, B28
 and B29 of the B chain is replaced by a naturally occurring amino acid
 residue which is selected from the group consisting of the neutral or of
 the acidic amino acids,
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula I, wherein at least one of the amino acid residues in the
 positions B27, B28 and B29 of the B chain is a naturally occurring amino
 acid residue which is selected from the group consisting of isoleucine
 (Ile), aspartic acid (Asp) and glutamic acid (Glu), preferably wherein at
 least one of the amino acid residues in the positions B27, B28 of the B
 chain is replaced by a naturally occuring amino acid residue which is
 selected from the group consisting of the neutral amino acids, or
 particularly preferably wherein at least one of the amino acid residues in
 the positions B27, B28 and B29 of the B chain is an isoleucine residue
 (Ile), or
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula I, wherein at least one of the amino acid residues in the
 positions B27, B28 and B29 of the B chain is a naturally occurring amino
 acid residue which is selected from the group consisting of the acidic
 amino acids, preferably wherein at least one of the amino acid residues in
 the positions B27, B28 and B29 of the B chain is an aspartic acid residue
 (Asp), preferably wherein the amino acid residue in position B27 or B28 of
 the B chain is an aspartic acid residue (Asp), or wherein at least one of
 the amino acid residues in the positions B27, B28 and B29 of the B chain
 is a glutamic acid residue (Glu).
 A preferred embodiment of the present invention is also an insulin
 derivative or a physiologically tolerable salt thereof of the formula I,
 wherein the amino acid residue in position B29 of the B chain is an
 aspartic acid residue (Asp).
 Further preferred embodiments are an insulin derivative or a
 physiologically tolerable salt thereof of the formula I, wherein the amino
 acid residue in position B27 of the B chain is a glutamic acid residue
 (Glu),
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula I, wherein the amino acid residue in position B28 of the B chain
 is a glutamic acid residue (Glu), or
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula I, wherein the amino acid residue in position B29 of the B chain
 is a glutamic acid residue (Glu).
 Very particularly preferably, an insulin derivative or a physiologically
 tolerable salt thereof is one which is distinguished in that the B chain
 has 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 Pro Glu Thr
 (SEQ ID NO 3), for example Lys (B3), Glu (B29)-human insulin, or
 an insulin derivative or a physiologically tolerable salt thereof which is
 distinguished in that the amino acid residue in position B27 of the B
 chain is an isoleucine residue (Ile), preferably an insulin derivative or
 a physiologically tolerable salt thereof which is distinguished in that
 the B chain has 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 Ile Pro Lys Thr
 (SEQ ID NO 5), for example Lys (B3), Ile (B27)-human insulin, or
 an insulin derivative or a physiologically tolerable salt thereof of the
 formula I, wherein the amino acid residue in position B28 of the B chain
 is an isoleucine residue (Ile), preferably an insulin derivative or a
 physiologically tolerable salt thereof which is distinguished in that the
 B chain has 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 Ile Lys Thr
 (SEQ ID NO 4), for example Lys (B3), Ile (B28)-human insulin.
 Particularly preferably, an insulin derivative or a physiologically
 tolerable salt thereof of the formula I, which is distinguished in that
 the amino acid residue in position B28 of the B chain is an isoleucine
 residue (Ile) and the amino acid residue in position A21 is an asparagine
 residue (Asp), is preferably one wherein the A chain has the sequence
 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Tyr Gln Leu
 Glu Asn Tyr Cys Asp (SEQ ID NO.: 9)
 and the B chain has 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 Pyr Thr Ile Lys Thr (SEQ ID NO.: 10)
 (Lys (B3), Ile (B28), Asp (A21)-human insulin).
 The insulin derivatives of the formula I can preferably be prepared by
 genetic engineering.
 The object set at the outset is accordingly further achieved by a process
 for the preparation of an insulin derivative or of a physiologically
 tolerable salt thereof of the formula I, comprising the construction of a
 replicable expression vehicle which contains a DNA sequence which codes
 for a precursor of the insulin derivative in which the amino acid residue
 in position A1 of the A chain is linked to the amino acid residue B30 of
 the B chain via a peptide chain of the formula II
EQU --R.sup.1.sub.n --Arg-- II
 in which R.sup.1.sub.n is a peptide chain having n amino acid residues and
 n is an integer from 0 to 34, and the B chain is prolonged in position B1
 by a peptide chain of the formula III
EQU Met-R.sup.2.sub.m --(Arg).sub.p -- III
 in which R.sup.2.sub.m is a peptide chain having m amino acid residues, m
 is an integer from 0 to 40, preferably from 0 to 9, and p is 0, 1 or 2,
 where for p=0 the peptide chain R.sup.2.sub.m preferably ends with Lys,
 expression in a host cell and release of the insulin derivative from its
 precursor using chemical and/or enzymatic methods.
 Preferably, the process is one wherein the host cell is a bacterium,
 particularly preferably one wherein the bacterium is E. coli.
 Preferably, the process is one wherein the host cell is a yeast,
 particularly preferably one wherein the yeast is Saccharomyces cerevisiae.
 For the preparation of an insulin derivative having the amino acid
 sequences SEQ ID NO.: 9 (A chain) and SEQ ID NO.: 10 (B chain), the
 precursor of this insulin derivative preferably has the sequence
 Met Ala Thr Thr Ser Thr Gly Asn Ser Ala 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 Ile Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu
 Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu
 Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln
 Ley Glu Asn Tyr Cys Asp (SEQ ID NO.: 11),
 a Lys (B3), Ile (B28), Asp (A21 )-preproinsuLin.
 For the preparation of an insulin derivative having the amino acid sequence
 SEQ ID NO 3, the precursor of this insulin derivative preferably has the
 sequence
 Met Ala Thr Thr Ser Thr Gly Asn Ser Ala Arg Phe Va 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 Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu
 Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu
 Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln
 Leu Glu Asn Tyr Cys Asn (Lys (B3), Glu (829)-preproinsulin)
 (SEQ ID NO 6).
 For the preparation of an insulin derivative having the amino acid sequence
 SEQ ID NO 5, the precursor of this insulin derivative preferably has the
 sequence
 Met Ala Thr Thr Ser Thr Gly Asn Ser Ala 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
 Ile Pro Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Gu Leu Gly
 Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln
 Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
 Glu Asn Tyr Cys Asn (Lys (B3), Ile (B27)-preproinsulin)
 (SEQ ID NO 8).
 For the preparation of an insulin derivative having the amino acid sequence
 SEQ ID NO 4, the precursor of this insulin derivative preferably has the
 sequence
 Met Ala Thr Thr Ser Thr Gly Asn Ser Ala 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 Ile Lys Thr Arg Arg Glu Ala Glu Asp Pro Gln Val Gly Gln Val Glu Leu
 Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu
 Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln
 Leu Glu Asn Tyr Cys Asn (Lys (B3), Ile (B28)-preproinsulin)
 (SEQ ID NO 7).
 The present invention accordingly also relates to said precursors of the
 preferred insulin derivatives, namely the peptides having the sequence
 numbers SEQ ID NO.: 11, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, the DNA
 sequences which code for said precursors, the expression vehicles which
 comprise these DNA sequences and the host cells which are transformed
 using these expression vehicles.
 The insulin derivatives of the formula I are mainly prepared by genetic
 engineering by means of site-directed mutagenesis according to standard
 methods.
 To do this, a gene structure coding for the desired insulin derivative of
 the formula I is constructed and expressed in a host cell--preferably in a
 bacterium such as E. coli or a yeast, in particular Saccharomyces
 cerevisiae--and--if the gene structure codes for a fusion protein--the
 insulin derivative of the formula I is released from the fusion protein;
 analogous methods are described, for example, in EP-A-0 211 299, EP-A-0
 227 938, EP-A-0 229 998, EP-A-0 286 956 and the DE patent application P 38
 21 159.
 The removal of the fusion protein component can be carried out chemically
 by cell disruption by means of cyanogen halide (see EP-A-0 180 920).
 In the preparation by means of a preproinsulin precursor which has a fusion
 protein component (presequence) according to U.S. Pat. No. 5,358,857, the
 removal of the fusion protein component takes place in a later stage
 together with the removal of the C peptide.
 The insulin precursor is then subjected to oxidative sulfitolysis according
 to the method described, for example, by R. C. Marshall and A. S. Inglis
 in "Practical Protein Chemistry--A Handbook" (Publisher A. Darbre) 1986,
 pages 49-53 and then renatured in the presence of a thiol with formation
 of the correct disulfide bridges, e.g. according to the method described
 by G. H. Dixon and A. C. Wardlow in Nature (1960), pages 721-724. The
 insulin precursors, however, can also be folded directly (EP-A-0 600 372;
 EP-A-0 668 292).
 The C peptide is removed by means of tryptic cleavage--e.g. according to
 the method of Kemmler et al., J. B. C. (1971), pages 6786-6791 and the
 insulin derivative of the formula I is purified by means of known
 techniques such as chromatography--e.g. EP-A-0 305 760--and
 crystallization.
 If n in formula II is 0, tryptic cleavage serves to sever the peptide bond
 between A and B chains.
 In this process, the B chain C terminal ends with arginine or two arginine
 residues. These can be removed enzymatically by means of carboxy-peptidase
 B.
 The insulin derivatives according to the invention have full biological
 activity. This was shown by intravenous administration to rabbits and the
 blood glucose fall resulting therefrom (Examples 5 and 6).
 The more rapid onset of action after subcutaneous administration was shown
 in fasting dogs using the euglycemic clamp technique (Example 7). 0.3
 IU/kg was administered. The reference preparation was human insulin. In
 the clamp technique, the blood glucose value is measured at short time
 intervals after insulin injection and exactly the amount of glucose to
 compensate the fall is infused. This has the advantage that no
 counter-regulation occurs with the animals, as would be the case with a
 severe fall in the blood glucose after the administration of insulin. The
 amount and the time course of the infused glucose characterize the action
 of the insulin. Lys(B3), Glu(B29)--(SEQ ID NO 3) and Lys(B3),
 Ile(B28)--(SEQ ID NO 4) insulin have a clearly more rapid onset of action
 than human insulin. The maximum action (glucose infusion rate) is achieved
 after 100 minutes with human insulin, after 80 minutes, however, with
 Lys(B3), Glu(B29)-insulin (SEQ ID NO 3) and already after 60 minutes with
 Lys(B3)--, Ile(B28)-insulin (SEQ ID NO 4). Therefore these analogs, when
 they are injected shortly before a meal, should compensate the
 postprandial rise in the blood glucose better than human insulin.
 The insulin derivatives described are therefore suitable both for the
 therapy of type I and of type II diabetes mellitus, preferably in
 combination with a basal insulin.
 The present invention therefore also relates to the use of the insulin
 derivative and/or its physiologically tolerable salt of the formula I for
 the production of a pharmaceutical preparation which has an insulin
 activity with a rapid onset of action.
 A suitable carrier medium which is physiologically acceptable and
 compatible with the insulin derivative is a sterile aqueous solution which
 is made isotonic with blood in the customary manner, e.g. by means of
 glycerol, sodium chloride, glucose, and in addition contains one of the
 customary preservatives, e.g. phenol, m-cresol or p-hydroxybenzoic acid
 ester. The carrier medium can additionally contain a buffer substance,
 e.g. sodium acetate, sodium citrate, sodium phosphate. For adjustment of
 the pH, dilute acids (typically HCl) or alkalis (typically NaOH) are used.
 The preparation can furthermore contain zinc ions.
 The insulin derivatives can be employed in the pharmaceutical preparations
 even in the form of their physiologically tolerable salts, as alkali metal
 or as ammonium salts. Any desired proportion of one or more insulin
 derivatives of the formula I or an insulin derivative of the formula I can
 be present in a mixture of other of these insulin derivatives
 independently of one another in each case in dissolved, amorphous and/or
 crystalline form.
 It is sometimes advantageous to add to the preparation according to the
 invention a suitable amount of a suitable stabilizer which prevents the
 precipitation of protein under thermomechanical stress on contact with
 various materials. Such stabilizers are disclosed, for example, in
 EP-A-18609, DE-A 32 40 177 or in WO-83/00288.
 The present invention further relates to a pharmaceutical preparation which
 comprises at least one insulin derivative and/or a physiologically
 tolerable salt thereof of the formula I, preferably in dissolved,
 amorphous and/or crystalline form.
 The insulin derivatives according to the invention have a rapid onset of
 action. In practical insulin therapy, it is customary under certain
 circumstances to mix rapid-acting insulins with preparations which contain
 a depot auxiliary (e.g. NPH insulin). Depending on the composition,
 preparations result from this whose profiles of action correspond to the
 superimposed individual profiles provided the individual components in the
 mixture are stable and are not mutually affected. When mixing an insulin
 derivative with human NPH insulin, however, it is to be expected that,
 particularly on long-term storage, an exchange takes place between the
 dissolved derivative and the crystalline NPH insulin. As a result of this
 both the pharmacodynamics of the depot insulin and those of the dissolved
 rapidly acting insulin are changed in an unforeseeable manner. In order to
 avoid this, it is sensible to prepare the rapidly acting derivative using
 a depot auxiliary--for example as NPH insulin. This depot form of the
 insulin derivative can then be mixed as desired with the dissolved rapidly
 acting form without the composition of one or the other form changing in
 the course of storage due to exchange.
 Although the invention in essence relates to rapidly acting insulin
 derivatives, it accordingly, however, also comprises the possibility of
 preparing derivatives of this type as a depot form for the purpose of
 miscibility, the depot auxiliary preferably being protamine sulfate and
 the insulin derivative and/or its physiologically tolerable salt being
 present with the protamine sulfate in a cocrystallizate.
 The present invention further relates to an injectable solution which
 comprises the pharmaceutical preparations described above in dissolved
 form.
 EXAMPLES
 Example 1
 Construction of Lys (B3)-proinsulin as a Starting Point for the Plasmids
 Relevant to the Invention Corresponding to Examples 2-4
 U.S. Pat. No. 5,358,857 describes the vector pINT 90d and the PCR primers
 Tir and Insu 11. These components serve as starting materials for the
 construction of a plasmid pINT 125d, which codes for the desired Lys
 (B3)-proinsulin.
 Additionally, the primers Insu 35 having the sequence
 5' TTT GTG AAG CAG CAC CTG 3' (SEQ ID NO: 12)
 and Insu 36 having the sequence
 5' CAG GTG CTG CTT CAC AAA 3' (SEQ ID NO: 12)
 are synthesized.
 A PCR reaction is carried out using the primers Tir and Insu 36 and a
 second reaction is carried out using the primers Insu 11 and Insu 35. The
 template used for this is pINT 90d DNA.
 The products of the two PCR reactions are partially complementary, such
 that when they are combined in a third PCR reaction with the primers Tir
 and Insu 11 they afford a fragment which codes for a proinsulin variant
 which contains the B chain lysine in position 3. This PCR fragment is
 precipitated in ethanol for purification, dried and then digested with the
 restriction enzymes Nco 1 and Sal 1 according to the instructions of the
 manufacturer. The reaction mixture is separated by gel electrophoresis and
 the desired Nco 1/Sal 1 fragment is isolated.
 The application cited describes a plasmid pINT 91d which codes for a
 mini-proinsulin. If the sequence coding for mini-proinsulin is excised by
 means of Nco 1 and Sal 1 and the residual plasmid DNA is isolated, this
 residual plasmid DNA can be reacted with the shown Nco 1/Sal 1 PCR
 fragment in a T 4 ligase reaction to give the plasmid pINT 125d. This is
 transformed by E. coli K12, replicated therein and reisolated. After
 verification of the plasmid structure by means of DNA sequence and
 restriction analysis, pINT 125d DNA is used as template DNA for the
 introduction of further mutations into this proinsulin variant.
 Example 2
 Construction of Lys (B3) Glu (B29)-proinsulin
 For the preparation of the mutein, the primers 329a having the sequence
 5' TTC TAC ACA CCC GAG ACC CGC GGC ATC G-3' (SEQ ID NO: 13)
 and 329b having the sequence
 5' GCC GCG GGT CTC GGG TGT GTA GAA GAA GC 3' (SEQ ID NO: 14)
 are synthesized.
 The template used is DNA of the plasmids pINT125d and pINT91d. Primer 329a
 is reacted with the primer Insu 11 on the template pINT91d and primer 329b
 is reacted with Tir (see above example) on the template pINT125d in a PCR
 reaction. Since both PCR products are partially complementary, they can be
 combined in a direct PCR reaction and reacted again with the primers Tir
 and Insu 11. A DNA fragment results which codes for the desired mutein.
 This fragment is double-digested using the restriction enzymes Nco 1 and
 Sal 1 and the resulting Nco 1/Sal 1 fragment is inserted into the pINT 91
 d residual plasmid DNA in a T4 ligase reaction.
 The plasmid pINT 329 results, which after amplification in E. coli K12 by
 means of restriction and DNA sequence analysis is verified with respect to
 the desired structure.
 The proinsulin derivative encoded by the plasmid is characterized by the
 two amino acid replacements and a C-bonding member which consists of the
 amino acid arginine.
 Example 3
 Construction of Lys (B3) Ile (B27)-proinsulin
 The construction is carried out according to the preceding example using
 the primer pairs
 KB3 JB 27A
 5' TTC TAC ATC CCC AAG ACC CGC CG 3' (SEQ ID NO: 15)
 and Insu 11
 and also
 K B3 J 27B
 5' CTT GGG GAT GTA GAA GAA GCC TCG 3' (SEQ ID NO: 16)
 and Tir.
 The template used in both PCR reactions is DNA of the plasmid pINT125d. The
 PCR products of both reactions are combined in a third reaction, as
 described in Example 1, and the product is cloned corresponding to the
 example.
 The plasmid pINT332 results.
 Example 4
 Construction of Lys (B3) Ile (B28)-proinsulin
 The construction is carried out according to Example 3 using the primer
 pairs:
 KB3 JB 28A
 5' TAC ACA ATC AAG ACC CGC CGG GAG-3' (SEQ ID NO: 17)
 and Insu 11
 and also
 KB J B28B
 5' GGT CTT GAT TGT GTA GM GM GCC TCG-3' (SEQ ID NO: 18)
 and Tir.
 The plasmid pINT 333 results.
 Expression of the constructed insulin variants
 The plasmids pINT 329, 332 and 333 are each transformed by way of example
 by E. coli K12 W3110. Recombinant bacteria which contain plasmids which
 encode the respective variants are then fermented according to Example 4
 of the US patent having the U.S. Pat. No. 5,227,293 and the desired raw
 material for the production of the respective insulin variants is thus
 produced.
 Example 5
 Construction of Lys (B3), Ile (B28), Asp (A21)-proinsulin
 Construction is carried out as in Example 3. instead of pINT125d, however,
 the template serving for the PCR reaction is DNA of the plasmid pINT333,
 which was constructed in Example 4. The following primer pair is used
 here:
 P-pint 365
 5'-TTTTTTGTCGACTATTAGTCGCAGTAGTTCTACCAGCTG-3' (SEQ ID NO: 19)
 and Tir.
 The plasmid pINT365 results.
 Example 6
 Biological Activity of Lys(B3),Glu(B29)-insulin After Intravenous
 Administration to Rabbits