NOVEL INSULIN DERIVATIVES AND PHARMACEUTICAL PREPARATIONS CONTAINING THESE DERIVATIVES

Abstract: Insulin derivatives in which one or more of the four amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain and pharmaceutical preparations containing these insulin derivatives.

Description: Novel insulin derivatives and pharmaceutical preparations containing these derivatives. TECHNICAL FIELD The present invention relates to novel insulin derivatives and to insulin preparations showing protracted action and comprising at least one of the novel insulin derivatives and, if desired, a fast acting insulin. BACKGROUND ART In severe or chronic cases the disease of Diabetes is us- ually treated with injection preparations containing insu¬ lin, e.g. porcine insulin, bovine insulin or human insulin. Soluble insulin preparations are usually fast acting, but in return the action ceases after few hours. Therefore, it is necessary to give frequent injections, normally several times a day. In order to overcome this disadvantage insulin preparations with protracted action have been formulated so that the ac¬ tion is maintained for several hours or even up to 24 hours or longer. Using such protracted preparations some diabetic patients only have to receive a small number of injections, e.g. a single or two injections during 24 hours. Such a protracted action can be achieved by converting the insulin to a slightly soluble salt, such as zinc insulin or protamin insulin. The slightly soluble insulin salts are used in the form of suspensions from which the insulin is gradually released e.g. after subcutaneous injection. Recently other methods have been invoked to achieve a pro- • tracted action. An example hereof is the encapsulation of insulin crystals in polymerized serum albumin. Another ex¬ ample is continuously acting infusion devices, so-called in¬ sulin pumps, which however may be uncomfortable and entail a risk to the patient. The specifications of Danish patent applications No. 3582/84 No. 3583/84 and No. 3757/84 disclose the preparation and use of insulin derivatives wherein the C-terminus of the B-chain is extended with an organic group carrying at least one positive charge, preferably Arg-OH or Arg-Arg-OH. Pre¬ parations containing suspensions of such insulin deriva¬ tives exhibit a protracted action. However, the preferred methods " for preparing the insulin derivatives disclosed in the specification of Danish patent application No. 3582/84 in an industrial scale involve the use of trypsin-like enzymes which implies that enzymatic ac¬ tivity may contaminate the final preparations, cf. examples of execution in the specification of Danish patent application No. 3757/84. The protracted action of the above-mentioned insulin derivatives wherein the C-terminus of the B-chain is extended with an organic group carrying at least one po¬ sitive charge results exclusively from the extension of the B-chain. However, the extended B-chain is sensitive to en¬ zymes which cleave the B-chain, especially at B29-lysine, and consequently, the protracted action may be reduced and even abolished. This enzymatic cleavage can also take place after subcutaneous injection, cf. the specification of Danish patent application No. 3583/84, page 15, line 18. As a result of the enzymatic activity the protracted action of the preparations containing the above-mentioned insulin derivatives wherein the C-terminus of the B-chain is ex¬ tended with an organic group carrying at least one posi¬ tive charge may vary considerably. DISCLOSURE OF THE INVENTION It has now surprisingly been found that by the use of in¬ sulin derivatives, wherein one or more of the four amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain, in insulin preparations, in parti¬ cular injection preparations, a hitherto unknown and de¬ sirably long-lasting protracted action is achieved.- Accordingly, the invention relates to insulin derivatives wherein one or more of the four amino acid residues of po- sitions A4, A17, B13 and B21 comprise an uncharged side chain. The invention relates specifically to insulin derivatives wherein one or two of the four amino acid residues of po¬ sitions A4, A17, B13 and B21 comprise an uncharged side chain. The invention relates in particular to insulin derivatives in which one of the four amino acid residues of positions A4, Al7, B13 and B21 comprises an uncharged side chain. The invention relates particularly to insulin derivatives wherein the amino acid residue of position A4 comprises an uncharged side chain. The invention relates specifically to insulin derivatives in which the amino acid residue of position A17 comprises an uncharged side chain. The invention relates in particular to insulin derivatives wherein the amino acid residue of position A4 is a glut- amine residue. The invention relates specifically to insulin derivatives wherein the amino acid residue of position A17 is a glut- amine residue. The invention also relates to insulin preparations contain¬ ing at least one insulin derivative wherein one or more of the four amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain and, if desired, also containing a fast acting insulin. A particularly advantageous embodiment of the insulin pre¬ paration of the invention is an injection preparation exhi¬ biting protracted insulin action and comprising a suspen¬ sion of at least one insulin derivative of the invention in an isotonic aqueous medium having a neutral pH and option¬ ally containing a buffer and/or a preserving agent and, if desired, a fast acting insulin. Insulins which are normally used for the treatment of Dia¬ betes, such as porcine, bovine, ovine, and human insulin, do all contain six free carboxylic groups, viz. in the A4-, A17-, A21-, B13-, B21- and B30-amino acid residues. The numbering is referring to the positions in the insulin A and B chain, respectively, starting from the N termini. The biological activity of insulin will usually decrease by an increasing degree of derivatization. However, the biologi¬ cal activity is influenced surprisingly little even at high degrees of derivatization of the free carboxylic acid groups but when all six free carboxylic acid groups are deriva- tized the biological activity is completely abolished, vide D. Levy: Biochem. Biophys. Acta, 310 (1973), pages 406-415. By means of the insulin preparations of the invention the insulin activity can be maintained, and at the same time a surprisingly protracted action can be achieved. This pro¬ tracting effect is dependent on the properties of the spe- cific derivative, e.g. the number of amino acid residues comprising an uncharged side chain and the chemical consti¬ tution of the uncharged side chain. Thus, it is possible to vary the protracted action at will. A considerable advan¬ tage and novel feature of the insulin derivatives of the invention is that the protracted action is relatively in¬ sensitive to tryptic activity irrespective of the origin of the tryptic activity. In the case of Insulin-Dependent Diabetes a frequently used therapy consists in two daily injections of a protracted insulin preparation, one in the morning and one in. the eve¬ ning just before bedtime, in order to create a basic insu¬ lin level. Additionally, three injections of a fast acting insulin preparation are given prior to the principal meals. The disadvantage of this therapy is that the late injection of the protracted preparation may result in a dangerously low blood glucose level in the course of the night. This situation may be avoided by injecting a mixed preparation of a protracted and a fast acting insulin before supper, whereby hypoglycemia will, if at all, occur during the eve¬ ning, where it can be averted by means of a light snack. However, this type of the therapy often results in hyper- glycemia during the morning, as the most used protracted ® R) r----- an d "Monotard" ^— do not act long enough. Therefore, it is desirable to provide the diabetic patient with insulin preparations that act longer than the preparations commonly in use, in particular if one injection of such preparations will suffice for one or even several days. Insulin preparations prepared accord- ing to the invention exhibit a protracted insulin action of the same or longer duration than that of the commonly used protracted insulin preparation "Insulatard" ( * R~') . The invention relates in particular to the following spe¬ cific compounds: Human insulin-Al7-gln Human insulin-A4-gln Porcine insulin-B21-gln Human insulin-Bl3-gln Human insulin-(A17,B21)-gin Human insulin-A4-ala Human insulin-B21-thr Human insulin-B13-val Human insulin-B21-thr-Al7-gln Human insulin-B21-methyl ester Human insulin-Al7-methyl ester When the insulin preparations of the invention contain a fast acting insulin, this insulin may e.g. be human or porcine insulin. By use of porcine, bovine, ovine, rabbit, or in particular human insulin, the insulin derivatives of the invention can be prepared in a manner known per se for the conversion of a carboxylic acid group into an uncharged group, e.g. by esterification or amidation. Thus, an insulin derivative wherein one or more of the carboxylic acid groups of posi- tions A4, A17, B13 and B21 are methylated, is e.g. prepared by reacting human insulin-B30-methyl ester in borontri- fluoride/methanol for a suitable time, followed by fractio- nation of the modified insulin derivatives on an anion ex¬ change column at a slightly basic pH value. The products are fractionated and isolated by preparative HPLC, where¬ after the B30 methyl ester group is removed by gentle hydro¬ lysis in an ice-cold solution of 0.1 N sodium hydroxide. The preparation of the insulin derivatives of the invention wherein one or more amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain can be carried out by use of biotechnological methods. Thus, some or all or the glutamic acids located at these positions may be replaced by naturally occuring amino acids carrying an uncharged side chain, such as glutamine. The preferred method to prepare these modified insulins is by biosynthesis, as by e.g. changing only a single base in the codon in the DNA strand coding for glutamic acid this codon can be made code for glutamine. By changing bases in the DNA strand coding for insulin (or proinsulin) it is thus possible to make modifications which would be diffi¬ cult or impossible to make chemically due " to limitations as to isolation. There exists a number of different methods to prepare human insulin by biosynthesis. It is common to all of them that the DNA strand coding for either the entire proinsulin, a modified version thereof or the A- and B-chain separately has been inserted into a replicable plasmide containing a suitable promotor. By transforming this system into a given host organism a product can be produced, which can be con¬ verted into authentic human insulin in a manner known per se. Some known methods for biosynthesis of proinsulin or A- ' and B-chain and their conversion into insulin are described below. In the well known Genentech process an intracellular prepa¬ ration in E. coli of polypeptides is made, wherein these polypeptides are a fusion between β-galactosidase and the A- and B-chain, respectively, of insulin. After fermentation the two products are isolated, the A- and B-chains are split off from ( -galactosidase by means of cyanogen brom¬ ide, whereafter the sulphitated chains are combined under reducing conditions in the presence of dithiothreitol at a pH value of 10.5. Finally human insulin is isolated from this reaction mixture. In the process disclosed in EPO 55945 the product is synthe- tized as a fusion protein with another protein separated by a cleaving site. This may be a protease cleaving site or a site which can be cleaved chemically (e.g. methionine) . The gene for proinsulin is e.g. cloned as a genefusion with another protein by means of recombinant DNA techniques. After isolation, the chimeric protein is cleaved by using a suitable enzyme or CNBr, whereupon the denatured proin- sulin is isolated in a sulphitolyzed form. This product is renatured under reducing conditions using J-mercapto ethanol at a pH of 10.5, as described by Frank, followed by trypsin/CpB cleavage as described by Kemmler (1971) . The resulting insulin is isolated in a manner known per se. Finally, proinsulin can be prepared biosynthetically by using the method disclosed in EPO 116201. In this method proteins are secreted into the culture medium by using the oL-factor system from Saccharamyces cerevisiae. By insert- ing the gene coding for proinsulin into this system, pro¬ insulin can be isolated from the culture medium by using the system described in Danish patent application No. 3091/84. Hereafter, proinsulin can be converted into insu¬ lin by the known methods described above. In the above, ' methods are described in which the product is either proinsulin or A- or B-rchain separately. Furthermore, these products are either synthetized together with a sig¬ nal sequence, the purpose of which is to bring the product through to the cell surface where it is split, or as a fu- sion with a protein, the purpose of which is to stabilize the product. Moreover it is possible to prepare modified proinsulins bio¬ synthetically. European Patent Application No. 82303071.3 discloses proinsulins wherein the C-chain is modified by means of recombinant DNA techniques. The above-mentioned modifications of insulin can be pre¬ pared by expressing the modified DNA sequence in a suitable expression system. The changes of the DNA sequence can be made by _in vitro mutagenesis of the insulin gene by a meth- od described by Thomas A. Kunkel, Proc. Natl. Acad. Sci. , USA, Vol _82_, pp. 448-492 (1985). According to this method the insulin gene is cloned into the single-stranded DNA bacteriophage M13. DNA from this phage is purified, and hereto a primer, typically a 15-25-mer, which contains the given mutation (one mismatch) as well as a homology on each side of this mutation is annealed. The primer is then ex¬ tended by use of a DNA polymerase in the total length of the phagegenome and thus provides the complete double- stranded molecule, wherein one strand contains the mutation, and the other contains the wild type gene. By introducing this molecule into an E. coli cell, the phage progeny will partly be phages containing the wild type gene, partly phages containing the mutation. Among these phages it is then possible to screen for the desired type by hybridiza¬ tion with the mutagenization primer to single-stranded DNA of the phage progeny. The primer will have a complete homo¬ logy to the mutated strand, but will be different from the wild type at a single nucleotide. After screening double- stranded DNA can be isolated from the E. coli cells, wherein the phage is replicated, and the modified insulin gene can be isolated herefrom. This gene is thereafter reinserted into the original expression system. The injection preparations can comprise the insulin deri¬ vatives of the invention as amorphous and/or crystalline suspensions, either separately or in combination with other such insulin derivatives and/or naturally occurring insu¬ lins. It has been found that a majority of these deriva- tives can be crystallized with zinc, either at a pH value being higher than their isoelectric points, or at a pH value about the isoelectric pH value, when the crystalli¬ zation medium contains a phenol. It is also possible to crystallize the insulin derivatives of the invention in the presence of protamin or an excess of zinc, which causes a further protracted activity. MODES FOR CARRYING OUT THE INVENTION The invention is further illustrated by means of the fol¬ lowing examples. Example 1 Preparation of human insulin-B21-methyl ester 500 mg of human insulin-B30-methyl ester were suspended in 50 ml of dry methanol. 2.0 ml of 20 % w/v borontrifluoride/- methanol were added with stirring. After standing at ambi¬ ent temperature for 2 hours the suspension was poured into 400 ml of ether and -centrifuged at 2500 x g at 4 C. for 20 minutes. The precipitate was washed twice with ether, resuspended in 150 ml of ether, evaporated in vacuo and dis- solved in 50 ml of 20 mM TRIS/HCl, 60 % by volume ethanol, pH 8.25. The sample was then applied to a 1.6 x 21 cm Q-Se- pharose ^-' CL-6B Fast Flow column equilibrated with the same buffer. After 1 hour of isocratic elution at a flow rate of 24 cm/hour the column was eluted with a sodium chloride gradient that increased form 0 to 0.1 M over the next 16 hours. The insulin dimethyl ester peak was divided into 3 main pools. The pool eluted first was further fractionated on a 4 x 250 mm LiChrosorb ^ RP18 column (Merck 50333) equili- brated with 0.125 M ammonium sulphate, pH value 4.0 and 38 % by volume acetonitrile at 45 C. After applying 1-2 mg of insulin dimethyl ester, the protein main fraction eluted at 20-25 minutes at a flow rate of 12 cm/min. 10 mg of the insulin dimethyl ester thus obtained were dis- solved in 1 ml of ice-cold 0.1 N sodium hydroxide solution. The reaction mixture was allowed to stand at 0 C. for 5 mi¬ nutes, whereafter the pH value was adjusted to 9 with hydro¬ chloric acid. The mixture was applied to a 0.5 x 5 cm Mono Q TR~') anion exchanger column and fractionated for 30 mi- nutes in 20 mM TRIS/HCl, 60 % by volume ethanol, pH 8.25 with a linear sodium chloride gradient increasing to 0.1 M at a flow rate of 5.1 cm/min. After removal of the ethanol in vacuo from the fraction containing human insulin-B21- -methyl ester, the protein was precipitated at a pH value of 6.3. Yield: 7 mg. The human-B21-methyl ester was characterized by Plasma De- sorption Mass Spectrometry showing the correct molecular weight and by decomposition with S.Aureus protease, whereby the B21 ester position was confirmed. Example 2 Preparation of human insulin-A14-methyl ester Human insulin-A17-methyl ester was isolated and identified as described in Example 1, however the second eluted insu¬ lin dimethyl ester pool isolated from the anion exchange chromatographical step was used for further fractionation. Hereby 6.5 mg of human insulin-Al7-methyl ester were ob¬ tained. Example 3 Formulation and biological effect of a preparation contain- ing human insulin-B21-dimethyl ester 25 ml of a medium containing 1.6 % (w/v) of glycerol. 0.33 % (w/v) of m-cresol, 1/75 M of sodium phosphate, pH value 7.3, were prepared. The solution was sterilized by filtration. 1.5 mg of human insulin-B21-methyl ester, prepared as de- scribed in Example 1, were suspended in 1.0 ml of medium. A protracted hypoglycemia was observed in guinea pigs after subcutaneous administration of this zinc-free preparation. The resulting protracted insulin action is comparable to the hypoglycemia observed after injection of a standard prota- min insulin preparation "Insulatard" • —' . Example 4 Formulation and biological effect of a crystalline insulin preparation containing human insulin-Al7-methyl ester 4.5 mg of human insulin-Al7-methyl ester were dissolved in 2.7 ml of 0.015 M phosphoric acid. 21 mg of sodium chloride and 4.1 μlitres of an aqueous solution of 152 mM zinc chlor¬ ide were added. The solution was sterilized by filtration. The pH value of the solution was adjusted td 8.0 under a- septic conditions with a sterile aqueous solution of sodium hydroxide. The pH value of the solution was then readjusted to 7.3 with sterile hydrochloric acid, and rhombohedral crystals with a mean size of 10 urn were formed with gentle ssttiirrrriinngg aatt 22l1' C. The volume was adjusted to 3 ml with sterile water. Moreover, a sterile solution of 3 ml of 13.5 mM phosphoric acid in admixture with 21 mg of sodium chloride and 9 μlit- res of m-cresol was prepared, and the pH value was ad¬ justed to 7.3 with a sterile aqueous solution of sodium hydroxide. The two solutions were combined. Such a solution induces strongly protracted hypoglycemia in guinea pigs at a dosage of 18 μg/kg. The protracted insulin action obtained is longer than the protracted ac¬ tion of the known injection preparation "Insulatard" -**" . The area below the blood glucose curve indicates that the insulin derivative exhibits nearly the same biological ac¬ tivity as human insulin. Example 5 Preparation of human insulin-A4-gln 150 mg of human proinsulin-A4-gln, produced by one of the biosynthetic methods described above, was dissolved in 30 ml of TRIS/hydrochloride, pH value 7.5, containing 3 mg of matrix-bound trypsin (Trypsin-Sepharose ^ Fast Flow) . The mixture was allowed to stand at room temperature for 1 hour, after which the resin was removed by filtration. The resulting des-B30-insulin-A4-gln was isolated by pre¬ cipitation at a pH value of 6.3 and freeze-dried. The pro¬ tein powder was dissolved in a mixture containing 200 mg of threonine methyl ester, 1000 μlitres of ethanol and 400 ulitres of distilled water. The pH value was adjusted to 6.3 with acetic acid, and 1 mg of matrix-bound trypsin was mixed in. After standing for 2 hours at room tempera- ture with gentle agitation, the protein was precipitated after filtration by adding 10 volumes of 2-propanol. The dried precipitate was redissolved in 20 mM TRIS/hydrochlor- ide, 60 % by volume ethanol, pH value 8.25, applied to a 1.6 x 20 cm Q-Sepharose -—•'" CL-6B Fast Flow column equili- brated with the said buffer and eluted with a linear so¬ dium chloride gradient increasing from 0 to 0.1 M over 15 hours at a flow rate of 24 cm/h. The ethanol was removed in vacuo from the fraction containing A4-gln-human insulin- -B30-methyl ester, and the protein was precipitated by ad¬ justing the pH value to 6.8. The B30-methyl ester was hy- drolyzed for 10 minutes in cold 0.1 M sodium hydroxide at a protein concentration of 10 mg/ml followed by adjustment of the pH value to 8.25. The rotein solution was applied to a 1.6 x 20 cm Q-Sepharose CL-6B Fast Flow column after dilution with 2 volumes of 20 mM TRIS/hydrochloride, pH value 8,25, and eluted as described above. The protein was precipitated at a pH value of 6.3 after removal of the eth¬ anol. 30 mg of human insulin-A4-gln was obtained after freeze-drying. The purity of the product was ascertained by reverse phase high pressure liquid chromatography, and the identity of the product was confirmed by amino acid analysis, multi- step Edman-degradation and plasma desorption mass spectro- etr . Example 6 15 mg of human insulin-A4-gln, obtained as described in Example 5, was dissolved in 10 ml of a solution containing 13.5 mM phosphoric acid, 0.3 % m-cresol and 1.6 % glycerol. The solution was sterilized by filtration, and the pH value was adjusted to 9,0 with a sterile aqueous solution of so- dium hydroxide. The pH value was readjusted to 8.2 with a sterile solution of hydrochloric acid, and human insulin- -A4-gln crystallized as rhombohedral crystals with gentle stirring. By subcutaneous injection of this crystalline suspension into guinea pigs at 0.5 IU/kg, the resulting protracted hypoglycemic effect is nearly identical both in timing and amplitude to the hypoglycemic effect caused by injection of "Insulatard" ®at 0.5 IU/kg.

Claims: CLAIMS 1. Insulin derivative, c h a r a c t e r i z e d in that one or more of the four amino acid residues of posi¬ tions A4, A17, B13 and B21 comprise an uncharged side chain. 2. Insulin derivative according to claim 1, c h a r a c ¬ t e r i z e d in that one or two of the four amino acid residues of positions A4, A17, B13 and B21 comprise an un¬ charged side chain. 3. Insulin derivative according to claim 1, c h a r a c - t e r i z e d in that one of the four amino acid residues of positions A4, A17, B13 and B21 comprises an uncharged side chain. 4. Insulin derivative according to claim 1, c h a r a c ¬ t e r i z e d in that the amino acid residue of position A4 comprises an uncharged side chain. 5. Insulin derivative according to claim 1, c h a r a c ¬ t e r i z e d in that the amino acid residue of position A17 comprises an uncharged side chain. 6. Insulin derivative according to claim 1, c h a r a c • t e r i z e d in that the amino acid residue of position A4 is a glutamine residue. 7. Insulin derivative according to claim 1, c h a r a c ¬ t e r i z e d in that the amino acid residue of position A17 is a glutamine residue. 8. Insulin preparation, c h a r a c t e r i z e d by containing at least one insulin derivative, wherein one or more of the four amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain, and, if de¬ sired, also containing a fast acting insulin. 9. Insulin preparation according to claim 8, wherein one or two of the four amino acid residues of positions A4, A17, B13 and B21 comprise an uncharged side chain. 10. Insulin preparation according to claim 8, wherein one of the four amino acid residues of positions A4, A17, B13 and B21 comprises an uncharged side chain. 11. Insulin preparation according to claim 8, wherein the amino acid residue of position A4 comprises an uncharged side chain. 12. Insulin preparation according to claim 8, wherein the amino acid residue of position A17 comprises an uncharged side chain. 13. Insulin preparation according to claim 8, wherein the amino acid residue of position A4 is a glutamine residue. 14. Insulin preparation according to claim 8, wherein the amino acid residue of position A17 is a glutamine residue. 15. Preparation according to claim 8, c h a r a c t e r ¬ i z e d by being an injection preparation exhibiting pro¬ tracted insulin action and comprising a suspension of at least one insulin derivative in an isotonic aqueous medium having a neutral pH and optionally containing a buffer and/or a preserving agent and, if desired, a fast acting insulin.

Inventors: BALSCHMIDT PER, HANSEN FINN BENNED, BALSCHMIDT, PER, HANSEN, FINN, BENNED
Applicants: NORDISK GENTOFTE, NORDISK GENTOFTE A/S
Application year: 1986
Main classification: C07K7
IPC codes: A61K38, C07K14, C12N15, C12P21