INSULIN PRECURSORS

Abstract: Insulin precursors having the amino acid sequence (I), wherein at least one of the groups R<B13>, R<B21>, R<A4>, and R<A17> is a neutral amino acid residue and the others, if any others, are Glu, the positions A6 and A11, A7 and B7, and A20 and B19, respectively, are connected by sulphur bridges, and Xn is a peptide bond or is a peptide chain of up to 40 members, the ultimate member being adjacent to Gly<A1> being Lys or Arg, are prepared in a known manner. The precursors can be converted into insulin derivatives or be used in pharmaceutical preparations in a known manner.

Description: Insulin precursors . TECHNICAL FIELD The present invention relates to novel insulin precursors. More specifically, the invention relates to novel proinsu- lin-like insulin precursors which can be used in the pre¬ paration of insulins showing an inherent protracted action, or which may be used per se in the treatment of Diabetes. Moreover, the invention relates to DNA sequences coding for such proinsulin-like insulin precursors as well as pharma- ceutical preparations comprising the insulin precursors of the invention. • 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, in¬ jections must be administered frequently, 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 also been invoked to achieve a protracted action. An Example hereof is the encapsula¬ tion of insulin crystals in polymerized serum albumin. An¬ other example is continuously acting infusion devices, so-called insulin pumps. Moreover, the specifications of Danish patent applications No. 3582/84 and No. 3583/84 disclose insulin derivatives wherein the C-terminus of the B chain is extended with an organic group of basic nature, such as Arg-OH or Arg-Arg-OH, as well as suspension preparations containing said insulin derivatives and showing protracted action. Insulins, which are normally used in the treatment of Dia¬ betes, such as porcine, bovine, ovine, or human insulin, contain six carboxylic acid groups, viz. in the A4, A17, A21, B13, B21, and " B30 positions. The numbering is refer¬ ring to the positions in the A and B chain, respectively, of the insulin, starting from the N-termini. The biological activity o-f insulin will usually decrease by an increasing degree of derivatization. However, the biological activity is influenced surprisingly little even at high degrees of derivatization of the free carboxylic acid groups. However, when all six free carboxylic acid groups are derivatized the biological activity is complete¬ ly abolished, vide D. Levy: Biochem. Biophys. Acta, 310 (1973), pages 406-415. DISCLOSURE OF THE INVENTION By the use of insulin derivatives, wherein one or more of the four amino acid residues of positions A4, A17, B13 and B21 contain an uncharged side chain, in insulin prepara- tions, in particular in injection preparations, the insu¬ lin activity can be maintained, and at the same time a surprisingly protracted action is achieved. This protrac¬ ted effect is dependent on the properties of the specific derivative, e.g. the number of substituted amino acid re- sidues and the chemical composition of the uncharged side chains of said residues. Thus, it is possible to vary the protracted action. It is a considerable advantage and a novel aspect of the insulin derivatives that the protracted action is relatively insensitive to tryptic activity, inde¬ pendent of the origin of said tryptic activity. The preparation of such insulin derivatives wherein one or more amino acid residues in the positions A4, A17, B13 and B21 contain an uncharged side chain can be carried out us- ing biotechnological techniques. Some or all of the glut¬ amic acid residues in said positions can thus be exchanged by naturally occurring amino acids carrying an uncharged side chain, e.g. glutamine. The preparation of such insulin derivatives wherein one or more of the glutamic acid residues in the positions A4, A17, B13, and B21 have been converted, into an amino acid residue carrying an uncharged side chain, can most easily be per¬ formed biotechnologically, via a single chain precursor. The present invention relates to such proinsulin-like in- sulin precursors characterized by the amino acid sequence: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-R Ala- Bl B2 B3 B4 B5 B6 B7 B8 B9 B10 Bll B12 B13 B14 Leu-Tyr-Leu-Val-Cys-Gly-R Arg-Gly-Phe-Phe-Tyr-Thr- B15 Blβ B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 Pro-Lys-X -Gly-Ile-Val-R—Gln-Cys-Cys-Thr-Ser-Ile-Cys- B28 B29 Al A2 A3 A4 A5 A6 A7 A8 A9 A10 All Ser-Leu-Tyr-Gln-Leu-R Asn-Tyr-Cys-Asn A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 wherei ■n a At. ileast one of * groups rR.B13, π RB21, π RA4, and, R A17 is a neutral amino acid residue and the others, if any others, are Glu, the positions A6 and All, A7 and B7, and A20 and B19, respectively, are connected by sulphur bridges, and X is a peptide bond or is a peptide chain of up to 40 members, the ultimate member being adjacent to Gly Al being Lys or Arg. In a manner known per se said precursors can be converted into the above-mentioned insulin derivatives by an enzyrrta- tically catalyzed semisynthesis. When X has the amino acid sequence, which connects B29-lysine and Al-glycine in human proinsulin, in which optionally one or more of the acidic amino acids in the positions X., X,-, X_, X 14/ or X-,- may be converted into a neutral amino acid, such an insulin precursor will per se be applicable in pharmaceu¬ tical preparations for the treatment of Diabetes. Recently, human proinsulin has acquired an increasing in¬ terest due to its use in the treatment of Diabetes, in spite of the relatively low specific biological strength of human proinsulin. This is not only due to the simpler bio echnological production, but' also to the appearance of some therapeutical advantages, over human insulin: - The duration of the hypoglycemic effect observed after subcutaneous injection is distinctly longer than after the injection of a dissolved insulin preparation of the same potency, however shorter than the effect of an insulin suspension preparation ("Insulatard" ^J , "Monotard" ^ ) (R.O.C. Adeniyi-Jones et al. : Diabetes Research and Clin¬ ical Practice, Suppl. 1 (1985) vide p. 3) , moreover, it suggests, however, that a better glycemic control can be achieved, as, apparently, proinsulin suppresses the hepa¬ tic glucose production rather than stimulates the periph¬ eral glucose conversion (R.R. Revers et al.: Diabetes _3_3 (1984) p. 762-770) . The above-mentioned therapeutically suitable insulin pre¬ cursors of the invention possess the same therapeutical activities as human proinsulin. Moreover, they offer the advantage that the protracted hypoglycemic activity may be extended substantially by choosing a suitable degree of derivatization. Of course, it is also possible to use the insulin precursors of the invention in admixture with ordi¬ nary insulin in preparations achieving combined effects. The preferred process for the production of the insulin precursors of the invention is by biosynthesis, as, e.g. by changing only a single base in the codon in the DNA strand coding for glutamic acid, this codon can be made code for glutamine. Thus, by changing or omitting bases in the DNA strand coding for proinsulin it is possible to make modifications which would be difficult and time-con¬ suming to make chemically. A number of different processes for the biosynthetic pro¬ duction of human insulin are known. Common to all of them is that the DNA strand coding for either the entire pro- insulin, a modified form hereof or for the A and B chain separately is inserted into a replicable plasmide contain¬ ing a suitable promotor. By transforming this system into a given host organism a product can be produced which can be converted into authentic human insulin in a manner known per se. Some known processes for biosynthesis of proinsulin or si¬ milar insulin precursors and their conversion to insulin are described below. In the specification of European Patent No. 55,945 the pro- duct is produced as a fusion protein with another protein separated by a cleaving site. This may be a protease cleav¬ ing 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 of the chimeric protein said protein is cleaved by using a suitable enzyme or CNBr, whereupon the denatured proinsulin is isolated in a sul- phitated form. This product is renatured under reducing conditions using 2-mercaptoethanol at a pH of 10.5 as de- scribed in U.S. Patent specification No. 4,430,266, fol¬ lowed by a trypsin/CpB cleavage as described by Kemmler, J. Biol. Che . 246, p. 6786 (1971) . The resulting insulin is isolated in a manner known per se. Proinsulin can be prepared biosynthetically by using the method disclosed in the specification of European Patent No. 116,201. In this method proteins are secreted into the culture medium by using the oL-factor system from Saccharo- myces cerevisiae. By inserting the gene coding for proinsu- lin into this system, proinsulin can be isolated from the culture medium by using the system described in the speci¬ fication of Danish Patent application No. 3091/84. Here¬ after, proinsulin can be converted into insulin by the known methods described above. DNA sequences coding for insulin precursors of the type B(l-29) - (Xn-Y)m -- A(l-21)__, wh.erein Xn is a peptide chain having n naturally occurring amino acid residues, Y repre¬ sents Lys or Arg, n is an integer from 0 to 33, m is 0 or 1, B(l-29) represents a shortened B chain of human insulin from Bl-Phe to B29-Lys, and (1-21) represents the A chain of human insulin provided that the peptide chain -X -Y- does not contain two adjacent basic amino acid residues, as well as the production of said precursors are disclosed in the specification of Danish Patent application No. 2385/85. However, in all essentials the process of the pro¬ duction of the insulin precursors is very similar to the process disclosed in European Patent specification No. 116,201. In the processes described above the product is either pro- insulin or proinsulin-like insulin precursors. Furthermore, these products are either produced together with a signal sequence the purpose of which is to carry the product to the cell surface where it is split off, or as a fusion with a protein for stabilizing the product. Moreover, it is possible to prepare modified proinsulins biosynthetically. European Patent application No. 82303071.3 discloses proinsulins wherein the C-chain is modified by means of recombinant DNA techniques. The insulin modifications described above can be produced by expressing the modified DNA sequence as a fusion with the gene encoding human SOD (superoxide dismutase) in the yeast Saccharomyces cerevisiae. SOD is expressed in high yields in yeast (Jabusch, Biochemistry, 1_9, 2310-2316, 1980) and is furthermore capable of stabilizing the expres¬ sion of proinsulin and variants of proinsulin which are unstable when expressed in microorganisms. As promotor for the transcription of the SOD-insulin gene is used the GAPDH-promotor (glyceraldehyde-3-phosphate de- hydrogenase) from Saccharomyces cerevisiae. By genetic en¬ gineering, the promotor is made regulatable by fusing to the GAPDH-proiribtor the regulatory region from the ADH2- 1 -promotor, also from Saccharomyces cerevisiae. When grown in a medium containing glucose, the promotor is inactive whereas it is active in absence of glucose. The modifications in the DNA sequence can be made using in vitro mutagenesis on the insulin gene. The procedure is de¬ scribed by T.A. Kunkel, Proc. Natl. Acad. Sci., USA, 82, 448-492 (1985) . According to this method, a sequence σon- taining the insulin gene is cloned into the single-strand¬ ed DNA bacteriophage M13. DNA (the template) from this hybrid phage is purified and a primer, typically a 15- to 25-mer, which contains the desired mutation and a homolo¬ gous region on each side of the mutation, is annealed to the template. Next step is to extend the primer along the whole phage genome using DNA polymerase I and in this way a double-stranded molecule is created, one strand contain¬ ing the mutation, the other strand being the wild- ype in¬ sulin gene. Among the phages it is possible to screen for desired type by hybridization of the mutagenization primer to the single-stranded DNA of the phage offspring. The primer will show complete homology to the mutated strand, but will differ by one single nucleotide from the wild- type. After the screening, double-stranded DNA can be iso- lated from the E. coli cells in which the phage has multi¬ plied, and from this DNA the modified insulin gene is re¬ covered. The gene is then re-inserted into the original expression system. MODES FOR CARRYING OUT THE INVENTION The invention is further illustrated in the following ex¬ amples: Example 1 Construction of a yeast strain, which expresses a modified (A4-Gln) -insulin precursor JPlasmid, which expresses the SOD-Met-PI fusion protein: The expression plasmid, which when present in the yeast Saccharomyces cerevisiae produces a fusion protein of the form SOD-Met-PI (superoxide dismutase-methionin-proinsulin) is shown in Figure 1. This plasmid is the basic plasmid for expression of modified insulin molecules because in vitro mutagenesis can be used to create expression plasmids which encode the modifications. In Fig. 1 the following abbreviations are used: 2μ: 2-micron. A DNA sequence isolated from Saccharo- myces cerevisiae. Responsible for replication of the plasmid in S. cerevisiae. LEU2d: Selectable marker. Encodes an enzyme in the biosyn¬ thesis of leucin. ADH2r: The regulatory part of the ADH2 promotor (alcohol dehydrogenase) (Shuster et al., Mol. Cell. Biol., 6_, 1894-1902 ( 1986 ) ) . GAPDHp: The glyceraldehyde-3-phosphate dehydrogenase pro¬ motor (Travis et al., J. Biol. Che . , 258, 4384-4389 (1985) ) . GAPDHt: The glyceraldehyde-3-phosphate dehydrogenase ter¬ minator (references as above) . pBR322: Bacterial sequence. Responsible for replication when propagating the plasmid in E. coli. B: Restriction endonuclease recognition site. Ba HI. N: Restriction endonuclease recognition site. Ncol. S: Restriction endonuclease recognition site. Sail. SOD: The superoxide dismutase gene. PI: The proinsulin gene. Construction of the structural SOD-PI gene: The plasmid pYSIl, an intermediate to the final expression plasmid pYASIl, encodes a SOD-Met-PI fusion. The SOD gene fragment was isolated as the big partial Sau3A restriction fragment from pSODNco5 (Hallewell et al., Nucleic Acids Res., 1_3_, 2017-2034, 1985) . Plasmid pins5 was used for the isolation of a Hindlll-Sall restriction fragment, which contains a synthetic proinsulin gene with preferred yeast codons with the exception " of the 13 aminoterminal amino acids. A synthetic 51 basepair Sau3A-HindIII linker encod¬ ing the 3 C-terminal amino acids of SOD, methionin and the 13 N-terminal amino acids of proinsulin were combined with the two purified fragments and an NcoI-Sall-cut pPGAP vec¬ tor (Travis et al., reference above) . Construction of- the ADH-GAPDH hybrid promotor: Plasmid pJS104, another intermediate of the plasmid pYASIl, encodes a promotor combined from ADH2 and GAPDH. A fragment containing the regulatory sequence of ADH2 was isolated from plasmid pADR2 (Beier and Young, Nature 300, 724-728, 1982) , which contains a BamHI-SphI fragment encoding ADH2 and its upstream regulatory region. Plasmid pADR2 was cut with EcoRV, which cuts in the position +66 from the ATG start-codon. The DNA was furthermore treated with Bal31 nuclease and synthetic Xhol-linkers were ligated to the ends. After digestion with Xhol and religation, the result¬ ing plasmid was transformed into E. coli. A plasmid, which contained an Xhol-linker in the position -232 from the ADH2 ATG initiation codon was digested with Xhol, treated with SI nuclease and digested with EcoRI creating a linear mole¬ cule with a blunt end at the Xhol-site in the ADH2 regula¬ tory region and an EcoRI-site originating from pBR322. A fragment containing the GAPDH promotor-region was isola-- ted by digestion with the restriction enzymes Alul and EcoRI of pPGAP (Travis et al.) and isolation of a 400 base- pair fragment. The hybrid promotor was now constructed by ligating the GAPDH promotor fragment with the linearized plasmid containing the ADH2 sequences. The fusion between the two fragments was verified by sequence analysis. Construction of the plasmid pYSAIl: The final expression plasmid was now constructed by liga¬ ting the BamHI/NcoI-fragment isolated from pJS104 and the NcoI/BamHI-fragment from pSIl, digesting the ligation pro¬ duct with BamHI and ligating the fragment to the yeast replicating vector pCl/1 (Brake et al., Proc. Natl. Acad. Sci. , J31., 4642-4646, 1984) treated with calf intestine phosphatase. Cloning of the insulin gene in the M13 phage: The 3034 basepair BamHI-fragment isolated from the ex- pression plasmid pYASIl was cloned into the replicative form of the M13 phage Ml3mpl8 digested with BamHI. After transformation of the E. coli strain JM101, the presence and orientation of the insert was verified by sequence analysis on DNA isolated as described by Messing and Viei- ra, Gene 1_9, 269-276, 1982. Oligodeoxyribonucleotide synthesis : The mutagenisation primer used was synthesized as described by Sanchez-Pescador and Urdea, DNA, _3 / 339-343, 1982. The sequence was: d (ACAACATTGTTGAACAATACC) . Kinasing of the oligodeoxyribonucleotide: The 21-mer was kinased at the 5 '-end in a 10 plitre volume containing 70 mM Hepes, pH 7.0, 10 mM MgCl 2 , 5 mM DTT, 1 itiM ATP, 50 pmol oligonucleotide and 3.6 units T4 polynucleo- tide kinase from Amersham. The incubation was carried out for 2 x 30 min. with addition of 1 plitre 10 mM ATP be¬ tween the two incubations. 5'- 32P-labellmg of oligonucleotide: The 21-mer was labelled at the 5'-end in a 50 μlitre vol¬ ume containing 70 mM Hepes, pH 7.0, 10 mM MgCl~, 5 mM DTT, 40 pmol oligonucleotide, 2.5 mM (ϊ- 32P) -ATP and 7 units of T4 polynucleotide kinase. The incubation was 30 min. at 37 C. After the incubation, non-incorporated nucleotide was separated from the labelled oligonucleotide on a 3 ml Seph- adex G-10 column in TE-buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 7.5) . Isolation of uracil-incorporated template-DNA: Template-DNA, in which a number of thymidin molecules have been replaced by uracil, can be isolated from the strain E. coli RZ1032 (T.A. Kunkel, reference given earlier) . The strain was infected with M13-phages containing the insulin g sequence in the following way: 10 M13-phages are mixed with 100 ml 2 x YT medium (yeast extract 5 g/litre, tryp- tone 8 g/litre, NaCl 5 g/litre) supplemented with 0.25 ug/ml uridine and 10 ml E. coli RZ1032-cells in mid-loga- rithmic phase. The culture was incubated with shaking at 37°C for 16 hours. From this culture single-stranded uracil- -containing DNA was purified according to Messing and Viei- ra (reference given earlier) scaled up to the greater vol- ume. Oligonucleotide-primer second-strand synthesis: Single-stranded Ml3mpl8 (0.13 pmol) containing the insulin sequence and isolated from the uracil-incorporating strain RZ1032 was incubated with the 5 '-kinased mutagenisation primer (5 pmol) in 20 mM Hepes, pH 7.3, 10 mM MgCl.,, 50 mM NaCl and 1 mM DTT. The mixture was heated to 85°C and then cooled to room temperature. To this mix was added 10 ulitres of a solution containing 20 mM Hepes, pH 7.3, 10 mM MgCl-, 1 mM DTT, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM dTTP, 1 mM ATP, 3 units T4 DNA ligase and 2 units DNA poly- merase I (Klenow fragment) . The final volume of 20 ulitres was incubated for 16 hours at 16 C. Transformation of JM101: 10 j ilitres of the above mixture were used for transforming CaCl -treated E.coli JM101. The cells were plated in mini- - • 7 al medium topagar containing 10 cells pr. ml. The trans- 4 formation procedure gave rise to about 10 plaques per pmol of uracil-containing template. Plaque-lift hybridization: The cooled (4 C) transformation plates were overla ed with a dry piece of nitrocellulose paper. After being wet- -through for about 4 min., the filters were bathed in 0.5 M NaOH, 1.5 M NaCl for 1 minute, in 0.5 M Tris-HCl, pH 8.0, 1.5 M NaCl for 1 minute and finally in 0.3 M NaCl, 0.03 M sodium citrate (2 x SSC) for 5 minutes. The filters were then baked in a vacuum-oven at 80 C for 2 hours and pre- -hybridized in 20 ml 0.9 M NaCl, 0.09 M sodium citrate, 0.2% serumalbumin, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.1% SDS and 50 pg/ml salmon-sperm DNA at 65°C for 2 hours. Finally, 10 7 cpm 32P-labelled mutagenisation primer was ad- ded and the hybridization took place for 16 hours at 31°C. After hybridization the filter was washed 3 times 15 min¬ utes at 55°C in 2 x SSC + 0.1% SDS. An autoradiograph of the filters revealed the positive plaques. The frequence of mutants was 25-40%. Purification of double-stranded M13 phage-DNA: The positive phages were used for infecting the E.coli strain JM101. About 10 phages and 5 colonies of JM101 were grown in 5 ml 2 x YT for 6 hours at 37 C and the double- -stranded, circular DNA was purified according to the meth¬ od described by Birnboim and Doly, Nucleic Acids Res., 7, ' 1513 (1979) . Isolation of the BamHI-fragment containing the mutation: The DNA-preparation (about 5 ug) was digested with 10 units of the restriction enzyme BamHI in 60 ulitres 100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 and 1 mM DTT for 2 hours at 37 C. The DNA fragments were separated on an ag< ose gel and the fragment of 3034 b-asepairs was purified. Ligation to the yeast replication vector pCl/1: The BamHI-fragment isolated above was ligated to the vector pCl/1 in the following reaction mixture: 0.6 ug fragment, 0.1 μg vector treated with BamHI and phosphatase, 50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 10 mM DTT and 1 mM ATP in a final volume of 20 μlitres. 5 μlitres of the ligation mix were transformed into the E.coli strain MC1061 in which the modified expression plasmid was propagated and identified. Transformation of the yeast strain P017: The yeast Saccharomyces cerevisiae P017 (a., Ieu2) was trans¬ formed according to a procedure described by Hinnen et al., Proc. Natl. Acad. Sci., USA, 75, 1929, 1978. Example 2 Expression of SOD-Met-Precursor in transformed yeast: A transformant was used to inoculate YPD medium (Sherman et al., Methods in yeast genetics, Cold Spring Harbor La- boratory, 1981) . Following growth at 30 C to stationary phase, the culture was diluted 20 times into YP-medium containing 1% ethanol and again grown to saturation at 30 C, Example 3 Preparation of a modified insulin precursor Purification of superoxide dismutase-met-precursor from Saccharomyces cerevisiae pellet 100 g pellet of homogenized (Manton Gaulin Homogenizer) yeast, transformed and grown as described in Examples 1 and 2, were triturated with 400 ml of water and centrifuged.at 6000 rpm. To the pellet was added 267 g of guanidinium hy- drochloride and 0.6 g of glycine, and the volume was slowly adjusted to 400 ml with water. The pH value was then ad¬ justed to 2.6 with 5 M hydrochloric acid, and the suspen¬ sion was gently agitated for 48 hours at 20°C. After centrifugation and decantation the pellet was dis¬ carded, and the supernatant was filtrated. In the filtrate was dissolved 1.42 g of disodium hydrogen phosphate, di- hydrate, and the pH value was adjusted to 8.0 with 5 M so¬ dium hydroxide. 422 μlitres of 2-mercaptoethanol were ad- ded, and the solution was gently agitated for 30 min. at 20°C. By gelfiltration on a column of Trisacryl^-' GF-05 the pro¬ tein was transferred to a buffer containing 0.05 M acetic acid, 0.005 M 2-mercaptoethanol, 7 M urea and adjusted to pH 5.5 with sodium" hydroxide, and the solution was appli- cated on a 5 x 15 cm column of SP-Trisacryl ^ M, equili¬ brated at 4 C with the said buffer. The protein was then eluted with a linear sodium chloride gradient in the same buffer increasing from 0 to 0.5 M over 20 hours at a flow rate of 100 ml per hour. The hybrid-protein containing fractions (detected by SDS- -PAGE) was diluted with 3 volumes of water, and the pro¬ tein was precipitated by addition of 390 g of ammonium sulphate per litre. The precipitation was isolated by cen- trifugation and after resuspension in water it was dia- lyzed against water and lyophilized. Cyanogen bromide cleavage of hybrid protein: 1 g of superoxide dismutase-met-precursor was dissolved in 80 ml of 70% (by volume) formic acid in a vacuum flask and 84.5 μlitres of 2-mercaptoethanol were added. The solution was left for 2 hours under a nitrogen blanket, and then 1.25 g of cyanogen bromide was added. The reaction mixture was now left for IS hours in the dark. The formic acid was evaporated unde_5 high vacuum, and after addition of 80 ml of water the residue was lyophilized. Preparation of sulphitated precursor: The lyophilized residue from the CNBr-cleavage was dis¬ solved at 37 C in 50 ml of 0.2 M disodium hydrogen phos¬ phate, 8 M urea, adjusted to pH 7.4 with 5 M acetic acid and 12.5 ml of 0.5 M sodium sulphite, 0.2 M EDTA, 8 M urea, adjusted to pH 7.4 with glacial acetic acid was added and the solution was left at 37 C. After 10 min. 6 ml of 0.5 M sodium tetrathionate, 8 M urea, adjusted to pH 7.4 with glacial acetic acid was added. After 30 min. 12.5 ml of the said sulphite solution and after 40 min. 7 ml of the said tetrathionate solution were added, and the reaction mixture was left for further 60 min. at 37 C. By gelfiltration on a column of Trisacryl (^R)GF-05 the pro¬ tein was transferred to a buffer containing 0.05 M acetic acid, 0.01 M sodium chloride, 7 M urea and adjusted to pH 4.7 with sodium hydroxide, and the solution was applied on a 5 x 15 cm column of SP-Trisacryl^M, equilibrated at 4°C with the said buffer. The protein was then eluted by the same buffer at a flow rate of 100 ml per hour, and the precursor containing fractions were pooled, desalted on a column of Trisacryl (^R- / )GF-05 in 0.05 M ammonium bicarb¬ onate and lyophilized. Renaturation and purification of precursor: 50 mg of sulphitated precursor were dissolved in 400 ml of oxygen-free 0.05 M glycine, adjusted to pH 10 with sodium hydroxide. Then 2 ml of 0.5% 2-mercaptoethanol in the same buffer were added, and the reaction mixture was left at 4°C for 24 hours under a nitrogen blanket. The reaction was then terminated by adjusting the pH value to 3 with 5 M hy¬ drochloric acid, and the protein was precipitated by addi- tion of 80 g of sodium chloride. After centrifugation the residue was redissolved by addition of 10 ml of water and the ' n dialyzed against water and lyophilized. The protein was? then redissolved in 0.02 M TRIS/hydrochloride, 60% eth- anol by adjusting the pH to 8.25, and applied to ' a 1.6 x 20 cm Q-Sepharose ( v ^CL-6B Fast Flow column, equilibrated with said buffer, and eluted with a linear gradient of so¬ dium chloride in the same buffer increasing from 0 to 0.1 M over 15 hours at a flow rate of 50 ml per hour. The ethanol was removed in vacuo from the fraction containing the pre- cursor, and the protein was isolated by dialysis against water and lyophilization. The following insulin precursors were prepared by the meth¬ ods described above: (A4-gln)-human proinsulin (A4,B21-gln)-human proinsulin (A4-gln)-insulin precursor, n=0 (X = peptide bond) (A4,B21-gln)-insulin precursor, n=3, X, = Thr, X„ = Lys, 3 = Arg) The identity of the precursors were confirmed by amino acid analysis and by multistep Edman degradation. Example 4 Preparation of (A4-gln) -human insulin: 150 mg of (A4-gln)-human proinsulin, produced by the meth- ods described in Examples 1, 2 and 3, were added to 30 ml of a suspension of 4 ml (settled volume) matrix-bound tryp- sin (Trypsin-Sepharose Fast Flow, 0.8 mg enzyme/ml gel) in 0.05 M TRIS/hydrochloric acid, pH 7.5, and the mixture was gently agitated for 60 minutes at 20 C. The resin was then removed by filtration, and the resulting solution of (A4-gln) -des- (B30)-insulin was isolated by precipitation at pH 6.3 and lyophilized. The protein powder was dissolved in a mixture containing 200 mg of threonine methyl ester, 1.0 ml of ethanol and 0.4 ml of distilled water. The pH value was adjusted to 6.3 with acetic acid, and 2 ml of Trypsin-Sepharose ^were ad¬ ded. After standing for 2 hours at 20 C with gentle agita¬ tion, the trypsin-matrix was removed by filtration, and the protein was precipitated by adding 10 volumes of 2-propanol. The air-dried precipitate was redissolved in 0.02 M TRIS/hydrochloride, 60% (by volume) ethanol, pH 8.25, applied to a 1.6 x 20 cm Q-Sepharose ^-~- CL-6B Fast Flow column, equilibrated with said buffer, and eluted with a linear sodium chloride gradient in the same buffer in- creasing from 0 to 0.1 M over 15 hours at a flow rate of 50 ml per hour. The ethanol was removed in vacuo from the fraction containing (A4-gln) -human insulin, B30-methyl ester, and the protein was precipitated by adjusting the pH value to 6.8. After centrifugation and lyophilization the B30-methyl ester was hydrolyzed 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 8.5. The solution was diluted with 2 volumes of 0.02 M TRIS/- hydrochloride, pH 8.5, and was then applied to a 1.6 x 20 cm Q-Sepharose ^ CL-6B Fast Flow column and eluted as described above. The protein was precipitated at a pH value of 6.3 after removal of the ethanol. 30 mg of (A4-gln) - -human insulin was obtained after lyophilization. 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 and multi- step Edman degradation. Example 5 Preparation of (A4-gln) -human insulin: 180 mg of (A4-gln)-insulin precursor (X = peptide bond), produced by the methods described in Examples 1, 2 and 3, were added to a 35 ml suspension containing 7 ml (settled volume.) of matrix-bound trypsin (Trypsin-Sepharose^-' Fast Flow, 0.8 mg enzyme per ml gel) in 0.05 M TRIS, 20% (by volume) ethanol, adjusted to pH 7.7 with hydrochloric acid, and the mixture was gently agitated for 2 hours at 20 C. The resin was then removed by filtration and the resulting solution mainly containing (A4-gln) -des-B30-insulin was ad¬ justed to pH 6.3. The hereby precipitated protein was iso¬ lated by centrifugation and lyophilization. The protein powder was redissolved in a mixture of 400 mg of threonine methyl ester, 2.0 ml of ethanol and 0.80 ml of distilled water. The pH value was adjusted to 6.3 with acetic acid, and 3.2 ml of Trypsin-Sepharose was added. After standing for 2 hours at 20 C with gentle agitation, the trypsin- -matrix was removed by filtration, and the protein was pre¬ cipitated by adding 10 volumes of 2-propanol. The air-dried precipitate was redissolved in 0.02 M TRIS/hydrochloride, 60% (by volume) ethanol, pH 8.25, applied to a 1.6 x 20 cm Q-Sepharose ^ CL-6B Fast Flow column, equilibrated with said buffer, and eluted with a linear sodium chloride gra¬ dient in the same buffer increasing from 0 to 0.1 M over 15 hours at a flow rate of 50 ml per hour. The ethanol was removed in vacuo from the_ contain¬ ing (A4-gϊn) -human insulin, B30-methyl ester, and the pro¬ tein was precipitated by adjusting the pH value to 6.8. Af¬ ter centrifugation and lyophilization the B30-methyl ester was hydrolyzed for 10 minutes in cold 0.1 M sodium hydrox¬ ide at a protein concentration of 10 mg/ l followed by ad¬ justment of the pH value to 8.5. The solution was diluted with 2 volumes of 0.02 M TRIS/hydrochloride, pH 8.5, and was then applied to a 1.6 x 20 cm Q-Sepharose ^ CL-6B Fast Flow column and eluted as described above. The protein was precipitated at a pH value of 6.3 after removal of the eth¬ anol. 34 mg of (A4-gin.) -human insulin were obtained after lyophilization. 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 and multi- step Edman degradation. Example 6 Preparation of / (A4,B21) -gln/-human insulin 200 mg of /_TA4,B21) -gin, Xl-thr, X2-lys, X3-arg_7-insulin precursor produced by the methods described in Examples 1, 2 and 3, were added to a 40 ml suspension containing 8 ml (settled volume) of matrix-bound trypsin (Trypsin-Sepha- rose ^R) Fast Flow, 0.4 mg enzyme per ml gel) in 0.05 M tri- ethyl a ine, 20% (by volume) ethanol, adjusted to pH 10 with hydrochloric acid, and the mixture was gently agitated for 3 hours at 4 C. The resin was then removed by filtra¬ tion, and the resulting solution mainly containing _/(A4,B21) -gln/-des-B30-insulin was adjusted to pH 6.5. The precipitated protein was isolated by centrifugation and lyophilization. The protein powder was redissolved in a mixture of 400 mg of threonine methyl ester, 2.0 ml of ethanol and 0.80 ml of 20 i 2 i h l it i the trypsin-matrix was removed by filtration, and the pro- tein was precipitated by adding 10 volumes of 2-propanol. The air-dried precipitate was redissolved in 0.02 M TRIS/- hydrochloride, 60% (by volume) ethanol, pH 8.25, applied to a 1.6 x 20 cm Q-Sepharose^CL-6B Fast Flow column, equili¬ brated with said buffer, and eluted with a linear sodium chloride gradient in the same buffer increasing from 0 to 0.1 M over 15 hours at a flow rate of 50 ml per hour. The ethanol was removed in vacuo from the fraction containing _/(A4,B21)-gin/-human insulin, B30-methyl ester, ≥nά the pro¬ tein was precipitated by adjusting the pH value to 7. After centrifugation and lyophilization the B30-methyl ester was hydrolyzed for 10 minutes in cold 0.1 M sodium hydroxide at a protein concentration of 10. mg/ml followed by adjust¬ ment o'f the pH value to 9. The solution was diluted with 2 volumes of 0.02 M TRIS/- hydrochloride, pH 9, and was then applied to a 1.6 x 20 cm Q-Sepharose ^-~- CL-6 Fast Flow column and eluted as de¬ scribed above. The protein was precipitated at a pH value of 6.5 after removal of the ethanol. 28 mg of _ (A4,B21)-gin/-human insulin were obtained after lyophili- zation. The purity of the product was ascertained by reverse phase high pressure liquid chroma ography, and the' identity of the product was confirmed by amino acid analysis and multi- step Ed an degradation. Example 7 Preparation of / 4,B21)-gln ~ -human insulin 170 mg of _/_(A4,B21)-gin/-human proinsulin, produced by the methods described in Examples 1, 2 and 3, was added to 34 ml of a suspension of 8 ml (settled volume) matrix-bound trypsin (Trypsin-Sepharose ^-^ Fast Flow, 0.8 mg enzyme/ml gel) in 0.05 M ammonium hydrogen carbonate, pH 8, and the mixture was gently agitated for 30 minutes at 20 C. The resin was then removed by filtration, and the resulting so- 5 lution of _/(A4,B21) -gln/-des (B30) -insulin was lyophilized. The protein powder was dissolved in a mixture containing 250 mg of threonine methyl ester, 1.25 ml of ethanol and 0.5 ml of distilled water. The pH value was adjusted to 6.3 with acetic acid, and 2 ml 10 ded. After standing for 2 hours at 20 C with gentle agita¬ tion, the trypsin-matrix was removed by filtration, and the protein was precipitated by adding 10 volumes of 2-propanol, The air-dried precipitate was redissolved in 0.02 M TRIS/- hydrochloride, 60% (by volume) ethanol, pH 8.25, applied 15 to a 1.6 x 20 cm Q-Sepharose ^CL-δB Fast Flow column, equilibrated with said buffer, and eluted with a linear sodium chloride gradient in the same buffer increasing ;-: from 0 to 0.1 M'over 15 hours at a flow rate of 50 ml per hour. 20 The ethanol was removed in vacuo from the fraction contain¬ ing _/(A4,B21)-gln/-human insulin, B30-methyl ester, and the protein was precipitated by adjusting the pH value to 7. After centrifugation and lyophilization the B30 methyl ester was hydrolyzed for 10 minutes in cold 0.1 M sodium 25 hydroxide at a protein concentration of 10 mg/ml followed by adjustment of the pH value to 9. The solution was di¬ luted with 2 volumes of 0.02 M TRIS/-hydrochloride, pH 9, and was then applied to a 1.6 x 20 cm Q-Sepharose ^CL-6B Fast Flow column and eluted as described above. The protein 0 was precipitated at a pH value of 6.5 after removal of the ethanol. 25 mg of _/ (A4,B21) -gin/-human insulin were ob¬ tained after lyophilization. The purity of the product was ascertained by reverse phase high pressure liquid chromatography, and the identity of 5 the product was confirmed by amino acid analysis and multi- step Edman degradation. Example 8 Preparation for injection containing (A4-gln)-human pro¬ insulin: 25 mg of (A4-gln)-human proinsulin were dissolved in 3 ml of 0.0225 M phosphoric acid containing 0.5% of m-cresol and 2.6% of glycerol, and the pH value was adjusted to 7.4 with sodium hydroxide solution. The volume was adjusted to 5.0 ml with water, and the solution was sterilized by fil- tration. By subcutaneous injection in Guinea Pigs (0.125 mg/kg) it is found that the timing of the maximum.hypoglycemic ef¬ fect is intermediate as compared to a regular insulin pre¬ paration (Velosulin * ) and a NPH suspension preparation (Insulatard® ) .

Claims: CLAIMS 1. Insulin precursors, c h a r a c t e r i z e d by the amino acid sequence: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-R -Ala- Bl B2 B3 B4 B5 B6 B7 B8 B9 BIO Bll B12 B13 B14 Leu-Tyr-Leu-Val-Cys-Gly-R -Arg-Gly-Phe-Phe-Tyr-Thr- B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 Pro-Lys-X -Gly-Ile-Val-R -Gln-Cys-Cys-Thr-Ser-Ile-Cys- B28 B29 Al A2 A3 A4 A5 A6 A7 A8 A9 A10 All Ser-Leu-Tyr-Gln-Leu-R -Asn-Tyr-Cys-Asn A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 wherein at least one of the groups R , R , R , and R A17 is a neutral amino acid residue and the others, if any others, are Glu, the positions A6 and All, A7 and B7, and A20 and B19, respectively, are connected by sulphur bridges, and X??is a peptide bond or is a peptide chain of up to 40 members, the ultimate member being adjacent to Gly Al being Lys or Arg. 2. Insulin precursor according to claim 1, c h a r - Bl3 a c t e r i z e d by at least one of the groups R , π RB21, π RA4, and, R π A17 b.ei.ng G_i,n. 3. Insulin precursor according to claim 1 or 2, c h a r a c t e r i z e d by the groups R Bl 3 , R B21 , and R A17 being Glu, and RA4 being Gin. 4. Insulin precursor according to claim 1 or 2, c h a r a c t e r i z e d by the groups R and R being Glu, and R A4 and RB21 being Gin. 5. Insulin precursor according to any of the claims 1 to 4, c h a r a c t e r i z e d by X having the amino acid sequence connecting Lys B29 and G n lyAl in human pro¬ insulin. 6. Insulin precursor according to claim 5, c h a r ¬ a c t e r i z e d by one or more of the acidic amino acid residues in the positions X., X fi , X 7 , X-.. or X-..- in X be¬ ing converted into a neutral amino acid residue. 7. DNA sequence, c h a r a c t e r i z e d by coding for the insulin precursor according to any one of claims 1 to 6. 8. Replicable expression agent, c h a r a c t e r ¬ i z e d by comprising a DNA sequence as defined according to claim 7. 9. Pharmaceutical preparation, c h a r a c t e r ¬ i z e d by comprising an insulin precursor according to claim 5 or 6 in admixture with a pharmaceutical acceptable carrier and optionally also a fast-acting insulin. -

Inventors: BALSCHMIDT PER, CLAUSEN IB GROTH, HANSEN FINN BENNED, HEJNAES KIM RY, BALSCHMIDT, PER, CLAUSEN, IB, GROTH, HANSEN, FINN, BENNED, HEJNAES, KIM, RY
Applicants: NORDISK GENTOFTE, NORDISK GENTOFTE A/S
Application year: 1987
Main classification: C07K7
IPC codes: A61K38, A61P3, C07K14, C12N9, C12N15, C12P21, C12R1