CS114290A3 - Specifically glycosylated insulin derivatives - Google Patents

Description

The present invention relates specifically to glycosylated insulin derivatives which can be processed by pharmaceutical compositions and processes for their preparation. In recent years, a number of insulin analogs have been proposed for the treatment of diabetes. The purpose of the proposed derivatives was to improve the treatment in which insulin is not necessarily replaced by using more available insulin analogues with faster or more prolonged efficacy compared to that of human insulin. The problem that is associated with the development of insulin analogues by substituting one or more native amino acid residues in native insulin is that nikající emerging substances can elicit an immunological response. In addition, there are often problems related to the unexpected solubility or instability of the obtained substance. ,

Insulin has a relatively short blood circulation half-life, but it cannot be ruled out that the glycosylation of a small proportion of insulin occurs not only in diabetes, as described in Nakayama et al., Nonenzyma-tic Glycosylation of Insulin, "Current topics in clinicaland experimental aspects of Diabetes Mellitus' (1985), 201, 204, Sakamoto, Min and Baba, Eds., Elsevier Science, Publishers BV, but also in people who suffer from this disease. insulin

It is further possible that some changes to the carbohydrate moiety may be made to mask the presence of antigen. *

Binding of glucose, mannose and some oligosaccharides to insulin has been subjected to a large number of in vitro studies to demonstrate whether the formation of glycosylated insulin in vivo can cause late complications in diabetics. Anzenbacljer et al., Biochemistry et Biophysica Acta 306 (1975), 603-607 disclose the results of studies with D-glucose binding to insulin using dialysis

In reaching steady state. Binding was not very specific, and eight molecules of glucose bound on average per molecule of insulin.

The interaction between insulin and glucose and mannose is described in Dolhofer et al., Feb. Letters 100: 1979, 133-136. The results show that both hexoses can bind covalently to the insulin molecule during in vitro incubation at 37 ° C. Under the selected reaction conditions, 3.6 + 0.39 glucose residues and 5.0 + 0.43 mannose residues per insulin molecule on average.

A glucose-controlled insulin delivery system was proposed by Brownlee and CeramiScience 206 (1979), 1190-1111, and Diabetes (1983), 499-55, for the synthesis of glycosylated insulin derivatives capable of competing with glucose at Maltose and other oligosaccharides have been reported to react with insulin.

Nakayama et al., In the aforementioned publication, observed non-enzymatic insulin invitro glycosylation and in vivo in diabetic patients and concluded that glucose was incorporated into the insulin molecule under parathological conditions. In experiments, there were three molecules, glucose, bound to one molecule of insulin. Lapolla et al., Diabes 37 (1988), 787-791, describe the reduced bioavailability of in vivo insulin glycosylated. Insulin was glycosylated in the presence of a high glucose concentration of Dolhofer in the aqueous medium for 17 hours at 37 ° C and pH 7.4. An average amount of 2 moles of glucose per mole of insulin was bound.

Since native insulin three free primary amino groups at the B1 (Phe), Al (Gly) and B29 (Lys) positions, it is evident that the above-described glycosylated insulin derivatives were a non-homogeneous mixture of glycosylated insulin molecules. Until now, no fractions have been undertaken to fractionate the above mixtures into their individual components.

Glycosylated insulin derivatives for self-regulating insulin release systems have been described by Kim et al., Journal of Controlled Release 2, (1984), 57-66 and U.S. Patent Nos. 4,483,792, 4,478,830, 4,478,746 and 4,489,063. In these glycosylated insulin derivatives, glucose or mannose was bound to insulin via a group derived from dicarboxylic acids, acid anhydride or phenylamine or a combination of these groups. European Patent Application No. 8 84200328, published under No. 119 65Ο, discloses galactosyl insulin derivatives which, like the glycosylatedinsulins described in Kim's above, contain a group through which these residues are bound between the galactosyl residue and the insulin molecule.

The object of the invention is to provide insulin derivatives with improved properties. In particular, they should be insulin derivatives that do not induce an immunological reaction. In addition, these derivatives should provide a faster onset of action than native insulin and improve the solubility of less soluble insulins, thus allowing the use of highly concentrated solutions, for example in so-called osmoticinsulin pumps. New derivatives should also not tend to form fibrils. Accordingly, the invention provides specifically glycosylated insulin derivatives. By this term is meant derivatives in which the carbohydrate moiety is in a specific position within the insulin molecule. As could not be expected, such specifically glycosylated insulin derivatives provide valuable advantages for therapeutic purposes, as will be appreciated from the following description and from the Examples. ——————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————————— glycosylated insulin derivatives, in the strict sense, are derivatives that are monoglycosylated at positions A1, 31 or B29, diglycosylated at positions A1 and B1, A1 and 329 or B1 and B29 'or triglycosylated at positions A1, B1 and B29.

Glycosylated insulins described by Kim and others are distinct from the insulin derivatives of the present invention in which it utilizes its own aldehyde sugar functions to form a covalent bond with insulin without the use of other auxiliary groups. Another advantage of the insulin derivatives of the invention compared to the derivatives of the publication is the conservative charge distribution of the insulin molecule. The amino groups involved in the glycosylation reaction are converted to secondary amino groups and continue to be able to protonate in conventional insulin.

The novel insulin derivatives may contain a monosaccharide or an oligosaccharide containing up to three sugar residues in each of the positions. Suitable mono-carbohydrates are glucose, mannose and galactose, suitable oligosaccharides maltose, isomaltose, lactose, maltotriose, meli-biose and cellobiose. Specific glycosylated insulin derivatives of the present invention may be used to treat diabetes. Special compositions of particular glycosylated compounds may also be used to treat insulin therapy.

Insulin is understood to mean a native form of insulin, for example, human, bovine and pig insulin, but also derivatives thereof, in which several amino acids or at least one amino acid have been replaced, added or deleted as compared to natural insulin as described in European patents No. 194,864A and 214,826A.

As mentioned, native sulphines contain three potential glycosylation sites, and the N-terminal amino acid residues in chain A and 3 and lysine residue at B29. It will be appreciated that the number of potential glycosylation sites in the insulin analog of the type described above may range from two (two N-terminal residues) to a greater number depending on how much glycosine is found in the modified insulin molecule whereas lysine is the only naturally occurring amino acid with a free primary amino group in the side chain.

Glycosylation schematically follows the following scheme, 3-glucose was used. - 8 -

D-glucose D-glucose pyranose chain structure

The reactive component is a chain-like structure.

Glucosoinsulin can also be expressed by the term 1-deoxy-D-fructosylinsulin;

The above reaction will also be carried out analogously using other monosaccharides or oligosaccharides which will contain a free aldehyde group. The following are specific examples of glycosylated insulin; they are always humaninsulin derivatives:

Phe (B1) glucosoinsulin,

Phe (Bl) mannosoinsulin

Phe (B1), Lys (B29) digalactosainsulin,

Gly (Al ^ Lys (B29), digalactosainsulin,

Phe (B1), Gly (Al) diisomsltosoinsulin,

Phe (B1), Lys (B29) diisomaltoseinsulin,

Gly (Al), Lys (B29) diisomaltoseinsulin,

Gly (Al), Phe (B1), Lys (B29) triglucoseinsulin,

Gly (AI), Phe (B1), Lys (B29) trimaltosinsulin,

Gly (AI) Phe (Bl), Lys (B29),

Gly (Al), Phe (B1), Lys (B29) trimaltotriosine,

Gly (AI), Phe (B1), Lys (B29) trimannosoinsulin, · "

Gly (Al), Phe (B1), Lys (B29) trigalactosine insulin,

Gly (Al), PheIlI), Lys (B29) triisomaltoseinsulin,

Phe (B1) glucose [AspB4] insulin,

Gly (Al), PheIlB) diglucose [ββ ^θ] insulin.

Also of interest are the specifically glycosylated insulins of other animal species, such as insulin pigs. Specifically glycosylated insulin derivatives can be obtained by reacting insulin or an analogue thereof with an excess of the selected monosaccharide or oligosaccharide in the appropriate organ-or outer-environment. 60 ° C. Lower organic acids, for example acid acetic and propionic, lower aliphatic alcohols, methanol, ethanol and 2-propanol, ethylene glycol and propylene glycol can be used from organic solvents, but such phenols can be used.

The reaction time and composition of the reaction mixture will depend on whether a monoglycosylated, digicycosylated or triglycosylated product is desired. The reaction can be monitored by high pressure liquid chromatography in the primary phase by HPLC to determine the point of maximum formation of individual glycosylated products.

The reaction is quenched by cooling, for example to -20 ° C, then the reaction mixture is evaporated to dryness, the major components of the reaction mixture are then isolated and purified by preparative HPLC. After salt removal, the resulting product can be characterized by mass spectrometry using fast atom bombardment (hereinafter FAB-MS), quantitative analysis of amino acids after reduction with borohydride and biological assays.

Glycosylated insulin derivatives and mixtures thereof may be used in place of human insulin or in known pharmaceutical compositions containing - 11 - insulin to form novel pharmaceutical compositions. The phytopharmaceutical compositions will comprise a glycosylated insulin or a salt thereof, pharmaceutically acceptable, preferably with a neutral pH. The aqueous medium may contain an isotonicity agent such as sodium chloride, sodium acetate, or glycerol. In addition, the aqueous medium may contain zinc ions, buffers such as acetate or phosphate buffer and preservatives such as cresol, methylparaben or phenol. The pH is adjusted to a suitable value and the resulting composition can be sterilized by filtration.

The novel insulin preparations according to the invention can be used in a manner similar to the known insulin preparations.

The following examples illustrate the invention. See Annex 1

Phe (B1) glucoseinsulin 0.1 mmol of human insulin was suspended in 30 ml of methanol and 5 ml of glacial acetic acid was added at room temperature. The mixture is gently mixed until the insulin dissolves. An additional amount of 35 mL of methanol was added and after addition of 2.2 mmol of β-glucose, the mixture was carefully stirred for 8 hours at 40 ° C, after which the resulting product became the major component of the reaction mixture.

The solution was concentrated to near dryness by narrative evaporation. The residue was dissolved in water and fractionated by preparative RP HPLC. It uses a 16 x 250 mm column with 7 µm 100 even particles.

The temperature is 30 ° C, the mobile phase. A: 0.04 M phosphoric acid, 0.2 M sodium sulfate, 10 56 acetonitrile, pH 2.5 after treatment with ethanolamine. B: 5056 acetonitrile.

The fraction corresponding to the central muzzle peak is freed from salt and lyophilized. The yield is 0.02 mmol. The product was characterized by FAB-MS and quantity. /ive analysis of amino acids after reduction with hydroborate. Amino acid analysis showed two phenylalanine residues, that is, one phenylalanine residue less than in human insulin, probably due to substitution on Phe (B1). The molecular weight was 5970, the theoretical value was 5970. - 13 - ................ P. r.kl ad .2 .. .......... ...... ...

Phe (B1) .gamma.v (Al) diglucoseinsulin

The above compound was obtained by the method of Example 1 except that the reaction time was 16 hours instead of 8 hours, resulting in the final product of the reaction mixture. The yield was 0.06 mmol.

Amino acid analysis showed one day Phe and one Gly less residue relative to the A1 and B1 substitution.

The molecular weight was 6132, the theoretical value being 6132.

Absorption after subcutaneous administration in pigs by injection of labeled Phe (B1)

Diglucoseinsulin Gly (Al) obtained by the method ofefrgensen et al., Diabetologia 19 (1980), 546-554.

Phe (B1), Gly (Al) diglucoseinsuline of human origin is shown in Table 1 below. Values and T2 'in Table 1 represent the time in hours that elapsed from injection of the sample to the measurement of radioactivity and its decrease at 75. 5% and 25% of the initial value. Table 1 shows that glycosylated insulin is absorbed faster than human insulin. - 14 ---- 1, and u 1 k and 1 * 5

..... «· * *» '.íw' <·; 4. · Human insulin human diglucosininsulin ** I * · .1.1, -, 1. Φ., | T, ^ ΙΙ , ^ Ι ^ Ι »» 1.12 2.33 3.690.65 1.47 2.76 Effect of human insulin (Actrapid®) and human Gly (Al), Phe (B1) diglucosinsulin after subcutaneous administration in pigs (average of 5 animals) at 0.1 unit / kg is shown in Table 2 below, which shows the glucose in mraol / 1 Table 2 time vh human insulin human diglucoseinsulin -0.33 5.30 5.24 0 5.36 5, 32 0.33 4.78 4.76 0.67 4.72 4.06 1 4.12 3.34 1.5 3.60 2.98 2 3.24 2.84 2.5 3.28 2, 96 3 3,18 3,14 4 3,34 3,96 - 15 - ... Table 2 shows a faster effect of diglucosinosulin compared to human insulin.

Immunological responses in rabbits (mean of 10 rabbits) for human insulin, insulin Aldelan Gly (A1), Phe (B1) diglucoseinsulin is shown in Table 3, where rabbit serum binding values are reported in the Schlichtkrull and more, Horm. Metab.Res. Suppl. ser. £ (1974), 134-143.

Table 3 time, days human insulin insulin cattle human diglu-kosoinsulin. 0 1.9 -0.4 1.2 · 13 2.3 0.6. 1,3 '' '27 3,0 28,5 1,5 41 3,2 29,7 1,4 55 4,3 30,7 1,1 69 3,9 32,7 2,2 Θ3 2,4 30.1 1.1 97 2.0 32.6 1.5 Table 3 shows that human diglucoseinsulin induces an unexpectedly low immunological response comparable to that of human insulin. - 16 - ....... · ----- 'Appendices 3'

The following compounds were obtained in an analogous manner. Molecular weight was determined by FAB-MS or by plasma mass spectrometry, these values, together with the calculated molecular weight, are given in the table for each substance. compound molecular weight calculated found

Phe (Bl) galactosoinsulin 5956 5970 Phe (Bl) maltosoinsulin 6119 6132 Phe (Bl) lactoseinsulin 6125 - '6132 Phe (Bl) maltotriosinsulin 6288 6294 Gly (AI), The (31) dimaltosoinsulin 6444 6456 Gly (Al) dilactosainsulin 6446 6456

Gly (AI), Phe (B1) dimaltotriosoinsulin 6771 6780

Gly (Al), Phe (B1), Lys (B29) Triglucose-Insulin Gly (Al), Phe (Bl) diglucose [AspB4 °) 6294 6294 insulin 6112 6110 They were always human insulin derivatives. hydroborate, followed by quantitative amino acid analysis.

Claims (11)

-17 - characterized in that it comprises one or more monosaccharide moieties or one or more oligosaccharide moieties with up to three sugar moieties. 2. The specifically glycosylated insulin of claim 1 comprising one mono-saccharide residue or one oligosaccharide moiety having up to three sugar moieties. 3. The specifically glycosylated insulin of claim 1 comprising two monosaccharide residues or two oligosaccharide moieties with up to three residues of sugar. 4. A specific glycosylated insulin according to claim 1 comprising three monosaccharide residues or three oligosaccharide s groups; up to three residues of sugar. 5 * according to claim 1, at position A1, B1 or B29. Specifically glycosylated insulin by being monoglycosylated - 18 - 6. A specific glycosylated insulin according to claim 3, wherein Se is diglycosylated at positions A1 and B1, A1 and B29 or B1 and B29. In particular, the glycosylated insulin according to claim 4, wherein it is triglycosylated at positions A1, B1 and B29. 8. The specifically glycosylated insulin according to clause 1 to 7, wherein Asp is present in the BIO position. 9. Specifically glycosylated insulin according to 1 to 8, wherein human insulin is derived. < 10. Human PheBl) Glucosoinsulin. 11. Human Fhe (B1), Gly (Al) diglucose insulin. 12. A composition comprising at least 90, preferably at least 95%, and in particular at least 99%, of specifically glycosylated insulin according to items 1 to 11. 13. A pharmaceutical composition comprising specifically glycosylated insulin according to 1 to 12, or a pharmaceutically acceptable salt thereof, optionally together with conventional excipients, additives and preservatives. 14. A process for producing a specifically glycosylated insulin according to any one of claims 1 to 9, wherein the corresponding insulin is reacted in an aqueous or organic medium with a monosaccharide containing a free aldehyde group or an oligosaccharide containing a free aldehyde group and up to three sugar residues, whereupon the resulting product is isolated from the reaction mixture. 59 O28 / K1 > Ifo.4 · • Jl AND