DK172632B1 - Drug for the treatment of diabetes mellitus and the use of insulin derivatives for the manufacture of such a drug - Google Patents

Abstract

1. Claims for the Contracting States : BE CH DE FR GB IT LI LU NL SE A medicament having a delayed action for the treatment of diabetes mellitus, comprising at least one insulin derivative of the formula I see diagramm : EP0137361,P16,F1 in which R**1 denotes H or H-Phe R**30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R**31 denotes a physiologically acceptable neutral group blocking the carboxyl group, with the exception of des-B30 human insulin Leu**B30 -NH2 and des-B30 human insulin Gly**B30 -NH2 and des-B30 human insulin Phe**B30 -NH2 and a pharmaceutically acceptable carrier. 1. Claims for the Contracting State : AT A process for the preparation of a medicament having a delayed action for the treatment of diabetes mellitus, which comprises bringing into a suitable dosage form at least one insulin derivative of the formula I see diagramm : EP0137361,P17,F1 in which R**1 denotes H or H-Phe R**30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R**31 denotes a physiologically acceptable neutral group blocking the carboxyl group, with the exception of des-B30 human insulin Leu**B30 -NH2 and des-B30 human insulin Gly**B30 -NH, and des-B30 human insulin Phe**B30 -NH2 Phe**B30 -NH2 and a pharmaceutically acceptable carrier.

Description

DK 172632 B1 i

The present invention relates to a medicament having a delayed effect for the treatment of diabetes mellitus and to the use of insulin derivatives for the manufacture of a medicament for the treatment of diabetes mellitus.

5 In the treatment of diabetes mellitus, today, preparations of the blood glucose lowering hormone insulin are usually administered parenterally. The special nature of the insulin and its metabolism mean that the duration of action of a simple solution is only very short, so that for sustained blood glucose control in the diabetic, either a durable Infusion with dosing devices, multiple daily injections or a delayed-action insulin preparation must be administered. Thus, as retention principles, such state forms of the insulin which are heavily soluble (e.g. crystalline or amorphous) at the injection site are of particular importance. For this, e.g. are considered zinc insulin crystals, (or prptamine insulin crystals, which undergo their slow dissolution over a certain period of time release insulin. ...

It has now proved extremely beneficial in the treatment of having 20 different insulin preparations available, soro in their effect characteristics comes as close to the individual patient's needs as possible. In the context of non-optimal setting, in addition to immediate effects such as hyper- or hypoglycemia, in particular the diabetic tendon complications associated with retinopathia, neuropathia, nephropathia, micro- and macroangiopathia are discussed.

·. Insulin deficiency in the diabetic means that the body can no longer achieve its natural hormonal equilibrium.

It is an object of the invention to provide a drug for the treatment of diabetes mellitus, with which one can better approach the natural hormonal equilibrium in a diabetic state and thereby maintain it better than by the administration of insulin in the usual 35 forms.

This object is fulfilled according to the invention by a doctor

delayed-action agent for the treatment of diabetes mellitus, consisting of at least one insulin derivative of formula I

5 Al rg ~~~ ^ Sl_A21

H-1 Gly A-kade ^ Asn -OH

J1 m 10 B1-Iqll ^ 829 l-R30-R31 wherein R1 means H or H-phe, R30 means the residue of a neutral, genetically encoded L-ara acid, if any OH group present may be free or protected by a physiologically acceptable group, and R31 means a physiologically acceptable neutral group which blocks the carboxy group, however Des-B30 human insulin-LeuB30-NH2r Des-B30 human insulin-GlyB30-NH2 · Des-B30 human insulin-PheB30 NH2, human insulin (B30) -0CH2CH2-NH2, human insulin (B30) -NHCH2CH2-NH2, human insulin- (B30) -OCH2CH2-N (CH3) 3 and human insulin- (B30) -NHCH2CH2-N (CH3) 3 are excluded , and a pharmaceutically acceptable carrier.

Some human ester ester ester positions 30 and methods for their preparation have already been described as intermediates in the semi-synthesis of human insulin. Thus, human insulin ThrB 30-OBut e.g. known from US Patent Nos. 4,320,196 and 4,320,197.

Tissue No. 2,069,502 discloses human insulin-Thr-OMe, human insulin-ThrB®-OEt, human insulin-ThrB®-o- (2,4,6-trimethylbenzyl), and human insulin-ThrB (Bufc) - OBufc.

Pra EP Publication No. 45 187 discloses human insulin - (B 30) amide.

JP Patent Application No. 55-144032 discloses Des-B30-porcine insulin-LeuB30-NH2 (Des-human insulin-LeuB30-NH2) DK 172632 B1 3 both useful in the treatment of diabetes in patients who are resistant to insulin.

In Chemical Abstracts, Volume 98 (1983) No. 161148, Volume 97 (1982) No. 138818, and Volume 95 (1981) No. 25640 describe five modifications of the B-chain terminal region of insulin.

In the prior art, there is no mention of a delayed (protracted) effect, and it is therefore considered surprising that the insulin derivatives of formula I are useful in the preparation of drugs having such a delayed effect, as will be done. more detail later in the description.

In Danish Patent Application No. 4398/84, the four insulin derivatives are written: human insulin (B30) -0CH2CH2-NH2 human insulin- (B30) -NHCH2CH2-NH2 human insulin- (B30) -OCH2CH2-N (ch3) 3 human insulin- (B30) -NHCH2CH2-N (CH3) 3 which is excluded from the scope of the invention.

'Genetically encodable are the following L-amino acids: 20' Gly, Ala. Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gin,

Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro (neutral amino acids are underlined).

Neutral groups which block the free carboxy function at the C-terminal end of the B chain of the 25 compounds herein are understood to be physiologically acceptable uncharged groups, especially ester and amide groups, or others. COOH protecting groups, such as are described in Bodanszky et al. Peptide Synthesis, 2nd Edition (1976),; John Wiley & Sons, preferably groups of formula 30 -NRaR4 or -ORC, wherein Ra and R3 are the same or different and represent hydrogen, (C1 -C8) alkyl, (C3-C8) -cycloalkyl, ( Cg-C ^g) -aryl, (CjC ^) -aralkyl, (C3-C ~) heteroaryl or - (CH₂-CH₂-O ^ R, wherein m «is 1 to about 120, and R = (Cj-C4 ) - alkyl, wherein these groups in the alkyl moiety may also be substituted with one or more identical or different substituents selected from halogen, nitro, (C, -C.) alkoxy,

a ^ * K

thylenedioxy and (C 1 -C 4) alkyl, or R and R taken together denote "CCH 2] n-, where n = 4-6, wherein a methylene group may also be replaced by 0 or NH, and Rc means (C ^-Cg) -alkyl, (C3-Cg) -cycloalkyl, (Cg-C1Q) -aryl, (Cy-Cj ^^) - aralkyl, (C3-C9) 5, wherein the aryl group may be substituted as by Ra and R.

As OH protecting groups for Ser, Thr and Tyr, physiologically acceptable groups selected from (C 1 -C 8 alkyl, (C 3 -C 10) -cycloalkyl, (C 6 -C 10) -aryl, (C 6 -C 4) can be used. aralkyl, (Cg-Cg) heteroaryl, (Cj-CgJ alkanoyl and (C7-C ^ ^ - aroyl or any other within the peptide chemistry common, physiologically acceptable group (cf. Eqs. Bodanszky et al. , loc, cit.

In the above context and also in the following, for example, by (C 1 -C 6) alkyl, e.g. are methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, amyl or hexyl, by (C C-C C) -cycloalkyl e.g. cyclopropyl, cyclobutyl, cyclohexyl, etc., by (Cg-C10)) aryl e.g. phenyl or naphthyl, preferably phenyl, at (C 7 -cn> -aralkyl e.g.

20 benzyl, phenethyl oSv., By (Cg-Cg) heteroaryl e.g.

pyridyl, pyrrolidyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, indolyl or quinolinyl, by (Cg-Cg) alkanoyl e.g. formyl, acetyl, propionyl, butyryl, etc., at (C 7 ~ C benzoyl, naphthoyl or toluyl.

Insulin derivatives carrying phenylalanine in position B1 are particularly preferred. Also preferred are those which exhibit Ala, Thr or Ser at position B30.

The A chain and chain (B 2 - 30) of the compounds of this invention suitably exhibit the sequence of bovine or porcine insulin, but in particular the sequence of human insulin.

31

Also preferred are insulin derivatives in which R is alkoxy or aralkoxy or -O (CH 2 CH 2 -O) mR.

Preferred OH protecting groups for Ser, Thr or Tyr are (Cg-Cg) alkyl, (Cg-Cg) -alkanoyl or (C7-Cgg) -35-alkyl.

DK 172632 B1 5

Among the insulin derivatives herein are mentioned, for example, the following compounds, without limiting the invention to them:

Des-Pheb ^ swine insulin- (b 30) -OBufc,

Bl t 5 Des-Phe -human insulin- (B 30) -OBu,

Des-PheB ^ -pigulin insulin- (B 30) -OCH, b

Bl J

Des-Phe -human insulin- (B 30) -OBZL,

Pig insulin (B 30) -O-cyclohexy1, Human insulin (B 30) -Q

Oxinsulin- (B 30) -OCHjr

Pig insulin (B 30) -O- (CH 2 -CH 2 -O-) 20C2H5f Human insulin (B 30) -NH-C2H,.

Des-ThrB 30-human insulin-ValB 30-O-C 2 H 5

Des-ThrB 30-human insulin-ValB 30-NH 2,

Human Insulin (B 30) -120 Human Insulin (B 30) -NH- (CH 2 -CH 2 -O) 30 CH 3

Human insulin- (B 30) -N (CH 3) 2,

Human insulin- (B 30) -OBZL,

Human Insulin- (B 30) -O-CH 2 -CH 2 - 25, Human Insulin-Thrk 3® (Et) OEt,

Human Insulin-Thrb (CH3) OCH3,

Human insulin (b 30) -O- [CH 2], - CH 3,

Human Insulin Thr (Ae) O-Et, r

Human Insulin-Thr (Bz) OBu, 30

Human insulin- (B 30) -NH ^ - ^ f

Human Insulin- (B 30) -0 ^ - ^, Human Insulin-Thr830 (FOR) -OPr, Human Insulin- (B 30) -NH2 · DK 172632 B1 6 The insulin derivatives of formula (I) can be prepared by

a) a Des-octapeptide (B23-30) insulin of formula XI

5 Al rS-S- | _A21

S Gly A chain Asn -OH

'| ; > (II) 10 f \ B2_ I_I B22

S ^ R1- Val B-chain Arg | -OH

wherein R 1 is Phe or a bond and S 1 is a proton solvolytic or (J-elimination leaving amino-protecting group, such as tert-butyloxycarbonyl - (Boc) -, tert-amyloxycarbony1 (Aoc) - The (Msc) group is condensed with a peptide of formula III

H-Gly-Phe-Phe-Tyr (S1) -Thr (S2) -Pro-Lys (S1) -R30-R31 (III) 30 wherein R or R have the above meanings, known per se, or

b) a Des-B30 insulin of formula I wherein R1 represents H or Η-Phe and the C-terminal grouping R1®-R1 R1 together represents OH, in the presence of trypsin or a trypsin-like endopeptidase, is reacted with a compound red formula IV

, H-R30-R31 (IV) 35 wherein R30 and R31 have the above meanings and any protecting groups present therefrom then. are decomposed in a manner known per se, and those according to a) or 2 to 3 S represent hydrogen, Bzl or Bu, and S means a urethane protecting group such as Boc, Moc, Fraoc or Z and present protecting groups are cleaved in i and DK (B) optionally prepared compounds are converted to their physiologically acceptable salts.

For example, in process variant a), for example, the NaA1, NaB1-Bis-Boc derivative of a Des-octapeptide (B23-30) -5 is reacted analogously to the process described in U.S. Patent No. 4,029,642 directly to one equivalent of a compound of formula III, using as a condensing agent dicyclohexylcarbodiimide in low deficit in the presence of 1-hydroxybenzotriazole.

As this method variant usually does not require any protection of the carboxyl groups, the damage to the insulin derivative is usually avoided both by the esterification and by the alkaline saponification. Unreacted des-octapeptide and a peptide by condensing the compound IV to Asp -OH, due to the different molecular size and the charge number can be easily separated by distribution chromatography on "Sephadex-LH 20" or by gel chromatography on "Sephadex - G 75" or "G 50 superfine".

20 TU cleavage of the tert-butyl protecting groups, the reaction product should only be treated with trifluoroacetic acid for 30-60 minutes at room temperature. This reaction does not damage the insulin derivative. When the methylsulfonylethyl-oxy-carbonyl group is selected as N-protecting group, an alkali treatment is required for cleavage by 3-elimination. The reaction conditions are such (eg 0.1 N NaOH, 0 ° C, 5 sec) that the insulin derivative is not damaged. The starting product used for ΝαΝ1, ΝαΒΒ - Bis-Boc-Des-B23_3Q ** octapeptide insulin from pigs is prepared, for example, in the following manner:

Pig insulin is reacted in a mixture of dimethylformamide, dimethylsulfoxide and water in the presence of N -; - ethylmorpholine with excess tert.butyloxycarbonyl-N - hydroxy-succinimide ester. Thereby, the expected 35 NaAl, NaBl'NéB29-Tris-Boc insulin is formed.

DK 172632 B1 8

The solution of this compound is now added: in dimethylformamide and tris buffer (pH 7.5) trypsin in small portions until no starting product can be seen in the electrophoresis. The NaA *, NaB1-Bis-Boc-Des-B23_3g “5-octapeptide insulin is purified by distribution chromatography on" Sephadex LH 20 ".

This compound is now reacted with 1 mole of the peptide of formula III, prepared in a manner known per se by the methods of peptide chemistry, 1-2 moles of 10. 1-hydroxybenzotriazole and ca. 0.9 mole of dicyclohexylcarbodiimide in dimethylformamide at a pH of approx. 7-8 (cf.

Chem. Ber. 103 (1970), page 788).

The crude product is purified by partition chromatography and freed from the protecting groups by treatment with trifluoroacetic acid / anisole at room temperature. After precipitation with ether, isoelectric precipitation from water and chromatography on "Sephadex-G 75" or "G 50 superfine" is the compound; electrophoretically pure and can be crystallized in a known manner. The insulin derivative thus obtained is biologically fully active.

bl

Des-Phe-Insulins as starting compounds for the process of the present invention are e.g. known from DE patent specification 20 05 658 or from EP disclosure no. 46 979.

The des-B30 insulins used as starting compounds in process variant b) are e.g. known from EP Publication No. 46,979 or from Hoppe-Seyler's 2. Physiol. Chem. 359 (1978) 799. The starting material used in variant b) of formula IV is prepared in a manner known per se by peptide chemistry methods.

The Des-B 30 Insulin and the compound of formula IV are condensed with one another analogously to the process described in U.S. Patent No. 4,320,196 in the presence of trypsin or a trypsin-like endopeptidase in an organic-35 aqueous solvent system at a pH of 5-9. and a temperature of 20-40 ° C. The resulting insulin derivative can be isolated by conventional peptide chemistry methods.

DK 172632 B1 9

Furthermore, the compounds of this invention can be synthesized analogously to those of e.g. GB submission no.

2 069 502, EP Publication No. 56 951, WO Patent Application No. 82/4069, DE Publication No. 31 29 404 5, or WO Patent Application No. 83/1074, disclosed semi-synthetic methods for the preparation of the known insulin - (B 30) esters.

Amino acid esters of formula IV, wherein R 31 represents an alkyl polyoxyethylene oxy chain, are prepared analogously to the process described in EP Publication No. 27,161.

Other compounds of formula IV are known or can be prepared by analogy to known methods.

All of the aforementioned insulin derivatives of formula I have in common that the molecule upon blocking of the (B 30) carboxy group "effectively obtains a further positive charge which provides it with an isoelectric point offset in the direction of the neutral point. Iso-electric points of between 5.8 and 7.3 are measured by isoelectric focusing, whereas the derivatives in the neutral range 20 are less soluble than natural insulin or proinsulin having their isoelectric point and thus the range of maximum insolubility at a pH of 5.4, while in the neutral region they are usually dissolved.

The solubility properties of Insulin and proinsulin 25 may be affected in the region above the Isoelectric point, ie.

in the therapeutically particularly interesting neutral range, by the addition of zinc ions. Zinc thereby acts as a deposition principle in that it stabilizes the hexameric state of the insulin as well as its crystallization tendency. IN

Thirty subcutaneous tissues redissolve these aggregates.

A further well-known deposition principle is the crystallization of the insulin or proinsulin as complex with jet basic protein, e.g. globin or proto miti.

When using the proinsulin I solution or I 35 'compound with one of the depot principles described, further proteolytic degradation is required to release natural, fully active Insulin. Intact proinsulin has only approx. 1/8 of the biological effect of the insulin, because, as is believed, part of the biologically active surface region, the receptor-binding region, is masked by the C-peptide present in the pro-insulin. As proinsulin for the treatment of diabetes, there is admittedly only homologous, ie only insulin with human sequence, on speech, cf. DE-A1 no. 32 32 036. Heterologous proinsulin has a substantial immunogenicity. In this context, it is remarkable that human proinsulins may also exhibit variations in the C-peptide moiety.

Surprisingly, it has been found that, unlike proinsulin in equimolar amount, the insulin derivatives of formula I, whose B chain is blocked C-terminally, exhibit an approx.

15 as high biological action as natural insulin.

The invention further relates to the use of insulin derivatives of formula I as defined above for the manufacture of a medicament for the treatment of diabetes mellitus.

The drugs herein represent novel principles of healing which can be put into effect without depot auxiliaries, such as zinc or protamine sulfate. The depot effect derives from an inherent, protein-chemically conditioned physical principle, the solubility of the insulin derivative 25 at its isoelectric point. Its redissolution under physiological conditions may be achieved by cleavage of the additional group (s), e.g. by enzymes with esterase activity. The cleaved group (s) are either purely physiological metabolites or also easily metabolizable, physiologically harmless substances.

Also, the insulin derivatives as active substances of these novel drugs are not, unlike the [intermediates described in the literature, which still exist. - holds parts of the heterologous C-peptide, more immunogenic than the corresponding insulin itself.

DK 172632 B1 11

The agents of this invention contain as active substance one or more of the insulin derivatives of formula I.

They preferably have a pH of 2.5-8.5, contain a suitable isotonic agent, a suitable preservative 5 and optionally a suitable buffer for a pH-value range of 5.0-8.5.

A typical application of the described derivatives is preparations which are below the isoelectric point as solutions in a physiologically harmless carrier.

Thus, the pH of the solution can typically be 4.5 and is thus clearly higher than the pH of acidic "old" insulins (typically pH - 3.0). A more neutral injection solution sometimes offers clear advantages in terms of its compatibility.

Another typical application is suspensions of amorphous or crystalline precipitates of the described derivatives in a physiologically harmless bar having an approx. neutral pH.

However, it is also possible to enhance the heavy solubility inherent in the derivatives in the physiological pH-20 range by further deposition principles, e.g. by the addition of zinc or protamine sulfate. The added zinc 2+ amount can thereby amount to up to 100 µg Zn / 100 insulin 2+ units, typically approx. 50 g Zn / 100 insulin units. The amount of prota min may be between 0.28 mg and 0.6 mg per day. 100 25 units (based on protamine sulfate). In this way, particularly long-acting preparations can be prepared, for which in the future there will be a broader use than hitherto, because "just a base dose of 'insulin seems therapeutically advantageous. This is already a recognition 30 from the treatment with insulin dosing.

As a physiologically harmless and acceptable medium with the insulin derivative, a sterile solution is suitable which is made Isotonic to blood in the usual manner, e.g. with glycerol, boiling salt or glucose, and further comprising one of the usual preservatives, e.g. phenol, m-cresol or p-hydroxybenzoic acid ester. The carrier medium may further contain a buffer substance, e.g. sodium acetate, sodium citrate or sodium phosphate. To adjust the pH, dilute acids (typically HCl) or bases (typically NaOH) are used.

The insulin derivatives can also be used as alkali metal or as ammonium salts in the present invention.

An arbitrary proportion of one or more insulin derivatives of formula I or an insulin derivative of formula I may be present in a mixture of other, of these insulin derivatives independently of one another in dissolved, amorphous and / or crystalline form.

It is sometimes advantageous to add a suitable amount of a suitable stabilizer to the present invention which prevents the precipitation of protein by thermal-mechanical loading upon contact with various materials. Such stabilizers are e.g. known from EP presenting publication no. 18609, from DE presenting publication no.

32 40 177 and from WO patent application 83/00288.

By the means of the present invention, which may also contain one of the known delaying principles, e.g. such as protamine sulphate, globin or zinc in suitable amounts, such a retention principle may be used in combination with the total proportion of active substance or with portions thereof or one or more insulin derivatives of formula I in admixture. An agent 25 may contain various insulin derivatives of formula I in combination with a variety of auxiliary agents which act delayingly.

In addition to the insulin derivatives of formula I, the present invention may also contain natural insulin 30 and / or proinsulin and / or des-Phe insulin independently of each other in dissolved, amorphous and / or crystalline form.

Thus, the therapeutic agents of the present invention can clearly achieve diverse and very finely tunable characteristics of action which, according to the previous comments made, should be associated with progress, especially with regard to late diabetic complications.

DK 172632 B1 13

The following examples serve to further describe the invention.

Preparation Example 1 5

Human Insulin (B30) -0-CH2-CH2-CH3 Dissolve 5 g of swine insulin in 45 ml of dimethylformamide, 25 ml of dimethylsulfoxide, 0.5 ml of N-ethylmorpholine and 2.5 ml of water. With stirring, 1.5 g of tert-butyloxycarbonyl-N-hydroxysuccinimide is added at room temperature and the reaction is continued for 6 hours. Then, by adding a drop of glacial acetic acid, the product is precipitated with ether and filtered off. The residue is dissolved in 360 ml of dimethylformamide and diluted with 320 ml of tris buffer (0.05 M, 0.01 M CaCl 2, pH 7.5). At 36 ° C, portions of 20 mg trypsin are added every 1 hour.

After a total of 12 additions, acetic acid is adjusted to a pH of 4.5 and the solution is evaporated. The subsequent cleaning of the material on a "Sephadex-LH

20 "column (8 x 200 cm) by distribution chromatography in the n-butanol / acetic acid water system (2: 1: 10) yields 3.25 g of NaA1, NaB3 - Bis-Boc-Des-B23_3Q "octapeptide insulin (swine), which in acidic and basic electrocoresis no longer" shows any starting material. The amino acid analysis of the substance is correct. After a deliberate cleavage of the BOC groups, there is no longer any insulin activity. This material (3.25 g) is dissolved in 30 ml of dimethylformamide together with 100 mg of 1-hydroxybenzotriazole, 750 mg of HCl-Gly-Phe-Phe-30-Tyr (But) -Thr-Pro-Lys (Boc) -Thr (But) -OCH2CH2-CH3 and 0.5 ml of N-ethylmorpholine. Then, at room temperature, 120 mg of dicyclohexylcarbodiimide is added and the reaction mixture is stirred for 24 hours. The separated dicyclohexylurea is filtered off and the product precipitates by ether addition.

35 DK 172632 B1 14

The precipitate is filtered off, washed with ether and dried.

The material is purified by distribution chromatography on "Sepha-dex-LH 20" in the above system. 2.6 g of material from the main peak is isolated by precipitation with acetone / ether. The 5 dried, yet unprotected derivative is reacted with a mixture of 5 ml of trifluoroacetic acid and 1 ml of anisole for 60 minutes at room temperature. Then, from the ice-cooled solution, the crude material is precipitated by the addition of ether. The dried precipitate is dissolved in water, precipitated with aqueous 10 ammonia and centrifuged. The product is purified in 10% acetic acid over "Sephadex-G 50 superfine" or "G 75". From the fractions of the desired peak, human insulin - (B30) -OCH₂CH₂CH₂ can be isolated by freeze-drying (yield after crystallization: 1.2 g). The insulin derivative thus obtained shows the biological test an effect that is equivalent to the action of human insulin.

The octapeptide of formula III is prepared according to the following condensation scheme according to conventional peptide condensation methods: 20 25 30 35 DK 172632 B1 15

Synthesis scheme for the octapeptide of formula III

Cly Phe_Phe Tyr Thr__Pro Lys Thr 5 Z-OH H OBufc DCCVHOBt Z ------ OBufc H / Pd Bufc BOC Bufc 10 Z -OH H ---- 03ufc 2 / oH H OHe Z Zqh j '/ orig

: DCC / BOBt DCC / J103: DCC / H03L

{Bufc [BOC I Bu1 Z -: - OBu1, zZ ---- Cite 2 L --- / oPr TFE H0 / Pd H0 / Pd 15 i 2 *. 2 .. ^ Bul BOC (ar Z - OM kZ —---- OMe Z-OH HcZ --- ^ Pr DCC / HOBt DCC / HOBt 'I Bufc I BOC Bub 20 .2 -: ---- -XL -— 1 CMe Z --.- ZZ) Pr

After OH lypd

Bufc | BOC I Bufc | Z ---- Z-1 — OH H --- Z -'- / orig! DCC / H03; 25; Bufc J BOC Bub Z _________ Z __________ Z __ / q Pr! i 2 / Pd

j Bufc I BOC BuU

30 IfijL -__ Jl_J__1 ___ L__J / 0Pr

Amino acid and elemental analysis corresponds to the theoretical values.

DK 172632 B1 16 Drugs Example 1

Pig insulin (B 30) -OCH3 (semi-synthetically produced from Des-B 30 pig insulin) in slightly acidic, dissolved formulation with 40 I.E. per. ml and its depot effect:

Dissolve in a total volume of 10 ml of water

Pig Insulin (B 30) -OCH3 14.5 mg 10 (27.5 I.E./mg)

Grape sugar (monohydrate) cryst. 540.0 mg of p-Hydroxybenzoic acid methyl ester 10.0 mg of pH is adjusted to 4.5 by addition of 1 N 15 HCl, respectively. 1 N NaOH.

Such a solution exhibits a marked depot effect at a dose of 0.4 I.E./-kg in rabbits. The area under the blood sugar curve is the same as for a standard 40 I.E./ml preparation.

20

Example 2

Human insulin-ThrB 30 (But) OBut (P = 6.8) prepared by semisynthesis from porcine insulin.1 neutral formulation with 40 I.E. per. 25 ml is dissolved in a total volume of 10 ml of water. Human insulin ThrB 30 (But) OBut 14.8 mg (27 IU / mg) Sodium dihydrogen phosphate dihydrate 21.0 mg 30 m-Cresol 27, 0 mg

Glycerol 160.0 mg pH is adjusted to 7.3 by the addition of 1 HCl, respectively. 1 N NaOH.

Such a suspension, at a dose of 0.4 I.E./kg in rabbits, exhibits a marked depot effect.

DK 172632 B1 17

Example 3

Human insulin (B 30} -NH2 prepared from porcine insulin by semisynthesis in the form of a crystalline NPH preparation with 40 I.E./ml and its highly delayed effect: 5 A total volume of 10 ml is dissolved with water:

Human Insulin (B 30) -NH 2 14.5 mg {27.5 I.E./mg)

Protamine sulphate 1.3 mg Sodium dihydrogen phosphate dihydrate 21.0 mg m-Cresol 15.0 mg

Phenol 6.0 mg

Glycerol 160.0 mg

The pH is adjusted to 7.3 by the addition of 1 N

HCl, respectively. 1 N NaOH ..

Such a crystal suspension at a dose of 0.4 I.E./kg in rabbits shows a highly delayed effect.

Example 4 B31

Mixture of human insulin-Arg- -OH and human insulin - ThrB 30Bufc (OBut) both prepared by semisynthesis from porcine insulin in the form of a zinc-containing suspension with 40 I.E./-ml and its highly delayed effect.

Dissolve in a total volume of 10 ml with water: B31

·. Human insulin-Arg- -OH

(27.5 I.E./mg) 7.3 mg

Human Insulin-ThrB 30But (OBut) 30 (27.0 I.E./mg) 7.4 mg

Zinc chloride, anhydrous _ 0.46 mg

Sodium acetate 14.0 mg p-Hydroxybenzoic acid methyl ester 10.0 mg

Boiling salt 80.0 mg The pH is adjusted to 7.0 by the addition of 1 N HCl and 10 ml. 1 N NaOH.

DK 172632 B1 18

Such a suspension, at a dose of 0/4 I * E / kg in rabbits, shows a highly delayed effect.

Example 5 Human insulin (B 30) -O- (CH 2 CH 2 -O-) 20C2H5 is ferro-sterilized by semisynthesis from porcine insulin in the form of a neutral preparation with 100 I.E./ml and its delayed effect:

Dissolve in a total volume of 10 ml with water: 10 Human insulin (B 30) -O- (CH 2 CH 2 -O-) 20 C 2 H 5 (24.0 I.E./mg) 41.7 mg

Sodium acetate 14.0 mg p-Hydroxybenzoic acid methyl ester 10.0 mg

Boiling salt 80.0 rag 15

A pH of 7.0 is adjusted by the addition of 1 N HCl, respectively. 1 N NaOH,

Such a suspension shows a delayed effect in rabbits.

20 25 30 35

Claims (16)

1. A drug with a delayed effect for the treatment of diabetes mellitus, consisting of at least one insulin derivative of the formula I Al S ~ l_A21 H l A-chain Asn -OH ^, l B2_l__I_ B29 R1-! Val B-Kgae | wherein R 1 is H or H-phe, R 30 is the residue of a neutral, genetically encoded L-amino acid, if any OH group present may be free or protected with a physiologically acceptable group, and R31 means a physiologically acceptable neutral group which blocks the carboxy group, however, des-BSO-human insulin-Leu ^ -NH * Des-B30-human insulin-GIyB30-NH2, Des-B30-human insulin-PheB30-NH2, human insulin- (B30 ) -OCH2CH2-NH2, human insulin- (B30) -NHCH2CH2-NH2l human insulin- (B30) -OCH2CH2-N (CH3) 3 and human insulin- (B30) -NHCH2CH2-N (CH3) 3 are excluded, and a pharmaceutically acceptable carrier. An agent according to claim 1, characterized in that in the insulin derivative of formula I, R 1 is H-Phe. An agent according to any one of claims 1 or 2, characterized in that in the insulin derivative of formula I, R30 means Ala, Thr or Ser. Agent according to one or more of claims 1 to 3, characterized in that in the insulin derivative of formula I, the A chain and the chain (B2-29) exhibit the sequence of human insulin. Agent according to one or more of claims 1-4, characterized in that in the insulin derivative of formula I, R 31 means a group of the formula -NR'Rb or -ORc wherein R 25 and R b are the same or different and represent hydrogen. C (1-6) -alkyl, C (3-8) -cycloalkyl. C (6-10) -aryl, C (7-11) -aralkyl, C (3-9) heteroaryl, or - (CH 2 -CH 2 -O) mR with m = 1 to ca. 120, and R = C (1-4) alkyl, these groups in the alkyl moiety may also be substituted with one or more identical or different substituents from the series halogen, nitro, C (1-4) alkoxy, methylenedioxy and C (1-4) -alkyl, or R * and Rb taken together represent - [CHJn with n »4-6, wherein a methylene group may also be replaced by 0 or NH and Rc means C (1-6) - alkyl, C (3-8) -cycloalkyl, C (6-10) -aryl, C (7-11) -aralkyl, C (3-9) heteroaryl, or the group (CH 2 CH 2 -O-) defined above wherein the aryl groups may be substituted as at R * and Rb. Agent according to one or more of claims 1-5, characterized in that in the insulin derivative of formula I, R31-O- (CH2-CH2 -O-) mR means, m and R having the meaning given in claim 5. Agent according to one or more of claims 1-6, characterized in that in the Insu line derivative of formula I, R31 means C (1-6) -alkoxy or C (7-11) -alkoxy. Agent according to one or more of claims 1-7, characterized in that in the insulin derivative of formula I, an OH group optionally present in R30 is free or protected with C (1-6) alkyl, C (1-6) -alkanoyl or C (7-11) -alkyl. 15 Θ. Agent according to one or more of claims 1-8, characterized in that it has a pH between 2.5 and 8.5, contains a suitable isotonizing agent and a suitable preservative, and that the insulin derivative of formula I is dissolved and / or in suspension. Agent according to one or more of claims 1-9, characterized in that this contains a suitable buffer and the pH is between 5.0 and 8.5. An agent according to one or more of claims 1-10, characterized in that it contains between 0 and 100 µg of zinc / 1001.U. 12. An agent according to one or more of claims 1 to 11, characterized in that the active substance of formula I is in the form of an alkali metal salt or ammonium salt. Agent according to one or more of claims 1-12, characterized in that any proportion of one or more insulin derivatives of formula I is present in a mixture of other of these insulin derivatives independently of one another in dissolved, amorphous and / or crystalline form. An agent according to one or more of claims 1 to 13, characterized in that it contains a suitable amount of an aid with a delaying effect. An agent according to claim 14, characterized in that this retention principle is used in combination with the total proportion of active substance or with parts thereof or one or more insulin derivatives of formula I in a mixture. DK 172632 B1 21 An agent according to one or more of claims 1 to 15, characterized in that it contains various insulin derivatives of formula I in combination with several different excipients with a delaying effect. Use of insulin derivatives of formula I as defined in one or more of claims 1-8 for the manufacture of a medicament for the treatment of diabetes mellitus.