IE842415L - Insulin derivatives - 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

7 6 6 9 2 In the therapy of diabetes mellitus today, in general preparations of the hypotensive hormone insulin are administered parenterally. The special nature of insulin and its metabolism mean that the duration of 5 action of a simple solution is only very short, i.e. that, for permanent control of the blood sugar in diabetics, it is necessary to administer either a continuous infusion ^ with metering apparatuses, multiple daily injections or a delayed-action insulin preparation. Particularly impor-10 tant principles with delayed action are those states of insulin which are sparingly soluble at the point of injection (e.g. crystalline or amorphous). These include, for example, zinc insulin crystals or protamine insulin crystals, which release insulin over a certain period as 15 they slowly redissolve.

It has proven extremely helpful in therapy to have available various insulin preparations whose characteristics of action meet the needs of the individual patient as closely as possible. In conjunction with non-20 optimum formulations, not only immediate effects, such as hyperglycemia or hypoglycemia, but also, in particular, the late diabetic complications, including retinopathy, neuropathy, nephropathy, microangiopathy and macroangio-pathy, will be discussed. 25 Lack of insulin in the diabetic results in the body being no longer able to achieve its natural hormonal balance.

It is the object of the invention to provide an insulin derivative or an appropriate pharmaceutical agent 30 by means of which the natural hormonal balance can be more closely approached in a diabetic state, and can be better maintained than by the administration of insulin in the hitherto conventional forms. « This object is achieved, in accordance with the 35 invention, by the provision of a medicament which contains at least one insulin derivative having a neutral, genetically codable amino acid in the B30 position of the B chain, the C-terminal carboxyl group of the B30 amino acid not being present in free form but being blocked 3 with a physiologically acceptable, neutral group -preferably an ester or amide group.

Some human insulins esterified in the B30 position, and processes for their preparation, have already been 5 described, these stated esterified human insulins being intermediates in the semisynthesis of human insulin. Thus, human insulin-Thr^-OBu6 is disclosed in, for example, US-A-4,320,196 and US-A-4,320,197. GB-A-2,069,502 discloses human insulin-ThrB30-OMe, human insulin-ThrW0-OEt, human 10 insulin-ThrB30-O- (2,4,6-trimethylbenzyl) and human insulin- Thr®30 (BU*) -OBu'. EP-A-45,187 discloses human insulin-(B30)-amide. However, the suitability of these insulin derivatives - without deblocking - for the treatment of diabetes mellitus is not described in the stated publica-15 tions.

However, in biological tests in connection with investigations into the disease diabetes mellitus, insulin derivatives having the blocked carboxyl group on the B30 amino acid have also been tested; these are in 20 particular des-B30 human insulin LeuB30-NH2, des-B30 human insulin GlyB30-NH2 and des-B30 human insulin PheB30-NH2; cf. M. Kobayashi et al.. Diabetes 1981, 30(6), 519-22, 25 ref. in C.A. Vol. 97 No. 17 (1982), page 95, ref. No. 1388l8g, JP-B-57-67548, ref. in C.A. Vol. 97 No. 23 (1982), page 616, ref. No. 198 580h, and T. Oka et al, Pept. Chem. 1980, page 157-162, ref. in 30 C.A. Vol. 95, No. 11 (1981), page 709, ref. No. 98302j.

These publications state nothing about the particular action profile of the insulin derivatives having a blocked carboxyl group on the B30 amino acid.

The invention relates then to a medicament having 35 a delayed action for the treatment of diabetes mellitus, comprising at least one insulin derivative of the formula I 4 Al r— S 1 — S——i A21 H- Gly A-Chain Asn J-OH (I) S S i I s s B2 I I B29 "'-EI B-Chain |— R^-R^1 in which R1 denotes H or H-Phe, R30 represents the radical of a neutral, genetically 5 codable L-amino acid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R31 denotes a physiologically acceptable neutral group blocking the carboxyl group, 10 with the exception of des-B30 human insulin LeuB30-NH2, des-B30 human insulin Gly*30-NH2 and des-B30 human insulin PheB30-NH2, and a pharmaceutically acceptable carrier.

The following L-amino acids are genetically 15 codable: Glv. Ala, Ser. Thr. Val. Leu. lie. Asp, Asn.

Glu, Gin. Cvs. Met. Arg, Lys, His, Tvr. Phe. Trp and Pro (neutral amino acids are underlined).

Neutral groups, which block the free carboyxl function at the C-terminal end of- the B chain in the 20 compounds of the formula X, are understood as meaning physiologically acceptable uncharged groups, especially ester and amide groups, or other COOH protective groups, as described in, for example, Bodanszky et al.. Peptide Synthesis, 2nd edition (1976) John Wiley & Sons, prefer-25 ably groups of the formula -NR*Rb or -ORc, wherein R" and Rb are identical or different and represent hydrogen, (Cj-Cb)-alkyl, (C3-C8)-cycloalkyl, (C8-C10)-aryl, (C7-Cu)-aralkyl, (C3-C0)-heteroaryl or -(CHg-CHs^O)^, in which m - 1 to about 120 and R » (Cx-C4)-alkyl, and these groups 30 may furthermore be substituted in the alkyl part by one or 5 more identical or different substituents from the series comprising halogen, nitro, (Cx-Cf)-alkoxy, methylenedioxy and (Ci-C*)-alkyl, or R* and Rb together denote -[CH2]n-, wherein n » 4 - 6 and a methylene group may also be 5 replaced by 0 or NH, and R° represents (Cj-Cg)-alkyl, (C3-Ce)-cycloalkyl, (CB-C10)-aryl, (C7-Cu)-aralkyl, (C,-C9)-heteroaryl or the above-defined radical (CH2CH2-0-)JR, wherein, as in the case of R* and Rb, the aryl radicals may be substituted. 10 In the above context, and also below, (Cx-C6)- alkyl is understood as meaning, for example, methyl, ethyl, propyl, isopropyl, butyl, tert.-butyl, amyl. or hexyl, (C3-C8)-cycloalkyl as meaning, for example, cyclo-propyl, cyclobutyl, etc., (Ce-C10)-aryl as meaning, for 15 example, phenyl or naphthyl, preferably phenyl, (C7-Cu)-aralkyl as meaning, for example, benzyl, phenethyl and the like, (C3-Cfl)-heteroaryl as meaning, for example, pyridyl, pyrrol idyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, indolyl or quinolinyl, (Cx-C8)-alkanoyl as 20 meaning, for example, formyl, acetyl, propionyl, butyryl, etc., and (C7-Cu)-aroyl as meaning, for example, benzoyl, naphthoyl or toluyl.

Medicaments with insulin derivatives of the formula I which carry phenylalanine in position B1 are 25 particularly preferred. Furthermore, the use of insulin derivatives which have Ala, Thr or Ser in position B30 is preferred.

The A chain and the chain (B2-30) of the insulin derivatives of the formula I advantageously have the 30 sequence of bovine or porcine insulin, but in particular of human insulin.

In the insulin derivatives of formula I R31 is preferably also -0(CH2CH2-0)BR (m and R, see above) or (Cx-C8)-alkoxy or (C7-Cu)-aralkoxy. 35 If the amino acids additionally have in the B30 position a phenolic hydroxyl group (which is the case for Ser, Thr and Tyr), this group may be free or protected by physiologically acceptable groups, preferably from the series comprising (Cj-Ce) -alkyl, (C,-C8) -cycloalkyl, 6 (CB-C10)-aryl, (Ce-CnJ-aralkyl, (C3-C9)-heteroaryl, (Cj-Ce)-alkanoyl and (C,-Cu)-aroyl; other physiologically acceptable radicals which are cdnmbnly used in peptide chemistry (cf. e.g. Bodanszky et al., loc. cit. ...) can also be 5 used as protective groups. However, (C^Cq)-alkyl, (C^-Cg)-alkanoyl and (C7-Cu) -aralkyl protective groups are particularly preferred.

In the series of the insulin derivatives of the formula I relevant to the medicaments according to the 10 invention, the following compounds may be mentioned by way of example, without restricting the invention to these: des-PheB1-porcine insulin- (B30) -OBufc des-PheB1-human insulin- (B30) -OBufc 15 des-PheB1-porcine insulin- (B30) -OCH3 des-PheB1-human insulin- (B30)-OBz porcine insulin- (B30) -O-cyclohexyl human insulin-(B30)-0 -0-CH, bovine insulin-(B30)-OCHa 20 porcine insulin-(B30)-0-(CH2-CH2-0-)20C2H3 human insulin-(BSOJ-NH-C^j des-Thr830-human insulin-Val^-O-CjHs des-Thr330-human insulin-ValB30-NH2 human 25 human human human human human 30 human human human human human 35 human human human insulin- (B30) - l(~/) insulin- (B30) -NH-(CH2-CH2-0-) 30-CH3 insulin- (B30) -N (CHS) 2 insulin-(B30)-OBz insulin- (B30) -0-CHa-CHa-^ insul in-Thr830 (Bt) OEt insulin-Thr830 (CHS) OCHs insulin- (B30) -0-[CH2]3CH3 insulin-Thr(Ac)O-Et insulin-Thr(Bz)OBu* insulin-(B30)-MHn TIE insulin- (B30) -0-tta V insulin-Thr8®0 (FOR) -Opr insulin-(B30)-NHa 7 The insulin derivatives of the formula I can be prepared by a) condensing a des-octapeptide (B23-30)-insulin of the formula II Al pS— S-) A21 s1- I Cly A-Chain Asn |—OH nr—i s s s 1 B2 j I B22 sW-rVal B-Chaln Arg |-0H in which R1 denotes Phe or a bond, and S1 denotes an amino protective group, such as the tert.-butyloxycarbonyl-(Boc), the tert. -amyloxycarbonyl- (Aoc) or the methyl-sulfonylethyloxycarbonyl-(Msc) radical, which can be 10 split off by proton solvolysis or by ^-elimination, with a peptide of the formula III H-Gly-Phe-Phe-Tyr (S2) -Thr (S2) -Pro-Lys (S3) -R30-R31 (III) in which R30 or R31 have the meanings defined above, S2 represents hydrogen, Bzl or Bufc, and S3 represents a 15 urethane protective group, such as Boc, Moc, Fmoc or Z, and splitting off any protective groups present in a manner which is known per se, or b) reacting a des-B30-insulin of the formula I, in which R1 represents H or H-Phe and the C-terminal end R30-R31 20 together represent OH, with a compound of the formula IV H-R30-R31 (IV) in which R30 and R31 have the meanings defined above, in the presence of trypsin or of a trypsin-like endopepti-dase, then splitting off any protective groups present in 25 a manner which is known per se and, if desired, converting the compounds obtained according to a) or b) into their physiologically acceptable salts. 8 In process variant a), for example, the N*1, NB1-bis-Boc derivative [sic] of a des-octapeptide-(B23-30)-insulin is reacted directly with one equivalent of a compound of the formula III, the procedure used being analogous 5 to that described in U.S. Patent 4,029,642, and the condensing agent used being dicyclohexylcarbodiimide in slight excess, in the presence of 1-hydroxybenzotriazole.

Since, in this process variant, the carboxyl group does not usually need to be protected, the insulin 10 derivative is also normally not damaged either in the esterification or in the alkaline hydrolysis. Unreacted des-octapeptide and peptide formed by condensation of IV with AspA21-0H differ in molecular size and in charge number and can therefore readily be separated by parti-15 tion chromatography over Sephadex * LH 20 or by gel chromatography over Sephadex G 75 or G 50 superfine.

To eliminate the tert.-butyl protective groups, it is only necessary to treat the reaction product with trifluoroacetic acid for 30 - 60 minutes at room tempera-20 ture. This reaction does not damage the insulin derivative. If the methylsulfonylethyloxycarbonyl radical is chosen as the N protective group, splitting off the latter by ^-elimination necessitates treatment with an alkali. The reaction conditions are such (e.g. 0.1 N 25 NaOH, 0°C, 5 sec.) that the insulin derivative is not damaged. The porcine N®*1, NaB1-bi8-Boc-des-B23-3o-octa-peptide-insulin used as the starting material is prepared, for example, in the following manner: Porcine insulin in a mixture of dimethyl-30 formamide, dimethyl sulfoxide and water is reacted with an excess of tert.-butyloxycarbonyl-N-hydroxysuccinimide ester in the presence of N-ethylmorpholine. This reaction gives the expected N®*1, N0®1, NtB29-tr is-Boc-insulin.

Small portions of trypsin are then added to the 35 solution of this compound in dimethyl formamide and tris buffer (pH 7.5) until starting material is no longer detectable in the electrophoresis. The N"*1, NaB1-bis-Boc-des-B23_30-octapeptide-insulin is purified by partition chromatography over Sephadex LH 20.

* Trade Mark 9 This compound is then brought to reaction with one mole of the peptide of the formula III, which is prepared in a manner known per se by the methods of peptide chemistry, 1-2 moles of 1-hydroxybenzotriazole and about 5 0.9 mole of dicyclohexylcarbodiimide in dimethyl formamide at about pH 7 - 8 (cf. Chem. Ber. 103 (1970), page 788).

The crude product is purified by partition chromatography, and is freed from the protective groups by treatment with trifluoroacetic acid/anisole at room 10 temperature. After precipitation with ether, isoelectric precipitation from water, and chromatography over Sephadex G 75 or 6 50 superfine, the compound is electrophorectically pure, and can be crystallized in a known manner. The insulin derivative thus obtained 15 possesses full biological activity.

Des-PheB1-insulins as starting compounds are disclosed in, for example, DE-B-2,005,658 or EP-A-46,979.

The des-B30-insulina used as starting compounds in process variant b) are disclosed in, for example, 20 EP-A-46,979 or Hoppe-Seyler' s Z. Physiol. Chem. 359 (1978), 799. The starting material of the formula IV which is used in variant b) is prepared in a manner known per se by the methods of peptide chemistry.

The des-B30-insulin and the compound of the 25 formula IV are condensed with one another analogously to the procedure described in U.S. Patent 4,320,196, in the presence of trypsin or of a trypsin-like endopeptidase, in an aqueous organic solvent system at pH 5 - 9 and a temperature of 20 to 40 *C. The insulin derivative 30 obtained can be isolated by the conventional methods of peptide chemistry.

The insulin derivatives of the formula I may furthermore by synthesized analogously to the semisynthetic procedure# for the preparation of the known 35 insulin-(B30)-esters, which procedures are described in, for example, GB-A-2,069,502, EP-A-56,951, WO 82/4069, DE-A-3,129,404 or WO 83/1074.

Amino acid esters of the formula IV, in which R31 represents an alkylpolyoxyethyleneoxy chain, are prepared 10 analogously to the procedure described in EP-A-27,161. Other compounds of the formula IV are known or can be prepared analogously to known processes.

All of the stated insulin derivatives of the 5 formula I have in common the fact that as a result of blocking the (B30)-carboxyl group, the molecule effectively receives an additional positive charge, which shifts its isoelectric point toward the neutral point. Depending on the derivative, isoelectric points between 10 5.8 and 7.3 are measured in the isoelectric focusing method. Thus, the derivatives are less soluble in the region of the neutral point than is natural insulin or proinsulin which have their isoelectric point, and hence the range of maximum insolubility, at pH 5.4, but are 15 usually present in solution in the region of the neutral point.

The solubility properties of insulin and pro-insulin in the range above the isoelectric point, i.e. in the therapeutically particularly interesting region of the 20 neutral point, can be influenced by the addition of zinc ions. In this context, zinc acts as a depot principle by virtue of the fact that it stabilizes the hexameric state of the insulin and its tendency to crystallize. These aggregates dissolve once again in subcutaneous tissue. 25 Another familiar depot principle is the crystal lization of the insulin or proinsulin as a complex with a basic protein, for example globin or protamine.

When the proinsulin is used in solution or in conjunction with one of the depot principles described, 30 a further proteolytic degradation is required in order to liberate fully active natural insulin. Intact proinsulin has only about 1/8 of the biological activity of insulin because, as is suggested, part of the biologically active surface region, the receptor-binding region, is masked by 35 the C peptide present in the proinsulin. However, only homologous proinsulin, i.e. only that which possesses a human sequence, is suitable for diabetes therapy (cf. for example DE-Al-3,232,036). Heterologous proinsulin possesses significant immunogenicity. In this context, it is 11 noteworthy that human proinsulins, too, can exhibit variations in the C peptide moiety.

Surprisingly, it has now been found that, in contrast to proinsulin, the insulin derivatives of the 5 formula I whose B chain is blocked at the C end have about the same biological activity as an equlmolar amount of natural insulin.

The medicaments according to the invention constitute completely novel delayed-action principles which 10 can be rendered effective without depot auxiliaries, such as zinc or protamine sulfate. The depot action is attributable to an inherent physical principle based on protein chemlstrys the poor solubility of the insulin derivative at its isoelectric point. It is possible that its 15 redissolution under physiological conditions is achieved by elimination of the additional group or groups, for example by means of enzymes with esterase activity. The particular group or groups which can be eliminated are either purely physiological metabolites or readily 20 metabolizable, physiologically acceptable substances.

Furthermore, in contrast to intermediates which are described in the literature and still contain moieties of the heterologous C peptide, the insulin derivatives, as active compounds in these novel medica-25 ments, are not as a rule, more highly immunogenic than the corresponding insulin itself.

The agents according to the invention contain, as active compound, one or more of the insulin derivatives of the formula I. 30 They preferably have a pH between 2.5 and 8.5, and contain a suitable isotonic agent, a suitable preservative and, if required, a suitable buffer for a pH range between 5.0 and 8.5.

A typical use form of the derivatives described 35 comprises preparations which, below the isoelectric point, are present as solutions in a physiologically acceptable excipient. The pH of the solution can be typically 4.5, and is therefore substantially higher than that of acidic former insulins (typical pH « 3.0). 12 In certain cases, a more neutral injectable solution offers substantial advantages in respect of its toleration.

Another typical use form comprises suspensions 5 of amorphous or crystalline precipitates of the derivatives described, in a physiologically acceptable exci-pient at about neutral pH.

However, it is also possible to reinforce the poor solubility in the physiological pH range, which is 10 an inherent property of the derivatives, by means of additional depot principles, for example by the addition of zinc or protamine sulfate. The amount of zinc added can be up to lOOpg of Zn*Vl00 insulin units, but is typically about 50/ig of Zn2+/100 insulin units. The amount 15 of protamine can be between 0.28 mg and 0.6 mg per 100 units (based on protamine sulfate). In this manner, it is possible to prepare preparations which are active for a particularly long time and which will have wider application in future than to date, since, in particular, a base 20 amount of insulin appears therapeutically advantageous.

This discovery has in fact already been made in therapy with insulin metering apparatuses.

A suitable excipient which is physiologically acceptable and compatible with the insulin derivative is 25 a sterile aqueous solution which is rendered isotonic with respect to blood in the usual manner, for example by means of glycerol, sodium chloride or glucose, and which additionally contains one of the usual preservatives, for example phenol, m-cresol or a p-hydroxybenzoate. The 30 excipient can additionally contain a buffer substance, for example sodium acetate, sodium citrate or sodium phosphate. The pH is adjusted using dilute acids (typically HC1) or alkali solutions (typically NaOH).

The insulin derivatives can also be employed in 35 the form of alkali metal or ammonium salts in the agents according to the invenion. Any desired amount of one or more insulin derivatives of the formula I, or an insulin derivative of the formula I, can be present in a mixture of other insulin derivatives from amongst these, 13 independently of one another and in each case in dissolved, amorphous and/or crystalline form.

It is sometimes advantageous to add a suitable amount of a suitable stabilizer to the preparation 5 according to the invention, the said stabilizer preventing the precipitation of protein under thermal-mechanical load during contact with various materials. Such stabilizers are disclosed in, for example, EP-A-18,609, DE-A-3,240,177 or WO-A-83/00,288. 10 In the agents according to the invention, which can also contain one of the known delayed-action principles, such as, for example, protamine sulfate, globin or zinc, in suitable amounts, a delayed-action principle of this type can be used in combination with the total 15 amount of active compound or with some of this, or with one or more insulin derivatives of the formula I, in a mixture. An agent can contain different insulin derivatives of the formula I in combination with several different auxiliaries having a delaying action. 20 In addition to containing the insulin derivatives of the formula I, the medicaments according to the invention can also contain natural insulin and/or proinsulin and/or des-Phe-insulin, independently of one another and in each case in dissolved, amorphous and/or crystalline form. 25 Obviously, therefore, a large variety of action characteristics which are very finely matched are achievable with the therapeutic agents according to the invention; according to the comments made at the outset, this should be associated with advances, particularly 30 with regard to late diabetic complications.

The examples which follow serve to illustrate the invention further: Preparation Example Is Human insulin-(B30)-0-CH2-CH2-CH3 35 5 g of porcine insulin are dissolved in 45 ml of dimethyl formamide, 25 ml of dimethyl sulfoxide, 0.5 ml of N-ethylmorpholine and 2.5 ml of water. 1.5 g of tert.-butyloxycarbonyl-N-hydroxysuccinimide are added at room temperature, while stirring, and the reaction is allowed 14 to continue for 6 hours. It is then terminated by adding one drop of glacial acetic acid, and the product is precipitated with ether and filtered off. The residue is dissolved in 360 ml of dimethyl formamide, and the solu-5 tion is diluted with 320 ml of tris buffer (0.05 M, 0.01 M of CaCl2, pH 7.5). Portions of 20 mg of trypsin are added at intervals of 1 hour, at 36"C.

After a total of 12 additions, the pH is brought to 4.5 with acetic acid, and the solution is evaporated 10 down. Subsequent purification of the material on a Sephadex LH 20 column (8 x 200 cm) by means of partition chromatography using the system n-butanol/glacial acetic acid/water (2:1:10) gives 3.25 g of N"*1, N®>1-bis-Boc-de823-30~octapeptide-insulin (porcine), in which the 15 starting material is no longer detectable in the acidic and basic electrophoresis. Analysis of the substance for amino acids gives the correct result. When the Boc groups are eliminated by way of a test, insulin activity is no longer found. This material (3.25 g) is dissolved in 30 ml 20 of dimethyl formamide, together with 100 mg of 1-hydro xy-benzotriazole, 750 mg of HC1.Gly-Phe-Phe-Tyr (Bufc) -Thr-Pro-Lys(Boc)-Thr(But)-OCH2-CH2-CH3 and 0.5 ml of N-ethylmorpho-line. 120 mg of dicyclohexylcarbodiimide are then added at room temperature, and the reaction mixture is stirred for 25 24 hours. The precipitated dicyclohexylurea is filtered off, and the product is precipitated by adding ether.

The precipitate is filtered off, washed with ether and dried. The substance is subjected to preliminary purification by partition chromatography over 30 Sephadex LH 20, using the above system. 2.6 g of material from the principal peak are isolated by precipitation with acetone/ether. The dried, still unprotected derivative is allowed to react with a mixture of 5 ml of trifluoroacetic acid and 1 ml of anisole for 60 minutes 35 at room temperature. The crude substance is then precipitated from the ice-cooled solution by adding ether. The dried precipitate is dissolved in water, the product is precipitated with aqueous ammonia, and the mixture is centrifuged. The product is purified in 10% strength 15 10 acetic acid, over Sephadex G 50 superfine or G 75. Human insulin-(B30)-OCH2CH2CH3 can be isolated from the fractions of the desired peak by freeze-drying (yield after crystallization: 1.2 g). In the biological test, the insulin derivative thus obtained exhibits an activity equivalent to that of human insulin.

The octapeptide of the formula III is prepared according to the following condensation diagram, by conventional methods of peptide condensation: Synthesis diagram for the octapeptide of the formula III Cly Phe Phe Tyr Thr Pro Lya Thr OH DCC/H0B& V fOH H DCC/HOBt! H H-OBu H,/Pd GBu €Bu TFE OBu OH Bu J/oh WOMc CMS DCC/HOBt I / Bu NaOH L ELs OMe Z-OH W-DCC/HOBt OMe Z.

OH H DCC/HOBt T I BOC OH DCC/HOStJ Bu ll/oP: BOcj BOC BOC / BOC V Bu 4)Pr Hg/Pd BOC | H_/Pd BOC 1 / 1 Bu £>Pr Bufc /r OPc Bu Bu1 ^Pr Bu11 ^ Pr Amino acid and elemental analysis correspond to theory 16 Medicaments Example 1s Porcine insulin-(B30)-OCH3 (prepared semi-synthetically from des-B30-porcine insulin), formulated 5 as a weakly acidic solution containing 40 I.U. per ml, and its depot activity: 14.5 mg of porcine insulin-(B30)-OCH3 (27.5 I.U./mg), 540.0 mg of crystalline dextrose (monohydrate) and 10.0 mg of methyl p-hydroxybenzoate 10 are dissolved in a total volume of 10 ml of water.

The pH is brought to 4.5 by adding 1 N HC1 or 1 N NaOH.

When administered in a dose of 0.4 I.U./kg to rabbits, a solution of this type exhibits a pronounced 15 depot activity. The area under the blood sugar curve is the same as that obtained with a standard preparation containing 40 I.U./ml.

Example 2: Human insulin-Thr830(Bu^OBu* (Pr[sic] ■ 6.8), pre-20 pared semisynthethically from porcine insulin, in a neutral formulation contining 40 I.U. per ml, and its depot activity# 14.8 mg of human insulin-Thr830(Bu1)08^(27 I.U./mg) 21.0 mg of sodium dihydrogen phosphate dihydrate, 25 27.0 mg of m-cresol and 160.Q mg of glycerol are dissolved in a total volume of 10 ml of water.

The pH is brought to 7.3 by adding 1 N HC1 or 1 N NaOH. 30 When administered in a dose of 0.4 I.U./kg to rabbits, a suspension of this type exhibits a pronounced depot activity.

Example 3: Human insulin-(B30)-NH2, prepared semi-35 synthetically from porcine insulin, in the form of a crystalline NPH preparation containing 40 I.U./ml, and its greatly delayed actions 14.5 mg of human insulin-(B30)-NH2 (27 I.U./mg), 1.3 mg of protamine sulfate. 17 21.0 mg of sodium dihydrogen phosphate dihydrate, 15.0 mg of m-cresol, 6.0 mg of phenol and 160.0 mg of glycerol 5 are dissolved in water to give a total volume of 10 ml.

The pH is brought to 7.3 by adding 1 N HC1 or 1 N NaOH.

When administered in a dose of 0.4 I.U./kg to rabbits, a crystal suspension of this type exhibits a 10 greatly delayed action.

Example 4s Mixture of human insulin-Arg-B31-OH and human insulin-ThrB30But(OBut), both prepared semisynthetically from porcine insulin, in the form of a zinc-containing 15 suspension with 40 I.U./ml, and its greatly delayed actions 7.3 mg of human insulin-Arg-B31-OH (27.5 I.U./mg), 7.4 mg of human insulin-ThrB30But(OBu') (27.0 I.U./mg) 0.46 mg of anhydrous zinc chloride, 20 14.0 mg of sodium acetate, 10.0 mg of methyl p-hydroxybenzoate and . 80.0 mg of sodium chloride are dissolved in water to give a total volume of 10 ml.

The pH is brought to 7.0 by adding 1 N HC1 or 25 IN NaOH.

When administered in a dose of 0.^ I.U./kg to rabbits, a suspension of this type exhibits a greatly delayed action.

Example 5s 30 Human insulin- (B30)-0-(CH2CH2-0-)20C2H3, prepared semisynthetically from porcine insulin, in the form of a neutral preparation containing 100 I.U./ml, and its delayed actions 41.7 mg of human insu 1 in- (B30) -0- (CH2CH2-0-) 2oC2H5 35 (24.0 I.U./mg), 14.0 mg of sodium acetate 10.0 mg of methyl p-hydroxybenzoate and 80.0 mg of sodium chloride are dissolved in water to give a total volume of 10 ml. 1 8 The pH is brought to 7.0 by adding 1 N HCl or 1 M NaOH.

In rabbits, a suspension of this type exhibits a delayed action. r i

Claims (18)

19 Patent Claimst

1. A medicament having a delayed action for the treatment of diabetes mellitus, comprising at least one insulin derivative of the formula I Al p~ S 5 —\ A21 H- Qly A-Chain Asn"|~OH B2 Val B-Chain S S (i) I ' S S B29 ~J-R30-r3 in which R1 denotes H or H-Phe, R30 represents the radical of a neutral, genetically codable L-amino acid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R31 denotes a physiologically acceptable neutral group blocking the carboxyl group, with the exception of des-B30 human insulin LeuB30-NH2, des-B30 human insulin GlyB30-NH2 and des-B30 human insulin PheB30-NH2, and a pharmaceutically acceptable carrier.

2. An agent as claimed in claim 1, wherein R1 is H-Phe in the insulin derivative of the formula I.

3. An agent as claimed in either of claims 1 or 2 wherein R30 represents Ala, Thr or Ser in the insulin derivative of the formula I.

4. An agent as claimed in one or more of claims 1 to 3, wherein, in the insulin derivative of the formula I, the A chain and the chain (B2-29) have the sequence of human insulin.

5. An agent as claimed in one or more of claims 1 to 4, wherein, in the insulin derivative of the formula 20 I, R31 denotes a group of the formula -NR*Rb or -0RC, wherein Ra and Rb are identical or different and represent hydrogen, (Cx-Ce)-alkyl, (C3-C8)-cycloalkyl, (C6-Cl0)-aryl, (C7-C11)-aralkyl, (C3-C9) -heteroaryl or -(CHj-CHj-OJ^R, 5 in which m = 1 to about 120 and R ■ (Ct-CA)-alkyl, and these groups may furthermore be substituted in thie alkyl f part by one or more identical or different substituents from the series comprising halogen, nitro, (Cx-C4)-alkoxy,

6. An agent as claimed in one or more of claims 1 to 5, wherein, in the insulin derivative of the formula I, R31 represents -0-(CH2-CH2-0-)BR, wherein m and R are as defined in claim 5. 2o

7. An agent as claimed in one or more of claims 1 to 6, wherein, in the insulin derivative of the formula I, R31 represents (Cj-CeJ-alkoxy or (C7-Cu)-aralkoxy.

8. 1 An agent as claimed in one or more of claims 1 to 7, wherein, in the insulin derivative of the formula I, 25 any OHi group which may be present in R30 is free or protected by (Ci-Ce) -alkyl, (Ci-Co)-alkanoyl or (C7-Cn)-aralkyl.

9. An agent as claimed in one or more of claims 1 to 8# which has a pH between 2.5 and 8.5, contains a suit- 30 able isotonic agent and a suitable preservative, and wherein the insulin derivative of the formula I is present in dissolved form and/or in suspension. *

10. An agent as claimed in one or more of claims 1 to 9, which contains a suitable buffer and has a pH between 35 5.0 and 8.5.

11. II. An agent as claimed in one or more of claims 1 to 10, which contains between 0 and 100 of zinc/100 I.U. 12. An agent as claimed in one or more of claims 1 to 11, wherein the active compound of the formula I is

12. V 21 present in the form of an alkali metal salt or of the ammonium salt.

13. An agent as claimed in one or more of claims 1 to 12, wherein any desired amount of one or more insulin derivatives of the formula I is present in a mixture of other insulin derivatives from amongst these, independently of one another and in each case in dissolved, amorphous and/or crystalline form.

14. An agent as claimed in one or more of claims 1 to 13, which contains a suitable amount of an auxiliary having a delaying action.

15. An agent as claimed in claim 14, wherein this delayed-action principle is used in combination with the total amount of active compound or with some of this, or with one or more insulin derivatives of the formula I, in a mixture.

16. An agent as claimed in one or more of claims 1 to 15, which contains different insulin derivatives of the formula I in combination with several different auxiliaries having a delaying action.

17. Use of the insulin derivatives of the formula I as defined in one or more of claims 1 to' 8, for the preparation of a medicament for the treatment of diabetes r mellitus. j i

18. A medicament as claimed in claim 1 ,j substantially as hereinbefore described and exemplified. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.