NZ209620A

NZ209620A - Insulin derivatives modified in the b30 position, processes for their preparation

Info

Publication number: NZ209620A
Application number: NZ209620A
Authority: NZ
Inventors: U Grau; R Geiger; R Obermeier
Original assignee: Hoechst Ag
Priority date: 1983-09-23
Filing date: 1984-09-21
Publication date: 1988-03-30
Also published as: EP0137361B1; PT79249A; EP0137361A2; DK172632B1; ATE52791T1; AU573624B2; IL73021A0; DK453084D0; JPS6094999A; FI843695L; KR850002287A; AU3341984A; DE3334407A1; FI843695A0; ES8505647A1; CA1247545A; IE57669B1; ZA847440B; HUT36843A; EP0137361A3

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

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £09620 Priority Data(s): 2.^-q -as Comp'ete Sp-scinc-st-ori Fiied. - aass: R& total*1?! Publication Date: P.O. Jcurnai, No: 3 0 MAR 19 ,,4 »»»•*»«* « 209620 N.Z.NO. NEW ZEALAND Patents Act, 1953 COMPLETE SPECIFICATION "INSULIN DERIVATIVES MODIFIED IN THE B 30 POSITION, PROCESSES FOR THEIR PREPARATION AND THEIR USE, AND PHARMACEUTICAL AGENTS FOR THE TREATMENT OF DIABETES MELLITUS." We, HOECHST AKTIENGESELLSCHAFT, a corporation « organized under the laws of the Federal Republic of Germany, of D-6230 Frankfurt/ Main 80. Federal Republic of Germany, do hereby declare the invention, for which we pray that a Patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - -1- - 2 - 2 09620 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. Parti-10 cularly important principles with delayed action are 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 15 certain period as 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 individua I 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 macroangiopathy, will be discussed. 209620 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 oran appropriate pharmaceutical agent 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 invention, by insulin derivatives whose C-terminal amino-acid in the B 30 position is esterified, or by a pharmaceutical agent which contains thin insulin derivative as an active compound. Some human insulins esterified in the B 30 position, and processes for their preparation, have already been described, these stated esterified human insulins being intermediates in the semi synthesi s of human insulin. Thus, human insulin-Thr8 ^®-0But is disclosed in, for example, U.S. Patents 4,320,196 and 4,320,197. GB-A- q 3 0 2,069,502 discloses human insulin-Thr -OMe, human insuIin-Thr8 ^®-0Et, human insulin-Thr8 ^-0-(2,4,6-trimethylbenzyl) and human insulin-Thr8 ^^(But)-0But. EP-A-45,187 discloses human insulin-(B 30)-amide. The invention relates to insulin derivatives of the formula I - 4 - 209620 H- A1 |— S S —^ A21 y A - c h a i n Asn ^J— OH (I) s s • I s s B2 I • 1329 Ji '~j Vai B- c h a i ri - n30-s31 i n whi ch a) R denotes H, represents the radical of a neutral, genetically 5 codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and 31 R denotes a physiologically acceptable neutral group blocking the carboxyl group, 10 b) R^ denotes H-Phe, b 1 ) R represents the radical of a neutral, genetically codable L-aminoacid, with the exception of Thr, and any OH group present can be free or protected by a physiologically acceptable group, and 31 15 R is defined as under a), b2> R^® represents Thr, and b2.1) the OH group of Thr8 ^ is present in unprotected form, and 31 R denotes a physiologically acceptable neutral 20 group -NRaRb or -0RC, wherein Ra and Rb are identical or different and represent (C1-C6)-alkyl, (C3-Cg)~cycloalkyl, <C^ — C-j q5 — aryt, CC7-Cl,)-araIkyI, <C3-C9>-heteroaryI or 209620 - 5 - -(CH2-CH2-O-)mR in which m = 1 to about 120 and R = (C<j-C4)-a I ky I, and Ra may furthermore be hydrogen, and these groups may furthermore be substituted in the aryl part by one or more identical or different substituents from the series comprising halogen, nitro, (C-j-C^-alkoxy, methylenedioxy and (C',-C4)-alkyl, or Ra and R*3 together denote -CCH2^n~ where n = 4 - 6 and a methylene group may also be replaced by 0 or NH, and Rc represents propyl, isopropyl, butyl, isobutyl, sec.-butyl, (C5-C^)-a I ky I, (Cj-CgJ-cycloalky I, (C£ — C^ q) — ary I, (Cy—C-j -j ) — a ra Iky I, (C3 —C^) — heteroaryl or (CH2-CH2-O-)mR in which m = 1 to about 120 and R = (C^-C^)-a I ky I, and these groups may furthermore be substituted in the aryl part by one or two identical or different substituents from the series comprising halogen, nitro, (C.j-C^)-a Ikoxy , methylenedioxy and (C-j-C^)-alky I, .2) Thr® is present as the tert.-butyl ether and R^ denotes a physiologically acceptable neutral group -NRaR^ or -0RC, wherein Ra and R*3 are defined as under b2.1) and Rb may furthermore denote hydrogen, and Rc represents (C^-C3)-a I ky I, butyl, isobutyl, sec.-butyl, -a Iky I, (Cg-Cg)-eyeloalky I, ( C ^ — C 1 Q ) — a r y I, ( C-jr — C^ ^ )—a ra Iky I, (C3-C9) — heteroaryl or (CH2_CH2-0-)mR, wherein m and R are as defined above, and these groups may be substituted in the aryl part by one or more substituents from the - 6 - series comprising halogen, nitro, (C-j-C^-alkoxy, methylenedioxy and (C-j-C^J-alkyl, b2.3) the OH group of Thr® ^ is protected by a physiologically acceptable group from the series comprising (C-j-CjJ-alkyl, butyl, isobutyl, sec.-butyl, ^ C 5 — C ^)-a Iky I, (Cj-Cg)-eye loa I ky I , (C^-C^q)-aryl, (Cg-C^)-ara Iky I, (C3-C9)-heteroary I, formyl, (C^-C^)-a I k anoy I and ^)~aroy I, which may be substituted in the aryl part as defined under b2.1) for Ra and or is protected by another physiologically acceptable radical conventionally used in peptide chemistry, and R is as defined under a), or b2.4) the OH group of Thr8 is protected by acetyl or benzyl, and 7«1 R 1 denotes a physiologically acceptable, neutral group - N R a R ^ or - 0 R c , wherein Ra and R ^ are as defined under b2.1), with the exception of R*3 = (Cy-C-j g)-a ry I,' and wherein Rc is as defined under b2.2), and their physiologically acceptable salts. The following L-aminoacids are genetically codable: Gly, Ala, Ser, Thr, VaI, Leu, lie. Asp, Asn, Glu, Gin, Cys, Wet, Arg, Lys, His, Tyr, Phe, Trp and Pro (neutral aminoacids are underlined). Neutral groups SN, which block the free carboxy I function at the C-terminal end of the B chain in the compounds according to the invention, are understood as meaning physiologically acceptable uncharged groups, 209620 especially ester and amide groups, or other C00H protec- ifr tive groups, as described in, for example, Bodanszky et ^ a I., Peptide Synthesis, 2nd edition (1976) John Wiley S Sons, preferably groups of the formula -NRaRb or -OR^, 5 wherein Ra and Rb are identical or different and represent hydrogen, (C-j-C^y-alkyl, (C3~Cg)-cycloalkyl, (C^-C-j g)-a ry I, (Cy-C <] <|)-a r a I ky I, (C3-C9) ~h et e ro-aryl or -(CH2~CH2~0)mR in which m = 1 to about 120 and R = ( -C^)-a Iky I , and these groups may-further--10 more be substituted in the alkylpart by one or more identical or different substituents from the series comprising halogen, nitro, (C^-C^)-alkoxy, methylenedioxy and (C-j-C^-alkyl, or Ra and Rb together represent -CCH23n-, wherein n = 4 - 6 and one methylene group 15 may furthermore be replaced by 0 or NH, and Rc represents C C >] ~C^>-a Iky I , ( €3-03) - cy c I oa I ky I , ( C^-C-j g)-a ry I, (C-jp-C-j-p-aralkyl, (C3~C9)-heteroaryl or the radical (CH2CH2~0~)mR defined above, and aryl radicals may be substituted as in the case of Ra and Rb. 20 Suitable OH protective groups for Ser, Thr or Tyr are physiologically acceptable groups from the series comprising (C-j-C5)-a I ky I, (C-j-Cg)-cy c loa I ky I, (C^-C-jg)-aryl, (Cg-C^-j)-a ra I ky I, (Cj-C^-heteroaryl, (C1-C6>-alk3noyl and ( Cy-C -j -j )-a r oy I, or another 25 physiologia I ly acceptable radical conventionally used in ^ peptide chemistry (cf. for example Bodanszky et al., loc. C T t • In the above context, and also below, (C^-C^)-alkyl is understood as meaning, for example, methyl, - 8 - ethyl, propyl, isopropyl, butyl, tert.-butyl, amyl or hexyl, (C3-C3)-cyc loa I ky 1 as meaning, for example, cyclopropyl, cyclobutyl, cyclohexyl, etc., (C^-C^q)-ary I as meaning, for example, phenyl or naphthyl, preferably 5 phenyl, (C^-C-j-jJ-aralkyl as meaning, for example, benzyl, phenethyl and the like, (C3-C9)-heteroary I as meaning, for example, pyridyl, pyrrolidyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, indolyl or quinolinyl, CC-j —C^) — alkanoyl as meaning, for example, formyl, acetyl, pro-10 pionyl, butyryl, etc., and (Cy-C-j -j)-a roy I as meaning, for example, benzoyl, naphthoyl or toluyl. Insulin derivatives which carry phenylalanine in position B1 are particularly preferred. Other preferred derivatives are those which have Ala, Thr or Ser in the 15 B30 position. The A chain and the chain (S 2 - 30) of the compounds according to the invention advantageously have the sequence of bovine or porcine insulin, but in particular of human insulin.-20 Other preferred insulin derivatives are those in which denotes alkoxy or aralkoxy or -0 (CH2CH2"*0) mR. Preferred OH protective groups for Ser, Thr or Tyr are (C-j-C^)-a I ky I, ( -C^)-a I kanoy I or (Cy-C-j 1 ) -a r a I ky I. 25 In the series of insulin derivatives according to the invention, the following compounds may be mentioned by way of example, without restricting the invention to these: 209620 - 9 - Des-Phe8 ^-porcine insulin-CB SOJ-OBu* Des-Phe8 ^-human insulin-CB 30)-0Bu* Des-Phe8 ''-porcine insulin-CB 30)-0CH3 Des-Phe8 ''-human insulin-CB 30)-0BZL porcine insulin-(B 30)-0-cyclohexyl human insulin-(B 30) -0-£> CH: bovine insulin-CB 30)-0CH3 porcine insulin-CB 30)-0-(CH2~CH2-0-)20c2H5 human insulin-(B 30)-NH-C2H5 10 Des-Thr8 ^-human insulin-Val8 ^^-0-C2H5 Des-Thr8 ^-human insu I in-Va I8 NH- human insulin-CB 30)-N b human insulin-CB 30)-NH-CCH2~CH2"0)3g-CH3 human insulin-CB 30)-NCCH3)2 15 human insulin-CB 30)-0BZL human insulin-CB 30)-0-C H2- human insulin-Thr8 30CEt)0Et human insulin-Thr8 ^CCH^OCh^ human insulin-CB 30)-O-CC^^g CH3 20 human insu I in-ThrCAc)0-Et human insuIin-ThrCBz)OBu* human insulin-CB 30)-NH-f—'~\ human insulin-CB 30)-0- human insulin-Thr83^CF0R)-0pr 25 human insulin-CB 30)-NH2 The invention furthermore relates to a process for the preparation of insulin derivatives of the formula 209620 -10 - I, wherein a) a Des-octapeptide (B23-30)-insuIin of the formula II A 1 S1- r A21 Gly A-chain Asn " | -OH S s i (II) B2 S1-R1- 10 Val B- chai n JB22 Arg"~~|-0H 1 1 in which R denotes Phe or a bond, and S denotes an amino protective group, such as the tert.-butyloxycarbon-yl-(Boc), the tert.-amy loxycarbonyI-(Aoc) or the methyl- su I fonyIethyloxycarbonyl~(Msc) radical, which can be \ split off by proton solvolysis or by p-e I imination,Ms condensed with a peptide of the formula III H -G1 y - Ph e - Ph e- Ty r () - Th r (S2) - P r o- !,y s (S 3 ) - R 3 0 - R 3 1 * (III) in which R3® and R3^ have the meanings defined above, v S2 represents hydrogen, Bzl or Bu*, and S3 represents a urethane protective group, such as Boc,Moc, Fmoc or Z , and any protective groups present are split off in a 15 manner which is known per se, or b) a Des~B30-insuLin of the formula I, in which R represents H or H-Phe and the C-terminal end R3^-R3^ together represents OH, is reacted with a compound of the formula IV H-R30-R31 (iv) 209620 in which and R^1 have the meanings defined above, in the presence of trypsin or of a trypsin-like endopep-t i das e, and any protective groups present are then split off in a manner which is known per se, and the compounds 5 obtained by a) or b) are, if required, converted to their physiologically acceptable salts. A1 In process variant a), for example, the N<?v , ^-b i s-Bo c derivative of a Des-octapepti de-(B23-30)-insulin is reacted directly with one equivalent of a com-10 pound of the formula III, the procedure used being analogous 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 15 groups do not usually need to be protected, the insulin derivative is also normally not damaged either in the esterification or in the alkaline hydrolysis. Unreacted Des-octapeptide and a peptide formed by condensation of A 1 IV with Asp -OH differ in molecular size and in charge 20 number and can therefore readily be separated by partition chromatography over SephadexRLH 20 or by gel chromatography over SephadexRG 75 or 6 50 superfine. To eliminate the tert.-butyl protective groups, it is only necessary to treat the reaction product with 25 trif luoroacetic acid for 30 - 60 minutes at room temperature. This reaction does not damage the insulin derivative. If a methylsulfonylethyloxycarbonyl radical is chosen as the N protective group, splitting off the latter by (^-elimination necessitates treatment with an 209^20 - 12 - alkali. The reaction conditions are such (e.g. 0.1 N N a 0 H , 0°C/ 5 sec) that the insulin derivative is not damaged. The porcine -b i s-Boc-D es-B23_3Q- octapeptide-insulin used as the starting material is pre-5 pared, for example, in the following manner: Porcine insulin in a mixture of dimethy Iform-amide, dimethyl sulfoxide and water is reacted with an excess of tert.-butyloxycarbonyl-N-hydroxysuccinimide ester in the presence of N-ethy Imorpho I ine. This reac-10 tion gives the expected Nca^, , NfJ^'-t r i s-Boc- insulin. Small portions of trypsin are then added to the solution of this compound in dimethyIformamide and tris buffer (pH 7.5) until starting material is no longer 15 detectable in the electrophoresis. The Nxf1 ^ , N^^-bis-Boc-DeS-B23_3q-octapeptide-insuIin i s purified by pai— tit ion chromatography over Sephadex^ LH 20. This compound is then brought to reaction with one mole of the peptide of the formula III, which is pre-20 pared in a manner known per se by the methods of peptide chemistry, 1 - 2 moles of 1-hydroxybenzotriazole and about 0.9 mole of dicyclohexyIcarbodiimide in dimethyl-formamide at about pH 7 - 8 <cf. Chem. Ber. 103 (1970), page 788). 25 The crude product is purified by partition chromatography, and is freed from the protective groups by treatment with trif luoroacetic acid/anisole at room temperature. After precipitation with ether, isoelectric precipitation from water, and chromatography over 2 096 2 0 SephadexR G 75 or 6 50 superfine, the compound is etectrophoretically pure, and can be crystallized in a known manner. The insulin derivative thus obtained possesses full biological activity. 5 Des-Phe^-insulins as starting compounds for the processes according to the invention are disclosed in, for example, German Patent 2,005,658 or EP-A-46,979. The Des-B30-insu I ins used as starting compounds in process variant b) are disclosed in, for example, EP-10 A-*46,979 or Hoppe-Seyler's Z. Physiol. Chem. 359 <1 978), 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-B 30-insulin and the compound of the 15 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 20 obtained can be isolated by the conventional methods of peptidechemistry. The compounds according to the invention may furthermore be synthesized analogously to the semisynthetic procedures for the preparation of the known 25 insulin-CB 30)-esters, which procedures are described in, for example, GB-A-2,069,502, EP-A-56,951, W0 82/4069, DE-A-3,129,404 or W0 83/1074. Aminoacid esters of the formula IV, in which represents an alkylpolyoxyethyleneoxy chain, are prepared - 14 - 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 (B 30)-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 20 the 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 hexa-meric state of the insulin and its tendency to crystallize. These aggregates dissolve once again in subcutarie-25 ous t i ssue. Another familiar depot principle is the crystallization 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 0 • 209620 - 15 - conjunction with one of the depot principles described, 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 5 because, as is suggested, part of the biologically active surface region, the receptoi—binding region, is masked by 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 10 example NZ 201,683). Heterologous proinsulin poss esses significant immunogenicity . In this context, it is noteworthy that human pro insulins, too, can exhibit variations in the C peptide moiety. Surprisingly, it has now been found that, in con-15 trast to proinsulin, the insulin derivatives of the formula I whose B chain is blocked at the C end have about the same biological activity as an equimolar amount of natural insulin. The invention furthermore relates to the use of 20 insulin derivatives of the formula I in which R^® 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 25 R denotes a physiologically acceptable neutral group SN blocking the carboxyl group, V , k', as remedies. • ' L- <v. The invention also relates to medicaments for the \\ i-'\\ 5SEP l%7 I treatment of diabetes mellitus, consisting of a pharma- 2096 2 0 - 16 - ceutically acceptable excipient and an insulin derivative of the formula I as an active compound. These medicaments according to the invention constitute completely novel delayed-action principles which 5 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 chemistry: the poor solubility of the insulin derivative at its isoelectric point. It is pos-10 sible that its 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 eliminated are either purely physiological metabolites or readily meta-15 bolizable, 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 medicaments, are not,, 20 as a rule, more highly immunogenic than the corresponding insulin itself. The agents according to the invention contain, as active compounds, one or more of the novel insulin derivatives of the formula I. 25 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 209620 - 17 - comprises preparations which/ below the isoelectric point7 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 5 that of acidic former insulins (typical pH 3.0). In certain cases, a more neutral injectable solution offers substantial advantages in respect of its toleration. Another typical use form comprises suspensions of amorphous or crystalline precipitates of the deriva-10 tives described/ in a physiologically acceptable excipient at about neutral pH. However, it is also possible to reinforce the poor solubility in the physiological pH range, which is an inherent property of the derivatives, by means of 15 additional depot principles, for example by the addition of zinc or protamine sulfate. The amount of zinc added ? + can be up to 100 jjg of Zn /100 insulin units, but is *? J. typically about 50 ^ig of Zn /100 insulin units. The amount of protamine can be between 0.28 mg and 0.6 mg per 20 100 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 amount of insulin appears therapeutically advan-25 tageous. 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 a sterile aqueous solution which is rendered isotonic 209620 - 18 - 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 5 excipient can additionally contain a buffer substance,' for example sodium acetate, sodium citrate or sodium phosphate. The pH is adjusted using dilute acids (typically HCl) or alkali solutions (typically NaOH). The insulin derivatives can also be employed in 10 the form of alkali metal or ammonium salts in the agents according to the invention. 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, 15 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 according to the invention, the said stabilizer prevent-20 ing 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-83/00,288. In the agents according to the invention, which 25 can also contain one of the known delayed-action principles, such as, for example, protamine sulfate, glob in or zinc, in suitable amounts, a delayed-action principle of this type can be used in combination with the total 2 096 2 0 - 19 - 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 5 different auxiliaries having a delaying action. 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-insu I in, independently of one another and 10 in each case in dissolved, amorphous and/or crystalline form. Obviously, therefore, a large variety of action characteristics which can be very finely matched are achievable with the therapeutic agents according to the 15 invention; according to the comments made at the outset, -this should be associated with advances, particularly with regard to late diabetic complications. The examples which follow serve to illustrate the invention further: 20 Preparation Example 1: Human i nsu li n-(330)-0-CH2~CH2""CH3 5 g of porcine insulin are dissolved in 45 ml of dimethylformamide, 25 ml of dimethyl sulfoxide, 0.5 ml of N-ethylmorpholine arid 2.5 ml of water. 1.5 g of tert.-25 butyloxycarbonyl-N-hydroxysuccinimide are added at room temperature, while stirring, and the reaction is allowed 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 209620 dissolved in 360 ml of dimethy Iformamide, and the solution is diluted with 320 ml of tris buffer (0.05 M, 0.01 • M of CaClg/ pH 7.5). Portions of 20 mg of trypsin are added at intervals of 1 hour, at 36°C. 5 After a total of 12 additions, the pH is brought to 4.5 with acetic acid, and the solution is evaporated down. Subsequent purification of the material on a SephadexR LH 20 column (8 x 200 cm) by means of partition chromatography using the system n-butanol/glacial 10 acetic acid/water (2:1:10) gives 3.25 g of ^ , tlx? ^ -bis-Boc-des-B23_3Q-octapeptide-insulin (porcine), in which the starting material is no longer detectable in the acidic and basic electrophoresis. Analysis of the substance for aminoacids gives the correct result. When 15 the B0C groups are eliminated by way of a test, insulin activity is no longer found. This material (3.25 g) is dissolved in 30 ml of dimethylformamide, together with 100 mg of 1-hydroxybenzotriazo le, 750 mg of HCl.Gly-Phe-Phe-Tyr(But)-Thr-Pro-Lys(Boc)-Thr(But)-0CH2CH2~CH3 20 and 0.5 ml of N-ethyI morpho I ine . 120 mg of dieye lohexy l-carbodiimide are then added at room temperature, and the reaction mixture is stirred for 24 hours. The precipitated dicyclohexylurea is filtered off, and the product is precipitated by adding ether. 25 The precipitate is filtered off, washed with ether and dried. The substance is subjected to preliminary purification by partition chromatography over Sepha-de xR LH 20, using the above system. 2.6 g of material from the principal peak are isolated, by precipitation 209620 - 21 - with acetone/ether. The dried, still unprotected derivative is allowed to react with a mixture of 5 ml of tri-fluoroacetic acid and 1 ml of anisole for 60 minutes at room temperature. The crude substance is then precipita-5 ted 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 p acetic acid, over Sephadex 6 50 superfine or G 75. 10 Human insulin-(B30)-0CH2CH2CH3 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. 15 The octapeptide of the formula III is prepared according to the following condensation diagram, by conventional methods of peptide condensation: m Gly 'M *Z- z Z-i H Synthesis diagram for the octapeptide of the formula 111 phe Phe Tyr ' , Thr • Pro- • Lys • •Thr ■ 2- -OH H-DCC/HOBtj I V OH H-OS« t DCC/H03t \!' H /Pd TFE OBu t -03u OBj t DCC/H03' Bu^ I iLJL Hp/Pd f Bu OH DCC/H031, Aminoacid and elemental analysis correspond to theory 2 096 20 - 23 - Medi cament s Examp Le 1 : Porcine insulin-(B 30)-0CH3 (prepared semi-synthetically from des-B 30-porcine insulin), formulated 5 as a weakly acidic solution containing 40 IU per ml, and its depot activity: 14.5 mg of porcine insulin-(B 30)-OCH-j (27.5 IU/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 HCl or 1 N NaOH. When administered in a dose of 0.4 IU/kg to rabbits, a solution of this type exhibits a pronounced depot acti-15 vity. The area under the blood sugar curve is the same as that obtained with a standard preparation containing 40 IU/ml. Example 2: Human insulin-ThrB "^(Bu^OBu* (pH = 6.8), prepared semisynthetica 11 y from porcine insulin, in a neutral for 20 mulation containing 40 IU per ml, and its depot activity: 14.8 mg of human insulin-Thr8 ^®(But)0But (27 IU/mg) 21.0 mg of sodium dihydrogen phosphate dihydrate, 27.0 mg of m-cresol and 160.0 mg of glycerol 25 are dissolved in a total volume of 10 ml of water. The pH is brought to 7.3 by addingl N HCl or 1 N NaOH. When administered in a dose of 0.4 IU/kg to rabbits, a suspension of this type exhibits a pronounced 209620 depot activity. Example 3: Human insulin-(B 30)-NH2, prepared semisyn-thetically from porcine insulin, in the form of a crystal-5 line NPH preparation containing 40 IU/ml, and its greatly delayedaction: 14.5 mg of human insulin-(B 30)-NH2 (27.5 IU/mg), 1.3 mg of protamine sulfate, 21.0 mg of sodium dihydrogen phosphate dihydrate, 10 15.0 mg of m-cresol, 6.0 mg of phenol and 160.0 mg of glycerol are dissolved in water to give a total volume of 10 ml. The pH is brought to 7.3 by adding 1 N HCl or 15 1 N NaOH. When administered in a dose of 0.4 IU/kg to rabbits, a crystal suspension of this type exhibits a greatly delayed action. Example 4: 20 Mixture of human insulin-Arg- -OH and human insulin-Thr® ^^Bu^ (OBu*) , both prepared semisyn-thetically from porcine insulin, in the form of a zinc-containing suspension with 40 IU/ml, and its greatly delayedaction: 25 7.3 mg of human i nsu I i n-A r g-®^-OH (27.5 IU/mg), 7.4 'mg of human insulin-Thr® "^Bu*(0Bu*) (27.0 IU/mg) 0.46 mg of anhydrous zinc chloride, 14.0 mg of sodium acetate, 10.0 mg of methyl p-hydroxybenzoate and </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 209620 - 25 - 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 HCl or 1 N NaOH. 5 When administered in a dose of 0.4 IU/kg to rabbits, a suspension of this type exhibits a greatly delayed action. Examp le 5: Human insulin-(B 30)-0-(CH2CH2~0-)20^2H5' 10 prepared semisyntheticaLly from porcine insulin, in the form of a neutral preparation containing 100 IU/rnl, and its delayed action: 41.7 nrg of human insulin-(B 30)-0-(C H2 C^-O-) 20 C2H5 (24.0 IU/mg), 15 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 HCl or 20 1 N NaOH. In rabbits, a suspension of this type exhibits a delayedaction. - 26 - 2 096 20 WHAT.WE CLAIM IS: 1. An insulin derivative of the formula I A 1 r HH A21 VI _ I e:1 \r A-chain Asn -OH S 1 S s I s B2 Val B29 B- x n a i n (I) - n30~-R3i in which 1 a) R denotes H, 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 3 1 R denotes a physiologically acceptable neutral group S ^ blocking the carboxyl group, b) R^ denotes H-Phe, b1> R^® represents the radical of a neutral, genetically codable L-aminoacid, with the exception of Thr, and any OH group present can be free or protected by a physiologically acceptable group, and * 1 R is defined as under a), b2> represents Thr, and btq b2.1) the OH group of Thr is present in.unprotected form, and "Z 1 denotes a physiologically acceptable neutral group -NRaRb or -0RC, wherein Ra and R ^ are identical or different and represent - •- ' 209620. - 27 - (C-j-C^)-alky I, (Cj-Cg)-cy c loa I ky L, (C^-C-jq)-aryl, C Cy—c ^ -j)-a ra t ky I, (C3-C9)-heteroaryI or - (CH^CHj-O-) mR in which m =. 1 to 120 and R = (C1-C4>-a Iky I, and Ra may furthermore be hydrogen, and these groups may furthermore be substituted in the aryl part by one or more identical or different substituents from the series comprising halogen, nitro, (C-j-C^J-a Ikoxy, methylenedioxy and (C-j-C^J-alkyl, or Ra and R*3 together denote -CC^U n~ where n = A - 6 and a methylene group may also be replaced by 0 or NH, and Rc represents propyl, isopropyl, butyl, isobutyl, sec.-butyl, (C5-C6>-a Iky I, (C3~Cg)-cycloaIky I, CC^ — C^q)—aryl, (Cy — C^^)—aralkyl, (C3 — C9) — heteroaryl or (CH2"CH2"0-)mR in which m = 1 to ; 120 and R = (C<j-C/)-alkyl, and these groups may furthermore be substituted in the aryl part by one or two identical or different substituents from the series comprising halogen, nitro, (C-j-C^J-alkoxy, methylenedioxy and (C-j-C^J-a Iky I, .
2) ThrB^® is present as the tert.-butyl ether and r31 denotes a physiologically acceptable neutral group -NRaR^ or -0RC, wherein Ra and R*3 are defined as under b2.1) and R*5 may furthermore denote hydrogen, and Rc represents (C^-Cj)-alkyl, butyl, isobutyl, sec.-butyl, (C5~C6>-a Iky I, (Cj-Cg)-cyc loaIky I, < c6"*c10^-a r>" I, ^7~C^-j)-aralkyl, (C3-C9)- heteroaryI or (CH_ —CH -0—) R/ wherein m and R are 2 2m. • 209620 - 28 - as defined above, and these groups may be substituted in the aryl part by one or more substituents from the series comprising halogen, nitro, (C-j-C^-a Ikoxy, methylenedioxy and (C<j-C^)-a I ky I, b2.3) the OH group of ThrB^ is protected by a physiologically acceptable group from the series comprising C C i-C-j)-a I ky I, butyl, isobutyl, sec.-butyl, (Cj-C^-a Iky I, ( Cj-Cg)-cy c loa Iky I, CC^-C-jq)-aryl, < Cg-C<] -j)-a r a I ky I, (C3-C9)-heteroary I, formyl, ( C3-C5)-a I kanoy I and (Cy-C-j-p-aroy I, which may be substituted in the aryl part as defined under _ and b2.1) for Ra and R^,|< is as defined under a), or b2.4) the OH group of Thr5^ is protected by acetyl or benzyl, and 7 A R denotes a physiologically acceptable, neutral group -NRaRk or -0RC, wherein Ra and R*3 are as defined under b2.1), with the exception of R*3 = (Cy-C-jQ)-aryl, and wherein Rc is as defined under b2.2) , and its physiologically acceptable salts. 2. An insulin derivative as claimed in claim 1, •* 1 wherein R represents alkoxy or aralkyloxy, which are defined as in claim 1. 3. An insulin derivative of the formula I, ■ w io : "-..X in whi ch R^ denotes H-Phe, . 29 . .20 9670 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^ denotes a physiologically acceptable neutral group SN blocking the carboxyl group, and its physiologically acceptable salts for use as remedies. 4. An insulin derivative as claimed in any one of claims 1 and 2, wherein R represents H. 5. An insulin derivative as claimed in any one of claims 1 to 4, wherein R^® represents Ala, Thr or Ser. 6. An insulin derivative as claimed in any one of claims 1 to 5, wherein the A chain and the chain (B 2 -29) have the sequence of human insulin. 7. An insulin derivative as claimed in any one of claims 1 and 3 to 6, wherein RJ1 represents -0-(CH2""CH2~0~)mR, wherein m and R are as defined in claim 1. 8. An insulin derivative as claimed in any one of claims 3 to 6, wherein R^ represents (C-j-C^)-a Ikoxy or ( Cy-C-j i )-a ra I koxy . 9. An insulin derivative as claimed in any one of claims 1-to 8, wherein any OH group which may be present in R^® is free or protected by (C-j-C^)-a Ikyl, CC-j-C5>"alkanoyl or (C7-C11>-ara Iky I. 10. An insulin derivative as claimed in any or*e of ■ in non-human animals claims 1 to 9, for the treatment of diabetes mellitus'. 11. A process for the preparation of an insulin ... . 209620 - 30 - derivative of the formula I as claimed in claim 1 or 3, wherein a) a Des-octapeptide (B 23-30)-insu I in of the formula II /»1 S'~ Gly B2 S^R1- Val r-S- S—j A-. cnain S I s B-c ha i n f S A21 Asn ■OH B22 Arg | - OH (II) in which R^ denotes Phe or a bond, and denotes amino protective group an which can be split off by proton solvolysis or by j^relimination, is condensed with a peptide of the formula III B~Gly-Phe-Phe-Tyr(S2)-Thr(S2)-Pra-Lys(S3)-R3°-R3'1 (III) in which r'® and R3' have the meanings defined in claims 1 or 3, represents hydrogen, Bzl or Bu*, and represents a urethane protective group and any protective groups present are split off in a manner which is known per se, or b) a Des-B30-insulin of the formula I, in which R^ represents H or H-Phe and the C-terminal end R^®-R*^ together represents OH, is reacted with a compound of the ,31 formula IV <*>!{ r+- It, -D30_d31 H-R (IV) - 31 - in which R^® and R^ have the meanings defined in claims 1 or 3, in the presence of trypsin or of a trypsin-like endopeptidase, and any protective groups present are then split off in a manner which is known per se, and the compound obtained by a) or b) is, if required, converted to one its physiologically acceptable salts. 12* A Process according to claim 11, wherein the said amino protective group denoted by S1 is selected from tert.-butyloxycarbon-yl-(BOC), the tert.-amyloxycarbonyl-(AOC) and the (2-methylsulfonyl)ethyloxycarbonyl-(MSC) radicals. 13' A Process according to claim 11 or claim 12, wherein the urethane protective group represented by S3 is selected from BOC, MOC, FMC or Z. i4_ A medicament consisting of a pharmaceuticaIly acceptable excipient and an active compound as claimed in any one of^ claims 1 to 10. 15. An agent as claimed in claim 14, which has a pH between 2.5 and 8.5, contains a suitable isotonic agent and a suitable preservative, and wherein the insulin derivative of the formula I is present in dissolved form and/or in suspension. 16. An agent as claimed in c lain* 14 or 15 , which contains a suitable buffer and has a pH between 5.0 and 8.5. 17. An agent as claimed in any one of claims 14 to 16, which contains between 0 and 100 fjg of zinc/100 I.U. 18. An agent as claimed in any one of claims 14 to 17, wherein the active compound of the formula I is 13 OCT 1987 X/ /present in the form of an alkali metal salt or of the \? ,5. o // c I M ammonium salt. m £o9 62i° - 32 - # 19. An agent as claimed in any one of claims 14 to 18 i fat least i two 1 wherein any desired amount ofl insulin derivatives of the formula I are present independently of one another and in each case in dissolved, amorphous and/or crystalline form. 20* An agent as claimed in any one of claims 14, to 19 i ', which contains a suitable amount of an auxiliary having a delaying action. 21. An agent as claimed in claim 20, 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. 22. An agent as claimed in any one of claims"14 to 21 which contains different insulin derivatives of the formula I in combination with several different auxiliaries having a delaying action. 23. a process for the preparation of an agent as claimed in any one of claims 14' to 22, wherein the active "\v compounds are brought into a suitable dosage form. 24. Use of an insulin derivative as claimed in any one of claims 1 to 10, or of an agent as claimed in any one of claims 14 to 22, for the treatment of diabetes mellitus in non-human animals. 25. An insulin derivative according to claim 1 substantially as herein described or exemplified. 26. A process according to claim 11 substantially as described or exemplified. - HOECHST AKTIENGESELLSCHAFT "3DtC19o7myJ By Their Attorneys HENRY HUGHES LltflTEQ^ </div>