to
`United States Patent
`5,656,722
`[11] Patent Number:
`Aug. 12, 1997
`[45] Date of Patent:
`Dorschug
`
`US005656722A
`
`Markussen et al., Soluble, prolonged—acting insulin deriva-
`tives, III. Degree ofprotraction, crystallizability and chemi-
`cal stability of insulins substituted in positions A21, B13,
`B23, B27, and B30, Protein Engineering 2(2):157-166
`(1988).
`Zinman, Bernard, The Physiologic Replacement of Insulin,
`Medical Intelligence, vol. 32, No. 6, pp. 363-370 (1989).
`Sundby, F., Separation and Characterization of Acid-in-
`duced Insulin Transformation Products by Paper Electro-
`phoresis in 7 M Urea, The Journal of Biological Chemistry,
`yol. 237, No. 11, pp. 3406-3411 (1962).
`Burgermeister, W., et al., The Isolation of Insulin from the
`Pancreas, Reprint from the Handbook of Experimental Phar-
`macology, pp. 715-727 (1975) Insulin Humanum, European
`Pharmacopeia 838 (1993).
`Primary Examiner—David Lukion
`Attorney, Agent, or Firm—Finnegan, Henderson, Farabow,
`Garrett & Dunner, L.L.P.
`
`[57]
`
`ABSTRACT
`
`New insulin derivatives, the use thereof, and a pharmaceu-
`tical composition containing them
`
`Insulin derivatives having an isoelectric point between 5 and
`8.5, or physiologically tolerated salts thereof, of the Formula
`st
`
`[54] A??-, B* - MODIFIED INSULIN
`DERIVATIVES HAVING AN ALTERED
`ACTION PROFILE
`
`(75]
`
`Inventor: Michael Dérschug, Bochum, Germany
`
`[73] Assignee: Hoechst Aktiengesellschaft, Frankfurt
`am Main, Germany
`
`[21] Appl. No.: 304,593
`
`[22] Filed:
`
`Sep. 12, 1994
`
`Related U.S. Application Data
`
`[63] Continuation of Ser. No. 46,481, Apr. 9, 1993, abandoned,
`which is a continuation of Ser. No, 929,510, Aug. 19, 1992,
`abandoned, which is a continuation of Ser. No. 431,844,
`Nov. 6, 1989, abandoned.
`
`[30]
`
`Foreign Application Priority Data
`
`Nov. 8, 1988
`[DE]
`Germany ......csee 38 37 825.6
`[S52]
`Tint, CS aeccccssssssstsscssseseseesssieecsteeee AGL 38/28
` [52] U.S. CR. oneeceeccsnseees
`se
`sees
`. 530/303; 530/304
`[58] Field of Search ...............05sersaesreeeee 530/303, 304;
`514/3, 12
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,701,440
`
`10/1987 Grand «0...ceccsseosesceescesenenetecsenseannees 5143
`
`FOREIGN PATENT DOCUMENTS
`
`62066/86
`75916/87
`13976/88
`0046979
`0133285
`0214826
`0254516
`
`5/1986 Australia .
`1/1988 Australia .
`9/1988 Australia .
`8/1981
`European Pat. Off. .
`2/1985 European Pat. Off.
`3/1987 European Pat. Off. .
`6/1987 European Pat. Off. .
`OTHER PUBLICATIONS
`
`...........+ 495/351
`
`Schomeet al. Diabetes vol. 27 (1978) 8-15.
`Geigee, Chem—Zeitung vol. 100 No. 3 pp. 54-56 (Jan.
`1976).
`Thompson et al Int’l J. Pept. Prot. Res. vol. 23 (1984)
`394-401.
`Schwartz et al PNAS vol. 84 pp. 6408-6411 (Sep. 1987).
`Volund Diabetic Medicine 8, 839, 1991.
`Neubauer. “The Immunogenicity of Different Insulins in
`Several Animal Species”, Diabetes, vol. 27, No. 1 (1977).
`“Galenics Of Insulin, The Physicochemical and Pharma-
`ceutical Aspects of Insulin and Insulin Preparations”, J.
`BRANGE:Springer-Verlag Berlin Heidelberg 1987. pp.
`35-36.
`Markussenet al., Soluble, prolonged-acting insulin deriva-
`tives. I. Degree of protraction and crystallizability of insu-
`lins substituted in positions A17, B8, B13, B27 and B30,
`Protein Engineering 1(3):215-223 (1987).
`
`H—Gly—L\oT
`—P——7
`
`A21
`R2
`
`(qt)
`
`BY
`
`R*®—RII
`
`§
`
`|
`
`B10
`
`Ai
`
`s——S
`
`B2
`
`R!—Val——B-chain- X
`
`in which:
`R!at position B1 denotes H or H-Phe;
`R? at position A21 denotes a genetically encodable L-amino
`acid selected from the group consisting of Gly, Ala, Val,
`Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Cys, Tyr, Asp, and
`Glu;
`R* represents the residue of a neutral genetically encodable
`L-amino acid selected from the group consisting of Ala,
`Thr, and Ser;
`R*! represents 1, 2, or 3 neutral or basic alpha amino acids,
`wherein at least one of the alpha amino acids is selected
`from the group consisting of Arg, Lys, Hyl, Orn, Cit, and
`His;
`X represents His at position B10; and
`the sequences Al to A20 and B1 to B29 in Formula If
`correspond to a mammalian insulin;
`excludingthoseinsulin derivatives in which simultaneously:
`R!at position B1 denotes Phe; and
`R? is one alpha amino acid having a terminal carboxyl
`group.
`
`15 Claims, No Drawings
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`5,656,722
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`2
`The abovementioned depotprinciple resulting from basic
`modification of the insulin has also been further utilized by
`the provision and corresponding use of other insulin deriva-
`tives with basic modifications, mainly within the A and B
`chains; cf. for example EP-A 0,194,864 and EP-A 0.254.
`516.
`
`In the insulin derivatives specified in EP-A 0,194,864, a
`basic amino acid is incorporated in the B27 position and/or
`a neutral amino acid is located at positions A4, A17, B13
`and/or B21; in addition, the C-terminal carboxyl group of
`the B chain is blocked by an amide orester residue.
`The insulin derivatives specified in EP-A 0,254,516 are
`very similar to those specified in the abovementioned EP-A;
`however,in this case, with the aim of increasingthe stability
`of the relevant pharmaceutical compositions at the weakly
`acid pH values, the amino acid Ashin position A21 can also
`be replaced by other amino acids which are more stable in
`acid medium, such as, for example, Asp. As is known, Ash
`(=asparagine) differs from Asp (=aspartic acid) by the block-
`ing of one of the two carboxyl groups by the amide group:
`COOH
`
`H)N4 —H
`CH,
`
`CONH
`asparagine
`COOH
`Ne—H
`bi
`OOH
`aspartic acid
`
`IC
`
`Rapid-actinginsulin derivatives are said to result from yet
`another modification of the insulin molecule in the A and B
`chain, in particular by replacing the amino acid His, which
`is responsible for the formation of a complex with zinc—and
`thus for a certain delaying action, in the B10 position by
`other appropriate amino acids; cf. EP-A 0,214,826.
`All the insulin derivatives specified in the 3 lastmentioned
`publications are mainly modified within the A and B chains;
`they are prepared by genetic engineering routes.
`
`SUMMARYOF THE INVENTION
`
`In the attempt to increase the stability in acid medium of
`the insulin derivatives with a basic modification on the
`C-terminal end of the B chain as specified in the European
`Patents EP-B 0,132,769 and EP-B 0,132,770 mentioned in
`the introduction, and, where appropriate, also to alter the
`action profile thereof, it has now been foundthat this object
`is achieved in an advantageous mannerby replacing Asn*?*
`by other genetically encodable amino acids which contain
`no amide group and, where appropriate, by replacing His*'°
`by other genetically encodable amino acids.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`Hence the invention relates to insulin derivatives of the
`formula I
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`
`1
`A”1., B* - MODIFIED INSULIN
`DERIVATIVES HAVING AN ALTERED
`ACTION PROFILE
`
`This application is a continuation, of application Ser. No.
`08/046,481 filed Apr. 9, 1993, abandoned, which is a con-
`tinuation of application Ser. No. 07/929,510,filed Aug. 19,
`1992, abandoned, which is a continuation of application Ser.
`No. 07/431,844,filed Nov. 6, 1989, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`As is known,insulin and insulin derivatives are required
`in considerable quantities for the treatment of the disease
`diabetes mellitus, and some of them are also produced on an
`industrial scale. Despite the considerable number ofinsulin
`compositions and modifications with different action pro-
`files which are already in existence, there is still a need,
`because of the variety of organisms with their inter- and
`intraindividual variations, for other insulin products which
`in turn have other properties and action characteristics.
`Insulin derivatives with a delayed action are described, for
`example, in EP-B 132,769 and EP-B 132,770. These are
`specifically derivatives with a basic modification in position
`B31 of the insulin B chain, of the following formula I:
`
`At
`
`S—-———Ss
`
`A2l
`
`®
`
`HoTpetot
`a=—~"
`|s
`|
`|
`
`R!—Val-——B-chain-His
`
`B10
`
`B29
`
`R30— R3t
`
`B2
`
`in which R! denotes H or H-Phe, R® represents the residue
`of a neutral, genetically encodable L-amino acid, and R**
`represents a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms, in whose
`structure 0 to 3 o-amino acids are involved and whose
`terminal carboxyl group which is present where appropriate
`can be free, in the form of an ester functionality, an amide
`functionality, a lactone or reduced to CH,OH.
`Characteristic of these insulin derivatives is an isoelectric
`point between 5.8 and 8.5 (measured by isoelectric
`focusing). The fact that the isoelectric point is shifted from
`the isoelectric point of unmodified natural insulin or proin-
`sulin (at pH=5.4) into the neutral range derives from the
`additional positive charge(s) located on the surface of the
`molecule as a result of the basic modification. This makes
`these insulin derivatives with a basic modification less
`soluble in the neutral range than, say, natural insulin or
`proinsulin, which are normally dissolved in the neutral
`Tange.
`The delaying or depot action of the insulin derivatives
`with a basic modification, of the formula I, derives from
`their sparing solubility at the isoelectric point. According to
`the two abovementioned publications, the redissolution of
`the insulin derivatives under physiological conditions is
`achieved by elimination of the additional basic groups,
`which is brought about, depending on the derivative, by
`trypsin or trypsin-like and/or carboxypeptidase B or carbox-
`ypeptidase B-like and/or esterase activity. The eliminated
`groups are in each case either purely physiological metabo-
`lites or else easily metabolized physiologically acceptable
`substances.
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`4
`suitable basic groups for this being, for example, the above-
`mentioned basic groups—in the case where no c-amino
`acids are involved in the structure of R°!. Of course, these
`basic ester or amide groups can also block the carboxyl
`group of basic o-amino acids. Also possible and suitable for
`blocking the carboxyl group of the basic a-amino acids
`are—if the blocking is desired—neutral ester or amide
`groups such as, for example, (C,-C,)-alkoxy,
`(C,—-C,)-
`cycloalkyloxy, NH, (C,-C,)-alkylamino or di-(C,—-C,)-
`alkylamino.
`Of course, the terminal carboxyl group can be in the form
`of a lactone only if the terminal amino acid is a
`hydroxyaminoacid.
`Moreover,
`the terminal carboxyl group can also be
`reduced to CH,OH.
`R*!is preferably composed of 1, 2 or 3 of the abovemen-
`tioned basic naturally occurring amino acids; R*! is particu-
`larly preferably Arg-OH or Arg-Arg-OH.
`Suitable genetically encodable L-amino acids—for
`X—are the same aminoacids as for R?, but the genetically
`encodable L-amino acids which contain an amide group—
`which are Ash and Gln—are also possible in this case; the
`latter—Asn and Gin—are in fact preferred in this case. If
`Asn or Gin is located in position B10, the amide groupis at
`least stable in weakly acid medium (in contrast to Asn or Gln
`in position A21). The sequences (Al~A20) and (B1-B9,
`B11—-B29)are preferably the sequences of human,porcine or
`bovine insulin, especially the sequences of humaninsulin.
`Examples of insulin derivatives of the formula II are:
`
`
`Asp“2}_Human insulin-Arg™”!—_OH
`Glu“?!Human insulin-Arg®>!—OH
`Gly?"Human insulin-Arg®*"_OH
`Ser“?!Human insulin-Arg®*!_OH
`Thr“?!Human insulin-Arg™?!—_OH
`Ala*?!-Human insulin-Arg™1—OH
`Asp*?!_Human insulin-Arg®?!—_Arg®*?__OH
`Gh*”"_Human insulin-Arg®?!Arg332__oH
`Gly*?!-Human insulin-Arg®?!Arg®?2__OH
`Ser-Human insulinArg?!—Arg>??_OH
`Thr*?*.Human insulin-Arg®?!—Arg?*?
`Ala“?"Human insulin-Arg™?!—Arg®°2__OH
`Asp*?!_Asn®!°_Human insulin-Arg®?!_OH
`Giu“?!_Asn®!°_Human insulin-Arg™?!_OH
`Gly“??__Asn?!°_Human insulin-Arg™?!_OH
`Ser“?!__Asn®!°Human insulin-Arg??!—OH
`ThrA24__Asn®!°_Human insulin-Arg®>"—_OH
`Ala‘?!—Asn?!°-Human insulin-Arg®*!-OH
`Asp*?}__asn®!°_Human insulin-Arg®?!Arg®??__OH
`Glu“?!Asn®*°Human insulin-Arg®?!—Arg™*?__OH
`Gly“?4—Asn®!°-Human insulin-Arg™?"—Arg®*?_OH
`Ser“2!__Asn®!°_Human insulin-Arg®?!_Arg®*?_OH
`Thr*?*_AsnP!°_Human insulin-Arg®?!——Arg™2__0H
`Ala*?*_AsnP!°-Human insulin-Arg™*—Arg®2__OH
`
`The insulin derivatives of the formula II are prepared
`mainly by a genetic manipulation by means ofsite-directed
`mutagenesis using standard methods.
`For this purpose, a gene structure coding for the desired
`insulin derivative of the formula II is constructed andits
`expression is brought about in a host cell—preferably in a
`bacterium such as E&.coli or a yeast, in particular Saccha-
`romyces cerevisiae—and—if the gene structure codes for a
`fusion protein—the insulinderivative of the formula II is
`liberated from the fusion protein; analogous methods are
`described, for example, in EP-A 0,211,299, EP-A 0,227,938,
`EP-A 0,229,998, EP-A 0,286,956 and German Patent Appli-
`cation P 38 21 159.9 dated Jun. 23, 1988 (HOE 88/F 158).
`After cell disruption, the fusion protein portion is elimi-
`nated either chemically using cyanogen halide—cf. EP-A
`0,180,920 or enzymatically using lysostaphin—cf. DE-A
`3,739,347.
`
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`A2l
`R2
`
`|s
`
`| Bx
`
`RH—R3t
`
`in which:
`R' denotes H or H-Phe,
`R? denotes a genetically encodable L-amino acid which
`contains no amide group,
`R*° represents the residue of a neutral genetically encod-
`able L-aminoacid,
`R*?represents a physiologically acceptable organic group
`which is basic in nature and has up to 50 carbon atoms,in
`whose structure 0 to 3 c-amino acids are involved and
`whose terminal carboxyl group which is present where
`appropriate can be free, in the form of an ester functionality,
`an amide functionality, a lactone or reduced to CH,OH,and
`X represents a genetically encodable L-amino acid, hav-
`ing an isoelectric point between 5 and 8.5, and the physi-
`ologically tolerated salts thereof.
`The new insulin derivatives and the physiologically tol-
`erated salts thereof are stable at the weakly acid pH values
`of appropriate pharmaceutical compositions even for
`extended periods and have—especially when His®!° has
`also been replaced by other amino acids—an altered
`(shorter) action profile compared with the known—
`unaltered—insulin derivatives with a basic modification of
`the formula I indicated in the introduction.
`R'in formula I is preferably H-Phe.
`Genetically encodable L-amino acids containing no
`amide group—for R? —are Gly, Ala, Ser, Thr, Val, Leu,Ile,
`Asp, Glu, Cys, Met, Arg, Lys. His, Tyr, Phe, Trp, Pro;
`Gly, Ala, Ser, Thr, Asp and Glu are preferred, especially
`Asp.
`Neutral genetically encodable L-amino acids—for R*°—
`are Gly, Ala, Ser, Thr, Val, Leu, He, Ash, Gln, Cys, Met, Tyr,
`Phe and Pro; Ala, Thr and Set are preferred.
`R*? is a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms and in
`whose structure 0-3 o-amino acids are involved. When no
`ot-aminoacids are involved in the structure of R®!, examples
`of suitable basic groups for this residue are the following:
`amino-(C,-C,)-alkoxy, (C,~C,)-alkylamino-(C,-C,)-
`alkoxy, di-(C,-C,)}-alkylamino-(C,-C,)-alkoxy. tri(C,-C,)
`ammonio-(C,-C,)-alkoxy, amino-(C,—-C,)-alkylamino,
`(C,-C,)-alkyl-amino]- (C,-C,)-alkylamino, di-(C,-C,)-
`alkylamino-(C,-C,)-alkylamino or [tri-(C,-C,)-
`alkylamino]-(C,-C,)-alkylamino, especially —O—[{CH,]
`—NR,and —O—[CH,]—N*°R,. —NH—{CH,],—NR,or
`—NH—/(CH,],—*Rs, in whichp is 2 to 6, and R is identical
`or different and represents hydrogen or (C,-C,)-alkyl.
`When up to 3 c-amino acids are involved in the structure
`of R*!
`.
`these are primarily neutral or basic naturally
`occurring L-amino acids and/or the D-amino acids corre-
`sponding thereto. Neutral naturally occurring amino acids
`are, in particular, Gly, Ala, Ser, Thr, Val, Leu, Tle, Ash, Gin,
`Cys, Met, Tyr, Phe, Pro and Hyp. Basic naturally occurring
`amino acids are, in particular, Arg, Lys, Hyl, Orn, Cit and
`His. If only neutral o-amino acids are involved, the terminal
`carboxyl group thereof cannot be free—in order for R?! to
`be basic in nature; on the contrary, the carboxyl group must
`in this case be amidated or esterified with a basic group,
`
`eTpn
`ee(f)eenOF}
`
`3
`
`Al
`
`s————S
`
`B2
`
`R!—Val—B-chain-X
`
`B10
`
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`5,656,722
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`5
`The insulin precursor is then subjected to oxidative sulfi-
`tolysis by the method described, for example, by R. C.
`Marshall and A. S.
`Inglis in “Practical Protein
`Chemistry—A Handbook” (edited by A. Darbre) 1986,
`pages 49-53, and subsequently renatured in the presence of
`a thiol with the formationof the correct disulfide bridges, for
`example by the method described by G. H. Dixon and A. C.
`Wardlow in Nature (1960), pages 721-724.
`The C peptide is removed by cleavage with trypsin—for
`example by the method of Kemmler et al., J. B. C. (1971),
`pages 6786-6791, and the insulin derivative of the formula
`II
`is purified by known techniques such as
`chromatography—cf., for example, EP-A-0,305,760—and
`crystallization.
`Theinsulin derivatives of the formula II with R?=Asp and
`X=His are expediently prepared by hydrolysis of the known
`insulin derivatives which have a basic modification and the
`formula lin aqueousacidic medium (because only the amide
`group ofthe asparagine in position A21 must be hydrolyzed
`in this case), preferably at pH values between about 2 and
`about 4, in particular of about 2.5, and at temperatures of
`about 0° to about 40° C., preferably at room temperature.
`Theinsulin derivatives of the formula II, according to the
`invention, and/or the physiologically tolerated salts thereof
`(such as, for example, the alkali metal or ammonium salts)
`are mainly used as active substances for a pharmaceutical
`composition for the treatment of diabetes mellitus.
`The pharmaceutical composition is preferably a solution
`or suspension for injection; it contains at least one insulin
`derivative of the formula TI and/or at least one of the
`physiologically tolerated salts thereof in dissolved, amor-
`phous and/or crystalline—preferably in dissolved—form.
`The composition preferably has a pH between about 2.5
`and 8.5, in particular between about 4.0 and 8.5, and
`contains a suitable tonicity agent, a suitable preservative
`and, where appropriate. a suitable buffer. as well preferably
`a certain zinc ion concentration, all, of course, in sterile
`aqueous solution. All the ingredients of the composition
`apart from the active substance form the composition
`vehicle.
`Examplesof suitable tonicity agents are glycerol, glucose,
`mannitol, NACI, and calcium or magnesium compounds
`such as CaCl,. MgCl, ete.
`The choice of the tonicity agent and/or preservative
`influences the solubility of the insulin derivative or the
`physiologically tolerated salt thereof at the weakly acid pH
`values.
`Examples of suitable preservatives are phenol, m-cresol,
`benzyl alcohol and/or p-hydroxybenzoic esters.
`Examples of buffer substances which can be used, in
`particular for adjusting a pH between about 4.0 and 8.5, are
`sodium acetate, sodium citrate, sodium phosphate etc.
`Otherwise, also suitable for adjusting the pH are physiologi-
`cally acceptable dilute acids (typically HCl) or alkalis
`(typically NaOH).
`When the composition contains zinc a content of 1 pg to
`2 mg, in particular from 5 pg to 200 pg, of zinc/ml is
`preferred.
`In order to vary the action profile of the composition
`according to the invention it is also possible to admix
`unmodified insulin, preferably bovine. porcine or human
`insulin, in particular human insulin.
`Preferred concentrations of active substance are those
`corresponding to about 1-1500, also preferably about
`5~1000, and in particular about 40-400, international units/
`ml.
`The invention is now explained in detail by the examples
`which follow.
`
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`6
`A) Preparation By Genetic Manipulation
`EXAMPLE 1
`
`Construction of a plasmid for the preparation of Gly
`{A21)-human insulin Arg (B31-OH)
`The plasmid pSW3 has been described in German Patent
`Application P 38 21 159.9 (HOE 88/F 158). The plasmid
`DNAis reacted with the restriction enzymes Pvull and Sail
`and subsequently treated with bovine alkaline phosphatase.
`The two resulting fragments are separated by gel
`electrophoresis, and the large fragment is isolated. This
`fragment is linked in a T4 DNAligase reaction with the
`following synthetic DNA sequence:
`
`5'- CTG GAA AAC TAC TGT GGT TGA TAG
`GAC CTT TTG ATG ACACCAACTATC AGCT-5'
`
`Competent E. coli W3110 cells are transformed with the
`ligation mixture. The transformation mixture is plated out on
`NAplates which contain 20 ug of Ap (=Ampicillin)/ml and
`incubated at 37° C. overnight. An overnight culture is
`obtained from single colonies, and plasmid DNAis obtained
`from this. This DNAis characterized by meansofrestriction
`analysis and DNA sequence analysis. Correct plasmids
`which encode the modified A chain are called pIK100.
`Expression is carried out in analogy to Example 3 of the
`abovementioned German Patent Application P 38 21 159.9.
`The modified mono-Arg-insulin is likewise prepared in
`analogy to the preparation of the unmodified mono-Arg-
`insulin described in this German Patent Application.
`EXAMPLE 2
`
`Construction of a plasmid for the preparation of Ser
`{A21)- human insulin (Arg B31-OH)
`The construction corresponds to the route described in the
`above example. The synthetic DNA sequence is, however,
`modified as follows:
`
`5'-CTG GAA AAC TAC TGT TCA TGA TAG
`GAC CTT TTG ATG ACA AGT ACT ATC AGCT-5'
`
`The plasmid pf{K110 which has an additional BspHI
`recognition sequence is obtained.
`EXAMPLE 3
`
`Construction of a plasmid for the preparation of Gly
`(A21)- Asn(B10)-human insulin Arg(B31-OH)
`DNA from the plasmid pIK100 is cleaved with the
`restriction enzymes Hpal and Dralll andtreated with bovine
`alkaline phosphatase. The tworesulting fragments are sepa-
`rated by gel electrophoresis, and the larger of the two
`fragments is isolated. The fragment is ligated with the
`synthetic DNA sequence
`
`55
`
`§'- AAC CAA CAC TTG TGT GGT TCT AAC TTG
`TIGGIT GTG AAC ACACCA AGA TTG
`
`-3
`
`and competentE. coli W3110cells are transformed with the
`ligation mixture. Further characterization of the resulting
`plasmid pIK101 is carried out as described in Example 1.
`EXAMPLE 4
`
`65
`
`Construction of a plasmid for the preparation of Ser
`(A21)- Asn(B10)-human insulin
`The construction corresponds to the cloning described in
`Example 3, but starting from DNA from the plasmid
`pIK110. The newly constructed plasmid is called pIK111.
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`

`

`7
`8
`EXAMPLE 5
`and 5 ml of a 1% strength zinc chloride solution per liter
`
`
`with a protein concentration of 5g/lat pH 6.0. The yield is
`390 mgof Asp*?4_human insalineAreeAngee *
`Construction of an expression plasmid for monkey pro-
`insulin
`
`5,656,722
`
`C) Preparation of an Injection Solution
`The insulin derivative from B is dissolved at a concen-
`tration of 1.4 mg/ml in a sterile vehicle solution of the
`following composition (per ml):
`18 mg of glycerol, 10 mg of benzyl alcohol, 80 pg of
`Zn", pH 4.0.
`D) Action Profile of an Asp4?'-Human Insulin-Arg”?!-
`Are”“OH Compositionin dogsby comparison with human
`insulin-Arg”*'-Arg”*?-OH and basal H insulin Hoechst“=
`an NPH (neutral protamine Hagedorn) composition contain-
`ing about 10 pg of Zn?*.
`
`
`Blood glucose as a % of the
`initial level in hours
`
` Product th 2h 3h Sh 7h
`
`
`According
`Asp“!human
`99
`62
`51
`75
`98
`to the
`[
`insulin
`
`52
`
`64
`
`85
`
`98
`
`Monkeyproinsulin differs from human proinsulin merely
`by replacement of a single amino acid in the C peptide
`(B37-Pro in place of Leu in this position of human
`proinsulin).
`The plasmid pSW3 is opened with HpaI and SalI and the
`remaining plasmid DNAis isolated. The DralII-Sall monkey
`proinsulin fragment
`is isolated from the plasmid pK50
`described in EP-A0,229,998. The two fragments are linked
`to the synthetic DNA fragment
`
`5 - AAC CAG CAC CTG TGC GGT TCT CAC CTA
`TIGGTC GTG GAC ACGCCA AGAGTG
`
`-3
`
`in aT4 DNAligasereaction. The plasmid pSW2is obtained,
`and its DNA is used hereinafter as starting material for the
`constructions of the expression plasmids encoding the
`di-Arg-human insulin derivatives.
`
`EXAMPLE 6
`
`Construction of a plasmid for the preparation of Gly(A21)
`-human insulin Arg(B31)-Arg(B32)-OH
`DNAofthe plasmid pSW2 is cleaved with Pvull and Sall
`in accordance with Example 1 and ligated with the synthetic
`DNAfrom Exampie 1; the result is the plasmid pSW21.
`
`EXAMPLE 7
`
`Constructionof a plasmid for the preparation of Ser(A21)
`-human insulin-Arg(B31)-Arg(B32)-OH
`The plasmid pSW22 is constructed starting from pSW2
`DNAin analogy to Example 2.
`
`EXAMPLE 8
`
`Construction of a plasmid for the preparation of Gly(A21)
`-Asn(B10)-human insulin-Arg(B31)-Arg(B32)-OH
`The plasmid pSW23 is constructedstarting from pSW21
`DNAin analogy to Example 3.
`The following sequence is used as synthetic DNA
`sequencefor this:
`
`3'- AAC CAACAC TTG TGT GGT TCT AAC CTA
`TTGGTT GTG AAC ACA CAA AGATTG-5'
`
`EXAMPLE 9
`
`Construction of a plasmid for the preparation of Set(A21)
`-Asn(B10)-human insulin-B31(Arg)-B32(Arg)-OH
`The plasmid pSW24 is constructed starting from pSW22
`DNAin analogy to Example 4 using the synthetic DNA
`sequence described in Example 8.
`B) Preparation of Asp*?'-Human Insulin-Arg®*-Arg??_
`OH From Human Insulin-Arg**' -Arg®?? -OH by Hydroly-
`sis
`
`1 g of human insulin-Arg**-Arg*?-OH is suspended in
`100 ml of H,,O.The pH is adjusted to 2.5 by addition of HCI,
`and the solution is left at 37° C. After one week about one
`half of the material has been converted into Asp*?!-human
`insulin-Arg”*'Arg®°?-OH. The product is separated from
`the starting material in a manner knownperse on an anion
`exchanger, is precipitated from the eluate andis crystallized
`in a buffer which contains 10.5 g ofcitric acid, 1 g of phenol
`
`10
`
`15
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`invention
`
`717
`
`Arg??!__Arg®°?__OH
`Human insulin
`Arg®?!_Arg™2_OH
`100
`49 9 83
`7h
`Basal H insulin
`Comparison
`Hoechst®
`
`
`H~Gly—Li“7R2
`ntee
`
`This example showsthat Asp*?!-human insulin-Arg??'-
`Arg”°?.OH has the same advantageous basal profile as
`human insulin-Arg®*!-Arg?°?-OH. In addition, Asp4?!-
`human-insulin-Arg”*'-Arg***-OH has the advantageous
`property that the compoundis stable for a long time under
`the chosen conditions.
`I claim:
`1. An insulin derivative having an isoelectric point
`between 5 and8.5, or a physiologically tolerated salt thereof,
`of the Formula II
`in which:
`
`Al
`
`5s————S
`
`A21
`
`8
`
`Ss
`
`RM—R3I
`
`R!—Val——B-chain- X
`
`B29
`|
`B10
`B2
`R!at position B1 denotes H or H-Phe;
`R? at position A21 denotes a genetically encodable
`L-aminoacid selected from the group consisting of Gly,
`Ala, Val,
`Leu,He, Pro, Phe, Trp, Met, Ser. Thr, Cys, Tyr, Asp, and
`Glu;
`R®™represents the residue of a neutral genetically encod-
`able L-amino acid selected from the groupconsisting of
`Ala, Thr, and Ser;
`R*? represents 1, 2, or 3 neutral or basic o-amino acids,
`wherein at least one of the o-amino acids is selected
`from the group consisting of Arg, Lys, Hyl, Orn,Cit,
`and His;
`X represents His at position B10; and the sequences A1 to
`A20 and B1 to B29 in Formula II correspond to a
`mammalian insulin;
`excluding those insulin derivatives in which simulta-
`neously:
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`

`

`5,656,722
`
`9
`R!at position B1 denotes Phe; and
`R*? is one alpha amino acid having a terminal carboxyl
`group.
`2. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1, wherein R'in formula I
`Tepresents H-Phe.
`3. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1, wherein R? in formula I
`represents Gly, Ala, Ser, Thr, Asp, or Glu.
`4. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1, wherein R®! in formula
`II represents Arg-Arg-OH.
`5. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1, wherein the sequences
`(Al to A20) and (B1 to B29) in formula II are the sequences
`of human, porcine, or bovineinsulin.
`6. A pharmaceutical composition that contains an effec-
`tive amountofat least one insulin derivative of the formula
`II, or at least one of the physiologically tolerated salts
`thereof. as claimed in claim 1, in dissolved, amorphous or
`crystalline form for the treatment of diabetes.
`7. A pharmaceutical composition as claimed in claim 6,
`which additionally contains 1 yg to 2 mg of zinc/ml.
`8. A pharmaceutical composition as claimed in claim 6,
`which additionally contains unmodified insulin.
`
`10
`
`15
`
`20
`
`10
`9. A methodfor treating a patient suffering from diabetes
`mellitus, which comprises administering to said patient a
`pharmaceutical composition as claimed in claim 6.
`10. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 3, wherein R? in formula
`represents Asp.
`11. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 5, wherein the sequences
`(A1 to A20) and (B1 to B29) in formula II are the sequences
`of human insulin.
`12. A pharmaceutical composition that contains an effec-
`tive amountofat least one insulin derivative of the formula
`Tl, or at least one of the physiologically tolerated salts
`thereof, as claimed in claim 8, in dissolved form for the
`treatment of diabetes.
`13. A pharmaceutical composition as claimed in claim 7,
`which additionally contains 5 pg to 200 pg of zinc/ml.
`14. A pharmaceutical composition as claimed in claim 8,
`wherein said unmodified insulin is unmodified human insu-
`lin.
`15. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 5, wherein R' represents
`H-Phe, R? represents Gly, R°° represents Thr, and R*!
`represents Arg-Arg-OH.
`ee #
`
`#
`
`®
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`