United States Patent
`
`[191
`
`[11] Patent Number:
`
`5,656,722
`
`
`
`[45] Date of Patent: Aug. 12, 1997
`Dfirschug
`
`USOOS656722A
`
`Markussen et al.. Soluble. prolonged—acting insulin deriva-
`tives. 1]]. Degree of protraaion. 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.
`vol. 237. No. 11. pp. 3406—3411 (1962).
`Burgermeister, W.. et al., The Isolation of Insan from the
`Pancreas. Reprint from the Handbook of Experimental Phar-
`macology, pp. 715-727 (1975) Insulin Humanurn. European
`Pharmacopeia 838 (1993).
`
`Primary Examiner—David Lukton
`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
`11
`
`A1 5—5 A21
`
`(If)
`
`[54] A"-, a” - 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 US. Application Data
`
`[63] Continuation of Ser. No. 46,481, Apr. 9, 1993, abandoned,
`which is acontinuation 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
`
`38 37 825.6
`
`Int. Cl.6
`[51]
`A61K 38/28
` . 530/303; 530/304
`[52] U.S.CI.
`530/303. 304',
`[58] Field of Search
`514/3. 12
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,701,440 10/1987 Gran ............................................ 514/3
`
`FOREIGN PATENT DOCUMENTS
`
`62066/86
`75916/87
`13976l88
`0046979
`0133285
`0214826
`0254516
`
`5/1986 Australia .
`111988
`9/1988
`8/1981
`European .Pat. Ofl".
`2/1985
`European Fat. 011‘.
`3/1987 European Pat. OE. .
`6/1987 European Fat. 03. .
`OTHER PUBLICATIONS
`
`.
`
`............... 495/351
`
`Schome et al. Diabetes vol. 27 (1978) 8—15.
`Geigee, Chem—Zeitung vol. 100 No. 3 pp. 54—56 (Jan.
`1976).
`Thompson et a] 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 Irnmuuogenicity of Different Insulins in
`Several Animal Species”. Diabetes. vol. 27. No. 1 (1977).
`“Galenics Of Insulin. The Physico—chemical and Pharma-
`ceutical Aspects of Insulin and Insan Preparations”. J.
`BRANGE: Springer—Verlag Berlin Heidelberg 1987. pp.
`35-36.
`Markussen et al.. Soluble. prolonged—acting insulin deriva-
`tives. 11. Degree of protraction and crystallizability of insu—
`lins substituted in positions A17. B8. B13. B27 and B30.
`Protein Engineering l(3):215—223 (1987).
`
`H—Gly—l—AIK-chain—l—‘—R2
`SI
`i
`
`s
`132
`I
`1310
`R1 —Va.l—B-chain- x
`
`s
`I
`
`329
`
`R30—R3I
`
`in which:
`
`R1 at position Bl denotes H or H—Phe;
`R2 at position A21 denotes a genetically encodable L—amino
`acid selected from the group consisting of Gly. Ala. Val.
`Len. 11e. Pro. Phe. Trp. Met, Ser, Thr, Cys. 'l‘yr. Asp. and
`Glu;
`R3° represents the residue of a neutral genetically encodable
`L—amino acid selected from the group consisting of Ala.
`Thr. and Ser;
`R31 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 1310; and
`the sequences A1 to A20 and B1 to B29 in Formula II
`correspond to a mammalian insulin;
`excluding those insulin derivatives in which simultaneously:
`R1 at position Bl denotes Phe; and
`R3 is one alpha amino acid having a terminal carboxyl
`group.
`
`15 Claims, No Drawings
`
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`
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`
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`

`

`1
`
`2
`
`5,656,722
`
`A21-, B” - MODIFIED INSULIN
`DERIVATIVES HAVING AN ALTERED
`ACTION PROFILE
`
`This application is a continuation. of application Ser. No.
`08/046481 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. 07/431344. 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 mellinrs. and some of them are also produced on an
`industrial scale. Despite the considerable number of insulin
`compositions and modifications with ditferent action pro-
`files which are already in existence, there is still a need.
`because of the variety of organisms with their inter— and
`intraindividnal 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 JET-B 132.770. These are
`specifically derivatives with a basic modification in position
`B31 of the insulin B chain. of the following formula I:
`
`Al 5—5
`
`A21
`
`(I)
`
`H—Gly—L-T-chain—-L——l—Asn—0H
`T
`T
`
`S
`
`s
`
`I
`BZ
`am
`kl-Val—B -chain-His
`
`I
`
`BE
`
`Elm—R“
`
`in which R1 denotes H or H-Phe, R3° represents the residue
`of a neutral. genetically encodable L-arnino acid. and R31
`represents a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms, in whose
`structure 0 to 3 ot—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 CHZOH.
`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
`range.
`
`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.
`
`The abovementioned depot principle resulting from basic
`modification of the insan 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 0.194.364 and EP-A 0.254.
`5 16.
`
`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 or ester 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 increasing the stability
`of the relevant pharmaceutical compositions at the weakly
`acid pH values. the amino acid Ash in 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) diflers from Asp (=aspartic acid) by the block-
`ing of one of the two carboxyl groups by the amide group:
`COOH
`l
`rim—(I: —HCH2
`CON-Hz
`asparagine
`COOH
`|
`
`I-[zN—(IJ—H
`i‘”COOH
`asparticacid
`
`Rapid-acting insulin derivan‘ves 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 B 10 position by
`other appropriate amino acids; cf. EP-A 0.214.826.
`All the insan derivatives specified in the 3 lastrnentioned
`publications are mainly modified within the A and B chains;
`they are prepared by genetic engineering routes.
`
`SUMMARY OF 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 found that this object
`is achieved in an advantageous manner by replacing Asn“21
`by other genetically encodable amino acids which contain
`no amide group and. where appropriate. by replacing Hisfilo
`by other genetically encodable amino acids.
`
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`Hence the invention relates to insulin derivatives of the
`formula I[
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Mylan EX. 1091
`
`Mylan V. Sanofi - IPR2018-01675
`
`Mylan Ex.1091
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`
`

`

`3
`
`5
`
`Al
`
`S
`
`H—Gly—|—T-cham
`T
`
`s
`
`32
`
`I
`
`Rl-Val—B-chain-X
`
`mo
`
`in which:
`R1 denotes H or H-Phe.
`R2 denotes a genetically encodable L-amino acid which
`contains no amide group.
`R30 represents the residue of a neutral genetically encod-
`able L-amino acid.
`R31 represents a physiologically acceptable organic group
`which is basic in nature and has up to 50 carbon atoms. in
`whose structure 0 to 3 ot-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 CHZOH. and
`X represents a genetically encodable L—arnino 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 HisBm 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.
`R1 in formula II is preferably H-Phe.
`Genetically encodable L-amino acids containing no
`amide group—for R2 —are Gly. Ala. Set. Thr. Val. Len. Ile.
`Asp. Glu. (his. 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 R30—
`are Gly. Ala. Ser. Thr. Val. Leu. lle. Ash. Gln. Cys. Met. Tyr.
`Phe and Pro; Ala. Thr and Set are preferred.
`R31 is a physiologically acceptable organic group which
`is basic in nature and has up to 50 carbon atoms and in
`whose structure 0—3 (Jr—amino acids are involved. When no
`
`u—amino acids are involved in the structure of R31 . examples
`of suitable basic groups for this residue are the following:
`arnino—(Cz-ngalkoxy.
`(C1-C4)-alkylamino-(C2—C5)-
`alkoxy. di-(Cl—C4)-alkylamino-(C2—C6)-alkoxy. tri-(Cl-CQ
`ammonio—(Cz-ngalkoxy. amino—(Cz—C6)-alkylamino.
`[(Cl—C4)-alkyl-amino]— (C2—C5)-alkylamino. di-(Cl—C4)-
`alkylamino-(C2—C6)-alkylamino or
`[tri-(Cl—C4)-
`alkylamino]~(C2—C6)-alkylamino. especially —O—[CH2]
`-—NR2 and ~—0—[CH2]—N3°R3. —NI-I—{CH2]p—NR2 or
`—NH——[CH2]p-—-+R3. in which p is 2 to 6. and R is identical
`or difl'erent and represents hydrogen or (C1—C4)-alkyl.
`When up to 3 (at-amino acids are involved in the structure
`of R31 .
`these are primarily neutral or basic naturally
`occurring L-amino acids and/or the D-arnino acids corre-
`sponding thereto. Neutral naturally occurring amino acids
`are. in particular. Gly. Ala. Ser. ThI. Val. Len. Ile. Ash. Gln.
`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 ot—amino acids are involved. the terminal
`carboxyl group thereof cannot be free—in order for R31 to
`be basic in nature; on the contrary. the carboxyl group must
`in this case be amidated or esterified with a basic group.
`
`5,656,722
`
`4
`
`(11)
`
`A21
`
`R2
`
`i
`
`s
`
`I
`
`329
`
`Rio—R3!
`
`suitable basic groups for this being. for example. the above,
`mentioned basic groups—in the case where no a—amino
`acids are involved in the structure of R31. Of course. these
`basic ester or amide groups can also block the carboxyl
`group of basic a-arnino 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. (C1—C5)-alkoxy. (C3-C6)-
`cycloalkyloxy. NH2.
`(C1—C5)-alkylamino or di—(C1—C6)-
`alkylamino.
`Of course, the terminal carboxyl group can be in the form
`of a lactone only if the terminal amino acid is a
`hydroxyamino acid.
`Moreover.
`the terminal earboxyl group can also be
`reduced to CH20H.
`R31 is preferably composed of 1. 2 or 3 of the abovemen-
`tioned basic naturally occurring amino acids; R31 is particu-
`larly preferably Arg-OH or Arg—Arg-OH.
`Suitable genetically encodable L-amino acids—for
`X—are the same amino acids as for R2. 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 Gln—are in fact preferred in this case. If
`Asn or Gln is located in position BIO. the amide group is at
`least stable in weakly acid medium (in contrast toAsn or Gln
`in position A21). The sequences (Al—A20) and (Bl—B9.
`B 1 1-329) are preferably the sequences of human. porcine or
`bovine insulin, especially the sequences of human insulin.
`Examples of insulin derivatives of the formula II are:
`
`
`“pm-Human insulin-Arg’m—OH
`Glu’m-Human insulin-Arg“‘—OH
`GlyMLHuman insulin-Argm—OH
`Wham insulin-Arg”‘—OI{
`'I‘hr“1~Hmnan insulin-Am"31—Oll'[
`Maul-Human insulin-Argml—OH
`Aspm-Hmn insulin-A133"~Argm—0H
`Glu‘2‘-Human main—Argm—Argm—OH
`Gly“‘-Human inaulin-Arg“‘——Arg"2—0H
`Mil-Hm mama—W411
`1111“2 -Human insulin-Ar;m —Arg'm—OH
`Mam-Human inmlin-Arg"'—Argm—OH
`Asp”‘—Asn"°—Human insulin-ArgB’I—OH
`Glam—Aan-Hmmn insulilt—Atgml—OH
`Glym—Amm-mman insulin—Argm‘—OH
`Ser‘21—Asn'w-Hmnan insulin-Argnfl—OH
`'l‘brAZ‘—Asn"°-Human insulin-Arg’"—0H
`Ala‘z‘—Asn:':-Humm insulin-ArgB“—-OH
`Asp
`1---Asn 1 Jim inmflin-Argna'—Arg”2——OH
`Glu“‘——Am"°-Human insadin—Arg”'—Arg‘”—OH
`Gly”‘—Am3‘°-Human instflin-Argn"——Arg’”—OH
`Sum—Amm-Hman insulin—Argw—Argm—OH
`M‘—Asn5'°-Human mum—Argw—Argm—OH
`Ala“1Hum“°-Human i11sulin-Arg331—Arg”2—OH
`
`The insulin derivatives of the formula II are prepared
`mainly by a genetic manipulation by means of site-directed
`mutagenesis using standard methods.
`For this purpose, a gene structure coding for the desired
`insulin derivative of the formula II is constructed and its
`expression is brought about in a host cell—prefaably in a
`bacterium such as E. Coli or a yeast. in particular Saccha-
`ramyces cerevisiae—and—if the gene structure codes for a
`fusion protein—the insulinderivative of the formula ]I 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
`3.739347.
`
`10
`
`15
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
`
`Mylan EX. 1091
`
`Mylan V. Sanofi - IPR2018-01675
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`

`

`5,656,722
`
`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 Handboo " (edited by A. Darbre) 1986,
`pages 49—53. and subsequently renatured in the presence of
`a thiol with the formation of 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.
`The insulin derivatives of the formula II with R2=Asp and
`X=His are expediently prepared by hydrolysis of the known
`insulin derivatives which have a basic modification and the
`formula I in aqueous acidic medium (because only the amide
`group of the 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.
`The insulin 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 II 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.
`Examples of suitable tonicity agents are glycerol. glucose.
`mannitol. NACl. and calcium or magnesium compounds
`such as CaCl2. MgCl2 etc.
`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 rig 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 adrnix
`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.
`
`6
`
`A) Preparation By Genetic Manipulation
`EXAMPLE 1
`
`Construction of a plasmid for the preparation of Gly
`(A21)-human insulin Arg (BS l-OH)
`The plasmid pSW3 has been described in German Patent
`Application P 38 21 159.9 (HOE 88/F 158). The plasmid
`DNA is reacted with the restriction enzymes Pvull and SalI
`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 DNA ligase reaction with the
`following synthetic DNA sequence:
`
`5'- CTG GAA AAC TAC TGT GGT TGA TAG
`GAC CTT TTG ATG ACA CCA ACT ATC AGCT - 5'
`
`Competent E. coli W3110 cells are transformed with the
`ligation mixture. The transformation mixture is plated out on
`NA plates which contain 20 ug of Ap (=Ampicillin)/ml and
`incubated at 37° C. overnight. An overnight culture is
`obtained from single colonies. and plasmid DNA is obtained
`from this. This DNA is characterized by means of restriction
`analysis and DNA sequence analysis. Correct plasmids
`which encode the modified A chain are called pIKlOO.
`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 ACAAGT ACT ATC AGCT - 5'
`
`5
`
`10
`
`15
`
`25
`
`30
`
`35
`
`The plasmid pIK110 which has an additional BspI-lI
`recognition sequence is obtained.
`EXAMPLE3
`
`45
`
`Construction of a plasmid for the preparation of Gly
`(A21)- Asu(BlO)-human insan Arg(B3 l-OH)
`DNA from the plasmid pIKIOO is cleaved with the
`restriction enzymes HpaI and DraIlI and treated with bovine
`alkaline phosphatase. The two resulting 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
`
`5'-AAC CAA CAC TTG TGT GGT TCT AAC TTG
`TTG GTT GTG AAC ACA CCA AGA TTG
`
`- 5‘
`
`and competent E. coli W3110 cells are transformed with the
`ligation mixture. Further characterization of the resulting
`plasmid le101 is carried out as described in Example 1.
`EXAMPLE 4
`
`65
`
`Construction of a plasmid for the preparation of Ser
`(A21)— Asn(BlO)«human insulin
`The construction corresponds to the cloning described in
`Example 3. but starting from DNA from the plasmid
`pIKllO. The newly constructed plasmid is called pIKlll.
`
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`

`7
`EXAMPLES
`
`5,656,722
`
`8
`
`and 5 ml of a 1% strength zinc chloride solution per liter
`with a
`tein concentration of 5
`at H 6.0. The ield is
`390 mgprgf AspAZI—human insulin-giltlrg’a-Arg’”.
`y
`C) Preparation of an Injection Solution
`The insulin derivative from B is dissolved at a concen-
`tration of 1.4 mglml 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 Asp‘m-Human Insulin-Arg‘m-
`Arggn-OH Composition in dogs by comparison with human
`insulin-Argm-Argm-orl and basal H insulin Hoechst®=
`an NPH (neutral protamine Hagedom) composition contain-
`ing about 10 pg of Zn“.
`
`
`Blood glueoseasa‘izof the
`initial levelinhours
`
`Product
`lb 211 an Sh 7h
`
`
`According
`AspM—human
`totlre
`[
`insulin
`invention
`Arg'm—Argmz—OH
`Humanimulin
`ArgB3‘—Arg”2—Ol{
`0mm“ [ Basalliinsulin
`Hoechstm
`
`
`99
`
`77
`
`71
`
`62
`
`51
`
`52
`
`49
`
`64
`
`59
`
`75
`
`as
`
`98
`
`98
`
`83100
`
`.
`
`This example shows that Asp‘zl-human insulin-Argm 1-
`Arg’m—OH has the same advantageous basal profile as
`human insulin-Argwl-Argmz-OH. In addition, Asp‘m-
`human-insulin—Argml-Argmz-OH has the advantageous
`property that the compound is stable for a long time under
`the chosen conditions.
`I claim:
`
`1. An insulin derivative having an isoelectric point
`between 5 and 8.5. or a physiologically tolerated salt thereof.
`of the Formula II
`in which:
`
`Al 5—5 A21
`
`(II)
`
`H—Gly—l—A—chlill‘l—II—R2
`|S
`l
`s
`l
`32
`BIO I BE
`Rl—Val—B-chain- X
`
`S
`|
`s
`
`R”—R31
`
`R1 at position Bl denotes H or H-Phe;
`R2 at position A21 denotes a genetically encodable
`L—amino acid selected from the group consisting of Gly.
`Ala. Val.
`
`Construction of an expression plasmid for monkey pro-
`insulin
`
`Monkey proinsulin differs from human proinsulin merely
`by replacement of a single amino acid in the C peptide
`(B37-Pro in place of Len in this position of human
`proinsulin).
`
`The plasmid pSW3 is opened with Hpal and SalI and the
`remaining plasmid DNA is isolated. The DraIlI-SalI monkey
`[n'oinsulin fragment
`is isolated from the plasmid p160
`described in EP—AO.229.998. The two fragments are linked
`to the synthetic DNA fragment
`
`5' - AAC CAG CAC CTG TGC GGT TCT CAC CTA
`TTG GTC GTG GAC ACG CCA AGA GTG
`
`- 5‘
`
`in a T4 DNA ligase reaction. The plasmid pSW2 is obtained,
`and its DNA is used hereinafter as starting material for the
`constructions of the expression plasmids encoding the
`di-Arg-human insulin derivatives.
`
`EXAMPLE6
`
`Construction of a plasmid for the preparation of Gly(A21)
`-human insulin Arg(B3l)-Arg(B32)-OH
`DNA of the plasmid pSW2 is cleaved with Pqu and SalI
`in accordance with Example 1 and ligated with the synthetic
`DNA from Example 1; the result is the plasmid pSW21.
`
`EXAMPLE7
`
`Construction of a plasmid for the preparation of Ser(A21)
`-human insulin-Arg(B31)—Arg(BBZ)-OH
`The plasmid pSW22 is constructed starting from pSW2
`DNA in analogy to Example 2.
`
`EXAMPLE8
`
`Construction of a plasmid for the preparation of Gly(A21)
`-Asn(B 10)-human insulin-Arg(B3 1)-Arg(B32)—OH
`The plasmid pSW23 is constructed starting from pSW21
`DNA in analogy to Example 3.
`The following sequence is used as synthetic DNA
`sequence for this:
`
`5' - AAC CAA CAC TTG TGT GGT TCT AAC CTA
`TTG GTT GTG AAC ACA CAA AGA TTG -5 ‘
`
`EXAMPLE9
`
`Construction of a plasmid for the preparation of Set(A21)
`-Asn(BlO)-human insulin-B31(Arg)—B32(Arg)—OH
`The plasmid pSW24 is constructed starting from pSW22
`DNA in analogy to Example 4 using the synthetic DNA
`sequence described in Example 8.
`B) Preparation of Asp‘m-Human Insulin-Arg"3 1-Arg"”—
`OH From Human Insulin-Arg'm -Arg"32 —0H by Hydroly-
`$15
`
`10
`
`15
`
`25
`
`30
`
`35
`
`45
`
`50
`
`1 g of human insulin-Arg31-Arg32-OH is suspended in
`100 ml of H20. The pH is adjusted to 2.5 by addition of HCl.
`and the solution is left at 37° C. After one week about one
`half of the material has been converted into Asp‘zl-human
`insulin-ArgmlArgmz-OH. The product is separated from
`the starting material in a manner known per se on an anion
`exchanger. is precipitated from the eluate and is crystallized
`in a bufer which contains 10.5 g of citric acid. 1 g of phenol
`
`65
`
`55
`
`Len. lle. Pro. Phe. 'Irp. Met. Set. Thr. Cys. 'Iyr. Asp, and
`Glu;
`
`R3° represents the residue of a neutral genetically encod-
`able L-amino acid selected from the group consisting of
`Ala. Tilt. and Ser;
`R31 represents 1, 2. or 3 neutral or basic u—amino acids.
`wherein at least one of the (rt-amino acids is selected
`from the group consisting of Arg. Lys. Hyl. Om. Cit.
`and His;
`
`X represents His at position B10; and the sequences Al to
`A20 and B1 to B29 in Formula II correspond to a
`mammalian insulin;
`excluding those insulin derivatives in which simulta-
`neously:
`
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`

`5,656,722
`
`9
`
`R1 at position B1 denotes Phe; and
`R31 is one alpha amino acid having a tm'minal carboxyl
`group.
`2. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1. wherein R1 in formula 11
`represents H-Phe.
`3. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1, wherein R2 in formula 11
`represents Gly. Ala. Ser. Thr. Asp. or Glu.
`4. An insulin derivative or the physiologically tolerated
`salts thereof as claimed in claim 1. wherein R31 in formula
`11 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 bovine insulin.
`6. A pharmaceutical composition that contains an effec-
`tive amount of at 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 pg to 2 mg of zinc/ml.
`8. A pharmaceutical composition as claimed in claim 6.
`which additionally contains unmodified insulin.
`
`10
`9. A method for 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 R2 in formula II
`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. Apharmaceutical composition fliat contains an effec-
`tive amount of at least one insulin derivative of the formula
`II, 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 R1 represents
`H-Phe. R2 represents Gly, R30 represents Thr. and 1131
`represents Arg-Arg-OH.
`*****
`
`10
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`15
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`20
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`Mylan EX. 1091
`
`Mylan V. Sanofi - IPR2018-01675
`
`Mylan Ex.1091
`Mylan v. Sanofi - IPR2018-01675
`
`