`
`111111111111111111111111 RIR1111111111111111111111111
`
`United States Patent [19]
`Dorschug
`
`[11] Patent Number:
`[45] Date of Patent:
`
`6,100,376
`*Aug. 8, 2000
`
`[54] A21, B30, MODIFIED INSULIN DERIVATIVES
`HAVING AN ALTERED ACTION PROFILE
`
`[75]
`
`Inventor: Michael Dorschug, Bochum, Germany
`
`[73] Assignee: Hoechst Aktiengesellschaft, Frankfurt
`am Main, Germany
`
`[ * ] Notice:
`
`This patent is subject to a terminal dis-
`claimer.
`
`[21] Appl. No.: 08/842,794
`
`[22] Filed:
`
`Apr. 16, 1997
`
`Related U.S. Application Data
`
`[62] Division of application No. 08/304,593, Sep. 12, 1994, Pat.
`No. 5,656,722, which is a continuation of application No.
`08/046,481, Apr. 9, 1993, abandoned, which is a continua-
`tion of application No. 07/929,510, Aug. 19, 1992, aban-
`doned, which is a continuation of application No. 07/431,
`844, Nov. 6, 1989, abandoned.
`
`[30]
`
`Foreign Application Priority Data
`
`Nov. 8, 1988
`
`[DE] Germany
`
` 3837825
`
`[51] Int. C1.7
`[52] U.S. Cl.
`
`[58] Field of Search
`
` A61K 38/28
` 530/303; 530/304; 514/3;
`514/12
` 530/303, 304;
`514/3, 12
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`8/1986 Grau
`4,608,364
`4,701,440 10/1987 Grau
`
` 514/4
` 514/3
`
`FOREIGN PATENT DOCUMENTS
`
`62066/86
`0 046 979
`0 194 864
`0 214 826
`0 254 516
`
`3/1987 Australia .
`8/1981 European Pat. Off. .
`9/1986 European Pat. Off. .
`3/1987 European Pat. Off. .
`1/1988 European Pat. Off. .
`
`OTHER PUBLICATIONS
`
`Sunby, 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 Insulin from the
`Pancreas, Reprint from the Handbook of Experimental Phar-
`macology, pp. 715-727 (1975).
`Neubauer, "The Immunogenicity of Different Insulins in
`Several Animal Species," Diabetes, vol. 27, No. 1 (1977),
`pp. 8-15.
`Markussen et al., "Soluble, prolonged-acting insulin deriva-
`tives. II. Degreee of protraction and crystallizability of
`insulins substituted in positions A17, B8, B13, B27 and
`B30," Protein Engineering 1(3):215-223 (1987).
`
`J. Brange; Springer-Verlag, "Galenics Of Insulin, The
`Physico-chemical and Pharmaceuticl Aspects of Insulin and
`Insulin Preparations," Berlin Heidelberg, pp. 35-36.
`
`Markussen et al., "Soluble prolonged-acting insulin deriva-
`tives. III. Degree of protraction, 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 Insu-
`lin," Medical Intelligence, vol. 32, No. 6, pp. 363-370
`(1989).
`
`Insulin Humanum, European Pharmacopeia 838 (1993).
`
`Primary Examiner-Michael P. Woodward
`Assistant Examiner-David Lukton
`Attorney, Agent, or Firm-Finnegan, Henderson, Farabow,
`Garrett & Dunner, L.L.P.
`
`[57]
`
`ABSTRACT
`
`New insulin derivatives of the formula II with an iso-electric
`point between 5 and 8.5, with improved stability in weakly
`acid aqueous medium and with a special action profile, and
`the physiologically tolerated salts of these insulin
`derivatives, for the treatment of diabetes mellitus; formula II
`is:
`
`Al
`H-Gly
`
`
`
`
`
`A21
`R2
`
`S
`
`B10
`B-chain-X
`
`S
`
`B29
`
`R30 R31
`
`B2
`R1-Val
`
`in which
`
`R1 denotes H or H-Phe,
`R2 denotes a genetically encodable L-amino acid which
`contains no amide group,
`R3° 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 a -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 CH2OH, and
`X represents a genetically encodable L-amino acid.
`
`12 Claims, No Drawings
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`

`

`6,100,376
`
`0
`
`2
`groups are in each case either purely physiological metabo-
`lites or else easily metabolized physiologically acceptable
`substances.
`The abovementioned depot principle resulting from basic
`5 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
`15 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
`20 acid pH values, the amino acid Asn 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, Asn
`(=asparagine) differs from Asp (=aspartic acid) by the block-
`ing of one of the two carboxyl groups by the amide group:
`
`1
`A21, B30, MODIFIED INSULIN DERIVATIVES
`HAVING AN ALTERED ACTION PROFILE
`
`This is a division of application Ser. No. 08/304,593,
`filed Sep. 12, 1994, now U.S. Pat. No. 5,656,722, which is
`a continuation of application Ser. No. 08/046,481, filed Apr.
`9, 1993, abandoned, which is a continuation 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 of insulin
`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:
`
`Al
`H-G1y
`
`B2
`R1-Val
`
`S
`
`
`
`S
`
`Pik-chain
`
`S
`
`S
`B10
`I
`B-chain-His
`
`S
`
`S
`
`25
`
`30
`
`(I)
`
`A21
`Asn OH
`
`B29
`
`R30 R31
`
`35
`
`COON
`
`coNH2
`asparagine
`
`COON
`
`H2N— C— H
`
`COON
`aspartic acid
`
`40
`
`in which R1 denotes H or H-Phe,
`R3° represents the residue of a neutral, genetically encod-
`able L-amino 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 a -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 CH2OH.
`Characteristic of these insulin derivatives is an iso-electric
`point between 5.8 and 8.5 (measured by iso-electric
`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 physio-logical conditions is
`achieved by elimination of the additional basic groups,
`which is brought about, depending on the derivative, by 65
`trypsin or trypsin-like and/or carboxypeptidase B or carbox-
`ypeptidase B-like and/or esterase activity. The eliminated
`
`Rapid-acting insulin 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
`45 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 last-
`mentioned publications are mainly modified within the A
`5° and B chains; they are prepared by genetic engineering
`routes.
`In the attempt to increase the stability in acid medium of
`the insulin derivatives with a basic modification on the
`55 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 AsnA21
`60 by other genetically encodable amino acids which contain
`no amide group and, where appropriate, by replacing Hiel°
`by other genetically encodable amino acids.
`
`SUMMARY OF THE INVENTION
`
`Hence the invention relates to insulin derivatives of the
`formula II
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`

`

`6,100,376
`
`3
`
`S
`
`I
`
`A chain
`
`S
`
`S
`B10
`I
`B chain -X
`
`Al
`1-1-Gly
`
`S
`
`I
`
`B2
`R1 Val
`
`A21
`R2
`
`I
`
`S
`
`S
`
`B29
`
`R30 R31
`
`in which
`R1 denotes H or H-Phe,
`R2 denotes a genetically encodable L-amino acid which
`contains no amide group,
`R3° 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 a -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 CH2OH, and
`x represents a genetically encodable L-amino acid, having
`an isoelectric point between 5 and 8.5, and the physi-
`ologically tolerated salts thereof.
`
`DETAILED DESCRIPTION
`
`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 HisBl° 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, 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 R3°
`—are Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met,
`Tyr, Phe and Pro; Ala, Thr and Ser 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 —30 a -amino acids are involved. When no
`a -amino acids are involved in the structure of R31, examples
`of suitable basic groups for this residue are the following:
`amino-(C2—C6)-alkoxy, (C1 4)-alkylamino-(C2—C6)-
`alkoxy, di-(C1—C4)-alkylamino-(C2—C6)-alkoxy, tri-
`(C1—C4)-ammonio-(C2—C6)-alkoxy, amino-(C2—C6)-
`alkylamino, [(C1—C4)-alkyl-amino]-(C2—C 6 )
`alkylamino, di-(C1—C4)-alkylamino-(C2—C 6)-
`alkylamino or [tri-(C1—C4)-alkylamino ]-(C2—C6)-
`alkylamino, especially —0—[CH2]P, NR2, [—O—]
`CH2 p — N e R 3, —NH—[CH2]—NR2 or —NH—
`[CH2]P—Ne R3, in which p is 2 to 6, and R is identical
`or different and represents hydrogen or (C1—C4)-alkyl.
`When up to 3 a -amino acids are involved in the structure
`of R31, these are primarily neutral or basic naturally occur-
`ring L-amino acids and/or the D-amino acids corresponding
`thereto. Neutral naturally occurring amino acids are, in
`particular, Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys,
`
`15
`
`4
`Met, Tyr, Phe, Pro and Hyp. Basic naturally occurring amino
`acids are, in particular, Arg, Lys, Hyl, Orn, Cit and His. If
`only neutral a -amino acids are involved, the terminal car-
`boxyl group thereof cannot be free—in order for R31 to be
`5 basic in nature; on the contrary, the carboxyl group must in
`this case be amidated or esterified with a basic group,
`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
`10 basic ester or amide groups can also block the carboxyl
`group of basic a -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, (C1—C6)-alkoxy, (C3—C6)-
`cycloalkyloxy, NH2, (C1—C6)-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 carboxyl group can also be
`reduced to CH2OH.
`R31 is preferably composed of 1, 2 or 3 of the above-
`mentioned basic naturally occurring amino acids; R31 is
`particularly 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 Asn and Gln—are also possible in this case; the
`latter—Asn and Gln—are in fact preferred in this case. If
`30 Asn or Gln is located in position B10, the amide group is at
`least stable in weakly acid medium (in contrast to Asn or Gln
`in position A21).
`The sequences (A1—A20) and (B1—B9, B11—B29) are
`preferably the sequences of human, porcine or bovine
`35 insulin, especially the sequences of human insulin.
`Examples of insulin derivatives of the formula II are:
`
`20
`
`25
`
`40
`
`45
`
`50
`
`55
`
`AspA21-Human
`Glu ~ 1-
`G1y^21-
`SerA21-
`Thr
`
`Glu` 21-
`oiyA21-
`serA21-
`ThrA21-
`AlaA21-
`AspA21-AsnB1°-Human
`GluA21-
`GlyA21-
`SerA21-
`ThrA21-
`AlaA21-
`AspAn-AsnB1°-Human
`Gle n -
`GiyA21-
`SerA21-
`ThrA21-
`AlaA21-
`
`insulin-ArgB31-0H
`
`insulin-ArgB31-ArgB32-0H
`
`insulin-Arem-OH
`
`insulin-ArgB31-ArgB32-0H
`
`60
`
`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
`65 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
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`

`

`6,100,376
`
`5
`fusion protein—the insulinderivative of the formula II is
`liberated from the fusion protein; analogous methods are
`described, for example, in EP-A0,211,299, EP-A0,227,938,
`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 eliminated
`either chemically using cyanogen halide—cf. EP-A 0,180,
`920 or enzymatically using lysostaphin—cf. DE-A 3,739,
`347.
`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 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.Biol. Chem.
`(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 as prefer-
`ably 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, NaCI, and calcium or magnesium compounds
`such as CaC12, MgC12 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 HC1) or alkalis
`(typically NaOH).
`When the composition contains zinc a content of 1µg to
`2 mg, in particular from 5µg to 200 ,ug, of zinc/ml is
`preferred.
`
`5
`
`6
`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
`10 which follow.
`A) Preparation by Genetic Manipulation
`
`15
`
`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
`DNA is reacted with the restriction enzymes PvuII and Sall
`and subsequently treated with bovine alkaline phosphatase.
`The two resulting fragments are separated by gel
`20 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
`30 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 pIK100.
`Expression is carried out in analogy to Example 3 of the
`abovementioned German Patent Application P 38 21 159.9.
`35 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.
`
`25
`
`EXAMPLE 2
`40 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 pIK110 which has an additional BspHI
`recognition sequence is obtained.
`
`45
`
`EXAMPLE 3
`50 Construction of a Plasmid for the Preparation of Gly(A21)-
`Asn(B1O)-human Insulin Arg(B31-OH)
`DNA from the plasmid pIK100 is cleaved with the
`restriction enzymes HpaI and DraIII and treated with bovine
`alkaline phosphatase. The two resulting fragments are sepa-
`55 rated by gel electrophoresis, and the larger of the two
`fragments is isolated. The fragment is ligated with the
`synthetic DNA sequence
`5'—AAC CAA CAC TTG TGT GGT TCT AAC TTG
`TTG GTT GTG AAC ACA CCA A&A TTG-5'
`and competent E. coli W3110 cells are transformed with
`the ligation mixture. Further characterization of the
`resulting plasmid pIK101 is carried out as described in
`Example 1.
`
`6
`
`65
`
`EXAMPLE 4
`Construction of a Plasmid for the Preparation of Ser(A21)-
`Asn(B10)-human Insulin
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`

`

`6,100,376
`
`8
`18 mg of glycerol, 10 mg of benzyl alcohol, 80 kig of
`Zn2+, pH 4.0.
`D) Action Profile of an AspA21-human Insulin-ArgB31-
`ArgB32-0H Composition in dogs by comparison with human
`insulin-ArgB31-ArgB32-0H and basal H insulin Hoechst(R)=
`an NPH (neutral protamine Hagedorn) composition contain-
`ing about 10 kig of Zn2'.
`
`Blood glucose as a % of the
`initial level in hours (h)
`
`Product
`
`1 h
`
`2h 3h
`
`5h
`
`7h
`
`AspA21-human
`According
`to the
`insulin
`ArgB31-ArgB32-0H
`invention
`Comparison Human insulin
`ArgB31-ArgB32-0H
`Basal H insulin
`Hoechst(R)
`
`99
`
`62
`
`51
`
`75
`
`98
`
`77
`
`71
`
`52
`
`49
`
`64
`
`59
`
`85
`
`83
`
`98
`
`100
`
`This example shows that AspA21-human insulin-ArgB31-
`ArgB32-0H has the same advantageous basal profile as
`human insulin-ArgB31-ArgB32-0H. In addition, AspAzi_
`human insulin-ArgB31-Arg32-0H 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
`
`Al
`H-Gly
`
` P -chain
`
`A21
`R2
`
`B2
`R1-Val
`
`B10
` B-chain-X
`
`B29
`
`R30 R31
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`7
`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.
`
`EXAMPLE 5
`Construction of an Expression Plasmid for Monkey Proin-
`sulin
`Monkey proinsulin 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 Sall and the
`remaining plasmid DNA is isolated. The DraIII-SalI monkey
`proinsulin fragment is isolated from the plasmid pl(50
`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
`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 plas-
`mids 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
`DNA of the plasmid pSW2 is cleaved with PvuII and Sall
`in accordance with Example 1 and ligated with the synthetic
`DNA from Example 1; the result is the plasmid pSW21.
`
`EXAMPLE 7
`Construction of a Plasmid for the Preparation of Ser(A21)-
`human Insulin-Arg(B31)-Arg(B32)-OH
`The plasmid pSW22 is constructed starting from pSW2
`DNA in 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 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'
`
`EXAMPLE 9
`Construction of a Plasmid for the Preparation of Ser(A21)-
`Asn(B1O)-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 AspA21-human Insulin-ArgB31-ArgB32-0H
`from Human Insulin-ArgA31-ArgB32-0H by Hydrolysis
`1 g of human insulin-ArgB31-ArgB32-0H is suspended in
`100 ml of H2O. The pH is adjusted to 2.5 by addition of HC1,
`and the solution is left at 37° C. After one week about one
`half of the material has been converted into AspA21-human
`insulin-ArgB31-Arg32-0H. 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 buffer which contains 10.5 g of citric acid, 1 g of phenol
`and 5 ml of a 1% strength zinc chloride solution per liter
`with a protein concentration. of 5 g/1 at pH 6.0. The yield is
`390 mg of AspA21-human insulin-ArgB31-ArgB32.
`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):
`
`45
`
`so
`
`in which:
`R1 at position B1 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, Leu, Ile, Pro, Phe, Trp, Met, Ser, Thr, Tyr,
`Asp, and Glu,
`R3° represents the residue of a neutral genetically encod-
`able L-amino acid selected from the group consisting of
`Tyr, Gly, Phe, and Thr,
`R31 represents 1, 2, or 3 neutral or basic a -amino acids,
`whose terminal carboxyl group can be free or in the
`form of an amide functionality;
`X represents His at position B10; and
`55 the sequences Al to A20 and B2 to B29 in Formula II
`correspond to a mammalian insulin.
`2. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R1 in formula II
`represents H-Phe.
`3. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R2 in formula II
`represents Gly.
`4. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R3° in formula II
`65 represents Thr.
`5. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein the sequences
`
`60
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`

`

`6,100,376
`
`9
`(Al to A20) and (B2 to B9 and B11 to B29) in Formula II
`are the corresponding sequences of human, porcine, or
`bovine insulin.
`6. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R3° in formula II
`represents Gly.
`7. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R3° in formula II
`represents Phe.
`8. An insulin derivative or the physiologically tolerated
`salt thereof as claimed in claim 1, wherein R3° in formula II
`represents Tyr.
`9. A pharmaceutical composition that contains an effec-
`tive amount of at least one insulin derivative of the formula
`
`10
`II, or at least one of the physiologically tolerated salts
`thereof, as claimed in claim 1, in dissolved, amorphous or
`crystalline form.
`
`5
`
`10. A pharmaceutical composition as claimed in claim 9,
`which additionally contains 1µg to 2 mg, of zinc/ml.
`
`11. A pharmaceutical composition as claimed in claim 9,
`which additionally contains unmodified insulin.
`
`10
`
`12. A method for treating a patient suffering from diabetes
`mellitus, which comprises administering to said patient a
`pharmaceutical composition as claimed in claim 9.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`Mylan Ex.1079
`Mylan v. Sanofi - IPR2018-01676
`
`