US007615532B2
`
`(12) United States Patent
`Jonassen et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,615,532 B2
`Nov. 10, 2009
`
`(54) INSULIN DERIVATIVES
`
`75 (75) Inventors: Ib Jonassen, Valby (DK); Thomas
`Hoeg-Jensen, Klampenborg (DK);
`Svend Havelund, Bagsvaerd (DK); Ulla
`Ribel-Madsen, Virum (DK); Tina
`Moller Tagmose, Ballerup (DK); Peter
`Madsen, Bagsvaerd (DK)
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DE)
`
`*) Notice:
`
`Subject to any disclaimer, the term of this
`y
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 316 days.
`(21) Appl. No.: 11/343,005
`
`(22) Filed:
`
`Jan. 30, 2006
`
`(65)
`
`Prior Publication Data
`US 2006/O183668A1
`Aug. 17, 2006
`
`Related U.S. Application Data
`(63) Continuation of application No. PCT/DK2004/
`000511, filed on Jul. 22, 2004.
`(60) Provisional application No. 60/495,451, filed on Aug.
`14, 2003.
`Foreign Application Priority Data
`(30)
`Aug. 5, 2003
`(DK) ............................... 2003 O1129
`
`(51) Int. Cl.
`(2006.01)
`A6 IK 38/28
`(52) U.S. Cl. ........................................... 514/3: 530/303
`
`(58) Field of Classification Search ....................... None
`See application file for complete search history.
`References Cited
`
`(56)
`
`U.S. PATENT DOCUMENTS
`
`3,528,960 A
`5,898,067 A
`6,251,856 B1
`2002/0045731 A1
`
`9, 1970 HaaS
`4/1999 Balschmidt et al.
`6/2001 Markussen et al.
`4/2002 Schaffer et al.
`
`FOREIGN PATENT DOCUMENTS
`
`2, 1997
`* 2/1999
`7, 1976
`2, 1991
`9, 1994
`7/1997
`
`EP
`894095
`EP
`O894095
`GB
`1492997
`WO
`91f12817
`WO
`95/O7931
`WO
`98/O2460
`* cited by examiner
`Primary Examiner Christopher R. Tate
`Assistant Examiner Roy Teller
`(74) Attorney, Agent, or Firm—Shelby J. Walker
`
`(57)
`
`ABSTRACT
`
`The present invention relates to insulin derivatives which are
`naturally occurring insulins or analogues thereof which have
`a side chain attached either to the C-amino group of the
`N-terminal amino acid residue of the B chain or to the
`e-amino group of a Lys residue present in the B chain of the
`parent insulin, the side chain being of the general formula:
`-W-X Y-Z
`wherein W, X, Y and Z are as defined in the disclosure.
`
`12 Claims, No Drawings
`
`MPI EXHIBIT 1096 PAGE 1
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`MPI EXHIBIT 1096 PAGE 1
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`

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`US 7,615,532 B2
`
`1.
`NSULIN DERVATIVES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of International Appli
`cation No. PCT/DK2004/000511, filed Jul. 22, 2004, which
`claims priority from Danish Patent Application No. PA 2003
`01 129 filed Aug. 5, 2003 and to U.S. Patent Application No.
`60/495,451 filed Aug. 14, 2003.
`
`FIELD OF THE INVENTION
`
`The present invention relates to novel human insulin
`derivatives which are soluble at physiological pH values and
`have a prolonged profile of action. The invention also relates
`to methods of providing such derivatives, to pharmaceutical
`compositions containing them, to a method of treating diabe
`tes and hyperglycemia using the insulin derivatives of the
`invention and to the use of such insulin derivatives in the
`treatment of diabetes and hyperglycemia.
`
`BACKGROUND OF THE INVENTION
`
`10
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`15
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`2
`The action profile of these complexes is only moderately
`prolonged and the bioavailability is reduced relative to human
`insulin.
`Human insulin has three primary amino groups: the N-ter
`minal group of the A-chain and of the B-chain and the
`e-amino group of Lys'. Several insulin derivatives which
`are substituted in one or more of these groups are known in the
`prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly) relates to
`N-carboxyaroyl insulins in which one, two or three primary
`amino groups of the insulin molecule has a carboxyaroyl
`group.
`According to GB Patent No. 1.492.997 (Nat. Res. Dev.
`Corp.), it has been found that insulin with a carbamyl substi
`tution at N’ has an improved profile of hypoglycaemic
`effect.
`JP laid-open patent application No. 1-254699 (Kodama
`Co., Ltd.) discloses insulin wherein a fatty acid is bound to the
`amino group of Phe' or to the e-amino group of LyS’’’ or to
`both of these. The stated purpose of the derivatisation is to
`obtain a pharmacologically acceptable, stable insulin prepa
`ration.
`Insulins, which in the B30 position have an amino acid
`having at least five carbonatoms which cannot necessarily be
`coded for by a triplet of nucleotides, are described in JP
`laid-open patent application No. 57-067548 (Shionogi). The
`insulin analogues are claimed to be useful in the treatment of
`diabetes mellitus, particularly in patients who are insulin
`resistant due to generation of bovine or porcine insulin anti
`bodies.
`WO95/07931 (Novo Nordisk A/S) discloses human insu
`lin derivatives wherein the e-amino group of LyS’’ has a
`lipophilic substituent. These insulin derivatives have a pro
`longed profile of action and are soluble at physiological pH
`values.
`EP 894095 discloses insulin derivatives wherein the N-ter
`minal group of the B-chain and/or the e-amino group of Lys in
`position B28, B29 or B30 has a substituent of the formula
`—CO W COOH where W can be a long chain hydrocar
`bon group. These insulin derivatives have a prolonged profile
`of action and are soluble at physiological pH values.
`However, there is still a need for insulins having a more
`prolonged profile of action than the insulin derivatives known
`up till now and which at the same time are soluble at physi
`ological pH values and have a potency which is comparable to
`that of human insulin.
`
`25
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`30
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`40
`
`45
`
`Currently, the treatment of diabetes, both type 1 diabetes
`and type 2 diabetes, relies to an increasing extent on the
`so-called intensive insulin treatment. According to this regi
`men, the patients are treated with multiple daily insulin injec
`tions comprising one or two daily injections of a long acting
`insulin to cover the basal insulin requirement Supplemented
`by bolus injections of a rapid acting insulin to cover the
`insulin requirement related to meals.
`Long acting insulin compositions are well known in the art.
`Thus, one main type of long acting insulin compositions
`comprises injectable aqueous Suspensions of insulin crystals
`35
`oramorphousinsulin. In these compositions, the insulin com
`pounds utilized typically are protamine insulin, Zinc insulin
`or protamine Zinc insulin.
`Certain drawbacks are associated with the use of insulin
`Suspensions. Thus, in order to secure an accurate dosing, the
`insulin particles must be suspended homogeneously by gentle
`shaking before a defined volume of the suspension is with
`drawn from a vial or expelled from a cartridge. Also, for the
`storage of insulin Suspensions, the temperature must be kept
`within more narrow limits than for insulin solutions in order
`to avoid lump formation or coagulation.
`While it was earlier believed that protamines were non
`immunogenic, it has now turned out that protamines can be
`immunogenic in man and that their use for medical purposes
`may lead to formation of antibodies. Also, evidence has been
`found that the protamine-insulin complex is itself immuno
`genic. Therefore, with some patients the use of long acting
`insulin compositions containing protamines must be avoided.
`Another type of long acting insulin compositions are solu
`tions having a pH value below physiological pH from which
`the insulin will precipitate because of the rise in the pH value
`when the solution is injected. A drawback with these solu
`tions is that the particle size distribution of the precipitate
`formed in the tissue on injection, and thus the release profile
`of the medication, depends on the blood flow at the injection
`site and other parameters in a somewhat unpredictable man
`ner. A further drawback is that the solid particles of the insulin
`may act as a local irritant causing inflammation of the tissue
`at the site of injection.
`WO91/12817 (Novo NordiskANS) discloses soluble insu
`lin compositions comprising insulin complexes of cobalt(III).
`
`SUMMARY OF THE INVENTION
`
`The present invention is based on the recognition that the
`overall hydrophobicity of an insulin derivative molecule
`plays an important role for the in vivo potency of the deriva
`tive.
`In one aspect the present invention relates to an insulin
`derivative which is a naturally occurring insulin or an ana
`logue thereof which has a side chain attached either to the
`C.-amino group of the N-terminal amino acid residue of the B
`chain or to the e-amino group of a Lys residue present in the
`B chain of the parent insulin, the side chain being of the
`general formula:
`
`wherein W is:
`an O-amino acid residue having a carboxylic acid group in
`the side chain which residue forms, with one of its car
`boxylic acid groups, an amide group together with the
`C-amino group of the N-terminal amino acid residue of
`
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`MPI EXHIBIT 1096 PAGE 2
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`MPI EXHIBIT 1096 PAGE 2
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`

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`US 7,615,532 B2
`
`3
`the B chain or together with the e-amino group of a Lys
`residue present in the B chain of the parent insulin;
`a chain composed of two, three or four C.-amino acid resi
`dues linked together via amide bonds, which chain—via
`an amide bond is linked to the C-amino group of the
`N-terminal amino acid residue of the B chain or to the
`e-amino group of a Lys residue present in the B chain of
`the parent insulin, the amino acid residues of W being
`Selected from the group of amino acid residues having a
`neutral side chain and amino acid residues having a
`carboxylic acid group in the side chain so that W has at
`least one amino acid residue which has a carboxylic acid
`group in the side chain; or
`a covalent bond from X to the C.-amino group of the N-ter
`minal amino acid residue of the B chain or to the e-amino
`group of a Lys residue present in the B chain of the
`parent insulin;
`X is:
`- CO—;
`CH(COOH) CO :
`- N(CHCOOH)CHCO :
`N(CHCOOH)CHCON(CHCOOH)CHCO :
`- N(CH-CHCOOH)CHCHCO :
`N(CHCHCOOH)CHCHCON(CHCHCOOH)
`CHCHCO :
`NHCH(COOH)(CH)NHCO :
`- N(CH-CHCOOH)CHCO-; or
`- N(CHCOOH)CHCHCO-.
`that
`a) when W is an amino acid residue or a chain of amino acid
`residues, via a bond from the underscored carbonyl carbon
`forms an amide bond with an amino group in W. or
`b) when W is a covalent bond, via a bond from the under
`scored carbonyl carbon forms an amide bond with the
`N-terminal C.-amino group in the B chain or with the
`e-amino group of a Lys residue present in the B chain of the
`parent insulin;
`Y is:
`—(CH), where m is an integer in the range of 6 to 32:
`a divalent hydrocarbon chain comprising 1, 2 or
`3-CH=CH-groups and a number of CH groups
`Sufficient to give a total number of carbon atoms in the
`chain in the range of 10 to 32:
`a divalent hydrocarbon chain of the formula —(CH),
`CH (CH2)— wherein V and w are integers or one of
`them is Zero so that the Sum of V and w is in the range of
`6 to 30; and
`
`10
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`15
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`25
`
`30
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`35
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`40
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`45
`
`4
`In another embodiment of the invention, side chain -W-
`X-Y-Z is attached to the e-amino group of a Lys residue
`present in the B chain of the parent insulin. In one more
`specific aspect of this embodiment, the side chain —W13
`X-Y-Z is attached to the e-amino group of a Lys residue
`present in position 28 of the B chain. In a further more specific
`aspect of this embodiment, the side chain -W-X Y-Z is
`attached to the e-amino group of a Lys residue present in
`position 29 of the B chain. In a further more specific aspect of
`this embodiment, the side chain-W-X—Y-Z is attached to the
`e-amino group of a Lys residue present in position 30 of the B
`chain.
`The substructure W of the side chain-W-X Y-Z can be a
`covalent bond. Alternatively, W can be a residue of an
`C.-amino acid having a carboxylic acid group in the side chain
`and comprising a total of from 4 to 10 carbonatoms. Specifi
`cally, W can be the residue of an C.-amino acid, that can be
`coded for by the genetic code. Thus, W can, for example, be
`selected from the group consisting of C.-Asp, B-Asp, O-Glu,
`and Y-Glu. Further options for Ware for example O.-hGlu and
`6-hGlu.
`In a further embodiment, W is a chain composed of two
`C.-amino acid residues of which one has from 4 to 10 carbon
`atoms and a carboxylic acid group in the side chain while the
`other has from 2 to 11 carbon atoms but no free carboxylic
`acid group. The C-amino acid residue with no free carboxylic
`acid group can be a neutral, codable C-amino acid residue.
`Examples of W according to this embodiment are: C.-Asp
`Gly; Gly-C.-Asp; B-Asp-Gly; Gly-B-Asp: C.-Glu-Gly; Gly-C.-
`Glu; Y-Glu-Gly; Gly-y-Glu, C.-hGlu-Gly; Gly-C.-hGlu;
`8-hGlu-Gly; and Gly-8-hGlu.
`In a further embodiment, W is a chain composed of two
`C.-amino acid residues, independently having from 4 to 10
`carbonatoms, and both having a carboxylic acid group in the
`side chain. One of these O-amino acid residues or both of
`them can be codable C.-amino acid residues. Examples of W
`according to this embodiment are: C.-Asp-O-Asp., C.-Asp-O-
`Glu, C.-Asp-O-hGlu, C.-Asp-f3-Asp., C.-Asp-y-Glu, C.-Asp-Ö-
`hGlu, B-Asp-O-Asp;B-Asp-O-Glu, B-Asp-C-hGlu, B-Asp-B-
`Asp; B-Asp-y-Glu, B-Asp-Ö-hGlu, C.-Glu-O-Asp, C.-Glu-C.-
`Glu; O-Glu-O-hGlu; O-Glu-f-Asp: C.-Glu-y-Glu; O-Glu-8-
`hGlu; Y-Glu-O-Asp; Y-Glu-O-Glu; Y-Glu-O-hGlu; Y-Glu-B-
`Asp; Y-Glu-y-Glu; Y-Glu-8-hGlu, C.-hGlu-O-Asp: C.-hGlu-O-
`Glu,
`C.-hGlu-O-hGlu,
`C.-hGlu-B-Asp:
`C.-hGlu-y-Glu;
`C.-hGlu-8-hGlu; 8-hGlu-O-Asp; 8-hGlu-O-Glu; 8-hGlu-C.-
`hGlu; 8-hGlu-B-Asp; 8-hGlu-y-Glu; and 8-hGlu-8-hGlu.
`In a further embodiment, W is a chain composed of three
`C.-amino acid residues, independently having from 4 to 10
`carbon atoms, the amino acid residues of the chain being
`selected from the group of residues having a neutral side
`chain and residues having a carboxylic acid group in the side
`chain so that the chain has at least one residue which has a
`carboxylic acid group in the side chain. In one embodiment,
`the amino acid residues are codable residues.
`In a further embodiment, W is a chain composed of four
`C.-amino acid residues, independently having from 4 to 10
`carbon atoms, the amino acid residues of the chain being
`selected from the group having a neutral side chain and resi
`dues having a carboxylic acid group in the side chain so that
`the chain has at least one residue which has a carboxylic acid
`group in the side chain. In one embodiment, the amino acid
`residues are codable residues.
`In one embodiment W can be connected to the e-amino
`group of the Lys residue in the B-chain via an urea derivative.
`The substructure X of the side chain-W-X Y-Z can be a
`group of the formula – CO— that, via a bond from the
`underscored carbonyl carbon, forms an amide bond with an
`
`Z is:
`COOH:
`—CO-Asp;
`- CO-Glu:
`- CO-Gly;
`—CO-Sar;
`-CH(COOH):
`- N(CHCOOH):
`—SOH; or
`- POH:
`and any Zn" complexes thereof, provided that when W is a
`covalent bond and X is —CO—, then Z is different from
`COOH.
`In one embodiment of the invention, the side chain-W-X—
`Y-Z is attached to the C-amino group of the N-terminal amino
`acid residue of the B chain of the parent insulin.
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`MPI EXHIBIT 1096 PAGE 3
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`MPI EXHIBIT 1096 PAGE 3
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`5
`amino group in Wor, when W is a covalent bond, with the
`N-terminal O-amino group in the B chain or with the e-amino
`group of a Lys residue present in the B chain of the parent
`insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula—CH(COOH)CO—that,
`via a bond from the underscored carbonyl carbon, forms an
`amide bond with an amino group in W or, when W is a
`covalent bond, with the N-terminal O-amino group in the B
`chain or with the e-amino group of a Lys residue present in the
`B chain of the parent insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula - N(CHCOOH)CH
`CO that, via a bond from the underscored carbonyl carbon,
`forms an amide bond with an amino group in Wor, when W
`15
`is a covalent bond, with the N-terminal O-amino group in the
`B chain or with the e-amino group of a Lys residue present in
`the B chain of the parent insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula - N(CH-CHCOOH)
`CHCO— that, via a bond from the underscored carbonyl
`carbon, forms an amide bond with an amino group in Wor,
`when W is a covalent bond, with the N-terminal O-amino
`group in the B chain or with the e-amino group of a Lys
`residue present in the B chain of the parent insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula - N(CHCOOH)
`CHCH-CO— that, via a bond from the underscored carbo
`nyl carbon, forms an amide bond with an amino group in W
`30
`or, when W is a covalent bond, with the N-terminal O-amino
`group in the B chain or with the e-amino group of a Lys
`residue present in the B chain of the parent insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula —N(CHCOOH)
`35
`CHCON(CHCOOH)CHCO - that, via a bond from the
`underscored carbonyl carbon, forms an amide bond with an
`amino group in Wor, when W is a covalent bond, with the
`N-terminal O-amino group in the B chain or with the famino
`group of a Lys residue present in the B chain of the parent
`insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula - N(CH-CHCOOH)
`CHCH-CO— that, via a bond from the underscored carbo
`nyl carbon, forms an amide bond with an amino group in W
`45
`or, when W is a covalent bond, with the N-terminal O-amino
`group in the B chain or with the e-amino group of a Lys
`residue present in the B chain of the parent insulin.
`In a further embodiment, the substructure X of the side
`chain can be a group of the formula - N(CH2CHCOOH)
`50
`CHCHCON(CH-CHCOOH)CH-CHCO - that, via a
`bond from the underscored carbonyl carbon, forms an amide
`bond with an amino group in Wor, when W is a covalent
`bond, with the N-terminal C.-amino group in the B chain or
`with the e-amino group of a Lys residue present in the B chain
`of the parent insulin.
`The substructure Y of the side chain-W-X Y-Z can be a
`group of the formula—(CH), where m is an integer in the
`range of from 6 to 32, from 8 to 20, from 12 to 20, or from
`12-16.
`In another embodiment, Y is a divalent hydrocarbon chain
`comprising 1, 2 or 3-CH=CH-groups and a number of
`—CH2— groups sufficient to give a total number of carbon
`atoms in the chain in the range of from 6 to 32, from 10 to 32,
`from 12 to 20, or from 12-16.
`In another embodiment, Y is a divalent hydrocarbon chain
`of the formula—(CH2)CH (CH2)— wherein V and w are
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`US 7,615,532 B2
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`10
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`6
`integers or one of them is Zero So that the Sum of V and w is in
`the range of from 6 to 30, from 10 to 20, or from 12-16.
`In one embodiment, the substructure Z of the side chain
`-W-X Y-Z is -COOH provided that when W is a covalent
`bond and X is —CO—, then Z is different from —COOH.
`In another embodiment, Z is —CO-Asp.
`In another embodiment, Z is —CO-Glu.
`In another embodiment, Z is —CO-Gly.
`In another embodiment, Z is —CO-Sar.
`In another embodiment, Z is —CH(COOH).
`In another embodiment, Z is N(CHCOOH).
`In another embodiment, Z is - SOH.
`In another embodiment, Z is —POH.
`In a further embodiment W is selected from the group
`consisting of C.-Asp, B-Asp, C.-Glu, and Y-Glu, X is —CO—
`or —CH(COOH)CO;Y is —(CH), where m is an integer
`in the range of 12-18 and Z is -COOH or -CH(COOH).
`The insulin moiety in the present text also referred to as
`the parent insulin—of an insulin derivative according to the
`invention can be a naturally occurring insulin Such as human
`insulin orporcine insulin. Alternatively, the parent insulin can
`be an insulin analogue.
`In one group of parent insulin analogues, the amino acid
`residue at position A21 is ASn.
`In another group of parent insulin analogues, the amino
`acid residue at position A21 is Gly. Specific examples from
`this group of analogues are Gly' human insulin, Gly''
`des(B30) human insulin; and Gly' Arg?' Arg’’ human
`insulin.
`In another group of parent insulin analogues, the amino
`acid residue at position B1 has been deleted. A specific
`example from this group of parent insulin analogues is des
`(B1) human insulin.
`In another group of parent insulin analogues, the amino
`acid residue at position B30 has been deleted. A specific
`example from this group of parent insulin analogues is des
`(B30) human insulin.
`In another group of parent insulin analogues, the amino
`acid residue at position B28 is Asp. A specific example from
`this group of parent insulin analogues is Asp human insu
`lin.
`In another group of parent insulin analogues, the amino
`acid residue at position B28 is Lys and the amino acid residue
`at position B29 is Pro. A specific example from this group of
`parent insulin analogues is Lys'Pro' human insulin.
`In another group of parent insulin analogues the amino acid
`residue in position B30 is Lys and the amino acid residue in
`position B29 is any codable amino acid except Cys, Met, Arg
`and Lys. An example is an insulin analogue where the amino
`acid residue at position B29 is Thrand the amino acid residue
`at position B30 is Lys. A specific example from this group of
`parent insulin analogues is Thr’LyS’’ human insulin.
`In another group of parent insulin analogues, the amino
`acid residue at position B3 is Lys and the amino acid residue
`at position B29 is Glu. A specific example from this group of
`parent insulin analogues is Lys' Glu’ human insulin.
`Examples of insulin derivatives according to the invention
`are the following compounds:
`N? (N' (HOOC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
`N’ (N' (HOOC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
`N’ (N' (HOOC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
`N? (N (HOOC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
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`MPI EXHIBIT 1096 PAGE 4
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`MPI EXHIBIT 1096 PAGE 4
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`des(B30)
`des(B30)
`des(B30)
`
`7
`N’ (N' (HOOC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
`N’ (N' (HOOC(CH2)CO)-y-Glu-N-(y-Glu)) des
`(B30) human insulin;
`N’ (N (Asp-OC(CH2)CO)-y-Glu)
`des(B30)
`human insulin;
`N’ (N (Glu-OC(CH),CO)-y-Glu)
`des(B30)
`human insulin;
`N’ (N'-(Glu-OC(CH2)CO ) des(B30) human
`insulin;
`N’ (N'-(Asp-OC(CH2)CO ) des(B30) human
`insulin;
`N’ (N' (HOOC(CH2)CO)-O-Glu-N-(B-Asp))
`des(B30) human insulin;
`N eB29 (N '-(Gly-OC(CH), CO)-y -Glu)
`human insulin;
`NeP29 (N ' (Sar-OC(CH),CO)-y-Glu)
`human insulin;
`N’ (N' (HOOC(CH),CO)-y-Glu)
`human insulin;
`(N’ (N' (HOOC(CH2)CO)-B-Asp)
`human insulin;
`des(B30)
`N’ (N' (HOOC(CH2)CO)-O-Glu)
`human insulin;
`N’ (N' (HOOC(CH),CO)-y-D-Glu) des(B30)
`human insulin;
`N’ (N' (HOOC(CH2)CO-B-D-Asp) des(B30)
`human insulin;
`N’ (N HOOC(CH2)CO-B-D-Asp)
`des(B30)
`human insulin
`N’ (N HOOC(CH2)CO-IDA) des(B30) human
`insulin;
`N’ -N-(HOOC(CH2)CO N-(carboxyethyl)-
`Gly des(B30) human insulin;
`N’ -N-(HOOC(CH2)CO)-N-(carboxyethyl)-
`Gly des(B30) human insulin; and
`N’ -N-(HOOC(CH2)CO)-N-(carboxymethyl)-
`B-Ala des(B30) human insulin.
`Insulin derivatives according to the invention may be pro
`vided in the form of essentially zinc free compounds or in the
`form of Zinc complexes. When Zinc complexes of an insulin
`derivative according to the invention are provided, two Zn"
`ions, three Zn" ions or four Zn" ions can be bound to each
`insulin hexamer. Solutions of Zinc complexes of the insulin
`derivatives will contain mixtures of Such species.
`In a further aspect of the invention, a pharmaceutical com
`position comprising a therapeutically effective amount of an
`insulin derivative according to the invention together with a
`pharmaceutically acceptable carrier can be provided for the
`treatment of type 1 diabetes, type 2 diabetes and other states
`that cause hyperglycemia in patients in need of Such a treat
`ment. An insulin derivative according to the invention can be
`used for the manufacture of a pharmaceutical composition for
`use in the treatment of type 1 diabetes, type 2 diabetes and
`other states that cause hyperglycaemia.
`In a further aspect of the invention, there is provided a
`pharmaceutical composition for treating type 1 diabetes, type
`2 diabetes and other states that cause hyperglycaemia in a
`patient in need of Such a treatment, comprising a therapeuti
`cally effective amount of an insulin derivative according to
`the invention in mixture with an insulin oran insulin analogue
`which has a rapid onset of action, together with pharmaceu
`tically acceptable carriers and additives.
`In one embodiment the invention provides a pharmaceuti
`cal composition being a mixture of an insulin derivative
`according to the invention and a rapid acting insulin analogue
`
`10
`
`des (B30)
`
`15
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`US 7,615,532 B2
`
`As28
`
`res
`
`8
`human insulin;
`selected group consisting of Asp
`Lys'Pro'humaninsulin and LyS'Glu’humaninsulin.
`In one embodiment the invention provides a pharmaceuti
`cal composition comprising N' N' (HOOC(CH),
`CO)-y-Glu) des(B30) human insulin and AspE28 human
`insulin together with pharmaceutically acceptable carriers
`and additives.
`The insulin derivative according to the invention and the
`rapid acting insulin analogue can be mixed in a ratio from
`about 90/10%; about 70/30% or about 50/50%.
`In a further aspect of the invention, there is provided a
`method of treating type 1 diabetes, type 2 diabetes and other
`states that cause hyperglycaemia in a patient in need of Such
`a treatment, comprising administering to the patient a thera
`peutically effective amount of an insulin derivative according
`to the invention together with a pharmaceutically acceptable
`carrier and pharmaceutical acceptable additives.
`In a further aspect of the invention, there is provided a
`method of treating type 1 diabetes, type 2 diabetes and other
`states that cause hyperglycaemia in a patient in need of Such
`a treatment, comprising administering to the patient a thera
`peutically effective amount of an insulin derivative according
`to the invention in mixture with an insulin or an insulin
`analogue which has a rapid onset of action, together with a
`pharmaceutically acceptable carrier and pharmaceutical
`acceptable additives.
`In a further aspect, the present invention relates to insulin
`derivatives which have an overall hydrophobicity which is
`essentially similar to that of human insulin.
`In further aspect, the present invention relates to insulin
`derivatives which have a hydrophobic index, k", which is in
`the range of from about 2 to about 200.
`In a further aspect, the insulin derivatives of the present
`invention have a hydrophobic index, k", which is in the
`range from about 0.02 to about 10, from about 0.1 to about 5:
`from about 0.5 to about 5; or from about 0.5 to about 2.
`According to one embodiment of the present invention the
`insulin derivatives will comprise a side chain-W-X Y-Z as
`defined above which has at least one hydrophilic and at least
`one hydrophobic region.
`According to another embodiment of the present invention,
`the insulin derivatives will comprise a side chain-W-X Y-Z
`as defined above which has at least one free carboxylic acid
`group and according to a further embodiment, the side chain
`will have at least two free carboxylic acid groups.
`In another embodiment, the invention relates to a pharma
`ceutical composition comprising an insulin derivative
`according to the invention which is soluble at physiological
`pH values.
`In another embodiment, the invention relates to a pharma
`ceutical composition comprising an insulin derivative
`according to the invention which is soluble at pH values in the
`interval from about 6.5 to about 8.5.
`In another embodiment, the invention relates to a pharma
`ceutical composition with a prolonged profile of action which
`comprises an insulin derivative according to the invention.
`In another embodiment, the invention relates to a pharma
`ceutical composition which is a solution containing from
`about 120 nmol/ml to about 2400 nmol/ml, from about 400
`nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to
`about 1200 nmol/ml, from about 600 nmol/ml to about 2400
`nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml
`of an insulin derivative according to the invention or of a
`mixture of the insulin derivative according to the invention
`with a rapid acting insulin analogue.
`
`MPI EXHIBIT 1096 PAGE 5
`
`MPI EXHIBIT 1096 PAGE 5
`
`

`

`US 7,615,532 B2
`
`Hydrophobicity Data on Insulin Derivatives According to the
`Invention.
`The hydrophobicity (hydrophobic index) of the insulin
`derivatives of the invention relative to human insulin, k",
`was measured on a LiChrosorb RP18 (5um, 250x4 mm)
`HPLC column by isocratic elution at 40°C. using mixtures of
`A) 0.1 M sodium phosphate buffer, pH 7.3, containing 10%
`acetonitrile, and B) 50% acetonitrile in water as eluents. The
`elution was monitored by following the UV absorption of the
`eluate at 214 nm. Void time, to was foundby injecting 0.1 mM
`10
`Sodium nitrate. Retention time for human insulin, t, was
`adjusted to at least2to by varying the ratio between the A and
`B Solutions. kei (ten-to)/(thin-to). kei found for a
`number of insulin derivatives according to the invention are
`given in Table 1.
`
`5
`
`10
`In a further embodiment of the invention the buffer is
`selected from the group consisting of sodium acetate, sodium
`carbonate, citrate, glycylglycine, histidine, glycine, lysine,
`arginine, sodium dihydrogen phosphate, disodium hydrogen
`phosphate, Sodium phosphate, and tris(hydroxymethyl)-ami
`nomethan, bicine, tricine, malic acid, Succinate, maleic acid,
`fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
`Each one of these specific buffers constitutes an alternative
`embodiment of the invention.
`In a further embodiment of the invention the formulation
`further comprises a pharmaceutically acceptable preservative
`which may be selected from the group consisting of phenol,
`o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, pro
`pyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxy
`benzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol,
`
`TABLE 1
`
`Insulin derivative
`N' (N HOOC(CH2)CO-y-Glu) des(B30) human insulin
`N' (N HOOC(CH2)6CO-y-Glu) des(B30) human insulin
`N' (N HOOC(CH2)CO-y-Glu) des(B30) human insulin
`N' (N HOOC(CH2)6CO-y-Glu-N-(y-Glu) des(B30) human insulin
`N' (N-(Asp-OC(C H2)6CO)-y-Glu) des(B30) human insulin
`N' (N-(Glu-OC(CH2)CO-y-Glu) des(B30) human insulin
`N' (N-(Glu-OC(CH2)CO-) des(B30) human insulin
`N’ (N HOOC(CH2)6CO-C-Glu)-N-(B-Asp) des(B30) human insulin
`N-29 (N-(Gly-OC(CH 2)3CO-y-Glu) des(B30) human insulin
`N' (N-(Sar-OC(CH2)CO-y-Glu) des(B30) human insulin
`N' (N HOOC(CH2)6CO-O-Glu)-N-(AspAsp) des(B30) human insulin
`NP29 (N-(G y-OC(CH2)CO-y-Glu) des(B30) human insulin
`N' (N HOOC(CH2)CO-Y-L-Glu) des(B30) human insulin
`N' (N HOOC(CH2)CO-B-L-Asp) des(B30) human insulin
`N' (N HOOC(CH2)CO-B-L-Glu) des(B30) human insulin
`N’ (N HOOC(CH2)CO-B-L-Asp) des(B30) human insulin
`N' (N HOOC(CH2)6CO-ö-L-Aad) des(B30) human insulin
`N' (N HOOC(CH2)6CO-y-D-Glu) des(B30) human insulin
`N' (N HOOC(CH2)CO-B-L-Asp) des(B30) human insulin
`N’ (N HOOC(CH2)6CO-C-L-Asp) des(B30) human insulin
`N' (N HOOC(CH2)6CO-C-L-Glu) des(B30) human insulin
`N' (N HOOC(CH2)CO-e-L-LysCO-) des(B30) human insulin
`N’ (N HOOC(CH2)6CO-B-L-Asp) des(B30) human insulin
`N-29 (N-(Gly-OC(CH 2)6CO-Y-L-Glu) des(B30) human insulin
`N' N—(HOOC(CH2)6CO-N-(carboxymethyl)-3-Alades(B30) human insulin
`N' N (HOOC(CH))NHCO(CH2)CO)-Y-L-Gludes(B30) human insulin
`
`rei
`
`O.87
`1.15
`O.45
`1.17
`O.70
`O.33
`1.17
`1.11
`O.S8
`O.63
`1.07
`O.88
`1.13
`O.69
`O.54
`O.47
`O.84
`1.4
`1.09
`1.49
`1.51
`O.90
`1.54
`1.57
`1.13
`O.42
`
`Pharmaceutical Compositions
`Pharmaceutical compositions containing an insulin deriva
`tive according to the present invention may be administered
`parenterally to patients in need of such a treatment. Parenteral
`administration may be performed by Subcutaneous, intramus
`cular or intravenous injection by means of a syringe, option
`ally a pen-like Syringe. Alternatively, parenteral administra
`tion can be performed by means of an infusion pump. Further
`options are to administer the insulin nasally or pulmonally,
`preferably in compositions, powders or liquids, specifically
`designed for the purpose.
`Injectable compositions of the insulin derivatives of the
`invention can be prepared using the conventional techniques
`of the pharmaceutical industry which involve dissolving and
`mixing the ingredients as appropriate to give the desired end
`product. Thus, according to one procedure, an insulin deriva
`tive according to the invention is dissolved in an amount of
`water which is somewhat less than