USOO88466 18B2
`
`(12) United States Patent
`Flink et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,846,618 B2
`Sep. 30, 2014
`
`(54) STABLE FORMULATION OF MODIFIED
`GLP-1
`
`(75) Inventors: James M. Flink, Klanpenborg (DK);
`Silke Moller Larsen, Charlottenlund
`(DK); Simon Bjerregaard Jensen,
`Frederiksberg (DK); Dorthe Kot
`Engelund, Holte (DK)
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`(21) Appl. No.: 12/785,861
`(22) Filed:
`May 24, 2010
`
`(65)
`
`Prior Publication Data
`US 2010/O234299 A1
`Sep. 16, 2010
`
`Related U.S. Application Data
`(63) Continuation of application No. 1 1/786,095, filed on
`Apr. 11, 2007, now abandoned, which is a continuation
`of application No. 10/185.923, filed on Jun. 27, 2002,
`now abandoned.
`(60) Provisional application No. 60/308,325, filed on Jul.
`27, 2001, provisional application No. 60/308,297,
`filed on Jul. 27, 2001.
`Foreign Application Priority Data
`
`(30)
`
`Jun. 28, 2001 (DK) ................................. 2001 O1010
`Jun. 28, 2001 (DK)......
`... 2001 O1011
`Jul. 4, 2001
`(DK)......
`... 2001 O1052
`Jul. 4, 2001
`(DK)......
`... 2001 O1053
`Jan. 18, 2002 (DK) ................................. 2002 OOO93
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`(51) Int. Cl.
`A6 IK38/26
`A6IP3/10
`A6IP 7/2
`C07K I4/605
`(52) U.S. Cl.
`CPC ................................... C07K 14/605 (2013.01);
`A6 IK38/26 (2013.01)
`USPC ......................................................... S14/117
`(58) Field of Classification Search
`CPC .............................. A61 K38/26: CO7K 14/605
`See application file for complete search history.
`
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`
`FOREIGN PATENT DOCUMENTS
`
`CA
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`
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`(Continued)
`OTHER PUBLICATIONS
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`
`(Continued)
`
`(56)
`
`References Cited
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`U.S. PATENT DOCUMENTS
`
`Primary Examiner — Marcela M Cordero Garcia
`(74) Attorney,
`Agent,
`or
`Firm — Rosemarie
`Wilk-Orescan
`
`R.
`
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`ABSTRACT
`(57)
`Pharmaceutical formulations of GLP-1 compounds and
`methods for preparation thereof.
`
`14 Claims, No Drawings
`
`MYLAN INST. EXHIBIT 1107 PAGE 1
`
`MYLAN INST. EXHIBIT 1107 PAGE 1
`
`

`

`US 8,846,618 B2
`Page 2
`
`(56)
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`
`MYLAN INST. EXHIBIT 1107 PAGE 2
`
`MYLAN INST. EXHIBIT 1107 PAGE 2
`
`

`

`US 8,846,618 B2
`Page 3
`
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`* cited by examiner
`
`MYLAN INST. EXHIBIT 1107 PAGE 3
`
`MYLAN INST. EXHIBIT 1107 PAGE 3
`
`

`

`1.
`STABLE FORMULATION OF MODIFIED
`GLP-1
`
`US 8,846,618 B2
`
`2
`tion of 1) glucagon itself, occupying positions 33-61 of pro
`glucagon (PG); 2) an N-terminal peptide of 30 amino acids
`(PG (1-30)) often called glicentin-related pancreatic peptide,
`GRPP; 3) a hexapeptide corresponding to PG (64-69); 4) and,
`finally, the so-called major proglucagon fragment (PG (72
`158)), in which the two glucagon-like sequences are buried.
`Glucagon seems to be the only biologically active product. In
`contrast, in the intestinal mucosa, it is glucagon that is buried
`in a larger molecule, while the two glucagon-like peptides are
`formed separately.
`While much attention has been focused on the pharmaco
`logical properties of acylated GLP-1 compounds, hitherto
`little is known about their physico-chemical and Solution
`structural properties. Such knowledge is a prerequisite for
`rational handling during e.g. production, purification and for
`mulation work and is eventually important for understanding
`of the structural basis for the protraction mechanism.
`It is an important technical challenge to ensure prolonged
`stability during storage (shelflife) of many protein based drug
`products due to the inherent lability of macromolecules.
`Hence, proteins are sensitive to both chemical and physical
`degradation unlike many Small molecules. Chemical degra
`dation involves covalent bonds. Such as hydrolysis, racem
`ization, oxidation or crosslinking. Physical degradation
`involves conformational changes relative to the native struc
`ture, which includes loss of higher order structure, aggrega
`tion, precipitation or adsorption to Surfaces. GLP-1 is known
`to be prone to instability due to aggregation. Both degradation
`pathways may ultimately lead to loss of biological activity of
`the protein drug.
`GLP-1 and analogues of GLP-1 and fragments thereof are
`potentially useful i.a. in the treatment of type 1 and type 2
`diabetes. However, solubility limitations and the low stability
`against the actions of endogenous diaminopeptidyl peptidase
`limits the usefulness of these compounds, and thus there still
`is a need for improvements in this field.
`In WO99/43341 are disclosed certain pharmaceutical for
`mulations comprising GLP-1 having a lipophilic Substituent.
`All of the disclosed formulations are maintained at pH 7.4.
`In WO 00/37098 are disclosed shelf-stable formulations
`comprising GLP-1, a preservative, and a tonicity modifier, at
`pH 8.2 to 8.8.
`Human GLP-1 is a 37 amino acid residue peptide originat
`ing from preproglucagon which is synthesised i.a. in the
`L-cells in the distal ileum, in the pancreas and in the brain.
`Processing of preproglucagon to give GLP-1 (7-36)amide,
`GLP-1 (7-37) and GLP-2 occurs mainly in the L-cells. A
`simple system is used to describe fragments and analogues of
`this peptide. Thus, for example, Val-GLP-1 (7-37) (or
`Val8GLP-1 (7-37)) designates a fragment of GLP-1 formally
`derived from GLP-1 by deleting the amino acid residues Nos.
`1 to 6 and Substituting the naturally occurring amino acid
`residue in position 8 (Ala) by Val. Similarly, Lys (N-tet
`radecanoyl)-GLP-1 (7-37) designates GLP-1 (7-37) wherein
`the e-amino group of the Lys residue in position 34 has been
`tetradecanoylated. For convenience the amino acid sequence
`of GLP-1 (7-37) is given below, wherein the N-terminal His is
`no. 7 and the C-terminal Gly is no. 37:
`
`(SEQ ID NO. :
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser
`
`1)
`
`Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe
`
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly.
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of U.S. application Ser.
`No. 1 1/786,095 filed on Apr. 11, 2007, which is a continua
`tion of U.S. application Ser. No. 10/185.923 filed on Jun. 27,
`2002 and claims priority under 35 U.S.C. 119 of Danish
`Application No. PA 2001 01010 filed Jun. 28, 2001; Danish
`Application No. PA 2001 01011 filed Jun. 28, 2001; Danish
`Application No. PA 2001 01052 filed Jul. 4, 2001; Danish
`Application No. PA 2001 01053 filed Jul. 4, 2001; and Danish
`Application No. PA 2002 00093 filed Jan. 18, 2002; and U.S.
`Provisional Applications No. 60/308.325 filed Jul. 27, 2001
`and 60/308.297 filed Jul. 27, 2001, the contents of which are
`fully incorporated herein by reference.
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`FIELD OF THE INVENTION
`
`The present invention relates to pharmaceutical formula
`tions comprising GLP-1 compounds, uses thereof and meth
`ods for preparing said formulations.
`
`BACKGROUND OF THE INVENTION
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`Peptides are widely used in medical practice, and since
`they can be produced by recombinant DNA technology it can
`be expected that their importance will increase also in the
`years to come.
`The hormones regulating insulin secretion belong to the
`so-called enteroinsular axis, designating a group of hor
`mones, released from the gastrointestinal mucosa in response
`to the presence and absorption of nutrients in the gut, which
`promote an early and potentiated release of insulin. The
`enhancing effect on insulin secretion, the so-called incretin
`effect, is probably essential for a normal glucose tolerance.
`Many of the gastrointestinal hormones, including gastrin and
`secretin (cholecystokinin is not insulinotropic in man), are
`insulinotropic, but the only physiologically important ones,
`those that are responsible for the incretin effect, are the glu
`cose-dependent insulinotropic polypeptide, GIP, and gluca
`gon-like peptide-1 (GLP-1). Because of its insulinotropic
`effect, GIP, isolated in 1973 immediately attracted consider
`able interest among diabetologists. However, numerous
`investigations carried out during the following years clearly
`indicated that a defective secretion of GIP was not involved in
`the pathogenesis of insulin dependent diabetes mellitus
`(IDDM) or non insulin-dependent diabetes mellitus
`(NIDDM). Furthermore, as an insulinotropic hormone, GIP
`was found to be almost ineffective in NIDDM. The other
`incretin hormone, GLP-1 is the most potent insulinotropic
`substance known. Unlike GIP, it is surprisingly effective in
`stimulating insulin secretion in NIDDM patients. In addition,
`and in contrast to the other insulinotropic hormones (perhaps
`with the exception of secretin) it also potently inhibits gluca
`gon secretion. Because of these actions it has pronounced
`blood glucose lowering effects particularly in patients with
`NIDDM.
`GLP-1, a product of the proglucagon, is one of the young
`est members of the secretin-VIP family of peptides, but is
`already established as an important gut hormone with regu
`latory function in glucose metabolism and gastrointestinal
`secretion and metabolism. The glucagon gene is processed
`differently in the pancreas and in the intestine. In the pan
`creas, the processing leads to the formation and parallel secre
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`MYLAN INST. EXHIBIT 1107 PAGE 4
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`MYLAN INST. EXHIBIT 1107 PAGE 4
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`

`

`US 8,846,618 B2
`
`3
`Where reference in this text is made to C-terminally extended
`GLP-1 analogues, the amino acid residue in position38 is Arg
`unless otherwise indicated, the optional amino acid residue in
`position 39 is also Arg unless otherwise indicated and the
`optional amino acid residue in position 40 is Asp unless
`otherwise indicated. Also, if a C-terminally extended ana
`logue extends to position 41, 42, 43, 44 or 45, the amino acid
`sequence of this extension is as in the corresponding sequence
`in human preproglucagon unless otherwise indicated.
`
`SUMMARY OF THE INVENTION
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`centration from 0.1 mg/ml to 100 mg/ml, and wherein said
`formulation has a pH from 7.0 to 10;
`provided that if an isotonic agent is present and pH is 7.4
`then mannitol or NaCl is not the isotonic agent.
`In another aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 1 mg/ml to 100 mg/ml, and wherein said
`formulation has a pH from 7.0 to 10;
`provided that if an isotonic agent is present and pH is 7.4
`then mannitol or NaCl is not the isotonic agent.
`In a further aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 0.1 mg/ml or above, and wherein said formu
`lation has a pH from 7.0 to 10.
`In a further aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 1 mg/ml or above, and wherein said formu
`lation has a pH from 7.0 to 10.
`In a further aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof, wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 0.1 mg/ml to 100 mg/ml, and wherein said
`formulation has a pH from 7.0 to 10.
`In a further aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof, wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 1 mg/ml to 100 mg/ml, and wherein said
`formulation has a pH from 7.0 to 10.
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, and a buffer, wherein said
`GLP-1 compound is present in a concentration from 0.1
`mg/ml or above, and wherein said formulation has a pH from
`7.0 to 10.
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, and a buffer, wherein said
`GLP-1 compound is present in a concentration from 1 mg/ml
`or above, and wherein said formulation has a pH from 7.0 to
`10.
`
`We have discovered that certain modified GLP-1 or ana
`logues thereof when formulated in aqueous solution together
`with a buffer, are physically stable at high concentrations of
`the modified GLP-1 or analogues thereof, when kept in the
`pH range from about 7 to about 10. The present formulations
`are physically stable within a given shelf life period at the
`recommended storage temperature (typically 2-3 years at
`2-8° C.). Furthermore, the present formulations are physi
`cally stable during in-use (typically 1 month at accelerated
`temperatures e.g. 25°C. or 37° C.). The formulations of the
`invention are also chemically stable thus rendering them
`shelf-stable and Suitable for invasive (eg. injection, Subcuta
`neous injection, intramuscular, intravenous or infusion) as
`well as non-invasive (eg nasal or pulmonary, transdermal or
`transmucosal e.g. buccal) means of administration. When the
`inventive formulation comprising a GLP-1 compound was
`compared to the same formulation comprising GLP-1 (7-37)
`substituted for the GLP-1 compound, the physical stability
`was increased considerably, and typically the shelf-life was
`increased from a few seconds to several months in the tests
`used.
`One object of the present invention is to provide a pharma
`ceutical formulation comprising a GLP-1 compound, and a
`buffer, wherein said GLP-1 compound is GLP-1 (7-37) or an
`analogue thereof wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`centration from 0.1 mg/ml to 100 mg/ml, and wherein said
`40
`formulation has a pH from 7.0 to 10;
`Another object of the present invention is to provide a
`method of preparing a physically stable pharmaceutical for
`mulation of a GLP-1 compound wherein said GLP-1 com
`pound is GLP-1 (7-37) or an analogue thereof wherein an
`45
`amino acid residue of the parent peptide has a lipophilic
`Substituent attached optionally via a spacer, comprising pre
`paring a formulation containing the GLP-1 compound, and a
`buffer, wherein said GLP-1 compound is present in a concen
`tration from 0.1 mg/ml to 100 mg/ml, and wherein said for
`mulation has a pH from 7.0 to 10.
`In one aspect of the invention the formulation contains a
`GLP-1 compound in a concentration from 1 mg/ml to 100
`mg/ml.
`In another aspect of the invention the formulation has a pH
`from 7.5 to 10.
`In one embodiment the GLP-1 compound is Arg, Lys’
`(N-e-(y-GlucN-O-hexadecanoyl)))-GLP-1 (7-37).
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`50
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`55
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`DESCRIPTION OF THE INVENTION
`
`60
`
`In one aspect the invention relates to a pharmaceutical
`formulation comprising a GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is GLP-1 (7-37) or an ana
`logue thereof wherein an amino acid residue of the parent
`peptide has a lipophilic Substituent attached optionally via a
`spacer, wherein said GLP-1 compound is present in a con
`
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`MYLAN INST. EXHIBIT 1107 PAGE 5
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`MYLAN INST. EXHIBIT 1107 PAGE 5
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`US 8,846,618 B2
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`10
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`55
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`5
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached 5
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, and a buffer, wherein said
`GLP-1 compound is present in a concentration from 0.1
`mg/ml to 100 mg/ml, and wherein said formulation has a pH
`from 7.0 to 10.
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached 15
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, and a buffer, wherein said
`GLP-1 compound is present in a concentration from 1 mg/ml
`to 100 mg/ml, and wherein said formulation has a pH from 7.0
`to 10.
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached 25
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, water, and a buffer, wherein
`said GLP-1 compound is present in a concentration from 0.1
`mg/ml to 100 mg/ml, and wherein said formulation has a pH
`from 7.0 to 10.
`30
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached 35
`optionally via a spacer, comprising preparing a formulation
`containing the GLP-1 compound, water, and a buffer, wherein
`said GLP-1 compound is present in a concentration from 1
`mg/ml to 100 mg/ml, and wherein said formulation has a pH
`from 7.0 to 10.
`40
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached
`optionally via a spacer, comprising preparing an aqueous
`Solution containing the GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is present in a concentration
`from 0.1 mg/ml to 100 mg/ml, and wherein said formulation
`has a pH from 7.0 to 10.
`50
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached
`optionally via a spacer, comprising preparing an aqueous
`Solution containing the GLP-1 compound, and a buffer,
`wherein said GLP-1 compound is present in a concentration
`from 1 mg/ml to 100 mg/ml, and wherein said formulation
`has a pH from 7.0 to 10.
`In a further aspect the invention relates to a method of
`preparing a physically stable pharmaceutical formulation of a
`GLP-1 compound wherein said GLP-1 compound is GLP-1
`(7-37) or an analogue thereof, wherein an amino acid residue
`of the parent peptide has a lipophilic substituent attached
`optionally via a spacer, comprising preparing a formulation
`co