(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2002/0061838A1
`Holmquist et al.
`(43) Pub. Date:
`May 23, 2002
`
`US 2002006 1838A1
`
`(54)
`
`PEPTIDE PHARMACEUTICAL
`FORMULATIONS
`
`(76)
`
`(21)
`(22)
`
`(63)
`
`Inventors: Barton Holmquist, Lincoln, NE (US);
`Daniel C. Dormady, Omaha, NE (US)
`Correspondence Address:
`Beth A. Burrous
`FOLEY & LARDNER
`Suite 500
`3000 K Street, N.W.
`Washington, DC 20007-5109 (US)
`Appl. No.:
`09/858,880
`Filed:
`May 17, 2001
`Related U.S. Application Data
`Non-provisional of provisional application No.
`60/205,377, filed on May 17, 2000. Non-provisional
`
`of provisional application No. 60/205,262, filed on
`May 19, 2000.
`
`Publication Classification
`
`(51) Int. Cl." .......................... A61K 38/18; A61K 38/29
`(52) U.S. Cl. .................................................................. 514/2
`
`(57)
`
`ABSTRACT
`
`A pharmaceutical composition for administration to a mam
`mal is disclosed. The composition includes a therapeutically
`effective amount of a peptide, Such as a GLP-1 molecule, a
`PTH molecule, or a GRF molecule. The composition further
`includes a buffer including a weak acid having an acid
`dissociation constant value of greater than about 1x10,
`Such as acetic acid. The composition also includes an
`excipient for making the composition generally isotonic,
`Such as D-mannitol.
`
`Novo Nordisk A/S Ex. 2012, P. 1
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 1 of 8
`
`US 2002/0061838A1
`
`
`
`ar s
`E
`
`eouequOSCW
`
`eoued JOSCW
`
`Novo Nordisk A/S Ex. 2012, P. 2
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 2 of 8
`
`US 2002/0061838A1
`
`CY)
`
`O
`of
`
`O
`O
`CN v.
`v.
`CN
`u/fu 'Aqn IOS
`
`O
`
`Novo Nordisk A/S Ex. 2012, P. 3
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 3 of 8
`
`US 2002/0061838A1
`
`
`
`O
`wn
`
`O.
`O O.
`O.
`O.
`O.
`Vr O O
`OO N CO
`v
`V
`?
`% 'AA3eo
`
`CY)
`
`O
`S
`S
`
`?
`
`O
`O.
`
`wim
`O
`O
`E
`ds
`o E
`Y
`
`5
`
`O)
`
`O
`O)
`
`OO
`
`% “Alund
`
`O
`
`Novo Nordisk A/S Ex. 2012, P. 4
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 4 of 8
`
`US 2002/0061838A1
`
`V
`O
`S
`
`?
`
`(
`
`s
`% "AIAIoeos
`
`()
`S
`e -
`2
`N
`CD
`E
`H
`
`f
`
`ed
`
`lif
`
`lf
`CN
`
`2 S
`%“Aund
`
`Novo Nordisk A/S Ex. 2012, P. 5
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 5 of 8
`
`US 2002/0061838A1
`
`3
`
`s
`
`i
`
`5
`% “Alund
`
`Novo Nordisk A/S Ex. 2012, P. 6
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 6 of 8
`
`US 2002/0061838A1
`
`i
`
`O
`O
`
`CO
`o
`
`N
`O
`
`O
`C)
`
`O)
`
`% 'epided go ee IW
`
`Novo Nordisk A/S Ex. 2012, P. 7
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May 23, 2002 Sheet 7 of 8
`
`US 2002/0061838A1
`
`O
`ve
`CN
`
`O
`CO
`ve
`
`S
`- E
`(N ds
`t
`is
`P
`
`O
`O
`
`O
`
`O
`
`GN
`
`ve
`
`Ifd-19) euSedle
`
`N
`O
`S
`S
`(vs
`?
`
`CY)
`
`Novo Nordisk A/S Ex. 2012, P. 8
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`Patent Application Publication May
`23, 2002. Sheet 8 of 8
`
`US 2002/0061838 A1
`
`O
`Y)
`
`O
`CN
`
`O
`V
`
`C
`S
`r
`O)
`E
`CN He
`
`OO
`O)
`S
`3
`?a
`
`(s
`
`<
`
`NU
`
`O O O
`O
`O O
`GN
`GN
`o
`se
`ulfu's eused
`
`v
`
`O
`O
`
`Novo Nordisk A/S Ex. 2012, P. 9
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`PEPTIDE PHARMACEUTICAL FORMULATIONS
`0001. This application claims priority to U.S. Ser. No.
`60/205,377, filed May 17, 2000 and U.S. Ser. No. 60/205,
`262, filed May 19, 2000, both of which are incorporated by
`reference.
`
`FIELD OF THE INVENTION
`0002 The present invention generally relates to pharma
`ceutical formulations for peptides. More Specifically, the
`present invention relates to pharmaceutical formulations of
`a peptide, Such as a glucagon-like peptide-1 (GLP-1), a
`parathyroid hormone (PTH) or a growth hormone releasing
`factor (GRF), or a pharmaceutically active derivative or
`analog of Such peptides, an acidic buffer and mannitol. The
`novel formulations, for example, are well-tolerated by
`humans, and are, for example, Surprisingly stable composi
`tions, the Soluble peptides do not dimerize or aggregate.
`
`BACKGROUND OF THE INVENTION
`0003) Peptides such as GLP-1, PTH, and GRF are known
`in the art to be useful for treating a variety of disorders. For
`example, GLP-1 (7-36)amide is useful for treating type II
`diabetes (also known as Non-Insulin Dependent Diabetes
`Mellitus, NIDDM). PTH(1-34) is useful for treating
`osteoporosis, as is GRF(1-44)amide. See U.S. Pat. No.
`4,870,054. A combination of PTH(1-34) and GRF(1-
`44)amide can also be used to treat Osteoporosis. See U.S.
`Pat. No. 5,164,368.
`0004. There is a variety of art-recognized problems asso
`ciated with formulating Such peptides into pharmaceutically
`acceptable compositions. It is important to have a Suffi
`ciently high concentration of peptide that is Soluble and that
`forms minimal peptide aggregates and peptide dimers. It is
`known in the art that the formation of Such aggregates and
`dimerS is a Significant problem encountered in making
`pharmaceutical formulations from peptides Such as GLP-1.
`For example, GLP-1 is known to gel and aggregate under
`numerous conditions, making it difficult to make Stable
`soluble peptide formulations. See EP 097.8565 A1.
`0005) A variety of pharmaceutical formulations compris
`ing GLP-1, PTH and GRF have been described in the art.
`Such peptides have generally been administered by dissolv
`ing the peptide in water containing albumin or other adju
`vants and injecting it into a human (Creutzfeldt et al.,
`Diabetes 19, 1 (1996); Ahren et al., J. Clin. Endo. Metab. 82,
`473 (1997)). This procedure has disadvantages because such
`peptides are not stable or Sufficiently Soluble under Such
`conditions (near neutral pH values), and adjuvants, Such as
`albumin, are unstable at acidic pH values.
`0006 Moreover, it is known in the art that it is desirable
`to use pharmaceutical formulations that are at physiological
`pH, to minimize adverse side effects and discomfort to
`patients. See Brazeau et al., J. Pharm. Sci., 87,667 (1998).
`However, at physiological pH (about pH 7.4), the solubili
`ties of GLP-1, PTH, and GRF are low. For example, the
`solubility of the peptide GLP-1 in water at a pH of about 7.4
`is less than about 0.2 mg/mL. The solubility of GLP-1 in
`physiological saline is also low. The solubilities of PTH and
`GRF at physiological pH are higher, up to 4 mg/mL.
`0007 To increase peptide solubility at physiological pH,
`prior art formulations have used various art-recognized
`agents, Such as detergents and Solvents. The use of Such
`agents is not desirable, however, because they can cause
`
`adverse side effects in patients. See Brazeau et al., J. Pharm.
`Sci. 87, 667 (1998). Also, human serum albumin has been
`used in GLP-1 formulations because of its buffering capa
`bilities and to reduce adsorption of GLP-1 to the storage
`container or devices used for administration. GLP-1 is a
`hydrophobic peptide that adsorbs to hydrophobic Surfaces
`that are found on, for example, tubing and Syringes. How
`ever, it is not desirable to use human Serum albumin because
`it can Stimulate adverse immune reactions in a patient. Also,
`great care must be taken to use highly purified albumin, to
`minimize contaminants that can also cause unwanted Side
`effects.
`0008. The stability of an amide bond generally is greatest
`at a pH in the range of about 4.0 to 4.5. However, such a pH
`range often cannot be used for formulations of therapeutic
`peptides. A low pH can result in denaturation of peptides that
`have tertiary or quaternary Structure and/or can result in
`peptide inactivation. Moreover, low pH pharmaceutical for
`mulations are known to cause discomfort to patients, upon
`injection. See Brazeau et al., J. Pharm. Sci. 87,667 (1998).
`0009 U.S. Pat. No. 5,705,483 describes a formulation of
`GLP-1 that is combined with distilled water and adjusted to
`a pH of about 6.0 to 9.0. The 483 patent states that
`D-mannitol is an example of a suitable excipient for GLP-1.
`However, the high pH recited in the 483 patent formulation
`may contribute to the instability of GLP-1.
`0010 PCT Application WO 98/19698 describes a com
`bination of 100 nmol GLP-1 (7-36)amide and 0.025 mL
`human albumin solution (20%), with the pH adjusted to 4
`using 5 Macetic acid. The volume of this formulation was
`brought to 1 mL using normal Saline for administration to
`the abdomen of a human making the concentration of GLP-1
`100 uM (about 0.3 mg/mL). However, as noted above, it is
`desirable to not use albumin in pharmaceutical formulations.
`0.011) The 1999 Physician's Desk Reference (pp. 532
`539) describes NEUPOGEN, commercially available from
`Amgen Inc., California. The PDR entry states that NEUPO
`GEN is the name of the drug product that is a formulation
`of filgrastim, a human granuloctye colony Stimulating factor
`(G-CSF) produced by recombinant DNA technology, Suit
`able for pharmaceutical use in Stimulating white blood cell
`production. The PDR entry states that NEUPOGEN is
`formulated in a 10 mM sodium acetate buffer at pH 4.0,
`containing 5% sorbitol and 0.004% TWEEN 80. TWEEN 80
`is an emulsifying, Wetting, and dispersing agent (i.e., deter
`gent), commercially available from Atlas Powder Company,
`Delaware. The PDR entry further states that the quantitative
`composition (per mL) of NEUPOGEN is: filgrastim 300
`mcg., acetate 0.59 mg, sorbitol 50 mg, TWEEN 800.004%,
`Sodium 0.035 mg, water for injection USP q.S. in 1.0 mL.
`G-CSF is a protein that is 175 amino acids long, and, as
`noted, the NEUPOGEN formulation contains detergent.
`0012. Accordingly, there is a need in the art for stable
`pharmaceutical formulations of relatively Small peptides,
`Such as GLP-1, PTH and GRF, that contain minimal levels
`of non-therapeutic adjuvants (Such as albumin, detergents,
`and Solvents) because this can cause adverse side effects. It
`would also be advantageous to provide effective Stable
`pharmaceutical formulations that are well tolerated by
`humans, i.e., cause minimal patient discomfort. It further
`would be advantageous to provide peptide formulations
`having acceptable concentrations, that are Soluble, and
`
`Novo Nordisk A/S Ex. 2012, P. 10
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`include minimal or no peptide dimers and/or aggregates. AS
`noted, GLP-1 is known to gel and aggregate under numerous
`conditions, making stable formulation difficult. See EP
`097.8565A1. Other advantages of the claimed invention will
`become apparent to those skilled in the art upon review of
`the Specification and the appended claims.
`
`SUMMARY OF THE INVENTION
`0013 To provide stable peptide pharmaceutical formula
`tions that are well tolerated by patients and that have
`minimal non-peptide components, the present inventors
`have developed pharmaceutical formulations comprising a
`peptide, a buffer, and a diluent. In particular, the present
`inventors have developed Stable pharmaceutical composi
`tions for administration to a mammal of peptides Such as
`GLP-1 (7-36)amide, PTH(1-34)OH, or GRF(1-44)amide,
`each prepared in acetic acid and D-mannitol.
`0.014.
`It is therefore an object of the present invention to
`provide a Stable unit dose of a pharmaceutical composition
`that provides for good Stability of the peptide for adminis
`tration to a mammal including a peptide, a buffer, and a
`diluent.
`0.015. It is another object of the present invention to
`provide a method for treating an illness or disease in a
`mammal using a pharmaceutical composition that is well
`tolerated by the mammal for administration to the mammal
`including a peptide, a buffer and a diluent. In accomplishing
`these and other objects, there has been provided in accor
`dance with one aspect of the present invention a unit dose of
`a pharmaceutical composition for administration to a mam
`mal. The composition includes a therapeutically effective
`amount of a peptide; the composition also includes a buffer
`comprising an acid having a pKa less than about 5, or leSS
`than 5. In particular, the inventive formulations comprise
`acetic acid. The formulations also include a diluent to make
`the composition isotonic. In particular, the inventive formu
`lations comprise D-mannitol.
`0016. In a preferred embodiment, the composition con
`Sists essentially of a peptide, a buffer comprising an acid
`having a pKa less than about 5, or less than 5, and a diluent
`Such as D-mannitol.
`0.017. In another preferred embodiment, the composition
`consists of peptide, a buffer comprising an acid having a pKa
`less than about 5 or less than 5, and a diluent.
`0.018. In one preferred embodiment, the inventive formu
`lations comprise a peptide, acetic acid, and D-mannitol. In
`another preferred embodiment, the inventive formulations
`consist essentially of a peptide, acetic acid, and D-mannitol.
`In another preferred embodiment, the inventive formulations
`consist of a peptide, acetic acid, and D-mannitol.
`0.019
`All of these formulations preferably have a pH
`between about 3.0 and about 5.0 or between 3.0 and 5.0,
`more preferably, between about 4.0 and about 5.0 or
`between 4.0 and 5.0; more preferably between about 4.5 and
`about 5.0 or between 4.5 and 5.0; most preferably between
`about 4.5 and about 4.7 or between 4.5 and 4.7. Other
`preferred embodiments have a pH of 4.5, 4.6, or 4.7.
`0020. In accordance with another aspect of the present
`invention, a System for administering an effective amount of
`a pharmaceutical formulation to a mammal is disclosed. The
`System includes an infusion pump for administering a unit
`dose of a pharmaceutical formulation of the invention. The
`unit dose includes a therapeutically effective amount of a
`
`peptide having a molecular weight of between about 200 to
`50,000 atomic mass units, including, for example, a GLP-1
`molecule, a GRF molecule, or a PTH molecule.
`0021. In accordance with another aspect of the present
`invention, a method for the treatment of a disease in a
`mammal having the disease is disclosed. The method
`includes administering to the mammal an effective amount
`of a pharmaceutical composition of the invention.
`0022. Further objects include the following. A pharma
`ceutical composition comprising (1) a molecule Selected
`from the group consisting of a GLP1 molecule, and GRF
`molecule, and a PTH molecule; (2) an acid having a disso
`ciation constant value of greater than 1x10; and (3) an
`excipient, wherein the pH of the composition is between
`about 3.0 and 5.0. The above composition, wherein the acid
`comprises acetic acid. The above composition, wherein the
`excipient is D-mannitol. The above composition wherein the
`acid is acetic acid and the excipient is D-mannitol. The
`above composition, wherein the composition comprises
`GLP-1 (7-36)amide. The above composition, wherein the
`composition comprises GRF(1-44)amide. The above com
`position, wherein the composition comprises PTH(1-34)OH.
`The above composition, wherein the composition is in unit
`dosage form. The above composition, wherein the compo
`Sition is Sterile. A System for administering a pharmaceutical
`composition comprising: an infusion pump for administer
`ing a unit dose of the above composition. The above System,
`wherein the composition is diluted up to about 40-fold with
`isotonic Saline prior to administration. A method for the
`treatment of a disease or condition in a mammal comprising
`administering to the mammal a pharmaceutically effective
`amount of an above composition. The method above,
`wherein the disease or condition is Selected from the group
`consisting of diabetes, exceSS appetite, obesity, Stroke,
`ischemia, reperfusion injury, disturbed glucose metabolism,
`Surgery, coma, Shock, gastrointestinal disease, digestive
`hormone disease, atherosclerosis, Vascular disease, gesta
`tional diabetes, liver disease, liver cirrhosis, glucorticoid
`excess, Cushings disease, the presence of activated coun
`terregulatory hormones that occur after trauma or a disease,
`hypertriglyceridemia, chronic pancreatitis, the need for
`parenteral feeding, osteoporosis, and a catabolic State fol
`lowing Surgery or injury. The above method, wherein the
`composition is administered to the mammal by a method
`Selected from the group consisting of intravenous, Subcuta
`neous, continuous, intermittent, parenteral, and combina
`tions thereof. The above composition, wherein the compo
`sition has a pH of about 4.5. The above composition,
`wherein the composition has a pH of about 4.7. The above
`composition, wherein the composition has a pH of between
`about 4.5 and 4.7. The above composition, wherein the
`composition has a pH of 4.5. The above composition,
`wherein the composition has a pH of 4.7. The above
`composition, consisting essentially of acetic acid, D-man
`nitol, and a molecule Selected from the group consisting of
`a GLP1 molecule, and GRF molecule, and a PTH molecule,
`wherein the composition is in liquid form. The above
`composition, consisting of acetic acid, D-mannitol, and a
`molecule Selected from the group consisting of a GLP1
`molecule, and GRF molecule and a PTH molecule, wherein
`the composition is in liquid form. The above composition,
`comprising acetate (about 10 mM) and D-mannitol (about
`50.7 mg/mL). The above composition, consisting essentially
`of acetate (about 10 mM), D-mannitol (about 50.7 mg/mL),
`and a molecule Selected from the group consisting of a GLP1
`molecule, and GRF molecule, and a PTH molecule. The
`above composition, comprising acetate (about 10 mM),
`
`Novo Nordisk A/S Ex. 2012, P. 11
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`D-mannitol (about 50.7 mg/mL), and GLP-1(7-36)amide
`(about 1 mg/mL). The above composition, consisting essen
`tially of acetate (about 10 mM), D-mannitol (about 50.7
`mg/mL), and GLP-1(7-36)amide (about 1 mg/mL). The
`above composition, wherein the composition comprises
`acetate (about 10 mM), D-mannitol (about 50.7 mg/mL),
`and GRF(1-44)amide (about 4 mg/ml). The above compo
`Sition, consisting essentially of acetate (about 10 mM),
`D-mannitol (about 50.7 mg/mL), and GRF(1-44)amide
`(about 4 mg/ml). The above composition, wherein the com
`position comprises acetate (about 10 mM), D-mannitol
`(about 50.7 mg/mL), and PTH(1-34)OH (about 50 mg/mL).
`The above composition, wherein the composition consists
`essentially of acetate (about 10 mM), D-mannitol (about
`50.7 mg/mL), and PTH(1-34)OH (about 50 mg/mL). The
`above System, wherein the pump is programmed to release
`the molecule at a rate of about 10 or more u per hour.
`0023. Further objects, features and advantages of the
`invention will be apparent from the following detailed
`description taken in conjunction with the accompanying
`drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`0024 Drawing 1
`0025) Examples of the use of reverse phase HPLC for
`peptide purity analysis and illustrating the capacity to moni
`tor the degradation of peptides. Samples were analyzed by
`reversed phase HPLC by elution with water/acetonitrile
`gradients in 0.1% trifluoroacetic acid. The HPLC system
`used was an HP 1100 chromatography system. Top Panel:
`GLP-1 stored at -20°C. (dotted line) and 50° C. (solid line)
`for one month in 10 mM acetic acid, 5.07% D-mannitol,
`adjusted to pH 4.5. Elution is with a gradient of from 33%
`to 95% acetonitrile in 22 min. with a Waters Symmetry
`Reverse Phase C18 column, 4.6x250 mm. Bottom panel:
`GRF stored at -20° C. (dotted line) and 37° C. (solid line)
`for one month in 10 mM acetic acid, 5.07% D-mannitol,
`adjusted to pH 4.7. The compositions of the HPLC buffers
`A and B were 20% and 50% (v/v) acetonitrile, respectively,
`and elution was with a gradient of from 25% to 55% B in 25
`min. 5 using a Zorbax 5 micron, 4.6x250 mm column.
`0026 Drawing 2
`0027 Solubility of GLP-1 in 10 mM acetate buffer con
`taining 5.07% D-mannitol as a function of pH at 25 C.
`Solutions were stirred with excess GLP-1 for four days.
`Following centrifugation, the amount of peptide in Solution
`was determined by ultraViolet absorption spectrophotom
`etry.
`0028) Drawing 3
`0029 Stability determined by HPLC (left panel) and
`bioactivity (right panel) of GRF as a function of Storage time
`in the preferred formulation, 4 mg/mL GRF dissolved in 10
`mM sodium acetate, 5.07% D-mannitol, adjusted to pH 4.7.
`Circles represent -20° C. and squares represent 4 C.
`0030) Drawing 4
`0031) Stability of GLP-1 in the preferred formulation (1
`mg/mL GLP-1 dissolved in 10 mM sodium acetate, 5.07%
`D-mannitol, adjusted to pH 4.5), as determined by HPLC
`analysis (left panel) and bioassay (right panel). Circles
`represent -20° C. and Squares represent 4 C.
`
`0032) Drawing 5
`0033 Stability of PTH (1 mg/mL PTH dissolved in 10
`mMSodium acetate, 5.07% D-mannitol, adjusted to pH 4.7),
`as determined by HPLC analysis. Circles represent -20° C.
`and Squares represent 4 C.
`0034) Drawing 6
`0035) Stability of GLP-1 by HPLC analysis of GLP-1
`formulated in 10 mM Sodium acetate, 5.07% D-mannitol at
`pH 4.5 at 1 mg/mL. Samples were stored in glass vials at 4
`C. (Solid circles), in glass Vials at 37 C. (Squares), in the
`MiniMed polypropylene reservoir at 37° C. (diamonds), and
`samples pumped with the MiniMed pump at 37° C. (tri
`angles).
`0036) Drawing 7
`0037 Response of rats to subcutaneous injections of 120
`Aug/kg of GLP-1 in the preferred formulation (1 mg/mL
`GLP-1 dissolved in 10 mM Sodium acetate, 5.07% D-man
`nitol, adjusted to pH 4.5). Values are the average of the
`response of 4 different animals.
`0.038) Drawing 8
`0039 Total GRF detected in the plasma of a rat following
`intravenous administration of 20 lug of GRF in the preferred
`formulation (4 mg/mL GRF dissolved in 10 mM sodium
`acetate, 5.07% D-mannitol, adjusted to pH 4.7).
`
`DETAILED DESCRIPTION OF THE
`PREFERRED EMBODIMENTS
`0040. In accordance with the present invention, pharma
`ceutical formulations of a peptide, an acidic buffer and a
`diluent may be used for injection into a mammal. The
`peptide may have a molecular weight of between about 200
`to 50,000 atomic mass units. According to a preferred
`embodiment, the peptide is a GLP-1 molecule, a PTH
`molecule, a GRF molecule, or a combination thereof.
`According to alternative embodiments, the peptide may be
`a derivative or an analog of GLP-1, PTH, GRF, or a
`combination thereof. According to a particularly preferred
`embodiment, the peptide is GLP-1(7-36)amide, PTH(1-
`34)OH, or GRF(1-44)amide.
`0041) The peptide concentration(s) (whether GLP-1,
`PTH, GRF, or combinations thereof) of the formulations are
`preferably in the range of about 25 ug to 5 mg per 1 mL of
`the combination of buffer and diluent.
`0.042 GLP-1
`0043. According to a preferred embodiment of the
`present invention, the peptide is a glucagon-like peptide
`1(7-36)amide. This molecule is a natural incretin hormone
`Secreted from the L-cells of the ileum. It assists in the
`regulation of insulin Secreatory rates and has a profound
`effect on glucose homeostasis. GLP-1 also acts Systemically
`to Suppress free fatty acids and to facilitate normalization of
`blood glucose levels through a large number of endocrine
`functions, including the control and expression of insulin
`from the pancreatic B-cells, and the Suppression of glucagon.
`The term “GLP-1 molecule” as used in the context of the
`present invention includes glucagon-like peptides, analogs
`of glucagon-like peptide-1, and derivatives of glucagon-like
`peptide-1, that bind to glucagon-like peptide-1 receptor
`proteins.
`
`Novo Nordisk A/S Ex. 2012, P. 12
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`Sequence of GLP-1 (7-36) amide (Sec. 1) :
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys
`
`Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH2.
`
`0044 According to an alternative embodiment of the
`present invention, an analog of GLP-1 may be used Such as
`the GLP-1 derivatives:
`
`5.0, the solubility of GLP-1 in 10 mM sodium acetate,
`5.07% D-mannitol is generally above 1 mg/mL, allowing
`effective doses for S.c. and i.v. injections. The present
`
`Sequence of GLP-1 (7-36.) OH (Seg. 2) :
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln
`Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-OH
`
`Sequence of GLP-1 (7-34) OH (Sec. 3) :
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln
`Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-OH
`
`Sequence of GLP-1 (7-37) OH (Seg. 4)
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln
`Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH
`
`0.045. Other GLP-1 analogs are known in the art. For
`example, U.S. Pat. No. 5,958,409 describes suitable GLP-1
`analogs. According to other alternative embodiments, the
`peptide may be a GLP-1 derivative such as alkylated or
`acylated GLP-1 derivatives or other analogs. Analogs of
`GLP-1 that are homologous, including the eXendins, Such as
`exendin 3 and 4, and GLP-2, are also included in the
`invention. According to a particularly preferred embodi
`ment, the GLP-1 molecule is GLP-1 (7-36)amide, having the
`amino acid Sequence Seq 1.
`0.046 A factor that may play a role in the stability of the
`GLP-1 formulations is the concentration of the GLP-1
`molecule. The solubility profile as a function of pH of
`GLP-1 is shown in Drawing 2. At pH values below about
`
`inventors have determined that a GLP-1 (7-36)amide con
`centration of about 1 mg/mL was Stable in the inventive
`formulations at pH 4.5, for up to 6 months at 25 C. with
`~4% degradation. This stability was evidenced by the mini
`mal amount of breakdown products (e.g., acid cleavage and
`beta shifts at aspartic acid) over time determined by HPLC
`methods. See Drawing 4. A particularly stable formulation
`includes about 0.1 to 4 mg/mL of a GLP-1 molecule.
`
`0047. Also included in “GLP-1 molecules” of the present
`invention are Six peptides in Gila monster Venoms that are
`homologous to GLP1. Their Sequences are compared to the
`sequence of GLP1 in the following table.
`
`TABLE
`
`Position 1
`
`a.
`
`H A E G T F T S D W S S Y L E G Q
`
`A K E F I A W L V K G R (NH)
`
`b
`
`H S D G T F T S D L S K Q M. E. E. E.
`
`V R L. F I E W L K N G G P S S G A P P P S (NH)
`
`c
`
`D L S K Q M. E. E. E.
`
`V R L. F I E W L K N G G P S S G A P P P S (NH)
`
`d
`
`H. G E G T F T S D L S K Q M. E. E. E.
`
`V R L. F I E W L K N G G P S S G A P P P S (NH)
`
`e H S D A T F. T. A. E. Y S K L. L. A. K. L.
`
`L Q KY L E S I L G S S T S P R P P S S
`
`f
`
`H S D A T F. T. A. E. Y S K L. L. A. K. L.
`
`L Q KY L E S I L G S S T S P R P P S
`
`g. H S D A F T E E Y S K L. L. A. K. L.
`
`L Q K Y L. A. S I L G S R T S P P P (NH)
`
`h. H S D A F T Q Q Y S K L L A K L
`
`L Q K Y L. A S I L G S R T S P P P (NH)
`
`= GLP-1 (7-36) amide.
`= exendin 3.
`= exendin 4 (9-39) (NH2).
`= exendin 4.
`held spectin I.
`= held spectin II.
`= helodermin.
`= Q8, Q9 helodermin.
`
`:
`
`Novo Nordisk A/S Ex. 2012, P. 13
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`0.048. The peptides c and h are derived from b and g,
`respectively. All 6 naturally occurring peptides (a, b, d, e, f,
`and g) are homologous in positions 1, 7, 11 and 18. GLP
`1(7-36)amide and exendins 3 and 4 (a, b, and d) are further
`homologous in positions, 4, 5, 6, 8, 9, 15, 22, 23, 25, 26 and
`29. In position 2, A, S and G are structurally similar. In
`position 3, residues D and E (Asp and Glu) are structurally
`similar. In positions 22 and 23, F (Phe) and I (Ile) are
`structurally similar to Y (Tyr) and L (Leu), respectively.
`Likewise, in position 26, Land I are structurally equivalent.
`0049. Thus, of the 30 residues of GLP1, exendins 3 and
`4 are identical in 15 positions and equivalent in 5 additional
`positions. The only positions where major Structural changes
`are evident are at residues 16, 17, 19, 21, 24, 27, 28 and 30.
`Exendins also have 9 extra residues at the carboxyl terminus.
`0050). PTH
`0051. According to another preferred embodiment of the
`present invention, the peptide is a PTH molecule. The term
`“PTH molecule” as used in the context of the present
`invention includes parathyroid hormones, analogs of par
`athyroid hormones, and derivatives of parathyroid hor
`mones. PTHs are regulatory factors in the homeostatic
`control of calcium and phosphate metabolism. The principal
`sites of PTH activity are believed to be the skeleton, kidneys,
`and gastrointestinal tract.
`
`Sequence of human PTH (1-34) (Seg. 5):
`
`Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu
`
`Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe
`
`0.052 According to an alternative embodiment of the
`present invention, an analog of PTH may be used. PTH
`analogs are known in the art. For example, U.S. Pat. No.
`5,840,837 describes suitable PTH analogs. According to
`other alternative embodiments, the peptide may be a PTH
`derivative such as PTH(1-84), PTH(1-37) and C-terminal
`amidated derivatives of PTH or its derivatives, as examples.
`According to a particularly preferred embodiment, the pep
`tide is PTH(1-34), a natural human PTH (Seq 5).
`
`0053. The present inventors have determined that a con
`centration of about 0.005 to 1.0 mg/mL of the PTH molecule
`was stable for 4 months at 4 C. in the inventive formula
`tions. A particularly stable formulation includes about 0.02
`to 0.10 mg/mL of PTH.
`
`0054 GRF
`0055 According to another preferred embodiment of the
`present invention, the peptide is GRF(1-44)amide (GRF).
`GRF is a peptide of 44 amino acids. GRF is one of a group
`of peptides Secreted by the hypothalamus, and is believed to
`Stimulate pituitary growth hormone release. GRF may be
`important in normal growth and development during child
`hood, and may mediate (together with Somatostatin) the
`neuroregulation of GH secretion. GRF is an attractive mol
`ecule for the treatment of postmenopausal osteoporosis, and
`other indications because it is relatively Small, and therefore
`can be effective when given by nasal insufflation using an
`appropriate vehicle.
`0056. The term “GRF molecule” as used in the context of
`the present invention includes growth hormone releasing
`factor, analogs of growth hormone releasing factor, and
`derivatives of growth hormone releasing factor, that bind to
`a growth hormone releasing factor receptor protein.
`
`Sequence of GRF (1-44) amide (Sec. 6):
`
`Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg
`
`Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg
`
`Ala-Arg-Leu-NH2.
`
`Novo Nordisk A/S Ex. 2012, P. 14
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`US 2002/0061838A1
`
`May 23, 2002
`
`0057 According to an alternative embodiment of the
`present invention, an analog of GRF may be used. GRF
`analogs that have biological activity are known in the art and
`generally contain about 27 to about 44 amino acids, but Such
`analogs may be somewhat less potent than GRF. For
`example, Kubiak et al., J. Med Chem. 36, 888 (1993)
`describes suitable GRF analogs. Examples of GRF analogs
`that are included are GRF(1-44)-OH, GRF(1-40)-OH,
`GRF(1-40)-NH,
`GRF(1-32)-NH,
`GRF(1-39)-NH,
`GRF(1-40)-Phe-NH, GRF(1-40)-Phe-OH, GRF(1-40)-Phe
`Gln-NH, GRF(1-29)-NH, and GRF(1-27)-NH, and com
`binations thereof. According to other alternative embodi
`ments, the peptide may be a GRF derivative such as detailed
`by Kubiak et al. above. According to a particularly preferred
`embodiment, the peptide is GRF (1-44) amide having the
`amino acid Sequence of Seq. 6. A particularly Stable formu
`lation for GRF includes about 1.0 to 10.0 mg/mL of GRF.
`0.058
`Buffer
`0059) The buffer of the formulations should have a pH
`that is slightly acidic. Without intending to be limited by any
`particular theory, it is known to those skilled in the art that
`acidic conditions increase the Stability of the amide bond of
`the peptide. Acidic conditions are provided by a generally
`weak acid. An acid is a generally weak acid if it has an acid
`dissociation constant value of greater than about 1x10, or
`greater than 1x10, i.e., a pKakabout 5, or a kPa-5. Such
`acids may include propionic, Succinic, malic acids, and
`combinations thereof. According to a particularly preferred
`embodiment, the acid is acetic acid. According to an alter
`native embodiment, th