(12) United States Patent
`Isaacs
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 7,056,886 B2
`Jun. 6, 2006
`
`US007056886B2
`
`(54) GLP-2 FORMULATIONS
`
`5,652,216 A * 7/1997 Kornfelt et a1. ............ .. 514/12
`
`5,912,229 A * 6/1999 Thim et a1. . . . . . . .
`
`. . . .. 514/12
`
`Inventor: Indu JI Isaacs, AndOVer, MA
`
`
`
`A * 5,997,856 A * 12/1999 Hora et a1. .............. .. 424/852 Drucker . . . . . . . ..
`
`
`
`(73) Assignee: NPS Allelix, Corp” Mississauga (CA)
`
`6,120,761 A * 9/2000 Yamazaki et a1. ....... .. 424/851
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U-S-C- 154(1)) by 628 days-
`
`(21) Appl. No.: 09/750,022
`(22) Filed:
`Dec. 29, 2000
`_
`_
`_
`Pnor Pubhcatlon Data
`US 2001/0027180 A1 Oct. 4, 2001
`
`(65)
`
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`97/39031
`10/1997
`98/03547
`1/1998
`99/43361
`9/1999
`
`OTHER PUBLICATIONS
`
`Buhl et al., Naturally Occurring Products of Proglucagon
`1114160 in the Porcine and Human Samll Intestine, J. Biol.
`Chem 263, 8621*8624 (1988)-*
`
`.
`.
`.
`.
`.
`D t
`F
`P
`t
`A l
`30
`orelgn PP lea Ion nonty a a
`(
`)
`Dec. 30, 1999
`(GB) ........................................... .. 9930882
`
`Lund et al. Angler?sh Islet Preiproglucagon II Nucleotide
`’
`’
`and corresponding amino acid Sequenmce of the cDNA, J.
`Biol_ Chem, 258, 328043284 (1983),*
`
`(51) Int. Cl.
`A61K 38/00
`A61K 38/26
`C07K 14/00
`
`(52) U 5 Cl
`
`(2006.01)
`(2006.01)
`2006.01
`(
`)
`514/12_ 530/308 530/399
`530/324; 435/4; 435/287.1
`
`~
`* ~t d b
`C1 e y exammer
`
`Primary Examinerilon Weber
`Assistant Examiner4Chih-Min Kam
`(74) Attorney, Agent, or FirmiFoley & Lardner LLP
`(57)
`ABSTRACT
`
`(58) Field of Classi?cation Search ................. .. 514/12;
`
`530/308, 399, 324; 435/4’ 2871
`See application ?le for Complete Search history
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`_
`
`_
`
`The 1nvent1on1s dlrected to formulatlons of GLP-2 peptldes
`and analogs thereof exhibiting superior stability following
`storage and/or exposure to elevated temperatures. The
`GLP-2 compositions comprise a GLP-2 peptide or an analog
`thereof, a phosphate buffer, L-histidine, and mannitol.
`
`_
`
`_
`
`_
`
`_
`
`4,985,244 A * l/l99l Makino et a1. ............. .. 424/89
`
`75 Claims, 6 Drawing Sheets
`
`CFAD Exhibit 1003
`
`1
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 1 6f 6
`
`US 7,056,886 B2
`
`Figure 1. Amino acids screening in buffers using heat stress.
`
`E‘
`‘a
`“a =\
`
`100
`
`95 _
`
`90 _
`
`1
`
`3
`
`7
`
`9
`
`11
`
`13
`
`‘Before heat 99.2 99.14 99.24 99.23 99.14 99.16
`‘After heat
`97.46 96.72 97.82 97.35 97.39 97.13
`Formulation
`
`Formulation
`
`1
`3
`
`7.
`9
`
`10 mM Phosphate, 10 mM Glu
`10 mM Phosphate, 10 mM Citrate
`
`10 mM Phosphate, 10 mM Ser
`10 mM Phosphate, 10 mM Pro
`
`11.
`13.
`
`10 mM Phosphate, 10 mM His
`10 mM Phosphate, 10 mM Gly
`
`2
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 2 0f 6
`
`US 7,056,886 B2
`
`Figure 2. Screening of buffers using heat stress.
`
`% Purit
`
`I 0 hours
`I 4 hours
`
`Phos
`
`Hist + Phos
`
`I 0 hours
`I 4 hours
`
`99.36
`99.18
`
`99.4
`93.35
`
`99.21
`97.43
`
`3
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 3 of 6
`
`US 7,056,886 B2
`
`Figure 3. Screening of Bulking agents analyzed by RP-HPLC.
`
`100
`
`3*
`‘i
`=1
`9.4
`°\°
`
`98.86 98.83 98.91 99.03 98.92 98.84 98.66
`RT
`!60 Deg. 94.26 94.11
`94
`93.65 89.5 91.88 91.64
`Formulation
`
`J
`
`Formulation
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`25 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`50 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`75 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`25 mM Histidine, 25 mM phosphate, 3% sucrose
`
`25 mM Histidine, 25 mM phosphate, 3% trehalose
`
`25 mM Histidine, 25 mM phosphate, 3% maltose
`
`25 mM Histidine, 25 mM phosphate, 3% lactose
`
`4
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 4 6f 6
`
`US 7,056,886 B2
`
`Figuxe 4. Bulking agents analyzed by SE-HPLC.
`
`3:‘
`;
`
`g
`9"
`
`100
`
`80
`
`20
`
`0
`
`“Main Peak
`
`‘Heat Stressed
`Main Peak
`
`‘HMW Pe k
`a
`
`'?ii‘ii‘éilf“
`
`100 100 100 100 100 93.9192.06
`Main Peak
`‘Heat Stressed 100 100 100 100 100 78.75 80.61
`Main Peak
`-
`‘HMW Peak
`.Heat Stressed
`HMW Peak
`
`6.09 7.94
`21.25 19.37
`
`_
`
`F0 m?a'ion
`
`Formulation
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`25 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`50 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`75 mM Histidine, 35 mM phosphate, 3% Mannitol
`
`25 mM Histidine, 25 mM phosphate, 3% sucrose
`
`25 mM Histidine, 25 mM phosphate, 3% trehalose
`
`25 mM Histidine, 25 mM phosphate, 3% maltose
`
`25 mM Histidine, 25 mM phosphate, 3% lactose
`
`5
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 5 6f 6
`
`US 7,056,886 B2
`
`Figure 5. Stability of liquid formulations stored at 4 °C.
`
`a‘
`-:
`5
`G-i
`°\°
`
`l
`g
`
`98.98
`98.62
`98.16
`98.28
`98.32
`97.85
`97.64
`
`2
`
`3
`
`98.65
`98.77
`79.73
`84.29
`68.95
`79.59
`55.95
`78.5
`47.35
`76.02
`39.44
`69.81
`30.05
`67.75
`Formulations
`
`98.93
`98.85
`99.02
`0
`88.16
`76.75
`71.18
`
`Formulations
`
`1.
`
`2.
`
`3.
`
`4.
`
`35 mM Phosphate, 50 mM Histidine, 3% Mannitol, pH 7.4
`
`35 mM Phosphate, 50 mM Histidine, 5% Sucrose, pH 7.4
`
`35 mM Phosphate, 25 mM Lysine, 3% Mannitol, pH 7.4
`
`35 mM Phosphate, 25 mM Lysine, 5% Mannitol, pH 7.4
`
`6
`
`

`

`U.S. Patent
`
`Jun. 6, 2006
`
`Sheet 6 0f 6
`
`US 7,056,886 B2
`
`Figure 6. Heat stressed samples.
`
`\
`
`i
`
`3- RT
`I 60 Deg.
`
`‘g
`:<
`
`I RT
`I 60 Deg.
`
`98.9
`96.52
`
`2
`
`3
`
`98.84
`99.06
`0.53
`43.78
`Furlu iatimr
`
`98.87
`30.98
`
`Formulations
`
`PPS"?
`
`35 mM Phosphate, 50 mM Histidine, 3% Mannito], pH 7.4
`35 mM Phosphate, 50 mM Histidine, 5% Sucrose, pH 7.4
`35 mM Phosphate, 25 mM Lysine, 3% Mannitol, pH 7.4
`35 mM Phosphate, 25 mM Lysine, 5% Mannitol, pH 7.4
`
`7
`
`

`

`US 7,056,886 B2
`
`1
`GLP-2 FORMULATIONS
`
`FIELD OF INVENTION
`The present invention provides formulations for GLP-2
`peptides and analogs thereof. In particular, the invention
`provides formulations of GLP-2 peptides and GLP-2 ana
`logs With improved stability.
`
`BACKGROUND OF THE INVENTION
`Administration of therapeutic peptides requires peptide
`formulations that remain stable during storage. In general,
`parenteral administration is used With peptides because of
`their increased siZe and subsequent di?iculty in crossing
`biological membranes. Peptides can be particularly dif?cult
`to formulate because of their tendency to degrade over time
`and/ or undergo aggregation and precipitation. Degradation,
`aggregation, and precipitation are all indicative of an
`unstable formulation. Such an unstable formulation is not
`commercially viable, as it cannot pass US. Food and Drug
`Administration approval.
`Formulation variables Which affect the degradation of
`peptides during storage include, but are not limited to, pH,
`the quantity of salts present, and the type and quantity of
`excipients. In addition, temperatures, pressures, and time for
`freeZing and drying cycles can affect the stability of a
`lyophiliZed peptide formulation. The role of most of these
`variables has been studied; hoWever, the synergistic effect of
`the variables is still poorly understood.
`Glucagon-like peptide-2 (GLP-2) is a 33 amino acid
`peptide having therapeutic applications in the treatment of
`diseases of the gastrointestinal tract. In particular, it has been
`determined that GLP-2 and analogs thereof act as trophic
`agents to enhance and maintain the functioning of the
`gastrointestinal tract and to promote groWth of intestinal
`tissue. See e.g., US. Pat. Nos. 5,834,428; 5,789,379; and
`5,990,077; and International Publication No. W0 98/ 52600.
`Commercial exploitation of GLP-2 or an analog thereof
`requires a stable GLP-2 formulation that can be readily
`prepared using a commercially acceptable process. Because
`GLP-2 is a protein, and thus far more labile than traditional
`small molecular Weight drugs, the formulation of GLP-2 or
`an analog thereof presents challenges not commonly
`encountered by the pharmaceutical industry. For example,
`methionine oxidation at position 10 and aspargine deami
`nation at position 11, 16, and/or 24 of GLP-2 are potential
`routes of degradation. Furthermore, GLP-2 or an analog
`thereof may also be adsorbed to surfaces to form aggregates
`and/ or precipitate, Which Would then render the formulation
`unstable.
`There is a need in the art for stable formulations of GLP-2
`peptides and analogs thereof Which can be prepared using a
`commercially acceptable process. The present invention
`satis?es these needs.
`
`SUMMARY OF THE INVENTION
`The present invention provides stable formulations of
`GLP-2 and analogs thereof, Which can be prepared using a
`commercially acceptable process.
`It has been discovered that relatively high concentrations
`of GLP-2 can be used in pharmaceutically acceptable for
`mulations. Moreover, it has been discovered that a pH of
`greater than about 5.5, more preferably greater than about 6,
`even more preferably from about 6.9 to about 7.9, and most
`preferably about 7.3 to about 7.4, is suitable for a stable
`formulation.
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`It has also been discovered that the GLP-2 analog h[Gly2]
`GLP-2 undergoes a phase transition between 40550 C.,
`depending upon the salt concentration, and becomes hydro
`phobic in the presence of salt. It has also been discovered
`that TWeen 80®, salt, and arginine are not suitable materials
`for producing a stable formulation for h[Gly2]GLP-2.
`According to one aspect of the present invention, there is
`provided a GLP-2 formulation comprising: (1) a medically
`useful amount of GLP-2; (2) a phosphate buffer su?icient to
`adjust the pH of the formulation to a pharmaceutically
`acceptable level, and in particular above about 6.0; (3) a
`stabiliZing amount of the amino acid L-histidine; and (4) a
`bulking agent selected from sucrose and mannitol.
`More particularly, there is provided a GLP-2 formulation
`comprising: (1) a medically useful amount of GLP-2 com
`prising from about 0.1 to about 50 mg/ml of GLP-2, pref
`erably about 5 to about 40 mg/ml, more preferably about 7
`to about 30 mg/ml, even more preferably about 10 to about
`20 mg/ml, and most preferably about 20 mg/ml; (2) a
`phosphate buffer to maintain the pH at a physiologically
`tolerable level, i.e., above 6; (3) a stabiliZing amino acid,
`particularly L-Histidine; and (4) a bulking agent, particu
`larly mannitol. All percentages described herein (except for
`percentages for Water) are Weight/volume of formulated
`product prior to lyophiliZation in gms/ml (x100). Percent
`ages for Water content are Weight/Weight of lyophiliZed
`product (x100).
`In one embodiment of the present invention, the GLP-2
`formulation is a h[Gly2]GLP-2 lyophiliZed formulation
`comprising in the reconstituted product: (1) phosphate buffer
`in an amount necessary to maintain the pH of the reconsti
`tuted product betWeen about 6.9479, and preferably in an
`amount to maintain a pH of about 7.3 to about 7.4; (2) about
`0.5 to about 1% L-histidine; (3) about 2 to about 5%
`mannitol, preferably about 2.5 to about 3.5% mannitol, and
`most preferably about 3% mannitol; and (4) from about 0.1
`to about 50 mg/ml of GLP-2 or an analog thereof, preferably
`about 5 to about 40 mg/ml, more preferably about 7 to about
`30 mg/ml, even more preferably about 10 to about 20 mg/ml,
`and most preferably about 20 mg/ml.
`In a more preferred embodiment of the invention, a
`h[Gly2]GLP-2 lyophiliZed formulation is provided compris
`ing in the reconstituted product: (1) about 7 to about 30
`mg/ml, preferably about 10 to about 20 mg/ml, and most
`preferably about 20 mg/ml of h[Gly2]GLP-2; (2) a phos
`phate buffer suf?cient to maintain the pH at about 7.3 to
`about 7.4; (3) about 0.5 to about 1% L-histidine; and (4)
`about 3% mannitol.
`In another aspect of the present invention there is pro
`vided a process for making the lyophiliZed formulation of
`GLP-2. Such a process comprises the folloWing steps:
`(a) preparing the GLP-2 formulation comprising GLP-2
`or an analog thereof, a phosphate buffer, L-histidine,
`and mannitol;
`(b) freeZing the formulation to about —400 C.;
`(c) performing a ?rst drying step at about —200 C.; and
`(d) performing a second drying step at +200 C.
`In a preferred embodiment the liquid formulation sub
`jected to the lyophiliZation process comprises:
`(1) the h[Gly2]GLP-2 analog; (2) 35 mM phosphate buffer
`to maintain the reconstituted product at a pH of about 6.9
`to about 7.9, and more preferably at a pH of about 7.3 to
`about 7.4; (3) about 0.5 to about 1% L-histidine; and (4)
`about 3% mannitol.
`According to another aspect of the present invention,
`there is provided a method for preparing a GLP-2 pharma
`
`8
`
`

`

`US 7,056,886 B2
`
`3
`ceutically acceptable formulation for parenteral
`administration, comprising the step of reconstituting the
`lyophiliZed GLP-2 formulation.
`There is further provided in accordance With the present
`invention a therapeutically useful kit comprising: (1) a
`sterile vial comprising a lyophiliZed GLP-2 formulation of
`the invention, (2) a vehicle suitable for reconstitution
`thereof, preferably sterile Water, (3) instructions for recon
`stitution; and (4) optionally instructions for administration.
`The kit may further comprise a device suitable for injection
`of the reconstituted preparation.
`Both the foregoing general description and the folloWing
`detailed description are exemplary and explanatory and are
`intended to provide further explanation of the invention as
`claimed. Other objects, advantages, and novel features Will
`be readily apparent to those skilled in the art from the
`folloWing detailed description of the invention.
`
`5
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`4
`eighteen months at 40 C. Storage stability is exhibited by
`minimal peptide degradation, preferably less than about 5%
`peptide degradation, more preferably less than about 3 to
`about 4% peptide degradation, and even more preferably
`less than about 1 to about 2% peptide degradation. Peptide
`degradation can be measured using standard reverse-phase
`HPLC (RP-HPLC) techniques.
`The naturally occurring GLP-2 peptides are highly con
`served peptides. Accordingly, GLP-2 peptides for use in the
`present invention include the various naturally produced
`forms of GLP-2, particularly vertebrate species (including
`piscine and avian species), more particularly mammalian
`(such as primate, rodent (including rat, mouse, degu,
`hamster, and guinea pig), porcine, and bovine,), and more
`particularly the human form. Desirably, but not essentially,
`the naturally occurring GLP-2 peptide selected for use is of
`the same species as the subject identi?ed for treatment.
`GLP-2 analogs potentially useful in the present invention
`include agonists and antagonists of the GLP-2 receptor.
`GLP-2 agonists activate the GLP-2 receptor by ?rst binding
`to the receptor, folloWed by stimulating an intracellular
`second messenger system coupled to the receptor. In one
`embodiment of the invention, the GLP-2 agonists act selec
`tively at the GLP-2 receptor. Selectively-acting GLP-2 ago
`nists are compounds that, in the context of a suitable GLP-2
`receptor binding or functional assay, bind to the GLP-2
`receptor With greater af?nity. Such greater af?nity is pref
`erably at least an order of magnitude greater relative to
`different receptor types, such as the GLP-1 receptor. In other
`embodiments, the GLP-2 analogs bind to the GLP-2 recep
`tor With an af?nity at least equivalent to the af?nity of
`naturally occurring GLP-2.
`In other embodiments of the invention, the GLP-2 peptide
`is an analog of natural GLP-2 that incorporates one or more
`amino acid substitutions, additions, deletions, or modi?ca
`tions and retains biological acitivity.
`The agonist activity of human GLP-2 and rat GLP-2 is
`believed to require an intact N-terminus, but various dele
`tions of up to several residues at the C-terminus are tolerated
`Without loss of agonist activity. Substitutions are tolerated at
`sites outside regions conserved across the various GLP-2
`species homologs. Similarly, substitutions are also tolerated
`at sites Within regions conserved across GLP-2 species. In
`preferred embodiments, the amino acid substitutions are
`conservative substitutions. For example, one member of an
`amino acid class can be substituted by another member, e. g.,
`the substitution of alanine by glycine, the substitution of
`asparagine by glutamine, the substitution of methionine by
`leucine or isoleucine, and the like.
`Antagonist activity of GLP-2 analogs in humans and rats
`is exhibited When the naturally occurring GLP-2 peptide is
`mutated in any one or more of the ?rst four N-terminal
`residues, in particular by deleting any one or more of these
`N-terminal residues. In addition, antagonist activity is exhib
`ited When naturally occurring hGLP-2 is substituted: (1)
`With an amino acid Which does not naturally occur at any of
`the folloWing positions: Aspls, Phe22, Thr29, Thr32 and/or
`Asp33i (2) and When Ala2 is replaced by anyone of the
`folloWing amino acids: Leu, Cys, Glu, Arg, Trp and PO3
`Tyr2. In addition, antagonists of GLP-2 analogs include any
`mutation or variation of the naturally occurring GLP-2
`peptide Which results in the inhibition of intestinotrophic
`activity of naturally occurring GLP-2 or GLP-2 analogs
`Which exhibit agonist acitivity. Structural analogs of GLP-2
`Which act as antagonists are speci?cally described in WO
`98/03547.
`
`20
`
`30
`
`35
`
`FIG. 1: ShoWs a bar graph of the effect of certain amino
`acid stabiliZers on a formulation of h[Gly2]GLP-2 using a
`heat stress test. The precent (%) purity is plotted for three
`different amino acid formulations, both before and after the
`application of heat;
`25
`FIG. 2: ShoWs a bar graph of the effect of L-histidine on
`a phosphate buffered formulation of h[Gly2]GLP-2. The %
`purity is plotted for three different formulations at 0 and at
`4 hours;
`FIG. 3: ShoWs a bar graph of the screening of bulking
`agents analyZed by reverse-phase high performance liquid
`chromatography (RP-HPLC) at room temperature and 60°
`C. The % purity is plotted for seven different amino acid
`formulations;
`FIG. 4: ShoWs a bar graph of the screening of bulking
`agents analyZed by siZe exclusion high performance liquid
`chromatography (SE-HPLC). “HMW” represents a high
`molecular Weight peak. The % purity is plotted for seven
`different formulations;
`FIG. 5: ShoWs a bar graph of the stability of mannitol and
`sucrose formulations of h[Gly2]GLP-2 in a liquid state,
`prior to lyophiliZation, Which have been stored at 40 C. The
`% purity is plotted for four different formulations at 0 min.
`through 49 min., at 7 min. intervals; and
`FIG. 6: ShoWs a bar graph of the stability of lyophiliZed
`mannitol and sucrose formulations of h[Gly2]GLP-2 Which
`have been stored at 60° C. The % purity is plotted for four
`different amino acid formulations.
`
`40
`
`45
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The invention relates to GLP-2 formulations Which
`exhibit superior storage stability. The term “GLP-2,” as used
`herein, means a naturally occurring GLP-2 peptide or a
`GLP-2 analog thereof (unless speci?cally indicated
`otherWise).
`The present GLP-2 formulations can be provided as liquid
`formulations suitable for administration, such as by
`injection, in unit or multi-dose amounts. The liquid formu
`lations can also serve as stock solution from Which lyo
`philiZed dosage forms can be prepared. Accordingly, the
`present GLP-2 formulations can also be provided in lyo
`philiZed form, e.g., as freeZe-dried poWders suitable for
`reconstitution and subsequent administration as injectable
`liquid formulations.
`LyophiliZed formulations of the present invention exhibit
`storage stability of six months at ambient temperature, and
`
`50
`
`55
`
`60
`
`65
`
`9
`
`

`

`US 7,056,886 B2
`
`5
`The GLP-2 receptor analogs can be identi?ed by screen
`ing peptides against cells genetically engineered to produce
`the GLP-2 receptor. The GLP-2 receptor has been cloned.
`See Munroe et. al., Proc. Natl. Acad. Sci. USA, 96(4):l569
`(1999). Cells functionally incorporating the GLP-2 receptor,
`and their use to screen GLP-2 analogs, are also described in
`International Publication No. WO 98/25955, published on
`Jun. 18, 1998.
`In a preferred embodiment, the GLP-2 analog With ago
`nist activity has been altered to confer resistance to degra
`dation by endogenous enzymes, such as DPP-IV. Such
`analogs suitably incorporate a replacement of the alanine
`residue at position 2. In speci?c embodiments, the Ala2
`residue is replaced by glycine or serine, or by other residues
`as described for example in Us. Pat. No. 5,789,379. In a
`preferred embodiment, the GLP-2 receptor agonist is [Gly2]
`GLP-2. For use in treating humans, the GLP-2 analog is
`desirably but not essentially a human GLP-2 peptide or
`analog, particularly including the Gly2 analog of human
`GLP-2.
`It Was discovered that the h[Gly2]GLP-2 analog precipi
`tated at a pH of less than 5.5, and that temperature pro?les
`suggested a heat-induced and salt-dependent transition tem
`perature of about 40° C. Based on pH solubility pro?les, it
`Was determined that a phosphate buffer provides optimal
`buffering capacity for GLP-2 peptides. Furthermore, the
`addition of L-histidine to the phosphate buffer Was found to
`effectively stabiliZe GLP-2 peptides, Whereas the addition of
`arginine citrate or lysine did not effectively stabiliZe GLP-2
`compositions. L-histidine acts as a stabiliZing amino acid
`that increases the length of time that the GLP-2 peptide
`remains intact prior to degradation.
`The lyophiliZed formulations of the present invention are
`preferably provided in a poWder form comprising not more
`than about 5% Water by Weight, preferably not more than 2%
`Water by Weight, and more preferably not more than about
`1% Water by Weight.
`The bulking agent incorporated in the preparation pro
`duces a non-crystalline amorphous cake. It Was found that
`lactose, trehalose, and maltose sugars did not effectively
`stabiliZe the GLP-2 formulation as Well as mannitol and
`sucrose. Mannitol Was found to be the preferred excipient
`for the GLP-2 formulations.
`The buffering agent incorporated in the formulation of the
`present invention is selected from those capable of buffering
`the preparation to a pH Within a physiologically tolerable
`range for administration to a patient. “Physiologically tol
`erable” formulations are those that elicit reactions, in a
`recipient, that are not so extreme as to preclude further
`administration of the formulation. acceptable range for
`administration to a patient. More particularly, it Was found
`that the pH of the formulation should by greater than about
`5.5, more preferably greater than about 6, even more pref
`erably of about 6.9 to about 7.9, and most preferably about
`7.3 to about 7.4. Preferably, the buffering agent is phosphate
`based. and most preferably a 35 mM phosphate buffer is
`used.
`The formulations of the present invention incorporate
`GLP-2 in a medically effective amount, namely an amount
`Which is useful either therapeutically or diagnostically. Such
`an amount can be determined based on the type of GLP-2
`peptide or analog selected and on the intended end-use of the
`preparation. Therapeutically useful amounts of GLP-2
`include those unit dosage amounts useful in a regimen to
`treat a subject that Would bene?t from GLP-2
`administration, as described more fully in Us. Pat. Nos.
`
`40
`
`45
`
`20
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`25
`
`30
`
`35
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`50
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`55
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`60
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`65
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`6
`5,834,428; 5,789,379; 5,990,077; and 5,952,301, and in
`International Publication No. WO 98/52600.
`In one application, the formulation maybe exploited for
`the treatment of gastrointestinal disease, particularly
`diseases, disorders or conditions of the intestine. Therapeu
`tically useful amounts also include multi-dose amounts of
`GLP-2, Which can be delivered to an intended subject.
`Diagnostically useful amounts of GLP-2 include those
`amounts useful as a calibrant When assessing endogenous
`levels of GLP-2 or levels of GLP-2 drug in a subject, for
`instance as a prelude to GLP-2 therapy, or during the course
`of GLP-2 treatment. Medically useful amounts of GLP-2
`thus can range Widely from a feW micrograms to many
`milligrams. The formulations of the present invention pref
`erably provide about 0.1 to about 50 mg/ml of GLP-2,
`preferably about 5 to about 40 mg/ml, more preferably about
`7 to about 30 mg/ml, even more preferably about 10 to about
`20 mg/ml, and most preferably about 20 mg/ml of GLP-2.
`In an embodiment of the invention, a liquid formulation
`of h[Gly2]GLP-2 suitable for lyophiliZation comprises: (1)
`preferably about 7 to about 30 mg/ml, even more preferably
`about 10 to about 20 mg/ml, and most preferably about 20
`mg/ml of h[Gly2]GLP-2; (2) about 2 to about 5% of
`mannitol, preferably about 2.5 to about 3.5%, most prefer
`ably about 3%; (3) about 0.5 to about 1% of an amino acid
`stabiliZer, Which is preferably L-histidine; and (4) a phos
`phate buffer in an amount capable of buffering the recon
`stituted product to a pH of about 6.9479, and preferably a
`pH of about 7.3 to about 7.4.
`The GLP-2 formulations of the present invention are
`preferably ?lled in individual vials to the desired volume
`and the vials are subjected to a lyophiliZation process. The
`lyophiliZation process includes a temperature cycling pro
`cess that is carefully controlled to ensure that drying pro
`ceeds uniformly. The drying process is continued until there
`is less than about 5% of Water, preferably less than about 2%
`of Water, and more preferably no more than about 1% of
`Water, in the GLP-2 formulation.
`A lyophiliZation process suitable for the present invention
`involves a freeZing step and a tWo-step drying process. In an
`exemplary freeZing process: (1) the formulation vials are
`?rst cooled from ambient temperature to about —1° C. at
`about 2 C/minute, and then held at about —1° C. for about 15
`minutes, (2) next the vials are cooled from about —1° C. to
`about —40° C. at about 20 C./minute, and then held at about
`—40° C. for about 4 hours.
`In an exemplary ?rst drying cycle, the temperature is
`increased from about —40° C. to about —20° C. at about 20
`C./minute, and then held at about —20° C. for about 14 hours
`under a vacuum of about 150 mT With a condenser tem
`perature of about —80° C. In an exemplary second drying
`cycle, the vials are Warmed from about —20° C. to about
`+20° C. at about 20 C./minute, and then held at about +20°
`C. for about 14 hours at a vacuum of about 150 mT and a
`condenser temperature of about —80° C. until there is less
`than about 5% of Water. preferably less than about 2% of
`Water, and more preferably no more than about 1% of Water.
`The vials are then preferably stored at about 4° C.
`The present invention also provides a medically useful kit
`comprising: (1) at least one vial containing the lyophiliZed
`freeZe-dried GLP-2 formulation of the invention; (2) at least
`one vial of sterile Water for reconstitution; (3) instructions
`directing reconstitution; and (4) optionally an injection
`device for administration. To use the kit, the user mixes the
`Water With the formulation vial, preferably by transferring
`the Water to the formulation vial. The lyophiliZed formula
`
`10
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`

`

`US 7,056,886 B2
`
`7
`tion of the present invention rapidly dissolves upon recon
`stitution and, When reconstituted, is stable for at least about
`12 hours, preferably up to about 24 hours, at 4° C. In a
`preferred embodiment, reconstitution of the lyophiliZed for
`mulation is carried out using sterile Water, preferably no
`more than about 1 mL of sterile Water per dose of GLP-2. To
`reconstitute, the sterile Water may be draWn into a syringe
`and then transferred to the vial containing the lyophiliZed
`GLP-2 formulation.
`The folloWing examples are given to illustrate the present
`invention. It should be understood, hoWever, that the inven
`tion is not to be limited to the speci?c conditions or details
`described in these examples. Throughout the speci?cation,
`any and all references to a publicly available document,
`including a US. patent, are speci?cally incorporated by
`reference.
`
`EXAMPLE 1
`
`Formulation and LyophiliZation of h[Gly2]GLP-2
`
`20
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`25
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`30
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`8
`EXAMPLE 2
`
`Screening of Amino Acid to Stabilize the
`Formulation
`
`The purpose of this example Was to determine the effect
`of various amino acid additives on the stability of GLP-2
`folloWing exposure to elevated temperatures.
`The h[Gly2]GLP-2 formulation Was tested With several
`amino acids as set out beloW. The tested formulations
`comprised: (1) h[Gly2]GLP-2 at a concentration of 10
`mg/ml; and (2) the additives listed beloW. The pH of the
`composition Was maintained betWeen 7.1475.
`1. 10 mM phosphate, 10 mM Glutamate
`. 10 mM phosphate, 10 mM Glutamate, 50 mM Arginine
`. 10 mM phosphate, 10 mM Citrate
`. 10 mM phosphate, 10 mM Citrate, 50 mM Arginine
`. 10 mM phosphate, 100 mM Citrate
`. 10 mM phosphate, 100 mM Citrate, 50 mM Arginine
`. 10 mM phosphate, 10 mM Serine
`. 10 mM phosphate, 10 mM Serine, 50 mM Arginine
`. 10 mM phosphate, 10 mM Proline
`. 10 mM phosphate, 10 mM Proline, 50 mM Arginine
`. 10 mM phosphate, 10 mM Histidine
`. 10 mM phosphate, 10 mM Histidine, 50 mM Arginine
`. 10 mM phosphate, 10 mM Glycine
`14. 10 mM phosphate, 10 mM Glycine, 50 mM Arginine
`15. 10 mM His, 10 mM Glycine
`16. 10 mM His, 10 mM Glycine, 50 mM Arginine
`Following preparation, the samples Were lyophiliZed
`according to the protocol of Example 1, stored at 40° C. for
`14 days, diluted to 0.4 mg/ml, and then heated at 60° C. for
`4 hours.
`All of the formulations containing arginine precipitated
`upon heating (Formulations 2, 4, 6, 8, 10, 12, 14, and 16).
`Formulation 5 (100 mM citrate) and Formulation 15
`(L-histidine and glycine) also precipitated. Formulations
`comprising L-histidine, 10 mM citrate, serine, proline,
`glutamate, and glycine (Formulations 1, 3, 7, 9, 11, and 13)
`shoWed similar stability When these compounds Were used
`Without the addition of other amino acids. (See FIG. 1.)
`As shoWn in FIG. 2, When L-histidine Was used as a
`stabiliZer in combination With a phosphate buffer, the GLP-2
`peptide remained stable folloWing heat stress for 4 hours at
`60° C.
`
`The purpose of this example Was to prepare a lyophiliZed
`formulation of the GLP-2 peptide h[Gly2]GLP-2.
`A base formulation buffer, comprising 35 mM sodium
`phosphate at pH 7.4, Was prepared as folloWs: (1) puri?ed
`Water Was added to a sterile, depyrogenated ?ask; (2)
`sodium heptahydrate Was added to the ?ask; and (3)
`monobasic sodium phosphate monohydrate Was added to the
`?ask. The buffer Was mixed and the pH Was veri?ed to be
`7.4102. The base formulation buffer Was then used to dilute
`the GLP-2 peptide h[Gly2]GLP-2 liquid bulk drug substance
`to a concentration of 10 mg/ml. L-histidine Was then added
`to a ?nal concentration of 7.76 gm/L, and mannitol Was
`added to a ?nal concentration of 30 gm/L.
`The preparation Was carefully mixed, folloWed by ?lter
`ing the preparation through a 0.22 pm ?lter into a sterile
`?lling tank. The GLP-2 preparation Was then aseptically
`?lled, in 1 ml aliquots, from the tank into 3 cc sterile USP
`Type I glass vials, Which Were then partially capped With
`sterile rubber stoppers and placed into lyophiliZation trays.
`The vials Were then loaded into the lyophiliZer, and the
`lyophiliZation cycle Was commenced by pre-freeZing the
`formulation to a temperature of —4012° C. for about 4 hours.
`In the freeZing step, the formulation vials Were ?rst cooled
`from ambient temperature to —1 ° C. at 2° C./ minute and then
`held at —1° C. for approximately 15 minutes. This ?rst
`freeZing step Was folloWed by cooling the vials from —1° C.
`to —40° C. at 2° C./minute, and the vials Were then main
`tained at —40° C. for 4 hours.
`In the ?rst and primary drying cycle, the temperature Was
`increased from —40° C. to —20° C. at 2° C./minute and then
`held at —20° C. for about 14 hours under a vacuum of 150
`mT With a condenser temperature of —80° C. In the second
`drying cycle, the vials Were Warmed from —20° C. to +20°
`C. at 2° C./minute and then held at +20° C. for about 14
`hours at a vacuum of 150 mT and a condenser temperature
`of —80° C. The second drying cycle Was continued until
`there is less than about 5% of Water, preferably less than
`about 2% of Water, and more preferably no more than about
`1% of Water, remaining in the GLP-2 formulation. The vials
`Were then stored at 4° C.
`At the end of the lyophiliZation cycle, the vials Were
`purged With ?ltered nitrogen and the rubber stoppers Were
`fully depressed into the vials. The stoppered vials Were
`removed from the lyophiliZer and permanently sealed With
`a crimped aluminum seal and c