l|||||||||\|||||i||1||||||l||||l|||||||||||||||||||ll|l||||||||||i||||i||||
`
`U$D0'7fl56336E2
`
`(.2;Un.1ted States Patent
`lsaacs
`
`(10) Patent 110.:
`(45) "Date of Patent:
`
`US 7,056,386 B2
`Jun. 6, 2006
`
`(54) GL1’-I FURNIULATTONS
`
`(75)
`
`lnvwiur:
`
`I11duJ.lsancs,.m1.dnvcr, MA (US)
`
`(73) A:1sigm°:1:: NPSA1lelh1,C0rp._. Mis3issnL1g:1({'_‘.’\)
`
`7/1997 Kornfizlt 1:1. ai
`5.652.216 A “
`5,912,219 A " WI999 Thim eta]
`51953-301 A "
`9/I999 Dfllfikel‘
`5,997,356 A “ 12.I'I9'99 Home-t
`51120175‘ A ' W200“ Y"""”‘*"k”" “L
`
`............ . Sldfll
`514313
`514512
`.. 424/852
`“M3511
`
`
`
`(*) Notice:
`
`Subject to any disclaimer.I}1etr:n:'10ft11.is
`pa-111:0! is e-11:1-cndezd 01' adjusIr.:d under 35
`U-3-C 1-5°1(h}by 533 119113
`
`we
`we
`WD
`
`FOR’?-ION PAIHMT “act-]Mi’iN"‘S
`97,3903;
`“M997
`90.-03547
`111903
`991M336!
`91’J 999
`
`(21) App]. No; 09I75D,022
`
`(22)
`
`Filed:
`
`Dec. 29, Zllflfl
`
`(553
`
`‘'‘''°' ‘’“‘’'‘'=‘’‘'”" D'“‘
`113 200110027130 AI 01:1.
`-1‘ 2001
`
`mm
`Dec, 30. 1999
`
`(GB)
`
`OTHER PU]-'3LlCA'l‘lONS
`
`liuhl at al., Naturally (J:x:un'i11g 1'md1a1::11'. 0|‘ J’n1gIL1t1-1q1,11n
`111-150 in the Porcine and Htunan Smnll Intestine, 1. 0101.
`C11‘=m 353‘ 3531--3634 £19881"
`
`9030832
`
`31.31, c11.«_\m_z5g, 31go_3234{1933)_1«
`
`'
`
`(51)
`
`,l4n1s'i1?1.502120
`AGIK 38/26
`.
`(‘WK “Mm
`(52) U5. CI.
`
`(2005.01)
`[200G.0i)
`(zmfim)
`514/12; 5301300; 53013510;
`53Ilf32=1; 4351'-4: =1135.'2$7.l
`(58) Field of Classification Search ................. 01 5]-IUIZ;
`53¢);-503, 399, 324; 43347 331]
`gm applgcminn fik. fur mmphm, scam], hgsm,-y_
`
`(55)
`
`References cued
`UISI PA_mN_]_ DOCUMENTS
`
`‘ “M by ‘’’‘‘‘“‘i‘‘‘"
`P1-1‘r11my I1‘xa1111'1rer—1 -Jon Weber
`As.r1‘sn:-111‘ Exam1‘n1er~—~C.hil1dMi11 K0111
`9”“ ”"°’”E'J" “'3"""- ‘" F"”"“F°"“3’ '5" L"""""‘" “*9
`(57)
`_,u;3'rR_.1,(;1'
`
`.
`_
`_
`_
`_
`_
`The Invention IS dim-1:tcd_t0 i"0nm1la110-us of G|i_.P-2‘p£api1(AlE:$
`and analog; th1.‘.r1.:11l‘e.1c111b1l1ng, siupnmur :;lnh111ly lnllnwmg,
`stnmgc andior cxposu.n':
`Io clcvatcd tcn1pcm111rcs. Thu
`GLI‘-2 compositions cc1n‘1pris1: :1 GLP-2 peptide or an mmlog,
`tlaereuf. a phnsphate bulfe.-r, L-hislidine, and rnanniml.
`
`4,985,244 A “
`
`1/1991 Makinu 01 :11.
`
`-124189
`
`75 Claims, 6 Drawing Sh-.-010
`
`5xHlB|T
`
`/0;
`egg}/\_,5’Z.
`
`%
`%
`
`CFAD Exhibit 1044
`CFAD Exhibit 1044
`CFAD v. NPS
`CFAD v. NPS
`IPR2015-01093
`IPR2015—O1093
`
`CFAD Exhibit 1003
`
`

`
`U.S. Patent
`
`Jun.6,2006
`
`Sheet 1 of 6
`
`US 7,056,886 B2
`
`Figure 1. Amino acids screening in buffers using heat stress.
`
`95
`
`90
`
`•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
`~~~~~--~~~~~~~o_rm~u_a_tio_n~~~~~~~~-
`
`J
`
`Formulation
`
`1.
`3.
`7.
`9.
`
`11.
`13.
`
`10 mM Phosphate, 10 mM Gin
`10 mM Phosphate, 10 mM Citrate
`10 mM Phosphate, 10 mM Ser
`10 mM Phosphate, 10 mM Pro
`10 mM Phosphate, 10 mM His
`10 mM Phosphate, 10 mM Gly
`
`2
`
`

`
`U.S. Patent
`
`Jun.6,2006
`
`Sheet 2 of 6
`
`US 7,056,886 B2
`
`Figure 2. Screening of buffers using heat stress.
`
`r
`
`i..
`
`:::
`=
`~
`~ C>
`
`100
`
`95
`
`90
`
`•O hours
`•4 hours
`
`His
`
`99.36
`99.18
`
`Phos
`
`99.4
`93.35
`
`Hist+ Phos
`
`99.21
`97.43
`
`·1
`
`•O hours
`•4 hours
`
`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.
`
`Formulation
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`1.
`
`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 of 6
`
`US 7,056,886 B2
`
`Figure 4. Bulking agents analyzed by SE-HPLC.
`
`mMain Peak
`
`•Heat Strc.,ed
`Main Peak
`
`.HMW Peak
`
`•Heat Stressed
`HMW Peak
`
`7
`92.06
`80.61
`
`7.94
`19.37
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`1
`4
`2
`6
`3
`1111Main Peak
`100 100 100 100 100 93.91
`100
`100 100 78.75
`•Heat Stressed 100 100
`Main Peak
`.HMW Peak
`•Heat Stressed
`HMW Peak
`
`;
`'6.09
`21.25
`
`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% trebalose
`
`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 of 6
`
`US 7,056,886 B2
`
`Figure 5. Stability of liquid formulations stored at 4 °C.
`
`!-----·----
`100
`
`.....
`;<:::
`= ~
`loo
`'$.
`
`80
`
`40
`
`20
`
`0
`
`gTO
`
`•T7
`•T14 j
`11T21 I
`~T28
`•T42
`•T49
`
`Formulations
`
`1
`98.98
`98.62
`98.16
`98.28
`98.32
`97.85
`97.64
`
`.TO
`•T7
`•T14
`11T21
`lliJT28
`mT42
`.T49
`
`4
`
`98.93
`98.85
`99.02
`0
`88.16
`76.75
`71.18
`
`2
`
`+
`
`3
`98.65
`98.77
`79.73
`84.29
`79.59
`68.95
`55.95
`78.5
`47.35
`76.02
`39.44
`69.81
`67.75
`30.05
`Formulations
`
`1.
`
`2.
`
`3.
`
`4.
`
`35 mM Phosphate, 50 mM Histidine, 3% Mannitol, pH 7.4
`
`35 mM Phosphate, SO 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 of 6
`
`US 7,056,886 B2
`
`Figure 6. Heat stressed samples.
`
`I.RT
`.60Deg.
`
`c
`
`'i::
`::I
`p..
`~ 0
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`.RT
`•60Deg.
`
`98.9
`96.52
`
`99.06
`43.78
`
`98.84
`0.53
`
`98.87
`30.98
`
`Formulations
`
`1.
`2.
`3.
`4.
`
`35 mM Phosphate, SO 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
`
`7
`
`

`
`US 7,056,886 B2
`
`1
`GLP-2 FORMULATIONS
`
`FIELD OF INVENTION
`'Ihe 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(cid:173)
`logs with improved stability.
`
`BACKGROUND OF THE INVENTION
`
`55
`
`SUMMARY OF THE 1NVENTION
`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(cid:173)
`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 65
`preferably about 7.3 to about 7.4, is suitable for a stable
`fonnulation.
`
`Administration of therapeutic peptides requires peptide
`fonnulations that remain stable during storage. In general,
`parenteral administration is used with peptides because of
`their increased size and subsequent difficulty in crossing
`biological membranes. Peptides can be particularly difficult !5
`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 fomrnlation is not
`commercially viable, as it cannot pass U.S. Food and Drng 20
`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 25
`freezing and drying cycles can affect the stability of a
`lyophilized peptide fonnulation. 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., U.S. Pat. Nos. 5,834,428; 5,789,379; and
`5,990,077; and International Publication No. WO 98i52600.
`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(cid:173)
`nation at position 11, 16, andior 24 of GLP-2 arc 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 50
`unstable.
`There is a need in the art for stable fomrn!ntions ofGLP-2
`peptides and analogs thereof which can be prepared using a
`commercially acceptable process. The present invention
`satisfies these needs.
`
`30
`
`35
`
`4
`
`2
`It has also been discovered that the GLP-2 analog h[Gly2]
`GLP-2 undergoes a phase transition between 40-55° C.,
`depending upon the salt concentration, and becomes hydro(cid:173)
`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 aniount of GLP-2; (2) a phosphate buffer sufficient to
`10 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 mam1itol.
`More particularly, there is provided a GLP-2 fonnulation
`comprising: (1) a medically useful amount of GLP-2 com(cid:173)
`prising from about 0.1 to about 50 mg/ml of GLP-2, pref(cid:173)
`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(cid:173)
`larly mannitol. All percentages described herein (except for
`percentages for water) are weight/volume of formulated
`product prior to lyophilization in gms/ml (xJOO). Percent(cid:173)
`ages for water content are weight/weight of lyophilized
`product (xlOO).
`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 butler
`in an amount necessary to maintain the pH of the reconsti-
`tuted product between about 6.9-7.9, and preferably in an
`amount to maintain a pH of about 7.3 to about 7.4; (2) about
`0.5 lo about 1% L-hislidine; (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 ofGLP-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,
`0 and most preferably about 20 mg/ml.
`In a more preferred embodiment of the invention, a
`h[Gly2]GLP-2 lyophilized fonnulation is provided compris(cid:173)
`ing in the reconstituted product: (1) about 7 to about 30
`mg/ml, preferably about 10 to about 20 mg/ml, and most
`45 preferably about 20 mg/ml of h[Gly2]GLP-2; (2) a phos(cid:173)
`phate buffer sufficient 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(cid:173)
`vided a process for making the lyophilized formulation of
`GLP-2. Such a process comprises the following steps:
`(a) preparing the GLP-2 fonnulation comprising GLP-2
`or an analog thereof, a phosphate buffer, L-histidine,
`and mannitol;
`(b) freezing the formulation to about -40° C.;
`(c) performing a first drying step at about -20° C.; and
`(d) performing a second drying step at +20° C.
`In a preferred embodiment the liquid fonnulation sub-
`60 jccted 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 l % L-histidine; and ( 4)
`about 3% mamlitol.
`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 use fol kit comprising: (1) a 5
`sterile vial comprising a lyophilizcd GLP-2 formulation of
`the invention, (2) a vehicle suitable for reconstitution
`thereof, preferably sterile water, (3) instrnctions for recon(cid:173)
`stitution; and ( 4) optionally instrnctions for administration.
`The kit may further comprise a device suitable for injection 10
`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 15
`be readily apparent to those skilled in the art from the
`following detailed description of the invention.
`
`BRIEF DESCRJPTION OF THE FIGURES
`FIG. 1: Shows a bar graph of the effect of certain amino
`acid stabilizers on a fonnulation 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;
`FIG. 2: Shows a bar graph of the effect of L-histidine on
`a phosphate buffered fomrnlation ofh[Gly2]GLP-2. The%
`purity is plotted for three different fonnulations at 0 and at
`4 hours;
`FIG. 3: Shows a bar graph of the screening of bulking
`agents analyzed by reverse-phase high perfonnm1ce 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 lyophilin1tion, which have been stored at 4° C. 'Jbe
`% purity is plotted for four difierent formulations at 0 min.
`through 49 min., at 7 min. intervals; and
`FIG. 6: Shows a bar graph of the stability oflyophilized
`mamiitol and sucrose formulations ofh[Gly2]GLP-2 which
`have been stored at 60° C. The % purity is plotted for four
`different amino acid formulations.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The invention relates to GLP-2 formulations which
`exhibit superior storage stability. The tenn "GLP-2," as used
`herein, means a natumlly occurring GLP-2 peptide or a
`GLP-2 analog thereof (unless specifically indicated
`otherwise).
`TI1e 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 fonnu(cid:173)
`lations can also serve as stock solution from winch lyo(cid:173)
`philized dosage forms can be prepared. Accordingly, the
`present GLP-2 formulations can also be provided in lyo(cid:173)
`philizcd fom1, e.g., as freeze-dried powders suitable for
`reconstitution and subsequent administration as injectable
`liquid formulations.
`Lyophilized formulations of the present jnvention exliibit
`storage stability of six months at ambient temperature, and
`
`4
`eighteen months at 4° 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 I 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(cid:173)
`served peptides. Accordingly, GLP-2 peptides for use in the
`present invention include the various natumlly produced
`forms of GLP-2, particularly vertebrate species (including
`piscine and avian species), more particularly manllllalian
`(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 identified for treatment.
`GLP-2 analogs potentially useful in the present invention
`include agonists and antagonists of the GLP-2 receptor.
`20 GLP-2 agonists activate the GLP-2 receptor by first 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 ag01iists act selec(cid:173)
`tively at the GLP-2 receptor. Selectively-acting GLP-2 ago-
`25 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 affinity. Such greater affinity is pref(cid:173)
`erably at least m1 order of magnitude greater relative to
`different receptor types, such as the GLP-1 receptor. Jn other
`30 embodiments, the GLP-2 analogs bind to the GLP-2 recep(cid:173)
`tor with an affinity at least equivalent to the affinity of
`naturally occurring GLP-2.
`In other embodiments of the invention, the GLP-2 peptide
`35 is an mialog of natural GLP-2 that incorporates one or more
`amino acid substitutions, additions, deletions, or modifica(cid:173)
`tions and retains biological acitivity.
`The agonist activity of human GLP-2 and rat GLP-2 is
`believed to require an intact N-tenninus, but various dele-
`40 tions of up to several residues at the C-tenninus are tolerated
`without loss of agonist activity. Substitutions are tolerated at
`sites outside regions conserved across the various GLP-2
`species homologs. Sin1ilarly, substitutions are also tolerated
`at sites within regions conserved across GLP-2 species. In
`45 preferred embodiments, the amino acid substitutions are
`conservative substitutions. For example, one member of ffi1
`amino acid class can be substituted by another member, e.g.,
`the substitution of almiine by glycine, the substitution of
`asparagine by glutmnine, the substitution of metlii01iine by
`50 leucine or isoleucine, and the like.
`Antagonist activity of GLP-2 analogs in humans m1d rats
`is exhibited when the naturally occurring GLP-2 peptide is
`mutated in m1y one or more of the first four N-terminal
`residues, in particular by deleting any one or more of these
`55 N-terminal residues. In addition, antagonist activity is exhib(cid:173)
`ited when naturally occurring hGLP-2 is substituted: (1)
`with an amino acid which does not naturally occur at any of
`the following positions: Asp 15
`, Phe22
`, Thr29
`, Thr3 2 and/or
`Asp33
`'. (2) and when Ala2 is replaced by anyone of the
`60 following amino acids: Leu, Cys, Glu, Arg, Trp and P03-
`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 natumlly occurring GLP-2 or GLP-2 analogs
`65 which exliibit agonist acitivity. Strnctural analogs of GLP-2
`which act as antagonists are specifically described in WO
`98/03547.
`9
`
`

`
`US 7,056,886 B2
`
`30
`
`5
`TI1e GLP-2 receptor analogs can be identified by screen(cid:173)
`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):1569
`(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(cid:173)
`nist activity has been altered to confer resistance to degra(cid:173)
`dation by endogenous enzymes, such as OPP-IV Such
`analogs suitably incorporate a replacement of the alanine
`residue at position 2. In specific embodiments, the Ala2
`residue is replaced by glycine or serine, or by other residues
`as described for example in U.S. 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(cid:173)
`tated at a pH of less than 5.5, and that temperature profiles
`suggested a heat-induced and salt-dependent transition tem(cid:173)
`perature of about 40° C. Based on pH solubility profiles, it
`was determined that a phosphate butter 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 eJJectively 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 fornrnlations of the present invention are
`preferably provided in a powder fom1 comprising not more
`than about 5% waler 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(cid:173)
`duces a non-crystalline amorphous cake. It was found that 40
`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 fomrnlations.
`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(cid:173)
`erable" fommlations 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 fomrnlation should by greater than about
`5.5, more preferably greater than about 6, even more pref(cid:173)
`erably of about 6.9 to about 7 .9, and most preferably about 55
`7.3 to about 7.4. Preferably, the buffering agent is phosphate
`based, and most preferably a 35 mM phosphate buffer is
`used.
`·me formulations of the present invention incorporate
`GLP-2 in a medically effective amount, namely an amount 60
`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. ·n1erapeutically useful amounts of GLP-2
`include those unit dosage amounts useful in a regimen to 65
`treat a subject that would benefit from GLP-2
`administration, as described more fully in U.S. Pat. Nos.
`
`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(cid:173)
`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
`10 levels of GLP-2 or levels of GLP-2 drng 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 microgran1s to many
`milligrams. 111e fommlations of the present invention pref-
`15 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.
`Jn an embodiment of the invention, a liquid fomrnlation
`20 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, prefernbly about 2.5 lo about 3.5%, most prefer-
`25 ably about 3%; (3) about 0.5 to about 1% of an amino acid
`stabilizer, which is preferably L-histidine; and ( 4) a phos(cid:173)
`phate buffer in an amount capable of buffering the recon(cid:173)
`stituted product to a pH of about 6.9-7 .9, and preferably a
`pH of about 7.3 lo about 7.4.
`The GLP-2 formulations of the present invention are
`preferably filled in individual vials to the desired vohune
`and the vials are subjected to a lyophilization process. The
`lyophilization process includes a temperature cycling pro-
`35 cess that is carefully controlled to ensure that drying pro(cid:173)
`ceec.ls uniformly. 111e drying process is continued until there
`is Jess 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 fornmlation.
`A lyophilization process suitable for the present invention
`involves a freezing step and a two-step drying process. In an
`exemplary freezing process: (J) the formulation vials are
`first cooled from ambient temperature to about -1 ° C. at
`about 2 C/minute, and then held at about -1° C. for about 15
`45 minutes, (2) next the vials are cooled from about -1° C. to
`about -40° C. at about 2° C./minute, and then held atabout
`-40° C. for about 4 hours.
`Jn an exemplary first c.lrying cycle, the temperature is
`increased from about -40° C. to about -20° C. at about 2°
`50 C./minute, and then held at about -20° C. for about 14 hours
`under a vacuum of about 150 mT with a condenser tem(cid:173)
`perature of about -80° C. In an exemplary second drying
`cycle, the vials are warmed from about -20° C. lo abnut
`+20° C. at about 2° 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, anc.I more preferably no more than about 1 % of water.
`The vials are then preferably stored at about 4° C.
`TI1e 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 fornrnlation vial. The lyophilized formula-
`10
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`

`
`US 7,056,886 B2
`
`8
`EXAMPLE 2
`
`Screening of Amino Acid to Stabilize the
`Formulation
`
`7
`tion of the present invention rapidly dissolves upon recon(cid:173)
`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(cid:173)
`mulation is carried out using sterile water, preferably no 5
`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 fonnulation.
`clbe following examples are given to illustrate the present
`invention. It should be nndcrstood, however, that the inven(cid:173)
`tion is not to be limited to the specific conditions or details
`described in these exan1ples. Throughout the specification,
`any and all references to a publicly available document,
`including a U.S. patent, are specifically incorporated by 15
`reference.
`
`EXAMPLE 1
`
`Formulation and Lyophilization of h[Gly2]GLP-2
`
`20
`
`The purpose of this example was to detem1ine the effect
`of various amino acid additives on the stability of GLP-2
`following exposure lo elevated temperatures.
`The h[Gly2]GLP-2 formulation was tested with several
`10 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 7.1-7.5.
`1. 10 mM phosphate, 10 mM Glutamate
`2. 10 mM phosphate, 10 mM Glutamate, 50 mM Arginine
`3. 10 mM phosphate, 10 mM Citrate
`4. 10 mM phosphate, 10 mM Citrate, 50 mM Arginine
`5. 10 mM phosphate, 100 mM Citrate
`6. 10 mM phosphate, 100 mM Citrate, 50 mM Arginine
`7. 10 mM phosphate, 10 mM Serine
`8. 10 mM phosphate, 10 mM Serine, 50 mM Arginine
`9. 10 mM phosphate, 10 mM Praline
`10. 10 mM phosphate, 10 mM Praline, 50 mM Arginine
`11. 10 mM phosphate, 10 mM Histidine
`12. 10 mM phosphate, 10 mM Histidine, 50 mM Arginine
`13. 10 mM phosphate, 10 mM Glycine
`14. 10 mM phosphate, 10 mM Glycine, 50 mM Arginine
`15. 10 mM His, 10 mM Glycine
`16. I 0 mM His, I 0 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 fonnulations containing arginine precipitated
`upon heating (Formulations 2, 4, 6, 8, 10, 12, 14, and 16).
`Formulation 5 (100 mM citrate) and Fonnulation 15
`(L-histidine and glycine) also precipitated. Fonnulations
`comprising L-histidine, 10 mM citrate, serine, praline,
`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.
`
`35
`
`40
`
`The purpose of this example was to prepare a lyophilized
`formulation of the GLP-2 peptide h[Gly2]GLP-2.
`A base fonnulation buffer, comprising 35 mM sodium
`phosphate at pH 7.4, was prepared as follows: (1) purified
`water was added to a sterile, depyrogenated flask; (2) 25
`sodium heptahydrate was added to the flask; and (3)
`monobasic sodium phosphate monohydrate was added to the
`flask. The buffer was mixed and the pH was verified to be
`7.4:t0.2. lbe base formulation buffer was then used to dilute
`the GLP-2 peptide h[Gly2]GLP-2 liquid bulk drng substance 30
`to a concentration of 10 mg/ml. L-histidine was then added
`to a final concentration of 7.76 gm/L, and mannitol was
`added to a final concentration of 30 gm/L.
`The preparation was carefully mixed, followed by filter-
`ing the preparation through a 0.22 µm filter into a sterile
`filling tank. The GLP-2 preparation was then aseptically
`filled, in 1 ml aliquots, from the tank into 3 cc sterile USP
`Type I glass vials, which were then partially capped with
`sterile rnbber stoppers and placed into lyophilization trays.
`The vials were then loaded into the lyophilizer, and the
`lyophili7.ation cycle was commenced by pre-freezing the
`formulation to a temperature of-40:t2° C. for about 4 hours.
`In the freezing step, the fonnulation vials were first cooled
`from ambient temperature to -1 ° C. at 2° C./minute and then 45
`held at -1° C. for approximately 15 minutes. This first
`freezing step was followed by cooling the vials from -1° C.
`to -40° C. at 2° C./minute, and the vials were then main(cid:173)
`tained at -40° C. for 4 hours.
`In the first and primary drying cycle, the temperature was 50
`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 55
`hours at a vacuum of 150 mT and a condenser temperature
`of -80° C. The second drying cycle was continued nntil
`there is less than about 5% of water, preferably less than
`a