Title of Invention

STABILIZED, SOLID-STATE POLYPEPTIDE PARTICLES.

Abstract The present invention includes solid-state polypeptide particles containing a polypeptide material that is stabilized against degradation at temperatures that approximate or exceed physiological conditions. In each embodiment, the polypeptide particles of the present invention incorporate a polypeptide material that is stabilized against degradation by one or more stabilizing condtions. Because the polypeptide particles of the present invention can be formulated to combine the addictive effects of two or more stabilizing conditions, when the polypeptide particles of the present invention include a stabilizing sugar, the amount of stabilizing sugar needed to achieve acceptable polypeptide stability is significantly reduced.
Full Text WO 2004/039392 PCT/US2003/034229
STABILIZED, SOLID-STATE POLYPETIDE PARTICLES
BACKGROUND
[0001] Field of the Invention: The invention is directed to polypeptide
formulations in solid-state, wherein the polypeptide is stabilized against degradation at
5 elevated temperatures over an extended period of time. In particular, tbe present
invention provides formulations and methods for preparing solid-state polypeptide
particles, wherein the polypeptides are stabilized through one or more stabilizing
conditions.
[0002] State of the Art: Implantable devices capable of delivering desired
10 doses of an active agent, such as a polypeptide, over extended periods of time are
known in the art. For example, U.S. patents 5,728,396, 5,985,305, 6,113,938,
6,156,331, 6,375,978, and 6,395,292 teach implantable osmotic devices capable of
delivering a stable active agent formulation, such as a solution or a suspension, and at a
desired rate over an extended period of time (i.e., a period ranging from about two
15 weeks to several months or more). Implantable drug delivery systems also include
depot-type materials, such as those described in U.S. patents 6,468,961, 6331,311, and
6,130,200, Depot materials typically sequester the active agent within a biodegradable
or bioerodible depot material such that the active agent is delivered from the implanted
depot material based on diffusion of the active agent or degradation or erosion of the
20 depot material, Exemplary depot materials include PLGA-based systems, which are
typically capable of delivering the active agent over periods ranging from about 2
weeks to 6 months. Because they can be designed to deliver a desired active agent at
therapeutic levels over an extended period of time, implantable delivery systems can
advantageously provide long-term therapeutic dosing of a desired active agent without
25 requiring frequent visits to a healthcare provider or repetitive self-medication.
However, delivering therapeutic polypeptides over an extended period of time using an
implantable drug delivery system presents various technical challenges.
[0003] In particular, it has proven challenging to maintain the stability of
therapeutic polypeptides loaded in an implantable delivery system designed to deliver
30 the polypeptide over a period of weeks or months. In order to achieve an implantable

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system of suitable size that provides delivery of therapeutic doses of a polypeptide over
an extended period of time, it is generally necessary to load the system with a solution
or suspension containing a high concentration of the polypeptide to be delivered.
However, if such a solution or suspension is exposed to temperatures that approach or
5 exceed physiological conditions (e,g., temperatures that approach or exceed 37° C) over
an extended period of time, the polypeptide contained in the solution or suspension will
degrade if the polypeptide is not stabilized. Degradation of polypeptides exposed to
temperatures that approximate or exceed physiological conditions can proceed through
various pathways and can alter, reduce or destroy the biological activity of the
10 polypeptide. Therefore, in order to achieve an implantable delivery system capable of
successfully delivering a therapeutic polypeptide over an extended period of time, the
polypeptide loaded therein must be stabilized against degradation such that the system
is capable of delivering therapeutic doses of biologically active polypeptide over the
functional life of the implantable system.
15 [0004] Sugars have been used in polypeptide formulations to stabilize the
polypeptides contained therein against degradation over time. In particular, sugars have
been used as lyoprotectants, working to inhibit polypeptide aggregation by reducing
molecular unfolding during the lyophilization process. Sugars also impart long-term
stability by limiting molecular mobility and by mimizing molecular interactions
20 during and after the lyophilization process. However, when using a sugar to stabilize a
polypeptide, it is often necessary to use large amounts in order to achieve a desired
degree of stabilisation. As is taught in U.S. patent 5,267,958 to Andya et al., as much
as 100 to 510 molar ratios of stabilizing sugar may be needed in order to achieve an
acceptable stabilization effect, and polypeptide formulations including such large
25 amounts of stabilizing sugar are not well suited for loading in an implantable drug
delivery system.
[0005] Where a high concentration of stabilizing sugar is needed to achieve
a desired degree of polypeptide stabilization within a delivery system, the overall bulk
of the polypeptide formulation contained within the system increases, while the
30 maximum concentration of polypeptide that can be loaded into the system decreases.
As the concentration of the polypeptide in the formulation loaded into an implantable
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system decreases, the amount of polypeptide formulation required to achieve a desired
dosing regimen and the minimum size of the implanted system increase. It would be an
improvement in the art, therefore, to provide formulations and methods for stabilizing
therapeutic polypeptides that reduce or altogether eliminate the need for a stabilizing
5 sugar. It would be a further improvement in the art if such formulations and methods
were capable of stabilizing polypeptides to such an extent that the polypeptides could
be loaded into an implantable deiivery system at a high concentration and still exhibit
acceptable stability and therapeutic activity after exposure to temperatures up to or
exceeding physiological conditions over an extended period of time.
10 SUMMARY OP THE INVENTION
[0006] The present invention includes solid-state polypeptide particles,
wherein the polypeptide contained within the particles is stabilized against degradation
at temperatures up to and exceeding physiological conditions. As they are used herein,
the term "physiological conditions" refers to environments having a temperature of
15 about 37° C and the term "polypeptide" includes oligopeptides and proteins and
encompasses any natural or synthetic compound containing two or more amino acids
linked by the carboxyl group of one amino acid to the amino group of another. In each
embodiment, the polypeptide particles of the present invention incorporate a
polypeptide material that is stabilized against degradation by one or more stabilizing
20 conditions. In particular, the polypeptide particles of the present invention are
formulated to stabilize the polypeptide contained therein by controlling one or more of
the following particle conditions: pH, sugar content, surfactant content, buffer content,
and metal ion concentration. Because the polypeptide particles of the present invention
can be formulated to combine the additive effects of two or more stabilizing conditions,
25 where the polypeptide particles of the present invention include a stabilizing sugar, the
amount of stabilizing sugar needed to achieve acceptable polypeptide stability is
significantly reduced. In a preferred embodiment, the polypeptide particles of the
present invention are formulated to stabilize the polypeptide contained therein without
the use of a stabilizing sugar.




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[0007] The present invention also includes aqueous stabilizing solutions. In
order to form the solid-state polypeptide particles of the present invention, an aqueous
stabilizing solution according to the present invention, which contains the polypeptide
to be stabilized, may be formed and subjected to a suitable particle formation process,
5 such as a lyophilization or spray drying process. An aqueous stabilizing solution
according to the present invention, therefore, is formulated such that, upon subjecting
the aqueous stabilizing solution to a particle formation process, the aqueous stabilizing
solution yields solid-state polypeptide particles that contain polypeptide material
stabilized by one or more stabilizing conditions. By controlling the pH, sugar content,
10 surfactant content, buffer content, metal ion concentration, or polypeptide concentration
of an aqueous stabilising solution according to the present invention, solid-state
polypeptide particles having a wide range of desired stabilization characteristics can be
produced.
[0008] The present invention further includes methods for producing
15 stabilised, solid-state polypeptide particles. The method of the present invention
includes dissolving the polypeptide to be stabilised in an aqueous stabilizing solution of
the present invention and reconstituting the polypeptide in solid-state polypeptide
particles. The formulation of the solid-state peptide particles produced by the method
of the present invention will depend on the aqueous stabilizing solution used. In one
20 embodiment, the method of the present invention includes dissolving a polypeptide to
be stabilized in an acidic stabilizing solution. In another embodiment, the method of
the present invention includes dissolving a polypeptide to be stabilized in an acidic
stabilizing solution in the presence of a stabilizing sugar, a metal ion, or both a
stabilizing sugar and a metal ion. In yet another embodiment, the method of the present
25 invention includes dissolving a polypeptide to be stabilized in a buffered, near neutral
stabilizing solution in the presence of a surfactant, a stabilizing sugar, a metal ion, or
both a stabilizing sugar and a metal ion. In each embodiment of the method of the
present invention, the step of reconstituting solid-state peptide particles may be carried
out by subjecting the aqueous stabilizing solution to a suitable particle formation
30 process, such as a lyophilization or spray drying process. Though the method of the
present invention may be varied to achieve particles having any one of a variety of
formulations, in each instance the method of the present invention is tailored to provide
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solid-state polypeptide particles, wherein the polypeptide is stabilized through one or
more stabilizing conditions.
[0009] The solid-state polypeptide particles of the present invention provide
excellent polypeptide stabilization, allowing recovery of up to 96% of the stabilized
5 peptide after two months storage at 60° C. Moreover, the solid-stale polypeptide
particles of the present invention can be loaded into suspension formulations at
relatively high concentrations (i.e., 25% polypeptide particle or more), which facilitates
the formulation of suspensions having relatively higher concentrations of the stabilized
polypeptide. The solid-state polypeptide particles of the present invention, therefore,
10 facilitate the loading of an acceptably- sized implant delivery system with a
concentration of stabilized, therapeutic polypeptide that is sufficiently high to enable
delivery of therapeutic doses of the stabilized, therapeutic polypeptide over an extended
period of time.
BRIEF DESCRIPTION OF THE DRAWINGS
15 [0010] FIG. 1 provides a graph illustrating the total degradation (RP-HPLC)
and Aggregation (SEC) of lyophized PACAP (ammonium acetate, app. pH 6.4) upon
storage at 40°C for 3 months.
[0011] FIG. 2 provides a graph illustrating the stability of lyophilized
PACAP stored at 4°C, 40°C and 60°C for 3 Months.
20 [0012] FIG. 3 provides graph illustrating the suppression of PACAP
aggregate formation at 40 0 C where PACAP is formulated in solid-state particles using
various excipients.
[0013] FIG. 4 provides graph illustrating the suppression of PACAP
aggregate formation at 60°C where PACAP is formulated in solid-state particles using
25 various excipients.
[0014] FIG. 5 provides a graph illustrating the stabilization effect of
Histidine, Succinate, and Sucrose on total PACAP degradation at 40°C.


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[0015] FIG. 6 provides a graph illustrating the stabilization effect of sucrose
on suppression of PACAP aggregate formation in BA/PVP Suspension at 40°C.
[0016] FIG. 7 provides a graph illustrating the stabilization effect of sucrose
on suppression of PACAP aggregate formation in LL/GML/PVP Suspension at 400C.
5 [0017] FIG. 8 provides a graph illustrating the total Degradation of PACAP
in different suspension vehicles upon storage at 40°C for 3 mouths.
[0018] FIG. 9 illustrates the total degradation, as determined by RP HPLC,
and aggregation, as determined by SEC, of PACAP contained within particles
lyophilized at an apparent pH of 2, an apparent pH of 4, and an apparent pH of 6 upon
10 storage at 600C for 2 months.
[0019] Table 1 sets forth the results of a study conducted to evaluate the
stability of PACAP dispersed in various suspension vehicles upon incubating the
PACAP suspensions at 65°C for 4 hrs.
[0020] Table 2 sets forth the results of a study conducted to evaluate the
15 stability PACAP incubated at 37°C for over a period of 17 days, wherein the PACAP
evaluated was lyophilised PACAP alone or PACAP dispersed within one of three
different suspension vehicles.
[0021] Table 3 sets forth the results of a study conducted to evaluate the
stability of PACAP incubated at 60°C over a period of 17 days, wherein the PACAP
20 evaluated was lyophilized PACAP alone or PACAP dispersed within one of three
different suspension vehicles.
[0022] Table 4 provides the estimated degradation and aggregation rates of
PACAP where PACAP is incorporated in solid-state particles including various
different excipients and such particles are incubated at 40°C and 60°C.
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[0023] Table 5 provides the estimated degradation and aggregation rates of
PACAP contained within solid-state particles formed from three different solutions
prepared in NH4OAc buffer at pH6, with each of the three solutions containing a
different amount of sucrose.
5 [0024] Table 6 sets forth the results of a study conducted to evaluate the
stabilization of PACAP achieved by formulating solid state solid-state PACAP particles
at varying pH and with one of three different sugars.
[0025] Table 7 sets fort the results of a study conducted to evaluate the
additive stabilization of PACAP achieved by formulating solid-state PACAP particles
10 at varying pH and with, one or more different excipients.
[0026] Table 8 sets forth the results of a study conducted to evaluate the
stability of PACAP achieved by formulating solid-state PACAP particles at varying pH
with CaCl2, with Histidine, and with both CaCl2 and Histidine.
15 DETAILED DESCRIPTION OF THE INVENTION
[0027] In a first embodiment, the solid-state polypeptide particles of the
present invention include a polypeptide that is stable at acidic pH and are formulated to
exhibit an acidic reconstitution pH. The pH of the aqueous stabilizing solution prior to
particle formation (the "apparent pH") controls the pH exhibited by the subsequently
20 formed particles (the "reconstitution pH") and defines the protonation state of the
polypeptide within the solid-stale polypeptide particles. For the purposes of the present
invention, particles exhibiting an acidic reconsitution pH include polypeptide particles
exhibiting a reconsttution pH below about pH 5, with particles exhibiting a
retconstitution pH below pH 4 being preferred and particles exhibiting a reconstitution
25 pH ranging from about pH 2 to about pH 4 being particularly preferred. Controlling the
pH exhibited by the salid-state polypeptide particles of the present invention allows
control over the protonation state of the polypeptide included in the solid-state particles,
and it has been found that formulating the solid-state polypeptide particles of the
present invention at an acidic pH imparts a substantial stabilization effect. It is believed
30 that formulating the solid-state polypeptide particles of the present invention at acidic
pH stabilizes the polypeptide because the acidic environment favors protonation of
amino groups included in the polypeptide. Often the amino groups included in a


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polypeptide are involved in the formation of reactive intermediates that are prone to
inter- or intra-molecular reactions that alter the chemical nature of the peptide and
function as a significant pathway of polypeptide degradation. By maintaining the
polypeptide in an environment that favors protonation of the amino groups included in
5 the polypeptide, it is believed that the polypeptide particles according to the first
embodiment limit the involvement of amino groups in the formation of reactive
intermediates and, as a result, limit the degradation of the polypeptide resulting from
inter- or intra-molecular reactions.
[0028] The addition of metal ions to solid-state particles according to the
10 first embodiment provides additive polypeptide stabilization. Where the solid-state
polypeptide particles according to the first embodiment are formulated to include metal
ions, the metal ions are preferably provided by a divalent metal salt, such, as, CaCl2,
MgCl2 or ZnCl2. The precise amount of metal ion included in a solid-state polypeptide
particle according to the first embodiment will depend on the particular peptide to be
15 stabilised, the pH of the particle, and the presence or absence of a stabilizing sugar.
However, where a solid-state peptide particle according to the first embodiment
includes a metal ion, the particle is generally formulated such that the molar ratio of the
metal ion to stabilized polypeptide is between about 1/1 and 10/1, with a molar ratio
ranging from about 2/1 to about 6/1 being preferred, and a molar ratio of about 4/1
20 being particularly preferred. The addition of metal ions, to the solid-state polypeptide
particles of the present invention reduces the amount of polypeptide dimerization over
time, and thereby imparts additive polypeptide stabilization. It is believed that the
metal ions work to reduce polypeptide dimerization through the formation of ionic
linkages or salt bridges with the polypeptide molecules, which ionic linkages or salt
25 bridges limit the opportunity for interaction between polypeptide molecules.
[0029] The addition of a stabilizing sugar to solid-state peptide particles
according to the first embodiment also provides additive polypeptide stabilization. The
inclusion of a stabilizing sugar imparts additive polypeptide stabilization by limiting
polypeptide molecular mobility and reducing intermolecular interaction between
30 polypeptide molecules. However, because the solid-state polypeptide particles of the
present invention can be formulated to stabilize the polypeptide via two or more


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additive stabilizing conditions, where solid-state polypeptide particles according to the
present invention are formulated to include a stabilizing sugar, the amount of sugar
required to achieve an acceptable level of stability is significantly reduced. In
particular, where solid-state polypeptide particles according to the first embodiment are
5 formulated to include a stabilizing sugar, the sugar is generally included in an amount
ranging from about 0.1/1 wt/wt to about 1/1 wt/wt relative to the polypeptide stabilized.
In preferred embodiments, solid-state polypeptide particles of the present invention
including a stabilizing sugar will include the stabilizing sugar in amounts ranging from
about 0.1/1 wt/wt to about 0.5/1 wt/wt or from about 0.1/1 wt/wt to about 0.25/1 wt/wt
10 relative to the polypeptide stabilized. Where solid-state polypeptide particles according
to the first embodiment are formulated to include both a stabilizing sugar and metal
ions, the amounts of sugar and metal ions required to achieve a desired degree of
polypeptide stabilization may be reduced relative formulations that include stabilizing
sugar without metal ions or metal ions without stabilizing sugar. Such a potential
15 advantage may be achieved due to the additive stabilizing effects of the sugar, the metal
ions, and acidic ecvironmental pH.
[0030] Though non-reducing sugars are generally useful as stabilizing
sugars in solid-state polypeptide particles according to the present invention, it has been
found that not all sugars are suitable for stabilization of a peptide particle according to
20 the first embodiment Specifically, it has been found that sucrose, a sugar commonly
used in the stabilization of polypeptides, is not suitable for use under acidic conditions.
In fact, when sucrose is used under such conditions, it has been found to produce a
destabilizing effect. It is believed that such a destabilizing effect is generated becuase
sucrose itself is not chemically stable in an acidic environment. In particular, sucrose
25 undergoes hydrolysis into glucose and fructose at acidic pH. Although, trehalose is a
disaccharide like sucrose, trehalose is chemically stable under acidic conditions and at
elevated temperatures, Methyi-mannopyranoside ("methyl-MP"), a monosaccharide, is
also chemically stable in an acidic environment. Moreover, both trehalose and methyl-
MP have been found to provide significant additive polypeptide stabilization when
30 included in solid-state polypeptide particles formulated to exhibit an acidic pH.
Therefore, where solid-state polypeptide particles according to the first embodiment are
formulated to include a stabilizing sugar, the sugar included in the formulation must be

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stable under acidic conditions and is preferably stable under acidic conditions at
temperatures up to and exceeding physiological conditions.
[0031] Solid-state polypeptide particles according to the first embodiment
can be prepared from an aqueous stabilising solution. To prepare an aqueous
5 stabilizing solution suitable for preparing solid-state polypeptide particles according to
the first embodiment, the polypeptide to be stabilized is dissolved in an acidic solution.
An acidic aqueous stabilizing solution may be achieved through the addition of a
suitable acid, such as HCl, in an amount that provides a solution having a desired pH.
The pH of an aqueous stabilizing solution according to the present invention plays a
10 crucial role in stabilizing the polypeptide contained within the solid-state polypeptide
particles that are produced using the aqueous stabilizing solution. In order to achieve
solid-state polypeptide particles according to the first embodiment (i-e., having an
acidic reconstitution pH), it may be necessary to prepare the aqueous stabilizing
solution at an apparent pH that is significantly lower than the targeted reconstitution
15 pH. An aqueous stabilizing solution suitable for preparing solid -state polypeptide
particles according to the first embodiment may also include a metal salt or stabilizing
sugar dissolved therein. The amount of metal salt or stabilizing sugar dissolved in an
aqueous stabilizing solution suitable for producing solid-state polypeptide particles
according to the first embodiment may vary. However, where the aqueous stabilizing
20 solution is to be used to prepare solid-state polypeptide particles including a stabilizing
sugar, the aqueous stabilising solution will preferably include the desired stabilizing
sugar at an amount ranging from about 0.1/1 wt/wt to about 1/1 wt/wt relative to the
polypeptide to be stabilised. Moreover, where the aqueous stabilizing solution is to be
used to prepare solid-state polypeptide particles including a metal ion, the aqueous
25 stabilizing solution is preferably prepared to include the desired metal ion at a molar
ratio of metal ion to polypeptide ranging from about 1/1 to about 10/1.
[0032] In order to form solid-state polypeptide particles according to the
first embodiment from an acidic aqueous stabilizing solution, the stabilizing solution is
subjected to a known particle formation process, For example, an aqueous stabilizing
30 solution according to the present invention may be processed using a lyophilization or
spray drying process to achieve solid-state peptide particles. In a specific embodiment,
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solid-state polypeptide particles according to the present invention are prepared from an
aqueous stabilizing solution according to the present invention using a lyophilization
cycle that includes freezing stabilizing solution at 4º C for 30 minutes and at -50º C
for 3 hours (with a cooling rate of 2.5°C/min). After freezing the aqueous stabilizing
5 solution as described, a primary drying cycle is conducted at a chamber pressure of 50
mT at -30ºC for 10 minutes and then at 0ºC for 10 hours. The primary drying cycle is
followed by a secondary drying cycle at a chamber pressure of 200 mT at 0ºC for 3
hours and then at 20 ºC for 12 hours and at 30ºC for 6 hours, with all temperature levels
ramped at 0.5ºC/min. However, the present invention is not limited to solid-state
10 polypeptide particles produced by the precise lyophilization process described herein.
Several suitable lyophilization and spray drying processes are known in the art of
protein particle formation and may be applied to provide solid-state polypeptide
particles according to the present invention.
[0033] In a second embodiment, the solid-state polypeptide particles of the
15 presesnt invention are formulated to exhibit a near neutral reconstitution pH. As it is
used herein, the term "near neutral" refers to a pH ranging from pH 5 to about pH 8.
Preferably, solid-state polypeptide particles according to the second embodiment are
formulated to exhibit a reconstitution pH of between about pH 5 and pH 7. Polypeptide
particles according to the second embodiment of the present invention include a buffer
20 and further include a surfactant, a stabilizing sugar, a metal ion, or both a stabilizing
sugar and a metal ion. It has been found that formulating solid-state polypeptide
particles to exhibit near neutral reconstitution pH and to include buffering excipieuts
significantly stabilises the polypeptide material included in the solid-state particles. It
is believed that at near neutral pH a buffer may serve to stabilize the polypeptide
25 included in solid-state particles through a counter ion effect. In particular, it is thought
that the buffer material may interact with or bind to one or more sites included in the
polypeptide that exhibit a tendency to react inter- or intra-molecularly in such a way
that such sites are no longer available to react
[0034] Buffers suitable for use in formulating solid-state polypeptide.
30 particles according to the second embodiment are those that buffer at near neutral pH.
Specific examples of buffers that may be used in solid-state polypeptide particles


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according to the second embodiment include amino acid or peptide buffers, such as
histidine buffer {His-6) or histidine-glutamic acid (His-Glu) buffer, and inorganic
buffers, such as succinate and citrate buffers. Solid-state polypeptide particles
according to the second embodiment are generally formulated with up to about 20%
5 buffer by weight, with polypeptide particles including up to about 15% buffer by weight
being preferred. However, the precise amount of buffer included in solid-state
polypeptide particles according to the second embodiment may vary according to the
amount and type of polypeptide included in the particles. Moreover, the amount and
type of buffer used to formulate solid-state polypeptide particles according to the
10 second embodiment may also be adjusted order to achieve solid-state polypeptide
particles exhibiting a desired reconstitution pH.
[0035] By formulating solid-state particles according to the second
embodiment to include a stabilising sugar, a metal ion, or both a stabilizing sugar and a
metal ion, the stabilization of the polypeptide included in the particles increases beyond
15 that achieved by simply formulating the polypeptide in a near neutral environment in
the presence of a buffer. Where solid-state polypeptide particles are formulated
according to the second embodiment and include a stabilizing sugar, a metal ion, or
both a stabilizing sugar and a metal ion, the amount of sugar and metal ion included in
the particles preferably falls within the ranges detailed in relation to the solid-state
20 peptide particles of the first embodiment. In particular, if solid-state polypeptide
particles according to the second embodiment are formulated to include a stabilizing
sugar, the amount of stabilizing sugar included in the particles preferably ranges from
about 0.1/1 wt/wt to about 1/1 wt/wt relative to the polypeptide to be stabilized. Again,
non-reducing sugars are generally useful as stabilizing sugars in solid-state polypeptide
25 particles according to the present invention, but because they are formulated at near
neutral pH, however, solid-state polypeptide particles according to the second
embodiment may incorporate stabilizing sugars, such as sucrose, that are not stable in
an acidic environment. Further, if solid-state polypeptide particles according to the
second embodiment are formulated to include a metal ion, the particles are preferably
30 formulated to include the desired metal ion at a molar ratio of metal ion to polypeptide
ranging from about 1/1 to about 10/1. Where solid-state polypeptide particles
according to the second embodiment are formulated to include both a stabilizing sugar
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and metal ions, the amounts of sugar and metal ions required to achieve a desired
degree of polypeptide stabilization may be reduced relative formulations that include
stabilizing sugar without metal ions or metal ions without stabilizing sugar. Such a
potential advantage may be achieved due to the additive stabilizing effects of the sugar,
5 the metal ions, and buffer conditions at near neutral pH.
[0036] Where solid-state peptide particles according to the second
embodiment are formulated to include a surfactant, the surfactant used is preferably an
anionic surfactant, such as sodium dedocyl sulfate (SDS). The amount of surfactant
included in solid-state polypeptide particles according to the second embodiment may
10 vary according to the amount and type of polypeptide to be stabilized as well as the
nature and amount of other excipients included in the particles, nevertheless, where
solid-state polypeptide particles according to the second embodiment are formulated
from an aqueous stabilising solution containing the desired surfactant in an amount
ranging from about 0.02 wt% up to about 0.2 wt%.
15 [0037] Solid-state polypeptide particles according to the second
embodiment can be prepared from an aqueous stabilizing solution. To prepare an
aqueous stabilizing solution suitable for preparing solid-state polypeptide particles
according to the second embodiment, the polypeptide to be stabilized is dissolved in a
solution exhibiting a near neutral apparent pH and including a surfactant, a metal salt, a
20 stabilizing sugar, or both a metal salt and a stabilizing sugar, as desired. A near neutral
stabilizing solution may be achieved through the addition of a suitable buffer, such as
those buffers already discussed, in an amount that provides a solution having a desired
apparent pH. As is true of aqueous stabilizing solutions used to prepare solid-state
polypeptide particles according to the first embodiment, the apparent pH of an aqueous
25 stabilizing solution used to create solid-state polypeptide particles according to the
second embodiment controls the recocstitution pH of the solid-state polypeptide
particles produced using the aqueous stabilizing solution. Therefore, the amount and
type of buffer used in an aqueous stabilizing soluion for producing solid-state
polypeptide particles according to the second embodiment may be varied in order to
30 achieve a desired apparent pH and a desired reconstitution pH. Moreover, in order to
achieve solid-state polypeptide particles that characterized by a buffer content within

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the ranges described herein, the amount of buffer included in the aqueous stabilizing
solution will vary depending, upon, among other factors, the type of buffer used, the
precise amount of buffer desired in the solid-state particles, and the nature and amounts
of the polypeptide and other excipients to be included in the solid-state particles. For
5 example, in the Examples that are detailed below, stabilizing solutions characterized by
a lOmM concentration of histidine buffer provided solid-state polypeptide particles
containing 14 wt% buffer material.
[0038] The amount of metal salt or stabilizing sugar dissolved in an aqueous
stabilizing solution suitable for producing solid-state polypeptide particles aaccording to
10 the second embodiment may also vary depending on the amount and nature of the
polypeptide to be stabilized as well as the nature of the metal salt or stabilising sugar.
However, where an aqueous stabilizing solution for preparation of solid-state
polypeptide particles according to the second embodiment includes a stabilizing sugar,
the aqueous stabilizing solution will preferably include tbe desired stabilizing sugar at
15 an amount falling within the ranges already described (i.e., irona about 0.1/1 wt/wt to
about 1/1 wt/wt relative to the polypeptide to be stabilised, with the ranges of from
about 0.1/1 wt/wt to about 0.5/1 wt/wt and from about 0.1/1 wt/wt and 0.25/1 wt/wt
being preferred). Moreover, where an aqueous stabilizing solution for preparation of
solid-state polypeptide particles according to the second embodiment includes a metal
20 ion, the aqueous stabilizing solution is preferably prepared to include the desired metal
ion at a molar ratio of metal ion to polypeptide falling within the ranges already
described (i.e., a molar ratio of metal ion to polypeptide ranging from about 1/1 to
about 10/1, with the range of about 2/1 to about 6/1 being preferred and a molar ratio of
about 4/1 being particularly preferred).
25 [0039] In order to form solid-state polypeptide particles according to the
second embodiment from a near neutral aqueous stabilizing solution, the near neutral
stabilizing solution is subjected to a known particle formation process. As described in
relation to the formation of particles according to the first embodiment, solid particles
according to the second embodiment may be formed from an appropriately formulated
30 aqueous stabilizing solution using a lyophilization or spray drying process. For
example, the lyophilizataou process described herein is suitable for providing solid-state
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polypeptide particles according to the second embodiment from an aqueous stabilizing
solution. Again, however, the present invention is not limited to the use of the
particular lyophilization process detailed. Particle formation through lyophilization and
spray drying processes is well known in the art and any suitable lyophilization or spray
5 drying process may be applied to the aqueous stabilizing solutions of the present
invention to provide solid-state polypeptide particles according to the present invention.
[0040] The solid-state polypeptide particles, the aqueous stabilizing
solutions, and the methods of the present invention are particularly well suited to the
stabilization of peptides belonging to the superfamily of peptides including Pituitary
10 Adenylate Cyclase Polypeptide ("PACAP") and glucagon. In humans, the
PACAP/glucagon superfamily of peptides includes at least nine different types of
bioactive peptides PACAP-27; PACAP-38; glucagon; glucagon-like peptides, such as
GLP-l and GLP-2; growth hormone releasing factor ("GRF"); vasoactive intestinal
polypeptide ("VIP"); peptide histidine methionine ("PHM"); secretin; and glucose-
15 dependent insulinotropic polypeptide ("GIP"). Eight of these nine different types of
bioactive peptides are found in the brain and are classified as neuropeptides. In
addition many members of the PACAP/glucagon superfamily are present in the
gastrointestinal, pancreatic and gonadal organs {See, The origin and function of the
pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamify,
20 Endocrine Reviews, 21 (6):619-670). The peptides included in the PACAP/glucagon
superfamily are related in structure by their N-Terminal amino acids, and the genes and
precursor molecules used in the cellular production of the various different peptides
included in the PACAP/glucagon superfamily are similar in structure.
[0041] Of particular interest is a synthetic PACAP-R3 agonist analog ("the
25 PACAP analog") manufactured by Bayer Corporation ("Bayer"). This synthetic peptide
is defined by the following 31 amino acid sequence: HSDAVFTDNY TRLRKQVAAK
KYLQSIKRY. Bayer engineered the PACAP analog for potency and selectivity to
the R3 receptor in tbe pancreas. To improve stability of the PACAP analog, the
original methionine at position 17 was replaced with a valine and the asparagine
30 originally located at position 24 was replaced with a glutamine. The PACAP analog is
useful in treating Type II Diabetes, and it would be desirable to deliver the PACAP


15

WO 2004/039392 PCT/US2003/034229
analog from an implantable drug delivery system capable of delivering the PACAP
analog to a subject (humans) at therapeutic doses over a period of at least three months,
and preferably at least 6 months. However, whether it is maintained in a solid state or
dissolved in aqueous or organic solvent solutions, unprotected PACAP analog is
5 unstable at temperatures that approach or exceed physiological conditions. Therefore,
in order to load the PACAP analog in a suspension suitable for delivery from an
implantable, extended release delivery systems, the solid-state PACAP analog particles
must be stabilised against the degradation that would otherwise result from extended
exposure to temperatures that approximate or exceed physiological conditions.
10 [0042] As solid-state PACAP analog is exposed to temperatures
approximating or exceeding physiological conditions, the most significant source of
degradation has been determined to be aggregate formation. In particular, using
lyophiized PACAP analog, it was detennined that the major degradation products for
unprotected, lyophilized PACAP analog are covalent dimers, dimer-OAc and PACAP-
15 OAc adducts. Presently it is believed that the degradation of PACAP analog into such
aggregate products proceeds via reactive intra-molecular cyclic imide formation
followed by nucleophilic attack by acetate ions, by another molecule of PACAP analog,
or by both. Likely sites of modification for these proposed degradation paths are at the
Asp3 Ala4 and the Gln24Ser25 amino acids of the PACAP analog molecule. It has
20 been further determined that N-terminal peptide bond cleavage also occurs to degrade
solid-state, unprotected PACAP analog. This proteolysis was found predominantly at
pH4 and presumably occurs via assisted intra-molecular ring closure of the Asp3
carboxyl side chain. However, as is detailed in the Examples that follow, various
embodiments of the solid-state polypeptide particles of the present invention are
25 effective in stabilising PACAP analog against degradation, even when the particles are
maintained at temperatures of 40° C and 60° C over a period of months.
[0043] Though several different embodiments of the method of the solid-
state polypeptide particles of the present invention, provide effective stabilization of
PACAP analog, three embodiments provide superior stabilization. In one embodiment,
30 solid-stale PACAP analog particles are lyophilized from a stabilizing solution
exhibiting an apparent pH of 2. The solvent for the stabilizing solution is formed using
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WO 2004/039392 PCT/US2003/034229

H2O having the pH adjusted to the desired value using diluted HCL. The solid-state
PACAP analog particles according to this embodiment allow 92% recovery of the
initial PACAP analog and permit only 1.1% dimer formation after two months of
storage at 60° C. Figure 9 presents the two-month stability results, which demonstrate
5 the greatly improved stability of solid-state PACAP particles prepared from a
stabilizing solution having an apparent pH of 2.
[0044] In a second eambodiment, the solid- state polypeptide particles are
formulated at acidic pH and include trehalose and PACAP analog at a 0.55/1 w/w ratio
(trehalose/PACAP analog). These particles allow 96% recovery of the initial PACAP
10 analog and allow only 0.7% dimer formation, even after two months of storage at 60°
C. To formulate solid-state PACAP analog particles characterized by an acidic pH and
including frehalose and PACAP analog at a 0.55/1 w/w ratio, an acidic aqueous
stabilizing solution may be used. Such a stabilizing solution can be prepared using a
suitable acid, such as diluted HCL. The pH of such a solution (the apparent pH) is
15 preferably adjusted to a pH of about 2 and the trehalose aud PACAP analog are
dissolved in the solution at the desired 0.55/1 w/w ratio. The prepared solution may
then subjected to a lyophilization or spray drying process 10 provide the desired solid-
stale PACAP analog particles.
[0045] In a third embodiment, the solid state polypeptide particles are again
20 formulated at acidic pH and include PACAP analog and Ca2+ ions at a 4/1 molar ratio
(Ca2+/PACAP analog). After two months of storage at 60° C, particles formed
according to this formulation provide 95% recovery of the initial PACAP analog and
allow only 0.8% dimer formation. To formulate solid-state PACAP analog particles
characterized by an acidic pH and including Ca2+ and PACAP analog at a 4/1 molar
25 ratio, an acidic aqueous stabilizing solution may be prepared. Again, such a solution
may be prepared using a suitable acid, such as diluted HCL, and the pH of the solution is
preferably adjusted to a of about pH 2. The appropriate amount of Ca2+ ions and
PACAP analog may be provided in solution by dissolving amounts of CaCl2 and
PACAP that result in a solution having a molar ratio of Ca2+ to PACAP analog of 4/1.
30 The prepared stabilizing solution may then be Subjected to a lyophilization or spray
drying process to provide the desired solid-state PACAP analog particles.

17

WO 2004/039392 PCT/US2003/034229

[0046] Though the stability of the PACAP analog contained within the
particles described herein has been evaluated for a maximum period of two months at a
maximum temperature of 60° C, the findings provided by such evaluations and a
comparison of degradation kinetics at 40º C indicate that the method of the present
5 invention is suitable for providing solid-state PACAP analog particles that are
stabilized against degradation well beyond two months at temperatures that
approximate physiologic temperature (i.e., 40º C). Specifically, it has been found that
the total rate of degradation of PACAP analog contained within particles is
approximately five times higher at 60° C than at 40° C. Moreover, the rate of
10 aggregation or dimerization within the solid-state PACAP particles produced according
to the present invention is ten times higher at 60° C than at 40ºC. Therefore, where
solid-state PACAP analog particles produced by the method of the present invention are
maintained at approximately 37° C, it is anticipated that the PACAP analog contained
within the particles will demonstrate acceptable stability for up to six months and likely
15 longer.
[0047] Though, the Examples that follow are directed to the stabilization of
PACAP analog, the present invention is not so limited. For instance, the method of the
present invention may be used to provide stabilized, solid-state particles of other
members of the PACAP/glucagon superfamily, particularly those family members, such
20 as human VIP and human growth hormone releasing factor, which that have
substantially homologous amino acid sequences. However, the method of the present
invention is also not limited to stabilization of members of the PACAP/glucagon
superfamily. The solid-state polypeptide particles, aqueous stabilizing solutions, and
methods of the present invention may be useful for stabilizing any polypeptide that
25 exhibits degradation due to proteolysis or aggregate formation or has primary structures
prone to degradation pathways, such as those observed for PACAP.
EXAMPLE 1
[0048] Due to the nature of the suspension vehicles used in implantable
drug delivery systems, mixing of the suspension formulation bulk and subsequent
30 loading of the suspension formulation into the implantable systems are often conducted
at elevated temperatures. To evaluate the stability of PACAP analog (or simply
18

WO 2004/039392 PCT/US2003/034229
"PACAP") suspended in various suspension vehicles at elevated temperatures,
unprotected, lyophilized PACAP was suspended in four different suspension vehicles
(LI/GML/PVP, BA/PVP, EHI/PVP, and PEG/PVP). To suspend the PACAP into the
suspension vehicles 3.3 mg PACAP acetate was manually mixed into each of the
5 different suspensions to achieve a PACAP content of approximately 3%. The stability
of the PACAP suspension formulations was then evaluated after incubation at 65° C for
four hours. The stability of the prepared PACAP suspensions was evaluated by RP-
HPLC and SEC, and as can be seen by reference to table 1, the results showed that
PACAP was stable throughout the four hour incubation period and, therefore, should
10 survive the temperature conditions typically associated with the suspension formulation
manufacturing processes.
[0049] The PACAP suspensions were then further incubated at 37° C and
65° C an additional 17 days. After the additional 17-day incubation period, a marginal
15 stability loss was observed in the suspensions incubated at 37ºC (Table 2), while a
relatively more aggressive degradation was observed in the suspensions incubated at
65 °C (Table 3). The results revealed covalently linked aggregation as the major
degradation pathway in the evaluated PACAP suspensions. The fact that aggregation
occurred with and without the presence of vehicle suggests that the suspension vehicle
20 was not responsible for the resultant self-association. In addition other chemical
degradation contributed to the loss of lyophilized PACAP.
EXAMPLE 2
25 [0050] The preliminary findings from the processing study led to the
evaluation of the stabilization effect of sugar (sucrose) and non-ionic surfactant (Tween
80) on PACAP lyophiles and suspensions. In order to carry out tbe evaluation, samples
were prepared with PACAP in 10 mM NH4OAc (pH6.4) at 3 levels of Tween 80 (0,
0.05, 0.2wt%) and sucrose (0,0.5/1,1/1, w/w). A solution of PACAP in 10 mM of
30 histidine (pH 6.4) and sodium succinate (pH 5.6) was also prepared. Each of the
PACAP solutions were lyophilized to obtain reconstituted, solid-state PACAP particles,
and individual vials were provided 3.3 mg samples of the material reconstituted from
each of the solutions, with the samples containing (1) no additive, (2) 0.2wt% Tween

19

WO 2004/039392 PCT/US2003/034229
80, (3) 0.2%wt Tween 80+0-5:1, w/w sucrose, and (4) 1:1, w/w sucrose. The various
3.3 mg samples were manually mixed with LL/GMI/PVP and BA/PVP to provide
suspension formulations having about 3% PACAP content, and the suspension
formulations were subjected to 4 hours of incubation at 65 °C. After the initial
5 incubation period, the various suspension formulations were stored as described below
and evaluated by RP-HPLC and SEC.

[0051] Figure 1 shows that sucrose suppressed PACAP aggregation while
the presence of Tween 80 had no added effect. The overall stability, assessed by
10 percent PACAP remaining, also increased accordingly. The extent of PACAP
aggregation was shown to be a function of storage temperature (Figure 2). Lyophilized
PACAP without a stabilizer was stable for at least 3 months at 4ºC and aggregated
substantially more rapidly at 60ºC compared to 40ºC. This study demonstrated that
sucrose stabilized lyophilized PACAP both at the physiological (40°C) and accelerated
15 (60ºC) temperatures.
[0052] Figures 3 and 4 show a linear increase of PACAP aggregate
formation over 3 months at 40 and 60°C, respectively, for the lyophilized formulations
prepared with various different excipieats. Histidine and sodium succinite proved to be
better stabilizers than ammonium acetate. Except in the samples containing 1:1 w/w
20 sucrose, the rate of aggregation a± 60° C was approximately 10 times faster than that
observed at 40° C, and the rate of total degradation was about 5 times faster at 60°C
than that observed at 40°C (Table 4). These results indicate that high sucrose content
increased thermal stability by decreasing overall molecular mobility and protein-protein
interaction.
25 [0053] The total degradation, on the other hand, followed a less linear
relationship over a 3-month period of time (Figure 5). This experiment demonstrated
that lyophilized PACAP (prepared at an apparent pH of 6.4 in ammonium acetate)
20

WO 2OO4/039392 PCT/US2003/034229
containing an equal amount of sucrose (w/w) resulted in the best (but not acceptable)
stability, with an estimated 8.4% the total degradation in 6 months at 40ºC.
[0054] In addition, this experiment showed that sucrose suppressed
aggregate formation for PACAP suspensions in BA/PVP and LL/GML/PVP (Figures 6
5 and 7) similarly to lyophilized PACAP formulations alone. The stabilization effect of
sucrose in respect to aggregate formation appeared to be reduced in the BA/PVP
suspension. Also a higher amount of sucrose was needed for a similar longer-term
stability of PACAP in BA/PVP (Figure 8).
[0055] In order to characterize the degradation products and elucidate the
10 degradation paths, the reconstituted stability sample (lyophilized PACAP stoned at
60ºC for 24 days) was analyzed by electrospray-time-of-flight mass spectrometry and
the degradation products were identified using an RP-HPLC process.
[0056] A molecular weight (MW) of 3743 amu was assigned to the intact
PACAP. Based on the KP-HPLC analysis, the major degradation products were
15 identified to be covalent dimer and dimer-OAc adducts. Sigificantly, a cluster of
peaks, which elute after the main peak but prior to the elution of the dimer peaks, all
have a mass is to that for PACAP-OAc adduct (i.e., 3785 amu,). The
combined results from the RP-HPLC and the electrospray-time-of-flight mass
spectrometry -analyses suggest that both degradations were rendered through same
20 reactive internediate(s), Based on this analysis, the potential degradation, mechanism
can be postulated as follows:
Scheme 1; Hypothesized Mechanism

25
[0057] It can be hypothesized that the degradation proceeds by a two-step
mechanism via the rate-limiting cyclic imide intermediate formation followed by a
nucleophilic addition of an acetate ion, another PACAP molecule, or both. Since the

21

WO 2004/039392 PCT/US2003/034229
water content was low for all samples ( addition of water was not predominant.
EXAMPIE3
5 [0058] Solid-state P ACAP paricles were prepared via a lyophilization
process (FTS Duro stop) from three different polypeptide solutions prepared in
NH4OAc buffer at pH6. Each of the three different solutions contained a different
amount of sucrose with the first containing no sucrose, the second containing a weight
ratio of sucrose to PACAP of 0.5/1, and the third containing a weight ratio of sucrose to
10 PACAP 1/1. Solid-state PACAP particles prepared from each of the three solutions
were stored at control temperature ranging from 2° C to 8º C, a temperature
approximating physiologic temperature (40° C), and a temperature exceeding
physiologic temperature (60° C). The stability of the PACAP contained within each
group of solid-state PACAP particles was assessed at 24 days and 3 months. To assess
15 PACAP stability within each group of particles, the particles were reconstituted in
water and analyzed by reversed-phase RP-HPLC (acelonitrile gradient elution with
mobile phase containing 0.1% trifluoroacetic acid, and a UV detection at 214 nm) and
size exclusion HPLX (SEC, isocreatic elution, 30% acetonitrile mixed wife 70% of
aqueous solution containing 0,1% trifluoroacetic acid and 200 mM of sodium chloride,
20 and a UV detection at 220 run).
[0059] The rate of aggregation (assessed by SEC) was found to bo 10 times
higher at 60° C than at 40° C for all formulations tested (Table 5). Except in solid- state
PACAP particles prepared with a PACAP to sucrose weight ratio of 1/1, the rate of
total degradation (assessed by RP HPLC) observed in the various solid-state PACAP
25 particles was about 5 times faster at 60° C that at 40° C.
EXAMPLE 4
[0060] The effect of pH on the stability of PACAP molecules over time was
evaluated. In order to make such an evaluation, PACAP raw material was first
dissolved in H2O in three different solutions, with the first solution having a pH of 2,
30 the second solution having a pH of 4, and the third solution having a pH of 6. The pH
22

WO 2004/039392 PCT/US2003/034229

of each solution was adjusted to the desired value using diluted HCL. Each of the three
solutions was placed in lyophinzation vials, and solid-state PACAP particles were
produced from each of the three solutions through the lyophilization process already
described herein. Particles from each of the three solutions were then stored for two
5 months at 60º C, and the stability of the PACAP included in the solid-state particles
was thereafter evaluated using RP-HPLC and SEC. Figure 9 presents the two-month
stability results, which demonstrate the greatly improved stability of solid-state PACAP
particles prepared at acidic pH.
10 EXAMPLE 5
[0061] The stability of PACAP included in solid-state PACAP particles
prepared at varying pH with an amount of potentially stabilizing sugar was evaluated.
In each imtance the solid-state PACAP particles consisted of a 7.3 molar equivalent of
15 one of three different sugars sucrose; trehalose; and methyl mannopyranoside (all from
Sigma). The solid-state PACAP particles evaluated were prepared from eight different
aqueous solutions. The first two aqueous solutions were prepared by dissolving
PACAP into a first solution having a pH adjusted to 2 and a second solution having a
pH adjusted to 6 (as before the pH of the various solutions described in this Example
20 was adjusted using diluted HCl). The third and fourth aqueous solutions were prepared
by dissolving both PACAP and methyl-MP into a solution having a pH adjusted to 2
and into a solution having a pH adjusted to 6. The fifth and sixth aqueous solutions
were prepared by dissolving both PACAP amd trehalose into a solution havhig a pH
adjusted to 2 and into a solution having a pH adjusted to 6. And the seventh and eighth
25 aqueous solutions were prepared by dissolving both PACAP and sucrose into a solution
having a pH adjusted to 2 and into a solution having a pH adjusted to 6.
[0062] Solid-state PACAP particles were prepared from each of the eight
solutions through lyphilization, as has already been described, and solid-state PACAP
particles produced from each of the eight solutions were then stored at 60° C for two
30 months, After exposing the various solid-state PACAP particles to 60º C over two
months, the stability of the PACAP included in various particles was evaluated using
RP HPLC and SEC processes.

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WO 2004/039392 PCT/US/2003/034229
[0063] The HPLC analyses indicated all three sugars investigated showed
improved stability when included in PACAP particles prepared at pH 6. In particular,
at pH 6, sucrose provided roughly a 6-fold improvement in preventing aggregate
formation and trehalose provided roughly a 4-fold improvement in the same measure.
5 Moreover, both trehalose and methyl MP provided additive stability to the PACAP
included in particles prepared at pH2 (Table 6) relative to PACAP particles prepared at
pH 2 without the addition of a stabilizing sugar. However, when included in PACAP
particles prepared at pH 2, sucrose exhibited a significant destabilizing effect. The
destabilization effect of sucrose for PACAP prepared at pH2 was believed to be a result
10 of sucrose decomposition during the process of producing the PACAP particles from a
solution having an acidic pH.
EXAMPLE 6
[0064] PACAP particles containing histidine buffer (Sigma) and PACAP
15 particles containing histidine buffer in combination with calcium chloride (CaCl2, JT
Baker), sucrose and sodium dedocyl sulfate (SDS, Pierce) were prepared and the
stabilization of PACAP provided by such particles was evaluated. To prepare the
PACAP particles evaluated, six different aqueous solutions were prepared. Each
solution included a desired amount of PACAP dissolved therein, and the pH of each of
20 the six solutions prepared was adjusted to 6 by the addition of diluted HCL The first
aqueous was prepared without additives (histidine buffer, CaCl2, sucrose, or SDS). The
second aqueous solution was prepared with a 10 mM concentration of histidine buffer.
The third aqueous solution was formulated with a 10 mM concentration of histidine
buffer as well as a 10 mM concentration of CaCl2. The fourth aqueous solution was
25 formulated with a 10 mM concentration of histidine buffer and included sucrose at a
0.5/1 weight ratio (sucrose/PACAP) relative to PACAP. The fifth aqueous solution
was prepared with a 10 mM concentration of histidine buffer, a 10 mM concentration of
CaCl2, and an amount of sucrose providing a 0.5/1 weight ratio (sucrose/PACAP)
relative to PACAP. The sixth aqueous solution was formulated with a 10 mM
30 concentration of histidine buffer and 0.02 wt% SDS. Solid-state PACAP particles were
then produced from each of the six solutions using the lycphilization process already
described herein. The PACAP particles produced from each solution were then stored
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WO 2004/039392 PCT/US2003/034229
at 60° C for two months, and the stability of the PACAP contained within the various
different particles was evaluated using RP-HPLC and SEC processes after such storage.
As can be seen in Table. 7, the presence of hitidine buffer greatly suppresses PACAP
aggregate formation (a roughly 6-fold decrease in aggregate formation). In addition,
5 sucrose, CaCl2 and SDS further stabilized the peptide, providing roughly 14 fold to 20
fold decreases in aggregate formation.
EXAMPLE 7
10 [0065] To evaluate the stabilizing effect of CaCl2 in PACAP particles
prepared at acidic and near neutral pH, four different types of particles were formulated,
stored at 60º C for two months, and then analyzed for PACAP degradation. The four
difierent particle formulations were prepared from four different aqueous solutions.
Each of the four solutions included a desired amount of PACAP dissolved therein, and
15 the pH of each solution was adjusted to the desired level using diluted HCl.The first
aqueous solution was formulated without additives (histidine or CaCl2), and the pH of
the first solution was adjusted to 2. Tht second aqueous solutioin was formulated with a
10 mM concentration of CaCl2, and the pH of the second solution was adjusted to 2.
The third aqueous solution was formulated with a 10 mM concentration of histidine
20 buffer, and the fourth aqueous solution was formulated with a 10 mM concentration of
histidine buffer as well as a 10 mM concentration of CaCl2, Solid-state PACAP
particles were prepared from each of the four aqueous solutions (the composition of
each of the different particles is outlined in Table 8) using the lyophilization process
already described, and PACAP particles produced from each of the four solutions were
25 stored at 60º C for two months. After storage of the particles at 60° C for two months,
the stability of PACAP included in particles was evaluated using RP-HPLC and SEC
processes. As can be seen in Table 8, the results of such evaluation demonstrated that
CaCl2 further improved the stability of PACAP particles at both pH 2 and 6 (Table 8).
30



35
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WO 2004/039392 PCT/US2003/034229
5 CLAIMS
We claim:
1. Stabilized polypeptide particles comprising a polypeptide and a stabilizing agent
selected from the group consisting of metal ions and sugars that are stable under acidic
10 conditions at temperatures up to or exceeding philological conditions, the polypeptide
particles being formulated to exhibit an acidic reconstitution pH.
2. The polypeptide particles of claim 1 , wherein the polypeptide particles comprise
a polypeptide, a metal ion and a sugar that is stable at acidic pH at temperatures up to
15 and exceeding physiological conditions.
3. The polypeptide particles of claim 1, wherein the stabilizing agent is selected
from disaccharides and monosaccharides that are stable under acidic conditions at
temperatures up to and exceeding physiological conditions.
20
4. The polypeptide particles of claim 3, wherein the stabilising agent is selected
from trehalose and methyl-maonopyranoside.
5. The polypeptide particles of claim 1, Wherein the stabilizing agent comprises a
25 stabilizing agentt selected from disaccharides and monosacchaarides that are stable under
acidic conditions at temperatures up to and exceeding physiological conditions and the
stabilizing agent is included in the polypeptide particles at a wt/wt ratio of stabilizing
agent to polypeptide that ranges between about 0.1/1 to about 1/1.
30 6. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a
stabilizing agent selected from disaccharides and monosaccharides that are stable under
acidic conditions at temperatures up to and exceeding physiological conditions and the
stabilizing agent is included in the polypeptide particles at a wt/wt ratio of stabilizing
agent to potypeptide that ranges between about 0.1/1 to about 0.5/1.
26

WO 2004/039392 PCT/US2003/034229
7. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a
stabilizing agent selected from disaccharides and monosaccharides that are stable under
acidic conditions at temperatures up to and exceeding physiological conditions and the
5 stabilizing agent is included in the polypeptide particles at a wt/wt ratio of stabilizing
agent to polypeptide that ranges between about 0,1/1 to about 0.25/1.
8. The polypeptide particles of claim 1, wherein the stabilising agent comprises a
metal ion derived from a divalent metal ion salt.
10
9. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a
metal ion derived from a divalent metal ion salt selected from the group consisting of
CaCl2, MgCl2, and ZnCl2.
15 10. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a
metal ion and the molair ratio of the stabilizing agent to the polypeptide included in the
polypeptide particles ranges from about 1/1 to about 10/1.
11. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a,
20 metal ion and the molar ratio of the stabilizing agent to the polypeptide included in the
polypeptide particles ranges from about 2/1 to about 6/1.
12. The polypeptide particles of claim 1, wherein the stabilizing agent comprises a
metal ion and the molar ratio of the stabilizing agent to the polypeptide included in the
25 polypeptide particles is about 4/1.
13. The polypeptide particles of claim 1, wherein the polypepude is selected from
the pituitary adenylate cyclase polypeptide/glucagon superfamily.
30 14. The polypeptide particles of claim 1, wherein the polypeptide is selected from
group consisting of pituitary adenylate cyclase polypeptides, glucagons, glucagons-like
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WO 2004/039392 PCT/US2OO3/034229
peptides, growth hormone releasing factor, vasoactive intestinal polypeptide, peptide
histidine methionine, secretin, and glucose-dependent insulinotropic polypeptide.
15. Stabilized polypeptide particles comprising a pituitary adenylate cyclase
5 polypeptide and a stabilizing sugar selected from trehalose and methyl-
mannopyranoside, wherein the wt/wt ratio of stabilizing sugar to pituitary adenylate
cyclase polypeptide included in the polypeptide particles is about 0.55/1 and the
polypeptide particles are formulated to exhibit an acidic recoustitution pH.
10 16. Stabilized polypeptide particles comprising a pituitary adenylate cyclase
polypeptide and a stabilizing metal ion selected from Ca2+, Mg2+, and Zn+, wherein
the molar ratio of metal ion to pituitary adenylate cyclase polypeptide included in the
polypeptide particles is about 4/1 and the polypeptide particles are formulated to exhibit
an acidic reconstitution pH.
15
17. Polypeptide particles comprising a polypeptide and two or more stabilizing
agents selected from the group consisting of metal ions, surfactants, buffers, and sugars
that are stable in near neutral pH environments at temperatures up to and exceeding
physiological conditions, the polypeptide particles being formulated to exhibit a near
20 neutral reconstitution pH.
18. The polypeptide particles of claim 18, wherein the polypeptide is selected from
the pituitary adenylate cyclase polypeptide/glucagon superfamily.
25 19. The polypeptide particles of claim 18, wherein the polypeptide is selected from
group consisting of pituitary adenylate cylylase polypeptides, glucagons, glucagons-like
peptides, growth hormone releasing factor, vasoactive intestinal polypeptide, peptide
histidine methionine, secretin, and glucose-dependent insulinotropic polypeptide.
30 20. The polypeptide particles of claim 18, wherein the polypeptide particles
comprise a stabilizing sugar and a buffer selected from amino acid buffers, peptide
buffers and inorganic buffers.
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WO 2004/039392 PCT/US2003/034229
21. The polypeptide particles of claim 21, wherein the wt/wt ratio of stabilizing
sugar to polypeptide ranges from about 0.25/1 to about 1/1.
22. The polypeptide particles of claim 18, wherein the polypeptide particles
5 comprise a buffer selected from amino acid buffers and peptide buffers and a stabilising
metal ion.
23. The polypeptide particles of claim 23, wherein the molar ratio of metal ion to
polypeptide ranges from about 2/1 to about 10/1.
10
24. The polypeptide particles of claim 18, wherein the polypeptide particles
comprise a buffer selected from amino acid buffers and peptide buffers and a surfactant.
25. The polypeptide particles of claim 25, wherein the surfactant accounts for
15 between about 0.02 wt% and about 0.2 wt% of the polypeptide particles.
26. The potypeptide particles of claim 18, wherein the polypeptide particles
comprise a buffer selected from amino acid buffers and peptide buffers, a surfactant, a
metal ion and a stabilizing sugar.
20
27. The polypeptide particles of claim 18, wherein the surfactant comprises sodium
dedocyl sulfate.
23. The polypeptide particles of claim 25, wherein the surfactant comprise sodium
25 dedocyl sulfate and accounts for between about 0.02 wt% and about 0.2 wt% of the
polypeptide particles
29. The polypeptide particles of claim 18, wherein the polypeptide is a pituitary
adenylate cyclase polypeptide and the polypeptide particles comprise a buffer selected
30 from amino acid buffers and peptide buffers and one or more additional stabilizing
agents selected from the group consisting of a stabilizing sugar included at a wt/wt ratio
of stabilising sugar to polypeptide that ranges from about 0.25/1 to about 1/1, a metal
29

WO 2004/039392 PCT/US2003/034229
ion included at molar ratio of metal ion to polypeptide that ranges from about 2/1 to
about 10/1, and a surfactant that accounts for between about 0.02 wt% and about 0.2
wt% of the polypeptide particles.
5 30. Stabilized polypeptide particles comprising a polypeptide that is stable under
acidic conditions, wherein the polypeptide particles are formulated to exhibit an acidic
reconstitution pH.
31. The stabilized polypeptide particles of claim 30, wherein the stabilized
10 polypeptide particles are formulated to exhibit a reconstitution pH below pH 5.
32. The stabilized polypeptide particles of claim 30, wherein the stabilized
polypeptide particles are formulated to exhibit a reconstitution pH between about pH 2
and pH 4.
15
33 . The stabilized polypeptide particles of claim 30, wherein the polypeptide
comprises a polypeptide selected from the group consisting of pituitary adenylate
cyolase polypeptides, glucagons, glucagons-like peptides, growth hormone releasing
factor, vasoactive intestinal polypeptide, peptide histidine methionine, secretin, and
20 glucose-dependent insnlinotropic polypeptide, and the particles are formulated to
exhibit a reconstitution pH below pH 5.
3 4. The stabiliszed polypeptide particles of claim 30, wherein the polypeptide
comprises a polypeptide selected from the group consisting of pituitary adenylate
25 cyclase polypeptides, glucagons, gluoagons-like peptides, growth hormone releasing
factor, vasoactive intestinal polypeptide peptide histidine methionine, secretin, and
glucose-dependent insulinotropic polypeptide, and the particles are formulated to
exhibit a reconstitution pH between about pH 2 and pH 4.
30 35. The stabilized polypeptid particles of claim 30, wherein the polypeptide
comprises a pituitary adenylate cyclase polypeptide analog and the particles are
formulated to allow recovery of greater than 90% of the initial pituitary adenylate


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WO 2004/039392 PCT/US2003/034229
cyclase polypeptide analog and permit less than 2% dimer formation after two months
of storage at 60° C.


















31

The present invention includes solid-state polypeptide particles containing a polypeptide material that is stabilized
against degradation at temperatures that approximate or exceed physiological conditions. In each embodiment, the polypeptide
particles of the present invention incorporate a polypeptide material that is stabilized against degradation by one or more stabilizing
condtions. Because the polypeptide particles of the present invention can be formulated to combine the addictive effects of two
or more stabilizing conditions, when the polypeptide particles of the present invention include a stabilizing sugar, the amount of
stabilizing sugar needed to achieve acceptable polypeptide stability is significantly reduced.


Documents:

00941-kolnp-2005-abstract.pdf

00941-kolnp-2005-claims.pdf

00941-kolnp-2005-description complete.pdf

00941-kolnp-2005-drawings.pdf

00941-kolnp-2005-form 1.pdf

00941-kolnp-2005-form 3.pdf

00941-kolnp-2005-form 5.pdf

00941-kolnp-2005-international publication.pdf


Patent Number 224707
Indian Patent Application Number 00941/KOLNP/2005
PG Journal Number 43/2008
Publication Date 24-Oct-2008
Grant Date 22-Oct-2008
Date of Filing 20-May-2005
Name of Patentee ALZA CORPORATION
Applicant Address 1900 CHARLESTON ROAD, M10-3B, MOUNTAIN VIEW, CA 94043, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 BENTZ JOHANNA 36036 BA YONNE DRIVE, NEWARK, CA 94560, U.S.A.
2 KANG LING-LING 4173 HUBBARTT DRIVE, PALO ALTO, CA 94306, U.S.A.
PCT International Classification Number A61K 38/16
PCT International Application Number PCT/US2003/034229
PCT International Filing date 2003-10-28
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/422,289 2002-10-29 U.S.A.