Note: Descriptions are shown in the official language in which they were submitted.
WO 95129664 218 9 2 5 4 P~~S95/OSS1I
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Mnnrn~TED RELEASE FROM BIOCOMPATIBLE POLXMERS
' Backctround
Many illnesses or conditions require a constant level
of medicaments or agents in vivo to provide the most
effective prophylactic, therapeutic or diagnostic results.
In the past, medicaments were given in doses at intervals
which resulted in fluctuating medication levels.
Attempts to control and steady medication levels have
more recently included the use of many biodegradable
substances, such as poly(lactide) or poly(lactide-co-
glycolide) microspheres containing the medicament. The
use of these microspheres provided an improvement in the
controlled release of medicaments by utilizing the
inherent biodegradability of the polymer to improve the
release of the medicament and provide a more even,
controlled level of medication. However, in some cases,
biodegradable polymers under in vivo conditions can have
an initial level of medicament release, which is too high
or too low, and after a period of hydration can
substantially degrade to thereby limit the effective life
of the controlled release microspheres. Therefore, a need
exists for a means of modulating the controlled release of
medicament from a biodegradable polymer to provide a
higher level of initial medicament release and to provide
longer periods of fairly consistent medicament release
levels in vi.vo.
s,~mmary of the Invention
The present invention relates to a composition for
the modulated release of a biologically active agent. The
composition comprises a biocompatible polymeric matrix, a
biologically active agent which is dispersed within the
polymeric matrix, and a metal cation component which is
separately dispersed within the polymeric matrix, whereby
CA 02189254 2006-07-21
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the metal ration component modulates the release of the
biologically active agent from the polymeric matrix.
The present invention also relates to a method for
modulating the release of a biologically active agent from
a polymeric matrix, comprising the steps of dissolving a
biocompatible polymer in a solvent to form a polymer
solution and also separately dispersing a metal ration
component and a biologically active agent within said
polymer solution. The polymer solution is then solidified
to form a polymeric matrix, wherein at least a significant
portion of the metal ration component is dispersed in the
polymeric matrix separately from the biologically active
protein, and whereby the metal ration component modulates
the release of the biologically active agent from the
polymeric matrix.
More particularly, the invention provides a method
for modulating the release of a biologically active agent
from a polymeric matrix, comprising:
a) dissolving a biocompatible polymer in a solvent
to form a polymer solution;
b) dispersing one or more metal ration components
wherein the metal ration component comprises a
ration selected from the group consisting of
Zn(II) and Mg(II), and wherein the metal ration
component is in said solvent;
c) separately dispersing a biologically active
agent, wherein the agent is not
adrenocorticotropic hormone, in said polymer
solution;
d) solidifying said polymer from said polymer
solution to form a polymeric matrix, whereby
the metal ration component modulates the
CA 02189254 2006-07-21
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release of the biologically active agent from
the polymeric matrix; and
e) selecting the metal cation and its mode and
manner of dispersion to provide a defined
release pattern of the biologically active
agent.
This invention has the advantage of modulating the
release of a biologically active agent in vivo from a
biodegradable polymer, thereby enhancing the control of
the level of prophylactic, therapeutic and diagnostic
agents released in vivo and lengthening the period during
which controlled release can be maintained for a single
dose.
Brief Description of the Drawings
Figure 1 is a plot of percent water uptake (% w/w) as
a function of time in 10 mM HEPES for the following
polymer films: a) blank poly(lactide-co-glycolide)
(hereinafter "PLGA"), b) PLGA containing glass beads, and
c) PLGA containing carbon black, illustrating the effect
of glass beads and carbon black on PLGA film water
absorption.
Figure 2 is a plot of percent water uptake (%w/w) as
a function of hydration time in 10 mM HEPES for the
following polymer films: a) blank PLGA, b) PLGA containing
2% MgC03, c) PLGA containing 5% MgC03, d) PLGA containing
WO 95129664 218 9 2 5 4 PCT~S95/O551I
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10% MgC03, e) PLGA containing 15% MgC03, and f) FLGA
containing 30% MgCO" illustrating the effect of MgC03 at
different concentrations on PLGA film water-absorption.
Figure 3 is a plot of percent water uptake (%w/w) as
S a function of hydration time in 50 tnM PBS for the
following polymer films: a) blank PLGA, b) PLGA containing
5% Mg(OH)=, c)PLGA containing 10% Mg(OH)2, and d) PLGA
containing 20% Mg(OH)2, illustrating the effect of Mg(OH)2
at different concentrations on PLGA film water absorption.
Figure 4 is a plot of percent water uptake (%w/w)
versus hydration time in 50 mM PBS for the following
polymer films: a) blank PLGA and b) PLGA, containing 10%
ZnC03, illustrating the effect of ZnC03 on PLGA film water
absorption.
Figure 5 is a plot of percent water uptake (%w/w)
versus hydration time in 10 mM HEPES for the following
polymer films: a) blank PLGA and b) PLGA, containing 5%
Mg(OAc)~, illustrating the effect of Mg(OAc)2 on PLGA film
water absorption.
Figure 6 is a plot of percent water uptake (%w/w)
versus hydration time in 10 mM HEPES for the following
polymer films: a) blank PLGA and b) PLGA, containing 5%
Zn(OAc)" illustrating the effect of Zn(OAc)a on PLGA film
water absorption.
Figure 7 is a plot of percent water uptake (%w/w)
versus hydration time in 10 mM HEPES for the following
polymer films: a) blank PLGA and b) PLGA, containing 15%
MgS04, illustrating the effect of MgS09 on PLGA film water
absorption.
Figure S is a plot of percent water uptake (%w/w)
versus hydration time in 10 mM HEPES for the following
polymer films: a) blank PLGA and b) PLGA, containing 10%
ZnSO" illustrating the effect of ZnS09 on PLGA film water
absorption.
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Figure 9 is a plot of-percent water uptake (%w/wj-
versus--hydration time in 1D mM HEPES for the following
polymerpellets: a) control blocked-PLGA and b) control
unblocked-PLGA, illustrating the effect of PLGA end group
characteristics an PLGA pellet water absorption.
Figure 10 is a plot of molecular weight as a function
of hydration time in 10 mM HEPES for the following polymer
films: a) blank PLGA, b) PLGA containing 10% MgC03, and c)
PLGA containing 2D% MgC03, illustrating the effects of
MgCO, at different concentrations on the changes in
molecular weight-of PLGA films due to hydration.
Figure 11 is a plot of molecular weight as a function
of hydration time in 10 mM HEPES for the following polymer
films: a) blank PLGA, b) PLGA containing 10% ZnC03, and c)
PLGA containing 20% ZnC03, illustrating the effects of
ZnC03 at different concentrations on the changes in
molecular weight of PLGA films due to hydration.
Figure 12 is a plot of molecular weight (Mw) as a
function of hydration time in 10 mM HEPES for the
following polymer films: a) blank PLGAand b) PLGA
containing 5% Mg(OAC)" illustrating the effectsof
Mg(OAc), on the molecular weight of PLGA.
Figure 13 is a plot of log molecularweight (Mw)_as a
function of hydration time in 10 mM HEPES for the
following polymer pellets: a) unblocked-PLGA and b)
blocked-PLGA, illustrating the effects of PLGA end group
characteristics on the molecular weight degradation of
PLGA due to hydration.
Figure 14 is a plot of glass transition temperature
(Tg) as a function of hydration time in 10 mM HEPES for
the following polymer films: a) blank PLGA, b) PLGA
containing 10% MgCO" and c) PLGA containing 20% MgC03,
illustrating the effects of MgCO, at, different
concentrations on-the changes in the glasstransition
temperature of PLGA due to hydration.
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Figure 15 is a plot of glass transition temperature
(Tg) as a function of hydration time in 10 mM HEPES for
' the following polymer films: a) blank PLGA, b) PLGA
containing 10% ZnC03, and c) PLGA containing 20% ZnCO;,
' 5 illustrating the effects of ZnCO, at different
concentrations on the changes in the glass transition
temperature of PLGA due to hydration.
Figure 16 is a plot of glass transition temperature
(Tg) as a function of hydration time in 10 mM HEPES for
the following polymer films: a) blank PLGA and b) PLGA
containing 5% Mg(OAc)" illustrating the effects of
Mg(OAc)2 on the changes in glass transition temperature of
PLGA due to hydration.
Figure 17 is a plot of percent weight loss as a
1 15 function of hydration time in 10 mM HEPES for the
following polymer films: a) blank PLGA, b) PLGA containing
5% MgC03, c) PLGA containing 10% MgCO" and d) PLGA
containing 15% MgC03, illustrating the effects of MgC03 at
different concentrations on the degradation of PLGA due to
hydration.
Figure 18 is a plot of percent mass loss as a
function of hydration time in 10 mM HEPES for the
following polymer pellets: a) blocked-PLGA, b) unblocked-
PLGA, c) blocked-PLGA containing 10% MgC03, d) blocked-
PLGA containing 10% ZnC03, e) unblocked-PLGA containing
10% MgC03, and f) unblocked-PLGA containing 10% ZnC03,
illustrating the effects of PLGA end group characteristics
and of salts on the degradation of PLGA due to hydration.
Figure 19 is a plot of molecular weight (Mw) as a
function of time of blocked-PLGA microspheres, containing
10% ZnCO~, a) in vitro in 50 mM HEPES and b) in vivo in
rats which were subcutaneously administered the
microspheres, illustrating the slower degradation rate of
blocked-PLGA microspheres containing 10% ZnCO, (for in
WO 95/29664 218 9 2 5 4 PCTIUS95105511
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vitro and in vivo), as compared to blocked-PLGA
microspheres of Figure 19, for in vitro miorospheres as
compared to in vivo microspheres. ,
Figure 20 is a plot-of molecular weight (Mw} as a
function of time of blocked-PLGA microspheres a) in vitro
in 50 mM HEPES and b) in vivo in rats which were
subcutaneously administered the microspheres, illustrating
the increased degradation rate for in vitro microspheres
as compared to in vivo microspheres.
Figure 21 is a plot of the cumulative percent release
of RNase-A in 10 mM HEPES from PLGA microspheres
containing 10% RNase-A and either 0% Mg(OH), or 10%
Mg(OH)z, illustrating the-effects Mg(OH)z onRNase-A -
release kinetics from PLGA microspheres due to hydration.
Figure 22 is a plot of the cumulative percent release
of RNase-A in 10 mM HEPES from PLGA microspheres
containing 10% RNase-A and-either 0% ZnC03, 5% ZnC03, -10%
ZnCO;, or 15% ZnC03, illustrating the effects 2nC03 on
RNase-A release kinetics from PLGA microspheres due to
hydration.
Figure 23 is a plot of the cumulative percent release
of adrenocorticotropin hormone (ACTH) in 50 mM PBS from
PLGA microspheres containing 10% ACTH and either 0% MgC03
or 15% MgC03, illustrating-theeffects MgCO, on ACTH
release kinetics from PLGA microspheres due to-hydration.
Figure 24 is a plot of the serum concentration
(IU/ml) of Interferon-a,2b in rats which were
subcutaneously administered a single injection of
microspherea containing zinc carbor~ate and Zn*a-stabilized
Interferon-a,2b in molar ratios of a) 1:1, b) 3:1 and c)
8:1 over a seven day period.
WO 95129664 2 1 ~ 9 2 5 4 P~~S95/05511
Detailed Description of the Invention
A modulated-release of a biologically active agent,
' as defined herein, is a release of a biologically active
agent from a biocompatible polymeric matrix containing a
dispersed metal cation component which is separate from
the biologically active agent. In a modulated release, at
least one release characteristic, such as initial release
level of said agent, subsequent agent release levels, the
amount of agent released and/or the zxtent of the release
period, are changed from the release characteristicis)
demonstrated for said biologically active agent from a
polymeric matrix not containing a dispersed metal cation
component by the selection of the type and amount of metal
cation component dispersed in the polymeric matrix.
A polymer of the polymeric matrix of this composition
is a biocompatible polymer which can be either a
biodegradable or non-biodegradable polymer, or blends or
copolymers thereof.
Biodegradable, as defined herein, means the
composition will degrade or erode in vivo to form smaller
chemical species. Degradation can result, for example, by
enzymatic, chemical and physical processes. Suitable
biocompatible, biodegradable polymers include, for
example, poly(lactide)s, poly(glycolide)s, poly(lactide-
co-glycolide)s, poly(lactic acids, poly(glycolic acids,
poly(lactic acid-co-glycolic acids, polyanhydrides,
polyorthoesters, polyetheresters, polycaprolactone,
polyesteramides, blends and copolymers thereof.
Biocompatible, non-biodegradable polymers suitable
for the modulated release composition of this invention
_ include non-biodegradable-polymers selected from the group
consisting of polyacrylates, polymers of ethylene-vinyl
acetates and other acyl substituted cellulose acetates,
non-degradable polyurethanes, polystyrenes, polyvinyl
WO 95129664 ~ ~ g 9 2 5 4 PCTlU595145511
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chloride, polyvinyl fluoride;=polyvinyl imidazole),
chlorosulphonate polyolefins, polyethylene oxide, blends
and copolymers thereof.
A polymer, or polymeric matrix, is-biocompatible if
the polymer, and any degradation products of--the polymer, - '
are non-toxic to the recipient and also present no
significant deleterious or untoward effects on the
recipient's body.
Further, the polymer can be blocked, unblocked or a
l0 blend of blocked and unblocked polymers. A blocked -
polymer is as classically defined in the art, specifically
having blocked carboxyl end groups. Generally the
blocking group is derived from the initiator of the
polymerization and is typically an alkyl radical. An
unblocked polymer is as classically defined in the art,
specifically having free carboxyl end groups.
Acceptable molecular weights for polymers used in
this invention can be determined by a person of ordinary
skill in the art taking into consideration factors such ae
the desired polymer degradation rate,-physical properties
such as mechanical strength, and rate of-dissolution of
polymer-in solvent. Typically, an acceptable range of
molecular weights is of about 2,00D Daltons to-about
2,000,000 Daltons. In a preferred embodiment, the polymer
is a biodegradable polymer or copolymer. In a more
preferred embodiment, the polymer is a poly(lactide-co-
glycolide) (hereinafter "PLGA") with a lactide:glycolide
ratio of about 1:1 and a molecular weight of about 5,000
Daltons to about 70,000 Daltons. In an even more
preferred_embodiment, the molecular weight of the PLGA
used in the present invention has a molecularweight of
about 5,000 Daltons to about 42,000 Daltons
A biologically active agent, as defined herein,-is an
agent which possesses therapeutic, prophylactic or -
diagnostic properties in vivo. Examples of suitable-
WO 95129664 218 9 2 5 4 PCT~595105511
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therapeutic and/or prophylactic biologically active agents
include proteins, such as hormones, antigens, growth
" factors, etc.; nucleic acids, such as antisense molecules;
and small molecules, such as antibiotics,--steroids,
decongestants, neuroactive agents, anesthetics and
sedatives. Examples of suitable diagnostic and/or
therapeutic biologically active agents include radioactive
isotopes and radiopaque agents.
In the modulated release composition of the present
invention, an effective amount of particles of a
biologically active agent is dispersed within a polymeric
matrix. An effective amount of a biologically active
agent is a therapeutically, prophylactically or
diagnostically effective amount, which can be determined
by a person of ordinary skill in the art taking into
consideration factors such as body weight; age; physical
condition; therapeutic, prophylactic or diagnostic goal
desired, type of agent used, type of polymer used, initial
burst and subsequent release levels desired, and release
rate desired. Typically, a polymeric matrix for
modulating the release of a biologically active agent will
contain from about 0.01% (w/w) biologically active agent
to about 50% (w/w) biologically active agent, by weight.
Particles of a biologically active agent include, for
example, crystalline particles, non-crystalline particles,
freeze dried particles and lyophilized particles. The
particles may contain only the biologically active agent
or may also contain a stabilizing agent and /or other
excipient.
In one embodiment, a biologically active agent is a
. protein. Preferred proteins for inclusion in a modulated
release composition include, for example, nucleases,
erythropoietin, human growth hormone, interferons,
interleukins, tumor necrosis factor, adrenocorticotropic
hormone, growth factors, and colony-stimulating factors.
WO 95129664 2 .t 8 9 2 5 4 PCTIU595105511
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A modulated controlled-re-lease composition may also
contain more than one biologically active agent, for
instance, two different proteins, such as erythropoietin
and granulocyte-macrophage colony-stimulating_factor.,__ .
A metal canon component, as defined-herein, is a ~
component containing at least one kind of multivalent
metal cation (having a valence-of +2 or more) in a non-
dissociated state, a dissociated state, or-a combination
of non-dissociated and dissociated states:- Suitable metal -
cation components include, for-instance, metal salts,
metal hydroxides, and basic (pH of about 7 or higher)
salts ofweak acids wherein the salt contains a metal
cation. It is preferred that the metal cation be
divalent. -
In the modulated release composition of the present
invention, a suitable concentration of a metal cation-
component is dispersed within a polymer matrix.-,-A -
auitable concentration of a metal cation component is--any
concentration of a metal cation component which will
modulate the release of a biologically active agent from a
polymeric matrix. In one embodiment, suitable proportions
of a metal cation component to be dispersed in a polymer
is between about 1% (w/w) to about 30% (w/w). The optimum
ratio depends upon the polymer, the metal cation component
and the biologically active agent utilized. In a -
preferred embodiment, suitable amounts of a metal cation
component to be dispersed in a polymer is between about 5%
(w/w) to about 20% (w/w).
In one embodiment, the metal cation component is
substantially insoluble in aqueous fluids. Substantial
insolubility in aqueous fluids, as defined herein means
that the-metal cation component is generally not soluble,
or is of low solubility, in water or fluids, such as PBS,
HEPES or alimentary track fluff-ds. Examples of--suitable
insoluble metal cation components include, or contain, for
WO 95/29664 ~, ~ 8 9 2 5 4 P~~S951055I I
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instance, Mg(OH)2, magnesium carbonate (such as
4MgC03~Mg(OH)2~5H20), zinc carbonate (such as
3Zn(OH)2~2ZnC03), CaC03, Zn3(C6H50,)z (hereinafter zinc
citrate) or combinations thereof.
In an alternate embodiment, the metal cation
component is substantially soluble in aqueous fluids.
Substantial solubility in aqueous fluids, as defined
herein means that the metal cation component is generally
soluble in water or fluids, such as PBS, HEPES or
alimentary track fluids. Suitable soluble metal cation
components include, or can contain, for example,
Mg (OAc) z, MgSO" Zn (OAc) ~ ZnS04, ZnCl2, MgCl2, Mg3 (C6H50,) z
(hereinafter magnesium citrate) and combinations thereof.
In yet another embodiment, the metal cation component
isa combination of substantially soluble and insoluble
components.
In one embodiment of the method for modulating the
release of a biologically active agent from a polymeric
matrix, a suitable polymer is dissolved in a solvent to
form a polymer solution. Examples of suitable solvents
include, for instance, polar organic solvents such as
methylene chloride, chloroform, tetrahydrofuran, dimethyl
sulfoxide and hexafluoroisopropanol.
Particles of at least one metal cation component are
then dispersed within the polymer solution. Suitable
means of dispersing a metal cation component within a
polymer solution include sonication, agitation, mixing and
homogenization. It is understood that a metal cation
component can be added directly to the polymer solution as
a solid, preferentially in particulate form, wherein the
metal cation component will either-then be suspended as
solid particles dispersed within the polymer solution or
the metal cation component will then dissociate within the
polymer solution to form free metal cations. It is also
understood that, before addition to a polymer solution, a
WO 95129664 ~ ~ 8 9 2- ~ ~ PCTIUS95/05511
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metal cation component can be suspended as solid particles
or dissolved in a second solvent, wherein the second-
solvent is then-added to the polymer solution_ A second
solvent is suitable if it is the same solvent as the -
polymer's solvent, or if the second solvent is--miscible
with the polymer's solvent and the polymer is soluble in
the second-solvent. An example of a suitable second
solvent is acetone.
In another embodiment, a metal cation component can
be suspended or dissolved in a solvent, after which, a
suitable polymer is then dissolved in said solvent.
At least one biologically active agent is also added
to the polymer solution separately from the addition of
the metal cation component, metal cation component
suspension, or metal cation component solution. In one
embodiment, the biologically active agent is dissolved in
a solvent, which is also suitable for the polymer, and
then mixed into the polymer solution.
It is to be understood that a metal cation component
and a biologically active agent can be added to the
polymer solution sequentially, in reverse order,
intermittently or through separate, concurrent additions.
It is also understood that a biologically active agent can
be suspended in. a-solution, or suspension, of a metal
cation component in a solvent before dissolving the
polymer in said solvent.-
The amount of a biologically active agent added to
the polymer solution can be determined empirically by
comparative in vitro tests ofpolymeric matrices
containing different concentrations of at least one metal
cation component and of at least one biologically active
agent. The amount used will vary depending upon the-
particular agent, the desired effect of the agent at the
planned release levels,-and the time span over which the
agent will be released.
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The formation of a polymeric matrix microparticles
for modulating the release of RNase-A, ACTH, Interferon-
a,2b and human growth hormone (hGH) are further described
in Examples IX, X, XI and XII. The effectiveness of the
method of modulating the in vitro release of RNase-A or
ACTH from polymeric microspheres is also described in
Example IX. Further, the effectiveness of the method of
modulating the in vivo release of Interferon-a,2b from a
polymeric microspheres is described in Example X.
Additionally,- the effectiveness of the method of
modulating the in vivo release of hGH from a polymeric
microspheres is demonstrated by Examples XI and XII.
In an alternate embodiment, the protein added to the
polymer solution can be mixed with an excipient, such as
at least one-stabilizing agent as is known in the art.
The polymeric matrix of this invention can be formed
into many shapes such as a film, a pellet, a cylinder, a
disc or a microparticle. A microparticle, as defined
herein, comprises a particle having a diameter of less
than about one millimeter containing particles of a
biologically active agent dispersed therein. A
microparticle can have a spherical, non-spherical or
irregular shape. The preferred microparticle shape is a
sphere.
In a preferred embodiment, the method includes
forming a modulated release polymeric matrix as a
microparticle. A suitable metal cation component is
dispersed ae solid particles or free dissociated cations,
and a biologically active agent is separately dispersed as
solid particles in a polymer solution containing about 5-
_ 30% polymer by weight. In a more preferred embodiment,
the polymer solution contains about 5-15% polymer by
weight. Biodegradable polymers are preferred, while PLGA
is more preferred.
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A microparticle is then formed from the polymer
solution. A suitable method for forming an acceptable
microsphere from a polymer solution is described in
U.S. Patent 5,019,400, issued to Gombotz et al.
In another embodiment, a modulated release
composition is prepared by the solvent evaporation method
described in U.S. Patent No. 3,737,337, issued to
Schnoring et al., U.S. Patent No. 3,523,337, issued to
Vranchen et al., U.S. Patent No. 3,691,090, issued to
Kitajima et al., or U.S. Patent No. 4,389,330, issued to
Tice et al.
In the solvent evaporation method a polymer
solution, which contains a dispersed metal cation
component and a dispersed biologically active agent, is
mixed in or agitated with a continuous phase, in which
the polymer's solvent is substantially immiscible, to
form an emulsion. The continuous phase is usually an
aqueous solvent. Emulsifiers are often included in the
continuous phase to stabilize the emulsion. The polymer's
solvent is then evaporated over a period of several hours
or more, thereby solidifying the polymer to form a
polymeric matrix having a metal cation component and a
biologically active agent separately dispersed therein.
In another embodiment, the method includes forming a
modulated release polymeric matrix as a film or any other
shape. A polymer solution and metal cation component, in
particulate or dissociated form, is mixed, for instance
by sonication, until the metal rations are generally
dispersed throughout the polymer solution. The polymer
solution is subsequently cast in a mold, such as a petri
dish. The solvent is then removed by means known in the
WO 95129664 PCT/US95105511
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art until a film or form, with a constant dry weight, is
obtained.- The formation of polymeric matrix films and
polymer pellets is further described in Examples I and II.
Several other methods of using the composition of
this invention can be used to modulate physical properties
of polymers. One embodiment of the method of use consists
of a method for modifying the water absorption, or
hydration capacity without significant polymer
degradation. The method comprises forming a solution of a
polymer and then dispersing a metal cation component into
the polymer solution. The polymer solution is then
solidified to form a polymer matrix wherein the metal
cation component is dispersed therein. See Example III
for-a further description of this method of enhancing
initial--hydration.
A further embodiment of the method of use consists of
a method for significantly stabilizing the glass
transition temperature for a polymer during hydration,
comprising the steps of forming a solution of a polymer
and a solvent and then dispersing a metal cation component
within said polymer solution. The polymer solution is
then solidified to form a polymer matrix wherein particles
of the metal cation component are dispersed therein.
Glass transition temperature (Tg) could be an
indirect indicator of polymeric degradation since Tg is a
function of the molecular weight of the polymer and
usually decreases as molecular weight decreases. Glass
transition temperature (Tg) is defined as the temperature
at which a polymer converts from a glass phase to a
rubbery phase. Tg is affected by the molecular weight of
the polymer. See Example V for further description of
this method of stabilizing Tg during polymer hydration.
In the-embodiment wherein the polymeric matrix is in the
WO 95129664 2 ~ g 9 2 5 4 PCT/US95105511
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form of microparticles, the stabilization of Tg maintains
the mechanical properties of the polymer, thereby
enhancing the control of-agent release.
Yet another embodiment of the method of use consists
of a method for increasing the porosity of a polymer
without significant polymer degradation. -This method
includes the steps of forming a solution of a polymer-and
a solvent and then dispersing -a metal cation component
into said polymer solution. -The polymer solution is then
solidified to form a polymer matrix wherein the metal
cation compound is dispersed therein and subsequently
hydrated to form at least one gap within said polymeric
matrix, thereby increasing the porosity of the polymer.
Gaps, as defined herein compri-se pores and/or voids. -See
Example VI for a further description of this method of
use.
An alternate embodiment of the method of use consists
of a method for slowing the rate of-degradation of a-
polymer. In this method a solution is formed of a polymer
and a metal cation component is then dispersed within said
polymer solution. The polymer solution is subsequently
solidified to form a polymeric matrix having a metal
cation component dispersed therein. Examples IV, VII and
VIII provide additional descriptions of-the modulating
polymeric degradation rate, both in vitro-and in vivo~ as
the result of the addition of metal cations to the polymer
and from the selection of polymer end groups.
The composition of this invention can be administered
to a human, or other animal, for example, by injection
and/or implantation subcutaneously, intramuscularly,
intraperitoneally, intradermally, intravenously, intra-
arterially or intrathecally; by administration to mucosal
membranes, such as intranasally or by means of a
suppository, or by in situ delivery to provide the desired
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2189254
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dosage of a biologically active agent based on the known
parameters for treatment of the various medical conditions
with said agent.
The invention will now be further and specifically
described by the following examples.
EXAMPLE I
prP.,a,-arson of Polymer Films Containina Salts
Blocked-PLGA (50:50) with a molecular weight of
42,000 Daltons (I. V. 0.7 dl/g Birmingham Polymers,
Birmingham AL.) was used for all film studies. The
polymer films were produced by a film casting technique.
The polymer was dissolvedin methylene chloride (5% w/v)
at room temperature for up to 24 hours.
Films were prepared using both water insoluble and
soluble salts containing divalent cations. The salts were
incorporated in the polymer either as particulates or by
cosolubilizing the salts with the polymer in an
appropriate cosolvent. The fabrication procedure is
described below.
Three salts with low water solubility, MgC03, Mg(OH)z
and ZnCO, (Spectrum Chemical MFG, Corp., Gardens, CA) and
two water soluble salts, MgS04 and ZnS04 (Spectrum
Chemical MFG, Corp., Gardens, CA) were incorporated into
films as particulates. MgC03, Mg(OH)2 and ZnC03 were
sieved prior to film casting using a 38 micron U.S.A.
standard testing sieve to control the particle size. The
average particle diameter of the sieved salts prior to
encapsulation is provided in Table 1.
WO 95/29664 2 ~ g 9 2 5 (~ PCT/US95105511
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a t Formula Diameter (um)
MgC03 4MgC03 ~ Mg (OH) z ~ SHOO 2 ~5
Mg(OH)z Mg(OH)a 2.5
ZnCO, 3Zn(OH)~~2ZnC0,- 4.0
As non-ionic water insoluble particulates, either
carbon black or glass particles (20 micron diameter,
Polysciences Inc:, Warrington,-PA) were used. --Polymer
films were prepared by adding the sieved salt to the-
polymer solution to a final concentration in the 0-30%
(w/w, salt/polymer) range. The salt polymer suspension
was sonicated for approximately four minutes to disperse
the salt particles. A sample of 100 ml of the suspension
was then cast in 9 x 5 x L inch teflon petri-dish (Plastic
Structures Co., Wilmington, MA.). The control polymer
film was the polymer containing 0.0% salt.
The films were cast in two layers to avoid settling
of the salt particles. The methylene -chloxide was
evaporated at room temperature in a hood for the first 24
hours at atmospheric pressure- The films were transferred-
to a vacuum oven and were dried at 30'C for 6 hours,-40'C
for 3 days, and then at 50'C for 3 days. No further-
reduction in dry weight was observed at the end of this
drying cycle.
Polymer films containing the water soluble salts
magnesium acetate and zinc acetate were prepared by
cosolubilizing the salts with PLGA in acetone. -A 10%
solution of polymer was prepared by dissolving-5 g of-
polymer in 50 ml of acetone at room temperature. A
solution of Mg(OAc), or Zn(OAc)2 was prepared by
dissolving 0.26 g of either salt in 50 ml of room
temperature acetone. Equal volumes of-the salt solution
and the polymer solution were--combined and the mixture was
WO 95!29664 218 9 2 5 4 P~~S951D5511
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sonicated for approximately four minutes. One hundred
milliliter samples of the salt-polymer solution were
poured into the teflon petri dishes. The methylene
chloride was evaporated as described previously.
EXAMPLE II-
preparation of Polymer Pellets Containing Salts
Blocked and unblocked-PLGA (50:50) polymers
(respectively, RG502 (Mw 12,700 Daltons) and RG502H (Mw
9,300 Daltons); Boehringer Ingelheim Chemicals, Inc.,
Montvale, NJ) were used for all polymer pellet studies.
Samples of the blocked-PLGA and unblocked-PLGA were mixed
with MgC03 (10% w/w) or ZnCO, (10% w/w)- and were
incorporated into the pellets as particulates. Prior to
mixing into the PLGA, the salts were sieved as described
in Example I.to control the particle size. Blocked-PLGA
and unblocked-PLGA polymer pellets containing 0.0% salt
were used as controls.
The polymer pellets of blocked-PLGA or unblocked-
PLGA, (containing 0.0% salt, 10% w/w MgC03 or 10% w/w
ZnC03) were prepared using a Carver Laboratory Press Model
C. Each polymer sample was heated-at 60 °C for 10 minutes
and then pressed for 1 minute at 15,000 pounds to form
polymer pellets.
EXAMPLE III
Water Uptake in Polymer Films and Pellets
Water uptake studies were conducted on the polymer
films of Example I and the control polymer pellets of
Example II. The buffer solutions used in this study were
_ HEPES (10 mM HEPES, 130 mM NaCl, 0.1% NaN" 0.1% Pluronics
F68, pH 7.3) or PBS (50 mM Sodium Phosphate, 78 mM NaCl,
0.1% NaN3, 0.1% Pluronics F68, pH 7.2). Polymer film
samples (50-80 mg) were incubated in buffer (0.5 ml/mg
film) at 37 °C. Polymer pellet samples (160 mg) were
WO 95129664 - 218 9 2 5 4 PCTIUS95105511
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incubated in buffer (.1 ml/mg of pellet) at 37 °C. When
incubated, the pellets were placed in scintillation vials.
Duplicate polymer film samples were utilized ~or each of
the time points to enable both dry and wet weight
measurements.
Samples were recovered at the specified time
intervals, the-surface water removed with absorbent paper
and the samples were weighed. Water-uptake (wet weight)
of the polymer samples was then determined
gravimetrically. _
The polymer film and pellet samples were then frozen
at -80 °C and subsequently lyophilized for 3-4--days until
a constant dry weight was achieved. The weights of the
dried-films were measured after lyophiliaation. Buffer
solution was replaced in full-for the film samples being
incubated for the later water uptake determinations.
Water uptake was calculated at each time point using
the following equation:
$H=0 Uptake= Wt. hydrated - Wt. dried X-100
Wt. dried
Values obtained for duplicate samples of films were
averaged.
The effects of different-salts on the water uptake of
blocked-PLGA films are shown in Figures 1-8. The control
films (blank films) without incorporated salts showed a
slow, gradual increase in the amount of water absorbed
during the first 15 to 20 days (Figure 1). After this
time, a large increase in water uptake was observed. This
secondary phase of water uptake was associated with
polymer degradation (see Example IV). Films containing
inert particles (carbon black-or glass particles) '
exhibited water uptake profiles similar to the control
polymer films (Figure 1). '
WO 95!29664 218 9 2 5 4 P~~595/OSS1I
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Films containing insoluble salts (MgC03, Mg(OH)z and
ZnCO,) all exhibited a greater initial water uptake than
control films (Figures 2-4). Following the initial uptake
phase, about 3 days, the amount of water.absorbed by the
films containing MgC03 and Mg(OH)2 did not change until
after 30 days. The second phase of water uptake occurred
approximately 2 weeks later than was observed with control
polymer films.
ZnCO, films exhibited a more continuous water uptake
of a magnitude greater than that of control films
(Figure 4). There was no clear distinction between
initial and secondary water uptake phases in the ZnC03
films.
Mg(OAc)~ containing films showed an initial water
uptake that was larger than the blank films (Figure 5),
but not as large as those with the insoluble magnesium
salts. No additional water uptake was observed until
after 21 days, when a second phase of water uptake took
place. The onset of secondary water uptake was delayed by
a few days relative to.the blank film. Water uptake
behavior by Zn(OAc)~, MgSO, and ZnSO, films was similar to
that of the Mg(OAc)2 film samples (Figures 6-8).
The comparative water uptakes of theblocked and
unblocked-PLGA pellets are shown in Figure 9. The initial
water uptake over the first 14 days was much greater for
the unblocked-PLGA pellet, wherein this pellet absorbed
water equal to its dry weight by day 7. By comparison,
the blocked-PLGA pellet had only absorbed 3% of its dry
weight by day 10. The wet mass of the unblocked polymer
could not be determined accurately after day 14 due to the
softening and degradation of the polymer pellet.
W095129664 ~ ~ 8 9 Z:5 4 PCT/US95105511
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EXAMPLE IV
Effect of Salts on Polymer Dectradation
The effects of encapsulated salts on polymer
degradation rates were assessed using molecular weight
determined by gel permeation chromatography (GPC). The
films of Example I and control pellets of-Example II were
hydrated as described in Example III. The polymer samples
were redissolved in chloroform (5-10 mg/m1) and were_
filtered through a 0.22 micron filter. GPC was conducted
using a MIXED column (300 x 10 mm, Polymer Labs) with
chloroform as eluent and refractive indeX -for detection.
Molecular weights were calculated using polystyrene as
standards (580 to 95D,OD0 Daltons) and the universal
calibration method.
The molecular weight of-the control films decreased
from 42DD0 to 3000 Daltons after 30.days as shown in
Figure 10.
In contrast, the rate of decrease in molecular weight
of the films containing MgC03 were smaller than for the
control film (see Figure 10). The molecular weight
decrease in films with ZnC03 was slower than in control
films (Figure 11), but more rapid than in films containing
MgCO,. Similar degradation kinetics were observed with
Mg(OAc)z containing films (Figure 12),_
Regarding the control pellets of Example II, shown in
Figure 13, the initial degradation rate of theunblocked-
PLGA pellet, as determined by linear least squares fit,
was about 6.5 times the initial degradation rate of the
blocked-PLGA pellet. After day 10, the degradation rate
of the blocked-PLGA pellet became approximately the same
as the unblocked-PLGA pellet, which corresponds to the
point where water absorption began to increase for the
unblocked-PLGA pellet. Thus for both control pellets,
polymer degradation correlated closely with increased-
water absorption into the control pellets.
WO 95!29664 2 1 8 9 2 5 4 PC'fIUS95105511
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F~XAMPLE V _
°~~ of Salts on Glass Transition Temperature
The glass transition temperature (Tg) of the films
was determined using a differential scanning calorimeter-
s (DSC) (DSC 7 Serial, Perkin Elmer,-Norwalk, CT) under
nitrogen and using indium as a standard. Each sample was
cooled to 0'C before heating to 60'C at 10'C/min. Tg
measurements were performed on the film samples after
lyophilization as described in Example III.
The time course of Tg decrease far control films is
plotted in Figure 14. The drop in Tg observed between 10
and IS days corresponds to the point at which the polymer
MW decreases to less than 20,000 Daltons.
In contrast, the rates of Tg decrease in polymer
films that contained Mg and Zn salts (Figures 14-16) were
either negligible (in the case of MgCO,; Figure 14), or
significantly slower (ZnCO, and Mg(OAc)z; Figures 15 and
16; respectively) than those of control films. In MgC03
and ZnC03 containing films, a trend toward a slower Tg
decrease with increasing salt content was observed.
EXAMPLE VI
FffPct of Salts on Film Porositv
SEM was used to observe qualitative changes in film
porosity and to monitor morphology changes of the film
surfaces and cross sections over time. Samples were
lyophilized as described in Example III. The dried
samples were sputter-coated with gold 200-300 A and the
samples observed using JEOL-6400 SEM.
A11 films displayed a dense structure with a few
pores scattered throughout the device prior to hydration.
However, the rate of water uptake was different depending
on the incorporated salt. Thus the increase in water
WO 95129664 218 9 2 5 4 PCTIUS95105511
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uptake was not dominated by initial porosity of the sample
but was a function of the type of salt dispersed in the
polymer film.
SEM evaluation of the control films without salts
demonstrated a dense and smooth structure up to 14 days of
hydration. Between 14 and 22-days, large pores became
visible on the film surface and throughout the sample
cross section. The appearance of-these pores coincides
with the secondary water uptake phase associated with
polymer degradation and erosion of the polymer- (see
Examples III- - V).
Films loaded with water insoluble salts exhibited
increasing porosity after hydration times as short as 24
hours- SEM analysis of 24 hour hydration samples of films
containing 2% MgC03 showed the formation of a porous
network within the film sample, concentrated at the film
surface. After 7 days, the film had become uniformly
porous across the cross section. Pores ranged in diameter
from approximately 1-20 Vim. No further increase in
porosity was observed between 7 days and 22 days. Similar
behavior was observed with films that contained higher
MgCO, percentages.
Films that contained 10% ZnCO, were also observed to
become highly porous within 3 daysofhydration. Three
day hydration samples showed the presence of a porous
network-extending throughout the entire film cross
section. The morphology of hydrated ZnC03 containing
films was similar to hydrated films with MgCO,.
Films that contained water soluble magnesium salts
also exhibited the formation of internal and surface pores
and voids well before-pore formation occurred in control
films. Pores rangingin-diameter from approximately-1-50
~m were visible in samples that had been hydrated for five _
days.
WO 95129664 ~ 2 ~ ~ PCTIU595105511
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There was some difference between the morphology of
the films loaded with soluble and insoluble salts that
were hydrated for 5 to 7 days. The films loaded with
Mg(OAc), seemed to display a lower porosity and a tendency
toward large voids (approximately 50 microns) compared to
films that contained insoluble salts. MgC03 and ZnCO,
films showed a higher porosity; a majority of the pore
volume was composed of pores of lesathan ten microns in
diameter.
EXAMPLE VII
Effect of Salts on Polymer Weicrht Loss
The effects of insoluble salts on polymer degradation
in hydrated polymer samples were also assessed by
monitoring the time course of polymer weight loss during
incubation. The films of Example I, and the pellets of
Example II, were-hydrated as described in Example III.
Samples were recovered at the indicated time intervals and
freeze-dried as described in Example III. The weights of
the dried polymer samples were gravimetrically measured
after lyophilization. Percent weight loss at different
times was computed according to the equation:
% Weight- LOBS (t) = 100 X (Wi"intai- -We) ~Winitial
where Winicisi is the initial weight of the polymer and Wt is
the weight of the sample at time point t.
The effects of different salts on the weight loss of
the PLGA films of Example I are shown in Figure 17.
As shown therein, the time course of weight loss in the
control film exhibited little weight loss until 14 days,
after which rapid weight loss takes place. This phase of
weight lose is associated with degradation and erosion of
the polymer, as evidenced by increased water uptake,
decreased molecular weight and Tg and the appearance of
R'O 95129664 218 9 2 5 4 PCTIUS95105511
_zs_
pores and voids in SEMs of film samples (see Examples III,
IV, V and VI). Also shown in Figure 1Z_are weight loss
profiles for polymer films that contain 5, 10 and 15% '
MgCO,. Instead;-weight loss ixi these films was more
gradual and of a lesser magnitude. - '
A portion of the weight loss occurring in-MgCO,-
containing films was due to dissolution of the
encapsulated salt particles. To assess how closely total
weight loss measurements approximate polymer weight loss
in salt-containing film samples, the polymer weight loss
was estimated according to the following two extreme ,
scenarios: (1) all of the encapsulated salt dissolved
between the initial hydration=and the first time point,
and (2) no salt dissolved throughout the entire study.
Regardless of which salt dissolution scenario was
selected, polymer weight loss-in control films exceeded
that of MgCO;-containing films, indicating that
incorporation of the insoluble salt prevented or delayed
erosion of the polymeric matrix.
The effects-of different-salts, and of the choice of
polymer end group, on polymeric weight lose for the
blocked-PLGA and unblocked-PLGA pellets of Examgle IL are
shown in Figure 18. As shown therein, the time course of
weight loss in the control blocked-PLGA pellet (blocked-
PLGA pellet with 0.0% salt) and the unblocked-PLGA pellet
(unblocked-PLGA with Q.0% salt) exhibited little weight
loss until day 10 and 20, respectively, after which rapid
weight loss takes place. Thus, polymeric degradation can
be substantially modulated by choice of the end group of
the PLGA. --
Also shown in Figure 18 are weight loss profiles for
unblocked-PLGA pellets that contain 10% MgC03 or 10%
ZnC03. Weight lose in the--unblocked-PLGA pellets -
containing ZnC03 was not substantially different from the
WO 95129664 2 ~ 8 l L-5 4 PCT/US95/05511
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control unblocked-PLGA. Weight Loss in the unblocked PLGA
pellets containing 10% MgCO" before day 25, was more
gradual.
Further shown in Figure 18 are weight loss profiles
for blocked-PLGA pellets that contain 10% MgC03 or 10%
ZnC03. Weight Loss in the blocked-PLGA pellets containing
MgC03 and ZnC03 was substantially more gradual and of a
lesser magnitude than the control blocked-PLGA pellet.
These blocked-PLGA pellets exhibited little weight loss
until after day 30.
EXAMPLE VIII
Comparison of the Effect of Zinc Carbonate on In Vivo and
In Vitro Degradation of Blocked-PLGA Microspheres
Microspheres of blocked--PLGA (50:50 PLGA, 1D,000
Daltons; Lot #115-56-1, Birmingham Polymers, Inc.,
Birmingham, AL), containing 6% w/w ZnC03, were formed by
the method described in U.S. Patent 5,019,4D0, issued to
Gombotz et a1. Specifically, the ZnCO; was added as a
particulate to a solution of PLGA in methylene chloride
whichwas sonicated at 4'C for 30 seconds to form a
suspension. The suspension was then sprayed into liquid
nitrogen which was overlaying frozen ethanol. The
methylene chloride was extracted into the ethanol at
-80'C. The microspheres were filtered and lyophilized to
produce a dry powder.
The effect of zinc carbonate upon in vitro molecular
weight degradation of blocked-PLGA was assessed. The
blocked-PLGA microspherea were incubated in HEPES buffer
(50 mM HEPES, pH 7.4) in a concentration of 10 mg
microspheres/ml at 37 °C. Microsphere samples were
recovered at the specified time intervals, and freeze
dried by freezing at -SD °C and subsequently lyophilized
for 2-3 days.
WO 95129664 2 ~ 8 Q 2 5 4 PCTIUS95/05511
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In addition, the effect of zinc carbonate-upon in
vivo molecular-weight degradation o~ blocked-PLGA was
assessed. PLGA microspheres containing O.Oa and 6% w/w
ZnCO, were administered to separate test groups of-normal
rats (Taconics, 7~c.), with three rats-in each test group.
Microsphere doses of 50 mg were injected, in 750 u1 of
vehicle-(3% carboxymethyl cellulose (low viscosity) and 1%
Tween-20 in saline), into the intrascapular region of the
rats.
Rats (Sprague-Dawley males) were anesthetized with a
halothane and oxygen mixture. The injection sites
(intrascapular region) were-shaven and marked-with a
permanent tatoo to provide far-the precise excision of
skin at the sampling time points. Each rat was injected
with an entire vial of microspheres using 18 to 21 gauge
needles.
On designated days (days 15 and 30 post-injection)
for animals receiving blocked-PLGA microspheres, the-rats
were sacrificed by asphyxiation with COZ gas and the skin -
at the injection sites (including microspheres) was
excised. Remaining microspheres were then collected.
The effects of ZnC03 on the in vitro and in vivo
polymer degradation rates of blocked-PLGA polymers were
assessed using molecular weight determined by gel
permeation chromatography (GPC) as described in Example
III. The results of these analyses are provided in
Figures 19-and 20. As shown therein, the addition of
ZnC03 substantially slowed molecular weight degradation of
blocked-PLGA for both in vitro and in vivo-microspheres.
2189254
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EXAMPLE IX
Effect of Salts on the Release of RNase-A or ACTH
from PLGA Microsbheres
A 10 mg/m1 RPIase-A solution was formed by dissolving
RNase-A (R5500; Sigma Chemicals) in deionized water. A
buffered-adrenocorticotropin hormone (ACTH) was formed by
dissolving lyophilized porcine ACTH powder (Diosynth,
Chicago, IL) in an aqueous 60 mM ammonium bicarbonate
buffer.
In separate procedures, each solution (RNase-A
solution and buffered ACTH solution) was then micronized
using an ultrasonic nozzle (Type V1A; Sonics and
Materials, Inc., Danbury, CT) and sprayed into liquid
nitrogen in a polypropylene tub (17 cm in diameter and 8
cm deep) to form frozen particles of RNase-A solution or
frozen particles of buffered ACTH solution. The
polypropylene tub was then placed into a -80 °C freezer
until the liquid nitrogen evaporated. The frozen RNase-A
solution particles or frozen buffered ACTH solution
particles were then lyophilized to form lyophilized RNase-
A or lyophilized buffered ACTH, respectively.
Lyophilized RNase-A was then micrnencapsulated into
5000 Dalton blocked-PLGA, (I. V. 0.15 dl/g Birmingham
Polymers, Birmingham, AL) with either ZnC03 or Mg(OH)2.
The method described in U.S. Patent 5,019,400, issued to
Gombotz et al., was used to encapsulate the lyophilized
RNase-A (10% w/w) in PLGA containing 0%, S%, 10% or 15%
w/w of salt. Specifically, the lyophilized RNase-A and
salt were added as particulates to a solution of PLGA in
methylene chloride which was sonicated at 4'C for 3D
seconds to form a suspension. The suspension was then
sprayed into liquid nitrogen which was overlaying frozen
WO 95129664 218 9 2 5 4 PCT/US95105511
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ethanol. The methylene chloride was extracted into the
ethanol at -SO'C. The microspheres were-filtered and
lyophilized o produce a dry powder.
Lyophilized buffered ACTH was also microencapsulated
into the same type PLGA with MgC03 by the method described
above.
The effect of the salts upon the in vitro release
kinetics of RNase-A and ACTH was asaeased_ Release -
studies were conducted by suspending 20 mg of microspheres
in 1 ml of 10 mM HEPES buffer-at 37 'C. Assays weradone
in 2 ml polypropylene Eppendbrf tubes. Release studies of-
ACTH were conducted in the same manner with the exception
of using PBS in lieu of H$PES buffer. At the specified
time points, the buffer was removed in full and replaced
with fresh buffer. The concentration of RNase-A in buffer
was measured using the BCA Protein Assay (Pierce,
Rockford, IL) and the concentration of ACTH was measured
using the Biorad Protein assay (Biorad, Richmond, CA).
The effects of Mg(OH)2 or ZnCO, on the release
kinetics of RNase-A are shown-in Figures 21 and 22.
RNase-A encapsulated into PLGA alone exhibited release of
the protein over the first 24 hours after which no further
release was observed until day twenty one. Mg(OH)2
resulted in continuous release of the protein over 14
days. ZnC03 resulted in continuous release of the protein
over thirty five days.
The effect of MgCO, on the release kinetics of ACTH
is shown in Figure 23. ACTH encapsulated into PLGA alone
exhibited approximately 40% release of the protein over
the first 24 hours after- which no further release was
observed.- MgCO; resulted in continuous release of the
protein-over the same period..
WO 95129664 218 9 2 .~ 4 p~~g95/05511
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EXAMPLE X
Effect of Salts on the In Vivo Release of Interferon-a 2 b
from PLGA Microspheres
The Interferon-a,2b, (IFN-a,2b) used herein is
identical to the IFN-a,2 described in Rubenstein et al.,
Biochem. Biophys. Acts, 695: 705-716 (1982), with the
exception that the lysine as position23 of IFN-a,2 is an
arginine in IFN-a,2b. The IFN-a,2b was dissolved in 10 mM
sodium bicarbonate buffer (pH 7.2) to form an IFN
solution. A 10 mM Zn'' solution was prepared from
deionized water and zinc acetate dihydrate, and then was
added to the IFN solution, at a molar ratio of 2.1
Zn'':IFN-a,2b to form a solution with a final IFN-a,2b
concentration of about 1.3 mg/ml. The pH of -the solution
was then adjusted to 7.1 by adding 1% acetic acid. A
cloudy suspended precipitate, comprising Zn"-stabilized
IFN then formed.
The suspension was micronized, frozen and
lyophilized, as described in Example IX, and then dried to
form an IFN powder. Zinc carbonate and IFN powder were
added in proportions, by mass, of about 1:1, 3:1 or 8:1,
respectively, to PLGA solutions containing about 0.4 g
PLGA in about 4 ml of methylene chloride, and then were
microencapsulated in the PLGA, also as described in
Example IX, to form IFN microspheres.
The effect of the zinc carbonate upon the in vivo
release kinetics of IFN, from lyophilized Zn-IFN
precipitate, was then assessed. Each type of IFN
microsphere was administered to separate test groups of
normal rats (Taconics, Inc.), with three rats in each test
group. Microsphere doses of 0.9 mg/kg were injected, in a
0.5% gelatin, 1% glycerol and 0.9% w/w NaCl vehicle, into
the intrascapular region of the rats. Blood samples were
then taken from thetail vein of each rat at 1, 2, 4, 8,
12, 24, 32, 28, 72, 96, 120, 144 and 168 hours after
WO 95129664 PCTIUS95105511
2189254
-32_
injection.- The IFN-a,2b concentrations in the-rat serum
samples were then determined using an IFN-a
immunoradiometric assay (Celltech, Slough, U.K.). The
assay results are presented in Figure 24, which shows that
the sustained release level of immunologically active IFN-
a,2b was modulated depending upon the ratio o~--ZnCO, to
lyophilized Zn-IFN in the PLGA polymer. Higher ratios of
ZnC03:lyophilized Zn-IFN demonstrated lower release rates
of IFN-a,2b from the microspheres as measured by IFN-a,2b
serum levels.
EXAMPLE XI . _ _. _.... - _
EFFECT OF END GROUPS ON IN VIVO PLGA DEGRADATION
Microspheres containing Zn'2-stabilized human growth
hormone (hGH), whose DNA sequence is described in U.S.
Patent 4,898,830, issued to Goeddel et al., were prepared -
from hydrophilic polymer RG502H having free carboxylQnd
groups (hereinafter-"unblocked-PLGA") (50-:50-PLGA, 9,300
Daltons; Boehringer Ingelheim Chemicals, Inc.) or a more
hydrophobic polymer having blocked carboxyl end groups
(hereinafter "blocked-PLGA") (50:50 PLGA, 10,000 Daltons;
Lot #115-56-1, Birmingham Polymers, Inc., Birmingham, AL).
The hGH was first Zn"-stabilized by forming an
insoluble complex with zinc.- A D.9 mM aqueous solution of
zinc acetate was added to a solution of hGH (10 mg/mI) in
bicarbonate buffer (0.336 mg/ml) to form an insoluble
complex having a Zn:hGH molar ratio-of--6:1. The pH of the
complex was adjusted to approximately 7.2 with 1% acetic
acid.
The method described in Example IX was used to form
microspheres by encapsulating 0% or 15% w/w hGH, in the
form of-Zn:hGH complex, and also 0%, 1% or 6%-w/w ZnCO,
salt, within blocked-PLGA and within unblocked-PLGA. In .-
vivo degradation of unblocked-PLGA microspheres versus
blocked-PLGA microspheres were compared by injecting
218925=4 .
WO 95129664 PCT/US95I05511
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samples of microspheres into rats and then analyzing the
microspheres remaining at the injection site at various
times post-injection. Three rats were assayed at each
time point for each microsphere sample. On the day of
administration of the microspheres, 750 ~C1 of vehicle (3%
carboxymethyl cellulose (low viscosity) and 1% Tween-20 in
saline) was added to vials containing 50 ~ 1 mg of
microspheres. Immediatel-y, the vials were shaken
vigorously to form a suspension which was then aspirated
into a 1.0 cc syringe without a needle.
Rats (Sprague-Dawley males) were anesthetized with a
halothane and oxygen mixture. The injection sites
(intrascapular region) were shaven and marked with a
permanent tatoo to provide for the precise excision of
skin at the sampling time points. Each rat was injected
with an entire vial of microspheres using 18 to 21 gauge
needles.
On designated days (days 15, 30, 59 and 90 post-
injection for animals receiving blocked-PLGA microspheres,
or days 7, 14, 21, 28 and 45 post-injection for animals
receiving unblocked-PLGA microspheres) the rats were
sacrificed by asphyxiation with COz gas and the skin at
the injection sites (including microspheres) was excised.
Since the microspheres tended to clump at the injection
sites, the presence or absence of microspheres was
determined visually.
The visual inspections found that the unblocked-PLGA
microspheres degraded substantially faster than the
blocked-PLGA microspheres, and than the addition of ZnCO;
to the blocked=PLGA substantially slowed polymeric
degradation. For example, in the rats injected with
unblocked-PLGA microspheres containing 0% hGH and 0% or 1%
ZnC03, no microspheres were visible on day 21. In
addition, for rats injected with blocked-PLGA microspheres
containing 0% hGH and 0% ZnC03, a few microspheres were
WO 95129664 2 l 8 9 2 5 4 PCTlU595/U5511
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visible on day 60 and none were visible on day 90.
Furthermore, for rats injected with blocked-PLGA
microspheres containing 0% or 15% hGH and 6% ZnC03,
microspheres were-visible on day 90.
EXAMPLE XII _
aee~y FOR hGH AFTER IN VIVO DEGRADATION __
OF Blocked-PLGA -Zn*~-STABILIZED hGH MICROSPHERES
Microspheres of blocked-PLGA, containing 16% w/v
Zn'2-stabilized hGH-and 0%, 6%, 10% or 20% ZnC03 were
formed by the method of Example IX. Groups of test rats
were injected with 50 mg samples ofthe different hGH
microspheres, also as described in Example XI. The rats
were sacrificed after 60 days-and the skin sample were
excised from the injection sites. The excised skin
samples were placed in 10% Neutral Buffered Formalin for
at least 24 hours. They were-then trimmed with a razor
blade to remove excess skin and placed in PBS. Tissue
samples were processed by Pathology Associates, Inc_-
(Frederick, MD). The skin samples were embedded in
glycomethacrylate, sectioned and assayed for the presence
of hGH using a HistoScan/LymphoScan Staining Kit (Product
#24-408M; Accurate Chemical & Scientific_Corp., Westbury,
NY) according to the manufacturer's instructions. Tissue
samples were scored for the presence or absence of
staining which was indicativeof the presence or absence
of hGH in the sample. A11 skin samples, associated with -
hGH microsphere injections, tested positive for the -
presence of hGH thus indicating that the blocked-PLGA
microspheres were still contained hGH after 60 days in
vivo.
