U.S. patent application number 11/826534 was filed with the patent office on 2008-05-29 for polymer-based sustained release device.
This patent application is currently assigned to Alkermes, Inc.. Invention is credited to Troy Christensen, Henry R. Costantino, Joyce M. Hotz, Rajesh Kumar, Michael E. Rickey, Steven G. Wright, Thean Y. Yeah.
Application Number | 20080125348 11/826534 |
Document ID | / |
Family ID | 34969591 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080125348 |
Kind Code |
A1 |
Wright; Steven G. ; et
al. |
May 29, 2008 |
Polymer-based sustained release device
Abstract
This invention relates to compositions for the sustained release
of biologically active polypeptides, and methods of forming and
using said compositions, for the sustained release of biologically
active polypeptides. The sustained release compositions of this
invention comprise a biocompatible polymer having dispersed
therein, a biologically active polypeptide and a sugar.
Inventors: |
Wright; Steven G.; (Madeira,
OH) ; Christensen; Troy; (Mason, OH) ; Yeah;
Thean Y.; (Foxboro, MA) ; Rickey; Michael E.;
(Loveland, OH) ; Hotz; Joyce M.; (Cincinnati,
OH) ; Kumar; Rajesh; (Marlborough, MA) ;
Costantino; Henry R.; (Woodinville, WA) |
Correspondence
Address: |
COVINGTON & BURLING, LLP;ATTN: PATENT DOCKETING
1201 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20004-2401
US
|
Assignee: |
Alkermes, Inc.
Cambridge
MA
|
Family ID: |
34969591 |
Appl. No.: |
11/826534 |
Filed: |
July 16, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11104877 |
Apr 13, 2005 |
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11826534 |
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60563245 |
Apr 15, 2004 |
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Current U.S.
Class: |
424/499 ;
514/16.4; 514/17.9; 514/7.2; 514/7.3; 514/9.9 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
3/08 20180101; A61P 9/00 20180101; A61K 47/38 20130101; A61P 3/04
20180101; A61P 43/00 20180101; A61K 38/2278 20130101; A61K 9/1647
20130101; A61P 7/12 20180101; A61K 47/34 20130101; A61K 9/1623
20130101; A61K 38/26 20130101; A61K 9/1694 20130101; A61K 47/26
20130101; A61K 9/0024 20130101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 38/00 20060101
A61K038/00 |
Claims
1. A composition for sustained-release of a polypeptide, consisting
essentially of: a poly(lactide-co-glycolide) polymer with a
lactide:glycolide ratio of about 1:1 having dispersed therein about
3%-5% (w/w) of the polypeptide and about 2% (w/w) of a sugar.
Description
BACKGROUND OF THE INVENTION
[0001] Numerous proteins and peptides, collectively referred to
herein as polypeptides, exhibit biological activity in vivo and are
useful as medicaments. Many illnesses or conditions require
administration of a sustained level of medicament to provide the
most effective prophylactic and/or therapeutic effects. Sustained
levels are often achieved by the administration of biologically
active polypeptides by frequent subcutaneous injections, which
often results in fluctuating levels of medicament and poor patient
compliance.
[0002] As an alternative, the use of biodegradable materials, such
as polymers, encapsulating the medicament can be employed as a
sustained delivery system. The use of biodegradable polymers, for
example, in the form of microparticles or microcarriers, can
provide a sustained release of medicament, by utilizing the
inherent biodegradability of the polymer to control the release of
the medicament thereby providing a more consistent, sustained level
of medicament and improved patient compliance.
[0003] However, these sustained release devices can often exhibit
high initial bursts of medicament and minimal release thereafter,
resulting in serum drug levels outside the therapeutic window
and/or poor bioavailability of the medicament. In addition, the
presence of polymer, physiological temperatures and body response
to the sustained release composition can cause the medicament to be
altered (e.g., degraded, aggregated) thereby interfering with the
desired release profile for the medicament.
[0004] Further, methods used to form sustained release compositions
can result in loss of activity of the medicament due to the
instability of the medicament and the degradative effects of the
processing steps. Degradative effects are particularly problematic
when the medicament is a polypeptide.
[0005] Therefore, a need exists for a means of administering
biologically active polypeptides in a sustained fashion wherein the
amount of polypeptide delivered is at therapeutic levels, and
retains activity and potency for the desired period of release.
While much work has been developed that addresses these problems,
novel solutions are required.
SUMMARY OF THE INVENTION
[0006] The invention relates to the discovery that superior release
profiles (such as those characterized by a ratio of C.sub.max to
C.sub.ave of about 3 or less) can be achieved with a formulation
containing few components by optimizing the silicone oil to polymer
ratio in the manufacturing process, thereby achieving a low pore
volume. This invention relates to compositions for the sustained
release of agents, such as biologically active polypeptides, and
methods of forming and using such compositions, for the sustained
release of biologically active polypeptides. The sustained release
compositions of this invention comprise a biocompatible polymer, an
agent, such as a biologically active polypeptide, and a sugar. The
polypeptide and sugar are preferably dispersed in the polymer. The
polypeptide and sugar can be dispersed separately or, preferably,
together. The sustained release composition provides a desirable
and consistent release profile. In a particular embodiment, the
profile is characterized as having a ratio of C.sub.max to
C.sub.ave of about 3 or less. In a preferred embodiment, the
biologically active polypeptide is an antidiabetic or
glucoregulatory polypeptide, such as GLP-1, GLP-2, exendin-3,
exendin-4 or an analog, derivative or agonist thereof, preferably
exendin-4. The sugar is preferably sucrose, mannitol or a
combination thereof. A preferred combination includes exendin-4 and
sucrose and/or mannitol.
[0007] Additionally or alternatively, the sustained release
composition comprises a biocompatible polymer, an agent, such as a
biologically active: polypeptide and a sugar wherein the
composition has a total pore volume of about 0.1 mL/g or less. In a
specific embodiment, the total pore volume is determined using
mercury intrusion porosimetry.
[0008] Additionally or alternatively, the sustained release
composition consists essentially of or, alternatively consists of,
a biocompatible polymer, exendin-4 at a concentration of about 3%
w/w and sucrose at a concentration of about 2% w/w. The
biocompatible polymer is preferably a poly lactide coglycolide
polymer.
[0009] The invention also includes a method for forming
compositions for the sustained release of biologically active
agents, such as polypeptides, which comprises forming a mixture by
combining an aqueous phase comprising water, an agent, such as a
water soluble polypeptide, and a sugar with an oil phase comprising
a biocompatible polymer and a solvent for the polymer; forming a
water-in-oil emulsion by, for example, sonicating or homogenizing,
the mixture; adding silicone oil to the mixture to form embryonic
microparticles; transferring the embryonic microparticles to a
quench solvent to harden the microparticles; collecting the
hardened microparticles; and drying the hardened microparticles. In
a particular embodiment, the silicone oil is added in an amount
sufficient to achieve a silicone oil to polymer solvent ratio of
about 1.5:1. Additionally or alternatively, the polymer is present
in the oil phase at about 10% w/v or less.
[0010] The agent or polypeptide, e.g. exendin-4, can be present in
the composition described herein at a concentration of about 0.01%
to about 10% w/w based on the total weight of the final
composition. In addition, the sugar, e.g. sucrose, can be present
in a concentration of about 0.01% to about 5% w/w of the final
weight of the composition.
[0011] The composition of this invention can be administered to a
human, or other animal, by injection, implantation (e.g.,
subcutaneously, intramuscularly, intraperitoneally, intracranially,
and intradermally), administration to mucosal membranes (e.g.,
intranasally, intravaginally, intrapulmonary or by means of a
suppository), or in situ delivery (e.g., by enema or aerosol
spray).
[0012] When the sustained release composition has incorporated
therein a hormone, particularly an anti-diabetic or glucoregulatory
peptide, for example, GLP-1, GLP-2, exendin-3, exendin-4 or
agonists, analogs or derivatives thereof, the composition is
administered in a therapeutically effective amount to treat a
patient suffering from diabetes mellitus, impaired glucose
tolerance (IGT), obesity, cardiovascular (CV) disorder or any other
disorder that can be treated by one of the above polypeptides or
derivatives, analogs or agonists thereof.
[0013] The use of a sugar in the sustained release compositions of
the invention improves the bioavailability of the incorporated
biologically active polypeptide, e.g, anti-diabetic or
glucoregulatory peptides, and minimizes loss of activity due to
instability and/or chemical interactions between the polypeptide
and other components contained or used in formulating the sustained
release composition, while maintaining an excellent release
profile.
[0014] The advantages of the sustained release formulations as
described herein include increased patient compliance and
acceptance by eliminating the need for repetitive administration,
increased therapeutic benefit by eliminating fluctuations in active
agent concentration in blood levels by providing a desirable
release profile, and a potential lowering of the total amount of
biologically active polypeptide necessary to provide a therapeutic
benefit by reducing these fluctuations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a graph showing the relationship between the
average pore diameter and the in vitro release for sustained
release compositions described herein (A.S.=Ammonium Sulfate).
[0016] FIG. 2 is a graph showing the effect of porosity on the in
vitro release of exendin-4 from microparticles and the impact that
the processing conditions, namely the ratio of silicone oil to
methylene chloride, has on the porosity of the microparticles
formed.
[0017] FIGS. 3A-3B are scans of cryogenic SEMs for selected
microparticle formulations described herein.
[0018] FIG. 4A-4D are scans of cryogenic SEMs for selected
microparticle formulations described herein.
[0019] FIG. 5 is a plot of % residual ethanol and methylene
chloride versus Tg for microparticle formulations described
herein.
[0020] FIG. 6 is a representative pharmacokinetic curve
(concentration, pg/ml v. time, days with inset showing
concentrations over first day) for Formulation 2-1 (3% exendin-4
and 2% sucrose), Formulation 1 (3% exendin-4 alone) and Formulation
4 (3% exendin-4 and 0.5% ammonium sulfate).
[0021] FIG. 7 is a graph of in vivo release profile for the three
microparticle Formulations 2, 2-1 and 2-2.
[0022] FIG. 8 is a graph of the pharmacokinetic data for
microparticle Formulations 5-1, 5-2 and 5-3.
[0023] FIG. 9 is a graph illustrating the relationship between
process parameters and the inner emulsion size achieved by the
process.
DETAILED DESCRIPTION OF THE INVENTION
[0024] This invention relates to compositions for the sustained
release of biologically active polypeptides, and methods of forming
and using said compositions, for the sustained release of
biologically active polypeptides. The sustained release
compositions of this invention comprise a biocompatible polymer,
and agent, such as a biologically active polypeptide, and a sugar.
The agent and sugar are dispersed in the biocompatible polymer
separately or, preferably, together. In a particular embodiment,
the sustained release composition is characterized by a release
profile having a ratio of maximum serum concentration (C.sub.max)
to average serum concentration (C.sub.ave) of about 3 or less. As
used herein, the terms a or an refer to one or more.
[0025] The Agent
[0026] In a preferred embodiment, the agent is a biologically
active polypeptide such as an antidiabetic or glucoregulatory
polypeptide, including GLP-1, GLP-2, exendin-3, exendin-4 or an
analog, derivative or agonist thereof. Most specifically, the
polypeptide is exendin-4. However, other agents can take advantage
of the discoveries made herein.
[0027] Biologically active polypeptides as used herein collectively
refers to biologically active proteins and peptides and the
pharmaceutically acceptable salts thereof, which are in their
molecular, biologically active form when released in vivo, thereby
possessing the desired therapeutic, prophylactic and/or diagnostic
properties in vivo. Typically, the polypeptide has a molecular
weight between 500 and 200,000 Daltons.
[0028] Suitable biologically active polypeptides include, but are
not limited to, glucagon, glucagon-like peptides such as, GLP-1,
GLP-2 or other GLP analogs, derivatives or agonists of Glucagon
Like Peptides, exendins such as, exendin-3 and exendin-4,
derivatives, agonists and analogs thereof, vasoactive intestinal
peptide (VIP), immunoglobulins, antibodies, cytokines (e.g.,
lymphokines, monokines, chemokines), interleukins, macrophage
activating factors, interferons, erythropoietin, nucleases, tumor
necrosis factor, colony stimulating factors (e.g., G-CSF), insulin,
enzymes (e.g., superoxide dismutase, plasminogen activator, etc.),
tumor suppressors, blood proteins, hormones and hormone analogs and
agonists (e.g., follicle stimulating hormone, growth hormone,
adrenocorticotropic hormone, and luteinizing hormone releasing
hormone (LHRH)), vaccines (e.g., tumoral, bacterial and viral
antigens), antigens, blood coagulation factors, growth factors (NGF
and EGF), gastrin, GRH, antibacterial peptides such as defensin,
enkephalins, bradykinins, calcitonin and muteins, analogs,
truncation, deletion and substitution variants and pharmaceutically
acceptable salts of all the foregoing.
[0029] Exendin-4 is a 39 amino acid polypeptide. The amino acid
sequence of exendin-4 can be found in U.S. Pat. No. 5,424,286
issued to Eng on Jun. 13, 1995, the entire content of which is
hereby incorporated by reference. AC2993 and exenatide are
synonymous with the term exendin-4. Exendin-4 has been shown in
humans and animals to stimulate secretion of insulin in the
presence of elevated blood glucose concentrations, but not during
periods of low blood glucose concentrations (hypoglycemia). It has
also been shown to suppress glucagon secretion, slow gastric
emptying and affect food intake and body weight, as well as other
actions. As such, exendin-4 and analogs and agonists thereof can be
useful in the treatment of diabetes mellitus, IGT, obesity,
etc.
[0030] The amount of biologically active polypeptide, which is
contained within the polymeric matrix of a sustained release
composition, is a therapeutically, diagnostically or
prophylactically effective amount which can be determined by a
person of ordinary skill in the art, taking into consideration
factors such as body weight, condition to be treated, type of
polymer used, and release rate from the polymer.
[0031] Sustained release compositions generally contain from about
0.01% (w/w) to about 50% (w/w) of the agent, e.g., biologically
active polypeptide (such as exendin-4) (total weight of
composition). For example, the amount of biologically active
polypeptide (such as exendin-4) can be from about 0.1% (w/w) to
about 30% (w/w) of the total weight of the composition. The amount
of polypeptide will vary depending upon the desired effect, potency
of the agent, the planned release levels, and the time span over
which the polypeptide will be released. Preferably, the range of
loading is between about 0.1% (w/w) to about 10% (w/w), for
example, 0.5% (w/w) to about 5% (w/w). Superior release profiles
were obtained when the agent, e.g. exendin-4, was loaded at about
3% w/w.
[0032] The Sugar
[0033] A sugar, as defined herein, is a monosaccharide,
disaccharide or oligosaccharide (from about 3 to about 10
monosaccharides) or a derivative thereof. For example, sugar
alcohols of monosaccharides are suitable derivatives included in
the present definition of sugar. As such, the sugar alcohol
mannitol, for example, which is derived from the monosaccharide
mannose is included in the definition of sugar as used herein.
[0034] Suitable monosaccharides include, but are not limited to,
glucose, fructose and mannose. A disaccharide, as further defined
herein, is a compound which upon hydrolysis yields two molecules of
a monosaccharide. Suitable disaccharides include, but are not
limited to, sucrose, lactose and trehalose. Suitable
oligosaccharides include, but are not limited to, raffinose and
acarbose.
[0035] The amount of sugar present in the sustained release
composition can range from about 0.01% (w/w) to about 50% (w/w),
such as from about 0.01% (w/w) to about 10% (w/w), such as from
about 0.1% (w/w) to about 5% (w/w) of the total weight of the
sustained release composition. Excellent release profiles were
obtained incorporating about 2% (w/w) sucrose.
[0036] Alternatively, the amount of sugar present in the sustained
release composition can be referred to on a weight ratio with the
agent or biologically active polypeptide. For example, the
polypeptide and sugar can be present in a ratio from about 10:1 to
about 1:10 weight:weight. In a particularly preferred embodiment,
the ratio of polypeptide (e.g., exendin-4) to sugar (e.g., sucrose)
is about 3:2 (w/w).
[0037] Combinations of two or more sugars can also be used. The
amount of sugar, when a combination is employed, is the same as the
ranges recited above.
[0038] When the polypeptide is exendin-4, the sugar is preferably
sucrose, mannitol or a combination thereof.
[0039] The Polymer
[0040] Polymers suitable to form the sustained release composition
of this invention are biocompatible polymers which can be either
biodegradable or non-biodegradable polymers or blends or copolymers
thereof. A polymer is biocompatible if the polymer and any
degradation products of the polymer are non-toxic to the recipient
and also possess no significant deleterious or untoward effects on
the recipient's body, such as a substantial immunological reaction
at the injection site.
[0041] Biodegradable, as defined herein, means the composition will
degrade or erode in vivo to form smaller units or chemical species.
Degradation can result, for example, by enzymatic, chemical and
physical processes. Suitable biocompatible, biodegradable polymers
include, for example, poly(lactides), poly(glycolides),
poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic
acid)s, polycarbonates, polyesteramides, polyanhydrides, poly(amino
acids), polyorthoesters, poly(dioxanone)s, poly(alkylene
alkylate)s, copolymers or polyethylene glycol and polyorthoester,
biodegradable polyurethane, blends thereof, and copolymers
thereof.
[0042] Suitable biocompatible, non-biodegradable polymers 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, polyvinylchloride, polyvinyl fluoride, poly(vinyl
imidazole), chlorosulphonate polyolefins, polyethylene oxide,
blends thereof, and copolymers thereof.
[0043] 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 as the desired polymer
degradation rate, physical properties such as mechanical strength,
end group chemistry and rate of dissolution of polymer in solvent.
Typically, an acceptable range of molecular weight is of about
2,000 Daltons to about 2,000,000 Daltons. In a preferred
embodiment, the polymer is biodegradable polymer or copolymer. In a
more preferred embodiment, the polymer is a
poly(lactide-co-glycolide) (hereinafter "PLG") with a
lactide:glycolide ratio of about 1:1 and a molecular weight of
about 10,000 Daltons to about 90,000 Daltons. In an even more
preferred embodiment, the molecular weight of the PLG used in the
present invention has a molecular weight of about 30,000 Daltons to
about 70,000 Daltons such as about 50,000 to about 60,000
Daltons.
[0044] The PLGs can possess acid end groups or blocked end groups,
such as can be obtained by esterifying the acid. Excellent results
were obtained with a PLG with an acid end group.
[0045] Polymers can also be selected based upon the polymer's
inherent viscosity. Suitable inherent viscosities include about
0.06 to 1.0 dL/g, such as about 0.2 to 0.6 dL/g, more preferably
between about 0.3 to 0.5 dL/g. Preferred polymers are chosen that
will degrade in 3 to 4 weeks. Suitable polymers can be purchased
from Alkermes, Inc. under the tradename Medisorb.RTM., such as
those sold as 5050 DL 3A or 5050 DL 4A. Boehringer Ingelheim
Resomer.RTM. PLGs may also be used, such as Resomer.RTM. RG503 and
503H.
[0046] The sustained release composition 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 polymer component having a diameter of less than about
one millimeter and having biologically active polypeptide dispersed
or dissolved therein. A microparticle can have a spherical,
non-spherical or irregular shape. Typically, the microparticle will
be of a size suitable for injection. A typical size range for
microparticles is 1000 microns or less. In a particular embodiment,
the microparticle ranges from about one to about 180 microns in
diameter.
[0047] Additional Excipients
[0048] While it is possible that additional excipients can be added
to the formulations of the claimed invention as is well known in
the art, a surprising discovery of the present invention is that an
excellent release profile can be achieved with the simple
formulation described herein. Such additional excipients can
increase or decrease the rate of release of the agent. Ingredients
which can substantially increase the rate of release include pore
forming agents and excipients which facilitate polymer degradation.
For example, the rate of polymer hydrolysis is increased in
non-neutral pH. Therefore, an acidic or a basic excipient such as
an inorganic acid or inorganic base can be added to the polymer
solution, used to form the microparticles, to alter the polymer
erosion rate. Ingredients which can substantially decrease the rate
of release include excipients that decrease the water solubility of
the agent.
[0049] A preferred embodiment of the described sustained release
formulations consists essentially of the biocompatible polymer, the
agent and the sugar. By "consists essentially of" is meant the
absence of ingredients which substantially increase the rate of
release of the active agent from the formulation. Examples of
additional excipients which would not be expected to substantially
increase or decrease the rate of release of the agent include
additional active agents and inert ingredients.
[0050] In yet another embodiment, the formulation consists of the
biocompatible polymer, the agent and the sugar. By "consists of" is
meant the absence of components or ingredients other than those
listed and residual levels of starting materials, solvents, etc.
from the process.
[0051] It has been a surprising discovery that buffering agents
such as acetate, citrate, phosphate or other biologically
compatible buffer was not necessary in the aqueous phase to achieve
a sustained release formulation with agent, e.g., exendin-4, with
good to excellent bioavailability. It was also a surprising
discovery that salting out salts were unnecessary to control burst
of the agent, e.g., exendin-4. As such, the compositions of the
invention also include compositions, as described herein, in the
substantial (or complete) absence of buffer and/or salting out
salts.
[0052] Alternatively or additionally, the sustained release
composition of the invention has low porosity. In such embodiments,
the sustained release composition comprises a biocompatible
polymer, a biologically active polypeptide and a sugar wherein the
composition has a total pore volume of about 0.1 mL/g or less. In a
specific embodiment, the total pore volume is determined using
mercury intrusion porosimetry, e.g., as described in more detail
below.
[0053] Administration
[0054] The compositions of the invention can be administered
according to methods generally known in the art. The composition of
this invention can be administered to a patient (e.g., a human in
need of the agent) or other animal, by injection, implantation
(e.g., subcutaneously, intramuscularly, intraperitoneally,
intracranially, and intradermally), administration to mucosal
membranes (e.g., intranasally, intravaginally, intrapulmonary or by
means of a suppository), or in situ delivery (e.g., by enema or
aerosol spray).
[0055] The sustained release composition can be administered using
any dosing schedule which achieves the desired therapeutic levels
for the desired period of time. For example, the sustained release
composition can be administered and the patient monitored until
levels of the drug being delivered return to baseline. Following a
return to baseline, the sustained release composition can be
administered again. Alternatively, the subsequent administration of
the sustained release composition can occur prior to achieving
baseline levels in the patient.
[0056] For example, when the sustained release composition has
incorporated therein a hormone, particularly an anti-diabetic or
glucoregulatory peptide, for example, GLP-1, GLP-2, exendin-3,
exendin-4 or agonists, analogs or derivatives thereof, the
composition is administered in a therapeutically effective amount
to treat a patient suffering from diabetes mellitus, IGT, obesity,
cardiovascular (CV) disorder or any other disorder that can be
treated by one of the above polypeptides or derivatives, analogs or
agonists thereof.
[0057] Other conditions which can be treated by administering the
sustained release composition of the invention include Type I and
Type II diabetes which can be treated with a sustained release
composition having insulin incorporated therein. In addition, when
the incorporated polypeptide is FSH or analogs thereof the
sustained release composition can be used to treat infertility. In
other instances, the sustained release composition can be used to
treat Multiple Sclerosis when the incorporated polypeptide is beta
interferon or a mutein thereof. As can be realized, the sustained
release composition can be used to treat disease which responds to
administration of a given polypeptide.
[0058] In a further embodiment, the sustained release composition
of the present invention can be coadministered with a
corticosteroid. Coadministration of the sustained release
composition of the invention with a corticosteroid can further
increase the bioavailability of the biologically active polypeptide
of the sustained release composition. Coadministration of a
corticosteroid in combination with sustained release compositions
is described in detail in U.S. Patent Application 60/419,430
entitled, "Method of Modifying the Release Profile of Sustained
Release Compositions" by Dasch et al. the entire content of which
is hereby incorporated by reference.
[0059] Corticosteroids, as defined herein, refers to steroidal
anti-inflammatory agents also referred to as glucocorticoids.
[0060] Suitable corticosteroids include, but are not limited to,
21-Acetoxypregnenolone, Alclometasone, Algestone, Amcinonide,
Beclomethasone, Betamethasone, Budesonide, Chloroprednisone,
Clobetasol, Clobetasone, Clocortolone, Cloprednol, Corticosterone,
Cortisone, Cortivazol, Deflazacort, Desonide, Desoximetasone,
Dexamethasone, Disflorasone, Diflucortolone, Difluprednate,
Enoxolone, Fluazacort, Flucloronide, Flumethasone, Flunisolide,
Flucinolone Acetonide, Fluocinonide, Fluocortin Butyl,
Flucortolone, Fluorometholone, Fluperolone Acetate, Fluprednidene
Acetate, Fluprednisolone, Flurandrenolide, Fluticasone Propionate,
Formocortal, Halcinonide, Halobetasol Propionate, Halometasone,
Halopredone Acetate, Hydrocortamate, Hydrocortisone, Loteprednol
Etabonate, Mazipredone, Medrysone, Meprednisone,
Methylprednisolone, Mometasone Furoate, Paramethasone,
Prednicarbate, Prednisolone, Prednisolone 25-Diethylamino-acetate,
Prednisolone Sodium Phosphate, Prednisone, Prednival, Prednylidene,
Rimexolone, Tixocortol, Triamcinolone (all forms), for example,
Triamcinolone Acetonide, Triamcinolone Acetonide 21-oic acid methyl
ester, Triamcinolone Benetonide, Triamcinolone Hexacetonide,
Triamcinolone Diacetate, pharmaceutically acceptable mixtures
thereof and salts thereof and any other derivative and analog
thereof.
[0061] In one embodiment, the corticosteroid can be co-incorporated
into the sustained release composition comprising the biocompatible
polymer and the biologically active polypeptide agent incorporated
therein.
[0062] In another embodiment, the corticosteroid can be separately
incorporated into a second biocompatible polymer. The second
biocompatible polymer can be the same or different from the first
biocompatible polymer which has the biologically active polypeptide
agent incorporated therein.
[0063] In yet another embodiment, the corticosteroid can be present
in an unencapsulated state but commingled with the sustained
release composition. For example, the corticosteroid can be
solubilized in the vehicle used to deliver the sustained release
composition. Alternatively, the corticosteroid can be present as a
solid suspended in an appropriate vehicle. Further, the
corticosteroid can be present as a powder which is commingled with
the sustained release composition.
[0064] It is understood that the corticosteroid is present in an
amount sufficient to increase the bioavailability of the
biologically active polypeptide from the sustained release
composition. Increased bioavailability refers to an increase in the
bioavailability of the biologically active polypeptide from the
sustained release composition when co-administered with a
corticosteroid in comparison to the administration in the absence
of corticosteroid over a time period beginning at two days post
administration and ending at the end of the release cycle for the
particular formulation.
[0065] As used herein, patient refers to a human, such as a human
in need of the agent or therapy, prophylaxis or diagnostic
method.
[0066] As defined herein, a sustained release of biologically
active polypeptide is a release of the polypeptide from the
sustained release composition of the invention which occurs over a
period which is longer than that period during which a biologically
significant amount of the polypeptide would be available following
direct administration of a solution of the polypeptide. It is
preferred that a sustained release be a release which occurs over a
period of at least about one week, such as at least about two
weeks, at least about three weeks or at least about four weeks. The
sustained release can be a continuous or a discontinuous release,
with relatively constant or varying rates of release. The
continuity of release and level of release can be affected by the
type of polymer composition used (e.g., monomer ratios, molecular
weight, block composition, and varying combinations of polymers),
polypeptide loading, and/or selection of excipients to produce the
desired effect.
[0067] As used herein, a therapeutically effective amount,
prophylactically effective amount or diagnostically effective
amount is the amount of the sustained release composition needed to
elicit the desired biological response following
administration.
[0068] C.sub.max as used herein is the maximum serum concentration
of drug which occurs during the period of release which is
monitored.
[0069] C.sub.ave as used herein, is the average serum concentration
of drug derived by dividing the area under the curve (AUC) of the
release profile by the duration of the release.
[0070] It is preferred that the ratio of C.sub.max to C.sub.ave, be
about 3 or less. This profile is particularly desirable of
anti-diabetic or glucoregulatory polypeptides, such as those
described above. A ratio of about 3 or less can provide a C.sub.ave
in a therapeutic window while avoiding adverse drug side effects
which can result from higher ratios.
[0071] Bioavailability, as that term is used herein, refers to the
amount of therapeutic that reaches the circulation system.
Bioavailability can be defined as the calculated Area Under the
Curve (AUC) for the release profile of a particular polypeptide
during the time period starting at post administration and ending
at a predetermined time point. As is understood in the art, the
release profile is generated by graphing the serum levels of a
biologically active agent in a subject (Y-axis) at predetermined
time points (X-axis). Bioavailability is often referred to in terms
of % bioavailability, which is the bioavailability achieved for a
particular polypeptide following administration of a sustained
release composition divided by the bioavailability achieved for a
particular polypeptide following intravenous administration of the
same dose of drug, multiplied by 100.
[0072] A modification of the release profile can be confirmed by
appropriate pharmacokinetic monitoring of the patient's serum for
the presence of the biologically active polypeptide agent. For
example, specific antibody-based testing (e.g., ELISA and IRMA), as
is well known in the art, can be used to determine the
concentration of certain biologically active polypeptide agents in
the patient's serum. An example of such testing is described herein
for exendin-4.
[0073] Pharmacodynamic monitoring of the patient to monitor the
therapeutic effects of the agent upon the patient can be used to
confirm retention of the biological activity of the released agent.
Methods of monitoring pharmacodynamic effects can be selected based
upon the biologically active polypeptide agent being administered
using widely available techniques.
[0074] Manufacture
[0075] A number of methods are known by which sustained release
compositions (polymer/biologically active polypeptide matrices) of
the invention can be formed, particularly compositions having low
porosity as described herein. Detailed procedures for some methods
of microparticle formation are set forth in the Working Examples.
In a preferred embodiment, the method of the invention for forming
a composition for the sustained release of biologically active
polypeptide includes forming a mixture by combining an aqueous
phase comprising water, agent, such as a water soluble polypeptide,
and a sugar with an oil phase comprising a biocompatible polymer
and a solvent for the polymer; forming a water-in-oil emulsion;
adding a coacervation agent, for example silicone oil, vegetable
oil or mineral oil to the mixture to form embryonic microparticles;
transferring the embryonic microparticles to a quench solvent to
harden the microparticles; collecting the hardened microparticles;
and drying the hardened microparticles. This process is generally
referred to herein as a water-oil-oil process (W/O/O).
[0076] Preferably, the polymer can be present in the oil phase in a
concentration ranging from about 3% w/w to about 25% w/w,
preferably, from about 4% w/w to about 15% w/w, such as from about
5% w/w to about 10% w/w. Excellent results were obtained herein
using a 6% w/w concentration of PLG in the oil phase.
[0077] The polymer is generally combined with a polymer solvent.
Where the polymer is a PLG, such as those preferred herein, the
polymer is added to a solvent for PLG. Such solvents are well known
in the art. A preferred solvent is methylene chloride.
[0078] The agent and sugar are added in the aqueous phase,
preferably in the same aqueous phase. The concentration of agent is
preferably 10 to 100 mg/g, preferably between 50 to 100 mg/g. The
concentration of sugar is preferably 10 to 50 mg/g and 30 to 50
mg/g.
[0079] The two phases are then mixed to form an emulsion. It is
preferred that the emulsion be formed such that the inner emulsion
droplet size is less than about 1 micron, preferably less than
about 0.7 microns, more preferably less than about 0.5 microns,
such as about 0.4 microns. Sonicators and homogenizers can be used
to form such an emulsion.
[0080] A coacervation agent as used herein refers to any oil in
which the polymer solution (polymer and solvent) is not readily
solubilized into and thereby forms a distinct phase with the
polymer solution. Suitable coacervation agents for use in the
present invention include, but are not limited to, silicone oil,
vegetable oil and mineral oil. In a particular embodiment, the
coacervation agent is silicone oil and is added in an amount
sufficient to achieve a silicone oil to polymer solvent ratio from
about 0.75:1 to about 2:1. In a particular embodiment, the ratio of
silicone oil to polymer is from about 1:1 to about 1.5:1. In a
preferred embodiment, the ratio of silicone oil to polymer is about
1.5:1.
[0081] The resulting mixture is added to a quench, which comprises
a polymer non-solvent. Polymer non-solvents are generally well
known in the art. A particularly preferred quench comprises a
heptane/ethanol solvent system.
[0082] Solid drug can also be encapsulated using a modified version
of the process described above. This modified process can be
referred to as a solid/oil/oil (S/O/O).
[0083] For example, solid exendin-4 was suspended in methylene
chloride containing 6% PLG and sonicated for about four minutes on
ice. Subsequent processing was conducted in a manner analogous to
the W/O/O method.
[0084] The invention will now be further and specifically described
by the following examples.
Exemplifications
Microparticle Preparation I
[0085] The sustained release compositions described herein were
prepared by a phase separation process. The general process is
described below for microparticles containing exendin-4 and sucrose
for a 1 kg batch size.
A. Inner Water-in-Oil Emulsion Formation
[0086] A water-in-oil emulsion was created with the aid of a
homogenizer. Suitable homogenizers include an in-line Megatron
homogenizer MT-V 3-65 F/FF/FF, Kinematica AG, Switzerland. The
water phase of the emulsion was prepared by dissolving exendin-4
and excipients such as sucrose in water. The concentration of drug
in the resulting solution can be from about 50 mg/g to about 100
mg/g. For example, when the drug is exendin-4, the concentration of
drug in solution can be from about 30 g to about 60 g per 600 g of
water. In a particular embodiment, 50 g exendin-4 and 20 g sucrose
were dissolved in 600 g water for irrigation (WFI). The specified
amounts listed above represent a nominal load without adjustment to
compensate for peptide content strength specific to the lot of
exendin-4 used. The oil phase of the emulsion was prepared by
dissolving PLGA polymer (e.g., 930 g of purified 50:50 DL4A PLGA
(Alkermes, Inc.) in methylene chloride (14.6 kg or 6% w/w).
[0087] The water phase was then added to the oil phase to form a
coarse emulsion with an overhead mixer for about three minutes.
Then, the coarse emulsion was homogenized at approximately 10,000
rpm at ambient temperature. This resulted in an inner emulsion
droplet size of less than 1 micron. It is understood that inner
emulsion formation can be achieved using any suitable means.
Suitable means of emulsion formation include, but are not limited
to, homogenization as described above and sonication.
B. Coacervate Formation
[0088] A coacervation step was then performed by adding silicone
oil (21.8 kg of Dimethicone, NF, 350 cs) over about a five minute
time period to the inner emulsion. This is equivalent to a ratio of
1.5:1, silicone oil to methylene chloride. The methylene chloride
from the polymer solution partitions into the silicone oil and
begins to precipitate the polymer around the water phase containing
exendin-4, leading to microencapsulation. The embryonic
microspheres thus formed are soft and require hardening.
Frequently, the embryonic microspheres are permitted to stand for a
short period of time, for example, from about 1 minute to about 5
minutes prior to proceeding to the microsphere hardening step.
C. Microsphere Hardening and Rinse
[0089] The embryonic microspheres were then immediately transferred
into a heptane/ethanol solvent mixture. The volume of
heptane/ethanol mixture needed can be determined based on the
microsphere batch size, typically a 16:1 ratio of methylene
chloride to heptane/ethanol solvent. In the present example, about
210 kg heptane and 23 kg ethanol in a 3.degree. C. cooled, stirred
tank were used. This solvent mixture hardened the microspheres by
extracting additional methylene chloride from the microspheres.
This hardening step can also be referred to as quenching. After
being quenched for 1 hour at 3.degree. C., the solvent mixture is
either decanted and fresh heptane (13 Kg) is added at 3.degree. C.
and held for 1 hour to rinse off residual silicone oil, ethanol and
methylene chloride on the microsphere surface or pumped directly to
the collection step.
D. Microsphere Drying and Collection
[0090] At the end of the quench or decant/wash step, the
microspheres were transferred and collected on a 12'' Sweco
Pharmasep Filter/Dryer Model PHI 2Y6. The filter/dryer uses a 20
micron multilayered collection screen and is connected to a motor
that vibrates the screen during collection and drying. A final
rinse with heptane (6 Kg at 3.degree. C.) was performed to ensure
maximum line transfer and to remove any excess silicone oil. The
microspheres were then dried under vacuum with a constant purge of
nitrogen gas at a controlled rate according to the following
schedule: 6 hours at 3.degree. C.; 6 hours ramping to 41.degree.
C.; and 84 hours at 41.degree. C.
[0091] After the completion of drying, the microspheres were
discharged into a collection vessel, sieved through a 150 .mu.m
sieve, and stored at about -20.degree. C. until filling.
[0092] For all microparticle formulations which were prepared
herein the amount of polypeptide, for example, exendin-4 and
excipients present in the prepared formulations is expressed as a %
(w/w) based on the final weight of the sustained release
composition. The % (w/w) is a nominal percentage, except where
indicated.
Microparticle Preparation II
A. Inner Water-in-Oil Emulsion Formation
[0093] A water-in-oil emulsion was created with the aid of a
sonicator. Suitable sonicators include Vibracell VCX 750 with model
CV33 probe head, Sonics and Materials Inc., Newtown, Conn. The
water phase of the emulsion was prepared by dissolving exendin-4
and excipients such as sucrose in water. The concentration of drug
in the resulting solution can be from about 50 mg/ml to about 100
mg/ml. For example, when the drug is exendin-4, the concentration
of drug in solution can be from about 3.28 g to about 6.55 g per
65.5 g of water. In a particular embodiment, 5.46 g exendin-4 and
2.18 g sucrose were dissolved in 65.5 g water for irrigation or
WFI. The specified amounts listed above represent a 4% overage to
target load in order to compensate for losses upon filter
sterilization of the components. The oil phase of the emulsion was
prepared by dissolving PLGA polymer (e.g., 97.7 g of purified 50:50
DL4A PLGA (Alkermes, Inc.)) in methylene chloride (1539 g or 6%
w/v).
[0094] The water phase was then added to the oil phase over about a
three minute period while sonicating at 100% amplitude at ambient
temperature. The water phase was pumped through a 1/4'' stainless
steel tube with a 1'' HPLC tube end (ID= 20/1000'') at 5 psig,
added below the sonication probe inside the sonication zone.
Reactor was then stirred at 1400 to 1600 rpm, with additional
sonication at 100% amplitude for 2 minutes, followed by a 30 second
hold, and then 1 minute more of sonication. This resulted in an
inner emulsion droplet size of less than 0.5 microns. It is
understood that inner emulsion formation can be achieved using any
suitable means. Suitable means of emulsion formation include, but
are not limited to, sonication as described above and
homogenization.
B. Coacervate Formation
[0095] A coacervation step was then performed by adding silicone
oil (2294 gr of Dimethicone, NF, 350 cs) over about a three to five
minute time period to the inner emulsion. This is equivalent to a
ratio of 1.5:1, silicone oil to methylene chloride. The methylene
chloride from the polymer solution partitions into the silicone oil
and begins to precipitate the polymer around the water phase
containing exendin-4, leading to microencapsulation. The embryonic
microspheres thus formed are soft and require hardening.
Frequently, the embryonic microspheres are permitted to stand for a
short period of time, for example, from about 1 minute to about 5
minutes prior to proceeding to the microsphere hardening step.
C. Microsphere Hardening and Rinse
[0096] The embryonic microspheres were then immediately transferred
into a heptane/ethanol solvent mixture. The volume of
heptane/ethanol mixture needed can be determined based on the
microsphere batch size. In the present example, about 22 kg heptane
and 2448 g ethanol in a 3.degree. C. cooled, stirred tank (350 to
450 rpm) were used. This solvent mixture hardened the microspheres
by extracting additional methylene chloride from the microspheres.
This hardening step can also be referred to as quenching. After
being quenched for 1 hour at 3.degree. C., the solvent mixture was
decanted and fresh heptane (13 Kg) was added at 3.degree. C. and
held for 1 hour to rinse off residual silicone oil, ethanol and
methylene chloride on the microsphere surface.
D. Microsphere Drying and Collection
[0097] At the end of the rinse step, the microspheres were
transferred and collected on a 6'' diameter, 20 micron multilayered
screen inside the cone shaped drying chamber which acted as a
dead-end filter. A final rinse with heptane (6 Kg at 4.degree. C.)
was performed to ensure maximum line transfer. The microspheres
were then dried with a constant purge of nitrogen gas at a
controlled rate according to the following schedule: 18 hours at
3.degree. C.; 24 hours at 25.degree. C.; 6 hours at 35.degree. C.;
and 42 hours at 38.degree. C.
[0098] After the completion of drying, the microspheres are
discharged into a teflon/stainless steel sterilized collection
vessel attached to the drying cone. The collection vessel is
sealed, removed from the drying cone and stored at -20.+-.5.degree.
C. until filling. Material remaining in the cone upon disassembly
for cleaning is taken for drug content analysis. The yield was
approximately 100 grams of microspheres.
[0099] For all microparticle formulations which were prepared
herein the amount of polypeptide, for example, exendin-4 and
excipients present in the prepared formulations is expressed as a %
(w/w) based on the final weight of the sustained release
composition. The % (w/w) is a nominal percentage, except were
indicated.
Polymer:
[0100] Examples of specific PLG polymers suitable for use are
listed below. All of the polymers employed in the following
examples are set forth in the list and all listed polymers were
obtained from Alkermes, Inc. of Cincinnati, Ohio and can be
described as follows: [0101] Polymer 2A:
Poly(lactide-co-glycolide); 50:50 lactide:glycolide ratio; 12.3 kD
Mol. Wt.; IV=0.15 (dL/g). [0102] Polymer 4A:
Poly(lactide-co-glycolide); 50:50 lactide:glycolide ratio; Mol. Wt.
45-64 kD; IV=0.45-0.47 (dL/g).
[0103] PURIFICATION OF PLG: It is known in the art (See, for
example, Peptide Acylation by Poly(.alpha.-Hydroxy Esters) by Lucke
et al., Pharmaceutical Research, Vol. 19, No. 2, p. 175-181,
February 2002) that proteins and peptides which are incorporated in
PLG matrices can be undesirably altered (e.g., degraded or
chemically modified) as a result of interaction with degradation
products of the PLG or impurities remaining after preparation of
the polymer. As such, the PLG polymers used in the preparation of
the majority of microparticle formulations described herein were
purified prior to preparation of the sustained release compositions
using art recognized purification methods.
Characterization Methods:
[0104] It has been determined that the following characterization
methods are suitable for identifying microparticles' which will
provide a desirable release profile of active agent.
SEM
[0105] SEM was used to assess the particle size, shape and surface
features of the microparticles. SEM imaging was performed on a
Personal SEM.RTM. system (ASPEX.TM., LLC). All samples were
deposited via spatula on standard SEM stubs covered with carbon
double-sided tape. Samples were sputter coated with Au for about 90
seconds at 18 mA emission current using a Model SC 7620 "Mini"
Sputter Coater (Energy Beam Sciences). All SEM imaging was
performed utilizing a 20 KeV electron beam over a magnification
range of approximately 250 to 2500.times..
Cryogenic SEM
[0106] The cross-section of microparticles was studied using
cryogenic SEM. The microparticle sample was mixed with HISTO
PREP.RTM. Solution (Fischer) and kept in a cryostat at -20.degree.
C. overnight. The hardened microparticles were mounted on a glass
cover slip and then sectioned using a metal knife. The sectioned
particles were mounted on aluminium stubs, sputter coated with
Platinum and Palladium and observed under a Scanning Electron
Microscope (Phillips 525M). Visual observation of the sections
provides a method of determining the degree of porosity for the
microparticles.
Porosity Measurement-Mercury Intrusion
[0107] Pore volume distribution in microparticles was determined
using a model SutoPor IV 9500 Moden Mercury Intrusion Porosimeter
(Micromeritics, Norcross, Ga.). Briefly, mercury was forced into a
known amount of microparticles in a penetrometer by applying
pressure in a step-wise manner up to a maximum pressure of 60,000
Psia. The volume of mercury intruded into the pores at various
pressures was measured. This method quantifies the pore
distribution in the microparticles. That is, the size of the pores
that are intruded is inversely related to the applied pressure. The
equilibrium of the internal and external forces on the
liquid-solid-vapor system can be described by the Washburn
equation. The relationship between applied pressure and the pore
size into which mercury is forced to enter is described by:
D = - 4 .gamma.cos.theta. P ##EQU00001##
Where
D=pore diameter
[0108] .gamma.=surface tension (constant)
[0109] .theta.=contact angle (constant)
[0110] P=Pressure
Therefore, the size of the pore into which mercury will intrude is
inversely proportional to the applied pressure. Assuming that all
pores are tight cylinders, the average pore diameter (D=4V/A) can
be calculated by dividing pore volume (V=.pi.D2h/4) by the pore
area (A=.pi.Dh).
Residual Solvents
[0111] A single method was used for quantitation of heptane,
ethanol and methylene chloride. The equipment consisted of an HP
5890 Series 2 gas chromatograph with an Rtx 1301, 30 cm.times.0.53
mm column. About 130 mg microparticles were dissolved in 10 ml
N,N-dimethylformamide. Propyl acetate was used as the internal
standard. The sample preparation was adjusted so that
concentrations of methylene chloride as low as 0.03% can be
quantitated.
Microparticle Preparation
[0112] The microparticle batches set forth in Table 1 were prepared
as described above at the 100 gram scale using the 4A polymer and a
ratio of silicone oil to methylene chloride of either 1.5:1 or 1:1
and the silicone oil had a viscosity of 350 cs. The amount of
exendin-4 and the excipients used in the formulation are also set
forth in Table 1.
TABLE-US-00001 TABLE 1 In Lot # Formulation vitro burst (%) Remarks
02-019-147(#1) 0% Sucrose, 0% AS 0.40 1.5:1 Si Oil:MeCl.sub.2
02-019-167(#2) 2% Sucrose (F16) 0.40 1.5:1 Si Oil:MeCl.sub.2
02-019-160(#2-1) 2% Sucrose (F16) 0.44 1.5:1 Si Oil:MeCl.sub.2
02-019-164(#2-2) 2% Sucrose (F16) 0.45 1.5:1 Si Oil:MeCl.sub.2
02-030-08(#2-3) 2% Sucrose (F16) 0.80 1:1 Si Oil:MeCl.sub.2
02-030-01(#2-4) 2% Sucrose (F16) 1.0 1:1 Si Oil:MeCl.sub.2
02-030-04(#2-5) 2% Sucrose (F16) 1.1 1:1 Si Oil:MeCl.sub.2
02-019-136(#3-1) 2% Sucrose, 0.5% AS (F14) 1.3 50:50 Quench
02-019-115(#3-2) 2% Sucrose, 0.5% AS (F14) 2.2 1.5:1 Si
Oil:MeCl.sub.2 02-019-170(#4) 0% Sucrose, 0.5% AS 3.8 1.5:1 Si
Oil:MeCl.sub.2 02-019-133A(#3-3) 2% Sucrose, 0.5% AS (F14) 12.7
100% Heptane Quench 02-019-185(#5) 2% sucrose (F17) 0.5 5% drug
load, (5% drug load) 1.5:1 Si Oil:MeCl.sub.2 02-019-64 (#3-4) 2%
Sucrose, 0.5% AS (F14) 0.5 1.5:1 Si Oil:MeCl.sub.2 02-019-10(#3-5)
2% Sucrose, 0.5% AS (F14) 1.30 1:1 Si Oil:MeCl.sub.2
02-001-196(#3-6) 2% Sucrose, 0.5% AS (F14) 2.70 1:1 Si
Oil:MeCl.sub.2 02-019-24(#3-7) 2% Sucrose, 0.5% AS (F14) 6.70 1:1
Si Oil:MeCl.sub.2 *ALL FORMULATIONS HAD 3% DRUG LOAD WITH THE
EXCEPTION OF #5 POROSITY
[0113] The total intrusion volume obtained from the mercury
intrusion porosimetry and the calculated average pore diameters are
given in TABLE 2. The relationship between the average pore
diameter and the in vitro release is shown in FIG. 1
TABLE-US-00002 TABLE 2 Total Pore Volume In vitro Average Pore Lot
# (mL/g) burst (%) Diameter (.mu.m) 02-019-147(#1) 0.033 0.40
0.0068 02-019-167(#2) 0.035 0.40 0.0069 02-019-160(#2-1) 0.037 0.44
0.0070 02-019-164(#2-2) 0.035 0.45 0.0070 02-030-08(#2-3) 0.036
0.80 0.0070 02-030-01(#2-4) 0.038 1.0 0.0073 02-030-04(#2-5) 0.039
1.1 0.0074 02-019-136(#3-1) 0.041 1.3 0.0073 02-019-115(#3-2) 0.039
2.2 0.0078 02-019-170(#4) 0.067 3.8 0.0125 02-019-133A(#3-3) 0.513
12.7 0.0277 02-019-64 (#3-4) 0.030 0.5 0.0060 02-019-10(#3-5) 0.060
1.30 0.0090 02-001-196(#3-6) 0.060 2.70 0.0100 02-019-24(#3-7)
0.180 6.70 0.0170
[0114] FIG. 1 shows the effect of ammonium sulfate on the in vitro
initial release. The data indicate that in vitro initial release is
correlated to the microparticle pore diameter. Formulations made
with ammonium sulfate showed varying levels of in vitro release and
variable porosity unlike the formulations without ammonium sulfate
which exhibited consistent porosity and release. During the
manufacturing of microparticles the presence of ammonium sulfate in
the aqueous phase can salt-out the drug substance during the
preparation of the inner-emulsion. The differences in the
micro-environment of the precipitates can contribute to the
differences in porosity and hence the variation in the initial
release. The effect was not observed in formulations prepared
without ammonium sulfate. Formulations with sucrose and exendin-4
show a more desirable and consistent level of initial release as
compared to formulations having exendin-4, sucrose and ammonium
sulfate.
[0115] FIG. 2 further demonstrates the effect of porosity on the in
vitro release and the impact that the processing conditions, namely
the ratio of silicone oil to methylene chloride, has on the
porosity of the microparticles formed. Briefly, microparticle
formulations prepared using a silicone oil-to-methylene chloride
ratio of 1:1 (Formulations 2-4 and 2-5 of Table 1) have a higher
initial release than the same formulations prepared using a
silicone-to-methylene chloride ratio of 1.5:1 (Formulations 2, 2-1
and 2-2 of Table 1). FIG. 2 suggests that a higher ratio of
silicone oil-to-methylene chloride results in a lower porosity
which results in a lower initial release.
Cryogenic SEM
[0116] Cryogenic SEM analysis was conducted as described above on
Formulations of the Types 2, 3 and 5 of Table 1. FIGS. 3A-3B are
scans of micrographs for selected formulations of Type 2
(Formulation 2-2, FIG. 3A) and of Type 5 (5% exendin-4, 2% sucrose,
FIG. 3B). FIGS. 4A-D are scans of micrographs for Formulations 3-4,
3-5, 3-6 and 3-7, respectively of Table 1. Again the variation in
porosity exhibited with the use of ammonium sulfate which can
contribute to the variability in initial release, can be seen in
the cryogenic SEM cross sections of FIGS. 4A-D.
Residual Solvent Levels
[0117] The level of residual solvents in a given formulation can
impact the Tg of the formulation. Residual solvent levels were
determined for microparticle formulations of Types 2 and 5 of Table
1. A single method was used for quantitation of heptane, ethanol
and methylene chloride. The equipment consisted of an HP 5890
Series 2 gas chromatograph with an Rtx 1301, 30 m.times.0.53 mm
column. About 130 mg microparticles were dissolved in 10 ml
N,N-dimethylformamide. Propyl acetate was used as the internal
standard. The sample preparation was adjusted so that
concentrations of methylene chloride as low as 0.03% can be
quantitated.
[0118] FIG. 5 is a plot of % residual ethanol and methylene
chloride for formulations of Types 2 and 5 of Table 1 (3 or 5%
exendin-4, 2% sucrose). FIG. 5 shows that the Tg decreases as the
amount of residual solvent increases.
Preparation of Microparticles Having 3% Exendin-4 and 2%
Sucrose
[0119] In view of the variation in porosity introduced by the
presence of ammonium sulfate in the microparticle formulations and
the identification of porosity as a characteristic which
significantly impacts initial release, ammonium sulfate was not
pursued in further discovery.
Impact of Inner Emulsion Droplet Size
[0120] The following study was done to determine the impact of
process parameters on forming the inner emulsion as well as
stability of the resulting emulsion and resulting 24 hour in vitro
release of microspheres produced using the different process
parameters. Inner emulsions of the water phase and solvent phase
were formed by either sonication as described above for the 100 gr
scale or homogenization using an MT5000 homogenizer with a 36/4
generator (Kinematica AG, Switzerland) at either a low speed
(10,800 rpm) or high speed (21,300 rpm). Following inner emulsion
formation by the different techniques, the emulsions were held in
the reactor with gentle agitation with an overhead stirrer for 5,
15 or 60 minutes prior to an aliquot being removed. Following the
designated hold times, the inner emulsion was further processed as
described above into microparticles and then the 24 hour in vitro
release determined for each batch as described below.
[0121] Inner Emulsion Droplet Size Characterization can be
Determined Using the Horiba Particle Size Analyzer
[0122] An aliquot of the inner emulsion was withdrawn from the
reactor using a glass pipet. Using a transfer pipet, .about.30
drops of the inner emulsion was added to .about.10 ml of 6%
Medisorb.RTM. 50:50 4A PLG polymer solution in a 20 cc screw-cap
scintillation vial followed by mixing. The 6% Medisorb.RTM. 50:50
4A PLG polymer solution also served as the reference blank
solution. About 9 ml of this diluted emulsion sample was then
transferred into a clean 10 ml Horiba sample holder. A cover was
placed on the sample holder to prevent rapid evaporation of the
polymer solvent. The prepared sample was within the acceptable %
transmission reading range of 0.65%-0.90% per the blue bar (Lamp).
A relative refractive index setting of 0.94-0.00i was selected in
the program setup. The sample was then measured by a Horiba
particle size analyzer such as model LA 910 for droplet size. The
data correlating the process parameters and the achieved inner
emulsion size over the 5, 15 and 60 minute hold times as well as
the resulting 24 hour in vitro release results (in parenthesis) are
shown in FIG. 9.
Microsphere Characterization
[0123] Exendin-4 microspheres were routinely characterized with
respect to drug content, particle size, residual solvents, initial
in vitro release, and PK characteristics in rats. Drug was
extracted to obtain a preliminary assessment of exendin-4 purity
post-encapsulation in selected batches.
In Vitro Initial Release
[0124] The initial release of exendin-4 was determined by measuring
the concentration of exendin-4 after 1 hour in release buffer (10
mM HEPES, 100 mM NaCl, pH 7.4). 150.+-.5 mg of microspheres were
placed in 5.0 mL of 10 mM HEPES, 100 mM NaCl, pH 7.4 buffer at room
temperature, vortexed for about 30 seconds to suspend the solution
and then placed in a 37.degree. C. air chamber for 1 hour. After 1
hour, the samples were removed from the chamber and inverted
several times to mix, followed by centrifuging at 3500 rpm for 10
minutes. The supernatant was removed and analyzed immediately by
HPLC using the following conditions: Column: TSK-GEL.RTM., 7.8
mm.times.30 cm, 5 m (TSOH BIOSEP PART #08540); Column Oven
Temperature: Ambient; Autosampler Temperature: 6.degree. C.; Flow
Rate: 0.8 mL/minute; Detection: 280 nm; Injection Volume: 10 L;
Mobile Phase: 35%. Acetonitrile/65% Water with 0.1% TFA/liter
(v/v); Run Time: Approximately 20 minutes. Exendin-4 bulk drug
substance, 0.2 mg/mL prepared in 30 mM Acetate Buffer, pH 4.5, was
used as a standard.
Animal Studies
[0125] All pharmacokinetic (PK) studies described herein were
conducted in adult male Sprague-Dawley rats weighing approximately
500.+-.50 g.
[0126] For PK characterization of the microparticle formulations,
each animal received a subcutaneous injection of microparticles
suspended in diluent (3% carboxymethylcellulose, 0.9% NaCl, 0.1%
Tween 20) to the inter-scapular region. Generally, the dose was
approximately 1.0 mg exendin-4 per rat in an injection volume of
0.75 mL. Blood samples were collected via lateral tail vein at 0.5,
2, 4, 6, 10, 24 hours, and 2, 4, 7, 10, 14, 17, 21, 24 and 28 days
post-dose. Blood samples were immediately placed in
MICROTAINER.RTM. tubes containing EDTA and centrifuged at about
14,000.times.g for about two minutes.
[0127] Plasma was then transferred to MICROTAINER.RTM. tubes
without additive and stored at -70.degree. C. until time of assay.
IRMA was used to determine plasma exendin concentrations.
In Vivo Release-Irma
[0128] The method for quantifying exendin-4 in plasma is a sandwich
immunoassay, with the analyte captured by a solid phase monoclonal
antibody EXE4:2-8.4 and detected by the radioiodinated monoclonal
antibody GLP-1:3-3. Counts bound are quantitated from a standard
calibration curve. This assay is specific for full length or intact
exendin-4 and does not detect exendin-4 (3-39). A typical standard
curve range is 30 pg/mL to 2000 pg/mL depending on the age of the
tracer antibody.
In Vitro and In Vivo Release
[0129] Formulations 2, 2-1 and 2-2 (3% exendin-4 and 2% sucrose)
were tested for initial release in vitro as described above. The in
vitro release was 0.4%, 0.4% and 0.5%, respectively. All three
batches also had a relatively low in vivo initial release in the
range of 1154 to 1555 pg/mL for C.sub.max 0-1 day. FIG. 6 is a
representative pharmacokinetic curve for the formulations having 3%
exendin-4 and 2% sucrose_(2-1) and also for 3% exendin-4 alone (1)
and 3% exendin-4 and 0.5% ammonium sulfate (4). A ratio of silicone
oil-to-methylene chloride of 1.5:1 was used and the viscosity of
the silicone oil was 350 cs.
[0130] From FIG. 6 it can be seen that the formulations not
containing ammonium sulfate exhibit a lower initial release.
Although the formulation having exendin-4 alone showed a suitable
initial release the post encapsulation purity of the drug was
decreased as compared to the formulation having the exendin-4 in
combination with the sucrose. The addition of sugar in the
formulations decreases degradation of the agent.
[0131] The in vivo release profile for the three formulations 2,
2-1 and 2-2 compared above, are shown in FIG. 7. All three batches
exhibited a relatively low initial release followed by a "trough"
(low serum levels between about day 4 to day 17), followed by a
sustained release over about day 21 to day 28. The low initial
release and the shape of the release profile were consistent for
the three formulations.
Formulation Using a 1:1 Ratio of Silicone Oil to Methylene
Chloride
[0132] Formulations 2-3, 2-4 and 2-5 from Table 1 (3% exendin-4, 2%
sucrose) were prepared using a 1:1 ratio of silicone oil to
methylene chloride. The initial release was higher for these
formulations than for formulations 2, 2-1 and 2-2 of Table 1 (3%
exendin-4, 2% sucrose with a 1.5:1 silicone to methylene chloride
ratio). Specifically the 1.5:1 ratio formulations provided an
average initial release about 0.4%, whereas the 1:1 ratio
formulations provided an average initial release about 1.0%. The
same trend was observed in vivo with C.sub.max 0-1 day in rats was
2288.+-.520 pg/mL for a 1:1 ratio, whereas the C.sub.max 0-1 day in
rats was 1300.+-.221 pg/mL for the 1.5:1 ratio.
Increased Drug Loading
[0133] Increasing the exendin-4 load to 4% while maintaining the
sucrose at 2% resulted in an initial release in vitro and in vivo
in the same range as for the 3% loading.
[0134] Three formulations of Type 5 from Table 1 were prepared (5%
drug load, 2% sucrose, 1.5:1 silicone oil-to-methylene chloride
ratio). The three batches, 5-1, 5-2 and 5-3 all exhibited a low in
vitro initial release ranging from 0.2 to 0.5%. Similarly, the in
vivo C.sub.max of the formulations was consistently low ranging
from 467 pg/mL to 1267 pg/mL. FIG. 8 shows a graph of the
pharmacokinetic data for the three batches tested. Compared to the
behavior of the 3% exendin-4 formulation having 2% sucrose, the 5%
formulations exhibited higher serum levels of drug over about day 1
and day 2. The remainder of the profile for the 5% formulations was
similar to the 3% formulations having a trough followed by release
of drug primarily over day 21 to day 28.
[0135] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *