Note: Descriptions are shown in the official language in which they were submitted.
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BIODEGRADABLE MICROPARTICLES WITH NOVEL ERYTHROPOIETIN STIMULATING PROTEIN
FIELD OF THE INVENTION
The present invention relates to a composition
for the sustained release of biologically active, novel
erythropoietin stimulating protein (NESP), and improved
methods of forming said composition. The composition
comprises polymeric microparticles within which
particles of NESP have been dispersed. The improved
method utilizes a cosolvent mixture to effect more
efficient and rapid removal of residual polymer solvents
during any drying process.
BACKGROUND OF THE INVENTION
For the past several years, there have been
extensive efforts directed to the development of
effective sustained-release formulations which could
provide a means of controlling blood levels of the
active ingredient, and also provide greater efficacy,
safety, patient convenience and patient compliance.
Unfortunately, the instability of most proteins (e. g.
denaturation and loss of bioactivity upon exposure to
heat, organic solvents, etc.) has greatly limited the
development and evaluation of sustained-release
formulations.
Methods of preparing microparticles in the
prior art have been described in both the patent and
scientific literature. In particular, various methods
are described for preparing biodegradable microparticles
of poly(lactic acid)(PLA) and poly(lactic-co-glycolic
acid (PLGA) for controlled release of water-soluble
drugs; see e.g., Wise et al., Contraception, 8:227-234
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(1973); Hutchinson et al., Biochem. Soc. Trans., 13:520-
523 (1985); and Jalil et al., J. Microencapsul.,
7:297-325 (1990); Putney et al., Nature Biotech.,
16:153-157 (1998); Burke, P., Handbook of Pharmaceutical
Controlled Release Technology, Klibanov, et al.
(editors)(in press). These methods include those
involving emulsions (phase separation, solvent
extraction and solvent evaporation) and those involving
atomization (spray drying, spray freezing).
The major disadvantages associated with the
above-referenced methods may include, under certain
circumstances: 1) high temperatures causing protein
inactivation; 2) exposure to organic solvents causing
protein inactivation; 3) inability to encapsulate
hydrophilic drugs due to loss of drug to the aqueous
phase which is used to extract the organic solvent; 4)
the large amount of organic solvents normally needed
during the processes, and which cannot be adequately
removed from the final product, i.e., high residual
solvent levels in final product; 5) poor protein loading
efficiencies; 6) poor overall yields; 7) problems with
drug leakage and/or high initial drug release upon
administration; and 8) expensive and complex to scale-
up. As specifically relates to item 4), the problems
associated with high levels of solvents in spray drying
processes have been described; see e.g., Clarke et al.,
Drug Devel. and Industrial Pharmacy,24:169-175 (1998);
Bitz and Doelker, Inter. Journal of Pharm., 131:171-181
(1996); and Takada et al., Journ. of Controlled
Release, 32:79-85 (1994).
There have been numerous reports in both the ,
patent and scientific literature regarding improved
processes for microparticle preparation. For example,
in Gombotz et al., U.S. Patent No. 5,019,400, a process
is disclosed for preparing microparticles wherein very
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cold temperatures are used to freeze polymer-
biologically active agent mixtures into polymeric
microspheres with high retention of biological activity
and material. In Ramstack et al., U.S Patent No.
5,650,173, a process is disclosed for preparing
microparticles wherein a blend of at least two non-toxic
solvents, free of halogenated hydrocarbons, was used to
dissolve the polymer and the active agent. The
resulting microparticles, though free of residual toxic
solvents, still had residual amounts of benzyl alcohol
and ethyl acetate, which had a negative effect on
product integrity. In Rickey et al., U.S. Patent No.
5,792,477, a process was described which alleviated the
residual solvent problem reported in Ramstack et al., by
including additional washing steps in the process to
effect adequate removal of solvents.
While these and other research efforts have
clearly furthered the technology, there still exists a
need for a more efficient, economical, broadly
applicable microparticle preparation process for use
with proteins, peptides, and small molecules, that is
amenable to aseptic processing and is scaleable.
Among the various proteins for which an
effective sustained release composition has been
reported is erythropoietin. Erythropoietin (EPO) is a
glycoprotein hormone involved in the maturation of
erythroid progenitor cells into erythrocytes. It is
produced in the kidney and is essential in regulating
levels of red blood cells in the circulation.
Conditions marked by low levels of tissue oxygen signal
increased production of erythropoietin, which in turn
stimulates erythropoiesis. A loss of kidney function as
is seen in chronic renal failure (CRF), for example,
typically results in decreased production of
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erythropoietin and a concomitant reduction. in red blood
cells.
Administration of recombinant human
erythropoietin (rHuEPO) is effective in raising red
blood cell levels in anemic patients with end stage
renal disease; Eschbach et al., New Eng. J. Med.,
316:73-38 (1987). Subsequent studies have shown that
treatment with rHuEPO can correct anemia associated with
a variety of other conditions; Fischl et al., New Eng.
J. Med., 322:1488-1493 (1990); Laupacis, Lancet,
341:1228-1232 (1993). Regulatory approvals have been
given for the use of recombinant human erythropoietin in
the treatment of anemia associated with CRF, anemia
related to therapy with AZT (zidovudine) in HIV-infected
patients, anemia in patients with non-myeloid
malignancies receiving chemotherapy, and anemia in
patients undergoing surgery to reduce the need of
allogenic blood transfusions. Current therapy for all
approved indications (except the surgery indication)
involves a starting dose of between 50-150 Units/kg
three times per week (TIW) administered either by an
intravenous (IV) or subcutaneous (SC) injection to reach
a suggested target hematocrit range of 30o to 360. For
the surgery indication, rHuEPO is administered every day
10 days prior to surgery, on the day of surgery, and
four days thereafter (EPOGEN~ Package Insert, 12/23/96).
In general, the current recommended starting doses for
rHuEPO raise hematocrit into the target range in about
six to eight weeks. Once the target hematocrit range
has been achieved, a maintenance dosing schedule is
established which will vary depending upon the patient,
but is typically three times per week for anemic
patients with CRF. The administration of rHuEPO
described above is an effective and well-tolerated
regimen for the treatment of anemia.
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In Zale et al., U.S. Patent No. 5,674,534,
sustained-release compositions of non-aggregated,
biologically active EPO are described. The compositions
comprise a polymeric matrix of a biocompatible polymer
and particles of biologically active, aggregation-
stabilized EPO, wherein said particles are dispersed
within the biocompatible polymer, and wherein said
microparticles are prepared using the process described
in U.S. Patent No. 5,019,400. The method described and
claimed utilizes a salting-out excipient to stabilize
the EPO. The formulation is said to have advantages
including longer, more consistent in vivo blood levels
of EPO, lower initial bursts of EPO, and increased
therapeutic benefits by eliminating fluctuations in
serum EPO levels.
Recombinant human erythropoietin expressed in
mammalian cells contains three N-linked and one 0-linked
oligosaccharide chains which together comprise about 400
of the total molecular weight of the glycoprotein.
N-linked glycosylation occurs at asparagine residues
located at positions 24, 38 and 83 while O-linked
glycosylation occurs at a serine residue located at
position 126; Lai et al., J. Biol. Chem., 261:3116
(1986); Broudy et al., Arch. Biochem. Biophys.,
265:329 (1988). The oligosaccharide chains have been
shown to be modified with terminal sialic acid residues.
Enzymatic treatment of glycosylated erythropoietin to
remove sialic acid residues results in a loss of in vivo
activity but does not affect in vitro activity; Lowy
et al., Nature, 185:102 (1960); Goldwasser et al.,
J. Biol. Chem., 249:4202 (1974). This behavior has been
explained by rapid clearance of asialo-erythropoietin
from the circulation upon interaction with the hepatic
asialoglycoprotein binding protein; Morrell et al.,
J. Biol. Chem., 243:155 (1968); Briggs, et al.,
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Am. J. Physiol., 227:1385 (1974); Ashwell.et al.,
Methods Enzymol., 50:287 (1978).
Novel erythropoietin stimulating protein
(NESP) is a hyperglycosylated erythropoietin analog
having five changes in the amino acid sequence of rHuEPO
which provide for two additional carbohydrate chains.
More specifically, NESP contains two additional N-linked
carbohydrate chains at amino acid residues 30 and 88
(numbering corresponding to the sequence of human
EPO)(see PCT Application No. US94/02957, herein
incorporated by reference in its entirety). NESP is
biochemically distinct from EPO, having a longer serum
half-life and higher in vivo biological activity; Egrie
et al., ASH 97, Blood, 90:56a (1997). NESP has been
shown to have ~3 fold increase in serum half-life in
mice, rats, dogs and man; Id. In mice, the longer serum
half-life and higher in vivo activity allow for less
frequent dosing (once weekly or once every other week)
compared to rHuEPO to obtain the same biological
response; Id.
A pharmacokinetic study demonstrated that,
consistent with the animal studies, NESP has a
significantly longer serum half-life than rHuEPO in
chronic renal failure patients, suggesting that a less
frequent dosing schedule may also be employed in humans;
MacDougall, et al., J American Society of Nephrology,
8:268A (1997). A less frequent dosing schedule would be
more convenient to both physicians and patients, and
would be particularly helpful to those patients involved
in self-administration. Other advantages to less
frequent dosing may include less drug being introduced
into patients, a reduction in the nature or severity of
the few side-effects seen with rHuEPO administration,
and increased compliance.
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The present invention is based upon the
discovery that NESP can be encapsulated in
microparticles and provide a pharmaceutical composition
with an even more dramatic sustained release profile,
allowing for a once every 4-6 week dosing for raising
hematocrit and treating anemia, and thus providing
tremendous therapeutic advantage. Additionally, the
NESP/PLGA system is such that the PLGA component is
cleared (biodegraded) up to at least one week prior to
the cessation of therapeutic effect, and thus allow for
repeated dosing with less concern of polymer and drug
build-up from dose to dose. Such a system may not be
possible with, for example, EPO/PLGA microparticles.
Additionally, the present invention provides
an improved, economical, scalable method for
microparticle preparation which is broadly applicable to
biologically active agents other than NESP, including
peptides and small molecules.
SUMMARY OF THE INVENTION
Accordingly, one aspect of the present
invention is a pharmaceutical composition for the
sustained-release of an active ingredient comprising a
biologically active ingredient contained within
polymeric microparticles. Importantly, the sustained-
release compositions of the present invention maintain
the activity, integrity and safety of the active
ingredient during encapsulation and release, which helps
to provide for longer periods of consistent release.
A second aspect of the present invention
relates to a new and improved process for preparing a
sustained-release composition comprising an active
ingredient contained within polymeric microparticles.
The process is economical, amenable to aseptic
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processing, scalable and broadly applicable to proteins,
peptides, and small molecules. The improved process can
be generally described as comprising the steps of: (a)
obtaining a dried powder of a specific active ingredient
or formulated active ingredient; (b) preparing a
polymer solution comprising a polymer dissolved in a
cosolvent mixture; (c) dispersing said dried powder in
said polymer solution to produce an active
ingredient/polymer mixture; (d) preparing active
ingredient-containing microparticles from said mixture;
(e) collecting said microparticles; and (f) finishing
said microparticles by secondary drying. Importantly,
the drying times associated with this process are
significantly reduced as compared to prior art methods.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic of the process of the
present invention for making the active ingredient
containing microparticles.
Figure 2 shows chromatograms depicting the
size exclusion chromatography results for a NESP
preparation prior to spray drying ( . . .), a spray
dried NESP protein powder (- -), and a typical result
for NESP after microparticle encapsulation ( ).
Figure 3 is a graph depicting NESP serum
levels for various microparticle preparations which had
been injected subcutaneously (360 ~tg/kg NESP peptide
dose) into rats. Serum concentration is plotted vs.
time (days).
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Figure 4 is a graph depicting hematocrit for
rats injected subcutaneously (360 ~,g/kg NESP peptide
dose) with various microparticle preparations.
Hematocrit (o) is plotted vs. time (days).
Figure 5 is a graph depicting NESP serum
levels for various microparticle preparations which had
been injected subcutaneously (360 ~.g/kg NESP peptide
dose) into rats. Serum concentration is plotted vs.
time (days).
Figure 6 is a graph depicting NESP serum
levels for various microparticle preparations which had
been injected subcutaneously (360 ~g/kg NESP peptide
dose) into rats. Serum concentration is plotted vs.
time (days).
Figure 7 is a graph depicting hemoglobin
levels for rats injected subcutaneously with a 10,000
ug/kg bolus injection of NESP vs. a 100 ~,g/kg (20 mg
microparticle) injection of a NESP microparticle
preparation (50:50, inherent viscosity 0.4 dL/g).
Hemoglobin levels (g/dL) is plotted vs. time (days).
Figure 8 is a graph depicting NESP serum
levels for various microparticle preparations which had
been injected subcutaneously (360 ~g/kg NESP peptide
dose) into rats. Serum concentration is plotted vs.
time (days).
Figure 9 is a graph depicting leptin serum
levels for rats injected with various leptin
preparations. Serum concentration is plotted vs. time
(hours).
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10
Figure 10 is a graph depicting o weight loss
for rats injected with various leptin preparations.
Body weight (mg) is plotted vs. time (days).
Figure 11 is a graph depicting NESP serum
levels for microparticle preparations which had been
injected subcutaneously (360 ~g/kg NESP peptide dose)
into rats. Serum concentration is plotted vs. time
(days).
Figure 12 is a graph depicting hematocrit for
rats injected subcutaneously (360 ~g/kg NESP peptide
dose) with microparticle preparations. Hematocrit
is plotted vs. time (days).
Figure 13 is a graph depicting NESP serum
levels for microparticle preparations which had been
injected subcutaneously (360 ~g/kg NESP peptide dose)
into rats. Serum concentration is plotted vs. time
(days).
Figure 14 is a graph depicting hematocrit for
rats injected subcutaneously (360 ~g/kg NESP peptide
dose) with microparticle preparations. Hematocrit (o)
is plotted vs. time (days).
DETAILED DESCRIPTION
Unless otherwise noted, the term
microparticles can be used to encompass microparticles,
microspheres, and microcapsules.
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As fully described below, the present
invention provides a pharmaceutical composition for the
sustained-release of an active ingredient comprising a
biologically active ingredient contained within
polymeric microparticles. A sustained-release
composition is defined as a release of biologically
active agent which results in measurable serum levels of
said agent for a period of time longer than that
obtained following direct administration of aqueous
biologically active agent. The sustained release can be
continuous or discontinuous, linear or non-linear, and
this can be accomplished using one or more polymer
compositions, drug loadings, selection of excipients, or
other modifications. In one embodiment of the present
invention, the sustained-release composition will
comprise the biologically active ingredient, NESP.
NESP of the present invention is a
hyperglycosylated EPO analog comprising two additional
glycosylation sites with an additional carbohydrate
chain attached to each site. NESP was constructed using
site-directed mutagenesis and expressed in mammalian
host cells. Details of the production of NESP are
provided in co-owned PCT Application No. US94/02957.
New N-linked glycosylation sites for rHuEPO were
introduced by alterations in the DNA sequence to encode
the amino acids Asn-X-Ser/Thr in the polypeptide chain.
DNA encoding NESP was transfected into Chinese Hamster
Ovary (CHO) host cells and the expressed polypeptide was
analyzed for the presence of additional carbohydrate
chains. In a preferred embodiment, NESP will have two
additional N-linked carbohydrate chains at residues 30
and 88. The numbering of the amino acid sequence is
that of human erythropoietin (EPO). The amino acid
sequence of EPO is that depicted in SEQ ID N0: 1. The
amino acid sequence of NESP is that depicted in SEQ ID
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N0: 2. It is understood that NESP will have the normal
complement of N-linked and 0-linked glycosylation sites
in addition to the new sites.
The NESP of the present invention may also
include conservative amino acid changes at one or more
residues in SEQ ID N0: 2. These changes do not result
in addition of a carbohydrate chain and will have little
effect on the biological activity of the analog.
Other active ingredients to be incorporated
into the microparticles of the present invention are
synthetic or natural compounds which demonstrate a
biological effect when introduced into a living
creature. Contemplated active agents include peptides,
small molecules, carbohydrates, nucleic acids, lipids,
proteins, and analogs thereof. Proteins contemplated
for use include potent cytokines, including various
hematopoietic factors such as G-CSF, GM-CSF, M-CSF,
MGDF, the interferons (alpha, beta, and gamma),
interferon consensus, the interleukins (1-12),
erythropoietin (EPO), fibroblast growth factor, KGF,
TNF, TNFbp, IL-lra, stem cell factor, nerve growth
factor, GDNF, BDNF, NT3, platelet-derived growth factor,
and tumor growth factor (alpha, beta), osteoprotegerin
(OPG), NESP, and OB protein. 0B protein will also be
referred to as leptin.
Also contemplated for incorporation into the
compositions of the present invention are derivatives,
fusion proteins, conjugates, analogs or modified forms
of the natural active ingredients. Chemical
modification of biologically active proteins has been
found to provide additional advantages under certain
circumstances, such as increasing the stability and
circulation time of the therapeutic protein and
decreasing immunogenicity. For example, Davis et al.,
U.S. Patent No. 4,179,337, discloses conjugation of
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water-soluble polypeptides such as enzymes and insulin
to polyethylene glycol (PEG); see also Kinstler et al.,
U.S. Patent No. 5,824,784.
In general, comprehended by the present
invention are pharmaceutical compositions comprising
effective amounts of protein or derivative products of
the invention together with pharmaceutically acceptable
diluents, stabilizers, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such
compositions include diluents of various buffer content
(e. g., Tris-HC1, phosphate), pH and ionic strength;
additives such as detergents and solubilizing agents
(e. g., Polysorbate 20, Polysorbate 80), anti-oxidants
(e. g., ascorbic acid, sodium metabisulfite),
preservatives (e.g., Thimerosol, benzyl alcohol) and
bulking substances (e. g., lactose, mannitol); see, e.g.,
Remington's Pharmaceutical Sciences, 18th Ed. (1990,
Mack Publishing Co., Easton, PA 18042) pages 1435-1712
which are herein incorporated by reference. An
effective amount of active ingredient is a
therapeutically, prophylactically, or diagnostically
effective amount, which can be readily determined by a
person skilled in the art by taking into consideration
such factors as body weight, age, therapeutic or
prophylactic or diagnostic goal, and release rate
desired.
As used herein, and when contemplating NESP-
containing microparticles, the term "therapeutically
effective amount" refers to an amount which gives an
increase in hematocrit that provides benefit to a
patient. The amount will vary from one individual to
another and will depend upon a number of factors,
including the overall physical condition of the patient
and the underlying cause of anemia. For example, a
therapeutically effective amount of rHuEPO for a patient
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suffering from chronic renal failure is 50. to 150
units/kg three times per week. The amount of rHuEPO
used for therapy gives an acceptable rate of hematocrit
increase and maintains the hematocrit at a beneficial
level (usually at least about 30~ and typically in a
range of 30o to 36~). A therapeutically effective
amount of the present compositions may be readily
ascertained by one skilled in the art using publicly
available materials and procedures.
The invention provides for administering NESP-
containing microparticles less frequently than NESP
and/or EPO. The dosing frequency will vary depending
upon the condition being treated, but in general will be
about one time per 4-6 weeks. It is understood that the
dosing frequencies actually used may vary somewhat from
the frequencies disclosed herein due to variations in
responses by different individuals to the NESP-
containing microparticles; the term "about" is intended
to reflect such variations.
The present invention may thus be used to
stimulate red blood cell production and correct
depressed red cell levels. Most commonly, red cell
levels are decreased due to anemia. Among the
conditions treatable by the present invention include
anemia associated with a decline or loss of kidney
function (chronic renal failure), anemia associated with
myelosuppressive therapy, such as chemotherapeutic or
anti-viral drugs (such as AZT), anemia associated with
the progression of non-myeloid cancers, and anemia
associated with viral infections (such as HIV). Also
treatable are conditions which may lead to anemia in an
otherwise healthy individual, such as an anticipated
loss of blood during surgery. In general, any condition
treatable with rHuEPO may also be treated with the NESP-
containing microparticles of the invention.
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The invention also provides for.administration
of a therapeutically effective amount of iron in order
to maintain increased erythropoiesis during therapy.
The amount to be given may be readily determined by one
skilled in the art based upon therapy with rHuEPO.
The present invention also provides an
improved method for preparing polymeric microparticles
containing an active ingredient, comprising utilization
of a cosolvent mixture to effect more rapid removal of
organic solvents during drying. The residual presence
of secondary organic solvent components in the mixture
during drying results in acceleration of the removal of
residual polymer solvent during any drying process.
This improved method provides several significant
advantages over the processes described in the art,
including, for example, 1) reduction of levels of
residual solvents in the polymer system after the
initial microparticle formation step; 2) reduction in
drying cycle times for polymer systems; and 3) enabling
the removal of toxic solvents to acceptable levels for
use in human pharmaceuticals. Importantly, these
advantages help make such processes commercially
practical.
The principal embodiment of the improved
method for making the protein loaded microparticles
comprises: (a) obtaining a dried powder of an active
ingredient or formulated active ingredient,
(b) dissolving a polymer in a cosolvent mixture to
produce a polymeric solution; (c) adding said dried
powder to said polymeric solution to produce a active
ingredient/polymer mixture; (d) spray drying said
mixture to produce the desired active ingredient loaded
microparticles; (e) collecting said microparticles; and
(f) finishing said microparticles by secondary drying.
The process is shown schematically in Figure 1.
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In one embodiment of the present. invention,
the active ingredient or formulated active ingredient
will be in the form of a spray dried powder. Spray
drying is a process wherein a solution is atomized to
form a fine mist and dried by direct contact with hot
carrier gases. For a detailed review of spray drying,
see e.g., Masters, K., "Spray Drying Handbook" (John
Wiley & Sons, eds., New York 1984). Provided herein is
an improved commercial scale spray dried protein powder
preparation method which results in significantly higher
yields and improved collection of the microparticles.
Polymer solvents contemplated for use in step
b) of the present processes include, for example,
chloroform, ethyl acetate, acetone, methylene chloride,
and dimethylsulfoxide. In one embodiment of the present
invention, the polymer solvent to be used is methylene
chloride. Non-solvents contemplated for use include
ethanol, ethyl formate, and heptane.
The microparticle preparation step (d) may
alternatively involve an emulsion based preparation, or
spray freezing. For the emulsions produced in the
processes of the present invention, the organic: aqueous
ratios contemplated for use are 1:1 to 12:1. In
general, the microparticles prepared by the methods of
the present invention will generally comprise 0.001-600
by weight of protein.
Secondary drying processes contemplated for
use in step (f) include gas bleed drying, fluidized bed
drying, lyophilization, vacuum drying, and tray drying.
In one embodiment of the present invention, gas bleed
drying is utilized in step (f).
Polymers contemplated for use may be selected
from the group consisting of biocompatible and/or
biodegradable polymers. As defined herein,
biodegradable means that the composition will erode or
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degrade in vivo to form smaller biocompatible chemical
species. Degradation may occur, for example, by
enzymatic, chemical or physical processes. Suitable
biodegradable polymers contemplated for use in the
present invention include poly(lactide)s,
poly(glycolide)s, poly(lactic acids, poly(glycolic
acids, polyanhydrides, polyorthoesters,
polyetheresters, polycaprolactone, polyesteramides,
polyphosphazenes, polyphosphoesters, pseudo-polyamino
acids; Langer, Nature, 392:5-10 (1998), blends and
copolymers thereof.
The range of molecular weights contemplated
for the polymers to be used in the present processes can
be readily determined by a person skilled in the art
based upon such factors the desired polymer degradation
rate. Typically, the range of molecular weight will be
2000 to 2,000,000 Daltons. Almost any type of polymer
can be used provided the appropriate solvent or
cosolvent system are found.
The term "PLGA" as used herein is intended to
refer to a polymer of lactic acid alone, a polymer of
glycolic acid alone, a mixture of such polymers, a
copolymer of glycolic acid and lactic acid, a mixture of
such copolymers, or a mixture of such polymers and
copolymers. PLGA's used may be in the free acid
("uncapped") form or in the terminal ester ("capped")
form. Preferably, the biodegradable polymer will be
poly lactide-co-glycolide (PLGA). The polymer
concentrations contemplated for use in the processes of
the present invention are in the range of 5-70 g/100mL
(g~) .
In general, an aqueous solution, a
suspension, or a solid form of the active agent can be
admixed with the organic solvent containing the polymer.
When an aqueous solution of active ingredient is used,
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an aqueous solution of active ingredient-in-polymer
solution emulsion, or a water-in-oil emulsion is formed
(containing the active ingredient in the aqueous phase
and the polymer in the organic phase) and used to
prepare microparticles. When a suspension or solid form
of active ingredient is used, suspensions of the solid
active ingredient in the polymer solution are formed and
used to prepare the microparticles. Alternatively, a
monophasic solution of active ingredient and polymer may
be used. In one embodiment of the present invention,
the active ingredient will be in the form of a spray
dried powder, the particle size of the protein powder
will be in the range of <10 ~.m. The protein
concentrations contemplated for use in the processes of
the present invention are in the range of 0.001-500
mg/mL when in the emulsion or suspension.
The active ingredient solution, suspension,
emulsion, or solid form may also be formulated, i.e.,
include a buffer, a surfactant, or an excipient which
serves to stabilize the active ingredient during drying,
e.g., trehalose, ammonium sulfate, 2-hydroxy propyl (3-
cyclodextrin, sucrose, or other protein-stabilizing
sugars or excipients.
A suspension of protein loaded microparticles
prepared in accordance with the present invention is
preferably administered by injection intraperitoneally,
subcutaneously, or intramuscularly. However, it would
be clear to one skilled in the art that other routes of
delivery could also be effectively utilized using the
compositions of the present invention.
The following examples are offered to more
fully illustrate the invention, but are not to be
construed as limiting the scope thereof. Example 1
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describes a method for preparing protein loaded
microparticles using spray drying for the microparticle
preparation. NESP (in the form of a spray dried protein
powder) is used as an example protein. Example 2
describes various characterization experiments performed
on the NESP-containing microparticles of Example 1.
Example 3 demonstrates the ability of NESP-containing
microparticles of Example 1 to provide for sustained
release of NESP in vivo. Example 4 describes a novel
method for preparing polymeric microparticles wherein a
cosolvent is utilized to effect more rapid and effective
removal of residual solvent. Example 5 describes a
method for preparing NESP-containing microparticles
using spray freezing for the microparticle preparation
step. Example 6 describes various characterization
experiments performed on the NESP-containing
microparticles of Example 5. Example 7 demonstrates the
ability of NESP-containing microparticles of Example 5
to provide for sustained release of NESP in vivo.
Example 8 describes the double emulsion/solvent
extraction and evaporation method for preparing PLGA
microparticles containing NESP, and demonstrates the
ability to provide for sustained release of NESP in
vivo. Example 9 describes the preparation of leptin-
containing microparticles using the process of Example 1
and demonstrates the ability of the leptin-containing
microparticles to provide for sustained release of
leptin in vivo. Example 10 describes a method for
preparing protein loaded microparticles using spray
drying for the microparticle preparation. Example 11
describes various characterization experiments performed
on the NESP-containing microparticles of Example 10.
Example 12 demonstrates the ability of NESP-containing
microparticles of Example 10 to provide for sustained
release of NESP in vivo. Example 13 demonstrates the
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ability of NESP-containing microparticles of Example 10
to provide for sustained release of NESP in vivo.
EXAMPLE 1
This example describes a method for preparing
protein loaded microparticles; specifically, the
preparation of poly(D,L-lactide-co-glycolide)
microspheres containing NESP, using spray drying for the
microparticle preparation step.
NESP was formulated at 46o NESP, 29o sodium
phosphate salts, 25~ trehalose (w/w/w) and spray dried
on a lab scale spray dryer (BUCHI 190) using the
following conditions: feed rate 2.0 ml/min, atomization
500 NL/hour, inlet temperature 135°C, outlet temperature
99°C, drying gas flow rate 800 SLPM. The protein powder
was collected and characterized as described in Example
2 below.
The protein powder was added to a PLGA in
dichloromethane (inherent viscosity 0.18, 11 kD) polymer
solution (40~ w/v) and the resulting suspension was
spray dried on a pilot plant scale spray dryer (Niro
Mobile MinorTM) using the following conditions: feed rate
50 ml/min, atomization flow rate (two-fluid nozzle) 3.0
kg/hr, inlet gas temperature 50°C, outlet temperature
30°C, drying gas flow rate 93 kg/hr. The resulting
NESP-containing microparticles (0.530 NESP) were then
collected and characterized after sieving (125 um mesh
size).
NESP-containing microparticles of various PLGA
composition were produced using this process. The PLGA
compositions differed in the copolymer ratio of
lactide:glycolide from 50:50 to 1000 lactide. The PLGA
polymers used had free acid polymer chain end groups.
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EXAMPLE 2
This example describes various
characterization experiments performed on the spray
dried NESP/trehalose protein powder and the NESP
containing microparticles described in Example 1.
The NESP/trehalose protein powder was
characterized by size exclusion chromatography under
native conditions; monomer percent was unchanged post
spray drying (>99.8o)(See Figure 2). No additional
aggregate were observed by sodium dodecyl polyacrylamide
gel electrophoresis (SDS-PAGE), using silver stain. The
protein powder was analyzed by reverse phase high
performance liquid chromatography (RP-HPLC); no change
from the unprocessed NESP was observed. The protein
powder was also characterized by an HPLC glycoform assay
and isoelectric focusing gel electrophoresis (IEF) and
IEF western blot; no change in glycoform distribution
was observed. Radioimmunoassay of the reconstituted
powder demonstrated full antibody recognition, and
tryptic mapping demonstrated no change in oxidation from
the unprocessed material. The particle size
distribution of the protein powder was determined by
Fraunhoffer diffraction to have mean volume distribution
of 4.7 ~.m.
The particle size of the NESP-containing
microparticles was 58 ~ 8 ~.m for the mean of the volume
distribution determined by Fraunhoffer diffraction,
averaged over the 5 lots. The post-encapsulation
integrity of NESP within these microparticles was
assessed by extracting the protein and analyzing the
extracts via anion exchange and size exclusion HPLC
under native conditions. To extract the NESP from the
microparticles, approximately 20 mg of microparticles
were placed in a tube with 1 mL acetonitrile. The
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samples were vortexed for 10-20 seconds and centrifuged
at 14,000 rpm for 2 minutes at 4°C to pellet the protein
and excipients. The supernatant was removed and the
pellet resuspended in 1 mL acetonitrile. The above
steps were performed three more times. After final
supernatant removal, samples were dried in a vacuum oven
for 2-3 hours at room temperature. The NESP pellet was
reconstituted in 20mM sodium phosphate, pH 6.0, with or
without 0.0050 Tween 80. After gentle flicking of the
tube the sample was incubated at room temperature for 2
hours to achieve full dissolution. Protein was
quantitated and integrity determined by anion exchange
in series with size exclusion HPLC. Protein recovery
was quantitative (>99~) and protein integrity was 98.2
1.0o monomeric by size exclusion HPLC averaged over the
5 lots, each lot characterized in triplicate (See Figure
2). The extract from the 750 lactide formulation was
additionally characterized by radioimmunoassay,
capillary electrophoresis, and peptide mapping. The RIA
resulted in protein recoveries consistent with the SEC
results, demonstrating antibody recognizable protein.
Capillary electrophoresis confirmed that the glycoform
distribution was unchanged from the unprocessed NESP.
The extent of oxidation as determined from peptide
mapping was equivalent to the unprocessed NESP.
EXAMPLE 3
This example demonstrates the ability of NESP-
containing microparticles prepared as described in
Example 1 to provide for sustained release of NESP in
vi v0 .
NESP-containing microparticles (360 ~.g/kg NESP
peptide dose) were injected subcutaneously at the nape
of the neck of Male Sprague Dawley rats (385 ~ 14 g). A
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subcutaneous, single injection bolus of NESP in a dose
equivalent to the microparticles was included as a
control. Blood samples were taken from the tail vein at
times post injection up to 8 weeks. The NESP
concentration in the rat serum was determined by ELISA
through 2 and 4 weeks. V~Thole blood was analyzed for
hematocrit, hemoglobin, and reticulocyte counts.
NESP serum levels from a single injection of
NESP-containing microparticles fabricated from 50:50
lactide:glycolide polymer were > 1 ng/mL for 15 days for
all three lots studied (See Figure 3). The NESP alone
given as a single bolus had elevated serum levels for 11
days (See Figure 3). The NESP-containing microparticles
elevated the hemoglobin and hematocrit above baseline
for 25 and 28 days, respectively (See Figure 4). The
NESP bolus had elevated hemoglobin and hematocrit for 25
days (See Figure 4).
NESP serum levels from a single injection of
NESP-containing microparticles fabricated from 75:25
lactide:glycolide polymer were > 1 ng/mL for 20 days
(See Figure 3). The NESP-containing microparticles
elevated the hemoglobin and hematocrit above baseline
for > 40 days (See Figure 4).
NESP serum levels from a single injection of
NESP-containing microparticles fabricated using a
solution mixture of 50:50 and 1000 lactide polymers to
create an overall average 750 lactide polymer were > 1
ng/mL for 18 days (See Figure 3). The NESP-containing
microparticles elevated the hemoglobin and hematocrit
above baseline for 35 days (See Figure 4).
EXAMPLE 4
This example describes the novel method for
preparing microparticles wherein a cosolvent is utilized
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to effect more rapid and effective removal of residual
solvent; specifically, the preparation of poly(D,L-
lactide-co-glycolide) microspheres utilizing an
ethanol/methylene chloride cosolvent mixture.
Two batches of 50:50 PLGA (11 kD)
microparticles were produced by spray drying. For batch
1, pure methylene chloride was used to dissolve a mass
of PLGA equal to 26~ of the solvent mass. For batch 2,
a cosolvent of methylene chloride (86. 4o by mass) and
ethanol (13.60 by mass) was used to dissolve a mass of
PLGA equal to 260 of the cosolvent mass). The resulting
solutions were spray dried on a pilot plant scale spray
dryer (Niro Mobile MinorTM) using the following
conditions: feed rate 50~ 5 ml/min, atomization flow
rate (two-fluid nozzle) 60 SLPM, inlet gas temperature
55°C, outlet temperature 33-36 °C, drying gas flow rate
2.1 lbs/min. The resulting microparticles were
collected and characterized.
The residual solvent concentration in Batch 1
following spray drying was 18550 ppm methylene chloride.
The residual solvent concentrations in Batch 2 following
spray drying were 6190 ppm methylene chloride, 3330 ppm
ethanol. Secondary drying was performed on both batches
as follows: the microparticles were placed in a 1.5"
diameter dryer on a retaining screen. The dryer was
sealed and nitrogen was flowed through the bed at 4.4
L/min. The dryer was submerged in a heating bath for
temperature control. After Batch 1 was dried for 73
hours, starting at 20°C and ending at 41°C, the residual
methylene chloride level was 750 ppm. Batch 2 was dried
for 40 hours, starting at 18°C and ending at 30°C.
These microparticles reached residual solvent levels of
487 ppm methylene chloride and 455 ppm ethanol.
Use of the cosolvent thus decreases the amount
of drying time necessary and improves overall residual
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solvents levels in the final microparticles, thus making
processes utilizing such solvents more commercially
practicable.
EXAMPLE 5
This example describes a method for preparing
protein loaded microparticles; specifically, the
preparation of poly(D,L-lactide-co-glycolide)
microspheres containing NESP, using spray freezing for
the microparticle preparation step.
Two formulations of NESP (trehalose
formulation from Example 1, and an ammonium sulfate
formulation) were spray dried on a lab scale spray dryer
using conditions described in Example 1. The ammonium
sulfate formulation for NESP was: 11~ NESP, 10~
phosphate salts, 79o ammonium sulfate (w/w/w).
Microparticles containing the NESP/trehalose
protein powder were prepared from unblocked PLGA
obtained from either Alkermes/Medisorb Wilmington, Ohio,
or Boehringer Ingelheim Chemicals, Inc., Montvale, N.J.
or blocked PLGA from Boehringer Ingelheim. The polymer
inherent viscosities ranged from 0.14 dL/g to 0.5 dL/g
(11 - 47 kD molecular weight), and the lactide contents
were from 50o to 1000. NESP/ammonium sulfate protein
powder was encapsulated in unblocked PLGA (50:50) with
an inherent viscosity of 0.18 dL/g (11 kD).
The method described by Gombotz et al. (U. S.
Pat. No. 5,019,400) was used to encapsulate the spray
dried NESP powder in PLGA. The protein powder was added
to a polymer solution of PLGA in dichloromethane (5 -
20~ w/v) to achieve a protein solids content in the
microparticles ranging from 1 - 5~ (w/w). A container
of ethanol was frozen by immersion in liquid nitrogen,
and overlayed with a layer of liquid nitrogen prior to
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the spray freezing step. The suspension of protein
powder in polymer solution was pumped via a syringe pump
to an ultrasonic nozzle placed above the frozen bath of
ethanol. The suspension was atomized into droplets that
froze upon contact with the liquid nitrogen and settled
onto the surface of the frozen ethanol, forming
microparticles. The frozen bath was transferred to -80
°C for 72 hours to allow the ethanol to melt and extract
the polymer solvent from the microparticles. The
resulting slurry of microparticles in ethanol was cold-
filtered (0.65 um PTFE) and the collected microparticles
were lyophilized. Following lyophilization the
microparticles (0.530 NESP peptide content) were sieved
(125 ~.m mesh size) prior to characterization.
EXAMPLE 6
This example describes various
characterization experiments performed on the spray
freeze NESP-containing microparticles described in
Example 5.
The NESP protein powders were characterized as
described in Example 2, with the results for the
NESP/trehalose already presented there. NESP/ammonium
sulfate (AS) was characterized by size exclusion
chromatography under native conditions; monomer percent
was significantly decreased post spray drying, by 2.50.
This was a dimer confirmed by sodium dodecyl
polyacrylamide gel electrophoresis (SDS-PAGE), and
demonstrated to be non-reducible, using silver stain.
The particle size distribution of the NESP/AS protein
powder was determined by Fraunhoffer diffraction to have
mean volume distribution of 4.3 ~,m.
The particle size of the NESP-containing
microparticles fabricated by spray freezing for 28
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unique lots of various formulations ranged from 20 to 45
~m for the mean of the volume distribution. The
residual methylene chloride levels were all < 500 ppm
for the 10 lots evaluated. The post-encapsulation
integrity of NESP within these microparticles was
assessed by extracting the protein and analyzing the
extracts as described in Example 2. Protein recovery
was quantitative and integrity (monomer o) was 97.2 ~
20, each lot characterized in at least duplicate. The
extracts from 9 select formulations were additionally
characterized by a radioimmunoassay, capillary
electrophoresis, and peptide mapping. The RIA resulted
in protein recoveries consistent with the SEC results,
demonstrating antibody recognizable protein. Capillary
electrophoresis confirmed that the glycoform
distribution was unchanged from the unprocessed NESP.
The extent of oxidation of the protein determined from
the peptide mapping ranged from 7 - 120, unprocessed
material is typically 8 ~ 20.
EXAMPLE 7
This example demonstrates the ability of NESP-
containing microparticles prepared in Example 5 to
provide for sustained release of NESP in vivo.
Male Sprague Dawley rats were treated as
described in Example 3 with different formulations of
NESP-containing microparticles fabricated by the spray
freeze process as described in Example 6. Serum NESP
concentration and whole blood analyses were performed
through 4-6 weeks as described in Example 3.
Figure 5 shows the results obtained for the
NESP serum levels using various copolymer formulations
of Example 5. All of the copolymer formulations
demonstrated a burst phase followed by a phase of zero-
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order release, and then a drop to below the assay
quantitation limit. Increasing the lactide content
decreased the NESP serum level through the zero order
phase by over 100-fold. Increasing the inherent
viscosity (molecular weight) likewise decreased this
NESP serum level, but only by 4-fold (See Figure 6).
Both increasing the inherent viscosity and increasing
the lactide content, whether by copolymer composition or
polymer solution mixture, increased the duration of
quantifiable serum NESP concentrations from 18 days to
as long as 35 days.
The pharmacodynamic effect, as measured by
elevated hematocrit above baseline levels, paralleled
the trends observed with the serum NESP concentration.
Preliminary data on NESP serum concentrations and the
elevation of reticulocytes counts suggest an efficacious
serum level in rats to be near 0.4 ng/mL for treatment
with NESP microparticles.
In a mouse pharmacodynamic study, male BDF1
mice (body weight 22 g) were treated with NESP/PLGA
microparticles (50:50, inherent viscosity 0.4 dL/g), in
a single, subcutaneous bolus injection into the nape of
the neck, at NESP doses of 6, 30 and 100 ug/kg. NESP
solution test groups were dosed in a single,
subcutaneous bolus injection into the nape of the neck,
at NESP doses of 100, 1,000 and 10,000 ~,g/kg. The study
design was such that blood was collected every 2-4 days
for whole blood analysis over a 35 day period, but any
individual animal was not bled more than twice in a 7
day period.
A dose response of the microparticle treated
groups was observed. Hemoglobin levels were elevated
above baseline for > 4weeks for the microparticle dose
of 100 ~.g/kg. The duration of the effect after
treatment with NESP solution bolus was only equivalent
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to that observed with the microparticles when given at
100-fold greater dose than the microparticles (See
Figure 7).
EXAMPLE 8
This example describes the double
emulsion/solvent extraction and evaporation method for
preparing PLGA microparticles containing NESP, and
demonstrates the ability to provide for sustained
release of NESP in vivo.
Two aqueous formulations of NESP were
prepared: formulation 1 = 5.5 mg/mL NESP (peptide
concentration) formulated with 20 mM sodium phosphate,
pH 6.0; formulation 2 - 5.2 mg/mL NESP (peptide
concentration) formulated with 20 mM sodium phosphate,
105 mg/mL 2-hydroxypropyl (3-cyclodextrin, pH 6Ø
Microparticles containing the above NESP
formulations were prepared from unblocked PLGA (50:50,
inherent viscosity 0.2, 11 kD) obtained from Boehringer
Ingelheim Chemicals, Inc., Montvale, N.,7. The double
emulsion followed by solvent extraction and evaporation
method was used as described below.
Approximately 2 g of polymer was dissolved in
6 mL of dichloromethane and homogenized at 25 krpm in an
18 x 150 mm glass test tube on ice. Protein solution
was added (1.0 mL) during homogenization which was
continued for an additional 30 seconds on ice to form
the primary emulsion. For the secondary emulsion, 40
mLs of the aqueous outer phase (18 mM sodium phosphate,
0.5o polyvinyl alcohol, pH 6.0) in a 100 mL beaker of
4.5 cm inner diameter, was prechilled to 15 C in a water
bath. A 1" Rushton impeller blade was immersed in the
chilled outer phase and mixing was initiated at 1480
rpm. The primary emulsion was added quickly to form the
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secondary emulsion, and mixing was continued at the same
speed for a total of 40 minutes.
The cured emulsion was washed with water twice
by transferring the solution to two 50 mL tubes,
centrifuging at 500 g-force for 30 seconds, decanting to
a volume of 10 mLs, and repeating. The final suspension
was transferred into vials and lyophilized. The final
dry powder was sieved (180 um mesh size) prior to
characterization by methods described in Example 2.
The process provided quantitative
encapsulation efficiency, poor product yield (500),
large particle size (120 ~.m), and acceptable residual
methylene chloride levels (near 500 ppm). The post-
encapsulation integrity of NESP within these
microparticles was assessed by extracting the protein
and analyzing the extracts as described in Example 2.
Protein recovery was quantitative and integrity (monomer
o) was 97.5% ~ 0.010 for formulation (1), and 96.7
0.3o for formulation (2); each lot was characterized in
triplicate.
Male Sprague Dawley rats were treated as
described in Example 3 with the two formulations of
NESP-containing microparticles fabricated by the double
emulsion process. Serum NESP levels demonstrated a
burst phase followed by a phase of zero-order release,
and then a drop to below the assay quantitation limit
after 18 days for formulation 1 and after 22 days for
formulation 2. The pharmacodynamic effect as measured
by hemoglobin elevated above baseline, lasted for 25 and
>28 days (end of study) for formulations (1) and (2),
respectively (See Figure 8).
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EXAMPLE 9
This example describes the preparation of
microparticles containing leptin and demonstrates the
ability of leptin-containing microparticles to provide
for sustained release of leptin in vivo.
Leptin-containing microparticles were prepared
using the procedure described in Example 1, and then
characterized as described in Example 2. It was
determined that protein recovery was >95o and the
integrity was >980. The process provided high
encapsulation efficiency (85-950), good product yield
(75-85~), low burst (<150), acceptable particle size
(--35 um), and acceptable residual solvent levels. It
was further demonstrated that the formulation exhibited
good storage stability.
"In vivo" bioactivity of leptin-containing
microparticles were evaluated in normal rats. A total
leptin dose of 50 mg/kg (corresponding to 150 mg
microparticles/kg) were administered as a single
injection at day 0. In addition, the following control
groups were included: daily leptin bolus (5 mg/kg/day x
10 days); dose dump control (50 mg/kg at day 0); placebo
microparticles administered at day ); and daily placebo
injection control. Animals were weighed daily, and
serum samples collected periodically to assess the serum
leptin concentration.
Serum leptin levels are shown in Figure 9.
The leptin concentration in the serum remains above
baseline for approximately 5 days. Weight loss is
presented in Figure 10. o body weight loss relative to
buffer control was determined for a 30 day period.
Daily injection of a leptin solution resulted in 4-6~
weight loss relative to placebo controls. A single
injection of leptin-containing microparticles resulted
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in 9-10~ weight loss relative to placebo microparticles,
and resulted in sustained weight loss in rats for a 25
day period.
After day 10, some animals were sacrificed for
histological examination of the injection site.
Histological examination of the injection site revealed
a localized minimal to mild inflammatory reaction, which
was fully reversible with biodegradation of the
microparticles over time.
Example 10
This example describes a method for preparing
protein loaded microparticles; specifically, the
preparation of poly(D,L-lactide-co-glycolide)
microspheres containing NESP, using spray drying for the
microparticle preparation step.
NESP was formulated at 46o NESP, 29o sodium
phosphate salts, 25o trehalose (w/w/w) and spray dried
on a pilot plant scale spray dryer (Niro Mobile MinorTM)
using the following conditions: feed rate 8.0 ml/min,
atomization gas 0.33 lbs/min (two-fluid nozzle), inlet
temperature 200°C, outlet temperature 100°C, drying gas
flow rate 2.0 lbs/min. The protein powder was collected
and characterized as described in Example 11 below.
The protein powder was added to a PLGA (high
molecular weight, (inherent viscosity 0.49 dL/g), 50~
lactide) in dichloromethane/ethanol polymer solution
(10~ w/v) and the resulting suspension was spray dried
on a pilot plant scale spray dryer (Niro Mobile MinorTM)
using the following conditions: feed rate 12 ml/min,
atomization (ultrasonic nozzle) 1.3 watts, inlet gas
temperature 55°C, outlet temperature 28°C, drying gas
flow rate 1.2 lbs/min. The resulting NESP-containing
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microparticles (0.530 NESP) were then collected and
secondary drying was performed as described in Example 4
to reduce residual solvent concentrations to less than
1000 ppm. The resulting microparticles were
characterized after sieving (125 ~.m mesh size).
Example 11
This example describes various
characterization experiments performed on the spray
dried NESP/trehalose protein powder and the NESP-
containing microparticles described in Example 10.
The NESP/trehalose protein powder was
characterized by size exclusion chromatography under
native conditions; monomer percent was 99.80 post spray
drying (1000 for un-processed material). No additional
aggregate was observed by sodium dodecyl polyacrylamide
gel electrophoresis (SDS-PAGE), using silver stain. The
protein powder was analyzed by reverse phase high
performance liquid chromatography (RP-HPLC); no change
from the unprocessed NESP was observed. The protein
powder was also characterized by an HPLC glycoform assay
and isoelectric focusing gel electrophoresis (IEF) no
change in glycoform distribution was observed. The
particle size distribution of the protein powder was
determined by Fraunhoffer diffraction to have a mean
size of 2.5 ~.m (volume distribution)
The NESP-containing microparticles were
determined by Fraunhoffer diffraction to have a mean
size of 45 ~ 1 ~,m (volume distribution). Residual
solvent concentrations of dichloromethane and ethanol
were determined by head space gas chromatography to be
638 ppm and <100 ppm, respectively.
The post-encapsulation integrity of NESP
within these microparticles was assessed by extracting
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the protein as described in Example 2 and analyzing the
extracts via anion exchange and size exclusion HPLC
under native conditions. Protein was quantitated and
integrity determined by anion exchange in series with
size exclusion HPLC. Protein recovery was quantitative
(>990) and protein integrity was 98.6 ~ 0.3o monomeric
by size exclusion HPLC. No additional aggregates were
observed by sodium dodecyl polyacrylamide gel
electrophoresis (SDS-PAGE), using silver stain. The
NESP microparticle protein extract was analyzed by
reverse phase high performance liquid chromatography
(RP-HPLC); no change from the NESP protein powder was
observed. The NESP microparticle protein extract was
also characterized by an HPLC glycoform assay and
isoelectric focusing gel electrophoresis (IEF) no change
in glycoform distribution was observed.
Example 12
This example demonstrates the ability of NESP-
containing microparticles prepared in Example 10 to
provide for sustained release of NESP in vivo.
Male Sprague Dawley rats were treated as
described in Example 3 with NESP-containing
microparticles fabricated as described in Example 10.
Serum NESP concentration and whole blood analyses were
performed through 4-8 weeks as described in Example 3.
NESP serum levels from a single injection of
NESP-containing microparticles were > 1 ng/mL for 20
days (See Figure 11). The NESP alone given as a single
bolus had elevated serum levels for 11 days (See Figure
11). The NESP-containing microparticles elevated
hematocrit above baseline for 34 days (See Figure 12).
The NESP bolus had elevated hematocrit for 20 days (See
Figure 12).
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Example 13
This example demonstrates the ability of NESP-
containing microparticles prepared in Example 10 to
provide for sustained release of NESP in vivo.
Male NIHRNU-M Nude rats were treated as
described in Example 3 with NESP-containing
microparticles fabricated as described in Example 10.
Serum NESP concentration and whole blood analyses were
performed through 4-8 weeks as described in Example 3.
NESP serum levels from a single injection of
NESP-containing microparticles were > 1 ng/mL for 21
days(See Figure 13). The NESP alone given as a single
bolus had elevated serum levels for less than 11 days
(See Figure 13). The NESP-containing microparticles
elevated hematocrit above baseline for 44 days days (See
Figure 14). The NESP bolus had elevated hematocrit for
18 days (See Figure 14).
Materials and Methods
The present NESP may be prepared according to
the above incorporated-by-reference PCT Application No.
US94/02957.
The present recombinant methionyl-human-OB
protein (leptin) may be prepared according to the above
incorporated-by-reference PCT publication, WO 96/05309
at pages 151-159. For the present working examples, a
human OB protein was used which has (as compared to the
amino acid sequence at page 158) a lysine at position 35
instead of an arginine, and an isoleucine at position 74
instead of an isoleucine. Other recombinant human OB
proteins may be prepared according to methods known
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generally in the art of expression of proteins using
recombinant DNA technology.
While the present invention has been described
in terms of certain preferred embodiments, it is
understood that variations and modifications will occur
to those skilled in the art. Therefore, it is intended
that the appended claims cover all such equivalent
variations which come within the scope of the invention
as claimed.
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1
SEQUENCE LISTING
<110> AMGEN INC.
<120> BIODEGRADABLE MICROPARTICLES FOR THE SUSTAINED DELIVERY
OF NOVEL ERYTHROPOIETIN STIMULATING PROTEIN
<130> A-626
<140> TO BE ASSIGNED
<141> 1999-10-22
<160> 2
<170> PatentIn Ver. 2.1
<210> 1
<211> 165
<212> PRT
<213> HUMAN
<400> 1
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His
20 25 30
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe
35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp
50 55 60
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80
Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp
85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 150 155 160
Cys Arg Thr Gly Asp
165
CA 02387229 2002-04-11
WO 01/30320 PCT/US00/29257
2
<210> 2
<211> 165 -
<212> PRT
<213> HUMAN
<400> 2
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu
1 5 10 15
Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Asn Glu Thr
20 25 30
Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe
35 40 45
Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp
50 55 60
Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80
Leu Val Asn Ser Ser Gln Val Asn Glu Thr Leu Gln Leu His Val Asp
85 90 95
Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
100 105 110
Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125
Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val
130 135 140
Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala
145 150 155 160
Cys Arg Thr Gly Asp
165
