U.S. patent application number 13/910328 was filed with the patent office on 2013-12-26 for therapeutic polymeric nanoparticles with mtor inhibitors and methods of making and using same.
This patent application is currently assigned to BIND Therapeutics, Inc.. The applicant listed for this patent is BIND Therapeutics, Inc.. Invention is credited to Mir Mukkaram Ali, Jeff Hrkach, Greg Troiano, James Wright, Stephen E. Zale.
Application Number | 20130344158 13/910328 |
Document ID | / |
Family ID | 41507663 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344158 |
Kind Code |
A1 |
Zale; Stephen E. ; et
al. |
December 26, 2013 |
Therapeutic Polymeric Nanoparticles with mTOR Inhibitors and
Methods of Making and Using Same
Abstract
The present disclosure generally relates to therapeutic
nanoparticles. Exemplary nanoparticles disclosed herein may include
about 1 to about 20 weight percent of a mTOR inhibitor; and about
70 to about 99 weight percent biocompatible polymer.
Inventors: |
Zale; Stephen E.;
(Hopkinton, MA) ; Troiano; Greg; (Pembroke,
MA) ; Ali; Mir Mukkaram; (Woburn, MA) ;
Hrkach; Jeff; (Lexington, MA) ; Wright; James;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIND Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
BIND Therapeutics, Inc.
Cambridge
MA
|
Family ID: |
41507663 |
Appl. No.: |
13/910328 |
Filed: |
June 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12485462 |
Jun 16, 2009 |
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13910328 |
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61061704 |
Jun 16, 2008 |
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61061697 |
Jun 16, 2008 |
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61061760 |
Jun 16, 2008 |
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61088159 |
Aug 12, 2008 |
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61105916 |
Oct 16, 2008 |
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61106777 |
Oct 20, 2008 |
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61169514 |
Apr 15, 2009 |
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61169519 |
Apr 15, 2009 |
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61169541 |
Apr 15, 2009 |
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61173790 |
Apr 29, 2009 |
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61175209 |
May 4, 2009 |
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61175219 |
May 4, 2009 |
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61175226 |
May 4, 2009 |
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Current U.S.
Class: |
424/501 ;
514/291 |
Current CPC
Class: |
A61K 31/436 20130101;
C07C 59/08 20130101; A61K 31/00 20130101; A61K 9/5146 20130101;
A61K 9/19 20130101; Y10S 977/773 20130101; A61K 31/435 20130101;
A61P 37/06 20180101; A61K 9/5153 20130101; A61P 35/00 20180101;
A61K 9/14 20130101 |
Class at
Publication: |
424/501 ;
514/291 |
International
Class: |
A61K 31/436 20060101
A61K031/436; A61K 9/14 20060101 A61K009/14 |
Goverment Interests
[0002] This invention was made with United States Government
support under Cooperative Agreement Number 70NANB7H7021 awarded by
the National Institute of Standard and Technology (NIST). The
United States Government has certain rights in the Invention.
Claims
1. A therapeutic nanoparticle having a hydrodynamic diameter of the
therapeutic nanoparticle of about 70 to about 130 nm; comprising:
about 5 to about 30 weight percent of a mTOR inhibitor; about 10 to
about 99 weight percent of a diblock poly(lactic)
acid-poly(ethylene)glycol copolymer, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
15 to about 20 kDa and poly(ethylene)glycol having a number average
molecular weight of about 4 to about 6 kDa; about 0.2 to about 10
weight percent of a polymer conjugate represented by:
PLA-PEG-ligand; wherein the ligand is covalently bound to the PEG
or covalently bound to the PEG through an alkylene linker, and
wherein PLA is poly(lactic)acid and PEG is poly(ethylene)glycol;
and wherein the therapeutic nanoparticle releases less than 10% of
the therapeutic agent over about one minute when placed in a
phosphate buffer solution at 37.degree. C.
2. The therapeutic nanoparticle of claim 1 wherein said mTOR
inhibitor is selected from the group consisting of sirolimus,
temsirolimus, and everolimus, and pharmaceutically acceptable salts
thereof.
3. The therapeutic nanoparticle of claim 1, wherein the
hydrodynamic diameter is about 70 to about 120 nm.
4. The therapeutic nanoparticle of claim 1, wherein the therapeutic
nanoparticle substantially retains the therapeutic agent for at
least 5 days at 25.degree. C.
5. The therapeutic nanoparticle of claim 1, comprising about 10 to
about 20 weight percent of the mTOR inhibitor.
6. The therapeutic nanoparticle of claim 1, comprising about 40 to
about 90 weight percent diblock poly(lactic)
acid-poly(ethylene)glycol copolymer.
7. The therapeutic nanoparticle of claim 1, wherein the particle
releases less than about 5% of the therapeutic agent over 1 hour
when placed in a phosphate buffer solution at room temperature.
8. The therapeutic nanoparticle of claim 1, wherein the particle
releases less than about 10% of the therapeutic agent over 24 hours
when placed in a phosphate buffer solution at room temperature.
9. The therapeutic nanoparticle of claim 1, wherein the ligand has
molecular weight of about 100 g/mol to about 6000 g/mol.
10. The therapeutic nanoparticle of claim 9, wherein the ligand has
a molecular weight of about 100 g/mol to about 500 g/mol.
11. The therapeutic nanoparticle of claim 10, wherein the
PLA-PEG-Ligand comprises a PLA having number average molecular
weight of about 10 kDa to about 20 kDa and a PEG having a number
average molecular weight of about 4 kDa to about 8 kDa.
12. The therapeutic nanoparticle of claim 1, wherein the diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(lactic acid) having a number average molecular weight of about
16 kDa.
13. The therapeutic nanoparticle of claim 12, wherein said diblock
poly(lactic) acid-poly(ethylene)glycol copolymer comprises
poly(ethylene)glycol having a number average molecular weight of
about 5 kDa.
14. A method of treating lymphoma comprising administering to a
patient in need thereof an effective amount of the therapeutic
nanoparticle of claim 13.
15. A pharmaceutical composition comprising: a plurality of
polymeric nanoparticles each having a hydrodynamic diameter of
about 60 nm to about 140 nm and comprising about 3 to about 40
weight percent of a mTOR inhibitor; about 10 to about 99 weight
percent of a diblock poly(lactic)acid-poly(ethylene)glycol
copolymer comprising poly(lactic)acid having a number average
molecular weight of about 15 to about 20 kDa and
poly(ethylene)glycol having a number average molecular weight of
about 4 to about 6 kDa, about 0.2 to about 10 weight percent of a
polymer conjugate represented by: PLA-PEG-ligand; wherein the
ligand is covalently bound to the PEG or covalently bound to the
PEG through an alkylene linker, and wherein PLA is poly(lactic)acid
and PEG is poly(ethylene)glycol and a saccharide; wherein said
nanoparticles are stable for at least 3 days when held at
25.degree. C. in said composition.
16. The pharmaceutical composition of claim 15, wherein the
saccharide is sucrose.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/485,462 filed Jun. 16, 2009, which claims priority to U.S. Ser.
No. 61/061,760, filed Jun. 16, 2008; U.S. Ser. No. 61/105,916,
filed Oct. 16, 2008, U.S. Ser. No. 61/106,777, filed Oct. 20, 2008;
U.S. Ser. No. 61/169,514, filed Apr. 15, 2009; U.S. Ser. No.
61/175,209, filed May 4, 2009; U.S. Ser. No. 61/061,704, filed Jun.
16, 2008; U.S. Ser. No. 61/169,519, filed Apr. 15, 2009; U.S. Ser.
No. 61/175,219 filed May 4, 2009; U.S. Ser. No. 61/061,697, filed
Jun. 16, 2008; U.S. Ser. No. 61/088,159, filed Aug. 12, 2008; U.S.
Ser. No. 61/169,541, filed Apr. 15, 2009; U.S. Ser. No. 61/175,226,
filed May 4, 2009; U.S. Ser. No. 61/173,790, filed Apr. 29, 2009;
each of which is hereby incorporated by reference in their
entirety.
BACKGROUND
[0003] Systems that deliver certain drugs to a patient (e.g.,
targeted to a particular tissue or cell type or targeted to a
specific diseased tissue but not normal tissue), or that control
release of drugs has long been recognized as beneficial. For
example, therapeutics that include an active drug and that are
capable of locating in a particular tissue or cell type e.g., a
specific diseased tissue, may reduce the amount of the drug in
tissues of the body that do not require treatment. This is
particularly important when treating a condition such as cancer
where it is desirable that a cytotoxic dose of the drug is
delivered to cancer cells without killing the surrounding
non-cancerous tissue. Further, such therapeutics may reduce the
undesirable and sometimes life threatening side effects common in
anticancer therapy. For example, nanoparticle therapeutics may, due
the small size, evade recognition within the body allowing for
targeted and controlled delivery while e.g., remaining stable for
an effective amount of time.
[0004] Therapeutics that offer such therapy and/or controlled
release and/or targeted therapy also must be able to deliver an
effective amount of drug. It can be a challenge to prepare
nanoparticle systems that have an appropriate amount of drug
associated each nanoparticle, while keeping the size of the
nanoparticles small enough to have advantageous delivery
properties. For example, while it is desirable to load a
nanoparticle with a high quantity of therapeutic agent,
nanoparticle preparations that use a drug load that is too high
will result in nanoparticles that are too large for practical
therapeutic use. Further, it may be desirable for therapeutic
nanoparticles to remain stable so as to e.g. substantially limit
rapid or immediate release of the therapeutic agent.
[0005] Accordingly, a need exists for new nanoparticle formulations
and methods of making such nanoparticles and compositions, that can
deliver therapeutic levels of drugs to treat diseases such as
cancer, while also reducing patient side effects.
SUMMARY
[0006] In one aspect, the invention provides therapeutic
nanoparticle that includes an active agent or therapeutic agent,
e.g. an mTOR inhibitor or pharmaceutically acceptable salts
thereof, and one, two, or three biocompatible polymers. For
example, disclosed herein is a therapeutic nanoparticle comprising
about 1 to about 20 weight percent of a therapeutic agent (such as
for example sirolimus, temsirolimus, or everolimus) and about 50 to
about 99 weight percent of a biocompatible polymer, e.g. about 70
to about 99 weight percent of a biocompatible polymer. For example,
the biocompatible polymer may be a diblock poly(lactic)
acid-poly(ethylene)glycol copolymer (e.g. PLA-PEG) or a diblock
(poly(lactic)-co-poly (glycolic) acid)-poly(ethylene)glycol
copolymer (e.g. PLGA-PEG), or the biocompatible polymer may two or
more biocompatible polymers, for example, the therapeutic
nanoparticles can also include a homopolymer such as poly(lactic)
acid homopolymer. For example, a disclosed therapeutic nanoparticle
may include about 1 to about 20 weight percent, e.g about 2 to
about 20 weight percent or about 10 to about 20 weight percent, of
a mTOR inhibitor; and about 70 to about 99 weight percent
biocompatible polymer, wherein the biocompatible polymer is
selected from the group consisting of a) a diblock poly(lactic)
acid-poly(ethylene)glycol copolymer, b) a diblock
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer, c) a combination of a) or b) and a poly (lactic) acid
homopolymer or poly(lactic)-co-(glycolic) acid; and d) a
combination of a) or b) and a poly (lactic) acid homopolymer or
poly(lactic)-co-(glycolic) acid.
[0007] The diameter of disclosed nanoparticles may be, for example,
about 60 to about 120 nm, or about 70 to about 120 nm. Disclosed
therapeutic nanoparticles may be stable for at least 5 days at
25.degree. C., e.g. may remain stable over 5 days in vitro, e.g. in
a sucrose solution. In another embodiment, disclosed particles may
substantially immediately release less than about 2% or less than
about 5%, or even less than about 10% of the therapeutic agent when
placed in a phosphate buffer solution at room temperature, or at
37.degree. C. For example, disclosed nanoparticles may
substantially retain the therapeutic agent for at least 5 days at
25.degree. C. In another embodiment, disclosed nanoparticles may
release the therapeutic agent over a period of at least 1 day or
more when administered to a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is flow chart for an emulsion process for forming
disclosed nanoparticle.
[0009] FIGS. 2A and 2B are flow diagrams for a disclosed emulsion
process.
[0010] FIG. 3 depicts the effect of coarse emulsion preparation on
quenched particle size. Placebo organic at 30% solids was used,
emulsified at 5:1 W:O using standard aqueous phase (1% sodium
cholate, 2% benzyl alcohol, 4% ethyl acetate).
[0011] FIG. 4 depicts the effect of feed pressure on resultant
particle size.
[0012] FIG. 5 depicts the particle size dependence on scale.
Placebo organic phase consisted of 25.5% polymer stock of 50:50
16.5/5 PLA/PEG:8.2 PLA. Organic phase was emulsified 5:1 O:W with
standard aqueous phase, and multiple discreet passes were
performed, quenching a small portion of emulsion after each pass.
The indicated scale represents the total solids of the
formulation.
[0013] FIG. 6 depicts the effect of solids concentration on
particle size.
[0014] FIG. 7 depicts the effect of solids concentration and
poly(lactic) homopolymer on loading percentage of sirolimus
(rapamycin).
[0015] FIG. 8 depicts in vitro release of sirolimus over time for
disclosed nanoparticles.
[0016] FIG. 9 depicts the effects of poly(lactic) homopolymer on
loading percentage of temsirolimus.
[0017] FIG. 10 depicts the effect of solids concentration on
particle size of temsirolimus containing particles.
[0018] FIG. 11 depicts in vitro release of temsirolimus over time
for disclosed nanoparticles
DETAILED DESCRIPTION
[0019] The present invention generally relates to polymeric
nanoparticles that include an active or therapeutic agent or drug,
and methods of making and using such therapeutic nanoparticles. In
general, a "nanoparticle" refers to any particle having a diameter
of less than 1000 nm, e.g. about 10 nm to about 200 nm. Disclosed
therapeutic nanoparticles may include nanoparticles having a
diameter of about 60 to about 120 nm, or about 70 to about 130 nm,
or about 60 to about 140 nm, or about 70 nm to about 140 nm.
[0020] Disclosed nanoparticles may include about 0.2 to about 35
weight percent, about 3 to about 40 weight percent, about 5 to
about 30 weight percent, about 1 to about 20 weight percent, about
10 to about 30 weight percent, about 5 to about 15 percent, about
15 to 25 weight percent, or even about 4 to about 25 weight
percent, e.g. about 10 weight percent of an active agent, such as
antineoplastic agent, e.g. a mTOR inhibiting agent (for example
sirolimus, temsirolimus or everolimus).
[0021] Nanoparticles disclosed herein include one, two, three or
more biocompatible and/or biodegradable polymers. For example, a
contemplated nanoparticle may include about 60 to about 99 weight
percent of one, two, three or more biocompatible polymers such as
one or more co-polymers (e.g. a diblock polymer) that include a
biodegradable polymer (for example poly(lactic)acid and
polyethylene glycol, and optionally about 0 to about 50 weight
percent of a homopolymer, e.g. biodegradable polymer such as
poly(lactic) acid.
Polymers
[0022] In some embodiments, disclosed nanoparticles include a
matrix of polymers. Disclosed nanoparticles may include one or more
polymers, e.g. a diblock co-polymer and/or a monopolymer. Disclosed
therapeutic nanoparticles include a therapeutic agent can may
associated with the surface of, encapsulated within, surrounded by,
and/or dispersed throughout a polymeric matrix.
[0023] A wide variety of polymers and methods for forming particles
therefrom are known in the art of drug delivery. In some
embodiments, the disclosure is directed toward nanoparticles with
at least one polymer, for example, a first polymer that may be a
co-polymer, e.g. a diblock co-polymer, and optionally a polymer
that may be for example a homopolymer.
[0024] Any polymer can be used in accordance with the present
invention. Polymers can be natural or unnatural (synthetic)
polymers. Polymers can be homopolymers or copolymers comprising two
or more monomers. In terms of sequence, copolymers can be random,
block, or comprise a combination of random and block sequences.
Contemplated polymers may be biocompatible and/or
biodegradable.
[0025] The term "polymer," as used herein, is given its ordinary
meaning as used in the art, i.e., a molecular structure comprising
one or more repeat units (monomers), connected by covalent bonds.
The repeat units may all be identical, or in some cases, there may
be more than one type of repeat unit present within the polymer. In
some cases, the polymer can be biologically derived, i.e., a
biopolymer. Non-limiting examples include peptides or proteins. In
some cases, additional moieties may also be present in the polymer,
for example biological moieties such as those described below. If
more than one type of repeat unit is present within the polymer,
then the polymer is said to be a "copolymer." It is to be
understood that in any embodiment employing a polymer, the polymer
being employed may be a copolymer in some cases. The repeat units
forming the copolymer may be arranged in any fashion. For example,
the repeat units may be arranged in a random order, in an
alternating order, or as a block copolymer, i.e., comprising one or
more regions each comprising a first repeat unit (e.g., a first
block), and one or more regions each comprising a second repeat
unit (e.g., a second block), etc. Block copolymers may have two (a
diblock copolymer), three (a triblock copolymer), or more numbers
of distinct blocks.
[0026] Disclosed particles can include copolymers, which, in some
embodiments, describes two or more polymers (such as those
described herein) that have been associated with each other,
usually by covalent bonding of the two or more polymers together.
Thus, a copolymer may comprise a first polymer and a second
polymer, which have been conjugated together to form a block
copolymer where the first polymer can be a first block of the block
copolymer and the second polymer can be a second block of the block
copolymer. Of course, those of ordinary skill in the art will
understand that a block copolymer may, in some cases, contain
multiple blocks of polymer, and that a "block copolymer," as used
herein, is not limited to only block copolymers having only a
single first block and a single second block. For instance, a block
copolymer may comprise a first block comprising a first polymer, a
second block comprising a second polymer, and a third block
comprising a third polymer or the first polymer, etc. In some
cases, block copolymers can contain any number of first blocks of a
first polymer and second blocks of a second polymer (and in certain
cases, third blocks, fourth blocks, etc.). In addition, it should
be noted that block copolymers can also be formed, in some
instances, from other block copolymers. For example, a first block
copolymer may be conjugated to another polymer (which may be a
homopolymer, a biopolymer, another block copolymer, etc.), to form
a new block copolymer containing multiple types of blocks, and/or
to other moieties (e.g., to non-polymeric moieties).
[0027] In some embodiments, the polymer (e.g., copolymer, e.g.,
block copolymer) can be amphiphilic, i.e., having a hydrophilic
portion and a hydrophobic portion, or a relatively hydrophilic
portion and a relatively hydrophobic portion. A hydrophilic polymer
can be one generally that attracts water and a hydrophobic polymer
can be one that generally repels water. A hydrophilic or a
hydrophobic polymer can be identified, for example, by preparing a
sample of the polymer and measuring its contact angle with water
(typically, the polymer will have a contact angle of less than
60.degree., while a hydrophobic polymer will have a contact angle
of greater than about 60.degree.). In some cases, the
hydrophilicity of two or more polymers may be measured relative to
each other, i.e., a first polymer may be more hydrophilic than a
second polymer. For instance, the first polymer may have a smaller
contact angle than the second polymer.
[0028] In one set of embodiments, a polymer (e.g., copolymer, e.g.,
block copolymer) contemplated herein includes a biocompatible
polymer, i.e., the polymer that does not typically induce an
adverse response when inserted or injected into a living subject,
for example, without significant inflammation and/or acute
rejection of the polymer by the immune system, for instance, via a
T-cell response. Accordingly, the therapeutic particles
contemplated herein can be non-immunogenic. The term
non-immunogenic as used herein refers to endogenous growth factor
in its native state which normally elicits no, or only minimal
levels of, circulating antibodies, T-cells, or reactive immune
cells, and which normally does not elicit in the individual an
immune response against itself.
[0029] Biocompatibility typically refers to the acute rejection of
material by at least a portion of the immune system, i.e., a
nonbiocompatible material implanted into a subject provokes an
immune response in the subject that can be severe enough such that
the rejection of the material by the immune system cannot be
adequately controlled, and often is of a degree such that the
material must be removed from the subject. One simple test to
determine biocompatibility can be to expose a polymer to cells in
vitro; biocompatible polymers are polymers that typically will not
result in significant cell death at moderate concentrations, e.g.,
at concentrations of 50 micrograms/10.sup.6 cells. For instance, a
biocompatible polymer may cause less than about 20% cell death when
exposed to cells such as fibroblasts or epithelial cells, even if
phagocytosed or otherwise uptaken by such cells. Non-limiting
examples of biocompatible polymers that may be useful in various
embodiments of the present invention include polydioxanone (PDO),
polyhydroxyalkanoate, polyhydroxybutyrate, poly(glycerol sebacate),
polyglycolide, polylactide, PLGA, polycaprolactone, or copolymers
or derivatives including these and/or other polymers.
[0030] In certain embodiments, contemplated biocompatible polymers
may be biodegradable, i.e., the polymer is able to degrade,
chemically and/or biologically, within a physiological environment,
such as within the body. As used herein, "biodegradable" polymers
are those that, when introduced into cells, are broken down by the
cellular machinery (biologically degradable) and/or by a chemical
process, such as hydrolysis, (chemically degradable) into
components that the cells can either reuse or dispose of without
significant toxic effect on the cells. In one embodiment, the
biodegradable polymer and their degradation byproducts can be
biocompatible.
[0031] For instance, a contemplated polymer may be one that
hydrolyzes spontaneously upon exposure to water (e.g., within a
subject), the polymer may degrade upon exposure to heat (e.g., at
temperatures of about 37.degree. C.). Degradation of a polymer may
occur at varying rates, depending on the polymer or copolymer used.
For example, the half-life of the polymer (the time at which 50% of
the polymer can be degraded into monomers and/or other nonpolymeric
moieties) may be on the order of days, weeks, months, or years,
depending on the polymer. The polymers may be biologically
degraded, e.g., by enzymatic activity or cellular machinery, in
some cases, for example, through exposure to a lysozyme (e.g.,
having relatively low pH). In some cases, the polymers may be
broken down into monomers and/or other nonpolymeric moieties that
cells can either reuse or dispose of without significant toxic
effect on the cells (for example, polylactide may be hydrolyzed to
form lactic acid, polyglycolide may be hydrolyzed to form glycolic
acid, etc.).
[0032] In some embodiments, polymers may be polyesters, including
copolymers comprising lactic acid and glycolic acid units, such as
poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide),
collectively referred to herein as "PLGA"; and homopolymers
comprising glycolic acid units, referred to herein as "PGA," and
lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid,
poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and
poly-D,L-lactide, collectively referred to herein as "PLA." In some
embodiments, exemplary polyesters include, for example,
polyhydroxyacids; PEGylated polymers and copolymers of lactide and
glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA, and
derivatives thereof. In some embodiments, polyesters include, for
example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated
poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine
ester), poly(4-hydroxy-L-proline ester),
poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives
thereof.
[0033] In some embodiments, a polymer may be PLGA. PLGA is a
biocompatible and biodegradable co-polymer of lactic acid and
glycolic acid, and various forms of PLGA can be characterized by
the ratio of lactic acid:glycolic acid. Lactic acid can be L-lactic
acid, D-lactic acid, or D,L-lactic acid. The degradation rate of
PLGA can be adjusted by altering the lactic acid-glycolic acid
ratio. In some embodiments, PLGA to be used in accordance with the
present invention can be characterized by a lactic acid:glycolic
acid molar ratio of approximately 85:15, approximately 75:25,
approximately 60:40, approximately 50:50, approximately 40:60,
approximately 25:75, or approximately 15:85.
[0034] In some embodiments, the ratio of lactic acid to glycolic
acid monomers in the polymer of the particle (e.g., the PLGA block
copolymer or PLGA-PEG block copolymer), may be selected to optimize
for various parameters such as water uptake, therapeutic agent
release and/or polymer degradation kinetics can be optimized.
[0035] In some embodiments, polymers may be one or more acrylic
polymers. In certain embodiments, acrylic polymers include, for
example, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl
methacrylate, amino alkyl methacrylate copolymer, poly(acrylic
acid), poly(methacrylic acid), methacrylic acid alkylamide
copolymer, poly(methyl methacrylate), poly(methacrylic acid
polyacrylamide, amino alkyl methacrylate copolymer, glycidyl
methacrylate copolymers, polycyanoacrylates, and combinations
comprising one or more of the foregoing polymers. The acrylic
polymer may comprise fully-polymerized copolymers of acrylic and
methacrylic acid esters with a low content of quaternary ammonium
groups.
[0036] In some embodiments, polymers can be cationic polymers. In
general, cationic polymers are able to condense and/or protect
negatively charged strands of nucleic acids (e.g. DNA, RNA, or
derivatives thereof). Amine-containing polymers such as
poly(lysine), polyethylene imine (PEI), and poly(amidoamine)
dendrimers are contemplated for use, in some embodiments, in a
disclosed particle.
[0037] In some embodiments, polymers can be degradable polyesters
bearing cationic side chains. Examples of these polyesters include
poly(L-lactide-co-L-lysine), poly(serine ester),
poly(4-hydroxy-L-proline ester). A polymer (e.g., copolymer, e.g.,
block copolymer) containing poly(ethylene glycol) repeat units can
also be referred to as a "PEGylated" polymer. Such polymers can
control inflammation and/or immunogenicity (i.e., the ability to
provoke an immune response) and/or lower the rate of clearance from
the circulatory system via the reticuloendothelial system (RES),
due to the presence of the poly(ethylene glycol) groups.
[0038] PEGylation may also be used, in some cases, to decrease
charge interaction between a polymer and a biological moiety, e.g.,
by creating a hydrophilic layer on the surface of the polymer,
which may shield the polymer from interacting with the biological
moiety. In some cases, the addition of poly(ethylene glycol) repeat
units may increase plasma half-life of the polymer (e.g.,
copolymer, e.g., block copolymer), for instance, by decreasing the
uptake of the polymer by the phagocytic system while decreasing
transfection/uptake efficiency by cells. Those of ordinary skill in
the art will know of methods and techniques for PEGylating a
polymer, for example, by using EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and
NHS (N-hydroxysuccinimide) to react a polymer to a PEG group
terminating in an amine, by ring opening polymerization techniques
(ROMP), or the like.
[0039] Particles disclosed herein may or may not contain PEG. In
addition, certain embodiments can be directed towards copolymers
containing poly(ester-ether)s, e.g., polymers having repeat units
joined by ester bonds (e.g., R--C(O)--O--R' bonds) and ether bonds
(e.g., R--O--R' bonds). In some embodiments of the invention, a
biodegradable polymer, such as a hydrolyzable polymer, containing
carboxylic acid groups, may be conjugated with poly(ethylene
glycol) repeat units to form a poly(ester-ether).
[0040] It is contemplated that PEG may include a terminal end
group, for example, when PEG is not conjugated to a ligand. For
example, PEG may terminate in a hydroxyl, a methoxy or other
alkoxyl group, a methyl or other alkyl group, an aryl group, a
carboxylic acid, an amine, an amide, an acetyl group, a guanidino
group, or an imidazole. Other contemplated end groups include
azide, alkyne, maleimide, aldehyde, hydrazide, hydroxylamine,
alkoxyamine, or thiol moieties.
[0041] In one embodiment, the molecular weight of the polymers can
be optimized for effective treatment as disclosed herein. For
example, the molecular weight of a polymer may influence particle
degradation rate (such as when the molecular weight of a
biodegradable polymer can be adjusted), solubility, water uptake,
and drug release kinetics. For example, the molecular weight of the
polymer can be adjusted such that the particle biodegrades in the
subject being treated within a reasonable period of time (ranging
from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks,
etc.). A disclosed particle can for example comprise a copolymer of
PEG and PLGA, the PEG can have a molecular weight of 1,000-20,000,
e.g., 5,000-20,000, e.g., 10,000-20,000, and the PLGA can have a
molecular weight of 5,000-100,000, e.g., 20,000-70,000, e.g.,
20,000-50,000.
[0042] For example, disclosed here is an exemplary therapeutic
nanoparticle that includes about 10 to about 99 weight percent
poly(lactic) acid-poly(ethylene)glycol copolymer or
poly(lactic)-co-poly (glycolic) acid-poly(ethylene)glycol
copolymer, or about 20 to about 80 weight percent, about 40 to
about 80 weight percent, or about 30 to about 50 weight percent, or
about 70 to about 90 weight percent poly(lactic)
acid-poly(ethylene)glycol copolymer or poly(lactic)-co-poly
(glycolic) acid-poly(ethylene)glycol copolymer. Exemplary
poly(lactic) acid-poly(ethylene)glycol copolymers can include a
number average molecular weight of about 15 to about 20 kDa, or
about 10 to about 25 kDa of poly(lactic) acid and a number average
molecular weight of about 4 to about 6, or about 2 kDa to about 10
kDa of poly(ethylene)glycol.
[0043] Disclosed nanoparticles may optionally include about 1 to
about 50 weight percent poly(lactic) acid or poly(lactic)
acid-co-poly (glycolic) acid (which does not include PEG, e.g a
homopolymer of PLA), or may optionally include about 1 to about 50
weight percent, or about 10 to about 50 weight percent or about 30
to about 50 weight percent poly(lactic) acid or poly(lactic)
acid-co-poly (glycolic) acid. For example, poly(lactic) or
poly(lactic)-co-poly(glycolic) acid may have a number average
molecule weight of about 5 to about 15 kDa, or about 5 to about 12
kDa. Exemplary homopolymeric PLA may have a number average
molecular weight of about 5 to about 10 kDa. Exemplary PLGA may
have a number average molecular weight of about 8 to about 12
kDa.
[0044] In certain embodiments, disclosed polymers of may be
conjugated to a lipid, e.g. "end-capped," for example, may include
a lipid-terminated PEG. As described below, the lipid portion of
the polymer can be used for self assembly with another polymer,
facilitating the formation of a nanoparticle. For example, a
hydrophilic polymer could be conjugated to a lipid that will self
assemble with a hydrophobic polymer.
[0045] Exemplary lipids include fatty acids such as long chain
(e.g., C.sub.8-C.sub.50), substituted or unsubstituted
hydrocarbons. In some embodiments, a fatty acid group can be a
C.sub.10-C.sub.20 fatty acid or salt thereof. In some embodiments,
a fatty acid group can be a C.sub.15-C.sub.20 fatty acid or salt
thereof. In some embodiments, a fatty acid can be unsaturated,
monounsaturated, or polyunsaturated. For example, a fatty acid
group can be one or more of butyric, caproic, caprylic, capric,
lauric, myristic, palmitic, stearic, arachidic, behenic, or
lignoceric acid. In some embodiments, a fatty acid group can be one
or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic,
gamma-linoleic, arachidonic, gadoleic, arachidonic,
eicosapentaenoic, docosahexaenoic, or erucic acid.
[0046] In a particular embodiment, the lipid is of the Formula
V:
##STR00001##
and salts thereof, wherein each R is, independently, C.sub.1-30
alkyl. In one embodiment of Formula V, the lipid is 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and salts
thereof, e.g., the sodium salt.
[0047] In one embodiment, optional small molecule targeting
moieties are bonded, e.g., covalently bonded, to the lipid
component of the nanoparticle. For example, contemplated herein is
also a nanoparticle comprising a therapeutic agent, a polymeric
matrix comprising functionalized and non-functionalized polymers, a
lipid, and a low-molecular weight targeting ligand, wherein the
targeting ligand is bonded, e.g., covalently bonded, to the lipid
component of the nanoparticle.
Targeting Moieties
[0048] Provided herein are nanoparticles that may include an
optional targeting moiety, i.e., a moiety able to bind to or
otherwise associate with a biological entity, for example, a
membrane component, a cell surface receptor, prostate specific
membrane antigen, or the like. A targeting moiety present on the
surface of the particle may allow the particle to become localized
at a particular targeting site, for instance, a tumor, a disease
site, a tissue, an organ, a type of cell, etc. The drug or other
payload may then, in some cases, be released from the particle and
allowed to interact locally with the particular targeting site.
[0049] In one embodiment of the instant invention, the targeting
moiety may be a low-molecular weight ligand, e.g., a low-molecular
weight PSMA ligand. For example, a targeting portion may cause the
particles to become localized to a tumor, a disease site, a tissue,
an organ, a type of cell, etc. within the body of a subject,
depending on the targeting moiety used. For example, a
low-molecular weight PSMA ligand may become localized to prostate
cancer cells. The subject may be a human or non-human animal.
Examples of subjects include, but are not limited to, a mammal such
as a dog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a
sheep, a goat, a rat, a mouse, a guinea pig, a hamster, a primate,
a human or the like.
[0050] Contemplated targeting moieties include small molecules. In
certain embodiments, the term "small molecule" refers to organic
compounds, whether naturally-occurring or artificially created
(e.g., via chemical synthesis) that have relatively low molecular
weight and that are not proteins, polypeptides, or nucleic acids.
Small molecules typically have multiple carbon-carbon bonds. In
certain embodiments, small molecules are less than about 2000 g/mol
in size. In some embodiments, small molecules are less than about
1500 g/mol or less than about 1000 g/mol. In some embodiments,
small molecules are less than about 800 g/mol or less than about
500 g/mol, for example about 100 g/mol to about 600 g/mol, or about
200 g/mol to about 500 g/mol. For example, a ligand may be a the
low-molecular weight PSMA ligand such as
##STR00002##
and enantiomers, stereoisomers, rotamers, tautomers, diastereomers,
or racemates thereof.
[0051] In some embodiments, small molecule targeting moieties that
may be used to target cells associated with prostate cancer tumors
include PSMA peptidase inhibitors such as 2-PMPA, GPI5232, VA-033,
phenylalkylphosphonamidates and/or analogs and derivatives thereof.
In some embodiments, small molecule targeting moieties that may be
used to target cells associated with prostate cancer tumors include
thiol and indole thiol derivatives, such as 2-MPPA and
3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives. In
some embodiments, small molecule targeting moieties that may be
used to target cells associated with prostate cancer tumors include
hydroxamate derivatives. In some embodiments, small molecule
targeting moieties that may be used to target cells associated with
prostate cancer tumors include PBDA- and urea-based inhibitors,
such as ZJ 43, ZJ 11, ZJ 17, ZJ 38 and/or and analogs and
derivatives thereof, androgen receptor targeting agents (ARTAs),
polyamines, such as putrescine, spermine, and spermidine,
inhibitors of the enzyme glutamate carboxylase II (GCPII), also
known as NAAG Peptidase or NAALADase.
[0052] In another embodiment of the instant invention, the
targeting moiety can be a ligand that targets Her2, EGFR, or toll
receptors. For example, contemplated the targeting moieties may
include a nucleic acid, polypeptide, glycoprotein, carbohydrate, or
lipid. For example, a targeting moiety can be a nucleic acid
targeting moiety (e.g. an aptamer, e.g., the A10 aptamer) that
binds to a cell type specific marker. In general, an aptamer is an
oligonucleotide (e.g., DNA, RNA, or an analog or derivative
thereof) that binds to a particular target, such as a polypeptide.
In some embodiments, a targeting moiety may be a naturally
occurring or synthetic ligand for a cell surface receptor, e.g., a
growth factor, hormone, LDL, transferrin, etc. A targeting moiety
can be an antibody, which term is intended to include antibody
fragments, characteristic portions of antibodies, single chain
targeting moieties can be identified, e.g., using procedures such
as phage display. Targeting moieties may be a targeting peptide or
targeting peptidomimetic has a length of up to about 50 residues.
For example, a targeting moieties may include the amino acid
sequence AKERC, CREKA, ARYLQKLN or AXYLZZLN, wherein X and Z are
variable amino acids, or conservative variants or peptidomimetics
thereof. In particular embodiments, the targeting moiety is a
peptide that includes the amino acid sequence AKERC, CREKA,
ARYLQKLN or AXYLZZLN, wherein X and Z are variable amino acids, and
has a length of less than 20, 50 or 100 residues. The CREKA (Cys
Arg Glu Lys Ala) peptide or a peptidomimetic thereof peptide or the
octapeptide AXYLZZLN are also contemplated as targeting moieties,
as well as peptides, or conservative variants or peptidomimetics
thereof, that binds or forms a complex with collagen IV, or the
targets tissue basement membrane (e.g., the basement membrane of a
blood vessel), can be used as a targeting moiety.
[0053] Exemplary targeting moieties include peptides that target
ICAM (intercellular adhesion molecule, e.g. ICAM-1).
[0054] Targeting moieties disclosed herein are typically conjugated
to a disclosed polymer or copolymer (e.g. PLA-PEG), and such a
polymer conjugate may form part of a disclosed nanoparticle. For
example, a disclosed therapeutic nanoparticle may optionally
include about 0.2 to about 10 weight percent of a PLA-PEG or
PLGA-PEG, wherein the PEG is functionalized with a targeting
ligand. Contemplated therapeutic nanoparticles may include, for
example, about 0.2 to about 10 mole percent PLA-PEG-ligand or poly
(lactic) acid-co poly (glycolic) acid-PEG-ligand. For example,
PLA-PEG-ligand may include a PLA with a number average molecular
weight of about 10 kDa to about 20 kDa and PEG with a number
average molecular weight of about 4,000 to about 8,000.
Nanoparticles
[0055] Disclosed nanoparticles may have a substantially spherical
(i.e., the particles generally appear to be spherical), or
non-spherical configuration. For instance, the particles, upon
swelling or shrinkage, may adopt a non-spherical configuration. In
some cases, the particles may include polymeric blends. For
instance, a polymer blend may include a first co-polymer that
includes polyethylene glycol and a second polymer.
[0056] Disclosed nanoparticles may have a characteristic dimension
of less than about 1 micrometer, where the characteristic dimension
of a particle is the diameter of a perfect sphere having the same
volume as the particle. For example, the particle can have a
characteristic dimension of the particle can be less than about 300
nm, less than about 200 nm, less than about 150 nm, less than about
100 nm, less than about 50 nm, less than about 30 nm, less than
about 10 nm, less than about 3 nm, or less than about 1 nm in some
cases. In particular embodiments, disclosed nanoparticles may have
a diameter of about 70 nm-200 nm, or about 70 nm to about 180 nm,
about 80 nm to about 130 nm, about 80 nm to about 120 nm.
[0057] In one set of embodiments, the particles can have an
interior and a surface, where the surface has a composition
different from the interior, i.e., there may be at least one
compound present in the interior but not present on the surface (or
vice versa), and/or at least one compound is present in the
interior and on the surface at differing concentrations. For
example, in one embodiment, a compound, such as a targeting moiety
(i.e., a low-molecular weight ligand) of a polymeric conjugate of
the present invention, may be present in both the interior and the
surface of the particle, but at a higher concentration on the
surface than in the interior of the particle, although in some
cases, the concentration in the interior of the particle may be
essentially nonzero, i.e., there is a detectable amount of the
compound present in the interior of the particle.
[0058] In some cases, the interior of the particle is more
hydrophobic than the surface of the particle. For instance, the
interior of the particle may be relatively hydrophobic with respect
to the surface of the particle, and a drug or other payload may be
hydrophobic, and readily associates with the relatively hydrophobic
center of the particle. The drug or other payload can thus be
contained within the interior of the particle, which can shelter it
from the external environment surrounding the particle (or vice
versa). For instance, a drug or other payload contained within a
particle administered to a subject will be protected from a
subject's body, and the body may also be substantially isolated
from the drug for at least a period of time.
[0059] For example, disclosed herein is a therapeutic polymeric
nanoparticle comprising a first non-functionalized polymer; an
optional second non-functionalized polymer; an optional
functionalized polymer comprising a targeting moiety; and a
therapeutic agent, In a particular embodiment, the first
non-functionalized polymer is PLA, PLGA, or PEG, or copolymers
thereof, e.g. a diblock co-polymer PLA-PEG. For example, exemplary
nanoparticle may have a PEG corona with a density of about 0.065
g/cm.sup.3, or about 0.01 to about 0.10 g/cm.sup.3.
[0060] Disclosed nanoparticles may be stable (e.g. retain
substantially all active agent) for example in a solution that may
contain a saccharide, for at least about 3 days, about 4 days or at
least about 5 days at room temperature, or at 25.degree. C.
[0061] In some embodiments, disclosed nanoparticles may also
include a fatty alcohol, which may increase the rate of drug
release. For example, disclosed nanoparticles may include a
C.sub.8-C.sub.30 alcohol such as cetyl alcohol, octanol, stearyl
alcohol, arachidyl alcohol, docosonal, or octasonal.
[0062] Nanoparticles may have controlled release properties, e.g.,
may be capable of delivering an amount of active agent to a
patient, e.g., to specific site in a patient, over an extended
period of time, e.g. over 1 day, 1 week, or more. In some
embodiments, disclosed nanoparticles substantially immediately
releases (e.g. over about 1 minute to about 30 minutes) less than
about 2%, less than about 4%, less than about 5%, or less than
about 10% of an active agent (e.g. a taxane) agent, for example
when places in a phosphate buffer solution at room temperature
and/or at 37.degree. C.
[0063] In one embodiment, the invention comprises a nanoparticle
comprising 1) a polymeric matrix and 2) an amphiphilic compound or
layer that surrounds or is dispersed within the polymeric matrix
forming a continuous or discontinuous shell for the particle, An
amphiphilic layer can reduce water penetration into the
nanoparticle, thereby enhancing drug encapsulation efficiency and
slowing drug release. Further, these amphipilic layer protected
nanoparticles can provide therapeutic advantages by releasing the
encapsulated drug and polymer at appropriate times.
[0064] As used herein, the term "amphiphilic" refers to a property
where a molecule has both a polar portion and a non-polar portion.
Often, an amphiphilic compound has a polar head attached to a long
hydrophobic tail. In some embodiments, the polar portion is soluble
in water, while the non-polar portion is insoluble in water. In
addition, the polar portion may have either a formal positive
charge, or a formal negative charge. Alternatively, the polar
portion may have both a formal positive and a negative charge, and
be a zwitterion or inner salt. Exemplary amphiphilic compound
include, for example, one or a plurality of the following:
naturally derived lipids, surfactants, or synthesized compounds
with both hydrophilic and hydrophobic moieties.
[0065] Specific examples of amphiphilic compounds include, but are
not limited to, phospholipids, such as 1,2
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcholine (DBPC),
ditricosanoylphosphatidylcholine (DTPC), and
dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of
between 0.01-60 (weight lipid/w polymer), most preferably between
0.1-30 (weight lipid/w polymer). Phospholipids which may be used
include, but are not limited to, phosphatidic acids, phosphatidyl
cholines with both saturated and unsaturated lipids, phosphatidyl
ethanolamines, phosphatidylglycerols, phosphatidylserines,
phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin,
and .beta.-acyl-y-alkyl phospholipids. Examples of phospholipids
include, but are not limited to, phosphatidylcholines such as
dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,
dipentadecanoylphosphatidylcholine dilauroylphosphatidylcholine,
dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC),
diarachidoylphosphatidylcholine (DAPC),
dibehenoylphosphatidylcho-line (DBPC),
ditricosanoylphosphatidylcholine (DTPC),
dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines
such as dioleoylphosphatidylethanolamine or
1-hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic
phospholipids with asymmetric acyl chains (e.g., with one acyl
chain of 6 carbons and another acyl chain of 12 carbons) may also
be used.
[0066] In a particular embodiment, an amphiphilic component may
include lecithin, and/or in particular, phosphatidylcholine.
Preparation of Nanoparticles
[0067] Another aspect of the invention is directed to systems and
methods of making disclosed nanoparticles. In some embodiments,
using two or more different polymers (e.g., a copolymer such as a
diblock copolymer and a homopolymer) properties of particles may be
controlled.
[0068] In a particular embodiment, the methods described herein
form nanoparticles that have a high amount of encapsulated
therapeutic agent, for example, may include about 1 to about 40
weight percent, or about 1 to about 30 weight percent, e.g. about
10 to about 25 weight percent or about 5 to about 20 weight percent
therapeutic agent.
[0069] In an embodiment, a nanoemulsion process is provided, such
as the process represented in FIGS. 1 and 2. For example, a
therapeutic agent, a first polymer (for example, PLA-PEG or
PLGA-PEG) and a second polymer (e.g. (PL(G)A or PLA), with an
organic solution to form a first organic phase. Such first phase
may include about 5 to about 50% weight solids, e.g about 5 to
about 40% solids, or about 10 to about 30% solids, e.g. about 10%,
15%, 20% solids. The first organic phase may be combined with a
first aqueous solution to form a second phase. The organic solution
can include, for example, acetonitrile, tetrahydrofuran, ethyl
acetate, isopropyl alcohol, isopropyl acetate, dimethylformamide,
methylene chloride, dichloromethane, chloroform, acetone, benzyl
alcohol, Tween 80, Span 80, or the like, and combinations thereof.
In an embodiment, the organic phase may include benzyl alcohol,
ethyl acetate, and combinations thereof. The second phase can be
between about 1 and 50 weight %, e.g., 5-40 weight %, solids. The
aqueous solution can be water, optionally in combination with one
or more of sodium cholate, ethyl acetate, and benzyl alcohol.
[0070] For example, the oil or organic phase may use solvent that
is only partially miscible with the nonsolvent (water). Therefore,
when mixed at a low enough ratio and/or when using water
pre-saturated with the organic solvents, the oil phase remains
liquid. The oil phase may bee emulsified into an aqueous solution
and, as liquid droplets, sheared into nanoparticles using, for
example, high energy dispersion systems, such as homogenizers or
sonicators. The aqueous portion of the emulsion, otherwise known as
the "water phase", may be surfactant solution consisting of sodium
cholate and pre-saturated with ethyl acetate and benzyl
alcohol.
[0071] Emulsifying the second phase to form an emulsion phase may
be performed in one or two emulsification steps. For example, a
primary emulsion may be prepared, and then emulsified to form a
fine emulsion. The primary emulsion can be formed, for example,
using simple mixing, a high pressure homogenizer, probe sonicator,
stir bar, or a rotor stator homogenizer. The primary emulsion may
be formed into a fine emulsion through the use of e.g. probe
sonicator or a high pressure homogenizer, e.g. by using 1, 2, 3 or
more passes through a homogenizer. For example, when a high
pressure homogenizer is used, the pressure used may be about 4000
to about 8000 psi, or about 4000 to about 5000 psi, e.g. 4000 or
5000 psi.
[0072] Either solvent evaporation or dilution may be needed to
complete the extraction of the solvent and solidify the particles.
For better control over the kinetics of extraction and a more
scalable process, a solvent dilution via aqueous quench may be
used. For example, the emulsion can be diluted into cold water to a
concentration sufficient to dissolve all of the organic solvent to
form a quenched phase. Quenching may be performed at least
partially at a temperature of about 5.degree. C. or less. For
example, water used in the quenching may be at a temperature that
is less that room temperature (e.g. about 0 to about 10.degree. C.,
or about 0 to about 5.degree. C.).
[0073] In some embodiments, not all of the therapeutic agent is
encapsulated in the particles at this stage, and a drug solubilizer
is added to the quenched phase to form a solubilized phase. The
drug solubilizer may be for example, Tween 80, Tween 20, polyvinyl
pyrrolidone, cyclodextran, sodium dodecyl sulfate, or sodium
cholate. For example, Tween-80 may added to the quenched
nanoparticle suspension to solubilize the free drug and prevent the
formation of drug crystals. In some embodiments, a ratio of drug
solubilizer to therapeutic agent is about 100:1 to about 10:1.
[0074] The solubilized phase may be filtered to recover the
nanoparticles. For example, ultrafiltration membranes may be used
to concentrate the nanoparticle suspension and substantially
eliminate organic solvent, free drug, and other processing aids
(surfactants). Exemplary filtration may be performed using a
tangential flow filtration system. For example, by using a membrane
with a pore size suitable to retain nanoparticles while allowing
solutes, micelles, and organic solvent to pass, nanoparticles can
be selectively separated. Exemplary membranes with molecular weight
cut-offs of about 300-500 kDa (-5-25 nm) may be used.
[0075] Diafiltration may be performed using a constant volume
approach, meaning the diafiltrate (cold deionized water, e.g. about
0.degree. C. to about 5.degree. C., or 0 to about 10.degree. C.)
may added to the feed suspension at the same rate as the filtrate
is removed from the suspension. In some embodiments, filtering may
include a first filtering using a first temperature of about 0 to
about 5.degree. C., or 0.degree. C. to about 10.degree. C., and a
second temperature of about 20.degree. C. to about 30.degree. C.,
or 15.degree. C. to about 35.degree. C. For example, filtering may
include processing about 1 to about 6 diavolumes at about 0 to
about 5.degree. C., and processing at least one diavolume (e.g.
about 1 to about 3 or about 1-2 diavolumes) at about 20.degree. C.
to about 30.degree. C.
[0076] After purifying and concentrating the nanoparticle
suspension, the particles may be passed through one, two or more
sterilizing and/or depth filters, for example, using .about.0.2
.mu.m depth pre-filter.
[0077] In exemplary embodiment of preparing nanoparticles, an
organic phase is formed composed of a mixture of a therapeutic
agent, e.g., sirolimus, and polymer (homopolymer, and co-polymer).
The organic phase may be mixed with an aqueous phase at
approximately a 1:5 ratio (oil phase:aqueous phase) where the
aqueous phase is composed of a surfactant and optionally dissolved
solvent. A primary emulsion may then formed by the combination of
the two phases under simple mixing or through the use of a rotor
stator homogenizer. The primary emulsion is then formed into a fine
emulsion through the use of e.g. high pressure homogenizer. Such
fine emulsion may then quenched by, e.g. addition to deionized
water under mixing. An exemplary quench:emulsion ratio may be about
approximately 8.5:1. A solution of Tween (e.g., Tween 80) can then
be added to the quench to achieve e.g. approximately 2% Tween
overall, which may serves to dissolve free, unencapsulated drug.
Formed nanoparticles may then be isolated through either
centrifugation or ultrafiltration/diafiltration.
Therapeutic Agents
[0078] According to the present invention, any agents including,
for example, therapeutic agents (e.g. anti-cancer agents),
diagnostic agents (e.g. contrast agents; radionuclides; and
fluorescent, luminescent, and magnetic moieties), prophylactic
agents (e.g. vaccines), and/or nutraceutical agents (e.g. vitamins,
minerals, etc.) may be delivered by the disclosed nanoparticles.
Exemplary agents to be delivered in accordance with the present
invention include, but are not limited to, small molecules (e.g.
cytotoxic agents), nucleic acids (e.g., siRNA, RNAi, and microRNA
agents), proteins (e.g. antibodies), peptides, lipids,
carbohydrates, hormones, metals, radioactive elements and
compounds, drugs, vaccines, immunological agents, etc., and/or
combinations thereof. In some embodiments, the agent to be
delivered is an agent useful in the treatment of cancer (e.g., an
anti-neoplastic agent).
[0079] In a particular embodiment, the drug may be released in a
controlled release manner from the particle and allowed to interact
locally with the particular patient site (e.g., a tumor). The term
"controlled release" is generally meant to encompass release of a
substance (e.g., a drug) at a selected site or otherwise
controllable in rate, interval, and/or amount. Controlled release
encompasses, but is not necessarily limited to, substantially
continuous delivery, patterned delivery (e.g., intermittent
delivery over a period of time that is interrupted by regular or
irregular time intervals), and delivery of a bolus of a selected
substance (e.g., as a predetermined, discrete amount if a substance
over a relatively short period of time (e.g., a few seconds or
minutes)).
[0080] The active agent or drug may be a therapeutic agent such as
an mTOR (mammalian target of rapamycin) inhibitor such as sirolimus
(rapamycin), temsirolimus, or everolimus, a taxane or diterpene
derivative such as paclitaxel (or its derivatives such as
DHA-paclitaxel or PG-paxlitaxel) or docetaxel. In another
embodiment, the active agent or drug may be a vinca alkaloid such
as vinorelbine, vinblastine, vincristine, or vindesine.
Pharmaceutical Formulations
[0081] Nanoparticles disclosed herein may be combined with
pharmaceutical acceptable carriers to form a pharmaceutical
composition. As would be appreciated by one of skill in this art,
the carriers may be chosen based on the route of administration as
described below, the location of the target issue, the drug being
delivered, the time course of delivery of the drug, etc.
[0082] The pharmaceutical compositions and particles disclosed
herein can be administered to a patient by any means known in the
art including oral and parenteral routes. The term "patient," as
used herein, refers to humans as well as non-humans, including, for
example, mammals, birds, reptiles, amphibians, and fish. For
instance, the non-humans may be mammals (e.g., a rodent, a mouse, a
rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In
certain embodiments parenteral routes are desirable since they
avoid contact with the digestive enzymes that are found in the
alimentary canal. According to such embodiments, inventive
compositions may be administered by injection (e.g., intravenous,
subcutaneous or intramuscular, intraperitoneal injection),
rectally, vaginally, topically (as by powders, creams, ointments,
or drops), or by inhalation (as by sprays).
[0083] In a particular embodiment, disclosed nanoparticles may be
administered to a subject in need thereof systemically, e.g., by IV
infusion or injection.
[0084] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables. In one
embodiment, the inventive conjugate is suspended in a carrier fluid
comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)
TWEEN.TM. 80. The injectable formulations can be sterilized, for
example, by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0085] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the encapsulated or unencapsulated conjugate is mixed with at least
one inert, pharmaceutically acceptable excipient or carrier such as
sodium citrate or dicalcium phosphate and/or (a) fillers or
extenders such as starches, lactose, sucrose, glucose, mannitol,
and silicic acid, (b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,
sucrose, and acacia, (c) humectants such as glycerol, (d)
disintegrating agents such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, (e) solution retarding agents such as paraffin, (f)
absorption accelerators such as quaternary ammonium compounds, (g)
wetting agents such as, for example, cetyl alcohol and glycerol
monostearate, (h) absorbents such as kaolin and bentonite clay, and
(i) lubricants such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof. In the case of capsules, tablets, and pills, the dosage
form may also comprise buffering agents.
[0086] Disclosed nanoparticles may be formulated in dosage unit
form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of nanoparticle appropriate for the patient to be
treated. For any nanoparticle, the therapeutically effective dose
can be estimated initially either in cell culture assays or in
animal models, usually mice, rabbits, dogs, or pigs. An animal
model may also used to achieve a desirable concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
Therapeutic efficacy and toxicity of nanoparticles can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED.sub.50 (the dose is
therapeutically effective in 50% of the population) and LD.sub.50
(the dose is lethal to 50% of the population). The dose ratio of
toxic to therapeutic effects is the therapeutic index, and it can
be expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions which exhibit large therapeutic indices may be useful
in some embodiments. The data obtained from cell culture assays and
animal studies can be used in formulating a range of dosage for
human use.
[0087] In an exemplary embodiment, a pharmaceutical composition is
disclosed that includes a plurality of nanoparticles each
comprising a therapeutic agent; and a pharmaceutically acceptable
excipient.
[0088] In some embodiments, a composition suitable for freezing is
comtemplated, including nanoparticles disclosed herein and a
solution suitable for freezing, e.g., a sugar (e.g. sucrose)
solution is added to a nanoparticle suspension. The sucrose may
e.g., act as a cryoprotectant to prevent the particles from
aggregating upon freezing. For example, provided herein is a
nanoparticle formulation comprising a plurality of disclosed
nanoparticles, sucrose and water; wherein, for example, the
nanoparticles/sucrose/water is are present with about
5-10%/10-15%/80-90% (w/w/w).
Methods of Treatment
[0089] In some embodiments, therapeutic particles disclosed herein
may be used to treat, alleviate, ameliorate, relieve, delay onset
of, inhibit progression of, reduce severity of, and/or reduce
incidence of one or more symptoms or features of a disease,
disorder, and/or condition. For example, disclosed therapeutic
particles, that include e.g., temsirolimus may be used to treat
renal cell carcinoma. In another embodiment, disclosed therapeutic
particles that include e.g. everolimus or temsirolimus may be used
to treat kidney cancer, glioblastoma multiforme, mantle cell
lymphoma, or dermal Kaposi's sarcoma.
[0090] Also contemplated here are methods of treating patients that
have been subject to organ transplantation, by administering
disclosed nanoparticles that e.g. include sirolimus. Other methods
contemplated herein include methods of treating patients having
tuberous sclerosis complex, and/or autism by administering an
effective amount of a disclosed nanoparticle.
[0091] Methods contemplated herein include, for example, a method
of preventing or deterring neointimal hyperplasia in a blood vessel
of a patient, for example, a patient receiving a bare metal stent
in a lesion of the blood vessel, is disclosed, comprising
administering a composition comprising disclosed therapeutic
particles such as those that include sirolimus or everolimus. Also
contemplated herein are methods of treating or preventing
restenosis (e.g. in a patient receiving a stent) comprising
administering disclosed nanoparticles having e.g. sirolimus or
everolimus to a patient.
[0092] Disclosed treatment methods may comprise administering a
therapeutically effective amount of the disclosed therapeutic
particles to a subject in need thereof, in such amounts and for
such time as is necessary to achieve the desired result. In certain
embodiments of the present invention a "therapeutically effective
amount" is that amount effective for treating, alleviating,
ameliorating, relieving, delaying onset of, inhibiting progression
of, reducing severity of, and/or reducing incidence of one or more
symptoms or features of e.g. a cancer being treated.
[0093] Also provided herein are therapeutic protocols that include
administering a therapeutically effective amount of an disclosed
therapeutic particle to a healthy individual (i.e., a subject who
does not display any symptoms of cancer and/or who has not been
diagnosed with cancer). For example, healthy individuals may be
"immunized" with an inventive targeted particle prior to
development of cancer and/or onset of symptoms of cancer; at risk
individuals (e.g., patients who have a family history of cancer;
patients carrying one or more genetic mutations associated with
development of cancer; patients having a genetic polymorphism
associated with development of cancer; patients infected by a virus
associated with development of cancer; patients with habits and/or
lifestyles associated with development of cancer; etc.) can be
treated substantially contemporaneously with (e.g., within 48
hours, within 24 hours, or within 12 hours of) the onset of
symptoms of cancer. Of course individuals known to have cancer may
receive inventive treatment at any time.
[0094] In other embodiments, disclosed nanoparticles may be used to
inhibit the growth of cancer cells, e.g., prostate cancer cells. As
used herein, the term "inhibits growth of cancer cells" or
"inhibiting growth of cancer cells" refers to any slowing of the
rate of cancer cell proliferation and/or migration, arrest of
cancer cell proliferation and/or migration, or killing of cancer
cells, such that the rate of cancer cell growth is reduced in
comparison with the observed or predicted rate of growth of an
untreated control cancer cell. The term "inhibits growth" can also
refer to a reduction in size or disappearance of a cancer cell or
tumor, as well as to a reduction in its metastatic potential.
Preferably, such an inhibition at the cellular level may reduce the
size, deter the growth, reduce the aggressiveness, or prevent or
inhibit metastasis of a cancer in a patient. Those skilled in the
art can readily determine, by any of a variety of suitable indicia,
whether cancer cell growth is inhibited.
[0095] Inhibition of cancer cell growth may be evidenced, for
example, by arrest of cancer cells in a particular phase of the
cell cycle, e.g., arrest at the G2/M phase of the cell cycle
Inhibition of cancer cell growth can also be evidenced by direct or
indirect measurement of cancer cell or tumor size. In human cancer
patients, such measurements generally are made using well known
imaging methods such as magnetic resonance imaging, computerized
axial tomography and X-rays. Cancer cell growth can also be
determined indirectly, such as by determining the levels of
circulating carcinoembryonic antigen, prostate specific antigen or
other cancer-specific antigens that are correlated with cancer cell
growth. Inhibition of cancer growth is also generally correlated
with prolonged survival and/or increased health and well-being of
the subject.
EXAMPLES
[0096] The invention now being generally described, it will be more
readily understood by reference to the following examples which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention in any way.
Example 1
Preparation of PLA-PEG
[0097] The synthesis is accomplished by ring opening polymerization
of d,l-lactide with .alpha.-hydroxy-.omega.-methoxypoly(ethylene
glycol) as the macro-initiator, and performed at an elevated
temperature using Tin (II) 2-Ethyl hexanoate as a catalyst, as
shown below (PEG Mn.apprxeq.5,000 Da; PLA Mn.apprxeq.16,000 Da;
PEGPLA M.sub.n.apprxeq.21,000 Da)
##STR00003##
[0098] The polymer is purified by dissolving the polymer in
dichloromethane, and precipitating it in a mixture of hexane and
diethyl ether. The polymer recovered from this step shall be dried
in an oven.
Example 2
Nanoparticle Preparation--Emulsion Process
[0099] An organic phase is formed composed of a mixture of
sirolimus and polymer (homopolymer, co-polymer, and co-polymer with
ligand). The organic phase is mixed with an aqueous phase at
approximately a 1:5 ratio (oil phase:aqueous phase) where the
aqueous phase is composed of a surfactant and some dissolved
solvent. In order to achieve high drug loading, about 30% solids in
the organic phase is used.
[0100] The primary, coarse emulsion is formed by the combination of
the two phases under simple mixing or through the use of a rotor
stator homogenizer. The rotor/stator yielded a homogeneous milky
solution, while the stir bar produced a visibly larger coarse
emulsion. It was observed that the stir bar method resulted in
significant oil phase droplets adhering to the side of the feed
vessel, suggesting that while the coarse emulsion size is not a
process parameter critical to quality, it should be made suitably
fine in order to prevent yield loss or phase separation. Therefore
the rotor stator is used as the standard method of coarse emulsion
formation, although a high speed mixer may be suitable at a larger
scale.
[0101] The primary emulsion is then formed into a fine emulsion
through the use of a high pressure homogenizer. The size of the
coarse emulsion does not significantly affect the particle size
after successive passes (1-3) through the homogenizer. M-110-EH
(FIG. 3).
[0102] Homogenizer feed pressure was found to have a significant
impact on resultant particle size. On both the pneumatic and
electric M-110EH homogenizers, it was found that reducing the feed
pressure also reduced the particle size (FIG. 4). Therefore the
standard operating pressure used for the M-110EH is 4000-5000 psi
per interaction chamber, which is the minimum processing pressure
on the unit. The M-110EH also has the option of one or two
interaction chambers. It comes standard with a restrictive
Y-chamber, in series with a less restrictive 200 .mu.m Z-chamber.
It was found that the particle size was actually reduced when the
Y-chamber was removed and replaced with a blank chamber.
Furthermore, removing the Y-chamber significantly increases the
flow rate of emulsion during processing.
[0103] After 2-3 passes the particle size was not significantly
reduced, and successive passes can even cause a particle size
increase. The results are summarized in FIG. 5.
[0104] The effect of scale on particle size showed surprising scale
dependence. The trend shows that in the 2-10 g batch size range,
larger batches produce smaller particles. It has been demonstrated
that this scale dependence is eliminated when considering greater
than 10 g scale batches. The amount of solids used in the oil phase
was about 30%. FIG. 6 depicts the effect of solids concentration on
particle size.
[0105] Table A summarizes the emulsification process
parameters.
TABLE-US-00001 TABLE A Parameter Value Observation Coarse emulsion
Rotor stator Coarse emulsion size does not affect final particle
size, but large formation homogenizer coarse emulsion can cause
increased oil phase retention in feed vessel Homogenizer feed
4000-5000 psi per Lower pressure reduces particle size pressure
chamber Interaction 2 .times. 200 .mu.m Z- 200 .mu.m Z-chamber
yields the smallest particle size, and allows for chamber(s)
chamber highest homogenizer throughput Number of 2-3 passes Studies
have shown that the particle size is not significantly homogenizer
passes reduced after 2 discreet passes, and size can even increase
with successive passes Water phase 0.1% [Sodium cholate] can
effectively alter particle size; value is [sodium cholate]
optimized for given process and formulation W:O ratio 5:1 Lowest
ratio without significant particle size increase is ~5:1 [Solids]
in oil phase 30% Increased process efficiency, increased drug
encapsulation, workable viscosity
[0106] The fine emulsion is then quenched by addition to deionized
water at a given temperature under mixing. In the quench unit
operation, the emulsion is added to a cold aqueous quench under
agitation. This serves to extract a significant portion of the oil
phase solvents, effectively hardening the nanoparticles for
downstream filtration. Chilling the quench significantly improved
drug encapsulation. The quench:emulsion ratio is approximately
5:1.
[0107] A solution of 35% (wt %) of Tween 80 is added to the quench
to achieve approximately 2% Tween 80 overall After the emulsion is
quenched a solution of Tween-80 is added which acts as a drug
solubilizer, allowing for effective removal of unencapsulated drug
during filtration. Table B indicates each of the quench process
parameters.
TABLE-US-00002 TABLE B Summary quench process parameters. Parameter
Value Observation Initial quench <5.degree. C. Low temperature
yields higher drug encapsulation temperature [Tween-80] solution
35% Highest concentration that can be prepared and readily
disperses in quench Tween-80:drug ratio 25:1 Minimum amount of
Tween-80 required to effectively remove unencapsulated drug Q:E
ratio 5:1 Minimum Q:E ratio while retaining high drug encapsulation
Quench .ltoreq.5.degree. C. (with Temperature which prevents
significant drug leaching during hold/processing temp current 5:1
Q:E quench hold time and initial concentration step ratio, 25:1
Tween- 80:drug ratio)
[0108] The temperature must remain cold enough with a dilute enough
suspension (low enough concentration of solvents) to remain below
the T.sub.g of the particles. If the Q:E ratio is not high enough,
then the higher concentration of solvent plasticizes the particles
and allows for drug leakage. Conversely, colder temperatures allow
for high drug encapsulation at low Q:E ratios (to .about.3:1),
making it possible to run the process more efficiently.
[0109] The nanoparticles are then isolated through a tangential
flow filtration process to concentrate the nanoparticle suspension
and buffer exchange the solvents, free drug, and drug solubilizer
from the quench solution into water. A regenerated cellulose
membrane is used with a molecular weight cutoffs (MWCO) of 300.
[0110] A constant volume diafiltration (DF) is performed to remove
the quench solvents, free drug and Tween-80. To perform a
constant-volume DF, buffer is added to the retentate vessel at the
same rate the filtrate is removed. The process parameters for the
TFF operations are summarized in Table C. Crossflow rate refers to
the rate of the solution flow through the feed channels and across
the membrane. This flow provides the force to sweep away molecules
that can foul the membrane and restrict filtrate flow. The
transmembrane pressure is the force that drives the permeable
molecules through the membrane.
TABLE-US-00003 TABLE C TFF Parameters Optimized Parameter Value
Effect Membrane Regenerated No difference in performance between RC
and PES, but Material cellulose - solvent compatibility is superior
for RC. Coarse Screen Membrane Molecular Weight 300 kDa No
difference in NP characteristics (i.e. residual Cut off
tween)Increase in flux rates is seen with 500 kDa membrane but 500
kDa is not available in RC Crossflow Rate 11 L/min/m.sup.2 Higher
crossflow rate led to higher flux Transmembrane 20 psid Open
channel membranes have maximum flux rates Pressure between 10 and
30 psid. Coarse channel membranes have maximum flux rates with min
TMP (~20 psid). Concentration of 30 mg/ml Diafiltration is most
efficient at [NP] ~50 mg/ml with Nanoparticle open channel TFF
membranes based on flux rates and Suspension for throughput. With
coarse channel membranes the flux rate Diafiltration is optimized
at ~30 mg/ml in the starting buffer. Number of .gtoreq.15 (based on
About 15 diavolumes are needed to effectively remove Diavolumes
flux increase) tween-80. End point of diafiltration is determined
by in- process control (flux increase plateau). Membrane Area ~1
m.sup.2/kg Membranes sized based on anticipated flux rates and
volumes required.
[0111] The filtered nanoparticle slurry is then thermal cycled to
an elevated temperature during workup. A small portion (typically
5-10%) of the encapsulated drug is released from the nanoparticles
very quickly after its first exposure to 25.degree. C. Because of
this phenomenon, batches that are held cold during the entire
workup are susceptible to free drug or drug crystals forming during
delivery or any portion of unfrozen storage. By exposing the
nanoparticle slurry to elevated temperature during workup, this
`loosely encapsulated` drug can be removed and improve the product
stability at the expense of a small drop in drug loading. 5
diavolumes is used as the amount for cold processing prior to the
25.degree. C. treatment.
[0112] After the filtration process the nanoparticle suspension is
passed through a sterilizing grade filter (0.2 .mu.m absolute).
Pre-filters are used to protect the sterilizing grade filter in
order to use a reasonable filtration area/time for the process.
Values are as summarized in Table D.
TABLE-US-00004 TABLE D Parameter O Value Effect Nanoparticle 50
mg/ml Yield losses are higher at higher [NP], but the ability to
filter at Suspension 50 mg/ml obviates the need to aseptically
concentrate after Concentration filtration Filtration flow ~1.3
L/min/m.sup.2 Filterability decreases as flow rate increases
rate
[0113] The filtration train is Ertel Alsop Micromedia XL depth
filter M953P membrane (0.2 .mu.m Nominal); Pall SUPRAcap with Seitz
EKSP depth filter media (0.1-0.3 .mu.m Nominal); Pall Life Sciences
Supor EKV 0.65/0.2 micron sterilizing grade PES filter.
[0114] 0.2 m2 of filtration surface area per kg of nanoparticles
for depth filters and 1.3 m2 of filtration surface area per kg of
nanoparticles for the sterilizing grade filters can be used.
Example 3
Cryoprotectant
[0115] Freezing a suspension of nanoemulsion nanoparticles in
deionized water alone results in particle aggregation. This is
believed to be due to crystallization and entanglement of PEG
chains on the nanoparticle surfaces. Sugar-based excipients
(sucrose, trehalose, or mannitol) can act to cryoprotect these
nanoparticles under freeze/thaw conditions, with a concentrations
as low as 1 wt % for dilute (.about.10 mg/ml) nanoparticle
suspensions. One formulation includes 10 wt % sucrose, which
contains excess sucrose to what is required and is the same
osmolality as physiological saline.
[0116] Table E shows that 16/5 PLA-PEG co-polymer is less
susceptible to freeze-thaw aggregation.
TABLE-US-00005 TABLE E Original Post-F/T Post-F/T Median PSD/
Median PS Poly- Post-F/T Description PD (nm) dispersity Baseline
Index 1:1 45/5 and PLA 143.4, 0.124 358.9 0.358 0.0/23.16%
(baseline) 16/5 PLA-PEG and PLA 186.7, 0.080 189.5 0.126 9.7/91.57%
(1:1) 2:1:1 16/5:PLA:cetyl 174.1, 0.084 232.7 0.146 0.0/61.19%
2:1:1 45/5:PLA:cetyl 111.0, 0.182 0 0 0.0/1.55% 16/5 PLA-PEG alone
218.8, 0.098 226.9 0.03 7.3/60.56% 16/5 PLA-PEG and PLA 222.2,
0.126 230.7 0.065 4.1/35.36% (3:1) 45/5 PLGA-PEG and 162.7, 0.099
178.6 0.091 7.7/95.41% PLA (3:1) 2:1:1 45/5 PLA- 115.9, 0.154 734.6
0.392 0.0/13.27% PEG:PLA:cetyl
Example 4
In Vitro Release
[0117] An in vitro release method is used to determine the initial
burst phase release from nanoparticles at both ambient and
37.degree. C. conditions. In order to maintain sink conditions and
prevent nanoparticles from entering the release samples, a dialysis
system was designed. After obtaining an ultracentrifuge capable of
pelleting 100 nm particles, the dialysis membranes were eliminated
and centrifugation was used to separate released drug from
encapsulated drug.
[0118] The dialysis system is as follows: 3 mL slurry of sirolimus
nanoparticles (approx 250 .mu.g/mL sirolimus PLGA/PLA
nanoparticles, corresponding to 2.5 mg/mL solid concentration) in
DI-water is placed into the inner tube of a 300 kDa MWCO dialyzer
by pipetting. The nanoparticle is suspension in this media. The
dialyzer is placed into a glass bottles containing 130 ml release
media (2.5% hydroxyl beta cyclodextrin in PBS), which is
continually stirred at 150 rpm using a shaker to prevent the
formation of an unstirred water layer at the membrane/outer
solution interface. At pre-determined time points, aliquot of
samples (1 mL) were withdrawn from the outer solution (dialysate)
and analyzed for sirolimus concentration by HPLC.
[0119] The centrifugal system is run using similar conditions at
lower suspension volumes without dialysis bags. Samples are
centrifuged at 60,000 g for 30 minutes and the supernatant is
assayed for sirolimus content to measured released sirolimus.
Example 5
Particle Size Analysis
[0120] Particle size is analyzed by two techniques--dynamic light
scattering (DLS) and laser diffraction. DLS is performed using a
Brookhaven ZetaPals instrument at 25.degree. C. in dilute aqueous
suspension using a 660 nm laser scattered at 90.degree. and
analyzed using the Cumulants and NNLS methods (TP008). Laser
diffraction is performed with a Horiba LS950 instrument in dilute
aqueous suspension using both a HeNe laser at 633 nm and an LED at
405 nm, scattered at 90.degree. and analyzed using the Mie optical
model (TP009). The output from the DLS is associated with the
hydrodynamic radius of the particles, which includes the PEG
`corona`, while the laser diffraction instrument is more closely
associated with the geometric size of the PLA particle `core`.
Example 6
[0121] Nanoparticle batches were prepared using the general
procedure of Example 2, with 80% (w/w) Polymer-PEG or Polymer-PEG
with homopolymer PLA at 40% (w/w) each, with a batch of % total
solids of 5%, 15% and 30%. Solvents used were: 21% benzyl alcohol
and 79% ethyl acetate (w/w). For each 2 gram batch size, 400 mg of
drug was used and 1.6 g of 16-5 Polymer-PEG or 0.8 g of 16-5
Polymer-PEG+0.8 g of 10 kDa PLA (homopolymer) was used. The diblock
polymer 16-5 PLA-PEG or PLGA-PEG (50:50 L:G) was used, and if used,
the homopolymer: PLA with a Mn=6.5 kDa, Mw=10 kDa, and
Mw/Mn=1.55.
[0122] The organic phase (drug and polymer) is prepared in 2 g
batches: To 20 mL scintillation vial add drug and polymer(s). The
mass of solvents needed at % solids concentration is shown
below:
[0123] i. 5% solids: 7.98 g benzyl alcohol+30.02 g ethyl
acetate
[0124] ii. 15% solids: 2.38 g benzyl alcohol+8.95 g ethyl
acetate
[0125] iii. 30% solids: 0.98 g benzyl alcohol+3.69 g ethyl
acetate
[0126] An aqueous solution is prepared with 0.5% sodium cholate, 2%
benzyl alcohol, and 4% ethyl acetate in water. To a 2 L bottle add
7.5 g sodium cholate, 1402.5 g of DI water, 30 g of benzyl alcohol
and 60 g of ethyl acetate, and mix on stir plate until
dissolved.
[0127] For the formation of emulsion, a ratio of aqueous phase to
oil phase of 5:1 is used. The organic phase is poured into the
aqueous solution and homogenized using IKA for 10 seconds at room
temperature to form course emulsion. The solution is fed through
the homogenizer (110S) at 9 Kpsi (45 psi on gauge) for 2 discreet
passes to form nanoemulsion.
[0128] The emulsion is poured into quench (D.I. water) at <5C
while stirring on stir plate. Ratio of quench to emulsion is
8:1.35% (w/w) Tween 80 is added in water to quench at ratio of 25:1
Tween 80 to drug. The nanoparticles are concentated through TFF and
the quench is concentrated on TFF with 500 kDa Pall cassette (2
membrane) to .about.100 mL. Diafiltering is used using .about.20
diavolumes (2 liter) of cold DI water, and the volume is brought
down to minimal volume then collect final slurry, .about.100 mL.
The solids concentration of unfiltered final slurry is determined
by the using tared 20 mL scintillation vial and adding 4 mL final
slurry and dry under vacuum on lyo/oven and the weight of
nanoparticles in the 4 mL of slurry dried down is determined.
Concentrated sucrose (0.666 g/g) is added to final slurry sample to
attain 10% sucrose.
[0129] Solids concentration of 0.45 um filtered final slurry was
determined by filteing about 5 mL of final slurry sample before
addition of sucrose through 0.45 nm syringe filter; to tared 20 mL
scintillation vial add 4 mL of filtered sample and dry under vacuum
on lyo/oven.
[0130] The remaining sample of unfiltered final slurry was frozen
with sucrose. Rapamycin (sirolimus) formulations:
TABLE-US-00006 Drug Release of Drug (t = hr) Name Polymer Size (nm)
Loading T = 0 T = 2 T = 4 T = 24 5% Solid 16/5 PLA/PEG 123.1 3.61%
ND ND ND ND 16/5 PLA/PEG + PLA 119.7 4.49% ND ND ND ND 15% Solid
16/5 PLA/PEG 82.1 4.40% ND ND ND ND 16/5 PLA/PEG + PLA 120.6 11.51%
ND ND ND ND 23% Solid 16/5 PLA/PEG 88.1 7.40% ND ND ND ND 16/5
PLA/PEG + PLA 118.3 7.8% ND ND ND ND 30% Solid 16/5 PLA/PEG 88.5
10.26% 8.5 17.3 22.4 64.2 16/5 PLA/PEG + PLA 118.3 10.18% 9.3 30.4
44.7 98.2
[0131] The effect of solid contents and the inclusions of
poly(lactic) acid homopolymer is shown in FIG. 7.
[0132] In-vitro release experiments are studied by dispersing
nanoparticles in PBS containing 10% (w/w) of Tween 20 (T20) at
37.degree. C. T20 was used to increase the solubility of rapamycin
in PBS to levels well detectable by HPLC as well as maintaining the
sink condition. 3 mL of drug-loaded nanoparticles were redispersed
in 130 mL of release medium in a jar at a known concentration
(approximately 2501 g/ml). These volumes were chosen to ensure that
the maximum concentration of the drug in the release medium would
always be less than 10% of the maximum solubility, i.e., sink
conditions. The media and nanoparticle suspension is stirred at 150
rpm. At pre-determined time points, 4 ml of aliquots were
centrifuged at 50,000 rpm (236,000 g) for 1 hr to separate the
nanoparticles from the elution media. The elution media is injected
in to a HPLC to determine drug released from the nanoparticles. The
release of rapamycin showed slow and sustained release, as shown in
FIG. 8.
Example 7
[0133] Nanoparticles were prepared as in Example 2 and 6, except
temsirolimus was used with 30% solid content in the organic phase
before emulsion:
TABLE-US-00007 Drug Release of Drug (t = hr) Name Lot # Polymer
Size (nm) Loading T = 0 T = 2 T = 4 T = 24 30% 45-48-1 16/5 PLA/PEG
97.5 9.9% 11.5 15.6 17.9 40.9 Solid 45-48-2 16/5 PLA/PEG + 112.8
14.2% 9.8 22.3 29.9 88.0 PLA 45-100-1 16/5 PLGA/PEG + 150.3 4.6 ND
ND ND ND PLA 50-52-6 16/5 PLGA/PEG + ND 6.9 10.6 35.7 45.8 87.0
PLA
[0134] FIG. 9 depicts the weight % of temsirolimus and FIG. 10
depicts the nanoparticle for the different polymeric nanoparticles
having temsirolimus. The results of an in-vitro release experiment
as in Example 6 shows the slow and sustained release of
temsirolimus showed slow and sustained release, as shown in FIG.
11.
EQUIVALENTS
[0135] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
INCORPORATION BY REFERENCE
[0136] The entire contents of all patents, published patent
applications, websites, and other references cited herein are
hereby expressly incorporated herein in their entireties by
reference.
* * * * *