U.S. patent application number 13/108361 was filed with the patent office on 2011-09-08 for long circulating nanoparticles for sustained release of therapeutic agents.
Invention is credited to Mir M. Ali, Jeff Hrkach, Susan Low, Greg Troiano, James Wright, Stephen E. Zale.
Application Number | 20110217377 13/108361 |
Document ID | / |
Family ID | 42288375 |
Filed Date | 2011-09-08 |
United States Patent
Application |
20110217377 |
Kind Code |
A1 |
Zale; Stephen E. ; et
al. |
September 8, 2011 |
Long Circulating Nanoparticles for Sustained Release of Therapeutic
Agents
Abstract
The present disclosure is directed in part to a biocompatible
nanoparticle composition comprising a plurality of non-colloidal
long circulating nanoparticles, each comprising a .alpha.-hydroxy
polyester-co-polyether and a therapeutic agent, wherein such
disclosed compositions provide a therapeutic effect for at least 12
hours.
Inventors: |
Zale; Stephen E.;
(Hopkinton, MA) ; Troiano; Greg; (Pembroke,
MA) ; Ali; Mir M.; (Woburn, MA) ; Hrkach;
Jeff; (Lexington, MA) ; Wright; James;
(Lexington, MA) ; Low; Susan; (Cambridge,
MA) |
Family ID: |
42288375 |
Appl. No.: |
13/108361 |
Filed: |
May 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12638297 |
Dec 15, 2009 |
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13108361 |
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61122479 |
Dec 15, 2008 |
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61249022 |
Oct 6, 2009 |
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61260200 |
Nov 11, 2009 |
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Current U.S.
Class: |
424/484 ;
514/249; 514/283; 514/291; 514/449; 977/773 |
Current CPC
Class: |
A61K 31/337 20130101;
A61K 47/593 20170801; A61K 47/6937 20170801; A61K 31/475 20130101;
A61K 47/542 20170801; B82Y 5/00 20130101; A61K 47/6925 20170801;
A61K 9/0019 20130101; A61P 35/00 20180101; A61K 31/519 20130101;
A61K 31/436 20130101; A61K 9/5153 20130101; A61K 9/1641 20130101;
A61K 47/60 20170801 |
Class at
Publication: |
424/484 ;
514/449; 514/291; 514/249; 514/283; 977/773 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/337 20060101 A61K031/337; A61K 31/436 20060101
A61K031/436; A61K 31/519 20060101 A61K031/519; A61K 31/437 20060101
A61K031/437; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[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 nanoparticle comprising a polymer matrix and a therapeutic
agent coupled to the polymer matrix wherein the polymer matrix is
biodegradable and/or biocompatible.
2. The nanoparticle of claim 1, wherein the therapeutic agent is an
anti-cancer agent.
3. The nanoparticle of claim 2, wherein the therapeutic agent is
docetaxel.
4. The nanoparticle of claim 1, wherein the therapeutic agent is a
peptide.
5. The nanoparticle of claim 1, wherein the polymer matrix
comprises an .alpha.-hydroxy polyester.
6. The nanoparticle of claim 5, wherein the polymer matrix is
selected from the group consisting of polylactide,
poly(lactide-co-glycolide), PEGylated polymer of lactide, PEGylated
copolymer of lactide and glycolide, or combinations thereof.
7. The nanoparticle of claim 2, wherein the docetaxel is coupled to
a PEGylated polymer of lactide.
8. The nanoparticle of claim 1, further comprising a
surfactant.
9. The nanoparticle of claim 1, comprising more than one
therapeutic agent.
10. The nanoparticle of claim 9, wherein the more than one
therapeutic agent each may be selected from the group consisting of
an agent useful in the treatment of cancer.
11. The nanoparticle of claim 10, wherein the polymer matrix is
coupled to a therapeutic agent selected from the group consisting
of: doxorubicin, gemcitabine, daunorubicin, procarbazine,
mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil,
vinblastine, vincristine, bleomycin, paclitaxel, docetaxel,
mitoxantrone, mitoxantrone hydrochloride, aldesleukin,
asparaginase, busulfan, carboplatin, cladribine, camptothecin,
dacarbazine, eniluracil, deoxycytidine, 5-azacytosine,
5-azadeoxycytosine, allopurinol, 2-chloroadenosine, trimetrexate,
aminopterin, oxaplatin, picoplatin, tetraplatin, satraplatin,
platinum-DACH, ormaplatin, epirubicin, etoposide phosphate,
9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin,
9-nitrocamptothecin, vindesine, L-phenylalanine mustard,
ifosphamidemefosphamide, perfosfamide, trophosphamide carmustine,
semustine, epothilones A-E, tomudex, 6-mercaptopurine,
6-thioguanine, amsacrine, etoposide phosphate, karenitecin,
acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine,
lamivudine, zidovudine, bevacizumab, trastuzumab, and
rituximab.
12. The nanoparticle of claim 10, wherein the polymer matrix is
coupled to a therapeutic agent selected from the group consisting
of docetaxel, oxaplatin, picoplatin, tetraplatin, satraplatin,
carboplatin, or camptothecin.
13. The nanoparticle of claim 12, wherein the nanoparticle includes
a therapeutic agent encapsulated within, surrounded by, or
dispersed throughout the polymer matrix.
14. The nanoparticle of claim 13, wherein the therapeutic agent is
docetaxel.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
12/638,297, filed Dec. 15, 2009, which claim priority to
provisional applications U.S. Ser. No. 61/122,479, filed Dec. 15,
2008, U.S. Ser. No. 61/260,200, filed Nov. 11, 2009, and U.S. Ser.
No. 61/249,022, filed Oct. 6, 2009, each of which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0003] Nanoparticles for the delivery of therapeutic agents have
the potential to circumvent many challenges associated with
traditional delivery approaches including lack of patient
compliance to prescribed therapy, adverse effects, and inferior
clinical efficacy due to lack of targeted delivery. Important
technological advantages of nanoparticles for drug delivery include
the ability to deliver water-insoluble and unstable drugs,
incorporation of both hydrophobic and hydrophilic therapeutic
agents, and ability to utilize various routes of administration.
Nanoparticle delivery systems may also facilitate targeted drug
delivery and controlled release applications, enhance drug
bioavailability at the site of action, reduce dosing frequency, and
minimize side effects.
[0004] Because of these possible advantages, nanoparticulate
systems have been examined for use as drug delivery vehicles,
including polymeric micelles, polymers, liposomes, low-density
lipoproteins, dendrimers, hydrophilic drug-polymer complexes, and
ceramic nanoparticles. Typical polymeric materials utilized in
polymeric particulate drug delivery systems include polylactic acid
(PLA), poly(D,L-glycolide) (PLG), and poly(lactide-co-glycolide)
(PLGA). PLA and PLGA are listed as Generally Recognized as Safe
(GRAS) under Sections 201(s) and 409 of the Federal Food, Drug, and
Cosmetic Act, and are approved for use in commercially available
microparticulate systems, including Decapeptyl.RTM., Parlodel
LA.RTM., and Enantone Depot.RTM., as well as in implant devices,
such as Zoladex.RTM..
[0005] However, certain nanoparticle systems, such as liposomes,
are not amenable for use with certain therapeutic agents. Polymeric
nanoparticles developed to date have limited effectiveness, in part
because such nanoparticles clear from the body quickly once
administered and/or may accumulate in healthy tissue where
treatment is not needed. Control of delivery of an active agent,
using nanosystems, remains a challenge.
[0006] Therefore there is a need for biocompatible compositions
capable of extended delivery of active agents, e.g.,
anti-neoplastic agents, that provide for prolonged and/or increased
plasma drug concentrations in a patient, especially as compared to
administration of an active agent alone.
SUMMARY
[0007] In one aspect of the invention, a nanoparticle composition
is provided that includes a biodegradable and/or biocompatible
polymer and a therapeutic agent, wherein the biodegradable and/or
biocompatible polymer matrix releases the therapeutic agent at a
rate allowing controlled release of the agent over at least about
12 hours, or in some embodiments, at least about 24 hours For
example, provided herein is a biocompatible nanoparticle
composition comprising a plurality of long circulating
nanoparticles, each comprising a biocompatible polymer and a
therapeutic agent, said composition providing an elevated plasma
concentration of the therapeutic agent for at least 12 hours when
the composition is administered to a patient, and an area under the
plasma concentration time curve (AUC) that is increased by at least
100% over the AUC provided when the therapeutic agent is
administered alone to a patient.
[0008] In an embodiment, disclosed herein is a biocompatible
nanoparticle composition comprising a plurality of long circulating
nanoparticles, each comprising a .alpha.-hydroxy
polyester-co-polyether and a therapeutic agent, said composition
providing an elevated plasma concentration of the therapeutic agent
for at least 6 hours, at least 12 hours, or at least 24 hours or
more when the composition is administered to a patient, to provide
an area under the plasma concentration time curve (AUC) that is
increased by at least 100%, or at least by 150%, over the AUC
provided when the therapeutic agent is administered alone to a
patient.
[0009] In some embodiments, disclosed nanoparticles may provide an
actual peak plasma concentration (C.sub.max) that is at least 10%
higher, or even at least 100% higher, as compared to a C.sub.max of
said therapeutic agent when administered alone. Disclosed
nanoparticles, for example, may provide a volume of distribution
when administered to the patient that is less than or equal to
about 5 plasma volumes. For example, disclosed nanoparticles and/or
compositions may decrease the volume of distribution (V.sub.z) by
at least 50% as compared to the V.sub.z of the patient when the
therapeutic agent is administered alone.
[0010] Disclosed biocompatible nanoparticle compositions may
include long circulating nanoparticles that may further comprise a
biocompatible polymer coupled to a targeting moiety, for example, a
targeting moiety that is selected from the group consisting of a
protein, peptide, antibody, antibody fragment, saccharide,
carbohydrate, small molecule, glycan, cytokine, chemokine,
nucleotide, lectin, lipid, receptor, steroid, neurotransmitter,
cell surface marker, cancer antigen, or glycoprotein antigen. An
exemplary targeting moiety may bind to prostate membrane specific
antigen (PMSA). For example, a disclosed nanoparticle may include a
biocompatible polymer coupled to a targeting moiety, e.g., a
nanoparticle may include
PLA-PEG-((S,S-2-{3-[1-carboxy-5-amino-pentyl]-ureido}-pentanedioic
acid. Disclosed long circulating nanoparticles may include 1 to
about 4% by weight, or 2% to about 4% by weight, of a biocompatible
polymer coupled to a targeting moiety
[0011] In some embodiments, a biocompatible nanoparticle may
include a biocompatible polymer such as PLA-PEG. For example, a
.alpha.-hydroxy polyester-co-polyether may be polylactic
acid-co-polyethylene glycol, and/or a .alpha.-hydroxy
polyester-co-polyether comprises about 16 kDa polylactic acid and
about 5 kDa polyethylene glycol.
[0012] Disclosed long circulating nanoparticles may be about 80 to
about 90 weight percent .alpha.-hydroxy polyester-co-polyether.
[0013] In some embodiments, disclosed long circulating
nanoparticles may further comprise a biodegradable polymer, such as
poly(lactic) acid. For example, long circulating nanoparticles may
have about 40 to about 50 weight percent poly(lactic)acid, and
about 40 to about 50 weight percent of .alpha.-hydroxy
polyester-co-polyether. Compositions that include such
biocompatible nanoparticles and a therapeutic agent may provide a
peak plasma concentration (C.sub.max) of a therapeutic agent at
least 100% higher than the C.sub.max of the therapeutic agent when
administered alone, and/or the area under the plasma concentration
time curve (AUC) may increased by at least 200% over the AUC of the
therapeutic agent when administered alone to the patient.
[0014] Disclosed nanoparticle compositions may include a
therapeutic agent such as one selected from the group consisting of
chemotherapeutic agents, diagnostic agents, prophylactic agents,
nutraceutical agents, nucleic acids, proteins, peptides, lipids,
carbohydrates, hormones, small molecules, metals, ceramics, drugs,
vaccines, immunological agents, and combinations thereof, for
example, a nanoparticle may include an anti-neoplastic agent such
as docetaxel, vincristine, methotrexate, paclitaxel, or sirolimus.
Disclosed nanoparticle compositions may further include an aqueous
solution of a saccharide.
[0015] Also provided herein is a method of treating a solid tumor
cancer, comprising administering disclosed nanoparticle composition
to a patient (e.g. a mammal or primate) in need thereof. Such
methods, may provide wherein at least 24 hours after
administration, a solid tumor has significant concentration of
therapeutic agent. Contemplated herein is a method of treating a
solid tumor in a mammal in need thereof, comprising administering a
nanoparticle composition comprising a plurality of nanoparticles
each comprising a .alpha.-hydroxy polyester-co-polyether and a
therapeutic agent, wherein the composition has an amount of
therapeutic agent effective to inhibit the growth of said tumor,
for example, a single dose of said composition may provide extended
elevated plasma concentrations of said therapeutic agent in the
patient for a least one day, (e.g. the peak plasma concentration
(C.sub.max) of the therapeutic agent after administration of the
composition to the mammal is at least 10% higher than the C.sub.max
of said therapeutic agent if administered in a non-nanoparticle
formulation.)
[0016] Also provided herein is a method of minimizing unwanted side
effects or toxicity of an active agent in a patient, comprising:
administering a nanoparticle composition comprising a plurality of
nanoparticles each comprising a .alpha.-hydroxy
polyester-co-polyether and a therapeutic agent, wherein said
composition is capable of delivery a higher plasma concentration of
therapeutic agent to the patient as compared to administering the
therapeutic agent alone, and wherein upon administering the
nanoparticle composition the volume distribution of the active
agent in the patient is reduced, as compared to the volume
distribution if the therapeutic agent was administered alone. A
method for modulating the plasma concentration of a therapeutic
agent in a patient, e.g. a primate (e.g. human) is also provided,
comprising:providing a polymeric nanoparticle comprising the
therapeutic agent and administering the polymeric nanoparticle to
the patient, thereby modulating the plasma concentration of the
human patient.
[0017] The invention may be more completely understood in
consideration of the following detailed description of various
embodiments of the invention in connection with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic illustration of a nanoparticle
according to one aspect of the present invention.
[0019] FIG. 2 is a block diagram of the emulsion process used in
the fabrication of nanoparticles in one aspect of the present
invention.
[0020] FIG. 3 depicts the in vitro release of docetaxel from
nanoparticles and conventional docetaxel.
[0021] FIG. 4 depicts the pharmacokinetics of docetaxel
encapsulated in nanoparticles and conventional docetaxel in
rats.
[0022] FIG. 5 depicts the distribution of radioactivity determined
in selected tissues of rats after IV administration of
nanoparticles containing .sup.14C-targeting polymer
(.tangle-solidup.) nanoparticles containing .sup.14C-docetaxel
(.box-solid.), and conventional .sup.14C-docetaxel
(.diamond-solid.).
[0023] FIG. 6 depicts docetaxel concentration in tumor tissue after
administration of docetaxel encapsulated in nanoparticles or
conventional docetaxel to LNCaP tumor bearing SCID mice.
[0024] FIG. 7 depicts the reduction in tumor volume in mice with
PSMA-expressing LNCaP xenografts when treated with docetaxel
encapsulated in nanoparticles or conventional docetaxel.
[0025] FIG. 8 depicts pharmacokinetics of vincristine encapsulated
in disclosed nanoparticles and conventional vincristine in
rats.
[0026] FIG. 9 depicts pharmacokinetics of methotrexate encapsulated
in disclosed nanoparticles and conventional methotrexate in
rats.
[0027] FIG. 10 depicts pharmacokinetics of paclitaxel encapsulated
in disclosed nanoparticles and conventional paclitaxel in rats.
[0028] FIG. 11 depicts pharmacokinetics of rapamycin (sirolimus)
encapsulated in disclosed nanoparticles and conventional rapamycin
in rats.
[0029] FIG. 12 depicts the tumor accumulation of docetaxel in
disclosed nanoparticles in a MX-1 mouse breast tumor model.
[0030] FIG. 13 depicts pharmacokinetics of docetaxel in a NHP model
using various disclosed nanoparticles.
DETAILED DESCRIPTION
[0031] It is to be understood that the invention is not limited to
the particular processes, compositions, or methodologies described,
as these may vary. It is also to be understood that the terminology
used in the description is for the purpose of describing particular
versions or embodiments only and is not intended to limit the scope
of the invention. All of the publications and references mentioned
herein are incorporated by reference. Nothing herein is to be
construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0032] As used herein the singular forms "a", "an" and "the"
include plural reference unless the context clearly dictates
otherwise. Further, unless defined otherwise, all technical and
scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art.
[0033] As used herein, the term "about" means plus or minus 10% of
the numerical value of the number with which it is being used.
Therefore, about 50% means in the range of 40%-60%.
[0034] An "effective amount" or "therapeutically effective amount"
of a composition, as used herein, is a predetermined amount
calculated to achieve a desired effect.
[0035] As used herein, the term "long-circulating" refers to
enhanced stability in the circulatory system of a patient,
regardless of biological activity.
[0036] As used herein, the prefix "nano" and the terms "nanophase"
and "nanosize" refer to a special state of subdivision implying
that a particle has an average dimension smaller than about 1000 nm
(1000.times.10.sup.-9 m).
[0037] As used herein, the terms "poly(ethylene glycol)" or "PEG"
and "poly(ethylene oxide)" or "PEO" denote polyethers comprising
repeat --CH.sub.2--CH.sub.2--O-- units. PEG and/or PEO can be
different polymers depending upon end groups and molecular weights.
As used herein, poly(ethylene glycol) and PEG describes either type
of polymer.
[0038] An ".alpha.-hydroxy polyester" refers to polymers having
monomers based on one or more .alpha.-hydroxy acid, such as
poly(lactic) acid, poly(glycolic) acid, poly-lactic-co-glycolic
acid, polycaprolactone.
[0039] The term "target", as used herein, refers to the cell type
or tissue to which enhanced delivery of the therapeutic agent is
desired. For example, diseased tissue may be a target for
therapy.
[0040] As used herein, the term "therapeutic agent" means a
compound utilized to image, impact, treat, combat, ameliorate,
prevent or improve an unwanted condition or disease of a
patient.
[0041] In an embodiment, disclosed long-circulating nanoparticles
include a therapeutic agent and biodegradable and/or biocompatible
polymeric particles, optionally functionalized with targeting
moieties. The nanoparticles are designed to circulate in a vascular
compartment of a patient for an extended period of time, distribute
and accumulate at a target, and release the encapsulated
therapeutic agent in a controlled manner. These characteristics can
result in an increased level of therapeutic agent in the target and
a potential reduction in off-target exposure. For example, the
disclosed nanoparticles remain in circulation longer because, upon
administration to a patient (e.g. a mammal, primate (e.g. human)),
the disclosed nanoparticles are substantially confined to the
vascular compartment of the patient, and are engineered to be
cleared very slowly.
[0042] The activity of many therapeutic agents is dependent on
their pharmacokinetic behavior. This pharmacokinetic behavior
defines the drug concentrations and period of time over which cells
are exposed to the drug. For most therapeutics, e.g.
anti-neoplastics, longer exposure times are preferred as this
results in increased killing of the cancer cells. In general,
several parameters are used to describe drug pharmacokinetics. Peak
plasma concentration, or maximum plasma concentration (C.sub.max)
and area under the curve (AUC) are examples. AUC is a measure of
plasma drug levels over time and provides an indication of the
total drug exposure. Generally, plasma concentration and plasma AUC
for a therapeutic agent correlate with increased therapeutic
efficacy.
[0043] The combination of long circulation time, confinement of
particles to the vascular compartment and controlled release of
drug results in higher circulating drug concentrations for longer
periods of time (as evidenced by higher AUC and lower Vd). than
drug alone, or, for example, drug in a PLA polymeric nanoparticles
that does not include PLA-PEG, or that do not include e.g. PLA
alone.
[0044] For example, provided herein, in an embodiment, is a
biocompatible nanoparticle composition comprising a plurality of
long circulating nanoparticles, each comprising a .alpha.-hydroxy
polyester-co-polyether and a therapeutic agent. Such compositions
may provide a therapeutic effect for at least 12 hours, at least 24
hours, or at least 36 hours, or 48 hours or more, upon
administration to a patient. In some embodiments, peak plasma
concentration (C.sub.max) of the therapeutic agent of such
nanoparticles, e.g. when the composition is administered to a
patient, may be least 10% higher, 20% higher, or about 10% to about
100% higher, or more, than the C.sub.max of the same therapeutic
agent when administered alone. Actual peak plasma concentration of
delivered therapeutic agent includes both agent that is released
from the nanoparticle (e.g. after administration) and therapeutic
agent remaining in any nanoparticle remaining in the plasma, e.g.
at a given time.
[0045] In another embodiment, disclosed nanoparticles may provide
upon administration to a patient, an area under the plasma
concentration time curve (AUC), that may be increased by at least
100%, at least 200%, or about 100% to about 500% or more, over the
AUC of the therapeutic agent when administered alone to the
patient. In another embodiment, a provided composition that
includes disclosed nanoparticles may decrease the volume of
distribution (V.sub.z) of distributed active agent, upon
administration, in a patient by at least 10%, or by at least 20%,
or about 10% to about 100%, as compared to the V.sub.z of the
patient when the therapeutic agent is administered alone. For
example, a provided nanoparticle composition may provide V.sub.z in
a patient that is on the same order of magnitude that the of plasma
volume and/or a volume of distribution less than about 10 plasma
volumes. For example, a disclosed nanoparticle composition may
provide a Vz that is less than, or about, 2 times the plasma
volume, or less than or about 8 plasma volumes. In an embodiment, a
disclosed nanoparticle composition may provide a V.sub.z in a
patient that is on about the same order of plasma volume, (e.g.
about 5 L for an exemplary 70 kg patient), e.g. about a V.sub.z
that indicates administered nanoparticles are substantially in the
patient's plasma and not substantially in other tissues.
[0046] In some embodiments, disclosed nanoparticles may be used as
a drug delivery vehicle based on the encapsulation of a therapeutic
agent in a polymer matrix with controlled porosity and/or a soluble
shell or matrix that upon dissolution releases the therapeutic
agent in the immediate vicinity of the targeted area. The
protection of the therapeutic agent provided by the polymer shell
or matrix allows for the delivery of therapeutic agents that are
water-insoluble or unstable. Furthermore, dissolution kinetics of
the polymer can be designed to provide sustained release of
therapeutic agents at a target for an extended period of time.
[0047] Disclosed nanoparticles can be used for a variety of
applications, such as, without limitation, drug delivery, gene
therapy, medical diagnosis, and for medical therapeutics for
cancer, pathogen-borne diseases, hormone-related diseases,
reaction-by-products associated with organ transplants, and other
abnormal cell or tissue growth.
[0048] Provided herein, in an embodiment, are methods for treating
a patient e.g. a mammal suffering from cancer, e.g. a solid tumor
cancer, prostate cancer, breast cancer or lung cancer using e.g.,
disclosed nanoparticles. However, contemplated diseases that may be
treated using disclosed nanoparticles include a broad range of
diseases and find limitation only by e.g. the therapeutic agent,
the availability of a marker and/or a targeting ligand for the
disease.
[0049] In other embodiments, a nanoparticle delivery system is
provided that mitigates against colloidal instability,
agglomeration, polydispersity in nanoparticle size and shape,
swelling, and leakage of encapsulated materials.
[0050] In yet another embodiment, nanoparticles for delivery of
therapeutic agents are provided that exhibit encapsulation
efficiency. Encapsulation efficiency is affected by factors
including, for example, material characteristics of the polymer
utilized as the carrier matrix, the chemical and physical
properties of the therapeutic agent to be encapsulated, and type of
solvents used in the nanoparticle fabrication process.
[0051] In yet another aspect, polymeric nanoparticles for delivery
of therapeutic agents are provided that exhibit particle
heterogeneity. Conventional polymeric nanoparticle fabrication
techniques generally provide multimodal particle size distributions
as a result of self-aggregation during nanoprecipitation of both
the polymer and the drug molecules.
[0052] Polymeric nanoparticles for delivery of therapeutic agents
are provided, in an embodiment, that may reduce or eliminate burst
release effects. Conventional polymeric nanoparticle carriers
frequently exhibit a bimodal drug release pattern with up to about
40-80% or more of the encapsulated drug released during the first
several hours. After 24 to 48 hours, drug release is significantly
reduced due to the increased diffusion barrier for drug molecules
located deep within the polymer matrix. In such conventional
nanoparticle carrier systems, poorly encapsulated drug molecules
diffuse quickly into solution, which may lead to significant
toxicity in vivo. Further, by the time the evacuated nanoparticles
arrive and accumulate at the targeted site (e.g., tumor tissue),
the nanoparticles generally have little or no remaining therapeutic
efficacy.
[0053] In an embodiment, polymeric nanoparticles for delivery of
therapeutic agents are provided that may evade rapid capture by the
reticuloendothelial system (RES), leading to extended circulation
time and elevated concentration of the nanoparticles in the blood.
Rapid capture and elimination is typically caused by the process of
opsonization in which opsonin proteins present in the blood serum
quickly bind to conventional nanoparticles, allowing macrophages to
easily recognize and remove these particulates before they can
perform their designed therapeutic function. The extent and nature
of opsonin adsorption at the surface of nanoparticles and their
simultaneous blood clearance depend on the physicochemical
properties of the particles, such as size, surface charge, and
surface hydrophobicity. In yet another embodiment, a nanoparticle
composition is provided including a biodegradable and/or
biocompatible polymer matrix and a therapeutic agent coupled to the
biodegradable and/or biocompatible polymer matrix wherein the
clearance rate of said therapeutic agent coupled to the
biodegradable and/or biocompatible polymer matrix is lower than the
clearance rate of said therapeutic agent when administered
alone.
[0054] In certain embodiments, methods are provided that mask or
camouflage nanoparticles in order to evade uptake by the RES. One
such method is the engineering of particles in which polyethers,
such as poly(ethylene glycol) (PEG) or PEG containing surfactants,
are deployed on the surface of nanoparticles. The presence of PEG
and/or PEG-containing copolymers, e.g. on the surface of
nanoparticles can result in an increase in the blood circulation
half-life of the nanoparticles by several orders of magnitude. This
method creates a hydrophilic protective layer around the
nanoparticles that is able to repel the absorption of opsonin
proteins via steric repulsion forces, thereby blocking and delaying
the first step in the opsonization process.
[0055] FIG. 1 schematically illustrates a nanoparticle according to
one aspect of the present invention. As shown in FIG. 1, docetaxel
100, an anti-neoplastic agent approved for the treatment of hormone
refractory prostate cancer (HRPC), is encapsulated in a matrix 110
derived from the biodegradable and/or biocompatible polymers PLA
and poly(lactide-b-ethylene glycol) (PLA-PEG). The polymer matrix
110 contains a targeting polymer (PLA-PEG-lys(urea)glu) 120 that is
end-functionalized (through the 5 amino moiety) with the
lysine-urea-glutamate heterodimer
(S,S-2-{3-[1-carboxy-5-amino-pentyl]-ureido}-pentanedioic acid
(lys(urea)glu) 130, a small molecule ligand that selectively binds
to PSMA, a clinically relevant prostate cancer cell surface
marker.
[0056] Once the nanoparticles, e.g. as provided herein are
administered, at least portions of the nanoparticle polymer(s) may
be biologically degraded by, for example, enzymatic activity or
cellular machinery into monomers and/or other moieties that cells
can either use or excrete. In certain aspects of the invention, the
dissolution or degradation rate of the nanoparticles is influenced
by the composition of the polymer shell or matrix. For example, in
some embodiments, the half-life of the polymer (the time at which
50% of the polymer is degraded into monomers and/or other
nonpolymeric moieties) may be on the order of days, weeks, months,
or years, depending on the polymer.
[0057] According to some aspects of the invention, nanoparticle
delivery characteristics such as water uptake, controlled release
of therapeutic agent, and polymer degradation kinetics may be
optimized through selection of polymer shell or matrix
composition.
[0058] Suitable polymers that may form some of the disclosed
nanoparticles may include, but are not limited to, biodegradable
.alpha.-hydroxy polyesters and biocompatible polyethers. In some
aspects, exemplary polyesters include, for example, PLA, PLGA, PEG,
PEO, PEGylated polymers and copolymers of lactide and glycolide
(e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA), and
derivatives thereof. In other aspects, suitable polymers include,
for example, polyanhydrides, poly(ortho ester) PEGylated poly(ortho
ester), poly(caprolactone), PEGylated poly(caprolactone),
polylysine, PEGylated polylysine, poly(ethylene inline), PEGylated
poly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine
ester), poly(4-hydroxy-L-proline ester),
poly[a-(4-aminobutyl)-L-glycolic acid], and combinations and
derivatives thereof.
[0059] In other aspects, a polymer matrix may comprise one or more
acrylic polymers. Exemplary acrylic polymers include, for example,
acrylic acid and methacrylic acid copolymers, methyl methacrylate
copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,
aminoalkyl methacrylate copolymer, poly(acrylic acid),
poly(methacrylic acid), methacrylic acid alkylamide copolymer,
poly(methyl methacrylate), poly(methacrylic acid polyacrylamide)
copolymer, aminoalkyl methacrylate copolymer, glycidyl methacrylate
copolymers, polycyanoacrylates, and combinations thereof. The
matrix may include dextran, acylated dextran, chitosan (e.g.,
acetylated to various levels), poly(vinyl) alcohol (for example,
hydrolyzed to various degrees), and/or alginate, e.g. alginate
complexed to bivalent cations such as a calcium alginate
complex.
[0060] Nanoparticles disclosed herein include one, two, three or
more biocompatible and/or biodegradable polymers. For example, a
contemplated nanoparticle may include about 10 to about 99 weight
percent of one or more block co-polymers that include a
biodegradable polymer and polyethylene glycol, and about 0 to about
50 weight percent of a biodegradable homopolymer. Exemplary
therapeutic nanoparticles may include about 40 to about 90 weight
percent poly(lactic) acid-poly(ethylene)glycol copolymer or about
40 to about 80 weight percent poly(lactic)
acid-poly(ethylene)glycol copolymer. Such poly(lactic)
acid-block-poly(ethylene)glycol copolymer may include poly(lactic
acid) having a number average molecular weight of about 15 to 20
kDa (or for example about 15 to about 100 kDa, e.g., about 15 to
about 80 kDa), and poly(ethylene)glycol having a number average
molecular weight of about 2 to about 10 kDa, for example, about 4
to about 6 kDa. For example, a disclosed therapeutic nanoparticle
may include about 70 to about 90 weight percent PLA-PEG and about 5
to about 25 weight percent active agent (e.g. docetaxel), or about
30 to about 50 weight percent PLA-PEG, about 30 to about 50 weight
percent PLA or PLGA, and about 5 to about 25 weight percent active
agent (e.g. doxetaxel). Such PLA ((poly)lactic acid) may have a
number average molecular weight of about 5 to about 10 kDa. Such
PLGA (poly lactic-co-glycolic acid) may have a number average
molecular weight of about 8 to about 12 kDa. It should be
appreciated that disclosed PLA-PEG copolymers may include a
chemical linker, oligomer, or polymer chain between the PLA and PEG
blocks, e.g., may include PLA-linker-PEG.
[0061] For example, disclosed nanoparticles may include about 10 to
15 weight percent active agent (e.g. about 10 weight percent
docetaxel), and about 86 to about 90 weight percent PLA-PEG (with
e.g. PLA about 16 kDa and PEG about 5 kDa, e.g. about 87.5% PLA-PEG
(16 kDa/5 kDa)), and optionally e.g. a PLA-PEG-lys(urea)-glu (e.g.
at 2.5 weight percent).
[0062] Alternatively, a disclosed nanoparticle, which may have slow
release properties, may include about 42 to about 45 weight percent
PLA-PEG (with e.g. PLA about 16 kDa and PEG about 5 kDa), (e.g.
43.25% PLA-PEG), about 42 to 45 weight percent PLA (e.g. about 75
kDa) (e.g. 43.25% PLA/75 kDa) and about 10 to 15 weight percent
active agent (e.g. docetaxel). For example, 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. In
an embodiment, disclosed nanoparticles may include two polymers,
e.g. PLA-PEG and PLA, in a weight ratio of about 30:60 to about
60:30, e.g, about 40:60, about 60:40, or about 50:50.
[0063] Such substantially homopolymeric poly(lactic) or
poly(lactic)-co-poly(glycolic) acid may have a weight average
molecular weight of about 10 to about 130 kDa, for example, about
20 to about 30 kDa, or about 100 to about 130 kDa. Such
homopolymeric PLA may have a number average molecule weight of
about 5 to about 90 kDa, or about 5 to about 12 kDa, about 15 to
about 30 kDa, or about 60 to about 90 kDa. Exemplary homopolymeric
PLA may have a number average molecular weight of about 80 kDa or a
weight average molecular weight of about 124 kDa. As is known in
the art, molecular weight of polymers can be related to an inherent
viscosity. In some embodiments, homopolymer PLA may have an
inherent viscosity of about 0.2 to about 0.4, e.g. about 0.3; in
other embodiments, PLA may have an inherent viscosity of about 0.6
to about 0.8. Exemplary PLGA may have a number average molecular
weight of about 8 to about 12 kDa.
[0064] In other embodiments, modified surface chemistry and/or
small particle size of disclosed nanoparticles may contribute to
the effectiveness of the nanoparticles in the delivery of a
therapeutic agent. For example, in one disclosed aspect,
nanoparticle surface charge may be modified to achieve slow
biodegradation and reduce clearance of the nanoparticles. In
another aspect, porosity of the polymer shell or matrix is
optimized to achieve extended and controlled release of the
therapeutic agent. For example, in one embodiment of the invention,
the nanoparticles may have porosity in the range of about 10 to
about 90 percent and/or a pore diameters in the range of about
0.001 to about 0.01 microns. Further, without wishing to be bound
by theory, because of their small size and persistence in the
circulation, the nanoparticles according to some embodiments of the
invention may be able to penetrate the altered and often
compromised vasculature of tumors via the enhanced permeability and
retention (EPR) effect resulting in preferential accumulation of
nanoparticles in tumor interstitium.
[0065] Examples of therapeutic agents that may form part of the
disclosed nanoparticles include, but are not limited to,
chemotherapeutic agents (e.g. anti-cancer agents), diagnostic
agents (e.g. contrast agents, radionuclides, and fluorescent,
luminescent, and magnetic moieties), prophylactic agents (e.g.
vaccines), nutraceutical agents (e.g. vitamins and minerals),
nucleic acids (e.g., siRNA, RNAi, and mircoRNA agents), proteins
(e.g. antibodies), peptides, lipids, carbohydrates, hormones, small
molecules, metals, ceramics, drugs, vaccines, immunological agents,
and/or combinations thereof. For example, the active agent or drug
may be a therapeutic agent such as an antineoplastic such as a mTor
inhibitor (e.g., sirolimus (rapamycin), temsirolimus, or
everolimus), vinca alkaloids such as vincristine, a diterpene
derivative, a taxane such as paclitaxel (or its derivatives such as
DHA-paclitaxel or PG-paxlitaxel), docetaxel, or methatrexate.
[0066] In some aspects of the invention, the therapeutic agent to
be delivered is an agent useful in the treatment of cancer (e.g., a
solid tumor cancer e.g., prostate or breast cancer). Such
therapeutic agents may include, for example, doxorubicin
(adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine,
mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil
(5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol),
docetaxel (taxotere), mitoxantrone, mitoxantrone hydrochloride,
aldesleukin, asparaginase, busulfan, carboplatin, cladribine,
camptothecin, CPT-I1,1O-hydroxy-7-ethylcamptothecin (SN38),
dacarbazine, S--I capecitabine, ftorafur, 5' deoxyfluorouridine,
UFT, eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,
allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,
methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin,
tetraplatin, satraplatin, platinum-DACH, ormaplatin, CI-973,
JM-216, and analogs thereof, epirubicin, etoposide phosphate,
9-aminocamptothecin, 10,11-methylenedioxycamptothecin, karenitecin,
9-nitrocamptothecin, TAS 103, vindesine, L-phenylalanine mustard,
ifosphamidemefosphamide, perfosfamide, trophosphamide carmustine,
semustine, epothilones A-E, tomudex, 6-mercaptopurine,
6-thioguanine, amsacrine, etoposide phosphate, karenitecin,
acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine,
lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab and
combinations thereof.
[0067] In some embodiments, contemplated nanoparticles may include
more than one therapeutic agent. Such nanoparticles may be useful,
for example, in aspects where it is desirable to monitor a
targeting moiety as such moiety directs a nanoparticle containing a
drug to a particular target in a subject.
[0068] Disclosed nanoparticles may be formed using an emulsion
process, e.g. as presented as a block diagram in FIG. 2. As shown
in FIG. 2, an organic polymer/drug solution containing docetaxel,
PLA, PLA-PEG, and PLA-PEG-lys(urea)glu dissolved in a co-solvent
mixture of ethyl acetate and benzyl alcohol is dispersed in an
aqueous solution of sodium cholate, ethyl acetate, and benzyl
alcohol to form a coarse emulsion. In some aspects the conditions
under which the emulsion process is performed favor the orientation
of the PEG and/or PEG-lys(urea)glu polymer chains toward the
particle surface. In other aspects, an orientation is achieved
where the PEG is folded within the nanoparticle polymer shell or
matrix.
[0069] As presented in FIG. 2, a coarse emulsion can be passed
through a high pressure homogenizer to reduce the droplet size,
forming a fine emulsion. The fine emulsion is diluted into an
excess volume of a quench solution of cold water containing
polysorbate 80. The presence of polysorbate 80 serves to remove
excess therapeutic agent that has not been encapsulated in the
nanoparticle. In some aspects of the present invention, polysorbate
80 may also be adhered or associated with a nanoparticle surfaces.
While not wishing to be bound by theory, polysorbate 80 coupled to
the nanoparticle surfaces may impact characteristics such as
controlled release of therapeutic agent and polymer degradation
kinetics. 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.).
[0070] In some embodiments, not all of the therapeutic agent (e.g.,
docetaxel) 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 (e.g., docetaxel) is
about 100:1 to about 10:1.
[0071] Ethyl acetate and benzyl alcohol are extracted from the
organic phase droplets, resulting in formation of a hardened
nanoparticle suspension. For example, docetaxel or other active
agent may be encapsulated at e.g. a loading level of 10% w/w;
corresponding to more than 10,000 drug molecules per
nanoparticle.
[0072] The nanoparticle suspension is processed using tangential
flow ultrafiltration/diafiltration (UF/DF) with cold water to
remove processing aids and to concentrate the nanoparticles to a
desired value. Residual precursor materials and excess organics
present in unwashed nanoparticle suspensions may have a detrimental
impact on biomedical applications as well as undesired toxic
effects on the physiological system. The washed nanoparticle
suspension is then passed through a prefilter and at least two
sterilizing-grade filters.
[0073] Once the nanoparticles have been prepared, they may be
combined with an acceptable carrier to produce a pharmaceutical
formulation, according to another aspect of the invention. As would
be appreciated by one of skill in this art, the carrier may be
selected based on factors including, but not limited to, the route
of administration, the location of the targeted disease tissue, the
therapeutic agent being delivered, and/or the time course of
delivery of the therapeutic agent. For example, as shown in FIG. 2,
a concentrated sucrose solution is aseptically added to the sterile
nanoparticle suspension to produce a pharmaceutical formulation.
The sucrose serves as a cryoprotectant and a tonicity agent. In
this embodiment, the resulting pharmaceutical formulation is a
sterile, aqueous, injectable suspension of docetaxel encapsulated
in nanoparticles comprised of biocompatible and biodegradable
polymers. The suspension is assayed for docetaxel content, and may
be aseptically diluted to the desired concentration. In some
embodiments, the particle suspension is aseptically filled and
sealed in glass vials. In other embodiments, the bulk drug product
suspension is stored frozen at -20.degree. C..+-.5.degree. C. prior
to filling into vials.
[0074] The fabrication methods for the nanoparticles of the
invention may be modified in some embodiments to achieve desired
drug-delivery features. For example, nanoparticle characteristics
such as surface functionality, surface charge, particle size, zeta
(.zeta.) potential, hydrophobicity, controlled release capability,
and ability to control immunogenicity, and the like, may be
optimized for the effective delivery of a variety of therapeutic
agents. Furthermore, the long-circulating nanoparticles produced
according to the emulsion process shown in FIG. 2 are well
dispersed and unagglomerated, which facilitates conjugation or
functionalization of the nanoparticle surfaces with targeting
moieties.
[0075] Disclosed nanoparticles may include optional targeting
moieties, which may be selected to ensure that the nanoparticles
selectively attach to, or otherwise associate with, a selected
marker or target. For example, in some embodiments, disclosed
nanoparticles may be functionalized with an amount of targeting
moiety effective for the treatment of prostate cancer in a subject
(e.g., a low-molecular weight PSMA ligand). Through
functionalization of nanoparticle surfaces with such targeting
moieties, the nanoparticles are effective only at targeted sites,
which minimizes adverse side effects and improves efficacy.
Targeted delivery also allows for the administration of a lower
dose of therapeutic agent, which may reduce undesirable side
effects commonly associated with traditional treatments of
disease.
[0076] In certain aspects, disclosed nanoparticles may be optimized
with a specific density of targeting moities on the nanoparticle
surface, such that e.g., an effective amount of targeting moiety is
associated with the nanoparticle for delivery of a therapeutic
agent. For example, the fraction of the biodegradable and/or
biocompatible polymer matrix functionalized with a targeting moiety
may be less than 80% of the total. According to another embodiment,
the fraction of the biodegradable and/or biocompatible polymer
matrix functionalized with a targeting moiety is less than about
50% of the total. Increased density of the targeting moiety may, in
some embodiments, increase target binding (cell binding/target
uptake).
[0077] Exemplary targeting moieties include, for example, proteins,
peptides, antibodies, antibody fragments, saccharides,
carbohydrates, glycans, cytokines, chemokines, nucleotides,
lectins, lipids, receptors, steroids, neurotransmitters and
combinations thereof. The choice of a marker may vary depending on
the selected target, but in general, markers that may be useful in
embodiments of the invention include, but are not limited to, cell
surface markers, a cancer antigen (CA), a glycoprotein antigen, a
melanoma associated antigen (MAA), a proteolytic enzyme, an
angiogenesis marker, a prostate membrane specific antigen (PMSA), a
small cell lung carcinoma antigen (SCLCA), a hormone receptor, a
tumor suppressor gene antigen, a cell cycle regulator antigen, a
proliferation marker, and a human carcinoma antigen. Exemplary
targeting moieties include:
##STR00001##
-lys-(urea)glu, which may be conjugated to PEG, e.g. a disclosed
nanoparticle may include PLA-PEG-targeting moiety, e.g.
S,S-2-{3-[1-carboxy-5-amino-pentyl]-ureido}-pentanedioic acid. For
example, disclosed nanoparticles may include about 10 to 15 weight
percent active agent (e.g. docetaxel), and about 86 to about 90
weight percent PLA-PEG (with e.g. PLA about 16 kDa and PEG about 5
kDa), and about 2 to about 3 weight percent PLA-PEG-lys(urea)glu
(16 kDa/5 kDa PLA-PEG). Alternatively, a disclosed nanoparticle may
include about 42 to about 45 weight percent PLA-PEG (with e.g. PLA
about 16 kDa and PEG about 5 kDa) about 42 to 45 weight percent PLA
(e.g. about 75 kDa), about 10 to 15 weight percent active agent
(e.g. docetaxel), and about 2 to about 3 weight percent
PLA-PEG-lys(urea)glu (16/5 PLA-PEG).
[0078] In other aspects of the invention, targeting moieties are
targeted to an antigen associated with a disease of a patient's
immune system or a pathogen-borne condition. In yet another aspect,
targeting moieties are targeted to cells present in normal healthy
conditions. Such targeting moieties may be directly targeted to a
molecule or other target or indirectly targeted to a molecule or
other target associated with a biological molecular pathway related
to a condition.
[0079] The amount of nanoparticles administered to a patient may
vary and may depend on the size, age, and health of the patient,
the therapeutic agent to be delivered, the disease being treated,
and the location of diseased tissue. Moreover, the dosage may vary
depending on the mode of administration.
[0080] Various routes of administration are contemplated herein. In
a particular aspect, the nanoparticles are administered to a
subject systemically. Further, in some aspects, methods of
administration may include, but are not limited to, intravascular
injection, intravenous injection, intraperitoneal injection,
subcutaneous injection, and intramuscular injection. According to
aspects of the present invention, the nanoparticles necessitate
only a single or very few treatment sessions to provide effective
treatment of disease, which ultimately may facilitate patient
compliance. For example, in some aspects, administration of the
nanoparticles can occur via intravenous infusion once every three
weeks.
[0081] Also contemplated herein are methods of treating solid
tumors, e.g. prostate, lung, breast or other cancers, comprising
administering a disclosed nanoparticle composition to a patient,
e.g. a mammal in need thereof. For example, after such
administration, e.g. at least after hours, 24 hours, 36 hours, or
48 hours, or more after administration, the solid tumor may have
significant concentration of therapeutic agent, e.g. may have an
increase in tumor drug concentration of at least about 20%, or at
least about 30% or more active agent (e.g. docetaxel) as compared
to the amount present in a tumor after administration of (e.g. the
same dosage) of therapeutic agent alone (e.g. not in a disclosed
nanoparticle composition).
[0082] Disclosed herein is a method of treating a solid tumor in a
mammal comprising administering a nanoparticle composition
comprising a plurality of nanoparticles each comprising a
.alpha.-hydroxy polyester-co-polyether and a therapeutic agent,
wherein the composition has an amount of therapeutic agent
effective to inhibit the growth of said tumor, for example, wherein
single dose of said composition provides extended release of said
therapeutic agent for a least one day. Such methods may provide an
actual peak plasma concentration (C.sub.max) of the therapeutic
agent after administration of the composition to the mammal that is
at least 10% higher, or at least 20% higher or 100% higher or more
than the C.sub.max of said therapeutic agent if administered in a
non-nanoparticle formulation. Disclosed methods may provide, upon
administration of nanoparticles, an area under the plasma
concentration time curve (AUC) in a patient that is increased by at
least 100% over the AUC provided when the therapeutic agent is
administered alone to a patient. In some embodiments, disclosed
methods may also, alone or in addition to the above plasma
parameters, decrease the volume of distribution (V.sub.z) of the
therapeutic agent upon administration by at least 50% as compared
to the V.sub.z of the patient when the therapeutic agent is
administered alone.
[0083] A method of minimizing unwanted side effects or toxicity of
an active or therapeutic agent in a patient is also provided
herein. For example, disclosed nanoparticles, may, upon
administration, provide a higher plasma concentration of
therapeutic agent as compared to administering an equivalent dosage
of therapeutic agent alone. However, upon administration, in some
embodiments, disclosed nanoparticles circulate substantially in the
vascular compartment, and therefore may not contribute
significantly to other areas that may cause toxicity or unwanted
side effects.
[0084] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting the invention in any
manner.
EXAMPLES
Example 1
In Vitro Release of Docetaxel from Nanoparticles
[0085] A suspension of docetaxel encapsulated in nanoparticles
fabricated according to the emulsion process depicted in FIG. 2 and
Example 12 using 87.5 weight percent PLA-PEG, 10 wt. % docetaxel,
and 2.5 wt. percent docetaxel (Formulation A) (all docetaxel
nanoparticle formulations used in these Examples were in a
composition of 5% nanoparticles, 65% water, and 30% sucrose). was
placed in a dialysis cassette and incubated in a reservoir of
phosphate buffered saline (PBS) at 37.degree. C. with stirring.
Samples of the dialysate were collected and analyzed for docetaxel
using reversed phase high performance liquid chromatography (HPLC).
For comparison, conventional docetaxel was analyzed under the same
procedure.
[0086] FIG. 3 presents the in vitro release profile of docetaxel
encapsulated in nanoparticles compared to conventional docetaxel.
Release of the encapsulated docetaxel from the polymer matrix was
essentially linear over the first 24 hours with the remainder
gradually released over a period of about 96 hours.
Example 2
Single Dose Pharmacokinetic Study of Docetaxel Encapsulated in
Nanoparticles and Conventional Docetaxel in Sprague-Dawley Rats
[0087] Six- to eight-week old male Sprague-Dawley rats were
administered a single bolus dose (5 mg/kg of docetaxel) of
docetaxel encapsulated in nanoparticles or conventional docetaxel
via a tail vein. The dose groups consisted of six rats each. Blood
was drawn at 0.083, 0.5, 1, 2, 3, 4, 6, and 24 hours post-dosing
and processed to plasma. The concentration of total docetaxel in
plasma was measured by a liquid chromatography-mass spectrometry
(LC-MS) method following extraction with methyl tert-butyl ether
(MTBE). The MTBE extraction does not differentiate
nanoparticle-encapsulated docetaxel from docetaxel that was
released from the nanoparticles into the plasma, and as such, the
LC-MS data does not distinguish between the two.
[0088] FIG. 4 and Example Table 2.1 present the observed
pharmacokinetic profiles and pharmacokinetic parameters,
respectively, of docetaxel encapsulated in nanoparticles and
conventional docetaxel. Example Table 2.1 further includes data
from the preclinical development of TAXOTERE.RTM. for comparative
reference (Bissery et al. 1995). The results for conventional
docetaxel were consistent with those reported in literature
(Bissery et al. 1995), indicating docetaxel was rapidly cleared
from the blood and distributed to tissues. The peak plasma
concentration (C.sub.max) was observed at the first sampling time
point for all treatments.
[0089] The C.sub.max and AUC of the docetaxel encapsulated in
nanoparticles were approximately 100 times higher than that for
conventional docetaxel. The difference in the C.sub.max may be
attributable to having missed the rapid initial tissue distribution
phase for conventional docetaxel. The data indicate that the
docetaxel encapsulated in nanoparticles largely remains in
circulation upon injection and is slowly cleared over a 24 hour
period. The data further shows that docetaxel is released from the
nanoparticles in a controlled manner during the 24 hour period
(e.g., rapid burst release is not observed). If the nanoparticles
were very quickly cleared from circulation, the large increase in
AUC would not be observed. Similarly, if there was rapid burst
release of docetaxel from the nanoparticles, the pharmacokinetic
profile would be expected to more closely resemble that of
conventional docetaxel.
TABLE-US-00001 EXAMPLE TABLE 2.1 Summary of Docetaxel Encapsulated
in Nanoparticles and Conventional Docetaxel Pharmacokinetic
Parameters Dose t.sub.max.sup.a C.sub.max t.sub.1/2
AUC.sub.0-.infin. CL Species (mg/kg) (min) (ng/mL) (h) (ng/mL h)
(L/h/kg) Conventional Sprague- 5 2 4,100 0.8.sup.b 910 5.5
Docetaxel Dawley Rats (Bissery et al. 1995) Conventional Sprague- 5
5 600 4.4.sup.c 623 2.33 Docetaxel Dawley Rats Docetaxel Sprague- 5
5 54,800 2.6.sup.c 57,300 0.01 Encapsulated in Dawley Rats
Nanoparticles .sup.aFor each treatment, t.sub.max equals the first
sampling time. .sup.bThe study duration was 6 hours. .sup.cThe half
life was determined from 2-12 hours.
Example 3
Tissue Distribution Study of Docetaxel Encapsulated in
Nanoparticles and Conventional Docetaxel in Sprague-Dawley Rats
[0090] Six- to eight-week old male Sprague-Dawley rats were
administered a single bolus intravenous dose of one of the
following: (1) docetaxel encapsulated in nanoparticles in which the
ligand of the PLA-PEG-lys(urea)glu targeting polymer was
.sup.14C-labeled, (2) docetaxel encapsulated in nanoparticles in
which the encapsulated docetaxel was .sup.14C-labeled, (3)
.sup.14C-labeled conventional docetaxel.
[0091] Blood was drawn at 1, 3, 6, 12, and 24 hours post-dosing and
processed to plasma. Immediately following blood collection, the
rats were euthanized by CO.sub.2 asphyxiation and tissues were
immediately collected, blotted, weighed, and frozen on dry ice.
Tissue samples were stored frozen (approximately -70.degree. C.)
until analysis for radioactivity by liquid scintillation (LS)
counting.
[0092] As shown in FIG. 5, the docetaxel encapsulated in
nanoparticles was gradually cleared from the plasma, exhibiting an
approximate 2-fold decrease in plasma concentration over the 24
hour period studied. These results are indicative of limited or
delayed nanoparticle clearance via the mononuclear phagocyte system
(MPS) relative to that often observed for particulate formulations.
Without wishing to be bound by theory, this difference in plasma
clearance times may be attributed to certain nanoparticle
characteristics, including particle size and surface properties
(e.g., surface charge and porosity).
[0093] The distinctions in plasma profiles of docetaxel
encapsulated in nanoparticles and conventional docetaxel indicate
that encapsulation of docetaxel in the nanoparticles prevents it
from being rapidly distributed from the plasma compartment,
resulting in significantly higher C.sub.max and AUC values relative
to conventional docetaxel.
[0094] The differences in the profiles of docetaxel encapsulated in
nanoparticles wherein the ligand of the PLA-PEG-lys(urea)glu
targeting polymer was .sup.14C-labeled and the docetaxel
encapsulated in nanoparticles wherein the encapsulated docetaxel
was .sup.14C-labeled are reflective of the controlled release of
docetaxel from the polymeric matrix of the nanoparticles. If
docetaxel was released very quickly from the nanoparticles, it
would be expected to be rapidly distributed from the plasma,
yielding a profile similar to that of conventional docetaxel.
Conversely, if docetaxel was retained in the nanoparticles over
this timeframe, the profiles of the docetaxel encapsulated in
nanoparticles wherein the ligand of the PLA-PEG-lys(urea)glu
targeting polymer was .sup.14C-labeled and the docetaxel
encapsulated in nanoparticles wherein the encapsulated docetaxel
was .sup.14C-labeled would be superimposable.
[0095] Example Tables 3.1, 3.2, and 3.3 present the tissue
distribution of radioactivity determined in rats after intravenous
(IV) administration of (1) docetaxel encapsulated in nanoparticles
in which the ligand of the PLA-PEG-lys(urea)glu targeting polymer
was .sup.14C-labeled, (2) docetaxel encapsulated in nanoparticles
in which the encapsulated docetaxel was .sup.14C-labeled, and (3)
.sup.14C-labeled conventional docetaxel, respectively. Example FIG.
5 contains the radioactivity concentration curves of the test
articles determined in plasma, liver, spleen, and bone marrow.
[0096] Lower levels of nanoparticles (i.e., radioactivity from the
.sup.14C-labeled targeting polymer) were detected in all tissues
relative to plasma except in the spleen, where nanoparticle
concentrations were higher than plasma at 12 and 24 hours. It
cannot be determined to what extent the radioactivity in tissues
reflect the content in blood contained within the tissues versus
the tissues themselves, because the tissues were not
exsanguinated.
[0097] At time points closely following administration, the
concentration of docetaxel encapsulated in nanoparticles was higher
in most tissues than conventional docetaxel. After 24 hours, the
concentration of docetaxel derived from the nanoparticles was lower
than or approximately the same as the concentration of conventional
docetaxel in all of the tissues evaluated, except the spleen.
[0098] Although the concentration of docetaxel encapsulated in
nanoparticles was higher than conventional docetaxel at early
timepoints and throughout the 24 hour period in the spleen, the
nanoparticles doped with docetaxel were well tolerated at
approximately 10 mg/kg docetaxel dose. In addition, body weight
changes and clinical observations in the Sprague-Dawley rats
indicate that the docetaxel encapsulated in nanoparticles was
tolerated as well as conventional docetaxel through a range of
acute doses (5-30 mg/kg docetaxel).
TABLE-US-00002 EXAMPLE TABLE 3.1 Tissue Distribution of
Radioactivity Determined in Rats after IV Administration of
Nanoparticles Containing .sup.14C-Targeting Polymer. Bone Small
Large Time Plasma Liver Spleen Heart Lungs Marrow Intestine
Intestine (h) (nCi/mL) (nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g)
(nCi/g) (nCi/g) 1 2341 .+-. 168 337 .+-. 14 829 .+-. 26 180 .+-. 23
294 .+-. 78 109 .+-. 25 56 .+-. 2.5 36 .+-. 3.1 3 2023 .+-. 58 334
.+-. 43 1141 .+-. 75 190 .+-. 62 264 .+-. 38 191 .+-. 122 50 .+-.
5.4 33 .+-. 3.6 6 2001 .+-. 71 364 .+-. 23 1789 .+-. 173 174 .+-.
25 263 .+-. 40 372 .+-. 8.7 48 .+-. 8.0 43 .+-. 10 12 1445 .+-. 59
375 .+-. 41 2079 .+-. 205 151 .+-. 21 266 .+-. 24 390 .+-. 58 71
.+-. 3.6 40 .+-. 6.1 24 998 .+-. 55 398 .+-. 59 2808 .+-. 238 119
.+-. 11 218 .+-. 26 594 .+-. 248 88 .+-. 17 38 .+-. 5.0
TABLE-US-00003 EXAMPLE TABLE 3.2 Tissue Distribution of
Radioactivity Determined in Rats after IV Administration of
Nanoparticles Containing .sup.14C-Docetaxel. Bone Small Large Time
Plasma Liver Spleen Heart Lungs Marrow Intestine Intestine (h)
(nCi/mL) (nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g) 1
753 .+-. 149 267 .+-. 45.sup. 889 .+-. 43 156 .+-. 15.sup. 277 .+-.
27 142 .+-. 20 409 .+-. 158 71 .+-. 24 3 265 .+-. 52 127 .+-.
12.sup. 999 .+-. 94 80 .+-. 3.6 154 .+-. 9.0 127 .+-. 17 219 .+-.
30.sup. 151 .+-. 37 6 140 .+-. 38 88 .+-. 9.7 972 .+-. 44 69 .+-.
9.8 118 .+-. 23 121 .+-. 5.9 119 .+-. 20.sup. 133 .+-. 38 12 24
.+-. 1.9 47 .+-. 6.3 854 .+-. 56 41 .+-. 2.1 .sup. 58 .+-. 8.4
.sup. 89 .+-. 9.3 50 .+-. 2.7 98 .+-. 14 24 5.7 .+-. 1.0 22 .+-.
3.1 634 .+-. 95 23 .+-. 1.3 .sup. 44 .+-. 2.5 .sup. 33 .+-. 8.2 43
.+-. 9.3 58 .+-. 4.9
TABLE-US-00004 EXAMPLE TABLE 3.3 Tissue Distribution of
Radioactivity Determined in Rats after IV Administration of
Conventional .sup.14C-Docetaxel. Bone Small Large Time Plasma Liver
Spleen Heart Lungs Marrow Intestine Intestine (h) (nCi/mL) (nCi/g)
(nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g) (nCi/g) 1 4.9 .+-. 0.4 78
.+-. 15 100 .+-. 9.4 71 .+-. 2.5 82 .+-. 9.6 97 .+-. 4.2 517 .+-.
99.sup. 54 .+-. 3.6 3 1.9 .+-. 0.2 49 .+-. 7.3 81 .+-. 7.5 39 .+-.
1.5 66 .+-. 0.7 83 .+-. 1.0 122 .+-. 43.sup. 166 .+-. 37 6 1.6 .+-.
0.5 49 .+-. 11 82 .+-. 4.6 33 .+-. 1.3 .sup. 993 .+-. 1605* 78 .+-.
1.9 62 .+-. 5.2 185 .+-. 82 12 0.8 .+-. 0.2 55 .+-. 7.4 77 .+-. 11
28 .+-. 1.7 1438 .+-. 1218* 62 .+-. 9.4 41 .+-. 4.9 83 .+-. 18 24
0.6 .+-. 0.1 43 .+-. 4.0 85 .+-. 8.6 24 .+-. 2.6 962 .+-. 99* 41
.+-. 6.8 47 .+-. 19 48 .+-. 34 *Samples likely contaminated during
collection/analysis
Example 4
Tumor Targeting of Docetaxel Encapsulated in Nanoparticles and
Conventional Docetaxel after a Single Dose in a Human Tumor
Xenograft Model (LNCaP)
[0099] Male severe combined immunodeficiency (SCID) mice were
subcutaneously inoculated with human LNCaP prostate cancer cells.
Three to four weeks after inoculation, the mice were assigned to
different treatment groups such that the average tumor volume in
each group was 300 mm.sup.3. At this time, a single intravenous
(IV) dose of 50 mg/kg docetaxel was administered as either
docetaxel encapsulated in nanoparticles or conventional docetaxel.
The test subjects were sacrificed 2 hour or 12 hour post-dose. The
tumors from each group were excised and assayed for docetaxel using
liquid chromatography-mass spectrometry (LC-MS).
[0100] The measured docetaxel concentrations in tumors excised from
the test subjects dosed with docetaxel encapsulated in
nanoparticles or conventional docetaxel are presented in Example
Table 4.1 and FIG. 6. At 12 hours post-dose, the tumor docetaxel
concentration in test subjects receiving docetaxel encapsulated in
nanoparticles was approximately 7 times higher than in the test
subjects receiving conventional docetaxel. These results are
consistent with the pharmacokinetic and tissue distribution data as
well as the proposed mechanism of action wherein the nanoparticles
doped with docetaxel are designed to provide extended particle
circulation times and controlled release of docetaxel from the
nanoparticles so that particles can be targeted to and bind with a
marker or target to increase the amount of docetaxel delivered to
the tumor.
TABLE-US-00005 EXAMPLE TABLE 4.1 Measured Docetaxel Concentration
in Tumors Treated with Docetaxel Encapsulated in Nanoparticles and
Conventional Docetaxel Docetaxel Concentration in the Tumor (ng/mg)
Docetaxel Encapsulated Time (h) Conventional Docetaxel in
Nanoparticles 2 12.9 .+-. 7.9 14.8 .+-. 6.5 12 3.6 .+-. 2.1 25.4
.+-. 15.1
Example 5
Anti-tumor Activity of Docetaxel Encapsulated in Nanoparticles and
Conventional Docetaxel after Repeated Doses in a Human Tumor
Xenograft Model (LNCaP)
[0101] Male severe combined immunodeficiency (SCID) mice were
subcutaneously inoculated with human LNCaP prostate cancer cells.
Three to four weeks after inoculation, the mice were assigned to
different treatment groups such that the average tumor volume in
each group was 250 mm.sup.3. Subsequently, the mice were treated
every other day (Q2D) for four doses, with an eight day holiday,
followed by another four doses at the Q2D schedule.
[0102] Average tumor volumes for each treatment group are shown in
Example FIG. 7. Treatment with either conventional docetaxel or
docetaxel encapsulated in nanoparticles resulted in appreciable
reduction in tumor volume. Tumor volume reduction was greater in
test subjects receiving docetaxel encapsulated in nanoparticles
compared to conventional docetaxel. These results suggest that the
increase in tumor docetaxel concentration in test subjects
receiving nanoparticles doped with docetaxel, compared to
conventional docetaxel, may result in a more pronounced cytotoxic
effect.
Example 6
Acute Dose Range Finding Study of Docetaxel Encapsulated in
Nanoparticles in Sprague-Dawley Rats
[0103] Sixty Sprague-Dawley rats (30/sex) were assigned to 10 dose
groups (3 rats/sex/group) and were administered a single dose of
either docetaxel encapsulated in nanoparticles (5.7, 7.5, 10, 15 or
30 mg/kg body weight) or conventional docetaxel (5.7, 7.5, 10, or
30 mg/kg body weight). The therapeutic compositions were
administered by intravenous (IV) infusion over a 30-minute period
on Day 1, after which the test subjects were observed for 7 days
prior to undergoing a gross necropsy.
[0104] All test subjects survived to their scheduled necropsy.
Clinical observations considered to be potentially related to
administration included piloerection, which appeared near the end
of the 7 day observation period, and discharges from the nose and
eyes. Piloerection was observed for one male rat dosed with 15
mg/kg of docetaxel encapsulated in nanoparticles, and for 5/9 male
rats and 1/9 female rats dosed with 10 mg/kg of conventional
docetaxel or higher. The nature and time of appearance of this
clinical sign were consistent with toxicity that would be expected
from cytotoxic drugs like docetaxel. Nasal and eye discharges
appeared with a pattern that was unrelated to dose level, test
article, sex of the animals, or time after dosing, and this
clinical sign was considered to be possibly related to docetaxel
and/or to stress from the dosing procedure. As shown in Example
Table 6.1, male and female rats dosed with either conventional
docetaxel or docetaxel encapsulated in nanoparticles showed
generally minor deficits in body weight gain or actual body weight
losses that were considered to be due to docetaxel toxicity. The
no-adverse effect level (NOAEL) of nanoparticles doped with
docetaxel in this study was considered to be 7.5 mg/kg.
TABLE-US-00006 EXAMPLE TABLE 6.1 Comparison of Body Weight Changes
in Males and Females Docetaxel Encapsulated Dose in Nanoparticles
Body Conventional Docetaxel Sex (mg/kg) Weight Change (%) Body
Weight Change (%) M 5.7 4.60 8.60 M 7.5 1.67 1.21 M 10 -3.15 -11.55
M 15 -6.23 -9.48 M 30 -7.16 -8.66 F 5.7 3.63 0.34 F 7.5 3.25 -0.11
F 10 -2.49 -0.17 F 15 -2.50 -6.86 F 30 -5.66 -5.89
Example 7
Pharmacokinetics of Vincristine Passively Targeted Nanoparticles in
Rats
[0105] Similar to the procedure in Example 2, rats were
intravenously dosed with 0.5 mg/kg with either nanoparticles
prepared as in FIG. 2 and Example 14 having vincristine and
PLA-PEG, and no specific targeting moiety (passively targeted
nanoparticles (PTNP); or vincristine alone. The release profiles
are shown in FIG. 8.
[0106] Plasma samples were analyzed using LC/MS and the PK analysis
was performed using WinNonlin software. A comparison of the
pharmacokinetics of the nanoparticles with vincristine alone is as
follows:
TABLE-US-00007 Comparison with vincristine alone C.sub.max (ng/mL)
69-fold .uparw. t.sub.1/2 (hr) 1.8-fold .dwnarw. AUC.sub.inf
(hr*ng/mL) 312-fold .uparw. V.sub.z (mL/kg) 592-fold .dwnarw. Cl
(mL/hr/kg) 322-fold .dwnarw.
Example 8
Pharmacokinetics of Methotrexate Passively Targeted Nanoparticles
in Rats
[0107] Similar to the procedure in Example 2, rats were
intravenously dosed with 0.5 mg/kg with either nanoparticles
prepared as in FIG. 2 and Example 15 having methotrexate and
PLA-PEG, and no specific targeting moiety (passively targeted
nanoparticles (PTNP); or methotrexate alone. The release profiles
are shown in FIG. 9.
[0108] Plasma samples were analyzed using LC/MS and the PK analysis
was performed using WinNonlin software. A comparison of the
pharmacokinetics of the nanoparticles with methotrexate alone is as
follows:
TABLE-US-00008 Comparison with methotrexate alone C.sub.max (ng/mL)
10-fold .uparw. t.sub.1/2 (hr) 16-fold .dwnarw. AUC.sub.inf
(hr*ng/mL) 296-fold .uparw. V.sub.z (mL/kg) 19-fold .dwnarw. Cl
(mL/hr/kg) 302-fold .dwnarw.
Example 9
Pharmacokinetics of Paclitaxel Passively Targeted Nanoparticles in
Rats
[0109] Similar to the procedure in Example 2, rats were
intravenously dosed with 1.0 mg/kg with either nanoparticles
prepared as in FIG. 2 having paclitaxel and PLA-PEG (formulation C)
and no specific targeting moiety (passively targeted nanoparticles
(PTNP); or paclitaxel alone. The release profiles are shown in FIG.
10.
[0110] Plasma samples were analyzed using LC/MS and the PK analysis
was performed using WinNonlin software. A comparison of the
pharmacokinetics of the nanoparticles with paclitaxel alone is as
follows:
TABLE-US-00009 Comparison with paclitaxel alone C.sub.max (ng/mL)
297-fold .uparw. t.sub.1/2 (hr) 3-fold .dwnarw. AUC.sub.inf
(hr*ng/mL) 600-fold .uparw. V.sub.z (mL/kg) 1512-fold .dwnarw. Cl
(mL/hr/kg) 516-fold .dwnarw.
Example 10
Pharmacokinetics of Rapamycin (Sirolimus) Passively Targeted
Nanoparticles in Rats
[0111] Similar to the procedure in Example 2, rats were
intravenously dosed with 2.0 mg/kg with either nanoparticles
prepared as in FIG. 2 and Example 16, having rapamycin and PLA-PEG
and no specific targeting moiety (passively targeted nanoparticles
(PTNP); or rapamycin alone. The release profiles are shown in FIG.
11.
[0112] Plasma samples were analyzed using LC/MS and the PK analysis
was performed using WinNonlin software. A comparison of the
pharmacokinetics of the nanoparticles with rapamcyin alone is as
follows:
TABLE-US-00010 Comparison with rapamcyin alone C.sub.max (ng/mL)
297-fold .uparw. t.sub.1/2 (hr) 3-fold .dwnarw. AUC.sub.inf
(hr*ng/mL) 600-fold .uparw. V.sub.z (mL/kg) 1512-fold .dwnarw. Cl
(mL/hr/kg) 516-fold .dwnarw.
Example 11
Tumor Accumulation of Docetaxel Nanoparticles in MX-1 Breast Tumors
in Mice
[0113] Mice with MX-1 breast tumors were randomized into three
groups, receiving docetaxel (3 mice), passively targeted
nanoparticles (Formulation A, without a targeting moiety, PTNP), or
Formulation A. The average tumor mass was 1.7 g (RSD 34%). Mice
were then injected with 10 mg/kg of the test article, then
euthanized 24 hours later and the tumors were removed and analyzed
for docetaxel content using LC/MS/MS. Results are depicted in FIG.
12. The percent of injected dose in the tumor was 3% (for docetaxel
alone), 30% for PTNP, and 30% Formulation A.
Example 12
Pharmacokinetics of Docetaxel Nanoparticles in Primates
[0114] Naive non human primates (3 male and 3 female) were
administered docetaxel, docetaxel nanoparticles (Formulation A) or
docetaxel nanoparticles (Formulation B: 43.25% PLA-PEG(16/5),
43.25% PLA (75 kDa), 10% docetaxel, 2.5% PLA-PEG-lys(urea)glu,
prepared as in Example 14), using and following appropriate ethical
guidelines at all times. 1 male and 1 female were used per dose
group. The dosing day was 1 day and the formulations were
administered by 30 minute IV infusion at 25 mg/m.sup.2 docetaxel or
50 mg/m.sup.2 docetaxel (animals were randomized and then dosed
with 50 mg/m2 on day 29 and PK, hematology and clinical chemistry
were measured for 21 days). At the end of the study, PK, hematology
and clinical chemistry collected over a 21 day period were
assessed. FIG. 12 depicts the results of male (M) and female (F)
PNP. A comparison of the pharmacokinetics of the nanoparticles of
Formulation A (25 mg/m.sup.2 dose) with docetaxel alone is as
follows:
TABLE-US-00011 Comparison with docetaxel alone C.sub.max (ng/mL)
180-fold .uparw. t.sub.1/2 (hr) 3-fold .dwnarw. AUC.sub.inf
(hr*ng/mL) 213-fold .uparw. V.sub.z (mL/kg) 617-fold .dwnarw. Cl
(mL/hr/kg) 212-fold .dwnarw.
[0115] The pharmokinetics were as follows for each NHP group:
A. Docetaxel Alone
TABLE-US-00012 [0116] 25 mg/m2 50 mg/m2 M F M F C.sub.max (ng/mL)
364 596 1210 835 C.sub.max/D 14.6 23.8 24.2 16.7 AUC (hr*ng/mL)
2553 2714 3285 3599 AUC/D 102 109 76.5 72 t.sub.1/2 (hr) 18 31 39
39 V.sub.z (mL/m2) 253783 412186 743682 788340 Cl (mL/hr/m2) 9794
9213 13073 13893
B. Formulation A
TABLE-US-00013 [0117] 25 mg/m2 50 mg/m2 M F M F C.sub.max (ng/mL)
89500 85500 95700 117000 C.sub.max/D 3580 3420 1914 2340 AUC
(hr*ng/mL) 495408 627216 352778 748073 AUC/D 19816 25089 7056 14961
t.sub.1/2 (hr) 7.6 9.1 5.6 6.8 V.sub.z (mL/m2) 554 526 1140 654 Cl
(mL/hr/m2) 50 40 142 67
C. Formulation B
TABLE-US-00014 [0118] 25 mg/m2 50 mg/m2 M F M F C.sub.max (ng/mL)
64500 101500 128000 116000 C.sub.max/D 2580 4060 2560 2320 AUC
(hr*ng/mL) 956312 1442885 1960145 1395580 AUC/D 38252 57715 39203
27912 t.sub.1/2 (hr) 13.9 17.8 17.8 15.5 V.sub.z (mL/m2) 525 445
657 803 Cl (mL/hr/m2) 26.1 17.3 25.5 35.8
Example 13
Preparation of Docetaxel Nanoparticles
[0119] An organic phase is formed composed of a mixture of
docetaxel (DTXL) 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.
[0120] 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.
[0121] The primary emulsion is then formed into a fine emulsion
through the use of a high pressure homogenizer.
[0122] After 2-3 passes the particle size was not significantly
reduced, and successive passes can even cause a particle size
increase. 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.
[0123] 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%. FIGS. 8 and 9 depicts the effect of solids
concentration on particle size and drug loading; with the exception
of the 15-175 series, all batches are placebo. For placebo batches
the value for % solids represents the % solids were drug present at
the standard 20% w/w.
[0124] Table A summarizes the emulsification process
parameters.
TABLE-US-00015 TABLE A Parameter Value Coarse emulsion formation
Rotor stator homogenizer Homogenizer feed pressure 4000-5000 psi
per chamber Interaction chamber(s) 2 .times. 200 .mu.m Z-chamber
Number of homogenizer passes 2-3 passes Water phase 0.1% [sodium
cholate] W:O ratio 5:1 [Solids] in oil phase 30%
[0125] 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.
[0126] 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-00016 TABLE B Summary quench process parameters. Parameter
Value Initial quench temperature <5.degree. C. [Tween-80]
solution 35% Tween-80:drug ratio 25:1 Q:E ratio 5:1 Quench
hold/processing temp .ltoreq.5.degree. C. (with current 5:1 Q:E
ratio, 25:1 Tween-80:drug ratio)
[0127] 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.
[0128] 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.
[0129] 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-00017 TABLE C TFF Parameters Parameter Value Membrane
Material Regenerated cellulose - Coarse Screen Membrane Molecular
Weight Cut off 300 kDa Crossflow Rate 11 L/min/m.sup.2
Transmembrane Pressure 20 psid Concentration of Nanoparticle 30
mg/ml Suspension for Diafiltration Number of Diavolumes .gtoreq.15
(based on flux increase) Membrane Area ~1 m.sup.2/kg
[0130] 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. By exposing
the nanoparticle slurry to elevated temperature during workup,
`loosely encapsulated` drug can be removed and improve the product
stability at the expense of a small drop in drug loading.
[0131] After the filtration process the nanoparticle suspension
(concentration 50 mg/ml), 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. Filtration flow rate is .about.1.3
L/min/m.sup.2.
[0132] 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. 0.2 m.sup.2
of filtration surface area per kg of nanoparticles for depth
filters and 1.3 m.sup.2 of filtration surface area per kg of
nanoparticles for the sterilizing grade filters can be used.
Example 14
Preparation of Nanoparticles with Long Release Properties
[0133] The nanoparticle preparation protocol described in Example
12 was modified to produce slow release nanoparticles.
[0134] A batch of nanoparticles was produced that incorporated a
50:50 ratio of 100 .quadrature.L 7E PLA (see Table 1) with the 16/5
PLA-PEG copolymer. The addition of high molecular weight PLA is
thought to decrease drug diffusion by increasing crystallinity,
raising the glass transition temperature, or reducing drug
solubility in the polymer.
TABLE-US-00018 TABLE 1 High Molecular Weight PLA Tested Molecular
Weight Molecular Weight PLA Manufacturer (Mn) (Mw) 100 DL 7E
Lakeshore Polymer 80 kDa 124 kDa
[0135] The addition of high molecular weight PLA resulted in larger
particle size when all other formulation variables were kept
constant. In order to obtain slow release nanoparticles with
comparable sizes as nanoparticles prepared without the high
molecular weight PLA, the concentration of solids in the oil phase
was reduced and the concentration of sodium cholate in the water
phase was increased. Table 2 illustrates the slow release
nanoparticle formulation.
TABLE-US-00019 TABLE 2 Slow Release Formulation Summary % Solids %
Sodium in Oil Cholate in % Drug Particle Polymers Used Phase Water
Phase Load Size (nm) 50% BI 16/5 PLA-PEG 20% 2.0% 11.7% 139.8 50%
Lakeshore 100 DL 7E PLA
Example 14
Nanoparticles with Vincristine
[0136] Nanoparticle batches were prepared using the general
procedure of Example 12, 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.
[0137] 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: 5% solids:
7.98 g benzyl alcohol+30.02 g ethyl acetate; 30% solids: 0.98 g
benzyl alcohol+3.69 g ethyl acetate
[0138] An aqueous solution is prepared with 0.5% sodium cholate, 2%
benzyl alcohol, and 4% ethyl acetate in water. Add to the bottle
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.
[0139] For the formation of emulsion, a ratio of aqueous phase to
oil phase is 5:1. 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.
[0140] The emulsion is poured into quench (D.I. water) at
<5.degree. C. 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 concentrated
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 liters) 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.
[0141] Solids concentration of 0.45 um filtered final slurry was
determined by filtering about 5 mL of final slurry sample before
addition of sucrose through 0.45 .mu.m syringe filter; to tared 20
mL scintillation vial add 4 mL of filtered sample and dry under
vacuum on lyo/oven.
[0142] The remaining sample of unfiltered final slurry was frozen
with sucrose.
Vincristine Formulations
TABLE-US-00020 [0143] Composition by Components Wt.(%)
mPEG(5k)-lPLA(16K)/Vincristine 96/4 mPEG(5k)-lPLA(16K)/Vincristine
95/5 mPEG(5k)-lPLA(16K)/Vincristine 96/4
mPEG(5k)-lPLA(16K)/lPLA(16K)/Vincristine 46/46/8
mPEG(5k)-lPLA(16K)/lPLA(16K)/Vincristine 47/47/6
[0144] Analytical Characterization of Vincristine Formulations:
TABLE-US-00021 Size (nm) Drug Load (%) Encapsulation Efficiency (%)
103 4.4 21.8 110 4.6 22.8 115 4.2 20.8 146 8.3 41.6 98 6.0 30.0
Example 15
Nanoparticles with Methotrexate
[0145] Drug was dissolved in the inner aqueous phase consisting of
water with 1-arginine or NaOH used for solubilizing the drug. The
polymer (16-5 PLA-PEG) was dissolved in the oil phase organic
solvent system, such as dichloromethane (DCM) at 20% solid
concentration. The outer aqueous phase consisted mainly of water
with 1% sodium cholate (SC) as surfactant, unless noted otherwise.
The w/o emulsion was prepared by adding the inner aqueous phase
into the oil phase under rotor stator homogenization or sonication
(using Branson Digital Sonifier) at a w/o ratio of 1:10. The coarse
w/o/w emulsion was also prepared by adding the w/o emulsion into an
outer aqueous phase under either rotor stator homogenization or
sonication at o/w ratio of 1:10. The fine w/o/w emulsion was then
prepared by processing the coarse emulsion through a Microfluidics
high pressure homogenizer (M110S pneumatic) at 45000 psi with a 100
.mu.m Z-interaction chamber. The fine emulsion was then quenched
into cold DI water at 10:1 quench:emulsion ratio. These w/o, o/w
and emulsion: quench ratios were maintained at 1:10 for all w/o/w
experiments, unless noted otherwise. Polysorbate 80 (Tween 80) was
then added as a process solubilizer to solubilize the
unencapsulated drug. No drug precipitation was observed at a
drug:Tween 80 ratio of 1:200. The batch was then processed with
ultrafiltration followed by diafiltration to remove solvents,
unencapsulated drug and solubilizer. The particle size measurements
were performed by Brookhaven DLS and/or Horiba laser diffraction.
To determine drug load, slurry samples were analyzed by HPLC and
solid concentration analysis. The slurry retains were then diluted
with sucrose to 10% before freezing. All ratios listed are on a w/w
basis, unless specified otherwise.
[0146] Using 16/5 PLA-PEG dissolved in ethyl acetate afforded
particles between 77-85 nm in size at .ltoreq.6% solid
concentration in an outer aqueous phase consisting of 1% SC in DI
water. Emulsions were formed under sonication at 30% amplitude. Gel
formation occurred in the initial w/o emulsion with .gtoreq.6%
solid concentration. The inner aqueous phase MTX concentration was
increased to 225 mg/ml using 1-arginine. The batch was made with
20% solids in the oil phase, consisting of 28/5 PLGA-PEG dissolved
in DCM. Here, both the inner w/o and outer w/o/w emulsions were
formed by rotor stator homogenization followed by 2 passes at 45 k
psi using a high pressure homogenizer. The nanoparticle suspension
was quenched in cold DI water followed by
ultrafiltration/diafiltration work-up. HPLC and PSD analysis was
used to determine that the drug load stayed at 0.38% for 131 nm
particles.
[0147] Three different batches can be prepared according to the
general procedure with the following modifications; Inner aqueous
phase MTX concentration was 225 mg/ml in 0.66N NaOH solution, i.e.,
a 1-arginine:MTX molar ratio of 1.45:1; Span 80/Tween 80 surfactant
mix (HLB=6.2) was used as the oil phase surfactant; Batch 55-101 C:
16/5 PLA-PEG was used instead of 28/5 PLGA-PEG. The emulsion
process for all three batches remained similar. The highest drug
load was obtained for the 16/5 PLA-PEG batch at 2.23% while the
drug load was 0.2% and 0.04% for other batches.
Example 16
Preparation of Sirolimus Nanoparticles
[0148] 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. 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.
[0149] The primary emulsion is then formed into a fine emulsion
through the use of a high pressure homogenizer. The process is
continued as in Example 12.
Representative Rapamycin (Sirolimus) Formulations:
TABLE-US-00022 [0150] Drug Name Polymer Size (nm) Loading 5% Solid
16/5 PLA/PEG 123.1 3.61% 16/5 PLA/PEG + PLA 119.7 4.49% 15% Solid
16/5 PLA/PEG 82.1 4.40% 16/5 PLA/PEG + PLA 120.6 11.51% 23% Solid
16/5 PLA/PEG 88.1 7.40% 16/5 PLA/PEG + PLA 118.3 7.8% 30% Solid
16/5 PLA/PEG 88.5 10.26% 16/5 PLA/PEG + PLA 118.3 10.18%
[0151] Although the invention has been described in considerable
detail with reference to certain preferred aspects thereof, other
versions are possible. Therefore the spirit and scope of the
appended claims should not be limited to the description and the
preferred versions contained within this specification.
INCORPORATION BY REFERENCE
[0152] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
EQUIVALENTS
[0153] Various modifications of the invention and many further
embodiments thereof, in addition to those shown and described
herein, will become apparent to those skilled in the art from the
full contents of this document, including references to the
scientific and patent literature cited herein. The subject matter
herein contains important information, exemplification and guidance
that can be adapted to the practice of this invention in its
various embodiments and equivalents thereof.
* * * * *