U.S. patent application number 11/632884 was filed with the patent office on 2008-12-04 for particulate constructs for release of active agents.
Invention is credited to Christine J. Allen, Lawrence D. Mayer, Robert K. Prud'Homme, Walid S. Saad.
Application Number | 20080299205 11/632884 |
Document ID | / |
Family ID | 35787676 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299205 |
Kind Code |
A1 |
Mayer; Lawrence D. ; et
al. |
December 4, 2008 |
Particulate Constructs For Release of Active Agents
Abstract
Particulate constructs stabilized by amphiphilic copolymers and
comprising at least one active coupled to a hydrophobic moiety
provide sustained release of the active in both in vitro and in
vivo environments.
Inventors: |
Mayer; Lawrence D.; (North
Vancouver, CA) ; Prud'Homme; Robert K.;
(Lawrenceville, NJ) ; Allen; Christine J.;
(Toronto, CA) ; Saad; Walid S.; (Princeton,
NJ) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
35787676 |
Appl. No.: |
11/632884 |
Filed: |
July 19, 2005 |
PCT Filed: |
July 19, 2005 |
PCT NO: |
PCT/US05/25549 |
371 Date: |
July 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60589164 |
Jul 19, 2004 |
|
|
|
Current U.S.
Class: |
424/489 ; 424/59;
514/772.3; 514/774; 514/785 |
Current CPC
Class: |
A61K 47/54 20170801;
A61K 47/59 20170801; A61K 47/60 20170801; A61K 45/06 20130101; A61Q
17/04 20130101; A61K 47/22 20130101; A61K 47/6935 20170801; A61K
8/85 20130101; A61K 47/55 20170801; A61K 47/551 20170801; B82Y 5/00
20130101; A61K 2800/57 20130101; A61K 47/593 20170801; A61K 47/6931
20170801; A61K 47/545 20170801; A61K 47/543 20170801; A61K 9/5153
20130101; A61K 47/552 20170801; A61K 47/58 20170801 |
Class at
Publication: |
424/489 ;
514/785; 424/59; 514/772.3; 514/774 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 47/14 20060101 A61K047/14; A61K 8/30 20060101
A61K008/30; A61K 47/30 20060101 A61K047/30; A61K 47/42 20060101
A61K047/42 |
Claims
1. A composition comprising particulate constructs wherein said
constructs comprise an amphiphilic stabilizer, and a conjugate of
the formula (active-linker).sub.n-hydrophobic moiety (1) wherein n
is an integer of 1-100; and wherein "active" refers to one or more
compounds that have a desired activity; "linker" is a covalent
bond, a divalent residue of an organic molecule or a chelator; and
"hydrophobic moiety" refers to the residue of an organic molecule
that is insoluble in aqueous solution; and (a) wherein the one or
more active is other than a pharmaceutical, nutritional compound or
diagnostic; or (b) wherein said constructs comprise at least two
different active therapeutic agents; or (c) wherein said
composition is obtainable by rapid micromixing of at least two jet
streams, one or more jet streams comprising solution(s) of the
amphiphilic stabilizer(s) and the conjugate(s) of formula 1 and the
other stream(s) comprising an anti-solvent for the conjugate and
the hydrophobic portion of the amphiphilic stabilizer, said jet
streams fed at high velocity; or (d) wherein said hydrophobic
moiety is a polymer having a molecular weight>800 Da or is a
natural product selected from hydrophobic vitamins, carotenoids,
retinols, folic acid, dihydrofolate, cholecalciferol, calcitriol
hydroxycholecalciferol, ergocalciferol, .alpha.-tocopherol,
.alpha.-tocopherol acetate, .alpha.-tocopherol nicotinate, and
estradiol.
2-36. (canceled)
37. The composition of claim 1, wherein the one or more active is
other than a pharmaceutical, nutritional compound or
diagnostic.
38. The composition of claim 1, wherein said constructs comprise at
least two different active therapeutic agents.
39. The composition of claim 1, wherein said composition is
obtainable by rapid micromixing of at least two jet streams, one or
more jet streams comprising solution(s) of the amphiphilic
stabilizer(s) and the conjugate(s) of formula 1 and the other
stream(s) comprising an anti-solvent for the conjugate and the
hydrophobic portion of the amphiphilic stabilizer, said jet streams
fed at high velocity.
40. The composition of claim 1, wherein said hydrophobic moiety is
a polymer having a molecular weight>800 Da or is a natural
product selected from hydrophobic vitamins, carotenoids, retinols,
folic acid, dihydrofolate, cholecalciferol, calcitriol,
hydroxycholecalciferol, ergocalciferol, .alpha.-tocopherol,
.alpha.-tocopherol acetate, .alpha.-tocopherol nicotinate, and
estradiol.
41. The composition of claim 1, wherein n is an integer of
2-100.
42. The composition of claim 37, wherein the one or more active is
a pigment, an ink, a toner, a pesticide, a viscoelastic agent, an
herbicide, a fluorescent probe, a sunscreen, a fragrance, or a
flavor compound.
43. The composition of claim 1, wherein the hydrophobic moiety is
vitamin E, vitamin A or vitamin K, or a retinol; or wherein the
hydrophobic moiety is polycaprolactone, polylactic acid,
polystyrene, polybutadiene, polycaproic acid, polymethylbenzylate,
poly(D,L-lactide), poly(D,L-lactide-co-glycolide), poly(glycolide),
poly(hydroxybutyrate), poly(alkylcarbonate) poly(orthoesters),
polyesters, poly(hydroxyvaleric acid), or copolymers thereof,
and/or wherein the amphiphilic stabilizer is methoxypolyethylene
glycol (mPEG)-polycaprolactone (PCL), mPEG-polystyrene,
mPEG-polybutadiene, polyacrylic acid-polybutylacrylate, or
mPEG-polylactate, block copolymers of polyethylene oxide,
polypropylene oxide, or polybutylene oxide, or is a copolymer or a
gelatin.
44. The composition of claim 1, wherein the linker is the residue
of a divalent organic molecule, and said molecule comprises a site
for hydrolytic cleavage and/or a site for enzymatic cleavage or a
site for photolytic cleavage, or wherein the linker is a
chelator.
45. The composition of claim 38, wherein said constructs comprise
at least two different active therapeutic agents that are
maintained at non-antagonistic ratio when administered to a
subject.
46. The composition of claim 1, wherein the particulate constructs
comprise 10.sup.3-10.sup.7 conjugates of formula (1).
47. The composition of claim 1, wherein the particulate constructs
have an average diameter less than 5.mu..
48. The composition of claim 1, which is in the form of an
emulsion.
49. A method to administer a combination treatment to a subject,
which method comprises administering a composition of claim 45 to a
subject in need of such treatment.
Description
RELATED APPLICATION
[0001] This application claims benefit of U.S. application Ser. No.
60/589,164 filed 19 Jul. 2004, which is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0002] The description relates to compositions and methods for
improved delivery and performance of active agents. More
particularly, the invention concerns particulate constructs
stabilized by an amphiphilic compound and comprising at least one
active agent coupled through a linker to a hydrophobic moiety,
which agent can be released from the construct by cleavage of the
linker.
BACKGROUND ART
[0003] Sustained release is desirable in many applications to
provide optimal use and effectiveness of active agents, including
pharmaceuticals, cosmetics, food, and fragrances. Attempts have
been made to solubilize, target, stabilize, and control the release
of substances, including use of microparticles, nanoparticles, and
polymer conjugation.
[0004] Approaches based on using polymer encapsulation to formulate
substances in microparticles or larger matrices have succeeded in
delaying their release. In such formulations, the release of the
encapsulated subsequence is controlled by diffusion out of the
polymer or by erosion of the matrix itself. This approach is not
effective in smaller particles, such as nanoparticles.
Nanoparticulate dimensions may be required in a number of
applications, such as drug delivery, in particular to tumors where
particulates in the size range<200 nm accumulate in tumors
whereas larger particles do not. While the art provides many
descriptions for preparation of nanoparticles containing active
agents, none is completely satisfactory. See, e.g., Mu, L., et al.,
Journal of Controlled Release (2003) 86:33-48; Fonseca, C., et al.,
Journal of Controlled Release (2002) 83:273-286.
[0005] Another strategy used to achieve controlled release has been
through the use of polymer conjugates of actives with cleavable
groups. (Frerot, E., et al., European Journal of Organic Chemistry
(2003) 967-971.) A common polymer used in drug delivery is poly
(ethylene glycol) (PEG) (Greenwald, R. B., et al., Critical Reviews
in Therapeutic Drug Carrier Systems (2000) 17:101-161; Greenwald,
R. B., Journal of Controlled Release (2001) 74:159-171).
Conjugation of drugs to PEG has been shown to provide long
circulation times in vivo, and increases the solubility of
hydrophobic drug. Pharmaceutically active proteins have also been
coupled to PEG, resulting in alteration of properties; increased
bioavailability, decreased immunogenicity, and enhanced solubility.
While this strategy provided sustained in vitro drug release
profiles, the drug release profiles in vivo showed significantly
faster rates. (Greenwald, R. B., et al., Journal of Medicinal
Chemistry (1996) 39:424-431.)
[0006] The literature with respect to controlled release systems
and particulate carriers for pharmaceuticals and other compounds is
extensive, and the following represent only illustrative
documents.
[0007] U.S. Pat. Nos. 6,429,200 and 6,673,612 describe reverse
micelles for carrying nucleic acids or other actives into cells.
U.S. Pat. No. 6,676,963 describes nanoparticulate formulations for
targeted drug delivery to tissues and organs. PCT publication WO
02/098465 describes lipid-based vehicles for delivery of
pharmaceuticals comprising an internalizing peptide. PCT
publication WO 03/028696 describes particulate delivery vehicles
for coordinating the release of combinations of drugs. A
multiplicity of liposomal formulations have been used for many
years to deliver drugs.
[0008] Chelators for release of platinum-containing antitumor
agents are described, for example, by Nishiyama, N., et al., J.
Controlled Release (2001) 74:83-94 and by Nishiyama, N., et al, J.
Cancer Res. (2003) 63:8977-8983.
[0009] Drug preparations have been formulated using mixed micellar
and emulsion type formulations, including the use of PEG-modified
phospholipids to stabilize oil in water emulsions. (Alkan-Onyuksel,
et al., Pharm. Res. (1994) 11:206-212, Lundberg, J. Pharm.
Pharmacol. (1997) 49:16-21; Wheeler, et al., Pharm. Sciences (1994)
83:1558-1564).
[0010] For example, U.S. Pat. No. 4,610,868 describes a matrix
material having a particle size in the range of 500 nm-100 .mu.m
which is composed of a hydrophobic compound and an amphipathic
compound. The resulting "lipid matrix carriers" encapsulate
biologically active agents and effect release from the matrix. U.S.
Pat. No. 5,869,103 describes particulate compositions in the size
range of 10 nm-200 .mu.m where the particles are formed by
combining emulsions of an active agent with mixtures of a
biodegradable polymer and a water-soluble polymer.
[0011] U.S. Pat. No. 5,145,684 describes particulate preparations
wherein a crystalline drug substance is itself coated with a
surface modifier. Similarly, U.S. Pat. No. 5,470,583 describes
nanoparticles having nonionic surfactants as a surface modifier
associated with a charged phospholipid. The biologically active
substance, itself having a particle size of <400 nm, is used as
the core of the particles.
[0012] U.S. Pat. No. 5,891,475 describes drug delivery vehicles
which contain hydrophilic cores such as those prepared from
polysaccharides. The particles are treated to contain an external
layer of fatty acids grafted onto the core by covalent bonds.
[0013] U.S. Pat. No. 5,188,837 describes microparticles which are
generally in the size range of 1-38 .mu.m which contain a solid
hydrophobic polymer as a core and a phospholipid, such as
phosphatidyl choline or lecithin as an exterior coating. According
to this disclosure, other phospholipids such as phosphatidyl
inositol and phosphatidyl glycerol are unworkable in this system.
U.S. Pat. No. 5,543,158 discloses 1 nm-1 .mu.m particles with
polymeric cores and a surface layer of PEG, which may be linked
covalently to a biologically active agent contained therein.
[0014] Perkins, W. R., et al., Int. J. Pharmaceut. (2000) 200:27-39
describe "lipocores" which are formed from a core of a poorly
water-soluble drug surrounded by a PEG-conjugated lipid.
[0015] Gref, R., et al, Coll and Surf B: Biointerfaces (2000)
18:301-313 describe the nature of protein absorption onto
PEG-coated nanoparticles formed from various polymers and
copolymers, including polycaprolactone. Although it is recognized
that such particles might be useful in pharmaceutical applications,
only the particles themselves were studied. Lemoine, D., et al.,
Biomaterials (1996) 17:2191-2197 reports studies of various
nanoparticles composed of, among other polymers, polycaprolactone.
While recognizing these as useful in delivery systems, only the
particles themselves were studied.
[0016] Lamprecht, A., et al., Int. J. Pharmaceut. (2000)
196:177-182 reports the study of the effect of the use of
microfluidizers on the particle size of nanoparticles obtained
using various hydrophobic polymers and copolymers.
[0017] Kim, S-Y., et al., J. Cont. Rel. (2000) 65:345-358 describe
copolymeric nanospheres of Pluronic.RTM. with polycaprolactone
(PCL). Nanospheres of Pluronic.RTM./PCL block copolymers having an
average diameter of <200 nm were loaded with endomethicin and
evaluated with regard to cytotoxicity, drug release, drug loading
efficiency and physical characteristics. The particles are formed
entirely of the block copolymer.
[0018] The literature regarding liposomal preparations for delivery
and release of drugs is extensive; suffice it to say that the
concept of encapsulating pharmaceuticals in liposomes is well
established and highly nuanced.
[0019] Particulate constructs for sustained or controlled delivery
of active agents is not confined to pharmaceuticals. For example,
U.S. Pat. No. 5,928,832 describes latex emulsions containing toner
for use in photocopying processes. U.S. Pat. No. 5,766,818
describes latex emulsions containing toner with hydrolyzable
surfactants. U.S. patent publication 2004/0221989 describes
surfactant compositions designed to decompose so as to reduce
viscosity of their surroundings. U.S. 2004/0152913 describes
cleavable surfactants for use in MALDI-MS analysis of hydrophobic
proteins. U.S. Pat. No. 6,559,243 describes glyoxylic compounds
coupled to active ingredients which are released on contact with an
aqueous medium.
[0020] Despite the substantial number of preparations of
microparticle, matrix chelator and nanoparticle formulations
designed for drug delivery and other applications, an ideal
composition has not been achieved.
[0021] One important application of controlled release delivery
systems relates to the administration of drug combinations where it
is desirable to coordinate the release of such drugs.
[0022] The progression of many life-threatening diseases such as
cancer, AIDS, infectious diseases, immune disorders and
cardiovascular disorders are influenced by multiple molecular
mechanisms. Due to this complexity, achieving cures with a single
agent has been met with limited success. Thus, combinations of
agents have often been used to combat disease, particularly in the
treatment of cancers. It appears that there is a strong correlation
between the number of agents administered and cure rates for
cancers such as acute lymphocytic leukemia and metastatic
colorectal cancer. To date, virtually all curative regimens for
cancer rely on drug combination cocktails in which optimal dosing
schedules of agents with differing toxicities were determined in
extensive post-marketing clinical trials.
[0023] Administration of free drug cocktails often results in rapid
clearance of one or all of the drugs before reaching the tumor
site. For this reason, many drugs have been incorporated into
delivery vehicles designed to `shield` them from mechanisms that
would otherwise result in their clearance from the bloodstream.
More relevant to the present invention, compositions wherein agents
are encapsulated or otherwise associated with particulate delivery
vehicles so that the vehicles control the pharmacokinetics and
assure coordinated delivery are described in PCT application
PCT/CA02/01500 as well as in PCT applications PCT/CA2004/000507 and
PCT/CA2004/000508. The formulations of the present invention offer
an alternative controlled release mechanism for these drug
combinations.
DISCLOSURE OF THE INVENTION
[0024] The present invention provides particulate constructs that
can be adapted to the release of active agents of various types
useful in both pharmaceutical and non-pharmaceutical applications.
These delivery systems provide high loading capacity for active
compounds as well as provide a means for controlled release of the
active, reduction in toxicity where relevant, and, if desired,
selective delivery to a target site. The active agents may include
various therapeutic agents such as platinum agents, taxanes and
antibiotics, actives important in other applications such as
pigments, dyes, fragrances and flavors, and may be applied in in
vivo therapeutic and diagnostic contexts, in agricultural
applications and in industrial uses.
[0025] Thus, in one aspect, the invention is directed to a
particulate construct comprising an amphiphilic stabilizer, and
[0026] a conjugate of the formula
(active-linker).sub.n-hydrophobic moiety (1)
[0027] wherein n is an integer of 1-100; and
[0028] wherein "active" refers to a compound that has a desired
activity;
[0029] "linker" is a covalent bond, a divalent residue of an
organic molecule or a chelator; and
[0030] "hydrophobic moiety" refers to the residue of an organic
molecule that is insoluble in aqueous solution.
[0031] In various embodiments, the active may be a fragrance, a
pharmaceutical, a diagnostic agent, a toner, or any compound with a
desirable activity. As noted in formula (I), a multiplicity of
active compounds may be coupled to the same hydrophobic moiety,
which may be a hydrophobic polymer with multiple linking sites, or
a smaller molecule, such as a vitamin or steroid. More than one
type of active agent may also be included, making the constructs
particularly useful for combination therapy. In any event, by
providing the delivery vehicle in this form, controlled release of
the active, either over time or at a desired site, is
facilitated.
[0032] Thus, in another aspect, the invention provides a method to
deliver active compounds in a controlled manner over time or at a
selected target.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 provides a depiction of poly (ethylene glycol) based
paclitaxel prodrug prepared by the method of Greenwald, et al, J.
Med. Chem. (1996) 39:424-431.
[0034] FIG. 2 provides a depiction of poly (ethylene glycol) based
cisplatin complex prepared by the method of Ohya, et al, Polymers
for Adv. Tech. (2000) 11:635-641.
[0035] FIG. 3 provides a depiction of an exemplary delivery vehicle
of the present disclosure including a combination of active
agents/drugs. Three steps are depicted, including (1) preparation
of the polymers, (2) mixture of the polymers and (3) administration
of the delivery vehicle.
MODES OF CARRYING OUT THE INVENTION
[0036] The present invention provides particularly advantageous
particulate constructs which are adaptable to the controlled
release of a wide variety of active agents. A single active agent
may be released from a single particulate construct, or a
multiplicity of such agents may be released. This may result from a
multiplicity of such actives linked in a single conjugate to a
hydrophobic moiety which can support covalent or chelator based
linkage to a multiplicity of agents, and/or a multiplicity of such
conjugates may be accommodated within a single particulate
construct.
[0037] In describing the invention, the following meanings are
attributed to the terms employed.
[0038] Unless defined otherwise, all terms of art, notations and
other scientific terms or terminology used herein have the same
meaning as is commonly understood by one of ordinary skill in the
art to which this invention belongs. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial
difference over what is generally understood in the art. Many of
the techniques and procedures described or referenced herein are
well understood and commonly employed using conventional
methodology by those skilled in the art. As appropriate, procedures
involving the use of commercially available kits and reagents are
generally carried out in accordance with manufacturer defined
protocols and/or parameters unless otherwise noted.
[0039] As used herein, "a" or "an" means "at least one" or "one or
more."
[0040] The term "active agent" or "active" as used herein refers to
chemical moieties used in a variety of applications including
therapy or diagnosis. Examples are therapeutic agents, imaging
agents, diagnostic agents, radionuclides, metal ions, inks,
fragrances, viscoelastic agents, flavors, and, indeed, any chemical
substance that has a desired behavior or activity. The solubility
range of the actives ranges from "insoluble" in water or buffer, to
those that are "sparingly soluble" or "soluble."
[0041] As used herein, "insoluble in aqueous medium" means that the
substance can be dissolved in an aqueous solution at physiological
ionic strength only to the extent of 0.05 mg/ml or less. It is
recognized that almost no substances are completely insoluble in
aqueous medium, and that the salt concentration or osmolality of
the medium may also influence solubility. "Insoluble in aqueous
medium," according to the present definition, assumes the
osmolality, ionic strength, and pH of physiologically compatible
solutions. Alternatively, "insolubility in pure water" may be used
as the standard if so specified. "Insolubility in water" is defined
as <0.05 mg/ml of pure water.
[0042] Similarly, "sparingly soluble" and "soluble" may be
described in terms of reference to either "aqueous medium" as
defined above or in "pure water." Substances that are "soluble" in
aqueous medium dissolve at least to the extent of being equal to or
greater than 1.0 mg/ml of the physiological solution; substances
that are "sparingly soluble" in aqueous medium dissolve only to the
extent of less than 1.0 mg/ml but more than 0.05 mg/ml of the
physiological solution.
[0043] "Hydrophobic moiety" is defined as a moiety which is
insoluble in aqueous solution as defined above. The hydrophobic
moiety may be a hydrophobic polymer such as polycaprolactone or may
be a hydrophobic small molecule such as a vitamin or a steroid. It
may be monovalent--i.e., have a suitable functional group for
coupling only to a single active through a linker--or may be
multivalent--i.e., able to couple to multiple actives through a
linker. Not all of the actives need be the same.
[0044] An "amphiphilic stabilizer" is a compound having a molecular
weight greater than about 500 that has a hydrophilic region and a
hydrophobic region. Preferably the molecular weight is greater than
about 1,000, or greater than about 1,500, or greater than about
2,000. Higher molecular weight moieties, e.g., 25,000 g/mole or
50,000 g/mole, may be used. "Hydrophobic" is defined as above.
"Hydrophilic" in the context of the present invention refers to
moieties that have a solubility in aqueous solution (i.e., a
physiological solution as defined above) of at least 1.0 mg/ml.
Typical amphiphilic stabilizers are copolymers of hydrophilic
regions and hydrophobic regions. Thus, in the amphiphilic
stabilizer, the hydrophobic region, if taken alone, would exhibit a
solubility in aqueous medium of less than 0.05 mg/ml and the
hydrophilic region, if taken alone, would exhibit a solubility in
aqueous medium of more than 1 mg/ml. Examples include copolymers of
polyethylene glycol and polycaprolactone.
[0045] A "linker" refers to any covalent bond, to a divalent
residue of a molecule, or to a chelator (in the case where the
active is a metal ion or organic metallic compound, e.g.,
cisplatin) that allows the hydrophobic moiety to be attached to the
active agent. The linker may be selectively cleavable upon exposure
to a predefined stimulus, thus releasing the active agent from the
hydrophobic moiety. The site of cleavage, in the case of the
divalent residue of a molecule may be at a site within the residue,
or may occur at either of the bonds that couple the divalent
residue to the agent or to the hydrophobic moiety. The predefined
stimuli include, for example, pH changes, enzymatic degradation,
chemical modification or light exposure. Convenient conjugates are
often based on hydrolyzable or enzymatically cleavable bonds such
as esters, carbonates, carbamates, disulfides and hydrazones.
[0046] In some instances, the conditions under which the active
performs its function are not such that the linker is cleaved, but
the active is able to perform this function while still attached to
the particle. In this case, the linker is described as
"non-cleavable," although virtually any linker could be cleaved
under some conditions; therefore, "non-cleavable" refers to those
linkers that do not necessarily need to release the active from the
particle as the active performs its function.
Exemplary Components
[0047] As noted previously, the members of the particulate
constructs of the invention include: (1) an active agent; (2) a
linker; (3) a hydrophobic moiety; and (4) an amphiphilic
stabilizer. Examples of each of these follow:
[0048] Active Agents
[0049] In one application of the constructs of the invention, the
constructs are used to deliver non-pharmaceutical or non-diagnostic
agents including but not limited to pigments, inks, pesticides,
herbicides, probes (including fluorescent probes), ingredients for
sunscreens, fragrances and flavor compounds.
[0050] In another application of the constructs of the invention,
the constructs are used to deliver pharmaceuticals or diagnostics
in vivo. In these cases, the active is a therapeutic agent or a
diagnostic agent.
[0051] A wide variety of therapeutic agents can be included. These
may be anti-neoplastic agents, anthelmintics agents, antibiotics,
anticoagulants, antidepressants, antidiabetic agents,
antiepileptics, antihistamines, antihypertensive agents,
antimuscarinic agents, antimycobacterial agents,
immunosuppressants, antithyroid agents, antiviral agents,
anxiolytic sedatives, astringents, beta-adrenoceptor blocking
agents, cardiac inotropic agents, contrast media, corticosteroids,
cough suppressants, diagnostic agents, diagnostic imaging agents,
diuretics, dopaminergics, haemostatics, immunological agents, lipid
regulating agents, muscle relaxants, parasympathomimetics,
parathyroid calcitonin, biphosphonates, protease inhibitors,
prostaglandins, radio-pharmaceuticals, sex hormones, steroids,
anti-allergic agents, stimulants, sympathomimetics, thyroid agents,
vasodilators and xanthines, disulfide compounds, antibacterials,
antivirals, nonsteroidal anti-inflammatory drugs, analgesics,
anticoagulants, anticonvulsants, antiemetics, antifungals,
antihypertensives, anti-inflammatory agents, antiprotozoals,
antipsychotics, cardioprotective agents, cytoprotective agents,
antiarrhythmics, hormones, immunostimulating agents, lipid-lowering
agents, platelet aggregation inhibitors, agents for treating
prostatic hyperplasia, agents for treatment of rheumatic disease,
or vascular agents (Compendium of Pharmaceuticals and Specialties
(35.sup.th Ed.) incorporated herein by reference).
[0052] "Anti-neoplastic agent" refers to moieties having an effect
on the growth, proliferation, invasiveness or survival of
neoplastic cells or tumors. Anti-neoplastic therapeutic agents
often include disulfide compounds, alkylating agents,
antimetabolites, cytotoxic antibiotics, drug resistance modulators
and various plant alkaloids and their derivatives. Other
anti-neoplastic agents are contemplated.
[0053] Anti-neoplastic agents include paclitaxel, an
etoposide-compound, a camptothecin-compound, idarubicin,
carboplatin, oxaliplatin, adriamycin, mitomycin, ansamitocin,
bleomycin, cytosine arabinoside, arabinosyl adenine,
mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan,
mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin,
mitomycin, plicamycin, aminoglutethimide, estramustine phosphate
sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen
citrate, testolactone, trilostane, amsacrine, asparaginase,
interferon, teniposide, vinblastine sulfate, vincristine sulfate,
bleomycin, methotrexate, valrubicin, carzelesin, paclitaxel,
taxotane, camptothecin, doxorubicin, daunomycin, cisplatin,
5-fluorouracil, methotrexate; anti-inflammatory agents such as
indomethacin, ibuprofen, ketoprofen, flubiprofen, dichlofenac,
piroxicam, tenoxicam, naproxen, aspirin, and acetaminophen; sex
hormones such as testosterone, estrogen, progestone, estradiol;
antihypertensive agents such as captopril, ramipril, terazosin,
minoxidil, and parazosin; antiemetics such as ondansetron and
granisetron; antibiotics such as metronidazole, and fusidic acid;
cyclosporine; prostaglandins; biphenyl dimethyl dicarboxylic acid,
carboplatin; antifungal agents such as itraconazole, ketoconazole,
and amphotericin; steroids such as triamcinolone acetonide,
hydrocortisone, dexamethasone, prednisolone, and betamethasone;
cyclosporine, and functionally equivalent analogues, derivatives or
combinations thereof.
[0054] Diagnostic agents may also be included as actives. These may
comprise, for example, chelated metal ions for MRI imaging,
radionuclides, such as .sup.99Tc or .sup.111In or other
biocompatible radionuclides. These may also be therapeutic
agents.
[0055] Linkers
[0056] The linker component, as described above, may be or may
include a cleavable bond.
[0057] The linker may be, for example, cleaved by hydrolysis,
reduction reactions, oxidative reactions, pH shifts, photolysis, or
combinations thereof; or by an enzyme reaction. Some linkers can be
cleaved by an intracellular or extracellular enzyme, or an enzyme
resulting from a microbial infection, a skin surface enzyme, or an
enzyme secreted by a cell, by an enzyme secreted by a cancer cell,
by an enzyme located on the surface of a cancer cell, by an enzyme
secreted by a cell associated with a chronic inflammatory disease,
by an enzyme secreted by a cell associated with rheumatoid
arthritis, by an enzyme secreted by a cell associated with
osteoarthritis, or by a membrane-bound enzyme. In some cases, the
linker can be cleaved by an enzyme that is available in a target
region. These types of linkers are often useful in that the
particular enzyme or class of enzymes may be present in increased
concentrations at a target region. The target tissue generally
varies based on the type of disease or disorder present in the
subject.
[0058] The linker may also comprise a bond that is cleavable under
oxidative or reducing conditions, or may be sensitive to acids.
Acid cleavable linkers can be found in U.S. Pat. Nos. 4,569,789 and
4,631,190; and Blattner, et al., Biochemistry (1984) 24:1517-1524.
Such linkers are cleaved by natural acidic conditions, or
alternatively, acid conditions can be induced at a target site as
explained in U.S. Pat. No. 4,171,563.
[0059] A non-limiting set of molecules that can form acid cleavable
bonds include cis-polycarboxylic alkenes (see U.S. Pat. No.
4,631,190), and amino-sulfhydryl cross-linking reagents which are
cleavable under mildly acidic conditions (see U.S. Pat. No.
4,569,789). The linker may comprise a time-release bond, such as a
biodegradable and/or hydrolyzable bond, such as esters, amides or
urethane bonds.
[0060] Examples of linking reagents which contain cleavable
disulfide bonds (reducible bonds) include
1,4-di-[3'-(2'-pyridyldithio)propionamido]butane;
N-succinimidyl(4-azidophenyl) 1,3'-dithiopropionate;
sulfosuccinimidyl (4-azidophenyldithio)propionate;
dithiobis(succinimidylpropionate);
3,3'-dithiobis(sulfosuccinimidylpropionate); dimethyl
3,3'-dithiobispropionimidate-2HCl (available from Pierce Chemicals,
Rockford, Ill.).
[0061] Examples of oxidation sensitive linking reagents include,
without limitation, disuccinimidyl tartarate; and disuccinimidyl
tartarate (available from Pierce Chemicals).
[0062] The linker may also comprise a small molecule such as a
peptide linker. Frequently, in such embodiments, the peptide linker
is cleavable by base, under reducing conditions, or by a specific
enzyme. The linker may be cleaved by an indigenous enzyme, or by an
non-indigenous enzyme administered after or in addition to the
presently contemplated compositions. A small peptide linker is pH
sensitive, for example, the linker may comprise linkers selected
from the group consisting of poly L-glycine; poly L-glutamine; and
poly L-lysine linkers.
[0063] For example, the linker may comprise a hydrophobic polymer
and a dipeptide, L-alanyl-L-valine (Ala-Val), cleavable by the
enzyme thermolysin. This linker is advantageous because
thermolysin-like enzyme has been reported to be expressed at the
site of many tumors. A linker may also be used that contains a
recognition site for the protease furin. Goyal, et al., Biochem. J.
(2000) 2:247-254.
[0064] The chemical and peptide linkers can be bonded between the
ligand and the agent by techniques known in the art for conjugate
synthesis, i.e., using genetic engineering or chemically.
[0065] Photocleavable linkers include, for example,
1-2-(nitrophenyl)-ethyl. A photocleavable linker often permits the
activation and action of the active agent in a very specific area,
for example at a particular part of the target tissue. Activation
(light) energy can be localized through a variety of means
including catheterization, via natural or surgical openings or via
blood vessels.
[0066] The linkers and techniques for providing coupling of the
active to the hydrophobic moiety are similar to those that have
been used previously to prepare conjugates to make actives more
soluble, in contrast to their application in the present invention.
In general, in the constructs of the invention, the active is
often, but not always, made less soluble in aqueous solution by
virtue of forming the conjugate. For example, the techniques
reviewed by Greenwald, et al., for attaching PEG to small organic
molecules can be adapted to the present invention. Some of these
techniques are described in Greenwald, R. B., Journal of Controlled
Release (2001) 74:159-171; Greenwald, R. B., et al., Journal of
Medicinal Chemistry (1996) 39:424-431; and Greenwald, R. B., et
al., Advanced Drug Delivery Reviews (2002) 55:217-250. In
particular, paclitaxel esters have been prepared via conjugation of
PEG acids to the .alpha.-position on the paclitaxel molecule. These
esters were demonstrated to be an especially effective linking
group, as hydrolysis of the ester carbonyl bond and the subsequent
release of the attached drug were shown to occur in a predictable
fashion in vitro. (Greenwald, R. B., et al., Critical Reviews in
Therapeutic Drug Carrier Systems (2000) 17:101-161.) The linker
chemistry as applied in the present invention does not enhance
solubility, but adapts the active for inclusion in the particulate
vehicles of the invention.
[0067] The covalent attachment of proteins, vaccines or peptides to
PEG can also be adapted to form the present conjugate. Such
techniques are reviewed in Katre, N. V., Advanced Drug Delivery
Reviews (1993) 10:91-114; Roberts, M. J., et al., Journal of
Pharmaceutical Sciences (1998) 87:1440-1445; Garman, A. J., et al.,
Febs Letters (1987) 223:361-365; and Daly, S. M., et al., Langmuir
(2005) 21:1328-1337. Coupling reactions between amino groups of
proteins and mPEG equipped with an electrophilic functional group
have been used in most cases for preparation of PEG-protein
conjugates. The most commonly used mPEG-based electrophiles,
referred to as `activated PEGs` are based on reactive aryl
chlorides, acylating agents and alkylating groups as described by
Zalipsky, S., Advanced Drug Delivery Reviews (1995) 16:157-182; and
Zalipsky, S., Bioconjugate Chem. (1995) 6:150-165. Tailoring the
number of ethylene groups in the linker can additionally be used to
adjust the hydrolysis rates of drug-linked ester bonds, to values
appropriate for once-a-week administration. For example,
Schoenmakers, et al., demonstrated the conjugation of a model
paclitaxel molecule to PEG using a hydrolysable linker based on
reaction between a thiol and an acrylamide. By changing the length
of the linker, the time of drug release was varied between 4 and 14
days. (Schoenmakers, R. G., et al., Journal of Controlled Release
(2004) 95:291-300.) Additionally, Frerot, et al., prepared a series
of carbamoyl esters of maleate and succinate and studied the rate
constants for neighboring group assisted alkaline ester hydrolysis.
The rates of hydrolysis were found to depend on the structure of
the neighboring nucleophile that attacks the ester function. (de
Saint Laumer, J. Y., et al., Helvetica Chimica Acta (2003)
86:2871-2899.) By taking account of the influence of structural
parameters on the rates of ester hydrolysis, hydrolysis rates may
be varied over several orders of magnitude and precursors yielding
the desired release profile may be designed.
[0068] In addition to ester linkages, enzymatically cleavable bonds
can be used to conjugate active agents to the hydrophobic moiety.
An enzymatically cleavable linker generally will comprise amino
acids, sugars, nucleic acids, or other compounds which have one or
more chemical bonds that can be broken via enzymatic degradation.
In a recent study, a variety of amino acid spacers were employed
for the conjugation of PEG to camptothecin, an anti-tumor drug.
Rates of amino acid linker hydrolysis were determined to vary
according to the type of amino acid spacer utilized. (Conover, C.
D., et al., Anti-Cancer Drug Design (1999) 14:499-506.)
[0069] Photocleavable linkers have also been extensively employed
for the synthesis of conjugates for release of actives. As an
example, keto-esters have been used as delivery systems for the
controlled release of perfumery aldehydes and ketones. Alkyl or
aryl .alpha.-keto esters of primary or secondary alcohols decompose
upon radiation at 350-370 nm, releasing the active aldehyde.
(Rochat, S., et al., Helvetica Chimica Acta (2000) 83:1645-1671.)
This mechanism has been shown to successfully sustain release of
the active agent. For drug delivery purposes, light energy can be
localized through a variety of means including catheterization, via
natural and surgical openings or via blood vessels.
[0070] As noted above, when the linker is the residue of a divalent
organic molecule, the cleavage "of the linker" may be either within
the residue itself, or it may be at one of the bonds that couples
the linker to the remainder of the conjugate--i.e., either to the
active or the hydrophobic moiety.
[0071] In some embodiments, it is unnecessary for the linker to be
cleavable. In particular, if the active is functional while still
coupled to the linker, there is no need to release the active from
the particulate moiety. One such example would be instances wherein
the active is printer's ink, which can remain in particulate form
when employed.
[0072] In instances where the linker need not be cleavable,
alternative organic moieties may be used to create the divalent
residue, or a covalent bond directly coupling the active to the
hydrophobic moiety may not be subject to cleavage under conditions
contemplated in use. (By "non-cleavable" is meant that the linker
will not release the active under the conditions wherein the
function of the active is being performed.) Examples of
non-cleavable linkers comprise, but are not limited to,
(sulfosuccinimidyl
6-[alpha-methyl-alpha-(2-pyridylthio)toluamido]hexanoate;
Azidobenzoyl hydrazide; N-Hydroxysuccinimidyl-4-azidosalicyclic
acid; Sulfosuccinimidyl
2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate;
N-{4-(p-azidosalicylamido) buthy}-3'(2'-pyidyldithio)propionamide;
Bis-[beta-(4-azidosalicylamido)ethyl]disulfide;
N-hydroxysuccinimidyl-4 azidobenzoate; p-Azidophenyl glyoxal
monohydrate;
N-Succiminidyl-6(4'-azido-2'-mitrophenyl-amino)hexanoate;
Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate;
N-5-Azido-2-nitrobenzyoyloxysuccinimide;
Sulfosuccinimidyl-2-(m-azido-o-mitrobenzamido)-ethyl-1,3'-dithiopropionat-
e; p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate; Succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate; Sulfosuccinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate;
m-Maleimidobenzoyl-N-hydroxysuccinimide ester;
m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester;
N-Succinimidyl(4-iodoacetyl)aminobenzoate;
N-Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate; Succinimidyl
4-(p-malenimidophenyl)butyrate; Sulfosuccinimidyl
4-(p-malenimidophenyl)butyrate; Disuccinimidyl suberate;
bis(sulfosuccinimidyl) suberate; Bis maleimidohexane;
1,5-difluoro-2,4-dinitrobenzene; dimethyl adipimidate 2HCl;
Dimethyl pimelimidate-2HCl; dimethyl suberimidate-2-HCl;
"SPDP"-N-succinimidyl-3-(2-pyridylthio)propionate;
Sulfosuccinimidyl 4-(p-azidophenyl)butyrate; Sulfosuccinimidyl
4-(p-azidophenylbutyrate);
1-9p-azidosalicylamido)-4-(iodoacetamido)butane;
4-(p-Azidosalicylamido)butylamine (available from Pierce
Chemicals).
[0073] Hydrophobic Moieties
[0074] A third component of the constructs of the invention is a
hydrophobic moiety. The hydrophobic moiety may include polymers or
natural products. Examples of suitable hydrophobic polymeric
moieties include but are not limited to polymers of the following:
acrylates including methyl acrylate, ethyl acrylate, propyl
acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl
acrylate, and t-butyl acrylate; methacrylates including ethyl
methacrylate, n-butyl methacrylate, and isobutyl methacrylate;
acrylonitriles; methacrylonitrile; vinyls including vinyl acetate,
vinylversatate, vinylpropionate, vinylformamide, vinylacetamide,
vinylpyridines, and vinylimidazole; aminoalkyls including
aminoalkylacrylates, aminoalkylmethacrylates, and
aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate,
cellulose acetate succinate, hydroxypropylmethylcellulose
phthalate, and the polymers poly(D,L lactide),
poly(D,L-lactide-co-glycolide), poly(glycolide),
poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),
polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene
terephthalate), poly(malic acid), poly(tartronic acid),
polyanhydrides, polyphosphazenes, poly(amino acids) and their
copolymers (see generally, Illum, L., Davids, S. S. (eds.) Polymers
in Controlled Drug Delivery, Wright, Bristol, 1987; Arshady, J.
Controlled Release (1991) 17:1-22; Pitt, Int. J. Phar. (1990)
59:173-196; Holland, et al., J. Controlled Release (1986)
4:155-180); hydrophobic peptide-based polymers and copolymers based
on poly(L-amino acids) (Lavasanifar, A., et al., Advanced Drug
Delivery Reviews (2002) 54:169-190), poly(ethylene-vinyl acetate)
("EVA") copolymers, silicone rubber, polyethylene, polypropylene,
polydienes (polybutadiene, polyisoprene and hydrogenated forms of
these polymers), maleic anhydride copolymers of vinyl-methylether
and other vinyl ethers, polyamides (nylon 6,6), polyurethane,
poly(ester urethanes), poly(ether urethanes), poly(ester-urea).
Particularly preferred polymeric hydrophobes include
poly(ethylenevinyl acetate), poly (D,L-lactic acid) oligomers and
polymers, poly (L-lactic acid) oligomers and polymers, poly
(glycolic acid), copolymers of lactic acid and glycolic acid, poly
(caprolactone), poly (valerolactone), polyanhydrides, copolymers of
poly (caprolactone) or poly (lactic acid) For non-biologically
related applications particularly preferred polymeric carriers
include polystyrene, polyacrylates, and butadienes. The polymers
must contain one or more functionizable groups which may be
incorporated into the polymer by derivitization or may be inherent
in the polymer chemistry. Polymers as hydrophobic moieties should
have molecular weights between 800 and 200,000. The preferred range
is 1,000 to 10,000 for polymers with mono or divalent functional
sites. For polymers with a multiplicity of functional sites for
derivation the preferred molecular weight of the polymer per
conjugated active is 1,000 to 10,000.
[0075] Natural products with functional groups or groups that can
be converted to functional groups for conjugation include:
hydrophobic vitamins (for example vitamin E, vitamins K and A),
carotenoids and retinols(for example beta carotene, astaxanthin,
trans and cis retinal, retinoic acid, folic acid, dihydrofolate,
retinyl acetate, retinyl palmitate), cholecalciferol, calcitriol,
hydroxycholecalciferol, ergocalciferol, .alpha.-tocopherol,
.alpha.-tocopherol acetate, .alpha.-tocopherol nicotinate, and
estradiol. The preferred natural product is vitamin E which can be
readily obtained as a vitamin E succinate, which facilitates
functionalization to amines and hydroxyls on the active
species.
[0076] Hydrophobic, non polymeric and moieties include hydrocarbon
molecules with solubilities less than 0.1 mg/ml that contain a
functional group for can be derivatized to incorporate a functional
group for conjugation. Molecules in this class include hydrophobic
dyes and plasticizers. Examples include, but are not limited to,
coumarin, diaminonaphthalene and other naphthalene derivatives,
anthracene and its derivatives, nile red. Further examples can be
found in Handbook of Dyes and pH Indicators. Examples of
hydrophobic plasticizes include dioctylphthalate, dibutylphthalate,
and its derivatives.
[0077] Depending on the nature of the hydrophobic moiety, it may be
able to accommodate more than one, including substantially more
than one active through a multiplicity of linking sites. Polymeric
moieties may have as many as 100 sites whereby actives could be
linked. Simpler hydrophobic moieties, such as Vitamin E, may
provide only one such site. Thus, the number of actives coupled to
a single hydrophobic moiety may be only 1, or may be 2, 5, 10, 25,
100 and more, and all integers in between. For instance, the
polymers set forth above can readily be provided with a
multiplicity of functional groups for coupling to the active.
Difunctional hydrophobic moieties would include the hydrophobic
polymer chains listed above that have two terminal OH, COOH, or
NH.sub.2 groups. Multifunctional hydrophobic moieties include all
of those listed above that have multiple OH, COOH, or NH.sub.2
groups on some or all of the monomer units on the polymer backbone.
These functional groups are merely illustrative; other moieties
which could form functional groups for linking include phenyl
substituents, halo groups, and the like. Typically, when the
hydrophobic moiety is a hydrophobic polymer, it may have multiple
sites for linkage. When the hydrophobic moiety is a relatively
small molecule, it will accommodate only the number of linkers for
which it has available functional groups.
[0078] Amphiphilic Stabilizers
[0079] The fourth component is an amphiphilic stabilizer.
Typically, the stabilizer is a copolymer of a hydrophilic block
coupled with a hydrophobic block. Nanoparticles formed by the
process of this invention can be formed with graft, block or random
amphiphilic copolymers. These copolymers can have a molecular
weight between 1,000 g/mole and 50,000 g/mole or more, or between
about 3,000 g/mole to about 25,000 g/mole, or at least 2,000
g/mole. Alternatively, the amphiphilic copolymers used in this
invention exhibit a water surface tension of at least 50
dynes/cm.sup.2 at a concentration of 0.1 wt %.
[0080] Examples of suitable hydrophobic blocks in an amphiphilic
copolymer include but are not limited to the following: acrylates
including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl
acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butyl
acrylate; methacrylates including ethyl methacrylate, n-butyl
methacrylate, and isobutyl methacrylate; acrylonitriles;
methacrylonitrile; vinyls including vinyl acetate, vinylversatate,
vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines,
and vinylimidazole; aminoalkyls including aminoalkylacrylates,
aminoalkylmethacrylates, and aminoalkyl(meth)acrylamides; styrenes;
cellulose acetate phthalate, cellulose acetate succinate,
hydroxypropylmethylcellulose phthalate, poly(D,L lactide), poly
(D,L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate),
poly(alkylcarbonate) and poly(orthoesters), polyesters,
poly(hydroxyvaleric acid), polydioxanone, poly(ethylene
terephthalate), poly(malic acid), poly(tartronic acid),
polyanhydrides, polyphosphazenes, poly(amino acids) and their
copolymers (see generally, Illum, L., Davids, S. S. (eds.) Polymers
in Controlled Drug Delivery, Wright, Bristol, 1987; Arshady, J.
Controlled Release (1991) 17:1-22; Pitt, Int. J. Phar. (1990)
59:173-196; Holland, et al, J. Controlled Release (1986)
4:155-180); hydrophobic peptide-based polymers and copolymers based
on poly(L-amino acids) (Lavasanifar, A., et al., Advanced Drug
Delivery Reviews (2002) 54:169-190), poly(ethylene-vinyl acetate)
("EVA") copolymers, silicone rubber, polyethylene, polypropylene,
polydienes (polybutadiene, polyisoprene and hydrogenated forms of
these polymers), maleic anhydride copolymers of vinyl methylether
and other vinyl ethers, polyamides (nylon 6,6), polyurethane,
poly(ester urethanes), poly(ether urethanes), poly(ester-urea).
Particularly preferred polymeric blocks include poly(ethylenevinyl
acetate), poly (D,L-lactic acid) oligomers and polymers, poly
(L-lactic acid) oligomers and polymers, poly (glycolic acid),
copolymers of lactic acid and glycolic acid, poly (caprolactone),
poly (valerolactone), polyanhydrides, copolymers of poly
(caprolactone) or poly (lactic acid) For non-biologically related
applications particularly preferred polymeric blocks include
polystyrene, polyacrylates, and butadienes.
[0081] Examples of suitable hydrophilic blocks in an amphiphilic
copolymer include but are not limited to the following: carboxylic
acids including acrylic acid, methacrylic acid, itaconic acid, and
maleic acid; polyoxyethylenes or poly ethylene oxide;
polyacrylamides and copolymers thereof with
dimethylaminoethylmethacrylate, diallyldimethylammonium chloride,
vinylbenzylthrimethylammonium chloride, acrylic acid, methacrylic
acid, 2-acrylamido-2-methylpropane sulfonic acid and styrene
sulfonate, polyvinyl pyrrolidone, starches and starch derivatives,
dextran and dextran derivatives; polypeptides, such as polylysines,
polyarginines, polyglutamic acids; poly hyaluronic acids, alginic
acids, polylactides, polyethyleneimines, polyionenes, polyacrylic
acids, and polyiminocarboxylates, gelatin, and unsaturated
ethylenic mono or dicarboxylic acids.
[0082] Preferably the blocks are either diblock or triblock
repeats. Preferably, block copolymers for this invention include
blocks of polystyrene, polyethylene, polybutyl acrylate, polybutyl
methacrylate, polylactic acid, polycaprolactone, polyacrylic acid,
polyoxyethylene and polyacrylamide A listing of suitable
hydrophilic polymers can be found in Handbook of Water-Soluble Gums
and Resins, R. Davidson, McGraw-Hill (1980).
[0083] In graft copolymers, the length of a grafted moiety can
vary. Preferably, the grafted segments are alkyl chains of 12 to 32
carbons or equivalent to 6 to 16 ethylene units in length. In
addition, the grafting of the polymer backbone can be useful to
enhance solvation or nanoparticle stabilization properties. A
grafted butyl group on the hydrophobic backbone of a diblock
copolymer of a polyethylene and polyethylene glycol should
increases the solubility of the polyethylene block. Suitable
chemical moieties grafted to the block unit of the copolymer
comprise alkyl chains containing species such as amides, imides,
phenyl, carboxy, aldehyde or alcohol groups. One example of a
commercially available stabilizer is the Hypermer family marketed
by Uniqema Co. The amphiphilic stabilizer could also be of the
gelatin family such as the gelatins derived from animal or fish
collagen.
[0084] Formation of the Particulate Constructs
[0085] A number of methods can be used to form the particulate
constructs of the invention. One particularly useful method is a
process termed "Nano Precipitation" as described by Johnson, B. K.,
et al., AIChE Journal (2003) 49:2264-2282 and U.S. 2004/0091546
incorporated herein by reference. This process is capable of
producing controlled size, polymer-stabilized and protected
nanoparticles of hydrophobic organics at high loadings and yields.
The Nano Precipitation technique is based on amphiphilic diblock
copolymer arrested nucleation and growth of hydrophobic organics.
Amphiphilic diblock copolymers dissolved in a good solvent can form
micelles when the solvent quality for one block is decreased. In
order to achieve such a solvent quality change, a tangential flow
mixing cell (vortex mixer) is used. The vortex mixer consists of a
confined volume chamber where one jet stream containing the diblock
copolymer and active agent dissolved in a water-miscible solvent is
mixed at high velocity with another jet stream containing water, an
anti-solvent for the active agent and the hydrophobic block of the
copolymer. The fast mixing and high energy dissipation involved in
this process provide timescales that are shorter than the timescale
for nucleation and growth of particles, which leads to the
formation of nanoparticles with active agent loading contents and
size distributions not provided by other technologies. When forming
the nanoparticles via Nano Precipitation, mixing occurs fast enough
to allow high supersaturation levels of all components to be
reached prior to the onset of aggregation. Therefore, the active
agent(s) and polymers precipitate simultaneously, and overcome the
limitations of low active agent incorporations and aggregation
found with the widely used techniques based on slow solvent
exchange (e.g., dialysis). The Nano Precipitation process is
insensitive to the chemical specificity of the components, making
it a universal nanoparticle formation technique.
[0086] In an exemplary procedure, the active agent conjugated
polymer and stabilizing diblock copolymer of methoxy poly(ethylene
glycol)-b-poly(.epsilon.-caprolactone) (mPEG-PCL, 5,000-2,900
g/mole, respectively), is dissolved in THF at a weight ratio of
1:1:1 to make a 0.3 wt % solution for each component. The resulting
solution is loaded into a 100 ml gas tight syringe, which is fixed
on a digitally controlled syringe pump. A 300 mM sucrose solution
is prepared, loaded into a 100 ml gas tight syringe, and fixed on a
second syringe pump. The syringes are connected to the vortex mixer
inlet, and pumped through at flow rates of 12 and 120 ml/min for
the active agent and the sucrose solution, respectively. At the
mixer outlet, two samples are collected. The first sample is
collected in a scintillation vial and analyzed for particle size by
dynamic light scattering (DLS), and the second sample is collected
in low temperature freezer vials, and freeze-dried. The
freeze-drying cycle is the following: -40.degree. C. overnight,
-10.degree. C. for a day, 4.degree. C. for a day, and then room
temperature for one day. DLS measurements are repeated at 1, 2, 8,
16 hours, and daily intervals for each sample to check for
stability. The samples are checked visually for crystals/aggregates
formation. The freeze-dried material are checked for the presence
of any residual solvent (THF). A freeze-dried sample is dissolved
in methanol to dissociate the nanoparticles, and the solution is
tested for the presence of THF using CGC.
[0087] Other processes that may be used to form the particulate
constructs of the invention include milling,
emulsification-diffusion and emulsification-evaporation. These are
well-known processes readily practiced by those of skill in the
art. Milling involves precipitating the conjugated active species
into a particulate form with a macroscopically large particle size.
The precipitate is then pulverized by mechanical means in the
presence of a grinding media and a stabilizing polymer or surface
active agent. The process is described in U.S. Pat. Nos. 4,726,955;
5,518,738 and 5,145,684).
[0088] In these processes, it may be useful to include, in addition
to the conjugate and the amphiphilic stabilizer, an excess of a
reactive form of the hydrophobic moiety coupled with linker so that
any excess free active can be captured, and additional stability
can be imparted to the resulting particles.
[0089] One conventional emulsification method of microencapsulating
an agent to form a microencapsulated product is disclosed in U.S.
Pat. No. 5,407,609. This method involves dissolving or otherwise
dispersing agents, liquids or solids, in a solvent containing
dissolved wall-forming materials, dispersing the
agent/polymer-solvent mixture into a processing medium to form an
emulsion and transferring all of the emulsion immediately to a
large volume of processing medium or other suitable extraction
medium, to immediately extract the solvent from the microdroplets
in the emulsion to form a microencapsulated product, such as
microcapsules or microspheres. The most common method used for
preparing polymer delivery vehicle formulations is the solvent
emulsification-evaporation method. This method involves dissolving
the polymer and drug in an organic solvent that is completely
immiscible with water (for example, dichloromethane). The organic
mixture is added to water containing a stabilizer, most often
poly(vinyl alcohol) (PVA) and then typically sonicated.
[0090] As indicated above, the particulate construct that results
may contain one or more than one of the conjugates described.
Typically, a nanoparticulate size construct will comprise
10.sup.3-10.sup.4 conjugates; larger microparticles might comprise
10.sup.5-10.sup.7 conjugates.
[0091] The resulting particles may have a variety of sizes
depending on the nature of the components and on the method used to
form them. Typically, the particles range in size from 50 nm to as
much as 5 .mu.m. For in vivo applications, nanometer size
particles, typically of the order of 200 nm or less are preferred.
For other applications, larger particles may be desirable. Thus,
the dimensions of the particles may range from as little as 50 nm
to 100 nm, 200 nm, 500 nm, 1 .mu.m or 5 .mu.m and the integers
between. A composition of these constructs may contain a variety of
sizes and can be described in terms of an average or median
diameter.
[0092] After the particulate constructs are formed, they may be
assessed for active agent loading content, size, and in vitro
active agent release. Methods are available to assess the degree of
polymer-active interaction or compatibility, including DSC, powder
X-ray diffraction, and FTIR.
[0093] As one example of a procedure to measure the content of
active agent, paclitaxel encapsulated in nanoparticles is assessed
as follows. A sample of the freeze-dried material is weighed and
dissolved in THF to solubilize the particles, then the sample is
placed in a semi-micro spectrophotometer cell, and the paclitaxel
concentration is determined using a UV spectrophotometer at 261 nm.
In addition, the absorbance is measured at 350 nm, and polymer only
solutions is run at 261 nm as controls. The results obtained by UV
analysis are confirmed using high performance liquid chromatography
(HPLC) with a C18 column, methanol and water as mobile phases
ranging from 10 to 100% methanol by volume, at a flow rate of 1
ml/min and 261 nm detection wavelength. The amount of cisplatin
encapsulated in the nanoparticles is determined using atomic
absorption spectrometry.
[0094] Particle size may be determined by DLS. For example, in one
illustration, measurements are performed using an Nd-YAG laser with
a 532 nm wavelength at a scattering angle of 90.degree.. The sample
collected in a scintillation vial from the mixer effluent is
inserted into the DLS sample cell containing decalin scintillation
fluid (maintained at 25.degree. C. using a temperature bath), and
left to equilibrate to the cell temperature. The run duration is 60
seconds, replicated three times. The particle size expressed as the
hydrodynamic diameter is obtained using an ALV 5000 correlator and
a second order cumulant fit.
[0095] In vitro release can also be measured. For the paclitaxel
containing particles described above, 10 mM phosphate, 150 mM NaCl
buffer solution is prepared, and active agent nanoparticles are
suspended in 2 ml of the buffer solution to form a 1 mg/ml to a 5
mg/ml solution. The solution is introduced into a 12-14K dialysis
membrane bag, and placed in 1 liter of the buffer solution at room
temperature. 0.05 ml aliquots are collected from the dialysis bag
and 1 ml of THF is added to dissociate the nanoparticles. The
resulting solution is placed in semi-micro spectrophotometer cells,
and the paclitaxel concentration is determined using a UV
spectrophotometer at 261 nm. In addition, the absorbance is
measured at 350 nm, and polymer only solutions is run at 261 nm as
controls. The cisplatin amounts are determined using atomic
absorption spectrometry. The measurements are repeated at intervals
of 1, 2, 4, 8, 16, 24, 48, and 72 hours.
[0096] The physical state of the active agent can also be studied
by DSC. DSC thermograms of pure active agent, empty polymer
nanoparticles or films and active agent-loaded polymer
nanoparticles or films are recorded. The concentration of active
agent ranges from 10-75% (w/w). The values for the heat of melting
(.DELTA.H.sub.m, J/g of active agent) of the active agent at each
active agent concentration are recorded and a plot of
.DELTA.H.sub.m versus concentration is prepared. The solid-state
solubility (saturation solubility) of the active agents in the
nanoparticles or films is determined by the y-intercept of the plot
(Puttipipatkhachorn, et al., J. Controlled Release (2001)
10:75(1-2):143-153). Below the solid-state solubility the active
agent is in a dissolved state while above that it exists in both a
dissolved state and a crystalline state.
[0097] The solid-state solubility of the active agent is dependent
on the molecular weight of the polymer. The higher the molecular
weight of the polymer, the greater the microviscosity of the medium
and the more difficult it is for the active agent to crystallize.
Therefore, an increase in polymer molecular weight should act to
increase the saturation solubility of an active agent.
[0098] Active agent-polymer interaction or compatibility is
frequently assessed by DSC. The active agent may act as a
plasticizer causing a decrease in the T.sub.g of the polymer or as
a reinforcing filler resulting in an increase in the T.sub.g of the
polymer. The criterion for polymer-active agent miscibility often
comprises the presence of a single concentration dependent T.sub.g
lying between the T.sub.g's of the individual components.
[0099] The miscibility of the polymer blends is frequently assessed
using DSC. The DSC thermograms of each polymer and the polymer
blend are recorded. The glass transition temperatures (T.sub.g) of
each component alone is compared to the T.sub.g value(s) in the
polymer blends. The criterion for polymer-polymer miscibility is
the same as that set out above for polymer-active agent
miscibility.
[0100] The state of the active agent in polymer films or
nanoparticles may be determined from diffractograms obtained from
Powder X-ray diffraction (PXRD) patterns of the pure active agent,
physical mixtures and the active agent-polymer blends. The presence
of sharp peaks in the diffractogram indicates that the active agent
is present in a crystalline state; while, a halo pattern indicates
an amorphous state.
[0101] Polymer-active agent interactions may be measured using FTIR
spectroscopy. The transmission infrared spectra of pure active
agent, physical mixtures of pure active agent and polymer as well
as films of the active agent-polymer blends are obtained.
Interactions in the blend will result in band shifts and broadening
in the FTIR spectrum when compared to the spectra for the pure
polymer and active agent.
[0102] Applications and Uses of the Invention Particulate
Constructs
[0103] In instances where the active is a therapeutic agent or a
diagnostic agent useful in vivo, the particulate constructs are
formulated into suitable veterinary or pharmaceutical compositions
and administered to subjects as appropriate. The subjects include
warm-blooded animals, including humans, domestic avian species,
fish and the like. For treatment of human ailments, a qualified
physician will determine how the compositions of the present
invention should be utilized with respect to dose, schedule and
route of administration using established protocols. Such
applications also frequently utilize dose escalation should agents
encapsulated in delivery vehicle compositions of the present
invention exhibit reduced toxicity to healthy tissues of the
subject.
[0104] The pharmaceutical or veterinary compositions of the present
invention may be administered parenterally, i.e., intraarterially,
intravenously, intraperitoneally, subcutaneously, or
intramuscularly, the pharmaceutical compositions are administered,
e.g., by a bolus or infusional injection. For example, see Rahman,
et al, U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410;
Papahadjopoulos, et al., U.S. Pat. No. 4,235,871; Schneider, U.S.
Pat. No. 4,224,179; Lenk, et al., U.S. Pat. No. 4,522,803; and
Fountain, et al., U.S. Pat. No. 4,588,578.
[0105] In other methods, the formulations of the invention can be
contacted with target tissue by direct application of the
preparation to the tissue. The application may be made by
"topical", "open" or "closed" procedures. By "topical," it is meant
the direct application of the multi-active agent preparation to a
tissue exposed to the environment, such as the skin, oropharynx,
external auditory canal, and the like. "Open" procedures are those
procedures that include incising the skin of a patient and directly
visualizing the underlying tissue to which the pharmaceutical
preparations are applied. This is generally accomplished by a
surgical procedure, such as a thoracotomy to access the lungs,
abdominal laparotomy to access abdominal viscera, or other direct
surgical approach to the target tissue. "Closed" procedures are
invasive procedures in which the internal target tissues are not
directly visualized, but accessed via inserting instruments through
small wounds in the skin. For example, the preparations may be
administered to the peritoneum by needle lavage. Alternatively, the
preparations may be administered through endoscopic devices,
pumping devices, stents, wafers, reservoirs, pastes or films.
[0106] Pharmaceutical or veterinary compositions comprising
delivery vehicles of the invention are prepared according to
standard techniques and may comprise water, buffered water, 0.9%
saline, 0.3% glycine, 5% dextrose, iso-osmotic sucrose solutions
and the like, including glycoproteins for enhanced stability, such
as albumin, lipoprotein, globulin, and the like. These compositions
may be sterilized by conventional, well-known sterilization
techniques. The resulting aqueous solutions may be packaged for use
or filtered under aseptic conditions and lyophilized, the
lyophilized preparation being combined with a sterile aqueous
solution prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, and the like.
[0107] Depending on the nature of the active agent, the formulation
similar to those above may also be applied for cosmetic purposes
and the excipients modified accordingly.
[0108] Combination Therapies
[0109] A particularly significant application of the present
techniques is its use to deliver combinations of therapeutic
agents, including multidrug resistance modulators, and imaging
agents. Where biologically active combinations are used, the
pharmacokinetics of the delivery are controlled by the particulate
constructs used to deliver them and the nature of the cleavable
linkers employed. Coordination of delivery of such agents to target
tissues or organs is assured by suitable control of these
parameters. It is particularly advantageous to deliver such agents
in a ratio that is non-antagonistic, and especially that is
non-antagonistic over a wide range of concentrations. As described
in PCT publication PCT/CA02/01500, algorithms are available such
that based on the results of in vitro tests, such non-antagonistic
ratios may be determined. As noted in this publication, coordinated
delivery of the specified ratio may be effected by including more
than a single active in a particulate construct, or separate
particulate constructs may be used. In the present case, if
separate constructs are employed, the pharmacokinetics and release
mechanisms of each construct are engineered to provide the desired
ratio maintenance.
[0110] These techniques are described in detail in the foregoing
publication; however, briefly, a preferred method is the
Chou-Talalay median-effect method which utilizes an equation
wherein the dose that causes a particular effect, f.sub.a, is given
by:
D=D.sub.m[f.sub.a/(1-f.sub.a)].sup.1/m
in which D is the dose of the active agent used, f.sub.a is the
fraction of cells affected by that dose, D.sub.m is the dose for
median effect signifying the potency and m is a coefficient
representing the shape of the dose-effect curve (m is 1 for first
order reactions).
[0111] This equation can be further manipulated to calculate a
combination index (CI) on the basis of the multiple active agent
effect equation as described by Chou and Talalay, Adv. Enzyme Reg.
(1984) 22:27-55; and by Chou, et al., in: Synergism and Antagonism
in Chemotherapy (1991)223-244, Chou and Rideout, eds., Academic
Press: New York. A computer program (CalcuSyn) for this calculation
is found in Chou and Chou ("Dose-effect analysis with
microcomputers: quantitation of ED50, LD50, synergism, antagonism,
low-dose risk, receptor ligand binding and enzyme kinetics":
CalcuSyn Manual and Software; Cambridge: Biosoft 1987).
[0112] A two-active agent combination may be further used as a
single pharmaceutical unit to determine synergistic or additive
interactions with a third agent. In addition, a three-agent
combination may be used as a unit to determine non-antagonistic
interactions with a fourth agent, and so on.
[0113] The underlying experimental data are generally determined in
vitro using cells in culture or cell-free systems. Preferably, the
combination index (CI) is plotted as a function of the fraction of
cells affected (f.sub.a) as shown in FIG. 1A which, as explained
above, is a surrogate parameter for concentration range. Preferred
combinations of agents are those that display synergy or additivity
over a substantial range of f.sub.a values. Combinations of agents
are selected that display synergy over at least 5% of the
concentration range wherein greater than 1% of the cells are
affected, i.e., an f.sub.a range greater than 0.01. Preferably, a
larger portion of overall concentration exhibits a favorable CI;
for example, 5% of an f.sub.a range of 0.2-1.0. More preferably 10%
of this range exhibits a favorable CI. Even more preferably, 20% of
the f.sub.a range, preferably over 50% and most preferably over at
least 70% of the f.sub.a range of 0.2 to 1.0 are utilized in the
compositions. Combinations that display synergy over a substantial
range of f.sub.a values may be re-evaluated at a variety of agent
ratios to define the optimal ratio to enhance the strength of the
non-antagonistic interaction and increase the f.sub.a range over
which synergy is observed.
[0114] While it would be desirable to have synergy over the entire
range of concentrations over which cells are affected, it has been
observed that in many instances, the results are considerably more
reliable in an f.sub.a range of 0.2-0.8. Thus, although the synergy
exhibited by combinations of the invention is set forth to exist
within the broad range of 0.01 or greater, it is preferable that
the synergy be established in the f.sub.a range of 0.2-0.8. Other
more sensitive assays, however, can be used to evaluate synergy at
f.sub.a values greater than 0.8 for example bioluminescence or
clonogenicity assays.
[0115] The optimal combination ratio may be further used as a
single pharmaceutical unit to determine synergistic or additive
interactions with a third agent. In addition, a three-agent
combination may be used as a unit to determine non-antagonistic
interactions with a fourth agent, and so on.
[0116] As set forth above, the in vitro studies on cell cultures
will be conducted with "relevant" cells. The choice of cells will
depend on the intended therapeutic use of the agent. Only one
relevant cell line or cell culture type need exhibit the required
non-antagonistic effect in order to provide a basis for the
compositions to come within the scope of the invention.
[0117] For example, in a frequent embodiment, the combination of
agents is utilized in anti-neoplastic therapy. Often, the
combination of agents is intended for leukemia or lymphoma therapy.
Appropriate choices will then be made of the cells to be tested and
the nature of the test. In particular, tumor cell lines are
suitable subjects and measurement of cell death or cell stasis is
an appropriate end point. As will further be discussed below, in
the context of attempting to find suitable non-antagonistic
combinations for other indications, other target cells and criteria
other than cytotoxicity or cell stasis could be employed.
[0118] For determinations involving antitumor agents, cell lines
may be obtained from standard cell line repositories (NCI or ATCC
for example), from academic institutions or other organizations
including commercial sources. Preferred cell lines would include
one or more selected from cell lines identified by the
Developmental Therapeutics Program of the NCI/NIH. The tumor cell
line screen used by this program currently identifies 60 different
tumor cell lines representing leukemia, melanoma, and cancers of
the lung, colon, brain, ovary, breast, prostate and kidney. The
required non-antagonistic effect over a desired concentration range
need be shown only on a single cell type; however, it is preferred
that at least two cell lines exhibit this effect, more preferably
three cell lines, more preferably five cell lines, and more
preferably 10 cell lines. The cell lines may be established tumor
cell lines or primary cultures obtained from patient samples. The
cell lines may be from any species but the preferred source will be
mammalian and in particular human. The cell lines may be
genetically altered by selection under various laboratory
conditions, and/or by the addition or deletion of exogenous genetic
material. Cell lines may be transfected by any gene-transfer
technique, including but not limited to, viral or plasmid-based
transfection methods. The modifications may include the transfer of
cDNA encoding the expression of a specific protein or peptide, a
regulatory element such as a promoter or enhancer sequence or
antisense DNA or RNA. Genetically engineered tissue culture cell
lines may include lines with and without tumor suppressor genes,
that is, genes such as p53, pTEN and p16; and lines created through
the use of dominant negative methods, gene insertion methods and
other selection methods. Preferred tissue culture cell lines that
may be used to quantify cell viability, e.g., to test antitumor
agents, include, but are not limited to, P388, L1210, HL-60,
MOLT-4, KBM-3, WeHi-3, H460, MCF-7, SF-268, HT29, HCT-116, LS180,
B16-F10, A549, Capan-1, CAOV-3, IGROV1, PC-3, MX-1 and
MDA-MB-231.
[0119] In one preferred embodiment, the given effect (f.sub.a)
refers to cell death or cell stasis after application of a
cytotoxic agent to a cell culture. Cell death or viability may be
measured using known techniques.
[0120] Because combination therapy is particularly useful in the
case of treatment of tumors, certain active agents are favored for
use in combination when the target disease or condition is cancer.
A non-limiting set of examples comprise the following: "Signal
transduction inhibitors" which interfere with or prevents signals
that cause cancer cells to grow or divide; "Cytotoxic agents";
"Cell cycle inhibitors" or "cell cycle control inhibitors" which
interfere with the progress of a cell through its normal cell
cycle, the life span of a cell, from the mitosis that gives it
origin to the events following mitosis that divides it into
daughter cells; "Checkpoint inhibitors" which interfere with the
normal function of cell cycle checkpoints, e.g., the S/G2
checkpoint, G2/M checkpoint and G1/S checkpoint; "Topoisomerase
inhibitors", such as camptothecins, which interfere with
topoisomerase I or II activity, enzymes necessary for DNA
replication and transcription; "Receptor tyrosine kinase
inhibitors" which interfere with the activity of growth factor
receptors that possess tyrosine kinase activity; "Apoptosis
inducing agents" which promote programmed cell death;
"Antimetabolites," such as Gemcitabine or Hydroxyurea, which
closely resemble an essential metabolite and therefore interfere
with physiological reactions involving it; "Telomerase inhibitors"
which interfere with the activity of a telomerase, an enzyme that
extends telomere length and extends the lifetime of the cell and
its replicative capacity; "Cyclin-dependent kinase inhibitors"
which interfere with cyclin-dependent kinases that control the
major steps between different phases of the cell cycle through
phosphorylation of cell proteins such as histones, cytoskeletal
proteins, transcription factors, tumor suppresser genes and the
like; "DNA damaging agents"; "DNA repair inhibitors";
"Anti-angiogenic agents" which interfere with the generation of new
blood vessels or growth of existing blood vessels that occurs
during tumor growth; and "Mitochondrial poisons" which directly or
indirectly disrupt mitochondrial respiratory chain function.
[0121] Especially preferred combinations for treatment of tumors
are the clinically approved combinations set forth hereinabove. As
these combinations have already been approved for use in humans,
reformulation to assure appropriate delivery is especially
important.
[0122] Preferred agents that may be used in combination include DNA
damaging agents such as carboplatin, cisplatin, cyclophosphamide,
doxorubicin, daunorubicin, epirubicin, mitomycin C, mitoxantrone;
DNA repair inhibitors including 5-fluorouracil (5-FU) or FUDR,
gemcitabine and methotrexate; topoisomerase I inhibitors such as
camptothecin, irinotecan and topotecan; S/G2 or G2/M checkpoint
inhibitors such as bleomycin, docetaxel, doxorubicin, etoposide,
paclitaxel, vinblastine, vincristine, vindesine and vinorelbine;
G1/early-S checkpoint inhibitors; G2/M checkpoint inhibitors;
receptor tyrosine kinase inhibitors such as genistein, trastuzumab,
ZD1839; cytotoxic agents; apoptosis-inducing agents and cell cycle
control inhibitors.
[0123] The mechanism of action of one or more of the agents may not
be known or may be incorrectly identified. All synergistic or
additive combinations of agents are within the scope of the present
invention. Preferably, for the treatment of a neoplasm,
combinations that inhibit more than one mechanism that leads to
uncontrolled cell proliferation are chosen for use in accordance
with this invention. For example, the present invention includes
selecting combinations that effect specific points within the cell
cycle thereby resulting in non-antagonistic effects. For instance,
active agents that cause DNA damage can be paired with those that
inhibit DNA repair, such as anti-metabolites. The present invention
also includes selecting combinations that block multiple pathways
that would otherwise result in cell proliferation.
[0124] Particularly preferred combinations are DNA damaging agents
in combination with DNA repair inhibitors, DNA damaging agents in
combination with topoisomerase I or topoisomerase II inhibitors,
topoisomerase I inhibitors in combination with S/G2 or G2/M
checkpoint inhibitors, G1/S checkpoint inhibitors or CDK inhibitors
in combination with G2/M checkpoint inhibitors, receptor tyrosine
kinase inhibitors in combination with cytotoxic agents,
apoptosis-inducing agents in combination with cytotoxic agents,
apoptosis-inducing agents in combination with cell-cycle control
inhibitors, G1/S or G2/M checkpoint inhibitors in combination with
cytotoxic agents, topoisomerase I or II inhibitors in combination
with DNA repair inhibitors, topoisomerase I or II inhibitors or
telomerase inhibitors in combination with cell cycle control
inhibitors, topoisomerase I inhibitors in combination with
topoisomerase II inhibitors, and two cytotoxic agents in
combination.
[0125] Specific agents that may be used in combination include
cisplatin (or carboplatin) and 5-FU (or FUDR), cisplatin (or
carboplatin) and irinotecan, irinotecan and 5-FU (or FUDR),
vinorelbine and cisplatin (or carboplatin), methotrexate and 5-FU
(or FUDR), idarubicin and araC, cisplatin (or carboplatin) and
taxol, cisplatin (or carboplatin) and etoposide, cisplatin (or
carboplatin) and topotecan, cisplatin (or carboplatin) and
daunorubicin, cisplatin (or carboplatin) and doxorubicin, cisplatin
(or carboplatin) and gemcitabine, oxaliplatin and 5-FU (or FUDR),
gemcitabine and 5-FU (or FUDR), adriamycin and vinorelbine, taxol
and doxorubicin, flavopuridol and doxorubicin, UCN01 and
doxorubicin, bleomycin and trichlorperazine, vinorelbine and
edelfosine, vinorelbine and sphingosine (and sphingosine
analogues), vinorelbine and phosphatidylserine, vinorelbine and
camptothecin, cisplatin (or carboplatin) and sphingosine (and
sphingosine analogues), sphingosine (and sphingosine analogues) and
daunorubicin and sphingosine (and sphingosine analogues) and
doxorubicin.
[0126] Preferred combinations in general include those set forth
hereinabove as already shown to be efficacious in the clinic as
recognized by the FDA and those further suggested based on
literature reports. While the candidate agents for use in the
method of the invention are not limited to these specific
combinations, those set forth hereinabove have been disclosed as
suitable combination therapies, and are thus preferred for use in
the methods and compositions of the present invention.
[0127] The therapeutic agents in the present compositions may be
formulated separately in individual compositions wherein each
therapeutic agent is stably associated with appropriate delivery
vehicles. These compositions can be administered separately to
subjects as long as the pharmacokinetics of the delivery vehicles
are coordinated so that the ratio of therapeutic agents
administered is maintained at the target for treatment. Thus, it is
useful to construct kits which include, in separate containers, a
first composition comprising delivery vehicles stably associated
with at least a first therapeutic agent and, in a second container,
a second composition comprising delivery vehicles stably associated
with at least one second therapeutic agent. The containers can then
be packaged into the kit.
[0128] The kit will also include instructions as to the mode of
administration of the compositions to a subject, at least including
a description of the ratio of amounts of each composition to be
administered. Alternatively, or in addition, the kit is constructed
so that the amounts of compositions in each container is
pre-measured so that the contents of one container in combination
with the contents of the other represent the correct ratio.
Alternatively, or in addition, the containers may be marked with a
measuring scale permitting dispensation of appropriate amounts
according to the scales visible. The containers may themselves be
useable in administration; for example, the kit might contain the
appropriate amounts of each composition in separate syringes.
Formulations which comprise the pre-formulated correct ratio of
therapeutic agents may also be packaged in this way so that the
formulation is administered directly from a syringe prepackaged in
the kit.
[0129] Therapeutic activity of delivery vehicle compositions
comprising two or more active agents may be measured after
administration into an animal model. Preferably, the animal model
comprises a tumor although delivery vehicle compositions may be
administered to animal models of other diseases. Rodent species
such as mice and rats of either inbred, outbred, or hybrid origin
including immunocompetent and immunocompromised, as well as
knockout, or transgenic models may be used.
[0130] Methods for evaluating efficacy in treatment of various
conditions, including tumors, are well known in the art.
[0131] Non-Pharmaceutical Applications
[0132] Many ink jet printing inks are based on soluble dyes. These
present two problems. First the soluble dyes are prone to
"bleeding" and are not as water-fast as is desired, and second, the
dye wicks into the paper prior to drying with a corresponding loss
in color intensity. To overcome these problems one strategy is to
use insoluble pigment particles. However, the range of colors
obtained from soluble dyes is not matched by the pigments in
particulate form. The current invention would allow the conjugation
of dyes to hydrophobic linkers that would allow incorporation into
nanoparticle form. The especially preferable embodiment of this
technology would couple Flash Nano Precipitation to form narrow
size distribution particles in the range of 200 nm with the
conjugation scheme to incorporate otherwise soluble dyes. The Flash
Nano-Precipitation process allows the incorporation of multiple
colors into a single particle to effect color blending.
[0133] In many industrial and biological applications it is
desirable to have fluorescently labeled particles in the size range
50 nm to 2,000 nm. These are most commonly made from polymeric
emulsion polymerized lattices into which dyes are imbibed.
Alternatively, fluorescent species are reacted onto the surface of
the particles. See Polysciences, Inc., Particle Catalog for a
listing of representative particles (website: polysciences.com).
The imbibing route has limitations as to the dyes that are
hydrophobic enough to be retained in the spheres, and the chemical
reacting route has limitations as to the number of fluorescent
molecules that can be attached to a single sphere. Furthermore, the
production of these tracer particles requires independent steps of
particle formation, and then post processing to introduce the
fluorescent species. In the present invention it is possible to
conjugate a wide range of fluorescent dyes to make them
hydrophobic. Using the Flash Nano Precipitation process it is then
possible to produce fluorescent particles with high levels of
fluorescence, narrow particle size distribution, controlled
particle size, and tailor surface functionality.
[0134] In many applications it is desirable to have fragrances that
are released over time. For example in laundry fabric conditioners,
spray deodorizers, and perfumes. With the present invention it is
possible to conjugate fragrances and to keep them in a particulate,
highly dispersed form. The fragrance can be released over time by
the hydrolytic cleavage of the linking bond, or by light cleavage
of a photocleavable bond.
[0135] Sunscreens for personal care operate by applying UV
absorbing species to the skin. See for example Chemical and
Engineering News (2005) 83:18-22. It has been found that
particulate systems have the added advantage of absorbing and
scattering UV light and, therefore, enhance the performance of
these formulations. Several efficient UV absorbers are too readily
soluble to remain on the skin after exposure to water. In the
present invention, UV absorbers can be conjugated with hydrophobic
moieties to enable incorporation into nanoparticles in long-lasting
formulation. By incorporation of appropriate hydrophilic blocks on
the particle surface, that include cationic and hydrogen bonding
monomers, it would be possible to have the nanoparticle formulation
adhere to the skin for prolonged periods of time.
[0136] The following examples are offered to illustrate but not to
limit the invention.
EXAMPLE 1
Conjugation of Paclitaxel to Vitamin E Succinate (VitES)
[0137] 280 mg of VitES were dissolved in 20 ml of dichloromethane
and brought to 0.degree. C. Then, 27 .mu.L of
diisopropylcarbodiimide were added, followed by 150 mg of
paclitaxel and 33 mg of dimethylaminopyridine. The reaction vessel
was warmed to room temperature, and left to react for 16 hours. The
reaction solution was washed with 0.1 N hydrochloric acid, dried
with magnesium sulfate, filtered, and dried in vacuo. The product
was characterized and verified to be paclitaxel-VitES by High
Performance Liquid Chromatography (HPLC) and Nuclear Magnetic
Resonance (NMR) analysis.
EXAMPLE 2
Conjugation of Paclitaxel to Polycaprolactone with Terminal
Carboxylic Acids
[0138] A. 79 mg of PCL (MW 2.2 kg/mole, PCL2.2) end-terminated with
carboxylic acid groups were dissolved in 20 ml of dichloromethane
and brought to 0.degree. C. 26 .mu.L of diisopropylcarbodiimide
were added, followed by 146 mg of paclitaxel and 32 mg of
dimethylaminopyridine. The reaction vessel was warmed to room
temperature, left to react for 16 hours, and washed with 0.1 N
hydrochloric acid, dried with magnesium sulfate, filtered, and
dried in vacuo. The amount of excess paclitaxel in the reaction
product was reduced by recrystallization from amyl acetate. The
product was characterized and verified as PCL-paclitaxel by HPLC
and NMR analysis.
[0139] B. The procedure of paragraph A was also carried out using
PCL1.45 and PCL3.5.
EXAMPLE 3
Nanoparticles with Methoxy Polyethylene Glycol-Polycaprolactone
(mPEG-PCL) and Paclitaxel-VitE
[0140] As a control, 15 mg of methoxy polyethylene glycol-(MW 5
kg/mole)-polycaprolactone (methoxy polyethylene glycol molecular
weight of 5 kg/mole, PCL molecular weight of 7 kg/mole)
(mPEG5-PCL7) in THF to make a 1 wt % solution (w:w) of mPEG5-PCL7.
Then, 8 mg of paclitaxel and 10 mg of VitES were added to the
solution and mixed using the vortex mixer at a flow rate of 12
ml/min against water at 120 ml/min. Crystals were visible about 20
minutes after mixing, and no particles were detected by Dynamic
Light Scattering (DLS).
[0141] 20 mg of mPEG5-PCL7 were dissolved in THF to make a 0.5 wt %
solution. Then, 23 mg of paclitaxel-VitES along with 17.4 mg VitES
prepared as described in Example 1 was added to make a 0.58 wt %
paclitaxel-VitES solution, and mixed using the vortex mixer at a
flow rate of 12 ml/min against water at 120 ml/min. Nanoparticles
with an average diameter of 126 nm, as determined by DLS, were
formed. The nanoparticles size after 17 hours was 134 nm. No
visible crystals or aggregates were observed in the sample.
EXAMPLE 4
Nanoparticles of Methoxy Polyethylene Glycol-Polycaprolactone and
Paclitaxel-PCL
[0142] mPEG5-PCL6 was dissolved in THF to make a 0.5 wt % solution
(w:w). Then, paclitaxel-PCL prepared as described in Example 2 was
added to the solution to make a 0.5 wt % (w:w) of the conjugate.
The resulting solution was mixed using the vortex mixer at a flow
rate of 12 ml/min against water at 120 ml/min, yielding
nanoparticles with an average diameter of 75 nm. The nanoparticles
size after 60 hours was 93 nm.
EXAMPLE 5
Rifampicin-VitES
[0143] Vitamin E succinate (1210 IU/g) (2273.0 mg, 4.28 mMol) was
dissolved in 20 ml of anhydrous methylene chloride. To this
solution at 0.degree. C. were added DIPC (654.9 .mu.l, 4.28 mMol),
rifampicin (1762.1 mg, 2.14 mMol) dissolved in 20 ml of anhydrous
methylene chloride, and DMAP (806.1 mg, 6.55 mMol). The resulting
solution was warmed to room temperature and left for 16 hours. The
reaction mixture was washed with 0.1 N HCl, dried, and evaporated
in vacuo to yield the product as a red powder. .sup.13C and .sup.1H
NMR and HPLC confirmed the function or Rifampicin-VitES.
EXAMPLE 6
Comparative Example
[0144] Attempts were made to obtain nanoparticles by vortex mixing
unconjugated rifampicin with block copolymer poly (ethylene
glycol)-b-poly (caprolactone) (PEG-b-PCL) (5k-5k). Only when the
initial concentration of rifampicin in solvent (dimethylformamide)
is over 12 wt %, could rifampicin be precipitated. Below this
concentration clear red solutions of the final product were
produced. Dynamic light scattering showed no nanoparticles above
the size of approximately 10 nm (the lower resolution limit of the
autocorrelator).
[0145] For unconjugated rifampicin at concentrations above 12 wt %
it was possible to make nanoparticles using a four stream vortex
mixer. The conditions of the experiment are given below.
[0146] Drug: rifampicin
[0147] Polymer: PS-b-PEO (1k-3k)
[0148] Solvent: DMF
[0149] Stream 1: 341 mg rifampicin (12 wt %) and 56.9 mg PW-b-PEO
(2 wt %) in 3 ml DMF.
[0150] Stream 2: pH 5.5 buffer solution.
[0151] Stream 3: pH 5.5 buffer solution.
[0152] Stream 4: pH 5.5 buffer solution.
[0153] Mixing conditions:
[0154] Stream 1: 12 ml/min
[0155] Stream 2, 3, 4: 36 ml/min
[0156] DLS results:
[0157] 2 hours after the mixing:
[0158] average radius=170.69 nm
[0159] width=108.09 nm
[0160] Polydispersity index=0.402
[0161] 4 hours after the mixing:
[0162] average radius=532.68 nm
[0163] width=335.68 nm
[0164] Polydispersity index=0.397
[0165] The particle growth from 170 nm after 2 hours to 532 nm
after four hours shows that the drug cannot form stable
nanoparticles in the unconjugated form.
EXAMPLE 7
Rifampicin-VitES Particles
[0166] Conjugated rifampicin from Example 5 was mixed with block
copolymer poly (ethylene glycol)-b-poly (caprolactone) (PEG-b-PCL)
(5k-5k) as described in Example 6. Stable particles were formed
when the initial concentration of conjugated rifampicin in DMF is 4
wt %. And the particles were stable as shown by DLS. The results
are shown in the table below,
TABLE-US-00001 Particle Formation with Rifampicin Drug solubility
in Anti-solvent mixed Sample # Drug polymer solvent (3 streams)
Ratio solution Flow rate description 1 Rifampicin PEG-b-PCL DMF pH
5.0 1:9 -- DMF: 12 ml/min Lighter orange color compare conjugated
with (5k-5k) citrite buffer Buffer: with others. Vit E succinate 2
wt % 36 ml/min each Most suspended in the 12 wt % solution. Some
residue. 2 Rifampicin No DMF pH 5.0 1:9 0.49 mg/ml DMF: 12 ml/min
Big junks quickly settle to the 12 wt % citrite buffer Buffer:
bottom. 36 ml/min each 3 Rifampicin PEG-b-PCL DMF pH 5.0 1:9 0.49
mg/ml DMF: 12 ml/min Day 1-2, clear red. No 12 wt % (5k-5k) citrite
buffer Buffer: particles observed by DLS. 2 wt % 36 ml/min Particle
size <10 nm; supersaturation S .apprxeq. 24. But after 3 days
will be the same as sample 4. 4 Rifampicin PEG-b-PCL DMF pH 5.5 1:9
0.52 mg/ml DMF: 12 ml/min Sample crystallized on the 12 wt %
(5k-5k) citrite buffer Buffer: glass walls after 3 days. 2 wt % 36
ml/min each 5 Rifampicin PEO-b-PS DMF pH 5.5 1:9 0.52 mg/ml DMF: 12
ml/min Most solids suspended in the 12 wt % (3k-1k) citrite buffer
Buffer: solution and stable. Some 2 wt % 36 ml/min each residues on
the bottom. (similar to PEG-b-PCL at pH = 4.0) 6 Rifampicin
PEG-b-PCL DMF pH 5.0 1:9 -- DMF: 12 ml/min Lighter orange color
compare conjugated with (5k-5k) citrite buffer Buffer: with others.
Vit E succinate 2 wt % 36 ml/min each Stable solution. 4 wt % 7
Rifampicin PEG-b-PCL DMF pH 4.0 1:9 -- DMF: 12 ml/min Similar to
sample 5. 12 wt % (5k-5k) citrite buffer Buffer: 2 wt % 36 ml/min
each
EXAMPLE 8
Conjugation of Rifampicin to Dicarboxyl PCL
[0167] Dicarboxyl PCL (5 kDa) (10700 mg, 2.14 mMol) was dissolved
in 20 ml of anhydrous methylene chloride. To this solution at
0.degree. C. were added DIPC (654.9 .mu.l, 4.28 mMol), rifampicin
(1762.1 mg, 2.14 mMol) dissolved in 20 ml of anhydrous methylene
chloride, and DMAP (806.1 mg, 6.55 mMol). The resulting solution
was taken out of the ice bath to warm to room temperature and left
for 16 hours. The reaction mixture was washed with 0.1 N HCl,
dried, and evaporated in vacuo to yield the product as a red
powder. .sup.13C and .sup.1H NMR and HPLC confirmed the structure
as rifampicin-PCL.
EXAMPLE 9
Nanoparticles from Poly (ethylene glycol)-b-poly (caprolactone) and
Rifampicin-PCL
[0168] Conjugated rifampicin from Example 8 above was mixed with
block copolymer poly (ethylene glycol)-b-poly (caprolactone)
(PEG-b-PCL) (5k-5k) as described in Example 6. Stable nanoparticles
were formed when the initial concentration of rifampicin-PCL in DMF
is 4 wt %. And the particles were stable as shown by DLS.
EXAMPLE 10
Estradiol-VitES Conjugation
[0169] .alpha.-Tocopherol succinate (530.8 g/mol) is dissolved in
anhydrous dichloromethane at 0.degree. C.
1,3-diisopropylcarbodiimide (DIPC, 152.9 g/mol), estradiol (272.39
g/mol) and 4-(dimethylamino)-pyridine (DMAP, 123.1 g/mol) are added
to the solution at a molar ratio of 1.5:0.25:1.5 with respect to
.alpha.-tocopherol succinate. The reaction mixture is warmed to
room temperature and aged for a period of 70 hours to achieve near
complete conversion to the conjugate. A 0.1 N HCl wash is employed
following reaction completion for the removal of residual DMAP. The
solution is evaporated to dryness and the solid product,
estradiol-VitES, isolated. The product estradiol-VitES was
characterized by High Performance Liquid Chromatography (HPLC) and
Nuclear Magnetic Resonance (NMR) analysis.
EXAMPLE 11
Estradiol-VitES Nanoparticle Formation
[0170] Control: Estradiol and methoxy-poly(ethylene
glycol)-b-poly(.epsilon.-caprolactone) (mPEG-PCL, 5,000-2,900
g/mole, respectively), are dissolved in THF at a weight ratio of
1:1 to make a 0.3 wt % solution for each component. The resulting
solution is loaded into a gas tight syringe, and impingement mixed
with an anti-solvent (water) using the Confined Impinging Jet (CIJ)
mixer at injection rates of 12 ml/min and 120 ml/min for THF and
water, respectively. Estradiol loaded nanoparticles were unstable
soon after particle formation (<30 minutes), as indicated by
visual observation of aggregates. DLS analysis could not be
performed due to the presence of these aggregates.
[0171] Standard mixing procedure was followed to produce
estradiol-VitES loaded nanoparticles with the Confined Impinging
Jet (CIJ) mixer. The estradiol-VitES along with the block copolymer
were dissolved in THF at the aforementioned weight ratios and
impingement mixed with DI water. The resulting nanoparticles
demonstrated a less than 10% increase in radius over the course of
48 hours, as indicated by Dynamic Light Scattering (DLS) analysis.
Additional stability was observed in particles where the reaction
mixture comprises a 3:1 molar ratio of VitES to conjugated to
estradiol-VitES with stability noted in excess of 30 days.
EXAMPLE 12
Nanoparticles Containing Estradiol-VitES and Paclitaxel-VitES
[0172] An estradiol-VitES conjugate is prepared as in Example 10. A
conjugate of paclitaxel-VitES is prepared as according to Example
1. Nanoparticles comprising both estradiol-VitES and
paclitaxel-VitES were made by first dissolving 30 mg of methoxy
polyethylene glycol-polycaprolactone (methoxy polyethylene glycol
molecular weight of 5 kg/mole, PCL molecular weight of 7 kg/mole)
(mPEG5-PCL7) in 3 ml of THF to make a 1 wt % solution (w:w) of
mPEG5-PCL7. Then, 8.5 mg of paclitaxel-VitES and 6.5 mg of
estradiol-VitES prepared as described in the previous examples were
added to the mPEG5-PCL7 solution in THF with 15 mg VitES. The
weight ratio of paclitaxel-VitES to estradiol-VitES is 1.27. The
resulting solution was mixed using the vortex mixer at a flow rate
of 12 ml/min against water at 120 ml/min, yielding nanoparticles
with an average diameter of 107 nm as determined by DLS (see plot
below). No visible crystals or aggregates were observed in the
sample.
EXAMPLE 13
Nanoparticles Containing Neoplastic Agents
[0173] A hydrophobic polymer-paclitaxel conjugate is prepared in
accordance with the methods set out in Greenwald, et al., supra
(1996), which addressed the formation of water soluble Taxol- poly
(ethylene glycol) prodrugs. Instead of using poly (ethylene
glycol), we will use poly (caprolactone) with a carboxylic acid end
group to form the active agent conjugate. The reaction scheme used
by Greenwald is shown in FIG. 1.
[0174] Complex 2, as depicted in FIG. 1, provided a hydrolysis
half-life of 2 h in human whole blood, as observed in Greenwald's
work. The hydrolysis kinetics in Greenwald's work vary from
t.sub.1/2>72 hours in DI water at pH 5.7 to t.sub.1/2>5.5
hours in PBS buffer at pH 7.4. In the present case, the prodrug is
encapsulated in the hydrophobic core of the nanoparticle, and
accordingly, different hydrolysis rates (e.g., slower) and
half-lives (e.g., longer) are provided, since the water activity is
lower in the hydrophobic core of the nanoparticle. Having
paclitaxel bound to a homopolymer in the nanoparticle core acts as
a crystallization site for free paclitaxel, resulting in increased
active agent retention.
[0175] The same rationale is used to form cisplatin-polymer
complexes. First, cisplatin is reacted with a mono acid or a diacid
end group of a hydrophobic homopolymer to form a cisplatin-polymer
complex, then the stabilizing diblock copolymer is added, and
finally PEG-protected nanoparticles are formed using the vortex
mixer. The cisplatin complex is prepared based on the work of Ohya,
et al. supra (2000), where poly (ethylene glycol)- cisplatin
complexes were prepared based on a 6-membered chelate-type
dicarboxylate coordination bond, as shown in FIG. 2.
EXAMPLE 14
Formulation of Synergistic Combinations in Polymer-Based Carriers:
where One or More of the Agents Has Both Unfavorable Water and
Lipid Solubilities
[0176] Four-two active agent combinations have been identified for
purposes of this example. These include combinations comprising
paclitaxel with cisplatin, etoposide with cisplatin, taxotere with
doxorubicin, and paclitaxel with doxorubicin. These are formulated
at particular ratios shown to be non-antagonistic.
[0177] Encapsulation Based on Hydrophobic Polymer-Active Agent
Conjugation
[0178] It is hypothesized that the formation of a hydrophobic
active agent-polymer conjugate using hydrophobic biodegradable
polymers will provide an active agent release rate determined by
the rate of chemical hydrolysis rather than diffusion. In addition,
covalent attachment of the active agent to the hydrophobic block of
the polymer will prevent Ostwald ripening of the nanoparticles and
improve the stability of the formulation. Thus, although selected
linkers are set forth below, a variety of other chemical linkers
between the active agent and the polymer are contemplated as set
forth herein. Coupling an active agent to a polymer using any of a
variety of these linkers is accomplished in accordance with the
presently described methods and others known and available in the
art.
[0179] Paclitaxel-Hydrophobic Polymer Conjugate Formation
[0180] Novel Paclitaxel-polymer conjugates are prepared using
hydrophobic polymers. The approach set out herein is based on the
conjugation of paclitaxel to a homopolymer backbone before
encapsulation using the diblock copolymer; First, paclitaxel is
reacted with a hydrophobic homopolymer to form a paclitaxel-polymer
conjugate, then the stabilizing diblock copolymer is added, and
finally PEG-protected nanoparticles is formed using the vortex
mixer. PCL and poly (lactide) with a terminal carboxylic acid group
(PLA-COOH) are specifically investigated, but other polymers that
are suitable are poly(lactide-co-glycolide)-COOH;
poly(lactide)-COOH; poly(.epsilon.-caprolactone)-COOH and
poly(.beta.-benzyl-aspartate)-COOH.
[0181] Paclitaxel-PLA conjugate is prepared following Greenwald's
procedure for making mPEG-paclitaxel prodrug. PLA-COOH (16,000
g/mole) is dissolved in dichloromethane to make a 3 wt % solution.
The resulting solution is brought to 0.degree. C., and
diisopropylcarbodiimide (DIPC) is added at a molar ratio of 1.36:1
DIPC:PLA-COOH. Paclitaxel is then added at a molar ratio of 1:1
paclitaxel:DIPC. The reaction mixture is. then warmed to room
temperature, and left to react for 16 hours. A 0.1 N HCl is used
for washing, and the solution is dried and evaporated in vacuo. The
resulting solid is crystallized from 2-propanol and the product is
analyzed using NMR. Nanoparticles of the active agent conjugate are
then formed following the method outlined in the Formulation and
Characterization section, using the active agent conjugate instead
of the pure active agent. The experiment is repeated for
paclitaxel-polymer: block copolymer weight ratios of 1:1, 1:3, and
1:10. The resulting nanoparticles are analyzed for size, active
agent content, and in vitro release rates as outlined in the
Formulation and Characterization section.
[0182] Cisplatin-Hydrophobic Polymer Complex Formation
[0183] Kataoka has demonstrated the formation of cisplatin
complexes in water with homopolymers of poly
(.alpha.,.beta.-aspartic acid) and poly(ethylene
glycol)-poly(glutamic acid) block copolymers, resulting in
nanoparticles with cisplatin release times of approximately 14
hours. See Nishiyama, N., et al., J. of Controlled Release (2001)
74:83-94; see also Nishiyama, N., et al., Cancer Research (2003)
63:8977-8983.
[0184] In another embodiment, cisplatin-polymer conjugates are
formulated using hydrophobic polymers. In this embodiment cisplatin
is conjugated to a homopolymer end group prior to encapsulation
using the diblock copolymer. PCL and poly (lactide) homopolymers
having terminal diacid groups are described below. The present
method is based on Ohya, Y., et al., (2000) (cited supra), to form
poly (ethylene glycol)-cisplatin complexes.
[0185] Diacid diethyl ester terminated poly (lactide)
(PLA-Da(Et).sub.2): 0.5 mMole of poly (lactide) is dissolved in 10
ml of anhydrous THF, and mixed with sodium and naphthalene (1.5
mMole). After refluxing under Ar atmosphere for 4 hours,
diethylchloropropylmalonate (3 nMole) in 10 ml of THF is added to
the reaction mixture, and refluxed for 4 hours under Ar. The final
product is obtained by concentration and re-precipitation using
diethyl ether. .sup.1H-NMR is used to confirm the structure.
[0186] Cisplatin attachment to PLA-Da(Et.sub.2): PLA-Da(Et.sub.2)
obtained from the above procedure is dissolved in 10 ml of ethanol
(aq., 95%) and 243 mg of NaOH, and refluxed for 90 min. The
resulting solution is subjected to an anion exchange resin column
(QAE-Sephadex A-25, water then 2M-NaCl at 1 ml/min effluent) after
refluxing for 90 minutes and re-precipitation using diethyl ether.
The solution is then freeze-dried as described in the Formulation
and Characterization section (above) to yield PLA-Da (Na salt).
.sup.1H-NMR is used to confirm the reaction. Cisplatin (50 mg) is
dissolved in water and stirred for 3 h at 60.degree. C., after
which 0.22 ml of a 0.1 M silver nitrate solution is added and mixed
at 60.degree. C. for 6 h. The solution is filtered to remove
precipitated silver chloride, and the filtrate dried in vacuo. The
product is dissolved in THF and PLA-Da (Na salt) is added and left
to react at 60.degree. C. for 24 h. Gel-filtration chromatography
is used to purify the sample, and the higher molecular weight
fraction is freeze-dried as described in the Formulation and
Characterization section. Atomic absorption spectrometry is used to
determine the amounts of platinum in the complex.
[0187] Co-Formulation of Paclitaxel and Cisplatin
[0188] Subsequent to the successful encapsulation of each
paclitaxel (or paclitaxel-polymer conjugate) and cisplatin-polymer
complex, both agents are encapsulated in one carrier.
[0189] The nanoparticles encapsulating both paclitaxel (or
corresponding polymer conjugate) and the cisplatin complex are
formed by the following procedure. Each component (in active agent
or complex form) is dissolved in THF to make a 0.3 wt % solution
for each active agent. mPEG-PCL is added at a weight ratio of 1:1
mPEG-PCL:active agents. The nanoparticles then form, as outlined in
the Formulation and Characterization section, using the
cisplatin-polymer complex and the paclitaxel (or paclitaxel-polymer
conjugate) instead of one active agent. The experiment is repeated
for cisplatin:paclitaxel molar ratios of 1:5 and 5:1, and for
active agents:block copolymer weight ratios of 1:1, 1:5 and 1:10.
The resulting nanoparticles are analyzed for size, active agent
content, and in vitro release rates as outlined in the Formulation
and Characterization section.
EXAMPLE 15
Controlled Delivery of Paclitaxel Using Stable Polymer-Based
Formulations
[0190] This example demonstrates the stability and controlled
release of paclitaxel from polymer-based nanoparticles formed via
the presently described methods.
[0191] Material Storage and Stability
[0192] A paclitaxel formulation is prepared, lyophilized and
redispersed in water to the primary particle size (determined
before freeze-drying) using sucrose at a weight ratio of 60:1
sucrose:nanoparticles.
[0193] The stability of the paclitaxel nanoparticles in
freeze-dried form is evaluated over a period of one month to
demonstrate long term storage of the lyophilized material.
[0194] Lyophilized material obtained through the nanoparticle
formation process as described in the Formulation and
Characterization section is stored at 4.degree. C. A sample is
collected every week for the first month, then monthly and analyzed
for size, active agent content, and in vitro release rate as
outlined in the Formulation and Characterization section. The same
procedure is repeated for a sample stored at room temperature.
[0195] In Vitro Active Agent Release Testing
[0196] The in vitro paclitaxel release rate from the nanoparticles
is controlled to provide release half lives of >4h.
[0197] Lyophilized nanoparticles containing paclitaxel are
dissolved at a target active agent concentration of 1-5 mg/ml in
water. The solution is diluted 2-10 fold in serum and incubated at
37.degree. C. Aliquots are collected at 1, 2, 4, 8, 16, and 24
hours intervals, and assayed for paclitaxel. A Biogel A-0.5M gel
filtration column is used to separate proteins and free active
agent from the polymer-associated active agent. The paclitaxel
concentration is determined as described in the Formulation and
Characterization section.
[0198] In Vivo Active Agent Release Testing
[0199] In vivo paclitaxel release rate are evaluated through the
injection of the active agent nanoparticles into mice IV at a
paclitaxel dosage of 10 mg/kg. The target paclitaxel half life is 4
hours or longer. The plasma active agent elimination properties of
polymer formulations are determined as a function of active
agent/polymer ratio as well as hydrophobic/block co-polymer
ratio.
[0200] Lyophilized nanoparticles containing paclitaxel are
dissolved at a target active agent concentration of 1-5 mg/ml in
water. The solution is diluted as necessary in saline to provide
paclitaxel doses of 10 mg/kg in a volume of 0.2 ml. Following i.v.
administration in mice at a dose of 20 mg/kg, plasma samples are
collected at 1, 2, 4, 8, 16, and 24 hours intervals, and assayed
for paclitaxel. The paclitaxel concentration is determined by HPLC
analysis of a solvent-extracted sample.
EXAMPLE 16
Matched PK for Polymer Formulation where the Synergistic Ratio of
the Active Agent Combination is Maintained after I.V. Injection to
Mice
[0201] Nanoparticles containing both cisplatin and paclitaxel are
investigated for in vivo release rates. The active agent ratios in
the nanoparticles are dictated by the in vitro release rate
results.
[0202] Lyophilized nanoparticles containing paclitaxel and
cisplatin are dissolved at a target paclitaxel concentration of 1-5
mg/ml in water. The solution is diluted as necessary in saline to
provide paclitaxel doses of 10 mg/kg in a volume of 0.2 ml.
Following i.v. administration in mice, plasma samples are collected
at 1, 2, 4, 8, 16, and 24 hours and assayed for paclitaxel. The
paclitaxel and cisplatin concentrations are determined by HPLC and
atomic absorption, respectively. The experiment is repeated at
various paclitaxel:cisplatin ratios and for various polymer
compositions until the synergistic ratio is maintained after i.v.
injection.
EXAMPLE 17
Evidence of Significant Antitumor Activity in Solid Tumor Model in
Pilot Efficacy Studies
[0203] In this embodiment, improved antitumor activity of a polymer
conjugate CombiPlex.TM. formulation compared to free active agent
cocktail in a human solid tumor xenograft model is evaluated.
[0204] A CombiPlex.TM. formulation containing polymer conjugated
paclitaxel and cisplatin at a molar ratio shown to be
non-antagonistic in vitro and also exhibiting matched release rates
for the two active agents is administered IV to mice bearing
100-200 mg solid tumors. The tumor selected is based on in vitro
screening data where significant non-antagonism and preferably
ratio dependency is observed. Dose range finding studies (3 mice
per group) are first performed to establish MTD's in non-tumor
bearing mice). For efficacy studies, mice (6 per group) are
administered by IV a minimum of two different dose levels of
CombiPlex.TM. and free active agent cocktail at approximate MTD's
and at a matched dose of active agents in the CombiPlex.TM.
formulation. Two treatment schedules are evaluated (weekly.times.3
and Q4D.times.3). Tumor weights are determined by measuring tumors
using calipers. Animals are also monitored for signs of toxicity
(weight loss and physical signs of stress).
EXAMPLE 18
Additional Polymeric Nanoparticle Formulations
[0205] In accordance with the above methods, additional
CombiPlex.TM. formulations are prepared utilizing a variety of
combinations of anti-neoplastic agents. Agent combinations such as
paclitaxel with etoposide, paclitaxel with taxotere, paclitaxel
with doxorubicin, cisplatin with etoposide, cisplatin with
taxotere, cisplatin with doxorubicin, etoposide with taxotere,
etoposide with doxorubicin, doxorubicin with taxotere are prepared
and evaluated. The chemical linkages are altered and adjusted to
provide for desired release rates.
[0206] One of skill in the art would understand that alternative
active agents can be substituted for those set forth in the above
Examples. For example, other platinum analogs such as Carboplatin,
Oxaliplatin, Tetraplatin, Platinum-DACH, Ormaplatin, among others,
can be substituted for Cisplatin. Frequently, an active agent is
substituted with another active agent within the same class, as
discussed above. Often, however, any of a variety of the active
agents set forth herein are combined in a nanoparticle formulation
in accordance with the present materials and methods. Frequently,
these nanoparticle formulations contain a combination of one or two
or three or more active agents.
* * * * *