U.S. patent application number 16/041821 was filed with the patent office on 2019-01-17 for nanoparticles containing a taxane and their use.
The applicant listed for this patent is ANP Technologies, Inc., Fulgent Therapeutics, Inc.. Invention is credited to Jing Pan, Yun Yen, Ray Yin, Yubei Zhang, Bingsen Zhou.
Application Number | 20190015350 16/041821 |
Document ID | / |
Family ID | 65000819 |
Filed Date | 2019-01-17 |
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United States Patent
Application |
20190015350 |
Kind Code |
A1 |
Yin; Ray ; et al. |
January 17, 2019 |
Nanoparticles Containing a Taxane and their Use
Abstract
Symmetrically and asymmetrically branched homopolymers are
modified at the surface level with functional groups that enable
forming aggregates with a taxane, such as, paclitaxel and
derivatives thereof, which are water insoluble or poorly water
soluble. The aggregates are formed by interaction of a taxane and a
homopolymer. Such aggregates improve drug solubility, stability,
delivery and efficacy.
Inventors: |
Yin; Ray; (Wilmington,
DE) ; Pan; Jing; (Newark, DE) ; Zhang;
Yubei; (Hockessin, DE) ; Zhou; Bingsen;
(Walnut, CA) ; Yen; Yun; (Arcadia, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ANP Technologies, Inc.
Fulgent Therapeutics, Inc. |
Newark
Temple City |
DE
CA |
US
US |
|
|
Family ID: |
65000819 |
Appl. No.: |
16/041821 |
Filed: |
July 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15430508 |
Feb 12, 2017 |
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16041821 |
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14765344 |
Aug 2, 2015 |
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PCT/US14/14336 |
Feb 1, 2014 |
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15430508 |
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61760890 |
Feb 5, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/337 20130101;
A61K 9/19 20130101; B82Y 5/00 20130101; A61K 9/0019 20130101; A61K
9/5146 20130101; A61K 9/513 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 31/337 20060101 A61K031/337; A61K 9/00 20060101
A61K009/00; B82Y 5/00 20060101 B82Y005/00; A61K 9/19 20060101
A61K009/19 |
Claims
1. An aggregate comprising: a) a polyoxazoline comprising at least
one first terminal group modified with a hydrophobic moiety,
wherein said polyoxazoline further comprises a linear portion, a
branched portion or both, said branched portion comprises a
symmetrically branched polymer, an asymmetrically branched polymer
or a combination thereof; and said polyoxazoline comprises a molar
ratio of monomer to initiator in a range of from 50:1 to 80:1, and
b) a taxane, wherein said aggregate comprises a weight ratio of
polymer to taxane of 6:1 to 8:1; is from about 50 nm to about 100
nm in size; and comprises a filtration rate through a 0.22 .mu.m
filter of from 50 to 100%.
2. The aggregate of claim 1, wherein said initiator comprises a
hydrophobic electrophilic molecule.
3. The aggregate of claim 1, wherein said initiator comprises a
hydrocarbon.
4. The aggregate of claim 1, wherein said initiator comprises an
aliphatic hydrocarbon, an aromatic hydrocarbon or a combination of
both.
5. The aggregate of claim 1, wherein said initiator comprises a
halide functional group.
6. The aggregate of claim 5, wherein said initiator comprises an
alkyl halide, an aralkyl halide, an acyl halide or combination
thereof.
7. The aggregate of claim 3, wherein said hydrocarbon comprises
from 1 to about 22 carbons, which may be saturated or
unsaturated.
8. The aggregate of claim 1, wherein said initiator comprises
methyl iodide, methyl bromide, methyl chloride, ethyl iodide, ethyl
bromide, ethyl chloride, 1-iodopropane, 1-bromopropane,
1-chloropropane, 1-iodobutane, 1-bromobutane, 1-chlorobutane,
1-iodopentane, 1-bromopentane, 1-chloropentane, 1-iodo hexane,
1-bromo hexane, 1-chloro hexane, 1-iodo dodecane, 1-bromo dodecane,
1-chloro dodecane, 1-iodo octadodecane, 1-bromo octadodecane,
1-chloro octadodecane, benzyl iodide, benzyl bromide, benzyl
chloride, allyl bromide, acyl iodide, acyl bromide, acyl chloride,
benzoyl bromide, benzoyl chloride or a combination thereof.
9. The aggregate of claim 1, wherein said initiator comprises a
tosyl group.
10. The aggregate of claim 1, comprising a size from 50 nm to about
100 nm before lyophilization.
11. The aggregate of claim 1, wherein the polyoxazoline further
comprises a second terminal group comprising a functional group
modified by an ethylenediamine (EDA) or an ethylenediamine
derivative thereof.
12. The aggregate of claim 11, wherein said ethylenediamine
derivative comprises diethylenetriamine, triethylenetetramine,
tetraethylenepentamine, pentaethylenehexamine, polyethylene amine
or tetramethylethylenediamine.
13. The aggregate of claim 1, wherein said taxane is associated
with said at least one first terminal group.
14. The aggregate of claim 1, wherein said polyoxazoline comprises
poly(2-oxazoline), poly(2-substituted oxazoline) or a combination
thereof.
15. The aggregate of claim 1, wherein said polyoxazoline comprises
poly(2-methyloxazoline), poly(2-ethyloxazoline),
poly(2-propyloxazoline) or poly(2-butyloxazoline) or a combination
thereof.
16. The aggregate of claim 1, further comprising a targeting
moiety.
17. The aggregate of claim 16, wherein said targeting moiety
comprises an antibody, an antigen-binding portion thereof, an
antigen, a cell surface receptor, a cytosolic receptor, a cell
receptor ligand or a lectin ligand.
18. The aggregate of claim 1, wherein said taxane comprises
paclitaxel, docetaxel or a combination thereof.
19. A pharmaceutical composition for treating a disease of a
subject in need thereof, wherein said pharmaceutical composition
comprises an aggregate comprising: a) a polyoxazoline comprising at
least one first terminal group modified with a hydrophobic moiety,
wherein said polyoxazoline further comprises a linear portion, a
branched portion or both, said branched portion comprises a
symmetrically branched polymer, an asymmetrically branched polymer
or a combination thereof; and said polyoxazoline comprises a molar
ratio of monomer to initiator in a range of from 50:1 to 80:1, and
b) a taxane, wherein said aggregate comprises a weight ratio of
polymer to taxane of 6:1 to 8:1; is from about 50 nm to about 100
nm in size; and comprises a filtration rate through a 0.22 .mu.m
filter of from 50 to 100%.
20. The pharmaceutical composition of claim 19, comprising a taxane
for treating a breast cancer, an ovarian cancer, a lung cancer,
NSCLC (Non-Small Cell Lung Cancer), a colon cancer, a gastric
cancer, a melanoma, a head and neck cancer, a pancreatic cancer or
a combination thereof.
Description
CROSS REFERENCE
[0001] The instant application is a continuation-in-part of U.S.
Ser. No. 15/430,508 filed on Feb. 12, 2017, which is a continuation
of U.S. Ser. No. 14/765,344 filed on Aug. 2, 2015, which is a 371
national stage application of PCT Ser. No. US14/14336 filed on Feb.
1, 2014, which claims benefit of U.S. Ser. No. 61/760,890, which
was filed on Feb. 5, 2013.
FIELD OF THE INVENTION
[0002] The present disclosure relates to a surface-modified
branched polymer (MBP) or a linear polymer, which can either be a
surface-modified symmetrically branched polymer (SBP); a
surface-modified asymmetrically branched polymer (ABP); or a linear
polymer with at least one chain end modified with a hydrophobic
group, which on exposure to a water insoluble or poorly water
soluble taxane forms a composite nanoparticle or nanoaggregate,
wherein the drug is dispersed or deposited at or near hydrophobic
domains, such as, at the surface or at structures where hydrophobic
portions, segments or sites are located. The particles or
aggregates of interest are stable, for example, can be desiccated
and rehydrated. The nanoparticles or nanoaggregates can range from
about 20 nm to about 500 nm in diameter. Hydrophobic,
electrostatic, metal-ligand interactions, hydrogen bonding and
other molecular interactions may be involved in the spontaneous
interactions between the water insoluble or poorly water soluble
taxane and the homopolymer to form aggregates. The particles or
aggregates of interest have a controlled release profile and thus
find utility, for example, as a carrier for the controlled release
of a taxane in a host for treating a suitable disorder; and the
like. For example, the present disclosure relates to the use of
such polymers for the in vivo delivery of a taxane, such as,
paclitaxel and derivatives thereof with lower toxicity, improved
solubility, greater bioavailability and enhanced efficacy in
treating cancers.
BACKGROUND
Symmetrically Branched Polymers
[0003] A new class of polymers called dendritic polymers, including
Starburst dendrimers (or Dense Star polymers) and Combburst
dendrigrafts (or hyper comb-branched polymers), recently was
developed and studied for various industrial applications. Those
polymers often possess: (a) a well-defined core molecule, (b) at
least two concentric dendritic layers (generations) with
symmetrical (equal length) branches and branch junctures and (c)
exterior surface groups, such as. polyamidoamine (PAMAM)-based
branched polymers and dendrimers described in U.S. Pat. Nos.
4,435,548; 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524;
and 5,714,166. Other examples include polyethyleneimine (PEI)
dendrimers, such as those disclosed in U.S. Pat. No. 4,631,337;
polypropyleneimine (PPI) dendrimers, such as those disclosed in
U.S. Pat. Nos. 5,530,092; 5,610,268; and 5,698,662; Frechet-type
polyether and polyester dendrimers, core shell tectodendrimers and
others, as described, for example, in, "Dendritic Molecules,"
edited by Newkome et al., VCH Weinheim, 1996, "Dendrimers and Other
Dendritic Polymers," edited by Frechet & Tomalia, John Wiley
& Sons, Ltd., 2001; and U.S. Pat. No. 7,754,500.
[0004] Combburst dendrigrafts are constructed with a core molecule
and concentric layers with symmetrical branches through a stepwise
synthetic method. In contrast to dendrimers, Combburst dendrigrafts
or polymers are generated with monodisperse linear polymeric
building blocks (U.S. Pat. Nos. 5,773,527; 5,631,329 and
5,919,442). Moreover, the branch pattern is different from that of
dendrimers. For example, Combburst dendrigrafts form branch
junctures along the polymeric backbones (chain branches), while
Starburst dendrimers often branch at the termini (terminal
branches). Due to the living polymerization techniques used, the
molecular weight distributions (Mw/Mn) of those polymers (core and
branches) often are narrow. Thus, Combburst dendrigrafts produced
through a graft-on-graft process are well defined with Mw/Mn ratios
often approaching 1.
[0005] SBP's, such as dendrimers, are produced predominantly by
repetitive protecting and deprotecting procedures through either a
divergent or a convergent synthetic approach. Since dendrimers
utilize small molecules as building blocks for the cores and the
branches, the molecular weight distribution of the dendrimers often
is defined. In the ease of lower generations, a single molecular
weight dendrimer often is obtained. While dendrimers often utilize
small molecule monomers as building blocks, dendrigrafts use linear
polymers as building blocks.
[0006] In addition to dendrimers and dendrigrafts, other SBP's
include symmetrical star-shaped or comb-shaped polymers, such as,
symmetrical star-shaped or comb-shaped polyethyleneoxide (PEO),
polyethyleneglycol (PEG), PEI, PPI, polyoxazoline (PDX),
polymethyloxazoline (PMOX), polyethyloxazoline (PEOX), polystyrene,
polymethylmethacrylate, polydimethylsiloxane or a combination
thereof.
Asymmetrically Branched Polymers
[0007] Unlike SBP's, asymmetrically branched polymers (ABP),
particularly asymmetrically branched dendrimers or regular ABP
(reg-ABP), often possess a core, controlled and well-defined
asymmetrical (unequal length) branches and asymmetrical branch
junctures as described in U.S. Pat. Nos. 4,289,872; 4,360,646; and
4,410,688.
[0008] On the other hand, a random ABP (ran-ABP) possesses: a) no
core, b) functional, groups both at the exterior and in the
interior, c) random/variable branch lengths and patterns (i.e.,
termini and chain branches), and d) unevenly distributed interior
void spaces.
[0009] The synthesis and mechanisms of ran-ABP's, such as those
made from PEI, were reported by Jones et al., J. Org. Chem. 9, 125
(1944), Jones et al., J. Org. Chem. 30, 1994 (1965) and Dick et
al., J. Macromol. Sci. Chem., A4 (6), 1301-1314, (1970)). Ran-ABP,
such as those made of PDX, i.e., poly(2-methyloxazoline) and
poly(2-ethyloxazoline), was reported by Litt (J. Macromol. Sci.
Chem. A9(5), 703-727 (1975)) and Warakomski (J. Polym. Sci. Polym.
Chem. 28, 3551 (1990)). The synthesis of ran-:BP's often can
involve a one-pot divergent or a one-pot convergent method.
Homopolymers
[0010] A homopolymer can relate to a polymer or to a polymer
backbone composed of the same repeat unit, that is, the homopolymer
is generated from the same monomer (e.g., PEI linear polymers, PDX
linear polymers, PEI dendrimers, polyamidoamine (PAA) dendrimers or
PDX dendrigrafts and randomly ranched polymers). The monomer can be
a simple compound or a complex or an assemblage of compounds where
the assemblage or complex is the repeat unit in the homopolymer.
Thus, if an assemblage is composed of three compounds, A, B and C;
the complex can be depicted as ABC. On the other hand, a polymer
composed of (ABC)-(ABC)-(ABC) . . . , is a homopolymer for the
purposes of the instant disclosure. The homopolymer may be linear
or branched. Thus, in the case of a randomly branched PEI, although
there are branches of different length and branches occur randomly,
that molecule is a homopolymer for the purposes of the instant
disclosure because that branched polymer is composed of a single
monomer, the ethyleneimine or aziridine repeat unit. Also, one or
more of the monomer or complex monomer components can be modified,
substituted, derivatized and so on, for example, modified to carry
a functional group. Such molecules are homopolymers for the
purposes of the instant disclosure as the backbone is composed of a
single simple or complex monomer.
Poorly Water Soluble Drugs: Taxanes
[0011] Paclitaxel is a water insoluble drug sold as TAXOL.RTM. by
Bristol-Myers Squibb. Paclitaxel is derived from the Pacific Yew
tree, Taxus brevifolia (Wan et al., J. Am. Chem. Soc. 93:2325,
1971). Taxanes, including paclitaxel and docetaxel (also sold as
TAXOTERE.RTM. under registered trademark of Aventis Pharma S.A.,
FR), are used to treat various cancers, including, breast, ovarian
and lung cancers, as well as colon, and head and neck cancers,
etc.
[0012] However, the poor aqueous solubility of paclitaxel has
hampered the widespread use thereof. Currently, TAXOL.RTM. and
generics thereof are formulated using a 1:1 solution of
ethanol:CREMAPHOR.RTM. (polyethyoxylated castor oil, registered
trademark of BASF, DE) to solubilize the drug. The presence of
CREMAPHOR.RTM. has been linked to severe hypersensitivity reactions
and consequently requires medication of patients with
corticosteroids dexamethasone) and antihistamines.
[0013] Alternatively, conjugated paclitaxel, for example,
ABRAXANE.RTM. (under registered trademark of Abraxis Bioscience,
NJ, USA), which is produced by mixing paclitaxel with human serum
albumin, has eliminated the need for corticosteroids and
antihistamine injections. However, ABRAXANE.RTM. generates
undesirable side effects, such as, severe cardiovascular events,
including chest pain, cardiac arrest, supraventricular tachycardia,
edema, thrombosis, pulmonary thromboembolism, pulmonary emboli,
hypertension etc., which prevents patients with high cardiovascular
risk from using the drug.
Delivery of Poorly Water Soluble Drugs
[0014] Although branched polymers, including SBP's and ABP'S, have
been used for drug delivery, those attempts are focused primarily
on the chemical attachment of the drug to the polymer, or physical
encapsulation of such drugs in the interior through unimolecular
encapsulation (U.S. Pat. Nos. 5,773,527; 5,631,329; 5,919,442; and
6,716,450).
[0015] For example, dendrimers and dendrigrafts are believed to
entrap physically bioactive molecules using unimolecular
encapsulation approaches, as described in U.S. Pat. Nos. 5,338,532;
5,527,524; and 5,714,166 for dense star polymers, and U.S. Pat. No.
5,919,442 for hyper comb-branched polymers. Similarly, the
unimolecular encapsulation of various drugs using SBP's to form a,
"dendrimer box," was reported in Tomalia et al., Angew. Chem. Int.
Ed. Engl., 1990, 29, 138, and in, "Dendrimers and Other Dendritic
Polymers," edited by Frechet & Tomalia, John Wiley & Sons,
Ltd., 2001, pp. 387-424.
[0016] Branched core shell polymers with a hydrophobic core and a
hydrophilic shell may be used to entrap a poorly water soluble drug
through molecular encapsulation. Randomly branched and
hyperbranched core shell structures with a hydrophilic core and a
hydrophobic shell have also been used to carry a drug through
unimolecular encapsulation and pre-formed nanomicelles (U.S. Pat.
No. 6,716,450 and Liu et al., Biomaterials 2010, 10, 1334-1341).
However, those unimolecular and pre-formed micelle structures are
generated in the absence of a drug.
[0017] In embodiments, block copolymers, such as, miktoarm polymers
(i.e., Y shaped/AB.sub.2-type star polymers) and linear
(A)-dendritic (B) block copolymers, were observed to form
stereocomplexes with paclitaxel (Nederberg et al.,
Biomacromolecules 2009, 10, 1460-1468 and Luo et al., Bioconjugate
Chem. 2010, 21, 1216). Those block copolymers closely resemble
traditional lipid or AB-type linear block copolymers, which are
well known surfactants used for the generation of micelles.
[0018] However, such branched block copolymers are difficult to
make and thus, are not suitable for mass production.
[0019] There is no description of modifying branched or linear
homopolymers with a hydrophobic group, which on exposure to a
poorly soluble or water insoluble drug, spontaneously form stable
aggregates which are suitable for controlled drug delivery.
SUMMARY
[0020] The present invention is directed to an aggregate
comprising:
[0021] a) a polyoxazoline comprising at least one first terminal
group modified with a hydrophobic moiety, wherein the polyoxazoline
further comprises a linear portion, a branched portion or both, and
the branched portion comprises a symmetrically branched polymer, an
asymmetrically branched polymer or a combination thereof; and the
polyoxazoline comprises a molar ratio of monomer initiator in a
range of, for example, from 50:1 to 80:1, and
[0022] b) a taxane,
[0023] wherein the polyoxazoline and the taxane has a weight ratio
of polymer to taxane of, for example, 6:1 to 8:1, and the aggregate
is from about 50 nm to about 100 nm in size, and
[0024] wherein the aggregate has a filtration rate through a 0.22
.mu.m filter in the range of from 50 to 100 percent.
[0025] In an aspect, the present disclosure is directed to use of
modified branched polymers (MBP) or linear polymers to increase the
solubility of taxanes, such as, paclitaxel, and derivatives
thereof.
[0026] In an aspect of the disclosure, the asymmetrically branched
polymer (ABP) has either random or regular, asymmetrical branches.
The random ABP can also have a mixture of terminal and chain
branching patterns.
[0027] In another aspect of the disclosure, both ABP's and SBP's
can be modified further with at least one molecule or group capable
of forming additional branches at a given time so that new material
properties can be achieved, wherein additional functional groups
further maybe attached. All of the modified polymers can be defined
as modified SBP's or ABP's.
[0028] In another aspect of the disclosure, the unmodified and
modified branched polymers either can be produced by a divergent or
a convergent method, and either a stepwise or a one-step synthetic
process can be used.
[0029] In another aspect of the disclosure, the SBP includes, but
is not limited to, PAA dendrimers; PEI dendrimers; PPI dendrimers;
polyether dendrimers; polyester dendrimers;
comb-branched/star-branched polymers, such as, PAA,
polyethyleneoxide (PEO), polyethyleneglycol (PEG), PMOX, PEOX,
polymethylmethacrylate (PMA), polystyrene, polybutadiene,
polyisoprene and. polydimethylsiloxane; comb-branched dendrigrafts,
such as, PEOX, PMOX, polypropyloxazoline (PPDX),
polybutyloxazoline, PEI, PAA; and so on.
[0030] In a further aspect of the disclosure, the SBP can have an
interior void space, while the ABP can have unevenly distributed
void spaces.
[0031] In another aspect of the disclosure, a hybrid branched
polymer comprising, the aforementioned SBP's, such as, dendrimers
or dendrigrafts, and ABP's, such as, regular and randomly branched
polymers, as well as star-branched and comb-branched polymers, or
combination thereof, also can be used for the generation of said
drug-induced aggregates or nanoparticles of interest.
[0032] In another aspect of the disclosure, the branched polymers
are modified with functional groups, such as, but not limited to,
NH.sub.2, NHR, NR.sub.2, NR.sub.3.sup.+, COOR, COOH, COO.sup.-, OH,
C(O)R, C(O)NH.sub.2, C(O)NHR or C(O)NR.sub.2, wherein R can be any
aliphatic group, aromatic group or combination thereof; an
aliphatic group (e.g., a hydrocarbon chain), which can be branched,
can contain one or more double and/or triple bonds and/or may be
substituted; an aromatic group, which may contain a plurality of
rings, which may be fused or separated, the rings may be of varying
size and/or may contain substituents; perfluorocarbon chains;
saccharides and/or polysaccharides, which may be of varying ring
sizes, the rings may contain a heteroatom, such as a sulfur or a
nitrogen atom, may be substituted, may contain more than one
species of saccharide, may be branched and/or may be substituted;
polyethylene glycols; and the like.
[0033] The molecular weight of the MBP's can range from about 500
to over 5,000,000; from about 500 to about 1,000,000; from about
1,000 to about 500,000; from about 2,000 to about 100,000.
[0034] In another aspect of the disclosure, the surface of the
SBP's and AR's is modified so that the physical properties of the
surface groups will be more compatible with a taxane, thus making
taxane more miscible with the surface group region, domain, portion
or segment of the MBP's.
[0035] In an embodiment, the modification of a branched polymer or
a linear polymer at a chain end is with a hydrophobic functional
group, such as, aliphatic chains (e.g., hydrocarbon chains
comprising 1 to about 22 carbons, whether linear or branched),
aromatic structures (e.g. containing one or more aromatic rings,
which may be fused) or combinations thereof.
[0036] In contrast to known drug carriers, the branched or linear
polymer structures of the instant invention do not physically
entrap taxane within each polymer molecule. Instead, a taxane
either can be located, at or dispersed in the domains/regions
containing functional groups of each branched or linear
polymer.
[0037] The resulting structures of interest optionally can be
preserved, for example, by lyophilization or other form of
desiccation, which may further stabilize the structures of
interest. Once redissolved in water or a buffer, nanoparticles with
sizes ranging from about 50 to about 500 nm in diameter can be
obtained.
[0038] The presence of multiple, often functionalized branches
enables the formation of intramolecular and intermolecular
crosslinks, which may stabilize the taxane-containing
nanoparticles. On dilution, said physical aggregate or nanoparticle
deconstructs releasing drug at a controlled rate.
[0039] In another aspect of the disclosure, a mixture of linear and
branched polymers also can be utilized to encapsulate a taxane. At
least one end group of said linear and/or branched polymer is
modified with a hydrophobic moiety or functional group. A
hydrophobic moiety or functional group can include, but is not
limited to, hydrocarbon chains (e.g., containing 1-22 carbons with
either saturated or unsaturated. chemical bonds) and hydrophobic
groups containing aralkyl, aromatic rings, fluorocarbons etc.
[0040] In another aspect of the disclosure, the branched or linear
polymer can comprise targeting moieties/groups including, but not
limited to, an antibody or antigen-binding portion thereof,
antigen, cognate carbohydrates (e.g., sialic acid), a cell surface
receptor ligand, a moiety bound by a cell surface receptor, such
as, a prostate-specific membrane antigen (PSMA), a moiety that
binds a cell surface saccharide, an extracellular matrix ligand, a
cytosolic receptor ligand, a growth factor, a cytokine, an
incretin, a hormone, a lectin, a lectin, ligand, such as, a
galactose, a galactose derivative, an N-acetylgalactosamine, a
matmose, a mannose derivative and the like, a vitamin, such as, a
folate or a biotin; avidin, streptavidin, neutravidin, DNA, RNA
etc. Such targeted nanoparticles release drug at the preferred
treatment locations, and therefore, enhance local effective
concentrations and can minimize undesired side effects.
[0041] In another aspect of the disclosure, a targeting
moiety/group and a functional group, including, hydrophobic,
hydrophilic and/or ionic functional groups, are attached to the
branched polymer prior to the formation of the composite
nanoparticle for targeted drug delivery.
[0042] In another aspect of the disclosure, specific ranges of
monomer:initiator and polymer:taxane ratios result in drug
nanoparticles of appropriate size to facilitate large scale
manufacturing of the drug nanoparticles, sterilization of drug
nanoparticles, and result in improved drug efficacy as compared to
other monomer:initiator and/or polymer:taxane ratios.
[0043] Additional features and advantages of the present disclosure
are described in, and will be apparent from, the following Detailed
Description and the attached Figures.
BRIEF DESCRIPTION OF THE FIGURES
[0044] The following description of the figures and the respective
drawings are non-limiting examples that depict various embodiments
that exemplify the present disclosure.
[0045] FIGS. 1A-D depict SBP's including a dendrimer (FIG. 1A), a
dendrigraft (FIG. 1B), a comb-shaped polymer (FIG. 1C) and a
star-shaped polymer (FIG. 1D). All have a core, whether globular or
linear.
[0046] FIGS. 2A-B depict a chemical structure of symmetrically
branched PPI dendrimers with 4 (FIG. 2A) and 8 (FIG. 2B)
branches.
[0047] FIG. 3 depicts chemical modification reactions of
symmetrically branched PPI dendrimers. The numbers, 8, 16, 32 and
so on indicate the number of reactive groups at the surface of the
dendrimer.
[0048] FIGS. 4A-B depict random (FIG. 4A) and regular (FIG. 4B)
APB's with asymmetric branch junctures and patterns.
[0049] FIG. 5 depicts a chemical structure of a random
asymmetrically branched PEI homopolymer.
[0050] FIGS. 6A-B depict synthetic schemes. FIG. 6A presents
chemical modification reactions of random asymmetrically branched
PEI homopolymers. FIG. 6B depicts a one-pot synthesis of
hydrophobically modified, randomly branched poly(2-ethyloxazoline)
with a primary amino group at the focal point of the polymer. The
initiator/surface group (I) is the brominated hydrocarbon. The
reaction opens the oxazoline ring.
[0051] FIGS. 7A-B illustrate a drug loaded in or at the surface
domain or region of the branched polymer SBP (FIG. 7A) and ABP
(FIG. 7B). In the and other figures, R indicates a surface group
and a solid circle depicts a drug of interest.
[0052] FIG. 8 illustrates one type of composite-based nanoparticles
containing both drug molecules and branched polymers.
[0053] FIGS. 9A-B illustrate an insoluble or poorly water soluble
drug that is loaded at hydrophobic surface groups of branched
polymers SBP (FIG. 9A) and ABP (FIG. 9B). In the and other figures,
a thin, wavy line depicts a hydrophobic surface group.
[0054] FIGS. 10A-B illustrate various drug-containing nanoparticles
also carrying at least one targeting group or moiety, such as, an
antibody, depicted herein and in other figures as a, "Y."
[0055] FIG. 11 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 25 mg/mL
and the drug concentration at 5 mg/mL in saline. The polymer is a
hydrophobically-modified, randomly-branched PEOX and the drug is
paclitaxel.
[0056] FIG. 12 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 2.5 mg/mL
and the drug concentration at 0.5 mg/mL in saline. The polymer is a
hydrophobically-modified, randomly-branched PEOX and the drug is
paclitaxel.
[0057] FIG. 13 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 250
.mu.g/mL and the drug concentration at 50 .mu.g/mL in saline. The
polymer is a hydrophobically-modified, randomly-branched PEOX and
the drug is paclitaxel.
[0058] FIG. 14 shows the size comparison of polymer-only and
polymer-drug aggregates with the polymer concentration at 25
.mu.g/mL and the drug concentration at 5 .mu.g/mL in saline. The
polymer is a hydrophobically-modified, randomly-branched PEOX and
the drug is paclitaxel.
[0059] FIG. 15 depicts normal cell survival on exposure to three
taxane formulations.
[0060] FIG. 16 depicts A549 lung cancer cell cytotoxicity on
exposure to three different taxane formulations.
[0061] FIG. 17 depicts MDA-MB-231 triple negative breast cancer
cytotoxicity on exposure to three different taxane
formulations.
[0062] FIG. 18 depicts OV-90 ovarian cancer cytotoxicity on
exposure to three different taxane formulations.
[0063] FIG. 19 depicts pharmacokinetic (PK) profiles of three
different taxane formulations depicting plasma concentration over
time.
[0064] FIG. 20 depicts A549 lung cancer tumor volume in a mouse
xenograft model with two control treatments and exposure to three
different taxane formulations,
[0065] FIG. 21 presents images of excised lung cancer cell tumors
grown as xenografts in a mouse and treatment of the mice with two
controls and two forms of taxane.
[0066] FIG. 22 depicts impact of two negative controls and three
formulations of taxane on ovarian cancer tumor size in a mouse
xenograft model.
[0067] FIG. 23 presents images of excised ovary cancer cell tumors
grown as xenografts in a mouse and treatment of the mice with two
controls and three forms of taxane.
DETAILED DESCRIPTION
[0068] Features and advantages of the present invention will be
more readily understood, by those of ordinary skill in the art,
from reading the following detailed description. It is to be
appreciated that certain features of the invention, which are
described above and below in the context of separate embodiments,
may also be provided in combination in a single embodiment.
Conversely, various features of the invention that are, for
brevity, described in the context of a single embodiment, may also
be provided separately or in any combination or sub-combination. In
addition, references in the singular may also include the plural
(for example, "a" and "an" may refer to one, or one or more) unless
the context specifically states otherwise.
[0069] Use of numerical values in the various ranges specified in
this application, unless expressly indicated otherwise, are stated
as approximations as though minimum and maximum values within the
stated ranges were both proceeded by the word, "about". In this
manner, slight variations above and below the stated ranges can be
used to achieve substantially the same results as values within the
ranges. Also, disclosure of ranges is intended as a continuous
range including every value between the minimum and maximum values
and including the minimum and maximum cited values.
[0070] The drug solubility in the instant disclosure is defined as,
relative to parts of solvent required to solubilize one part of
drug, <30 (soluble), 30-100 (poorly soluble) and >100
(insoluble). Taxane, such as paclitaxel and its derivatives, are
water insoluble or poorly water soluble, when water is used as a
solvent.
[0071] For the purposes of the instant disclosure, the words, such
as, "about," "substantially," and the like are defined as a range
of values no greater than 10% from the stated value or figure.
"Homopolymer," is as described hereinabove.
Drug of Interest
[0072] The drug of interest described is a taxane and comprises
paclitaxel and other taxane derivatives, such as, docetaxel.
Paclitaxel is water-insoluble and has well-defined performance
characteristics, such as, a low maximum tolerated dose (MTD), PK
profile and limited efficacies in treating various types of cancer.
The present disclosure covers the use of, for example, ABP's, as
previously described, in improving those performance
characteristics.
Nanocomposite, Nanoparticle or Nanoaggregate
[0073] A nanocomposite is a physical mixture of two or more
materials or components (e.g., polymer and a taxane). In the
instant disclosure, such a mixture could contain different
nanoscopic phases or domains formed between a taxane and a branched
homopolymer molecule in either solid or liquid state.
Nanocomposites can include a combination of a bulk matrix (e.g.,
branched homopolymers and a taxane) and nanodimensional phase(s),
which may exhibit different properties due to dissimilarities of
structure and chemistry (e.g., the domain formed by a taxane and
the surface groups of branched polymer, as well as the domains
formed by the interior of the branched polymers). Since the
solubility of the domains/phases may be different, on dissolving
the nanocomposite in an aqueous solution, one of the phases may
dissolve faster than the other or others, resulting in a gradual
breakdown of the composite aggregate resulting in a graded and
controlled release of the composite components and optionally,
reformation of one or more of the components into a novel form,
such as, a new aggregate. The terms, "nanocomposite,"
"nanoparticle" and "nanoaggregate," are equivalent and are used
interchangeably herein.
[0074] The size of the aggregates described in the disclosure
ranges from between about 10 to about 500 nm in diameter, from
about 30 nm to about 300 nm in diameter. Aggregates may exhibit
size-related properties that differ significantly from those
observed for microparticles or bulk materials.
[0075] SBP's are depicted in FIG. 1A-1D, with symmetric branches,
wherein all the homopolymers of interest possess a core and exhibit
symmetric branch junctures consisting either of terminal or chain
branches throughout the homopolymer. The functional groups are
present predominantly at the exterior.
[0076] The modified SBP's can be obtained, for example, through
chemically linking functional groups on, for example, symmetrically
branched PAMAM or PPI dendrimers, commercially available from
Aldrich, polyether dendrimers, polyester dendrimers,
comb-branched/star-branched polymers, such as, those containing
PEO, PEG, PMOX or PEOX, polystyrene, and comb-branched
dendrigrafts, such as, those containing PEOX, PMOX or PEI.
[0077] The synthetic procedures for making such SBP's/dendrimers
are known (see, for example, "Dendrimers and Other Dendritic
Polymers," Frechet & Tomalia, eds., John Wiley & Sons,
Ltd., 2001) using commercially available reagents (for example,
various generations of PPI dendrimers, such dendrimer-4 (FIG. 2A)
and dendrimer-8 (FIG. 2B)) or a number of SBP's are commercially
available. The synthesis of comb-branched and Combburst polymers is
known (see, for example, U.S. Pat. Nos. 5,773,527; 5,631,329; and
5,919,442). Symmetrically branched PPI dendrimers can be chemically
modified, for example via reactions illustrated in FIG. 3. The
numbers, 8, 16, 32, 64 or 128 indicate the number of reactive
groups at the surface of the dendrimer.
[0078] The higher branching densities of SBP's render the polymers
molecularly compact with a well-defined interior void space, which
makes such molecules suitable as a carrier for a taxane entrapped
or encased, therein.
[0079] The surface modifications can enhance the properties and
uses of the resulting modified SBP's. For example, with suitable
modification, a water insoluble SBP can become water soluble, while
an SBP with a high charge density can be modified to carry very low
or no charge on the polymer or at the polymer surface. On the other
hand, a water soluble SBP cm be modified with hydrophobic surface
groups to enhance the ability to solubilize water insoluble or
poorly water soluble drugs at the surface thereof. Modification can
occur at any site of a polymer, for example, at a terminus, a
branch, a backbone residue and so on.
[0080] In one embodiment of the instant disclosure, the SBP (for
example, either a symmetrically branched PEI dendrimer, a PH
dendrimer, a PAMAM dendrimer or a symmetrically branched PEI
dendrigraft, for example) can be modified with different kinds of,
for example, primary amine groups through, for example, Michael
addition or an addition of acrylic esters onto amine groups of the
homopolymer. Thus, for example, through a Michael addition
reaction, methyl acrylate can be introduced onto the primary and/or
secondary amino groups of PEI, PPI and polylysine (PLL)
homopolymers. The ester groups then can be derivatized further, for
example, by an amidation reaction. Thus, for example, such an
amidation reaction with, for example, ethylenediamine (EDA), can
yield the addition of an amino group at the terminus of the newly
formed branch. Other modifications to the homopolymer can be made
using known chemistries, for example, as provided in, "Poly(amines)
and Poly(ammonium salts)," in, "Handbook of Polymer Synthesis,"
(Part A), Kricheldorf ed., New York, Marcel Dekker, 1994; and,
"Dendrimers and Other Dendritic Polymers" Frechet & Tomalia,
eds., John Wiley & Sons, Ltd., 2001. Derivatives of EDA also
can be used and include any molecular entity that comprises a
reactive EDA, a substituted EDA or, for example, other members of
the polyethylene amine family, such as, diethylenetriamine,
triethylenetetramine, tetraethylenepentamine,
pentaethylenehexamine, and so on including polyethylene amine,
tetramethylethylenediamine and so on.
[0081] In embodiments, a modification can comprise a moiety that
contributes to or enhances hydrophobicity of a polymer or a portion
of a polymer. For example, hydrophobic functional groups, such as,
aliphatic chains (e.g., hydrocarbon chains comprising 1 to about 22
carbons, whether saturated or unsaturated, linear, cyclic or
branched), aromatic structures (e.g. containing one or more
aromatic rings, which may be fused) or combinations thereof, can be
used as a modifying agent and added to a polymer as taught herein
practicing chemistries as provided herein.
[0082] On such addition, a modified SBP, such as, a modified PEI,
PPI, PAMAM dendrimer or PEI dendrigraft, is formed. As an extension
of the SBP, such as PPI and PEI, the resulting modified SBP also is
symmetrically branched. Depending on the solvent environment (i.e.
pH or polarity), the surface functional groups can carry different
charge and/or charge density, and/or hydrophobic groups. The
molecular shape and surface functional group locations (i.e.,
surface functional group back folding) then can be tuned further,
based on those characteristic properties.
[0083] In another embodiment of the disclosure, the modified SBP's
can be produced using any of a variety of synthetic schemes that,
for example, are known to be amenable to reaction with a suitable
site on the homopolymer. Moreover, any of a variety of reagents can
be used in a synthetic scheme of choice to yield any of a variety
of modifications or additions to the homopolymer backbone. Thus,
for example, in the case of the Michael addition reaction to an
amine described above, the addition of any of a variety of
substituents can be used, for example, at the alkylation stage,
using for example, any of a variety of acrylate reagents, such as,
an acrylate comprising a hydrocarbon substituent, such as saturated
or unsaturated hydrocarbons comprising 1 to about 22 carbons, which
may be substituted, aliphatic, aromatic, ringed, saturated at one
or more bonds or a combination thereof. Thus, suitable reactants
include, methyl acrylate, ethyl acrylate, propyl acrylate, butyl
acrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octyl
acrylate, nonyl acrylate, decyl acrylate, undecyl acrylate, dodecyl
acrylate and so on, and mixtures thereof. Similarly, at the
amidation stage in the example exemplified above, any of a variety
of amines can be used. For example, EDA, monoethanolamine,
tris(hydroxymethyl)aminomethane, alkyl amine, allyl amine or any
amino-modified polymer, including those comprising PEG, PEO,
perfluoropolymers, polystyrene, polyethylene, polydimethylsiloxane,
polyacrylate, polymethylmethacrylate and the like, and mixtures
thereof, can be used.
[0084] Such a synthetic strategy would allow not only symmetric
growth of the molecule, where more branches with different chemical
compositions can be introduced, but also the addition of multiple
functional groups at the exterior of the structure. The precursor
homopolymer can be modified, and continuously, using the same or a
different synthetic process until the desired SBP's with
appropriate molecular weight and functional groups are attained. In
addition, the hydrophobic and hydrophilic properties, as well as
charge densities of such polymers, can be tailored to fit specific
application needs using appropriate monomers for constructing the
homopolymer and suitable modification reactions.
[0085] In another embodiment of the disclosure, if a divergent
synthetic procedure is used, the chain end of symmetrically
star-branched or comb-branched homopolymer, such as, a
poly(2-substituted oxazoline), including, for example,
poly(2-methyloxazoline), poly(2-ethyloxazoline),
poly(2-propyloxazoline) and poly(2-butyloxazoline, etc.), PEI,
PEO/glycol, polyvinylpyrrolidone (PVP), polyphosphate, polyvinyl
alcohol (PVA) or polystyrene, can be modified with another small
molecule or polymer to generate various functional groups at the
homopolymeric chain ends including a primary, secondary or tertiary
amine, carboxylate, hydroxyl, aliphatic (e.g., hydrocarbon chain),
aromatic, fluoroalkyl, aryl, PEG, PEO, acetate, amide and/or ester
groups. Alternatively, various initiators also can be utilized so
that the same type of functional groups can be introduced at the
chain end if a convergent synthetic approach is utilized
("Dendritic Molecules," Newkome et al., eds., VCH, Weinheim, 1996;
"Dendrimers and Other Dendritic Polymers," Frechet & eds., John
Wiley & Sons. Ltd., 2001; and J. Macromol. Sci. Chem. A9(5),
pp. 703-727 (1975)).
[0086] The initiator can be a hydrophobic electrophilic molecule,
including hydrocarbons, aliphatic hydrocarbons, aromatic
hydrocarbons or a combination of both, along with a halide
functional group, such as, alkyl halides, aralkyl halides, acyl
halides or combinations thereof. Examples of such compounds are
monofunctional, initiators such as hydrocarbons containing from 1
to about 22 hydrocarbons with either saturated or unsaturated
chemical bonds, such as, methyl iodide/bromide/chloride, ethyl
iodide/bromide/chloride, 1-iodo/bromo/chloro butane,
1-iodo/bromo/chloro hexane, 1-iodo/bromo/chloro dodecane,
1-iodo/bromo/chloro octadodecane, benzyl iodide/bromide/chloride
and so on. Other initiators include allyl bromides/chlorides. Acyl
halides, such as, acyl bromide/chloride, benzoyl bromide/chloride
and tosyl group-containing compounds, such as, p-toluenesulfonic
acid, methyl tosylate and other tosylate esters can also be used.
Any one or more initiators can be used in combination.
[0087] During polymerization, an initiator can be used to start
polymerization. When used, various molar ratios of monomer to
initiator can be used to obtain particular polymers. The particular
polymers can have differing properties, such as, molecular size.
Hence, suitable monomer to initiator molar ratios can be 20:1 to
80:1, such as, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1,
65:1, 70:1 or 75:1 including 21:1, 22:1, 23:1, 24:1, 26:1, 27:1,
28:1, 29:1, 31:1, 32:1, 33:1, 34:1, 36:1, 37:1, 38:1, 39:1, 41:1,
42:1, 43:1, 44:1, 46:1, 47:1, 48:1, 49:1, 51:1, 52:1, 53:1, 54;1,
56:1, 57:1, 58:1, 59:1, 61:1, 62:1, 631, 64:1, 66:1. 67:1, 68:1,
69:1 :1, 72:1, 73:1, 741, 76:1, 77:1, 78:1, 79:1 and any other
ratios within the range.
[0088] ABP's are depicted in FIG. 4A and FIG. 4B with asymmetric
branches, wherein some of the polymers of interest possess no core
and exhibit asymmetrical branch junctures consisting of both chain
and terminal branches throughout the entire homopolymer. The
junctional groups often are present both at the exterior and in the
interior. However, when a larger functional group (e.g., a large
hydrophobic or hydrophilic group) is used, the functional groups
often can be attached preferentially and perhaps necessarily at the
exterior of the ABP, for example, possibly due to steric effects.
Therefore, such surface MBP's can be utilized for solubilization of
or aggregate formation with an insoluble or poorly soluble
drug.
[0089] The modified ABP's can be obtained, for example, through
chemically linking functional groups on regular ABP's, such as,
polylysine (e.g., branched PLL), on random ABP's, such as, PEI's
(commercially available from Aldrich, Polysciences, or BASF under
the trade name, LUPOSAL.TM.) or polyoxazolines, which can be
prepared according to the procedure of Litt (J. Macromol. Sci.
Chem. A9(5), pp. 703-727 (1975)). Other ABP's can include, but are
not limited to, polyacrylamides, polyphosphates, PVP's, PVA's
etc.
[0090] A variety of known starting materials can be used. For
making such modified ABPs. Such monomers and polymers are available
commercially in large quantities at modest cost. For example, one
such precursor monomer that can be used to synthesize a homopolymer
of interest is PEI. The synthesis of random asymmetrically branched
PEI's is known (Jones et al., J. Org. Chem. 9, 125 (1944)). PEI's
with various molecular weights are available commercially from
different sources, such as, Aldrich, Polysciences and BASF (under
the trade name LUPOSAL.TM.). The random asymmetrically branched
PEI's are produced primarily through cationic ring opening
polymerization of ring-strained cyclic imine monomers, such as,
aziridines (ethyleneimine) and azetidines (propyleneimine), with
Lewis or Bronsted acids as initiators (Dernier et al.,
"Ethylenediamine and Other Aziridines," Academic Press, New York,
(1969); and Pell, J. Chem. Soc. 71 (1959)). Since many of the
methods are essentially one-pot processes, large quantities of
random ABP's can be produced readily. Randomly branched
poly(2-substituted oxazoline) polymers can be prepared using the
procedure of Litt (J. Macromol. Sci. Chem. A9 (5), pp. 703-727
(1975)).
[0091] The synthetic processes for making ABP's often generate
various branch junctures within the macromolecule. In other words,
a mixture of terminal and chain branch junctures is distributed
throughout the molecular structure. The branching densities of the
random ABP's can be lower, and the molecular structure can be more
open when compared with dendrimers and dendrigrafts. Although the
branch pattern is random, the average ratio of primary, secondary
and tertiary amine groups can be relatively consistent with a ratio
of about 1:2:1, as described by Dick et al., J. Macromol. Sci.
Chem., A4 (6), 1301-1314 (1970) and Lukovkin, Eur. Polym. J. 9,
559(1973).
[0092] The presence of the branch junctures can make the random
ABP's, such as, asymmetrically branched PEI's, form, macromolecules
with a possible spherical, ovoid or similar configuration. Within
the globular structure, there are various sizes of pockets formed
from the imperfect branch junctures at the interior of the
macromolecule. Unlike dendrimers and dendrigrafts where interior
pockets are always located around the center core of the molecule,
the pockets of random ABP's are spread unevenly throughout the
entire molecule. As a result, random ABP's possess both exterior
and unevenly distributed interior functional groups that can be
reacted further with a variety of molecules, thus forming new
macromolecular architectures, a modified random ABP of
interest.
[0093] Although having a core, the functional groups of the regular
ABP are also distributed both at the exterior and in the interior,
which is very similar to the random ABP. One such homopolymer is
PLL, which can be made as described in U.S. Pat. Nos. 4,289,872;
4,360,646; and 4,410,688. Such homopolymers also can be modified in
a manner similar as that for random ABP's, as taught herein, and as
known in the art.
[0094] In an embodiment of the disclosure, the ABP (for example,
either a random asymmetrically branched PEI or a regular
asymmetrically branched PLL) is modified with different kinds of
primary amine groups through, for example, Michael addition or an
addition of acrylic esters onto amines of the polymer. Thus, for
example, through a Michael addition reaction, methyl acrylate or
other acrylates as provided herein can be introduced onto the
primary and/or secondary amino groups of, for example, PEI and PLL
homopolymers. The ester groups then can be further derivatized, for
example, by an amidation reaction. Thus, for example, such an
amidation reaction with, for example, EDA, can yield the addition
of an amino group at the terminus of the newly formed branch. Other
modifications to the polymer can be made using known chemistries,
for example, as provided in "Poly(amines) and Poly(ammonium salts)"
in "Handbook of Polymer Synthesis" (Part A), Kricheldorf, ed., New
York, Marcel Dekker, 1994. An example of a random asymmetrically
branched PEI homopolymer is shown in FIG. 5.
[0095] On such addition, a modified ABP, such as, a modified PET or
PLL homopolymer, is formed. As an extension of the ABP, such as PEI
and PLL, the resulting modified ABP also is branched,
asymmetrically. Depending on the solvent environment (i.e. pH or
polarity), the surface functional groups can carry different charge
and charge density. The molecular shape and functional group
locations (i.e., functional group back folding) then cars be
further tuned, based on those characteristic properties.
[0096] In another embodiment, the modified ABP's can be produced
using any of a variety of synthetic schemes that, for example, are
known to be amenable to reaction with a suitable site on the
homopolymer. Moreover, any of a variety of reagents can be used in
a synthetic scheme of choice to yield any of a variety of
modifications or additions to the polymer backbone. Thus, for
example, in the case of the Michael addition reaction to an amine
described above, the addition of any of a variety of substituents
can be used at the alkylation stage, as provided hereinabove, for
example, with an acrylate, which can comprise a saturated or
unsaturated hydrocarbon, such as one comprising one carbon to about
22 carbons, which may be aliphatic, branched, saturated, aromatic,
ringed or combination thereof. Suitable reactants include methyl
acrylate, ethyl acrylate, propyl, acrylate, butyl acrylate, pentyl
acrylate, hexyl acrylate, heptyl acrylate, octyl acrylate, nonyl
acrylate, decyl acrylate, undecyl acrylate, dodecyl acrylate and
the like, and mixtures thereof. Similarly, at the amidation stage
in the example exemplified above, any of a variety of amines can be
used in the methods provided herein and known in the art. For
example, EDA, monoethanolamine, tris(hydroxymethyl)aminomethane,
alkyl amine, allyl amine or any amino-modified polymers, including
PEG, perfluoropolymers, polystyrene, polyethylene,
polydimethylsilixane, polyacrylate, polymethylmethacrylate and the
like, and mixtures thereof, can be used. In addition, the linking
of the hydrophobic groups, including aliphatic (e.g., hydrocarbons
from C.sub.1 to about C.sub.22) groups, aromatic groups,
polyethylene polymers, polystyrene polymers, perfluoropolymers,
polydimethlylsiloxanes, polyacrylates, polymethylmethacrylates, as
well as, hydrophilic groups, including a OH group, hydrophilic
polymers, such as, PEOX, PEG, PEO etc. to a modified ABP can be
achieved by using, for example, epoxy reactions, amidation
reactions, Michael addition reactions, including using an --SH or
an --NH.sub.2 group reacted with maleimide,
aldehyde/ketone-amine/hydrazide coupling reactions,
iodo/iodoacetyl-SH coupling reactions,
hydroxylamine-aldehyde/ketone coupling reactions etc. Such
synthetic strategies allow not only asymmetric growth of the
molecule, where more pockets are introduced, but also the addition
of multiple functional groups at both the interior and the exterior
of the structure. The homopolymer can be modified further using the
same or a different synthetic process until the desired ABP's with
appropriate molecular weight and functional groups are attained. In
addition, the hydrophobic and hydrophilic properties, as well as
charge density of such homopolymers, can be tailored to fit
specific application needs using appropriate monomers for
constructing the homopolymer and suitable modification
reactions.
[0097] In another embodiment of the disclosure, a focal point
(merged from various reactive chain ends during a convergent
synthesis) of a random ABP, such as, PDX, can be terminated or
reacted with another small molecule to generate various functional
groups at the homopolymeric chain ends, including primary,
secondary or tertiary amines, carboxylate, hydroxyl, alkyl,
fluoroalkyl, aryl, PEG, acetate, amide and/or ester groups.
Alternatively, various initiators also can be utilized so that the
same type of functional group can be introduced at the surface
groups where a polymerization begins during a convergent synthesis
(J. Macromol. Sci. Chem. A9 (5), pp. 703-727(1975)).
[0098] An alkyl surface-modified, randomly branched
poly(2-ethyloxazoline) with a primary amine group at the focal
point of the branched polymer can be prepared using the Litt and
Warakomski procedures, supra. For example,
CH.sub.3(CH.sub.2).sub.17-Br can be utilized as an initiator for
2-ethyloxazoline polymerization through a cationic ring opening
process to generate a randomly branched polymer, followed by
quenching with N-ten-butyloxycarbonylpiperazine (N-Boc-piperazine)
or EDA. The termination with a large excess of EDA allows the
hydrophobically modified branched poly(2-ethyloxazoline) polymer to
be functionalized with a primary amine group at the focal point
(FIG. 6B). Alternatively, N-Boc-piperazine-terminated
hydrophobically-modified branched poly(2-ethyloxazoline) polymer
also can be deprotected to generate a free amino group at the focal
point. If not terminated, the focal point of the polymer can be
hydrolyzed to, for example, a hydroxyl group on dissolving in water
(e.g., containing, for example, 1N Na.sub.2CO.sub.3).
[0099] While the introduction of a primary amine group to a
hydrophobically-modified branched poly(2-oxazoline) homopolymer
enhances drug solubility and produces taxane-induced aggregates,
the primary amine group also allows the attachment of various
targeting groups, such as, an antibody, antigen-binding portion
thereof an antigen or a member of a binding pair, such as, to the
hydrophobically modified branched poly(2-oxazoline) polymer (FIG.
10). Such aggregates or nanoparticles containing such targeting
groups and modifications thereto can provide a targeting ability on
the aggregate with a taxane and enable taxane to be released
preferentially or solely at the desired treatment location.
[0100] As taught herein, the MBP's, such as, a
hydrophobically-modified homopolymers, including both SBP's and
ABP's, can be used to generate an encapsulating polymer or
nanocapsule for solubilizing water insoluble or poorly water
soluble taxanes, or for forming taxane-induced nanoparticles with
water insoluble or poorly water soluble taxanes, such as,
paclitaxel (FIG. 7A-7B, FIG. 8 and FIG. 9). In an organic solvent
environment, the hydrophilic or amphiphilic interior can be
poly(2-oxazoline), poly(2-substituted oxazolines), including
poly(2-methyloxazoline, poly(2-ethyloxazoline),
poly(2-propyloxazoline) and poly(2-butyloxazoline) etc., PEG, PEO,
polyphosphonate and the like. The hydrophobic exterior can comprise
aliphatic hydrocarbons (such as, from C.sub.1 to about C.sub.22),
aromatic hydrocarbons, polyethylene polymers, polystyrene polymers,
perfluoropolymers, polyditnethylsiloxanes, polyacrylates,
polymethylmethacrylates and the like. In an aqueous environment,
the reverse is true. In the drug-induced aggregates in an aqueous
environment, the drug molecules such as taxanes are associated with
the hydrophobic groups/domains of the MBP's (FIG. 9). The branching
density (e.g., from low generation, such as, star and comb
homopolymers, to high generation of dendrimers and dendrigrafts),
as well as the amount of hydrophobic surface group coverage (e.g.,
from 0% to 100% coverage) of the branched homopolymers can affect
significantly homopolymer solubility, which in turn, also affects
the ability to dissolve or to adsorb/absorb a taxane. For example,
the increase in branching density and the amount of hydrophobic
group coverage will make the homopolymer more compatible with a
taxane.
[0101] In some cases, the ABP's and SBP's with from about 0.1 to
about 30% or more surface hydrophobic component by weight are
effective at solubilizing or dispersing poorly water soluble or
water insoluble taxanes, such as, paclitaxel. In addition, the
branched homopolymers utilized, for example, a PDX, a PEOX, a PMOX,
PEO/PEG, polyacrylamides, polyphosphates, PVP's and PVA's are
soluble in both water and in various organic solvents, thereby
facilitating forming various taxane-containing nanoparticles or
aggregates. The good water solubility along with good hydrophobic
drug miscibility in an aqueous solution, with or without other
organic solvents, makes such homopolymers useful for enhancing the
solubility of poorly water soluble taxanes. For example, the
homopolymers of interest simplify manufacturing processes and
decrease production cost by reducing formulation steps, processing
time, as well as the need to use complex and expensive equipment
currently used in the pharmaceutical industry. If additional
branching densities are needed, the SBP's or ABP's first can be
modified with additional groups as described herein, and then, for
example, attached with additional hydrophobic functional groups for
enhancing taxane solubility.
[0102] On mixing hydrophobically-modified SBP's or ABP's with a
water insoluble or poorly water soluble taxane, such as,
paclitaxel, a distinct physical aggregate is formed of size
distinct from aggregates formed only of polymer (FIG. 11-FIG. 14).
When the homopolymer and taxane concentrations decrease, the size
and distribution of the polymer/taxane aggregates become much more
similar to that of polymer only aggregates suggesting taxane is
released from the induced aggregates or nanoparticles. The broad
size distribution of polymer-only aggregates is similar to that
observed for other structures composed of lipid, whether or not
associated with a taxane. On the other hand, the taxane-induced
aggregates of interest are of a particular size of narrower
distribution, that is, unique aggregates of certain size are
produced. As taxane concentration in the aggregate decreases,
homopolymer concentration in the aggregate decreases, aggregate
concentration decreases or any combination thereof, the aggregates
of interest release paclitaxel, as evidenced by a reduction of
aggregate size and/or a broader distribution of aggregate size. The
broader distribution may result from a mixture of homopolymer-only
aggregates and polymer/taxane aggregates of varying size due to
taxane release, until the only aggregates observed are those which
have the characteristics of those Which are homopolymer only. In
other words, taxane is released gradually after introduced into a
host, such as, in the circulatory system. That mechanism is
important for various drug delivery applications including,
intravenous (IV), oral, transdermal, ocular, intramuscular and the
like modes of administration, and where a delayed release or
sustained release profile may be desirable.
[0103] For simplicity, the term "polymer" used in describing
aggregate, polymer-drug nanoparticles, polymer aggregate, or
polymer to drug ratio, etc., include SBP and ABP disclosed in this
application, and include modified polyoxazoline, such as,
hydrocarbon modified polyoxazolines including those further
modified with EDA or EDA derivative disclosed herein.
[0104] Suitable weight ratios of polymer to taxane are 6:1 to 8:1,
such as, 6.5:1, 7:1 or 7.5:1, including each and all of 6.11,
6.2:1, 6.3:1, 6,4:1, 6.5:1, 6.6:1, 6.7:1, 6.8:1, 6.9:1, 7.0:1,
7.1:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1, 7.6:1, 7.7:1, 7.8:1, 7.9:1,
8.0:1, and all ratios within the range.
[0105] Applicants unexpectedly discovered that the combination of
the molar ratio of monomer to initiator in the polymerization and
the weight ratio of polymer to taxane in the nanoparticles can
affect large scale manufacturability of the drug nanoparticles,
nanoparticle size, and efficacy as a tumor-reducing treatment. As
an example, taxane-induced aggregates prepared with a
polymer:taxane weight ratio of 5:1, using a polymer synthesized
with 100:1 monomer:initiator molar ratio results in larger
nanoparticles, for example, in the 120-140 nm range before
lyophilization. Such large nanoparticles are difficult to pass
through a 0.22 .mu.m filter (a required sterilization step for
injectables) when manufactured in large quantities.
[0106] In comparison, when a polymer synthesized using a
monomer:initiator molar ratio of 60:1 is mixed with taxane with, a
polymer:taxane weight ratio of 7:1, the nanoparticles formed can be
70-100 nm in size before lyophilization, which allows the particles
to pass through a 0.22 .mu.m filter with little difficulty.
[0107] Smaller nanoparticles at about 100 nm or less in size before
lyophilization reduced tumors at lower dose concentrations than did
larger particles. For example, smaller nanoparticles achieve the
same cancer treatment efficacy with only 1/5 of the taxane content
when compared to larger nanoparticles. Thus, lower doses of drug
can be used and the risk of side effects is minimized.
[0108] The taxane-induced aggregates also can be linked with a
targeting moiety or group including, but not limited to, an
antibody (or antigen-binding portion thereof), antigen, cognate
carbohydrates (e.g., sialic acid), a cell surface receptor ligand,
a moiety that binds a cell surface receptor, such as,
prostate-specific membrane antigen (PSMA), a moiety that binds a
cell surface saccharide, an extracellular matrix ligand, a
cytosolic receptor ligand, a growth factor, a cytokine, an
incretin, a hormone, a lectin, a lectin target, such as, a
galactose, a galactose derivative, an N-acetylgalactosamine, a
mannose, a mannose derivative and the like, a vitamin, such as, a
folate, a biotin and the like, an avidin, a streptavidin, a
neutravidin, a DNA, an RNA etc. to form a conjugate so that the
targeting group(s) are incorporated with nanocomposite particle of
interest (FIG. 10).
Drug Formulation and Nanoparticle Preparation
[0109] Taxane and modified homopolymer can be suspended
individually in suitable buffers and/or solvents, such as, a
buffer, methanol, acetone, ethanol and the like, at suitable
concentrations, such as, those which are established for in vivo
use, generally in milligram or nanogram quantities. Then, the two
solutions are mixed at a suitable temperature, such as, room
temperature or at another temperature known to be acceptable for
maintaining integrity of the taxane and homopolymer, for a suitable
period of time, such as, one hour, two hours and so on. Other
incubation times can vary from minutes to hours as the aggregates
of interest are stable once formed. The aggregates can be
concentrated or collected practicing methods known in the art, for
example, by filtration, centrifugation, evaporation,
lyophilization, dialysis and the like. The aggregates can be
desiccated for extended shelf life.
[0110] For example, a taxane, such as, paclitaxel, was dissolved in
methanol or ethanol in various amounts of up to 40 mg/mL. A
hydrocarbon (CH.sub.3(CH.sub.2).sub.17)-modified randomly branched
PEOX60 (monomer to initiator molar ratio=60:1) was prepared as
taught herein and dissolved at varying concentrations of up 100
mg/mL in methanol or ethanol.
[0111] The two solutions then were mixed in various volumes to
result in final homopolymer to taxane weight ratios in the mixtures
ranging from 2:1 to 10:1 and rotary evaporated to dryness. The
mixtures then were redissolved in water or saline, followed by
sterile filtration by a 0.22 .mu.M filter and lyophilization for 20
to 72 hours depending on volume to yield a dry powder.
[0112] The size of the aggregates or nanoparticles, as measured by
light scattering, can range from about 50 to about 100 nm, from
about 60 to about 95 nm and from about 70 to about 90 nm (e.g., at
3 mg paclitaxel per mL) before lyophilization. The size of
aggregates can range from about 110 to about 150 nm, from about 115
to about 145 nm, from about 120 to about 140 nm (e.g., at 5 mg
paclitaxel per mL) in diameter after lyophilization.
[0113] In an aspect, the invention is directed to an aggregate
comprising:
[0114] a) a polyoxazoline comprising at least one first terminal
group modified with a hydrophobic moiety, wherein the polyoxazoline
further comprises a linear portion, a branched portion or both, and
the branched portion comprises a symmetrically branched polymer, an
asymmetrically branched polymer or a combination thereof; and the
polyoxazoline comprises a molar ratio of monomer to initiator in a
range of from 50:1 to 80:1, and
[0115] b) a taxane,
[0116] wherein the polyoxazoline and the taxane comprises a weight
ratio of polymer to taxane of 6:1 to 8:1, and the aggregate is from
about 50 nm to about 100 nm in size, and
[0117] wherein the aggregate comprises a filtration rate through a
0.22 .mu.m filter of from 50 to 100 percent.
[0118] Any aforementioned polyoxazoline polymer can be suitable.
The aggregate can be formed as described herein.
[0119] Filtration rate of the polymer-drug aggregates through a
filter of known pore size can be measured according to following
procedure:
[0120] a polymer-drug aggregate sample can be dissolved in water,
saline, PBS, or a solvent as described herein at a predetermined
final concentration. Based on filter composition, a
polymer-aggregate can be dissolved in water, saline, PBS, other
aqueous solution, a solvent and so on. In an example, a
polymer-drug aggregate sample is prepared based on weight of the
polymer. In an example, a polymer-drug aggregate sample is prepared
based on weight of the drug, such as, paclitaxel. In an example, a
polymer-drug aggregate sample is prepared based on a final
paclitaxel concentration, such as, mg/mL.
[0121] A sample can be passed through the filter by, for example,
gravity or by pressure. It is common for pressure to be applied to
compel a sample to pass through a filter. An optional pressure
gauge can be used. In an example, a container is sealed and a
sample is exposed to pressurized air. In embodiments, a sealed
container is configured to accept a vacuum in a collection portion
of the sealed container to draw sample through an integral
filter.
[0122] In an example, a syringe with a plunger is used in
conjunction with a filter assembly which interfaces with the nozzle
portion of the syringe. A sample is loaded into the barrel of the
syringe and the sample therein is forced to pass through the filter
assembly by depressing the plunger of the syringe with pressure. A
constant pressure is applied to the plunger until all of the sample
is expelled from the barrel. If filter clogging occurs which is
evident by back pressure and reduced passage of fluid through the
filter into the collection portion of the device or a collection
vessel, pressure is removed as excessive pressure on the plunger
can rupture the membrane or can disrupt the assembly.
[0123] A predetermined volume, V.sub.0, of a sample is loaded into
a container. Then, the container is attached or is exposed to a
filter assembly. For a 0.22 .mu.m filter assembly, a V.sub.0 of
9-10 ml can be selected. Any sample volume remaining in the
container, V.sub.1, is recorded. Filtered volume, V.sub.f, can be
calculated with the formula, V.sub.f=V.sub.0-V.sub.1.
Alternatively, V.sub.f can be determined by measuring the volume of
the filtrate or of the collected filtered sample.
[0124] Filtration rate, R.sub.f, of a sample can be calculated with
the formula:
R.sub.f=V.sub.f/V.sub.0.
[0125] R.sub.f can be expressed as a fraction or the fraction can
be converted to a percentage by multiplying the fraction by
100.
[0126] It has been discovered that polymer-drug aggregates prepared
from, for example, C.sub.18-PEOXABP60 and paclitaxel, unexpectedly
can be passed through a 0.22 .mu.m filter with a filtration rate of
50%-100%. As a comparison, polymer-drug aggregates prepared from
C.sub.18-PEOXABP100 and paclitaxel only have a filtration rate of
less than 50%.
[0127] In embodiments, a filtration rate must be at least 50%, at
least 60%, at least 70%, at least 80%, at least 90% or more; must
be in a range of 50-100%, 60-100%, 70-100%, 80-100%, 90-100%,
55-100%, 65-100%, 75-100%, 85-100%, 95-100%, 55-95%, 65-95%,
75-95%, 85-95%, 50-95%, 60-95%, 70-95%, 80-95%, 90-95%, 50-90%,
60-90%, 70-90%, 80-90%, 55-90%, 65-90%, 75-90%, 85-90%; or, in any
rate, must be 50% or greater.
[0128] Since polymer-drug aggregates, in embodiments, are intended
for pharmaceutical use, and sterilization by filtration through a
0.22 .mu.m filter is a common processing requirement or step, such
a discovery of the correlation between monomer:initiator ratio and
polymer:drug ratio, and aggregate size provides advantages at least
for ease of manufacturing and reducing waste.
[0129] Selection of specific monomer to initiator molar ratios and
specific polymer:drug, ratios to yield certain sized aggregates is
unexpected and surprising. Polyoxazoline polymers with lower
monomer to initiator molar ratios, such as, C.sub.18-PEOXABP20, are
unsuitable for drug formulation due to manufacturing limitations
and variations in cytotoxicity in some cell bioassays. Polymers
with a monomer to initiator molar ratio of from about 50:1 to 80:1
provide drug aggregates comprising ease of manufacturing, reduced
cytotoxicity and high filtration rate using a 0.22 .mu.m
filter.
[0130] The first terminal group modified with a hydrophobic moiety
of the polyoxazoline polymer can comprise a hydrophobic
electrophilic molecule including hydrocarbons, aliphatic
hydrocarbons, aromatic hydrocarbons or a combination of both, along
with a halide functional group, such as, alkyl halides, aralkyl
halides, acyl halides or combinations thereof and can be provided
by an initiator. Polymerization of an initiator and one or more
monomers of choice can produce a polyoxazoline of this invention
haying at least one first terminal group modified with a
hydrophobic moiety. As disclosed throughout this application, the
initiator can comprise a hydrophobic electrophilic molecule
including a hydrocarbon. The hydrocarbon can comprise from 1 to
about 22 carbons, which may be saturated or unsaturated. In one
embodiment, the initiator can comprise an aliphatic hydrocarbon, an
aromatic hydrocarbon or a combination of both. The initiator can
comprise a halide functional group. In one example, the initiator
can comprise an alkyl halide, an aralkyl halide, an acyl halide or
combination thereof.
[0131] The initiator can comprise alkyl halides containing from 1
to about 22 carbons, including, but not limited to, methyl iodide,
methyl bromide, methyl chloride, ethyl iodide, ethyl bromide, ethyl
chloride, 1-iodopropane, 1-bromopropane, 1-chloropropane,
1-iodobutane, 1-bromobutane, 1-chlorobutane, 1-iodopentane,
1-bromopentane, 1-chloropentane, 1-iodo hexane, 1-bromo hexane,
1-chloro hexane, 1-iodo dodecane, 1-bromo dodecane, 1-chloro
dodecane, 1-iodo octadodecane, 1-bromo octadodecane, 1-chloro
octadodecane, as well as benzyl iodide, benzyl bromide, benzyl
chloride, allyl bromide, acyl iodide, acyl bromide, acyl chloride,
benzoyl bromide or benzoyl chloride, in a further example, the
initiator comprises a tosyl group.
[0132] As disclosed herein, the phrase, "polyoxazoline comprises a
molar ratio of monomer to initiator in a range of from," means that
the polyoxazoline is produced by reacting an initiator and at least
one monomer at a monomer to initiator ratio (molar ratio) in that
specific range including the starting and the ending points.
Monomers, such as, oxazoline and substituted oxazoline disclosed
herein can be suitable. Initiators disclosed above and hereafter
can be suitable.
[0133] The aggregate can comprise a size from 50 nm to about 100 nm
before lyophilization. The term, "size," refers to a size of
particles of the polymer-drug aggregate in solution as measured
using dynamic light scattering method described hereafter. The size
of the polymer-drug aggregate can be the size measured in an
aqueous solution in one example, in water, in another example, in
saline, in yet another example, in a buffer, such as, a phosphate
buffer (PBS), in yet another example, in a combination of saline
and buffer in a further example.
[0134] In the aggregate of this invention, the polyoxazoline can
further comprise a second terminal group comprising a functional
group modified by an ethylenediamine (EDA) or an ethylenediamine
derivative thereof. The ethylenediamine derivative can comprise
diethylenetriamine, triethylenetetramine, tetraethylenepentamine,
pentaethylenehexamine, polyethylene amine or
tetramethylethylenediamine.
[0135] In the aggregate of this invention disclosed herein, the
taxane can be associated with the at least one first terminal group
modified with a hydrophobic moiety. The taxane can be associated
with the first terminal group via a covalent bond, a non-covalent
link, or a combination thereof. In one example, the taxane is
associated with the hydrocarbon via a non-covalent link. Not
wishing to be bound by a particular theory or mechanism, Applicants
believe that hydrophobicity of the hydrocarbon of the polyoxazoline
provides desired interaction between the polymer and taxane, which
is water insoluble or poorly water soluble.
[0136] The aggregate disclosed herein can further comprise a
targeting moiety. The targeting moiety can comprise an antibody, an
antigen binding portion thereof, an antigen, a cell surface
receptor, a cytosolic receptor, a cell receptor ligand or a lectin
ligand.
[0137] The polyoxazoline of this invention can comprise
poly(2-oxazoline), poly(2-substituted oxazoline) or a combination
thereof. In one example, the polyoxazoline can comprise
poly(2-methyloxazoline), poly(2-ethyloxazoline),
poly(2-propyloxazoline), poly(2-butyloxazoline) or a combination
thereof. The polyoxazoline of this invention can be polymerized
from at least one monomer comprising oxazoline, 2-substituted
oxazoline, or a combination thereof. The oxazoline can be
2-oxazoline. The 2-substituted oxazoline can comprise
2-methyloxazoline, 2-ethyloxazoline, 2-propyloxazoline,
2-butyloxazoline or a combination thereof.
[0138] In the aggregate of this invention, the taxane can comprise
paclitaxel, docetaxel or a combination thereof.
[0139] The aggregate of this invention can be sterile. In one
example, the aggregate can be sterilized by filtration through a
0.22 .mu.m filter.
[0140] This invention is further directed to a pharmaceutical
composition comprising an aggregate disclosed herein. In
embodiments, the pharmaceutical composition comprises an aggregate
comprising:
[0141] a) a polyoxazoline comprising at least one first terminal
group modified with a hydrophobic moiety, wherein the polyoxazoline
further comprises a linear portion, a branched portion or both, and
the branched portion comprises a symmetrically branched polymer, an
asymmetrically branched polymer or a combination thereof; and the
polyoxazoline comprises a molar ratio of monomer to initiator in a
range of, for example, from 50:1 to 80:1, and
[0142] b) a taxane,
[0143] wherein the polyoxazoline and the taxane has a weight ratio
of polymer to taxane of, for example, 6:1 to 8:1, and the aggregate
is from about 50 nm to about 100 nm in size, and
[0144] wherein the aggregate has a filtration rate through a 0.22
.mu.m filter in a range of from 50 to 100 percent.
[0145] A polyoxazoline as known or described herein can be suitable
polymer. The initiator for the polyoxazoline can comprise a
hydrophobic electrophilic molecule, for example, a hydrocarbon,
such as, an aliphatic hydrocarbon, an aromatic hydrocarbon or a
combination of both. The hydrocarbon can comprise from 1 to about
22 carbons, which may be saturated or unsaturated. The initiator
can comprise a halide functional group, an alkyl halide, an aralkyl
halide, an acyl halide or combination thereof. In one example, the
initiator can comprise methyl iodide, methyl bromide, methyl
chloride, ethyl iodide, ethyl bromide, ethyl chloride,
1-iodopropane, 1-bromopropane, 1-chloropropane, 1-iodobutane,
1-bromobutane.sub.; 1-chlorobutane, 1-iodopentane, 1-bromopentane,
1-chloropentane, 1-iodo hexane, 1-bromo hexane, 1-chloro hexane,
1-iodo dodecane, 1-bromo dodecane, 1-chloro dodecane, 1-iodo
octadodecane, 1-bromo octadodecane.sub.; 1-chloro octadodecane,
benzyl iodide, benzyl bromide, benzyl chloride, allyl bromide, acyl
iodide, acyl bromide, acyl chloride, benzoyl bromide, benzoyl
chloride or a combination thereof. In a further example, the
initiator comprises a tosyl group. The polyoxazoline can further
comprise a second terminal group comprising a functional group
modified by EDA or an ethylenediamine derivative thereof, wherein
the ethylenediamine derivative comprises diethylenetriamine,
triethylenetetramine, tetraethylenepentamine,
pentaethylenehexamine, polyethylene amine or
tetramethylethylenediamine. The polyoxazoline can comprise
poly(2-oxazoline), poly(2 substituted oxazoline) or a combination
thereof. In a particular example, the polyoxazoline can comprise
poly(2-methyloxazoline), poly(2-ethyloxazoline),
poly(2-propyloxazoline) or poly(2-butyloxazoline) or a combination
thereof.
[0146] In the pharmaceutical composition, the aggregate can
comprise particles having a size from 50 nm to about 100 nm before
lyophilization.
[0147] In the pharmaceutical composition, the taxane can be
associated with said at least one first terminal group.
[0148] The aggregate can further comprise a targeting moiety,
wherein the targeting moiety can comprise an antibody, an antigen
binding portion thereof, an antigen, a cell surface receptor, a
cytosolic receptor, a cell receptor ligand or a lectin ligand, as
disclosed above.
[0149] In the pharmaceutical composition, the taxane can comprise
paclitaxel, docetaxel or a combination thereof.
[0150] The pharmaceutical composition can further comprise a
carrier. Carriers disclosed herein can be suitable.
[0151] The pharmaceutical composition can be a cancer treatment
drug for treating breast cancers, ovarian cancers, lung cancers, an
NSCLC (Non-Small Cell Lung Cancer), colon cancers, gastric cancers,
melanomas, head and neck cancers, pancreatic cancers or a
combination thereof. In pharmaceutical composition can be
administered to a patient via parenteral, e.g., intravenous,
intradermal, subcutaneous, oral (e.g., inhalation), transdermal
(topical), transmucosal or rectal administration, or a combination
thereof.
[0152] It was discovered that unexpectedly polyoxazoline polymers
polymerized with a specific range of monomer to initiator molar
ratio provide an advantage in filtration for the manufacturing:
polymers having monomer to initiator molar ratios in a range of
from 50:1 to about 80:1, such as, C.sub.18-PEOXABP60, can produce
polymer-drug aggregates that can pass through a 0.22 .mu.m membrane
filter with an R.sub.f of about 50% to 100% for producing sterile
drug aggregate preparations, while polymers having monomer to
initiator molar ratios of, for example, 100:1 (C.sub.18-PEOXABP100)
or greater produce polymer-drug aggregates that have a filtration
rate of less than 50%. Such a low filtration rate leads to lower
yield of product, material waste and longer filtration process
resulting in reduced productivity for manufacturing.
[0153] Not wishing to be bound by any particular theory or
mechanism, it is believed polymer-drug aggregates produced from a
polymer having a monomer to initiator molar ratio of 100:1 or
greater have undesirable aggregate size and aggregate distribution,
and are susceptible to inter-particle interaction, particle-filter
interaction, or a combination thereof, so when particles are forced
into proximity or close to filter materials, such as, when passed
through a 0.22 .mu.m filter under pressure, aggregates may further
aggregate or interact causing the filter to be clogged leading to a
lower filtration rate. Unexpectedly, it was discovered that a
monomer to initiator molar ratio, for example, in the 50:1 to 80:1
range overcomes the filter clogging problem leading to easier
manufacturing, reduced waste and increased productivity.
[0154] A pharmaceutical composition of this disclosure for use as
disclosed herein is formulated to be compatible with intended
routes of administration. Examples of routes of administration can
include parenteral, e.g., intravenous, intradermal, subcutaneous,
oral (e.g., inhalation), transdermal (topical), transmucosal and
rectal administration. Solutions or suspensions used for
parenteral, intradermal or subcutaneous application can include a
sterile diluent, such as, water for injection, saline, oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents, such as, benzyl alcohol
or methyl parabens; antioxidants, such as, ascorbic acid or sodium
bisulfite; chelating agents, such as, EDTA; buffers, such as,
acetates, citrates or phosphates; and agents for the adjustment of
tonicity, such as, sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as, HCl or NaOH. The parenteral
preparation can be enclosed in ampoules, disposable syringes or
multiple dose vials made of glass or plastic as an article of
manufacture.
[0155] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation, of sterile injectable
solutions or dispersions. For intravenous administration, suitable
carriers include physiological saline, bacteriostatic water or
phosphate-buffered saline (PBS). The composition generally is
sterile and is fluid to the extent that syringability exists. The
composition must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as, bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, liquid
PEG, polysorbates and the like) and suitable mixtures thereof.
Proper fluidity can be maintained, for example, by use of a
coating, such as, a lecithin, by maintenance of required particle
size in the case of dispersion, use of a thickener and by use of
surfactants. Prevention of action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid and the like.
Isotonic agents, for example, sugars, polyalcohols, such as,
mannitol, sorbitol or sodium chloride, can be included in the
composition. Prolonged absorption of injectable compositions can be
brought about by including in the composition, an agent that delays
absorption, for example, aluminum monostearate or gelatin.
[0156] Sterile injectable solutions can be prepared by
incorporating active compound in the required amount of an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filter sterilization.
Generally, dispersions are prepared by incorporating an active
compound in a sterile vehicle that contains a basic dispersion
medium and the required other ingredients, for example, from those
enumerated above, and as known in the art. In the case of sterile
powders for preparation of sterile injectable solutions, the
preparation can be treated by, for example, lyophilization, vacuum
drying or freeze drying, that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. A preparation of interest can be
stored and reconstituted with a suitable liquid for use.
[0157] Oral compositions generally include an inert diluent,
flavorant, odorant or an edible carrier. The composition can be
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the active compound can
be incorporated with excipients and used in the form of tablets,
troches or capsules. Oral compositions also can be prepared using a
fluid carrier to yield a syrup or liquid formulation, or for use as
a mouthwash, wherein the compound in the fluid carrier is applied
orally and swished and expectorated or swallowed.
[0158] Pharmaceutically compatible binding agents and/or adjuvant
materials can be included, as part of the composition. Tablets,
pills, capsules, troches and the like can contain a binder, such
as, microcrystalline cellulose, gum tragacanth or gelatin; an
excipient, such as, starch or lactose, a disintegrating agent, such
as, alginic acid, PRIMOGEL.RTM. (a modified corn starch, a
trademark of DFE pharma, DE) or corn starch; a lubricant, such as,
magnesium stearate or Sterotes (U.S. Pat. No. 8,933,193); a
glidant, such as, colloidal silicon dioxide; a sweetening agent,
such as, sucrose or saccharin; or a flavoring agent, such as,
peppermint, methyl salicylate or orange flavoring.
[0159] For administration by inhalation, the compound is delivered
in the form of, for example, a wet or dry aerosol spray from a
pressurized container or dispenser that contains a suitable
propellant, e.g., a gas, such as, carbon dioxide or a nebulizer, or
a mist.
[0160] Systemic administration also can be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants generally are known in the art and
include, for example, for transmucosal administration, detergents,
bile salts and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels or creams as
generally known in the art. A suitable carrier includes
dimethylsulfoxide.
[0161] The compound also can be prepared in the form of
suppositories (e.g., with conventional suppository bases, such as,
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0162] In one embodiment, the active compound is prepared with
carriers that will protect the compound against rapid elimination
from the body, such as, a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as, ethylene vinyl
acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters and polylactic acid.
[0163] Methods for preparation of such formulations will be
apparent to those skilled in the art. The materials also can be
obtained commercially, for example, from Alza Corporation and Nova
Pharmaceuticals, Inc.
[0164] The instant aggregates can be used in topical forms, such
as, creams, ointments, lotions, unguents, other cosmetics and the
like. Pharmaceutically active agents (PAAs), such as, the taxanes
of interest and other bioactive or inert compounds, can be carried,
and include emollients, bleaching agents, antiperspirants,
pharmaceuticals, moisturizers, scents, colorants, pigments, dyes,
antioxidants, oils, fatty acids, lipids, inorganic salts, organic
molecules, opacifiers, vitamins, pharmaceuticals, keratolytic
agents, UV blocking agents, tanning accelerators, depigmenting
agents, deodorants, perfumes, insect repellants and the like.
[0165] It can be advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form," as used herein refers to
physically discrete units suited as unitary dosages for a subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce a desired therapeutic
endpoint.
[0166] The dosages, for example, preferred routes of administration
and amounts are obtainable based on empirical data obtained from
preclinical and clinical studies, practicing methods known in the
art. The dosage and delivery form can be dictated by and can be
dependent on the characteristics of the PAA, the polymer, the
particular therapeutic effect to be achieved, the characteristics
and condition of the recipient and so on. For repeated
administrations over several days or longer, depending on the
condition, the treatment can be sustained until a desired endpoint
is attained. An exemplary dosing regimen is disclosed in WO
94/04188.
[0167] The progress of the therapy can be monitored by conventional
techniques and assays, as well as patient input.
[0168] The pharmaceutical compositions can be included in a
container, pack or dispenser together with instructions for
administration.
[0169] Another method of administration comprises the addition of a
compound of interest into or with a food or drink, as a food
supplement or additive, or as a dosage form taken on a prophylactic
basis, similar to a vitamin. The aggregate of interest can be
encapsulated into forms that will survive passage through the
gastric environment. Such forms are commonly known, for example,
enteric coated formulations. Alternatively, the aggregate of
interest can be modified to enhance half-life, such as, chemical
modification or combination with agents known to result in delayed,
sustained or controlled release, as known in the art.
[0170] The instant disclosure now will be exemplified in the
following non-limiting examples.
EXAMPLES
[0171] The present invention is further defined in the following
Examples. It should be understood that the Examples, while
indicating embodiments of the invention, are given by way of
illustration only. From the above discussion and the Examples, one
skilled in the art can ascertain the essential characteristics of
the invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt to various uses and conditions,
Materials
[0172] Symmetrically branched PPI dendrimers were purchased from
Sigma-Aldrich. Symmetrically branched PEI dendrimers and
dendrigrafts were prepared according to procedures provided in U.S.
Pat. Nos. 4,631,337, 5,773,527, 5,631,329 and 5,919,442. All of the
antibodies were purchased from Sigma-Aldrich, Biodesign or
Fitzgerald. Different generation PAMAM dendrimers were purchased
from Dendritech, Inc.
Modified Symmetrically Branched PPIs with Amino Functional Groups
(m-SB-PPI-NH.sub.2-1.0)
[0173] The following reagents, including symmetrically branched PH
(SB-PPI-4, 8, 16, 32, 64, MW 316, 773, 1,687, 3,514 and 7,168),
methyl acrylate (MA, FW=86.09), EDA (FW=60.10) and methanol, were
utilized.
[0174] To a round bottom flask were added 1.0 g PPI-64 dendrimer
(MW 7168) and 20 ml methanol (solution A). To a separate round
bottom flask were added 2.4 g methylacrylate (MA) and 10 ml
methanol (solution B). Solution A was then slowly dropped into
solution B while stirring at room temperature. The resulting
solution was allowed to react at 40.degree. C. for 2 hours. On
completion of the reaction, the solvent and unreacted MA monomer
were removed by rotary evaporation and the product, 2.5 g of
MA-functionalized PPI, then was redissolved in 20 ml of
methanol.
[0175] To a round bottom flask were added 160 g EDA and 50 ml of
methanol, followed by a slow addition of MA-functionalized PPI at
0.degree. C. The solution then was allowed to react at 4.degree.
C.' for 48 hours. The solvent and the excess EDA were removed by
rotary evaporation. The crude product then was precipitated from an
ethyl ether solution and further purified by dialysis to give about
2.8 g of primary amine-functionalized symmetrically branched PPI
(m-SB-PPI-NH.sub.2-1.0) with a molecular weight of about 21,760.
The product was characterized by .sup.1H and .sup.13C nuclear
magnetic resonance (NMR) and size exclusion chromatography
(SEC).
[0176] Other MA or primary amine-modified symmetrically branched
PPI dendrimers and symmetrically branched PEI dendrigrafts with
various molecular weights were prepared in a similar manner.
Modified Symmetrically Branched PPIs with Mixed Hydroxyl and Amino
Functional Groups (mix-m-SB-PPI-64-NH.sub.2/OH-2)
[0177] Amino-functionalized symmetrically branched PPI
(m-SB-PPI-64-NH.sub.2-1.0), MA, EDA, monoethanolamine (MEA,
FW=61.08) and methanol were utilized.
[0178] To a round bottom flask were added 1.0 g amino-modified. PPI
or m-SB-PPI-NH.sub.2-1.0 produced from the previous procedure and
20 ml of methanol (solution A). To a separate round bottom flask
were added 2.4 g of MA and 10 ml methanol (solution B). Solution A
then was dripped slowly into solution B while stirring at room
temperature. The resulting solution was allowed to react at
40.degree. C. for 2 hours. On completion of the reaction, the
solvent and unreacted monomer MA were removed by rotary evaporation
and, the product, 2.5 g of MA-functionalized m-SB-PPI-64-MA-1.5,
then was redissolved in 20 ml of methanol.
[0179] To a round bottom flask were added 32 EDA, 130 g MEA and 100
ml methanol (the molar ratio of EDA:MEA was 20:80), followed by
slow addition of m-SB-PPI-64-MA-1.5 at 0.degree. C. The solution
then was allowed to react at 4.degree. C. for 48 hours. The solvent
and the excess EDA were removed by rotary evaporation. The crude
product then was precipitated from an ethyl ether solution and
further purified by dialysis to give about 2.8 g of mixed hydroxyl
and amino functionalized (mixed surface) SBP
(mix-m-SB-PPI-64-NH.sub.2/OH-2.0, with an average of 20% NH.sub.2
and 80% OH surface groups and a molecular weight of about
21,862).
[0180] Other modified random AB-PEI and regular AB-PLL molecules
with varying ratios of hydroxyl and amino groups, as well as
different molecular weights, were prepared in a similar manner.
[0181] Random asymmetrically branched PEI's were purchased from
Aldrich and Polysciences. Regular ABP's were prepared according to
procedures provided in U.S. Pat. No. 4,289,872. All of the
antibodies were purchased from Sigma-Aldrich, Biodesign or
Fitzgerald.
Modified Random Asymmetrically Branched PEI's with Amino Functional
Groups (m-ran-AB-PEI-NH.sub.2-1.0)
[0182] Random asymmetrically branched PEI (ran-AB-PEI, MW 2,000,
25,000 and 75,000), MA, EDA and methanol were utilized.
[0183] To a round bottom flask were added 1.0 g PEI (MW 2,000) and
20 ml methanol (solution A). To a separate round bottom flask were
added 3.0 g MA and 10 ml methanol (solution B). Solution A then was
dripped slowly into solution B while stirring at room temperature.
The resulting solution was allowed to react at 40.degree. C. for 2
hours. On completion of the reaction, the solvent and unreacted MA
were removed by rotary evaporation and the product,
MA-functionalized PEI, then was redissolved in 20 ml of
methanol.
[0184] To a round bottom flask were added 80 g EDA and 50 ml of
methanol, followed by a slow addition of MA-functionalized PEI at
0.degree. C. (1 g MA dissolved in 20 ml methanol). The solution
then was allowed to react at 4.degree. C. for 48 hours. The solvent
and excess EDA were removed by rotary evaporation. The crude
product then was precipitated from an ethyl ether solution and
further purified by dialysis to give about 3.0 g of primary
amine-functionalized random asymmetrically branched PEI
(m-ran-AB-PEI-NH.sub.2-1.0) with a molecular weight of about 7,300.
The product was characterized by .sup.1H and .sup.13C NMR and
SEC.
[0185] Other MA or primary amine modified random asymmetrically
branched PEI and regular asymmetrically branched PLL polymers with
various molecular weights were prepared in a similar manner.
Modification of Branched Polymers with Hydrocarbon Chains
[0186] The modification of a randomly branched PEI with 10%
hydrocarbon chains is used as an example. One gram of branched PEI
(FW=25000) was dissolved in 10 mL methanol. To the solution were
added 0.23 g of 1,2-epoxyhexane (FW=100.16) and the mixture was
heated at 40.degree. C. for 2 hours. The solvent then was rotary
evaporated and the residue redissolved in water. After dialysis
(3,500 cutoff), the modified PEI was generated.
[0187] Other MBP's, such as, PAMAM, PEI and PPI dendrimers and
dendrigrafts, and asymmetric PLL with various percentages and
lengths (e.g., C.sub.4, C.sub.12, C.sub.18 and C.sub.22) of
hydrocarbon chains were prepared in a similar manner.
Modified Random Asymmetrically Branched PEI's with Mixed Hydroxyl
and Amino Functional Groups (m-ran-AB-PEI-NH.sub.2/OH-2)
[0188] Amino-functionalized random asymmetrically branched. PEI
(m-ran-AB-PEI-NH.sub.2-1.0), MA, EDA, monoethanolamine (MEA,
FW=61.08 and methanol were utilized.
[0189] To a round bottom flask were added 1.0 g ammo-modified PEI
or m-ran-AB-PEI-NH.sub.2-1.0 produced from the previous procedure
and 20 ml of methanol (solution A). To a separate round bottom
flask were added 3.0 g of MA and 10 ml methanol (solution B).
Solution A then was slowly dripped into solution T3 while stirring
at room temperature. The resulting solution was allowed to react at
40.degree. C. for 2 hours. On completion of the reaction, the
solvent and unreacted MA were removed by rotary evaporation and the
product, MA-functionalized m-ran-AB-PEI-MA-1.5, then was
redissolved in 20 ml of methanol.
[0190] To a round bottom flask were added 60 g EDA, 244 g MEA and
100 ml methanol (the mole ratio of EDA:MEA was 20:80), followed by
slow addition of m-ran-AB-PEI-MA-1.5 at 0.degree. C. (1 g MA
dissolved in 20 ml of methanol). The solution then was allowed to
react at 4.degree. C. for 48 hours. The solvent and excess EDA were
removed by rotary evaporation. The crude product then was
precipitated from an ethyl ether solution and further purified by
dialysis to give about 2.4 g of mixed hydroxyl and amino
functionalized random ABP (m-ran-AB-PEI-NH.sub.2/OH-2.0, with an
average of 20% NH.sub.2 and 80% OH surface groups and the molecular
weight was about 18,000).
[0191] Other modified random AB-PEI and regular AB-PLL polymers
with various ratios of hydroxyl and amino groups, as well as
different molecular weights were prepared in a similar manner.
Alkyl-Modified Random Asymmetrically Branched
Poly(2-ethyloxaxoline) (PEOX) with Primary Amine Chain End
Group
[0192] The synthesis of CH.sub.3--(CH.sub.2).sub.11-PEOX-ABP100
(C.sub.12ABP100 is an arbitrary.sup., name to denote the molar
ratio of monomer to initiator in the initial reaction) is provided
as a general procedure for the preparation of core shell
structures. A mixture of CH.sub.3(CH.sub.2).sub.11--Br (2.52 g) in
500 ml of toluene was azeotroped to remove water with a
distillation head under N.sub.2 for about 15 min. 2-Ethyloxazoline
(100 g) was added dropwise through an addition funnel and the
mixture was allowed to reflux between 24 and 48 hours. On
completion of the polymerization, 12.12 g of EDA were added to the
reactive polymer solution (A) to introduce the amine function
group. The molar ratio of PDX chain end to EDA was 1 to 20.
[0193] Alternatively, N-Boc-piperazine or water (e.g., with 1N
Na.sub.2CO.sub.3) can be added to terminate the reaction.
Morpholine or PEI also can be added to the reactive polymer
solution (A) to terminate the reaction. The crude product was
redissolved in methanol and then precipitated from a large excess
of diethyl ether. The bottom layer was redissolved in methanol and
dried by rotary evaporation and vacuum to give an asymmetrically
random branched. PEOX polymer as a white solid (101 g).
[0194] Other asymmetrically randomly branched polymers, such as,
C.sub.6-PEOX ABP20, 50, 100, 200, 300 and 500, C.sub.12-PEOX ABP20,
50, 200, 300 and 500, C.sub.22-PEOX ABP20, 50, 100, 200, 300 and
500, and polystyrene-PEOX etc., as well as, non-modified and
modified poly(2-substituted oxazoline), such as,
poly(2-methyloxazoline), were prepared in a similar manner. All the
products were analyzed by SEC and NMR.
Alkyl-Modified Random Asymmetrically Branched
Poly(2-ethyloxazoline) (PEOX) with Primary Amine Chain End
Group
[0195] The synthesis of CH.sub.3--(CH.sub.2).sub.17-PEOX-ABP60
(C.sub.18-PEOXABP60 is an arbitrary name to denote the molar ratio
of monomer to initiator in the initial reaction) is provided as a
general procedure for the preparation of core shell structures. A
mixture of CH.sub.3(CH.sub.2).sub.17--Br (5.61 g) in 500 ml of
toluene was azeotroped to remove water with a distillation head
under N.sub.2 for about 15 min. 2-Ethyloxazoline (100 g) was added
dropwise through an addition funnel and the mixture was allowed to
reflux between 24 and 48 hours. On completion of the
polymerization, 10.1 g of EDA were added to the reactive polymer
solution (A) to introduce the amine function group. The molar ratio
of polyoxazoline reactive chain end to EDA was 1 to 10.
[0196] Alternatively, N-Boc-piperazine or water (e.g., with 1N
Na.sub.2CO.sub.3) can be added to terminate the reaction.
Morpholine or PEI also can be added to the reactive polymer
solution (A) to terminate the reaction. The crude product was
redissolved in methanol and then precipitated from a large excess
of diethyl ether. The bottom layer was redissolved in methanol and
dried by rotary evaporation and vacuum to give an asymmetrically
random branched PEOX polymer as a white solid.
[0197] Other asymmetrically randomly branched polymers, such as,
C.sub.18-PEOX ABP20, 40, 50, 70, 80, 100, 120, 200, 300, 500 etc.
as well as, non-modified and modified poly(2-substituted
oxazoline), such as, poly(2-methyloxazoline), were prepared in a
similar manner. All the products were analyzed by SEC and NMR.
Mixed Surface Modified Symmetrical Branched Polymer-IgG
Conjugates
[0198] The preparation of mixed surface (OH/NH.sub.2 mix) modified
symmetrically branched PPI-IgG conjugates
(mix-m-SB-PPI-64-NH.sub.2/OH-2-IgG conjugates) is provided as a
general procedure for the preparation of polymer antibody.
[0199] Other conjugates, such as, m-SB-PPI-4-NH.sub.2-1-IgG,
m-SB-PPI-8-NH.sub.2-1IgG, m-SB-PPI-16-NH.sub.2-1-IgG,
m-SB-PPI-32-NH.sub.2-1-IgG, m-SB-PPI-4-NH.sub.2-2-IgG,
m-SB-PPI-8-NH.sub.2-2-IgG, m-SB-PPI-16-NH.sub.2-2-IgG,
m-SB-PPI-32-NH.sub.2-2-IgG, m-SB-PPI-4-NH.sub.2-3-IgG,
m-SB-PPI-8-NH.sub.2-3-IgG, m-SB-PPI-16-NH.sub.2-3-IgG,
m-SB-PPI-32-NH.sub.2-1-IgG, mix-m-SB-PPI-4-NH.sub.2/OH-1
(OH/NH.sub.2 mix)-IgG, mix-m-SB-PPI-8-NH.sub.2/OH-1 (OH/NH.sub.2
mix)-IgG, mix-m-SB-PPI-16-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-32-NH.sub.2/OH-1 (OH/NH.sub.2mix)-IgG,
mix-m-SB-PPI-4-NH.sub.2/OH-2 (OH/NH.sub.2
mix-m-SB-PPI-8-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-16-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-32-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-4-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI8-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG,
mix-m-SB-PPI-16-NH.sub.2/OH-3 (OH/NH.sub.2
mix-m-SB-PPI-32-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG, as well as
primary amine and mix OH/NH.sub.2 modified Combburst PEI
dendrigrafts (Generation 0-5) also were obtained in a similar
manner. The synthesis of other targeting moieties attached to a
modified SBP of interest also was obtained in a similar manner.
LC-SPDP-Mixed Surface m-SB-PPI-64-NH.sub.2/OH-2
[0200] To the mixed surface randomly branched
mix-m-SB-PPI-64-NH.sub.2/OH-2 (4.times.10.sup.-7 mol) in 400 .mu.l
of phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH 7.5) were
added 4.0.times.10.sup.-6 mol of sulfo-LC-SPDP (Pierce, Ill.) in
400 .mu.L of water. The mixture was vortexed and incubated at
30.degree. C. for 30 minutes. The
LC-SPDP-mix-m-SB-PPI-64-NH.sub.2/OH-2 was purified by gel
filtration chromatography and equilibrated with buffer A (0.1 M
phosphate, 0.1 M NaCl and 5 mM EDTA, pH 6.8). The product was
concentrated further to yield 465 .mu.L, of solution with a
concentration of approximately 0.77 nmol.
Thiolated mix-m-SB-PPI-64-NH.sub.2/OH-2
[0201] The LC-SPDP mix-m-SB-PPI-64-NH.sub.2/OH-2 (50 nmol in 65
.mu.L of buffer A) was mixed with 100 .mu.L of dithiothreitol (DTT)
(50 mM in buffer A) and was incubated at room temperature for 15
minutes. Excess DTT and byproducts were removed by gel filtration
with buffer A. The product was concentrated in a 10 K Centricon
Concentrator to yield 390 pi of thiolated
mix-m-SB-PPI-64-NH.sub.2/OH-2 that was used for conjugation with
activated antibody.
Maleimide R (MAL-R)-Activated Antibody
[0202] To the antibody in PBS (310 .mu.L, 5.1 mg or 34 nmol) were
added 20.4 .mu.L of a MAL-R-NHS (N-hydroxysuccinimide) solution (10
mM in water). The mixture was vortexed and incubated at 30.degree.
C. for 15 minutes. The product was purified by gel filtration with
buffer A. The maleimide-R-activated antibody was used for
conjugation with the thiolated mix-m-SB-PPI-64-NH.sub.2/OH-2.
Mix-m-SB-PPI-64-NH.sub.2/OH-2-Antibody Conjugate
[0203] To the thiolated mix-m-SB-PPI-64-NH.sub.2/OH-2 (310 .mu.L or
35.7 nmol) was added the MAL-R-activated antibody (1.8 ml, or 34
nmol). The reaction mixture was concentrated to approximately 800
.mu.L and then allowed to incubate overnight at 4.degree. C. and/or
at room temperature for about 1 hr. On completion, the reaction was
quenched with 100 .mu.L of ethyl maleimide (50 mM solution) and the
conjugate then was fractionated on a carboxymethyl cellulose (CM
cellulose) column (5 mL) with a sodium chloride step gradient in 20
mM phosphate buffer at pH 6. The conjugate was eluted with a sodium
chloride gradient and characterized by cationic exchange
chromatography, UV spectroscopy and polyacrylamide gel
electrophoresis.
Conjugation Via Reductive Coupling-Reduction of Antibody
[0204] To the antibody, 2.1 mg or 14 nmol in 160 .mu.L of buffer B
(containing 0.1 M sodium phosphate, 5 mM EDTA and 0.1 M NaCl, pH
6.0) were added 40 .mu.L of DTT (50 mM in buffer B). The solution
was allowed to stand at room temperature for 30 min. The product
was purified by gel filtration in a Sephadex G-25 column
equilibrated with buffer B. The reduced antibody was concentrated
to 220 .mu.L and was used for conjugation.
MAL-R-Mixed Surface Modified SBP
[0205] To the mixed surface modified SBP in 400 .mu.L
(400.times.10.sup.-9 mols) at pH 7.4 were added 400 .mu.L of
MAL-R-NHS (10 mM in water). That was mixed and incubated at
30.degree. C. for 15 min. On termination, the product was purified
on a Sephadex G-25 column equilibrated with buffer B. The
MAL-R-mixed surface modified SBP was collected and stored in
aliquots in the same buffer at -40.degree. C.
Mixed Surface Modified SBP-Antibody Conjugate
[0206] To the reduced antibody (14 nmols in 220 .mu.L) was added
the MAL-R-mix-m-SB-PPI-64-NH.sub.2/OH-2 (154 .mu.L, 16.6 nmols)
with stirring. The pH was adjusted to about 6.8 by the addition of
1.2.5 .mu.L of sodium carbonate (1.0 M solution), the reaction was
continued for 1 hr at room temperature and terminated with the
addition of 100 .mu.L of cysteamine (0.4 mM solution). The
conjugation mixture was purified on a CM cellulose column with a
sodium chloride gradient elution.
IgG-Asymmetrical Randomly Branched Polymer Conjugates
[0207] The preparation of randomly branched mixed surface
(OH/NH.sub.2 mix) m-ran-AB-PEI-NH.sub.2/OH-2-IgG conjugates is
provided as a general procedure for the preparation of
polymer-antibody conjugates.
[0208] Other conjugates, such as, PEI-IgG,
m-ran-AB-PEI-NH.sub.2-1-IgG, m-ran-AB-PEI-NH.sub.2-2-IgG,
m-ran-AB-PEI-NH.sub.2-3-IgG, m-ran-AB-PEI-NH.sub.2-4-IgG, as well
as m-ran-AB-PEI-NH.sub.2/OH-1 (OH/NH.sub.2 mix)-IgG,
m-ran-AB-PEI-NH.sub.2/OH-2 (OH/NH.sub.2 mix)-IgG,
m-ran-AB-PH-NH.sub.2/OH-3 (OH/NH.sub.2 mix)-IgG, regular polylysine
polymer, alkyl-modified randomly branched poly(2-ethyloxazoline)
with primary amine chain ends were all synthesized in a similar
manner. The synthesis of various protein conjugates with
asymmetrically randomly branched PEOX polymers also is conducted in
a similar manner.
LG-SPDP-Mixed Surface m-ran-AB-PEI-NH.sub.2/OH-2
[0209] To the mixed surface randomly branched
m-ran-AB-PEI-NH.sub.2/OH-2 (4.times.10.sup.-7 mol) in 400 .mu.L of
phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH 7.5) were
added 4.0.times.10.sup.-6 mol of sulfo-LC-SPDP (Pierce, Ill.) in
400 .mu.l of water. That was vortexed and incubated at 30.degree.
C. for 30 minutes. The LC-SPDP-m-ran-AB-PEI-NH.sub.2/OH-2 was
purified by gel filtration chromatography and equilibrated with
buffer A (0.1 M phosphate, 0.1 M NaCl and 5 mM EDTA, pH 6.8). The
product was concentrated further to yield 465 .mu.l of solution
with a concentration of approximately 0.77 nmol.
Thiolated m-ran-AB-PEI-NH.sub.2/OH-2
[0210] The LC-SPDP m-ran-AB-PEI-NH.sub.2/OH-2 (50 nmol in 65 ml of
buffer A) was mixed with 100 .mu.L, of dithiothreitol (DTT) (50 mM
in buffer A) and was allowed to incubate at room temperature for 15
minutes. Excess DTT and byproducts were removed by gel filtration
with buffer A. The product was concentrated in a 10 K Centricon
Concentrator to yield 390 .mu.L of the thiolated
m-ran-AB-PEI-NH.sub.2/OH-2 that was used for conjugation with
activated antibody.
[0211] Maleimide-R-activated antibody made as described above was
used for conjugation with the thiolated
m-ran-AB-PEI-NH.sub.2/OH-2.
m-ran-AB-PEI-NH.sub.2/OH-2-Antibody Conjugate
[0212] To the thiolated m-ran-AB-PEI-NH.sub.2/OH-2 (310 .mu.L or
35.7 nmol) was added the MAL-R-activated antibody (4.8 mL or 34
nmol). The reaction mixture was concentrated to approximately 800
.mu.L and allowed to incubate overnight at 4.degree. C. and/or at
room temperature for about 1 hr. On completion, the reaction was
quenched with 100 .mu.L of ethyl maleimide (50 mM solution) and the
conjugate then was fractionated on a CM cellulose column (5 ml)
with a sodium chloride step gradient in 20 mM phosphate buffer at
pH 6. The conjugate was eluted with a sodium chloride gradient and
characterized by cationic exchange chromatography, UV spectroscopy
and polyacrylamide gel electrophoresis.
Paclitaxel Formulation and Nanoparticle Preparation
[0213] As a general procedure, paclitaxel was dissolved in methanol
to a concentration of up to 40 mg/mL. A C.sub.18-PEOXABP60 polymer
was separately dissolved to a concentration of up to 100 mg/mL in
methanol. The two solutions were then mixed at various volumes to
result in final polymer to paclitaxel weight ratios in the mixtures
ranging from 3:1 to 10:1. The mixtures subsequently were
lyophilized for 20 to 96 hours depending on volume.
[0214] The size of the aggregates as measured by light scattering
ranged from about 70 nm to 90 nm in diameter before lyophilization
and 120-140 nm after lyophilization.
[0215] Alternatively, both paclitaxel and the C.sub.18-PEOXABP60
polymer can be dissolved in a common solvent, such as, acetone,
methanol or ethanol, and then dropwise added to water while being
stirred or sonicated, followed by sterile filtration with a 0.22
.mu.m filter. The final product then can be generated by
lyophilization and the size of the aggregates measured by light
scattering.
[0216] Other taxane-induced aggregates or nanoparticles using
various hydrophobically surface-modified branched polymers, such
as, C.sub.4, C.sub.6, C.sub.12 or C.sub.22 hydrocarbon-modified
randomly branched PEOX, PEI and PPI polymers: C.sub.4, C.sub.6,
C.sub.12, C.sub.18 and C.sub.22 hydrocarbon-modified PAMAM, PEI and
PPI dendrimers and dendrigrafts; and C.sub.4, C.sub.5, C.sub.12,
C.sub.18 and C.sub.22 hydrocarbon-modified branched PLL/polymers
can be prepared in a similar manner.
Nanoparticle with a 7:1 C.sub.18-PEOXABP60 Polymer:Paclitaxel
Ratio
[0217] Paclitaxel (700 mg) was dissolved in 9.33 mL of methanol to
yield a 75 mg/mL solution. A 15 mg/mL solution of paclitaxel was
also prepared by dissolving 100 mg in 6.67 mL of methanol. The two
solutions were mixed for 20 minutes resulting in a solution
containing 6.25 mg paclitaxel and 43.75 mg polymer per MI providing
a solution with a 7:1 polymer:drug ratio. The mixture was placed on
a rotary evaporator and the methanol removed to dryness. The
resultant solid was redissolved with stirring in 33.3 ml of water
to a final paclitaxel concentration of 3 mg/mL. The solution
preparation was passed through a 0.8 .mu.m filter and then a 0.22
.mu.m filter. The filtrate was lyophilized over a 24-72 hour period
depending on the amount used. The vial was stoppered and the
ready-to-use white powder was stored at room temperature. That
preparation was designated as FID-007. Nanoparticles with
C.sub.18-PEOXABP80 and C.sub.18-PEOXABP50 and Paclitaxel
[0218] Polymers C.sub.18-PEOXABP80 and C.sub.18-PEOXABP50 were used
to produce polymer-drug according to the same procedure described
above at 7:1 polymer:drug ratio. Particle sizes were measured and
shown in Table 1.
Nanoparticles of Comparative Polymers C.sub.18-PEOXABP200 and
C.sub.18-PEOXABP100 and Paclitaxel
[0219] With the same procedure, two comparative polymers having
different monomer/initiator ratios, C.sub.18-PEOXABP200 and
C.sub.18-PEOXABP100, were used to produce comparative aggregates at
two different weight ratios of polymer:drug, 5:1 and 7:1. Aggregate
sizes were measured, and data are shown in Table 1.
Nanoparticle Measurement
[0220] The size of various polymers, polymer-only aggregates, as
well as drug-induced polymer aggregates was measured by a dynamic
light scattering method using a Malvern Zetasizer Nano-ZS Zen3600
particle size analyzer (Malvern Panalytical Inc., Westborough,
Mass.).
[0221] As shown in Table 1, a polymer having a monomer to initiator
molar ratio of 100:1 or higher yielded polymer-drug aggregates over
100 nm in size, both at 5:1 and 7:1 polymer:drug weight ratios.
Polymer-drug aggregates produced with a polymer having a monomer to
initiator molar ratio at 80:1, 60:1, 50:1 or 20:1 yielded aggregate
having particle sizes in a range of from about 70 to 100 nm.
[0222] D(v,0.9) value (also known as D.sub.90) can be obtained
using, for example, a Malvern Zetasizer Nano-ZS Zen3600 particle
size analyzer. The D.sub.90 value is that where 90% of particles
have a size (in diameter) below, smaller than or lower than that
value. D.sub.90 values for polymer-drug aggregates prepared from
C.sub.18-PEOXABP100 (comparative) and C.sub.18-PEOXABP60
(invention) are shown in Table 1.
TABLE-US-00001 TABLE 1 Particle size of polymer-drug aggregates.
Polymer/Drug Aggregate Polymer Ratio Size (d, nm) D.sub.90
C.sub.18-PEOXABP200 (Comparative) 5 to 1 >200 --
C.sub.18-PEOXABP100 (Comparative) 5 to 1 159 -- C.sub.18-PEOXABP100
(Comparative) 7 to 1 120 209 C.sub.18-PEOXABP80 7 to 1 94 --
C.sub.18-PEOXABP60 7 to 1 99 173 C.sub.18-PEOXABP50 7 to 1 89 --
C.sub.18-PEOXABP20 7 to 1 75 --
Measurement of Filtration Rate
[0223] Polymer-drug aggregates prepared above were measured for
R.sub.f according to the following procedure.
[0224] Each of the polymer-drug aggregate samples was prepared and
dissolved in water as described herein to a same final
concentration. A final paclitaxel concentration of 3 mg/mL was used
for samples with data presented in Table 2.
[0225] A starting volume (V.sub.0) of each of samples was loaded
into a sterile syringe. A single use 25 mm sterile 0.22 .mu.m
syringe filter assembly (Pall Corp., Ann Arbor, Mich.) was affixed
to the syringe. A V.sub.0 volume of 9 ml was used for each sample.
For a 25 mm filter, starting volume can be in a range of from 9 to
10 ml.
[0226] Each sample was then made to pass through the 0.22 .mu.m
filter by depressing the plunger of the syringe with a constant
thumb pressure until all of the sample volume passed through the
filter or the plunger no longer moved forward under the same
pressure.
[0227] Sample volume remaining in the syringe (V.sub.1) was
recorded. The filtered volume (V.sub.f) or filtrate was calculated
based on the formula, V.sub.f=V.sub.0-V.sub.1. Alternatively, the
sample that passed through the filter can be collected and V.sub.f
was measured as the volume of filtrate or collected, filtered
sample.
[0228] R.sub.f for each sample was calculated based on the formula,
R.sub.f=V.sub.f/V.sub.0.
[0229] For polymer-drug aggregates prepared from C.sub.18-PEOXABP60
and paclitaxel, all 9 ml passed through the filter, V.sub.f=9,
resulting in R.sub.f=1 or 100%.
[0230] A comparative polymer-drug aggregate was prepared from
C.sub.18-PEOXABP100 and paclitaxel. Only 4.4 ml passed through the
filter. Hence, V.sub.f=4.4 resulting in R.sub.f=4.4/9 or 0.489 or
48.9%. Data are shown in Table 2.
TABLE-US-00002 TABLE 2 Filtration rate of polymer-drug aggregates.
Example Comparative Sample Polymer C.sub.18-PEOXABP60
C.sub.18-PEOXABP100 Polymer:Drug Ratio 7:1 7:1 Filter Pore Size
0.22 .mu.m 0.22 .mu.m Filtration Rate (R.sub.f) 100% 48.9%
Activity Testing
[0231] Metabolism in viable cells produces, "reducing equivalents,"
such as, NADH or NADPH. Such reducing compounds pass electrons to
an intermediate electron transfer reagent that can reduce the
tetrazolium product, MTS (Promega), into an aqueous, soluble
formazan product, which is colored. At death, cells rapidly lose
the ability to reduce tetrazolium products. The production of the
colored formazan product, therefore, is proportional to the number
of viable cells in culture.
[0232] CellTiter 96.RTM. AQ.sub.ueous products (Promega, Madison,
Wis.) are materials and methods for conducting MTS assays for
determining the number of viable cells in culture. A single reagent
added directly to the assay wells at a recommended ratio of 20
.mu.l reagent to 100 .mu.l of culture medium was used. Cells were
incubated 1-4 hours at 37.degree. C. and then absorbance was
measured at 490 nm.
Toxicity and Efficacy of Nanoencapsulated Paclitaxel/ABP60
(FIB-007)
[0233] As previously described, nanoencapsulated paclitaxel was
prepared using C.sub.18-PEOXABP60 polymer with a polymer to
paclitaxel ratio of 7:1. That preparation, given the designation
FID-007, was compared to TAXOL and ABRAXANE in cytotoxicity studies
with normal human dermal fibroblast cell lines and various cancer
cell lines, and in in vivo studies of toxicity (maximum tolerated
dose, MTD) and inhibition of tumor growth in three mouse xenograft
models.
In Vitro Activity of FID-007
[0234] FID-007 was tested with TAXOL and ABRAXANE on normal human
fibroblast cells and on various cancer cell lines in in vitro
cytotoxicity experiments. While FID-007 inhibits the proliferation
of a range of human cancer cell lines in vitro including lines
originating from breast, ovarian and lung cancer cells, FID-007
exhibited lower toxicity on normal cells, similar to the levels
observed with TAXOL and ABRAXANE (FIG. 15). Overall, FID-007 was 10
times less toxic to normal cells than to tumor cells, exhibiting a
very high EC.sub.50 (concentration of drug that yields a half
maximal response), greater than 100 .mu.M. FID-007 was active in a
72 h toxicity assay in human lung cancer cell line A549 with an
EC.sub.50 of 2.8 ng/mL (FIG. 16). FID-007 was cytotoxic to
MDA-MB-231 (triple negative breast cancer cells) with an EC.sub.50
of 4.9 ng/mL (FIG. 17). FID-007 was cytotoxic to OV-90 (ovarian
cancer cells) with an EC.sub.50 of 5.0 ng/mL (FIG. 18). With all
three cancer cell lines, FID-007 cytotoxicity was comparable to
that of TAXOL and ABRAXANE.
In Vivo Activity of FID-007
[0235] A series of experiments was performed to determine in vivo
tolerability, activity and basic pharmacokinetics of FID-007
administered intravenously (IV) in mice, as compared to TAXOL and
ABRAXANE. HD-007 was well tolerated up to 150 mg/kg daily dosing.
To confirm antineoplastic activity, FID-007 was administered IV
daily at well-tolerated doses to mice in three different mouse
xenograft models (including lung, ovarian and breast cancers). In
general, FID-007 was better tolerated in mouse xenograft models
than standard cytotoxic agents that have similar targets, such as,
TAXOL and ABRAXANE, and selectively inhibited the growth of
tumors.
[0236] Half-life of FID-007 in mice was determined, using an
optimized HPLC method, to be approximately 9.3 hours. Liver and
spleen, followed by blood were the organs with the highest
concentration of HD-007 at 1 hour. The PK profiles of FID-007,
TAXOL and ABRAXANE are shown in FIG. 19.
[0237] The single dose MTD of FID-007 was compared to that of TAXOL
and ABRAXANE in a study wherein various doses of the drugs were
administered through the tail vein of healthy CD-1 mice and SCID
(immune deficient) mice over the course of several weeks. Control
mice were administered saline. The single dose MTD for TAXOL,
ABRAXANE, and FID-007 on CD-1 mice was found to be 20 mg/kg, 240
mg/kg, and 175 mg/kg, respectively. No major side effects were
observed in all the mice that survived. However, weight gain was
observed in all the treatment groups of ABRAXANE and FID-007 as
compared to the control groups (treated with saline). ABRAXANE at
120 mg/kg and above caused a dose-dependent increase in weight. The
same was observed with FID-007 at closes of 150 mg/kg and
higher.
[0238] The multiple dose MTD of FID-007 was determined similarly by
administering FID-007 (100 and 150 mg/kg) to healthy CD-1 and SCID
mice (10 weeks, females) via the tail vein at day 0, day 3 and day
6. Animals were monitored twice per day and weighed every 3 days.
The multiple dose MTD for FID-007 in CD-1 mice was determined to be
100 mg/kg and was 30 mg/kg in SCID mice with some side effects
immediately after injection. The FID-007 multiple dose groups did
not have excessive weight gain as compared to the control
group.
[0239] The in vivo efficacy of FID-007 in inhibiting tumor growth
was compared to that of TAXOL and ABRAXANE in tumor xenograft mouse
models of human lung, breast and ovarian cancer. Sixty female and
male SCID mice (6-8 weeks, 20-26 g, Charles River, 40 female mice
for breast and ovarian cancer, 20 male mice for lung cancer) were
injected on each side of the torso (left and right) with 0.1 mL of
suspension of lung A549, breast MDA-MB-231 or ovarian OV90 cells in
serum-free medium. Cells were cultured previously in a humidified
incubator (37.degree. C., 5% CO.sub.2, 95% air). Doses of
3.times.10.sup.6 (A549), 10.sup.7 (MDA-MB-231) and 5.times.10.sup.6
(OV-90) cells were used per mouse tumor. The tumors were allowed to
grow for 7 to 9 days before treatment started, and all tumor volume
measurements were obtained using a digital caliper (VWR The tumor
volumes were calculated by the formula (W.sup.2.times.L)/2, where W
is the maximum tumor width and L is the maximum tumor length. Tumor
and body weight measurements were obtained on the same day prior to
the first treatment, then every three days. Day 0 was designated as
the first day of treatment. On day 0, the animals that developed
tumors were divided randomly into five groups [about 4 mice (8
tumors) per group], with each treatment group representing a wide
range of tumor sizes. Volumes of OV-90 xenograft tumors are shown
in FIG. 22. ABRAXANE (80 mg/kg), FID-007 (20 mg/kg), TAXOL (20
mg/kg) and C.sub.18-ABP60 polymer starting material, designated as
NanoCarrier 001-B (20 mg/kg), were prepared fresh for each
injection. Saline was used as a vehicle control. The drugs or
saline were administered through tail vein injection every three
days. Drug doses were chosen to be equitoxic for all treatment
groups based on previously determined single and multiple dose
MTD's. Lung, breast and ovarian cancer groups each received a total
of four injections. Injection volume for control, ABRAXANE and
FID-007 was 0.1 mL per injection throughout the entire study. Due
to viscosity of the TAXOL formulation, 0.2. mL per injection were
administered for the 20 mg/kg dose. Average body weight and tumor
volume measurements were calculated by averaging across the animals
within the same group. The mice were euthanized with isoflurane 21
days from the last treatment for lung cancer and ovarian cancer,
and 10 days for breast cancer. Blood and isolated serum, as well as
tumor tissues and liver were collected and stored at -80.degree.
C.
[0240] For the lung cancer (A549) xenograft group, overall, no
deaths occurred in any of the treatment groups. Probably due to the
toxicity of TAXOL, heavy breathing and inactivity were observed in
the first 30 minutes post treatment in a couple of mice. Average
body weight and tumor volume measurements were calculated by
averaging across the animals within the same group. The overall
average body weight gains for saline control, TAXOL, FID-007 and
Nano vehicle control were 6.05%, 5.87%, 6.38% and 12.3%,
respectively. However, all mice in the ABRAXANE group developed
neurotoxicity and lost>20% weight. Those mice were sacrificed at
13 days. Tumor volume increased by 1827 mm.sup.3 for the saline
control group and 1311 mm.sup.3 for the NanoCarrier-001B vehicle
control group, and by 305.8 mm.sup.3 for the TAXOL group. However,
FID-007 groups had a reduction in tumor volume by 39.7 mm.sup.3
(FIG. 20). FIG. 21 and FIG. 23 show representative images of tumors
of the treatment groups.
[0241] For the breast cancer (MDA-MB-231) xenograft group, no
deaths occurred in any of the treatment groups. Possibly due to the
toxicity of TAXOL, heavy breathing and inactivity were observed in
the first 30 minutes post treatment. In the ABRAXANE group, all
mice showed side effects of weak hind legs and 20% body weight loss
after three treatments, leading to a decision to stop the 4.sup.th
treatment for that group. Average body weight and tumor volume
measurements were calculated by averaging across the animals within
the same group. The overall average body weight gains for saline,
TAXOL, FID-007 and NanoCarrier-001B were 3.76%. 0.46%, 1.8%, and
4.2%, respectively. For the ABRAXANE group, average body weight
loss was 7.66%. Tumor volume increased by 328.6 mm.sup.3 and 458.8
mm.sup.3 in the saline and NanoCarrier-001B groups, respectively.
In the FID-007, TAXOL and ABRAXANE groups, tumor volumes decreased
by 108.7 mm.sup.3, 75.5 mm.sup.3 and 70.2 mm.sup.3, respectively.
Tumor volume observations are shown in FIG. 23.
[0242] For the ovarian cancer (OV-90) xenograft group, the TAXOL
treatment group showed some toxicity with heavy breathing and
inactivity observed in the first 30 minutes post treatment in two
mice. The average body weight gain was 3.23%, 17.1%, 13.5%, 15.4%
and 2.24% in the saline control, TAXOL, ABRAXANE, FID-007 and
NanoCarrier-001B control groups, respectively. Tumor volume
increased by 652.7 mm.sup.3, 271.9 mm.sup.3 and 9.1 mm.sup.3 in
saline control, NanoCarrier-001B control and TAXOL groups,
respectively, while there was a decrease in tumor volume in the
FID-007 group by 93.1 mm.sup.3 and in the ABRAXANE group (80 mg/kg)
by 72.4 mm.sup.3.
[0243] FID-007 demonstrated in vitro cytotoxicity to lung, breast
and ovarian cell lines similar to the established antineoplastic
drugs TAXOL and ABRAXANE, while maintaining a low level of toxicity
to normal cells. The in vivo efficacy of FID-007 in inhibiting
tumor growth and reducing tumor mass was as good as or
significantly better than the two approved drugs in mouse xenograft
models of human lung, breast, and ovarian cancers.
[0244] All references cited herein are herein incorporated by
reference in entirety.
[0245] It will be appreciated that various changes and
modifications can be made to the teachings herein without departing
from the spirit and scope of the disclosure.
* * * * *