U.S. patent application number 14/977480 was filed with the patent office on 2016-04-21 for method of delivering an anti-cancer agent to a cell.
This patent application is currently assigned to Agency for Science, Technology and Research. The applicant listed for this patent is Agency for Science, Technology and Research. Invention is credited to Joo Eun Chung, Motoichi Kurisawa, Jackie Y. Ying.
Application Number | 20160106706 14/977480 |
Document ID | / |
Family ID | 40579792 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106706 |
Kind Code |
A1 |
Ying; Jackie Y. ; et
al. |
April 21, 2016 |
METHOD OF DELIVERING AN ANTI-CANCER AGENT TO A CELL
Abstract
There is provided a delivery vehicle comprising an anti-cancer
agent together with a conjugate of a delivery agent containing a
free aldehyde and a flavonoid, having the delivery agent conjugated
at the C6 and/or the C8 position of the A ring of the flavonoid.
The resulting delivery vehicles may be used to deliver an
anti-cancer agent to a cell.
Inventors: |
Ying; Jackie Y.; (Singapore,
SG) ; Chung; Joo Eun; (Singapore, SG) ;
Kurisawa; Motoichi; (Singapore, SG) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agency for Science, Technology and Research |
Singapore |
|
SG |
|
|
Assignee: |
Agency for Science, Technology and
Research
Singapore
SG
|
Family ID: |
40579792 |
Appl. No.: |
14/977480 |
Filed: |
December 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12739668 |
Aug 20, 2010 |
9248200 |
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PCT/SG08/00409 |
Oct 23, 2008 |
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14977480 |
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60960969 |
Oct 23, 2007 |
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Current U.S.
Class: |
424/133.1 ;
435/375 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 31/353 20130101; A61K 9/1075 20130101; A61K 2039/505 20130101;
C08L 2203/02 20130101; B82Y 5/00 20130101; A61K 47/6919 20170801;
C08B 37/0072 20130101; A61K 39/39558 20130101; A61P 35/00 20180101;
C08G 65/3317 20130101; C08G 65/3326 20130101 |
International
Class: |
A61K 31/353 20060101
A61K031/353; A61K 9/107 20060101 A61K009/107; A61K 39/395 20060101
A61K039/395 |
Claims
1.-32. (canceled)
33. A micellar nanocomplex comprising: an anti-cancer agent that is
a protein; and a conjugate of: (i) aldehyde-terminated polyethylene
glycol; and (ii) a flavonoid that is either monomeric
(-)-epigallocatechin gallate or oligomeric (-)-epigallocatechin
gallate, the micellar nanocomplex having the polyethylene glycol
conjugated at the C6 and/or the C8 position of the A ring of the
flavonoid by attachment of the polyethylene glycol via reaction of
the free aldehyde group with the C6 and/or C8 position of the A
ring of the flavonoid, and exhibiting an anti-cancer effect on a
cell as a result of a synergistic effect between the anti-cancer
effect of the anti-cancer agent and the anti-cancer effect of the
flavonoid, wherein when the flavonoid is monomeric
(-)-epigallocatechin gallate, the micellar nanocomplex further
comprises non-conjugated oligomeric (-)-epigallocatechin gallate in
an inner core of the micellar nanocomplex, wherein the anti-cancer
agent is complexed with either the conjugated or non-conjugated
oligomeric (-)-epigallocatechin gallate, whichever is present.
34. The micellar nanocomplex of claim 33, wherein the
aldehyde-terminated polyethylene glycol is conjugated to oligomeric
(-)-epigallocatechin gallate.
35. The micellar nanocomplex of claim 34, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through
enzyme-catalyzed oxidative coupling.
36. The micellar nanocomplex of claim 34, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through
aldehyde-mediated oligomerization.
37. The micellar nanocomplex of claim 34, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through a
carbon-carbon linkage between the C6 or C8 position on the A ring
of a first monomeric unit to the C6 or C8 position on the A ring of
a second monomeric unit.
38. The micellar nanocomplex of claim 33, wherein the
aldehyde-terminated polyethylene glycol is conjugated to monomeric
(-)-epigallocatechin gallate and the micellar nanocomplex comprises
an inner core containing non-conjugated oligomeric
(-)-epigallocatechin gallate.
39. The micellar nanocomplex of claim 38, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through
enzyme-catalyzed oxidative coupling.
40. The micellar nanocomplex of claim 38, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through
aldehyde-mediated oligomerization.
41. The micellar nanocomplex of claim 38, wherein the oligomeric
(-)-epigallocatechin gallate is formed from (-)-epigallocatechin
gallate monomers that have been oligomerized through a
carbon-carbon linkage between the C6 or C8 position on the A ring
of a first monomeric unit to the C6 or C8 position on the A ring of
a second monomeric unit.
42. The micellar nanocomplex of claim 33, wherein the anti-cancer
agent is a protein that is a peptide, an antibody, a hormone, an
enzyme, a growth factor, or a cytokine.
43. The micellar nanocomplex of claim 33, wherein the anti-cancer
agent is a protein that is an antibody directed against a tumour
cell-surface marker, an immunoregulatory peptide, a cytokine or a
growth factor.
44. The micellar nanocomplex of claim 33, wherein the anti-cancer
agent is a protein that is a cytokine.
45. The micellar nanocomplex of claim 33, wherein the anti-cancer
agent is an antibody directed against a tumour cell-surface
marker.
46. A pharmaceutical composition comprising the micellar
nanocomplex according to claim 33.
47. A pharmaceutical composition comprising the micellar
nanocomplex according to claim 34.
48. A pharmaceutical composition comprising the micellar
nanocomplex according to claim 38.
49. A method of delivering trastuzumab to a cell comprising
contacting the micellar nanocomplex of claim 33 with the cell.
50. The method according to claim 48, wherein the cell is in
vitro.
51. The method according to claim 49, wherein the cell is in vivo
and the method comprises administering the micellar nanocomplex to
a subject in need of anti-cancer treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of, and priority from, U.S.
provisional patent application No. 60/960,969, filed Oct. 23, 2007,
the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a method of
delivering a bioactive agent to a cell, including a tumour cell in
a subject.
BACKGROUND OF THE INVENTION
[0003] Flavonoids are one of the most numerous and best-studied
groups of plant polyphenols. The flavonoids consist of a large
group of low-molecular weight polyphenolic substances naturally
occurring in fruits and vegetables, and are an integral part of the
human diet. Dried green tea leaves can contain as much as 30%
flavonoids by weight, including a high percentage of flavonoids
known as catechins (flavan-3-ol derivatives or catechin-based
flavonoids), including (-)-epicatechin, (-)-epigallocatechin,
(+)-catechin, (-)-epicatechin gallate and (-)-epigallocatechin
gallate.
[0004] In recent years, these green tea catechins have attracted
much attention because they have been recognized to have biological
and pharmacological properties, including antibacterial,
antineoplastic, anti-thrombotic, vasodilatory, antioxidant,
antimutagenic, anti-carcinogenic, hypercholesterolemic, antiviral
and anti-inflammatory properties, which have been demonstrated in
numerous human, animal and in vitro studies (Jankun J., et al.
Nature 387, 561 (1997); Bodoni A. et al. J. Nutr. Biochem. 13,
103-111 (2002); Nakagawa K. et al. J. Agric. Food Chem. 47,
3967-3973 (1999)). These biological and pharmacological properties
are potentially beneficial in preventing diseases and protecting
the stability of the genome.
[0005] Many of the beneficial effects of catechins are thought to
be linked to the antioxidant actions of the catechins (Terao J., et
al. Arch. Biochem. Biophys. 308, 278-284 (1994)). Among the
catechins, (-)-epigallocatechin gallate (EGCG), which is a major
component of green lea, is thought to have the highest activity,
possibly due to the trihydroxy B ring and the gallate ester moiety
at the C3 position (Isemura M., et al. Biofactors 13, 81-85 (2000);
Ikeda I., et al. J. Nutr. 135, 155 (2005); Lill G., et al. FEBS
Letters 546, 265-270 (2003); Sakanaka S. and Okada Y. J. Agric.
Food Chem. 52, 1688-1692 (2004); Yokozawa T., et al., J. Agric.
Food Chem. 48, 5068-5073 (2000)).
[0006] In general, the activity half-life of flavonoids is limited
to a few hours inside the body; metabolism of these compounds has
not yet been established. Despite the favorable anti-oxidation and
anti-cancer properties of the catechins including EGCG, it is
impractical to achieve a therapeutic level of this compound in the
body by directly ingesting a large amount of green tea, due to the
inherent volume constraint. That is, in order to obtain a
therapeutic or pharmacological benefit from flavonoids through diet
alone, it would be necessary to ingest an amount of food and
beverage that is larger than is practical to consume. Moreover,
pro-oxidant activity has been reported for several flavonoids
including EGCG, making ingesting crude green tea directly a less
effective means of delivering EGCG (Yen G. C., et al. J. Agric.
Food Chem. 45, 30-34 (1997); Yamanaka N., et al. FEBS Lett. 401,
230-234 (1997); Roedig-Penman A. and Gordon M. H. J. Agric. Food
Chem. 1997, 45, 4267-4270).
[0007] On the other hand, a relatively high-molecular fraction of
extracted plant polyphenols (procyanidins) and synthetically
oligomerized (+)-catechin and rutin have been reported to exhibit
enhanced physiological properties such as antioxidant and
anti-carcinogenic activity compared to low-molecular weight
flavonoids, (Zhao J., el al. Carcinogenesis, 1999, 20, 1737-1745;
Ariga T. and Hamano M. Agric. Biol. Chem. 54, 2499-2504 (1990);
Chung J. E., et al. Biomacromolecules 5, 113-118 (2004); Kurisawa
M., et al. Biomacromolecules 4, 1394-1399 (2003); Hagerman A. E.,
et al. J. Agric. Food Chem. 46, 1887 (1998)) and without
pro-oxidant effects (Hagerman A. E., et al. J. Agric. Food Chem.
46, 1887 (1998); Li C. and Xie B. J. Agric. Food Chem. 48, 6362
(2000)). However, neither naturally occurring nor synthesized high
molecular weight flavonoids are expected to be absorbed and
transported to other tissues after ingestion, since these compounds
are typically large, form strong complexes with proteins and are
resistant to degradation (Zhao J., et al. Carcinogenesis, 1999, 20,
1737-1745).
[0008] In cases of flavonoids consumed via oral intake of foods and
beverages, the flavonoids may play a role as antioxidants to
protect the digestive tract from oxidative damage during digestion.
However, flavonoids can be expected to remain only in the digestive
tract and thus their beneficial physiological activities are not
likely to be utilized to other tissues. Moreover, their strong
hydrophobicity as well as their tendency to form complexes with
proteins makes parenteral delivery of these compounds
difficult.
SUMMARY OF THE INVENTION
[0009] The present invention provides a method of delivering
bioactive anti-cancer agents to a cell, including a tumour cell,
using flavonoid conjugates and delivery agents previously described
in published International application WO 2006/124000 and published
US application 2008/102052.
[0010] The present invention is based on the surprising discovery
that use of the flavonoid-based delivery agents to deliver
anti-cancer agents to a cell results in a synergistic effect
between the anti-cancer bioactive agent and the flavonoids. Thus,
these conjugates and delivery agents combine the synergistic
therapeutic effects arising from the flavonoid portion that acts as
a carrier and the bioactive agent that is to be delivered.
[0011] In one aspect, there is provided a delivery vehicle
comprising an anti-cancer agent and a conjugate of a delivery agent
containing a free aldehyde and a flavonoid, having the delivery
agent conjugated at the C6 and/or the C8 position of the A ring of
the flavonoid.
[0012] In another aspect, there is provided a method of delivering
an anti-cancer agent to a cell comprising contacting the delivery
agent as described herein with the cell.
[0013] In another aspect, there is provided a pharmaceutical
composition comprising the delivery vehicle as described
herein.
[0014] In another aspect, there is provided use of the
micelle-protein complex as described herein for delivering an
anti-cancer agent to a cell in a subject.
[0015] Other aspects and features of the present invention will
become apparent to those of ordinary skill in the art upon review
of the following description of specific embodiments of the
invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] In the figures, which illustrate, by way of example only,
embodiments of the present invention,
[0017] FIG. 1 is a graph depicting cytotoxicity of (.quadrature.)
EGCG, (.tangle-solidup.) OEGCG and (.smallcircle.) PEG-EGCG on
HMEC. N=5 for samples and N=8 for control;
[0018] FIG. 2 is a graph depicting the size of the micellar complex
in the (.smallcircle.) absence and ( ) presence of serum. N=3;
[0019] FIG. 3 is a graph depicting the fluorescene intensity of
(.quadrature.) free FITC-BSA, (.smallcircle.) FITC-BSA and OEGCG
complex, (.DELTA.) (FITC-BSA+OEGCG) in PEG-EGCG complex in the
presence of proteinase K; and (.box-solid.) free FITC-BSA in the
absence of proteinase K. N=3;
[0020] FIG. 4 is a graph depicting in vitro cell proliferation of
SKBR-3 cells in the presence of (.quadrature.) PEG-EGCG,
(.box-solid.) OEGCG, (.DELTA.) Herceptin, ( ) (Herceptin+OEGCG) in
PEG-EGCG complex, (.tangle-solidup.) (BSA+OEGCG) in PEG-EGCG
complex, and (.smallcircle.) control (untreated cells). N=5 for
samples and N=8 for control;
[0021] FIG. 5 is a graph depicting the size of the
(Herceptin+OEGCG) in PEG-EGCG complex subjected to suspension
dilution. N=3;
[0022] FIG. 6 is a graph depicting in vitro proliferation of SKBR-3
cells in the presence of OEGCG, PEG-EGCG, Herceptin,
(Herceptin+OEGCG) in PEG-EGCG complex, and (BSA+OEGCG) in PEG-EGCG
complex (bars from left to right in each group). Percentage of cell
proliferation was normalized with the control at each time point.
N=5 for samples and N=8 for control; and
[0023] FIG. 7 is a graph depicting tumour size in tumour-containing
Balb/nude mice treated twice weekly for one month with (.DELTA.)
micellar complex plus Herceptin, (.quadrature.) Herceptin alone, or
(.smallcircle.) PBS control.
DETAILED DESCRIPTION
[0024] Flavonoids are known to have a variety of biological
properties, including having anti-cancer effects, inhibiting growth
of cancer cells. Delivery vehicles comprising flavonoids were
previously described in published international application WO
2006/124000 and published US application 2008/102052.
[0025] Surprisingly, the inventors have found that the combination
of the flavonoid delivery vehicle and bioactive anti-cancer agent
has a synergistic anti-cancer effect, greater than the combined
effects of each of the flavonoid delivery vehicle and bioactive
anti-cancer agent when used alone. Thus, such delivery vehicles
loaded with an anti-cancer agent provide an effective way of
delivering anti-cancer agents to a cell, taking advantage of the
synergistic effect between the anti-cancer activity of the
flavonoid portion of the delivery vehicle and the anti-cancer
effect of the anti-cancer agent.
[0026] The inventors developed a delivery vehicle containing an
anti-cancer agent such as a protein, nucleic acid or drug, by
self-assembly using flavonoids and conjugates of flavonoids such as
EGCG. The delivery vehicles were formed by self-assembly. For
example, delivery vehicles were synthesised by (i) two-step
self-assembly process involving assembly of oligomeric
(-)-epigallocatechin-3-O-gallate (OEGCG) and the anti-cancer agent,
and then assembly of the pre-formed OEGCG-anti-cancer agent complex
with a conjugate of poly(ethylene glycol) (PEG) and EGCG
(PEG-EGCG); and (ii) one-step self-assembly process involving
assembly of a conjugate of PEG and OEGCG (PEG-OEGCG) and the
anti-cancer agent. Both delivery vehicles formed as stable and
highly oriented micellar complexes loaded with the anti-cancer
agent via spontaneous self-assembly in a mild aqueous solution. The
resulting micellar nanocomplexes characteristically display a PEG
outer shell, with an inner core comprised of OEGCG-anti-cancer
agent complex.
[0027] Since the formation of a micellar nanocomplex is mainly
driven by hydrophobic interaction and hydrogen bonding, the
delivery vehicles described herein can be used to load a wide
variety of anti-cancer agents for improved delivery to a cell.
[0028] The delivery vehicles are designed to take advantage of the
enhanced permeability and retention (EPR) effect, and to avoid
reticuloendothelial system (RES) uptake when administered in vivo,
providing for selective accumulation at cancer sites. Ideally, the
delivery vehicles preserve the stability of both the flavonoid
molecules and the anti-cancer agent from loss of activity and from
degradation during delivery, exerting a synergistic therapeutic
effect when delivered to a cell. The delivery vehicle may mask the
activity of the flavonoid and the anti-cancer agent activity by
sequestering these moieties within the inner core of the delivery
vehicle until successfully delivered and released at the cell,
restoring the respective activities only upon dissociation of the
delivery vehicle at the site of a cell.
[0029] To demonstrate the synergistic therapeutic effects by the
delivery vehicle, the anti-cancer protein Herceptin (trastuzumab)
was loaded into the micellar complex, as described in Example 1
below. The micellar complex kept its integrity and showed good
stability in the presence of serum without size change as a
function of time, and the protein was safely protected from
proteolysis within the micellar complex. A greater inhibition was
demonstrated with the Herceptin-loaded delivery vehicle as compared
to the individual components (OEGCG, PEG-EGCG and Herceptin).
[0030] Since the formation of the delivery vehicle is mainly driven
by hydrophobic interaction, it can be dissociated by hydrophobic
competitors, such as amphiphilic molecules. Thus, the delivery
vehicle may be dissociated by bio-amphiphilic molecules (such as
plasma membrane lipids) to release the contained therapeutic
flavonoid and anti-cancer compounds when the delivery vehicle
accumulates at the targeted cell site.
[0031] Thus, there is provided a method of delivering an
anti-cancer agent to a cell. A delivery vehicle containing a
flavonoid and an anti-cancer agent is used, as previously described
in published international application WO 2006/124000 and published
US application 2008/102052, and as described below. The delivery
vehicle is then contacted with the cell to which the anti-cancer
agent is to be delivered.
Flavonoid Conjugates and Delivery Vehicles
[0032] In order to increase the availability of beneficial
flavonoid compounds, the conjugation of flavonoids to various
delivery agents through a free aldehyde group on the delivery agent
to the A ring of the flavonoid allows for modification of the
physical properties of the flavonoid without disrupting the
polyphenol structure of the flavonoid, while augmenting the
biological and pharmacological properties of the flavonoid.
[0033] That is, the aldehyde-mediated conjugation of a delivery
agent to the flavonoid results in attachment of the delivery agent
at the C6 and/or C8 position of the flavonoid A ring, and does not
disrupt or affect the B and C rings of the flavonoid or the various
hydroxyl groups on the flavonoid.
[0034] Conjugation of a delivery agent to a flavonoid can provide a
composition that is suitable for administration to a subject by
incorporating the flavonoid into a particular vehicle formed with
the delivery agent, and can allow for administration of higher
concentrations of flavonoids than can be obtained through diet. The
delivery agent can provide stability to the composition, resulting
in a composition that is metabolized or degraded more slowly, and
which thus may have a longer half-life in the body than the
unconjugated flavonoid alone. For example, the delivery agent may
be of such a nature that the flavonoid is incorporated into a
composition that enhances the water-solubility of the flavonoid,
which can avoid uptake by the reticuloendothelial system and
subsequent clearance by the kidneys, resulting in a longer
half-life in the body. Conjugation of other delivery agents may
protect the flavonoid from enzyme degradation.
[0035] A delivery agent may be conjugated to a flavonoid by
reacting the delivery agent with the flavonoid in the presence of
an acid catalyst, the delivery agent having a free aldehyde group,
or a group that is able to be converted to a free aldehyde group in
the presence of acid.
[0036] The flavonoid may be any flavonoid from the general class of
molecules derived from a core phenylbenzyl pyrone structure, and
includes flavones, isoflavones, flavonols, flavanones,
flavan-3-ols, catechins, anthocyanidins and chalcones. In a
particular embodiment the flavonoid is a catechin or a
catechin-based flavonoid. A catechin, or a catechin-based flavonoid
is any flavonoid that belongs to the class generally known as
catechins (or flavan-3-ol derivatives), and includes catechin and
catechin derivatives, including epicatechin, epigallocatechin,
catechin, epicatechin gallate and epigallocatechin gallate, and
including all possible stereoisomers of catechins or catechin-based
flavonoids. In particular embodiments, the catechin-based flavonoid
is (+)-catechin or (-)-epigallocatechin gallate.
(-)-epigallocatechin gallate (EGCG) is thought to have the highest
activity among the catechin-based flavonoids, possibly due to the
trihydroxy B ring and gallate ester moiety at the C3 position of
this flavonoid.
[0037] The delivery agent is any chemical group or moiety that
contains a free aldehyde or group, or a functional group that can
be converted to a free aldehyde group in the presence of acid, for
example an acetal group. The delivery agent is capable of being
formed into a delivery vehicle, thus allowing for the incorporation
of a conjugated flavonoid into the delivery vehicle without
compromising the biological or pharmacological properties of the
flavonoid. As well, the delivery agent should be biocompatible, and
may be biodegradable in some embodiments.
[0038] The following discussion refers to an embodiment in which
the flavonoid is a catechin-based flavonoid and in which the
delivery agent is a polymer. However, it will be understood that
the aldehyde condensation reaction between an aldehyde-containing
chemical group and a flavonoid is applicable to conjugation of any
delivery agent having a free aldehyde group, including following
acid treatment of the delivery agent, to any flavonoid, as
described above.
[0039] Thus, the reaction may involve conjugation of a polymer
containing a free aldehyde group or a group that is able to be
converted to a free aldehyde group in the presence of acid to a
catechin-based flavonoid.
[0040] The catechin-based flavonoid may be a single monomeric unit
of a catechin-based flavonoid or it may be an oligomer of one or
more catechin-based flavonoids. As stated above, conjugation of a
polymer to a flavonoid results in augmentation of the flavonoid's
biological or pharmacological properties. As well, an oligomer of
the catechin-based flavonoid tends to have amplified or augmented
levels of the biological and pharmacological properties associated
with catechin-based flavonoids, and may even have reduced
pro-oxidant effects that are sometimes associated with monomeric
catechin-based flavonoids. Thus, the catechin-based flavonoid is an
oligomerized catechin-based flavonoid having amplified or augmented
flavonoid properties.
[0041] Oligomers of catechin-based flavonoids are known, including
oligomers prepared through enzyme-catalyzed oxidative coupling and
through aldehyde-mediated oligomerization. An aldehyde-mediated
oligomerization process results in an unbranched oligomer that has
defined linkages, for example through carbon-carbon linkages such
as CH--CH.sub.3 bridges linked from the C6 or C8 position on the A
ring of one monomer to the C6 or C8 position on the A ring of the
next monomer, including in either possible stereoconfiguration,
where applicable. Thus, the CH--CH.sub.3 linkage may between the C6
position of the A ring of one monomer and either of the C6 or C8
position of the next monomer or it may be between the C8 position
of the A ring of the first monomer and either of the C6 or C8
position of the next monomer.
[0042] The oligomer of the catechin-based flavonoid may be of 2 or
more monomeric units linked together. In certain embodiments, the
catechin-based flavonoid oligomer has from 2 to 100 flavonoid
monomer units, from 10 to 100, from 2 to 80, from 10 to 80, from 2
to 50, from 10 to 50, from 2 to 30, from 10 to 30, from 20 to 100,
from 30 to 100 or from 50 to 100 monomeric units.
[0043] The polymer may be any polymer having a free aldehyde group
prior to conjugation with the catechin-based flavonoid, or having a
group that is converted to an aldehyde group in the presence of
acid, for example an acetal group. Furthermore, it will be
understood that the polymer should be non-toxic, biocompatible and
suitable for pharmacological use. The polymer may also have other
desirable properties, for example, the polymer may have low
immunogenicity, and it may be biodegradable or non-biodegradable
depending on the desired biological application of the composition,
for example, for controlled release of catechin-based flavonoids
and the anti-cancer agent at a particular site in a body.
[0044] The polymer may be chosen based on its particular
characteristics and its ability to form certain types of delivery
vehicles. For example, the polymer may be an aldehyde-terminated
poly (ethylene glycol), or it may be hyaluronic acid derivatized
with an aldehyde group, or a derivative of such polymers.
Alternatively, the polymer may be a phenoxymethyl(methylhydrazono)
dendrimer (PMMH), for example, cyclotriphosphazene core PMMH or
thiophosphoryl core PMMH. The polymer may also be any biological
polymer, modified to contain a free aldehyde group or a group that
is convertible to an aldehyde in the presence of acid, for example
an aldehyde-modified protein, peptide, polysaccharide or nucleic
acid. In one particular embodiment the polymer is an
aldehyde-terminated poly (ethylene glycol) (PEG-CHO). In another
particular embodiment, the polymer is aldehyde-derivatized
hyaluronic acid, hyaluronic acid conjugated with
aminoacetylaldehyde diethylacetal, or either of the aforementioned
hyaluronic acid polymers derivatized with tyramine.
[0045] The free aldehyde group on the polymer allows for the
conjugation of the polymer in a controlled manner to either the C6
or the C8 position of the A ring, or both, of the flavonoid
structure, thus preventing disruption of the flavonoid structure,
particularly the B and C rings of the flavonoid, and thus
preserving the beneficial biological and pharmacological properties
of the flavonoid.
[0046] The polymer is conjugated to the catechin-based flavonoid
via a reaction of the aldehyde group of the polymer with the C6
and/or the C8 position of the A ring of the catechin-based
flavonoid.
[0047] The conjugate is synthesized using acid catalysis of a
condensation of the aldehyde group of the polymer with the
catechin-based flavonoid, or using acid to convert a functional
group on the polymer to a free aldehyde prior to condensation of
the aldehyde group with the catechin-based flavonoid.
[0048] To conjugate the polymer and the catechin-based flavonoid,
the polymer and the catechin-based flavonoid may be separately
dissolved in a suitable solvent. The polymer with the free aldehyde
is added, for example by dropwise addition, to the solution
containing the catechin-based flavonoid, in the presence of an
acid. The reaction is allowed to go to completion. Following the
conjugation reaction, excess unreacted polymer or catechin-based
flavonoid can be removed from the conjugated composition, for
example by dialysis or by molecular sieving.
[0049] The ratio of catechin-based flavonoid to polymer may be
varied, so that there is only one polymer moiety attached to the
catechin-based flavonoid portion of the polymer, or so that there
is a catechin-based flavonoid portion attached at more than one
position on the polymer, or so that the catechin-based flavonoid
portion has two polymer portions attached, one at either of the C6
and C8 positions of the catechin-based flavonoid.
[0050] The ratio of polymer to catechin-based flavonoid in the
final composition can be controlled through the ratio of starting
reagents. For example, when the molar ratio of polymer moiety to
catechin-based flavonoid moiety is about 1, a single polymer moiety
will be attached to a single catechin-based flavonoid moiety
(either monomeric or oligomeric may be used). However, at higher
concentrations of polymer, for example at a 10:1 molar ratio of
polymer to catechin-based flavonoid, a composition having a
tri-block structure of polymer-flavonoid-polymer may be
obtained.
[0051] A conjugate of a polymer containing a free aldehyde and a
catechin-based flavonoid, having the polymer conjugated at the C6
and/or the C8 position of the A ring of the flavonoid is also
contemplated.
[0052] Conjugation of the polymer also allows for the incorporation
of catechin-based flavonoids into various compositions or vehicles.
By selection of the particular polymer containing a free aldehyde
group based on the physical properties of the polymer, it is
possible to incorporate flavonoids into a variety of different
vehicle types, allowing for the delivery of high concentrations of
flavonoids in different contexts to various targeted areas of the
body.
[0053] Thus, the conjugate resulting from the above-described
reaction may be formed into a delivery vehicle, depending on the
nature of the polymer portion of the conjugate. The delivery
vehicle may be used to deliver the catechin-based flavonoid to a
body, including a particular targeted site in a body, depending on
the nature of the delivery vehicle.
Anti-Cancer Agents
[0054] An anti-cancer agent is included in the delivery vehicle,
which is then contacted with a cell to which the anti-cancer agent
is to be delivered. Thus, there is provided a delivery vehicle
comprising a catechin-based flavonoid conjugated to a polymer
through a free aldehyde group on the polymer, the delivery vehicle
further comprising an anti-cancer agent.
[0055] The anti-cancer agent may be any agent that has an
anti-cancer effect on a cell, including an anti-tumour effect, such
as a cytotoxic, apoptotic, anti-mitotic anti-angiogenesis or
inhibition of metastasis effect. The anti-cancer effect is intended
to include inhibition or reduction of tumour cell growth,
inhibition or reduction of carcinogenesis, killing of tumour cells,
or inhibition or reduction of carcinogenic or tumourogenic
properties of a cell, including a tumour cell.
[0056] An anti-cancer agent includes a protein, a nucleic acid, a
small molecule or a drug. An anti-cancer agent that is a protein
may be a peptide, an antibody, a hormone, an enzyme, a growth
factor, or a cytokine. An anti-cancer agent that is a nucleic acid
may be single stranded or double stranded DNA or RNA, a short
hairpin RNA, an siRNA, or may comprise a gene encoding an
anti-cancer product. Also included in the scope of anti-cancer
agent is a chemotherapeutic agent or an angiogenesis inhibitor. The
anti-cancer agent may be an antibody, including a monoclonal
antibody, directed against a tumour cell-surface marker, an
immunoregulatory peptide, a cytokine or a growth factor. The
anti-cancer agent may be Herceptin (trastuzumab) or TNP470.
Formation of Delivery Vehicle Containing Anti-Cancer Agent
[0057] In one particular embodiment, the delivery vehicle is a
micellar nanocomplex, which is suitable for parenteral delivery of
a catechin-based flavonoid and an anti-cancer agent to a cell,
including a cell located at a particular site within a body of a
subject.
[0058] To form the delivery vehicle containing the anti-cancer
agent, the polymer is chosen to have properties that allow it to
assemble with the catechin-based flavonoid portion of the
composition, protecting the flavonoid from the solution
environment. If a suitable solvent is chosen in which the polymer
portion of the conjugate is soluble and is more soluble than the
catechin-based flavonoid, the conjugate will self-assemble,
excluding the solution from the flavonoid core, thus allowing for
assembly of micellar complexes.
[0059] In a particular embodiment of the micellar nanocomplex
delivery vehicle, the polymer chosen is aldehyde-terminated PEG, or
a derivative thereof. PEG is a polymer widely used as a
pharmacological ingredient, and possesses good hydrophilic,
non-toxic, non-immunogenic and biocompatibility characteristics
with low biodegradability.
[0060] By conjugating PEG-CHO to a catechin-based flavonoid, a
conjugate is formed that has strong self-assembly tendencies. In
one embodiment, PEG is conjugated to a monomer of a catechin-based
flavonoid, to form a PEG-flavonoid. The delivery vehicle is formed
together with non-conjugated catechin-based flavonoids and the
anti-cancer agent. Thus, the central core contains relatively high
concentrations of a flavonoid and the anti-cancer agent, while the
external shell of the micellar nanocomplex comprises the conjugated
PEG-monomeric flavonoid, and is assembled in a two-step process. In
a particular embodiment, the central core is oligomeric EGCG
(OEGCG) and the external core is made up of conjugated
PEG-EGCG.
[0061] Formation of this two-step assembly of the delivery vehicle
results in temporary partial or complete masking of the biological
activities of the oligomeric flavonoid that is incorporated into
the core of the delivery vehicle, as well as protecting the
anti-cancer agent from degradation prior to delivery to the cell.
For example, while assembled into core of the delivery vehicle, the
augmented properties of the oligomerized EGCG are less available,
due to physical interactions with other molecules in the assembled
core portion of the delivery vehicle. Upon release from the
delivery vehicle, for example by fusion of the vehicle with a
cellular phospholipid membrane, the components of the delivery
dissociate, unmasking the biological properties of the oligomeric
catechin-based flavonoid, and releasing the anti-cancer agent for
delivery into the cell.
[0062] This embodiment of the delivery vehicle is well suited to
deliver the anti-cancer agent. Since the catechin-based flavonoids
have a rigid, multi-ring core structure, these molecules associate
well with anti-cancer agents such as proteins and nucleic acids, as
well as other molecules containing ring structures, likely by
stacking of the catechin rings with the ring or rings on the
anti-cancer agent. Thus, an oligomeric catechin-based flavonoid can
be used to associate with the anti-cancer agent prior to assembly
in the micellar nanocomplex.
[0063] The concentration of the anti-cancer agent is chosen
depending on the total amount of anti-cancer agent that is to be
delivered to a particular site in a body, and on the amount of
anti-cancer agent that can be included in the micellar nanocomplex
without destabilizing the micellar structure. In certain
embodiments, the anti-cancer agent may constitute up to 50%, or up
to 40%, w/w of the micellar complex.
[0064] The biological activity of the anti-cancer agent is also
temporarily partially or completely masked while incorporated into
the present delivery vehicles. As with the oligomeric
catechin-based flavonoid, the biological properties of the
anti-cancer agent are masked or sequestered, making them less
available while the anti-cancer agent is assembled in the delivery
vehicle, meaning that the anti-cancer agent is not able to exert
anti-cancer activity or interact with other molecules in a
bioactive manner while contained in the delivery vehicle, and is
also protected from activity of other molecules. Upon release of
the anti-cancer agent from the delivery vehicle, the biological
properties of the anti-cancer agent are once again available, and
the anti-cancer agent is able to exert an anti-cancer effect once
delivered to the cell.
[0065] In another embodiment, PEG is conjugated to an oligomeric
catechin-based flavonoid. This embodiment of the delivery agent has
strong self-assembly properties and can be self-assembled in a
single step process. As with the two-step assembly micellar
nanocomplex above, the single-step assembling micellar nanocomplex
includes the anti-cancer agent.
[0066] The above micellar nanocomplexes are of nanoscale
dimensions, and may be from about 1 nm to about 10000 nm in
diameter, or from about 20 nm to about 4000 nm in diameter, or from
about 20 nm to about 100 nm in diameter. The size of the micellar
nanocomplexes can be varied by varying the length of the
oligomerized catechin-based flavonoid, the length of the polymer,
and the concentration of unconjugated oligomerized catechin-based
flavonoid. The size of the micellar nanocomplex may be pH
dependent, depending on the polymer used. For example, in micellar
nanocomplexes in which the conjugated polymer is PEG, the diameter
of the micelles tends to decrease with increasing pH.
[0067] Generally, the micellar nanocomplexes containing the
anti-cancer agent undergo self assembly and thus little synthesis
is required. For the two step process, the components that are to
form the core, including the anti-cancer agent, are dissolved in a
suitable solvent, for example in diluted DMSO or methanol, and are
allowed to assemble. The solvent is a solvent in which the core
components are soluble, and which may be miscible in water, or
which may be volatile, or from which the assembled micelles can
otherwise be isolated or extracted. As indicated above, the core
components include an anti-cancer agent and a catechin-based
flavonoid, for example an oligomeric catechin-based flavonoid. The
polymer-catechin-based flavonoid conjugate that is to form the
outer shell is then added to the solution and the micellar complex
is allowed to form.
[0068] For the one step self-assembly process, the
polymer-catechin-based flavonoid conjugate together with the
anti-cancer agent, is dissolved in a suitable buffer as described
for the two-step process and the micellar nanocomplex is allowed to
assemble.
[0069] This micellar nanocomplex system provides the ability to
achieve controlled biodistribution of catechin-based flavonoids and
prolonged circulation half-life in bloodstream due to the PEG outer
shell, as well as amplified pathological activities of the
catechin-based flavonoid compound, with the added benefit that such
compounds may be accompanied by therapeutic effect of the
anti-cancer agent loaded in the inner core of the micelle. Where
the anti-cancer agent is a sensitive molecule such as a protein,
the nanoscale micelles offer a convenient delivery vehicle with the
advantage of a gentle, self-assembly method that does not involve
the mechanical, thermal and chemical stresses that can be
associated with conventional encapsulation techniques currently
used, which conventional techniques may lead to denaturation of
sensitive bioactive anti-cancer agents such as proteins.
[0070] In another particular embodiment, the delivery vehicle is a
hydrogel, which can be used as a dressing, for sustained release
delivery of an anti-cancer agent, or as a support for tissue
regeneration.
[0071] The polymer is chosen to have good swellability
characteristics and to have appropriate groups available for
cross-linking of the polymer moieties, and to be non-toxic and
biocompatible, and in some embodiments to be biodegradable.
[0072] In a particular embodiment of the hydrogel, the polymer is
aldehyde derivatized hyaluronic acid, or a derivative of hyaluronic
acid such as hyaluronic acid aminoacetylaldehyde diethylacetal
conjugate, or a tyramine derivative of aldehyde-derivatized
hyaluronic acid or hyaluronic acid aminoacetylaldehyde
diethylacetal conjugate.
[0073] Conjugates comprising a hyaluronic acid-catechin-based
flavonoid can be readily cross-linked to form a hydrogel, without
disruption of the biological or pharmacological properties of the
flavonoid. Such hydrogels also comprise an anti-cancer agent as
described above, for release of the anti-cancer agent to a cell at
the site where the hydrogel is applied.
[0074] The hyaluronic acid-flavonoid conjugate is synthesized by
reacting the hyaluronic acid with the catechin-based flavonoid
under acidic conditions, for example from about 1 to about 5, or
for example at pH of about 1. The conjugated polymer-flavonoid is
then purified, for example by dialysis, and then mixed with the
anti-cancer agent and a cross-linking agent, such as hydrogen
peroxide. A cross-linking catalyst is added, for example
horseradish peroxidase, and the hydrogel may then be quickly poured
in to a mold to form a desired shape before the cross-linking
reaction is completed. For example, the hydrogel may be formed into
a slab suitable for application as a wound dressing.
[0075] The components of the hydrogel may also be injected and
reacted to form the hydrogel in vivo, for example by injecting an
uncrosslinked conjugate together with an anti-cancer agent,
together with a cross-linking agent, such as hydrogen peroxide and
a cross-linking catalyst, for example, horseradish peroxidase. Such
a hydrogel is useful for drug delivery to a specific site in a
body, or for tissue engineering.
[0076] Since hyaluronic acid has multiple sites that may react with
the flavonoid during the conjugation reaction, by varying the
concentration of the catechin-based flavonoid in the starting
reaction, it is possible to vary the degree of conjugation between
the hyaluronic acid polymer and the catechin-based flavonoid. For
example, the ratio of reactants may be adjusted so that the
resulting conjugate has from about 1% to about 10% of the sites on
the polymer conjugated with the flavonoid. Alternatively,
additional hyaluronic acid that has not been conjugated can be
added to the mixture prior to cross-linking of the hydrogel so that
some of the polymer molecules in the hydrogel will not be
conjugated to the flavonoid.
Delivery of Anti-Cancer Agent to a Cell
[0077] In order to deliver the anti-cancer agent to a cell using
the flavonoid-containing delivery vehicle, the delivery vehicle
comprising the anti-cancer agent is contacted with a cell, which
may allow for uptake of the anti-cancer agent into the cell.
[0078] Thus, delivery of the anti-cancer agent to a cell comprises
contacting the delivery vehicle containing the anti-cancer agent
with the surface of a cell. Without being limited to any particular
theory, the delivery system is dissociated by amphiphilic molecules
such as lipids of plasma membranes, and thus the anti-cancer agent
is released at the site of the cell by passive targeting, and may
be released into the cell.
[0079] The cell to which the anti-cancer agent is to be delivered
may be any cell, including an in vitro cell, a cell in culture, or
an in vivo cell within a subject. The term "cell" as used herein
refers to and includes a single cell, a plurality of cells or a
population of cells where context permits, unless otherwise
specified. The cell may be an in vitro cell including a cell
explanted from a subject or it may be an in vivo cell in a subject.
Similarly, reference to "cells" also includes reference to a single
cell where context permits, unless otherwise specified.
[0080] The cell may be derived from any organism, for example an
animal including a mammal including a human.
[0081] When delivered to the cell, the anti-cancer agent retains
its anti-cancer function, as described above, and may be delivered
in to the cell. A skilled person can readily determine whether the
anti-cancer agent has been delivered into the cell using known
methods and techniques, including protein detection methods,
immunoassays and fluorescence labelling techniques.
[0082] The above described compositions and delivery vehicles are
well-suited for controlled and targeted delivery of anti-cancer
agents together with catechin-based flavonoids to particular sites
within the body of a subject. The flavonoids can provide
antibacterial, antineoplastic, anti-thrombotic, vasodilatory,
antioxidant, antimutagenic, anti-carcinogenic,
hypercholesterolemic, antiviral and anti-inflammatory activity at
the targeted site. In addition, the delivery vehicles include an
anti-cancer agent, making the delivery vehicles useful in the
treatment of cancer. For example, immunoregulatory peptides and
proteins including cytokines and growth factors have emerged as an
important class of drugs for the treatment of cancer.
[0083] Thus, there is presently provided a method of delivering
anti-cancer agent to a subject comprising administering a delivery
vehicle comprising conjugate of a polymer containing a flee
aldehyde and a catechin-based flavonoid, having the polymer
conjugated at the C6 and/or the C8 position of the A ring of the
flavonoid is also contemplated, as described above. In certain
embodiments, the conjugate is formed into a delivery vehicle, such
as a micellar nanocomplex or a hydrogel, as described above.
[0084] The subject is any animal, including a human, in need of
treatment with an anti-cancer agent.
[0085] Therefore, there is provided a pharmaceutical composition
comprising a delivery vehicle containing the anti-cancer agent and
flavonoid as described above. The pharmaceutical composition may
further include a pharmaceutically acceptable diluent or carrier.
The pharmaceutical composition may routinely contain
pharmaceutically acceptable concentration of salts, buffering
agents, preservatives and various compatible carriers. For all
forms of delivery, the delivery vehicle may be formulated in a
physiological salt solution.
[0086] The proportion and identity of the pharmaceutically
acceptable diluent or carrier is determined by the chosen route of
administration, compatibility with biologically active proteins if
appropriate, and standard pharmaceutical practice.
[0087] The pharmaceutical composition can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions suitable for administration to subjects, such that an
effective amount of the delivery vehicle and any additional active
substance or substances is combined in a mixture with a
pharmaceutically acceptable vehicle. An effective amount of
delivery vehicle is administered to the subject. The term
"effective amount" as used herein means an amount effective, at
dosages and for periods of time necessary to achieve the desired
result, for example to deliver the anti-cancer agent to the target
cell or cell population within the subject, including a desired
amount of the anti-cancer agent to the cell based on factors
including the effect of the anti-cancer agent, the effect of the
flavonoid and the synergistic effect of the anti-cancer agent and
the flavonoid together.
[0088] Suitable vehicles are described, for example, in Remington's
Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., USA 1985). On this basis, the
compositions include, albeit not exclusively, solutions of the
delivery vehicle, in association with one or more pharmaceutically
acceptable vehicles or diluents, and contained in buffer solutions
with a suitable pH and iso-osmotic with physiological fluids.
[0089] Under ordinary conditions of storage and use, such
pharmaceutical compositions may contain a preservative to prevent
the growth of microorganisms, and that will maintain any biological
activity of the anti-cancer agent. A person skilled in the art
would know how to prepare suitable formulations. Conventional
procedures and ingredients for the selection and preparation of
suitable formulations are described, for example, in Remington's
Pharmaceutical Sciences and in The United States Pharmacopeia: The
National Formulary (USP 24 NF19) published in 1999. Alternatively,
the delivery vehicle may be formulated at a time sufficiently close
to use by mixing the components, without the need for
preservatives.
[0090] The delivery vehicle may be administered using known
methods, which will depend on the form of the delivery vehicle.
Non-oral routes are preferred. If the delivery vehicle is
formulated as a solution, or in the form of micellar nanoparticles,
the delivery vehicle may be delivered parenterally, including
intravenously, intramuscularly, or by direct injection into a
targeted tissue or organ. If the delivery vehicle is formulated as
a hydrogel, the conjugate may be applied topically or by surgical
insertion at a wound site.
[0091] When administered to a subject, the delivery vehicle is
administered in an amount effective and at the dosages and for
sufficient time period to achieve a desired result. For example,
the delivery vehicle may be administered in quantities and dosages
necessary to deliver an anti-cancer agent which may function to
alleviate, improve, mitigate, ameliorate, stabilize, prevent the
spread of, slow or delay the progression of or cure a disease or
disorder, or to inhibit, reduce or impair the activity of a
disease-related enzyme. A disease-related enzyme is an enzyme
involved in a metabolic or biochemical pathway, which when the
pathway is interrupted, or when regulatory control of the enzyme or
pathway is interrupted or inhibited, the activity of the enzyme is
involved in the onset or progression of a disease or disorder, for
example, cancer.
[0092] The effective amount of delivery vehicle to be administered
to a subject can vary depending on many factors such as the
pharmacodynamic properties of the delivery vehicle, including the
anti-cancer agent, the polymer moiety and the catechin-based
flavonoid moiety, the mode of administration, the age, health and
weight of the subject, the nature and extent of the disorder or
disease state, the frequency of the treatment and the type of
concurrent treatment, if any, and the concentration and form of the
delivery vehicle.
[0093] One of skill in the art can determine the appropriate amount
based on the above factors. The delivery vehicle may be
administered initially in a suitable amount that may be adjusted as
required, depending on the clinical response of the subject. The
effective amount of delivery vehicle can be determined empirically
and depends on the maximal amount of the delivery vehicle that can
be administered safely. However, the amount of delivery vehicle
administered should be the minimal amount that produces the desired
result.
[0094] There is also provided a delivery vehicle as described above
containing the anti-cancer agent and flavonoid.
[0095] There is also provided use of the above-described delivery
vehicle for delivering the anti-cancer agent to a cell, or use of
the above-described delivery vehicle for the manufacture of a
medicament for delivering the anti-cancer agent to a cell,
including when the cell is an in vivo cell in a subject.
[0096] The invention is further illustrated by the following
non-limiting example.
EXAMPLE
Example 1
Synergistic Therapeutic Effects of Protein-Loaded Micellar Complex
Composed of Green Tea Derivatives
[0097] Materials and Methods
[0098] Synthesis of OEGCG and PEG-EGCG:
[0099] OEGCG was synthesized by the Baeyer reaction of EGCG (Kurita
Ltd.) and acetaldehyde (pH 2). To synthesize PEG-EGCG, the
aldehyde-terminated PEG (Mw 5000, NOF Co.) and EGCG were reacted at
pH=2.
[0100] The micellar complexes were formed as previously described
in published international application WO 2006/124000 and published
US application 2008/102052.
[0101] Cytotoxicity Test:
[0102] Cytotoxicity of EGCG derivatives was examined with human
normal mammary epithelial cells (HMEC, Cambrex, USA), and compared
to that of intact EGCG. Cells were plated (1.times.10.sup.4 cells
in mammary epithelial growth medium/well) in quintuplicate and
octuplicate for samples and control, respectively, in 96-well
microplates, and allowed to adhere overnight. After the cells were
treated with different concentrations of EGCG, OEGCG or PEG-EGCG
for 2 days, the cell viability was estimated using Alamar Blue, a
dye that would be reduced by the cytochrome c activity of
cells.
[0103] Proteolysis Assessment:
[0104] Protein degradation was determined by monitoring the
fluorescence intensity increment in FITC-BSA when samples were
subjected to proteinase K (0.05 mg/ml), using a fluorescence
spectrophotometer (Hitachi, Japan, .lamda..sub.ex=490 nm and
.lamda..sub.em=530 nm). All measurements were run in
triplicate.
[0105] Cancer Cell Proliferation Assessment:
[0106] SKBR-3 cells (ATCC, HTB3O, USA) were plated
(1.times.10.sup.4 cells in McCoy's 5A medium/well) in quintuplicate
and octuplicate for samples and control, respectively, in 96-well
microplates and allowed to adhere overnight. The culture media were
then replaced by media containing the samples (OEGCG, PEG-EGCG,
Herceptin (0.5 mg/ml), Herceptin-loaded micellar complex, and
BSA-loaded micellar complex), and the cells were incubated at
37.degree. C. in 5% of CO.sub.2. At the time points indicated, the
culture media were replaced by the phenol red-free media containing
10% of Alamar Blue. Cell proliferation was determined from the
reduction in dye absorbance at 570 and 600 nm after 4 h of
incubation.
[0107] Results
[0108] The micellar complexes were synthesised by (i) two-step
self-assembly process involving assembly of oligomeric
(-)-epigallocatechin-3-O-gallate (OEGCG), including with the
relevant protein (fluorescent marker protein or anti-cancer agent)
where applicable, and then assembly of the pre-formed OEGCG (plus
protein) complex with a conjugate of poly(ethylene glycol) (PEG)
and EGCG (PEG-EGCG); or (ii) one-step self-assembly process
involving assembly of a conjugate of PEG and OEGCG (PEG-OEGCG) (and
the relevant protein where applicable). In both instances, the
micellar complexes immediately formed as stable and highly oriented
micellar complexes, including loaded with the relevant protein, via
spontaneous self-assembly in a mild aqueous solution and displayed
a PEG outer shell with an inner core comprised of OEGCG (plus
protein) complex.
[0109] The PEG-EGCG showed no cytotoxicity, while the OEGCG and
EGCG displayed low cytotoxicity for HMEC (see FIG. 1). The micellar
complex kept its integrity, and demonstrated good stability in the
presence of serum without size change (see FIG. 2).
[0110] The micellar complex was loaded with a fluorescence-labeled
protein (FITC-BSA) and subjected to a protease (proteinase K) to
investigate protein degradation over time (FIG. 3). The
fluorescence intensity of free FITC-BSA greatly increased with
time, indicating protein degradation by proteinase K. In contrast,
the fluorescence intensity of FITC-BSA loaded in the micellar
complexes was kept very low (even lower than that of free FITC-BSA
in the absence of proteinase K), illustrating that the protein was
robustly protected from proteolysis in these systems.
[0111] The inhibition of cancer cell growth was explored in vitro
for the micellar complex loaded with Herceptin (trastuzumab), which
is a humanized monoclonal antibody against the HER2/neu (erbB2)
receptor that induces regression of HER2-overexpressing metastatic
breast cancer tumors (FIG. 4). The Herceptin-loaded micellar
complex showed no size reduction, tested up to a thousand-fold
dilution (FIG. 5). When SKBR-3 (HER2-overexpressing human breast
cancer cell line) was treated with either free Herceptin or the
carrier components (OEGCG or PEG-EGCG), cell growth was observed to
be inhibited. The micellar complex loaded with BSA showed more
inhibition effect than OEGCG or PEG-EGCG alone (whereas BSA itself
showed no effect). Notably, the Herceptin-loaded micellar complex
showed an impressively greater inhibition effect, as compared to
the individual components delivered (OEGCG, PEG-EGCG or Herceptin)
and the BSA-loaded micellar complex.
[0112] Since the formation of micellar nanocomplex is mainly driven
by hydrophobic interaction, the complex would dissociate by
hydrophobic competition of the surfactants. The complex could
gradually dissociate and release components by interaction with
bio-amphiphilic molecules, such as lipids of cell membrane, while
the complex was retained with the cells. The micellar complex
showed a time lag before exhibiting the therapeutic effect (FIG.
6), i.e. no effect was displayed at early time points (e.g. at 10
h), whereas the free protein and the individual carrier components
started to exert anticancer effects immediately. This illustrated
the structural advantage of the micellar complex, which was loaded
with protein within a PEG outer shell. Besides the delayed release,
the micellar complex provided for sustained release of the
therapeutic molecules (proteins and carrier components) by
dissociation resulting from interaction with cells. These features
would be particularly useful and advantageous in light of the fact
that the carrier would need to travel from the point of
administration to the intended target sites before exerting the
therapeutic activities.
[0113] Discussion
[0114] A core-shell micellar nanocomplex carrier has been
synthesized with two EGCG derivatives that were designed to bind
with proteins in a spatially ordered structure. These complexes
formed micellar nanocomplexes through spontaneous self-assembly in
an aqueous solution (FIG. 1). The micellar nanocomplexes were
derived by two self-assembly processes: (i) via the complexation
between oligomerized EGCG (OEGCG) and proteins to form the core,
and (ii) via the complexation of poly(ethylene glycol)-EGCG
(PEG-EGCG) surrounding the pre-formed core to form the shell. The
resulting micellar nanocomplex displayed a PEG shell with a core of
OEGCG-protein complex. This structural feature can serve to reduce
protein immunogenicity and prevent rapid renal clearance and
proteolysis of protein by reticuloendothelial system uptake,
decreasing the need for frequent injections or infusion therapy.
The highly water-soluble shell of PEG and the tailored size
(<100 nm) may not only prolonged plasma half-life, but also
provide enhanced permeability and retention effect, resulting in
selective accumulation at tumors and sites of infection and
inflammation.
[0115] The affinity of EGCG for protein was utilized to load the
micellar complexes with an anticancer protein. The protein-loaded
complexes demonstrated a much greater anticancer effect than the
free protein or the carrier itself. This micellar system may offer
improved delivery of various biological molecules, so as to exploit
the advantages of the synergistic therapeutic effects and the
delivery effects associated with the versatile
flavonoid-derivatized carrier.
Example 2
Treatment of Balb/Nude Mice with Micellar Complexes Containing
Herceptin
[0116] Balb/nude mice were induced with tumours as follows. A
17.beta.-estradiol pellet (0.72 mg, 60 day-release) was
administered to each mouse by subcutaneous injection. The following
day, a suspension of BT474 cells (8.1.times.10.sup.6 cells/100 uL
of Matrigel) was injected subcutaneously in each mouse. Tumours
were allowed to develop for two weeks.
[0117] Two weeks following injection of the BT474 cells, the mice
were treated by intravenous injection, twice weekly for a one-month
period, with one of micellar nanocomplexes loaded with Herceptin,
Herceptin alone or PBS as a control. After the treatment regimen,
tumour size was assessed. The results are shown in FIG. 7.
[0118] As can be understood by one skilled in the art, many
modifications to the exemplary embodiments described herein are
possible. The invention is intended to encompass all such
modification within its scope, as defined by the claims.
[0119] All documents referred to herein are fully incorporated by
reference.
[0120] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. All technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art of this invention, unless defined otherwise.
REFERENCES
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