U.S. patent application number 16/687789 was filed with the patent office on 2020-03-12 for polymeric systems and uses thereof in theranostic applications.
This patent application is currently assigned to Ramot at Tel-Aviv University Ltd.. The applicant listed for this patent is Ramot at Tel-Aviv University Ltd.. Invention is credited to Hemda BAABUR-COHEN, Rachel BLAU, Yana EPSHTEIN, Shiran FERBER, Einat KISIN-FINFER, Orit REDY-KEISAR, Ronit SATCHI-FAINARO, Doron SHABAT.
Application Number | 20200078475 16/687789 |
Document ID | / |
Family ID | 54071045 |
Filed Date | 2020-03-12 |
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United States Patent
Application |
20200078475 |
Kind Code |
A1 |
SATCHI-FAINARO; Ronit ; et
al. |
March 12, 2020 |
POLYMERIC SYSTEMS AND USES THEREOF IN THERANOSTIC APPLICATIONS
Abstract
Polymeric systems useful for theranostic applications are
disclosed. The polymeric systems comprise a fluorescent or
fluorogenic moiety and a therapeutically active agent, each
attached to the same or different polymeric moiety. The polymeric
systems are designed such that a fluorescent signal is generated in
response to a chemical event, preferably upon contacting an analyte
(e.g., an enzyme) that is over-expressed in a diseased tissue or
organ. Probes useful for inclusion in such polymeric systems,
processes of preparing such probes and the polymeric systems, and
uses thereof in diagnostic and/or theranostic applications are also
disclosed.
Inventors: |
SATCHI-FAINARO; Ronit;
(Tel-Aviv, IL) ; SHABAT; Doron; (Tel-Aviv, IL)
; BLAU; Rachel; (Tel-Aviv, IL) ; EPSHTEIN;
Yana; (Tel-Aviv, IL) ; BAABUR-COHEN; Hemda;
(Tel-Aviv, IL) ; FERBER; Shiran; (Tel-Aviv,
IL) ; REDY-KEISAR; Orit; (Tel-Aviv, IL) ;
KISIN-FINFER; Einat; (Hod-HaSharon, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ramot at Tel-Aviv University Ltd. |
Tel-Aviv |
|
IL |
|
|
Assignee: |
Ramot at Tel-Aviv University
Ltd.
Tel-Aviv
IL
|
Family ID: |
54071045 |
Appl. No.: |
16/687789 |
Filed: |
November 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15124360 |
Sep 8, 2016 |
10532113 |
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PCT/IL2015/050269 |
Mar 13, 2015 |
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16687789 |
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61952259 |
Mar 13, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/593 20170801;
A61K 47/65 20170801; A61K 49/0032 20130101; A61K 49/0054 20130101;
A61K 31/337 20130101; A61K 47/58 20170801 |
International
Class: |
A61K 49/00 20060101
A61K049/00; A61K 31/337 20060101 A61K031/337; A61K 47/65 20060101
A61K047/65; A61K 47/59 20060101 A61K047/59; A61K 47/58 20060101
A61K047/58 |
Claims
1. A polymeric system comprising a first polymeric moiety
comprising a polymeric backbone composed of a plurality of backbone
units and having attached to at least a portion of said backbone
units a fluorogenic moiety, said fluorogenic moiety being attached
to said backbone units via a first cleavable linking moiety such
that upon cleavage of said linking moiety, a fluorescent signal is
generated, the system further comprising a therapeutically active
agent, such that: (i) said fluorogenic moiety is attached to one
portion of said backbone units and said therapeutically active
agent is attached to another portion of said backbone units; (ii)
said therapeutically active agent forms a part of said fluorogenic
moiety; (iii) said therapeutically active agent is attached to said
first cleavable linking moiety; or (iv) the system further
comprises a second polymeric moiety comprising a second polymeric
backbone composed of a plurality of backbone units and having
attached to at least a portion of said backbone units a
therapeutically active agent.
2. The polymeric system of claim 1, wherein upon said cleavage, a
fluorescent moiety is generated.
3. The polymeric system of claim 2, wherein said fluorescent moiety
is or comprises a cyanine dye.
4. The polymeric system of claim 1, wherein said first cleavable
linking moiety is a first biocleavable linking moiety.
5. The polymeric system of claim 1, wherein said first polymeric
moiety further comprises a quenching agent.
6. The polymeric system of claim 5, wherein said fluorogenic moiety
is attached to one portion of said backbone units and said
quenching agent is attached to another portion of said backbone
units.
7. The polymeric system of claim 1, wherein said first polymeric
moiety is represented by Formula IA: ##STR00014## wherein: A.sub.1,
A.sub.2 and A.sub.4 are backbone units forming said polymeric
backbone; F is said fluorogenic moiety; Q is a quenching agent;
L.sub.2 is said first cleavable lining moiety; S.sub.2 is a first
spacer, linking said fluorogenic moiety to L.sub.2, or is absent;
L.sub.4 is a cleavable or non-cleavable third linking moiety,
linking said quenching agent to respective backbone units, or is
absent; S.sub.4 is a third spacer linking said quenching agent to
said linking moiety L.sub.4, or is absent; w is an integer having a
value such that w/(x+s+w) multiplied by 100 is in the range of from
0 to 99.9; x is an integer having a value such that x/(x+s+w)
multiplied by 100 is in the range of from 0.1 to 100; and s is an
integer having a value such that s/(x+s+w) multiplied by 100 is in
the range of from 0 to 99.9, such that each
[A.sub.2-L.sub.2-S.sub.2-F] independently represents a backbone
unit having attached thereto the fluorogenic moiety; each
[A.sub.4-L.sub.4-S.sub.4-Q] independently represents a backbone
unit having attached thereto the quenching agent; and each of said
backbone units A.sub.1, A.sub.2 and A.sub.4 is independently a
terminal unit, attached to one other unit, or is attached to two
other units, which can be the same of different.
8. The polymeric system of claim 7, wherein s is 0.
9. The polymeric system of claim 7, wherein s is a positive
integer.
10. The polymeric system of claim 9, wherein a ratio of s to x
which is in a range of from 20:1 to 1:20, or from 10:1:10, or from
5:1 to 1:5, or from 2:1 to 1:2, or is 1:1.
11. The polymeric system of claim 5, wherein said quenching agent
forms a part of said fluorogenic moiety.
12. The polymeric system of claim 10, wherein said fluorogenic
moiety comprises a fluorescent moiety linked by said first
cleavable linking moiety or by a degradable spacer to said
quenching agent.
13. The polymeric system of claim 12, wherein said fluorogenic
moiety is represented by formulae III or III*: ##STR00015##
wherein: the curled line indicates an attachment point to said
first cleavable linking moiety, or to a spacer that is linked to
said first linking moiety; F* is said fluorescent moiety; Q is said
quenching agent; S' is a spacer, or is absent; S''' is a spacer, or
is absent; and S'' is a multifunctional spacer which connects said
fluorogenic moiety to said first cleavable moiety, or to an
additional spacer which is connected to said cleavable linking
moiety.
14. The polymeric system of claim 13, wherein at least S'' in
Formula III is a degradable spacer.
15. The polymeric system of claim 13, wherein at least S'' and S'
in Formula III* is a degradable spacer.
16. The polymeric system of claim 1, wherein said fluorogenic
moiety is attached to one portion of said backbone units and said
therapeutically active agent is attached to another portion of said
backbone units, and wherein said therapeutically active agent is
attached to said backbone units via a second cleavable linking
moiety.
17. The polymeric system of claim 16, being represented by Formula
I: ##STR00016## wherein: A.sub.1, A.sub.2, A.sub.3 and A.sub.4 are
each backbone units covalently linked to one another and forming
said polymeric backbone; D is said therapeutically active agent; F
is said fluorogenic moiety; Q is said quenching agent; L.sub.2 is
said first linking moiety; L.sub.3 is said second linking moiety or
absent; L.sub.4 is a linking moiety linking said quenching agent,
or is absent; each of S.sub.2, S.sub.3 and S.sub.4 is independently
a spacer or absent; w is an integer having a value such that
w/(x+y+w+s) multiplied by 100 is in the range of from 0 to 99.9; x
is an integer having a value such that x/(x+y+w+s) multiplied by
100 is in the range of from 0.1 to 100; y is an integer having a
value such that y/(x+y+w+s) multiplied by 100 is in the range of
from 0 to 99.9; and s is an integer having a value such that
s/(x+y+w+s) multiplied by 100 is in the range of from 0 to 99.9,
such that each [A.sub.3-L.sub.3-S.sub.3-D] independently represents
a backbone unit having attached thereto said therapeutically active
agent; each [A.sub.2-L.sub.2-S.sub.2-F] independently represents a
backbone unit having attached thereto said fluorogenic moiety; and
each [A.sub.4-L.sub.4-S.sub.4-Q] independently represents a
backbone unit having attached thereto said quenching agent, wherein
when s is 0, said quenching agent forms a part of said fluorogenic
moiety, and when y is 0, said therapeutically active agent forms a
part of said fluorogenic moiety.
18. The polymeric system of claim 16, wherein said second linking
moiety is a biocleavable linking moiety.
19. The polymeric system of claim 1, wherein the system further
comprises a second polymeric moiety comprising a second polymeric
backbone composed of a plurality of backbone units and having
attached to at least a portion of said backbone units a
therapeutically active agent, and wherein said therapeutically
active agent is attached to said backbone units via a second
cleavable linking moiety.
20. The polymeric system of claim 19, wherein said second linking
moiety is a biocleavable linking moiety.
21. The polymeric system of claim 1, wherein said therapeutically
active agent forms a part of said fluorogenic moiety, or is
attached to said first cleavable linking moiety, and wherein upon
said cleavage, said therapeutically active agent is released.
22. The polymeric system of claim 21, wherein upon said cleavage, a
fluorescent moiety is generated.
23. The polymeric system of claim 22, wherein said fluorescent
moiety is or comprises a cyanine dye.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 15/124,360 filed on Sep. 8, 2016, which is a National
Phase of PCT Patent Application No. PCT/IL2015/050269 having
International Filing Date of Mar. 13, 2015, which claims the
benefit of priority under 35 USC .sctn. 119(e) of U.S. Provisional
Patent Application No. 61/952,259 filed on Mar. 13, 2014. The
contents of the above applications are all incorporated by
reference as if fully set forth herein in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention, in some embodiments thereof, relates
to therapy and diagnosis (theranostic) and, more particularly, but
not exclusively, to polymeric systems in which a labeling moiety
(e.g., a fluorescent or fluorogenic moiety) or a labeling moiety
and a therapeutically active agents are attached to a polymeric
backbone, and to uses thereof in diagnostic and theranostic
applications.
[0003] In the past few years, tremendous efforts have been employed
in monitoring cancer treatment, detecting response to drugs and
measuring real-time accumulation of the drug within the tumor.
Numerous nanocarrier systems have been developed (e.g., polymers,
liposomes, micelles, dendrimers, etc.) and studied as delivery
vehicles for anticancer drugs to improve the drugs'
biodistribution, solubility, and half-life, and thus to exhibit
enhanced efficacy and reduced toxicity. Clinically-available
fluorescence-based imaging contrast agents (e.g., indocyanine green
and fluorescein) hold many of the limitations attributed to
chemotherapeutic agents, including low molecular weight, short
half-life and poor selectivity. Consequently, monitoring slow
processes, such as drug accumulation at the tumor site, is
challenging.
[0004] Combining therapeutic and diagnostic modalities on the same
delivery system, thereby forming a theranostic (therapy and
diagnostic) nanomedicine, may overcome these limitations, while
enabling simultaneous monitor and treatment of
angiogenesis-dependent diseases, like cancer [Kelkar, S. S. and T.
M. Reineke, Theranostics: Combining Imaging and Therapy. Bioconjug
Chem, 2011. 22(10): p. 1879-1903]. Information obtained from
theranostic nanomedicine is exploited for fine tuning the
therapeutic dose, while monitoring the progression of the diseased
tissue, treatment efficacy and delivery kinetics [Janib et al. Adv
Drug Deliv Rev, 2010. 62(11): p. 1052-1063; McCarthy, J. R., The
future of theranostic nanoagents. Nanomedicine, 2009. 4(7): p.
693-695]. This, from a clinical prospective, should enhance early
diagnosis and treatment and may decrease drugs under- or
over-dosing, resulting in a more personalized treatment.
[0005] Among different imaging modalities (e.g., radiography,
magnetic resonance imaging and ultrasound), optical imaging holds
several advantages. Fluorescent molecular probes are highly
sensitive, possess a high spatial resolution, enable simultaneous
multicolor imaging and specificity, by signal activation in the
tissue of interest, they may possess high target to background
ratio (TBR), and are relatively inexpensive. Furthermore, they do
not hold long term health risks, like other commonly-used computed
tomography (e.g., PET-positron emission tomography and
SPECT-single-photon emission computed tomography), which expose the
patient to ionizing radiation.
[0006] An ideal theranostic nanomedicine system should hold (i)
long circulation time in the body, (ii) high specificity to the
target tissue, (iii) an efficient release mechanism, (iv) an
imaging probe that enables monitoring its activity, (v) deep tissue
penetration, and (vi) high target-to-background (TBR) ratio. High
specificity can be obtained via passive targeting, by exploiting
the enhanced permeability and retention (EPR) effect or via an
additional functional targeting moiety.
[0007] In contrast to thin layer imaging of cells or surfaces, the
signal from fluorescent probes in vivo is impeded by the emitted
fluorescence from tissues and biomolecules (e.g., water, melanin,
proteins and hemoglobin), which absorb photons in the wavelengths
range of 200-650 nm (i.e., low signal-to-noise ratio). In addition,
tissues contribute to reflection, refraction and scattering of
incident photons, thus increasing the background and blur of the
obtained image. The `imaging wavelength window` left for intravital
imaging in order to overcome these obstacles is at the near
infra-red (NIR) range (i.e., 650-1450 nm). In this range,
auto-fluorescence is minimal and scattering of light is reduced,
enabling deep tissue penetration and facilitating non-invasive
monitoring.
[0008] One way to maximize the signal from the target and to
minimize the signal from background (i.e., high TBR ratio), is the
use of activatable optical probes. The fluorescent signal is
silenced/"OFF" under physiological conditions, and is turned-ON at
a designated site and/or under specific conditions [Lee et al.,
Activatable molecular probes for cancer imaging. Vol. 10. 2010.
1135-44].
[0009] Although numerous classes of Turn-ON optical probes have
been described in the literature for detection of chemical and
biological factors [Karton-Lifshin, N., et al., J Am Chem Soc,
2011. 133(28): p. 10960-5; Kobayashi, H., et al., Chem Rev, 2010.
110(5): p. 2620-40; Lee, S., et al., Chem Commun (Camb), 2008(36):
p. 4250-60; Redy-Keisar, O., et al., Nat Protoc, 2014. 9(1): p.
27-36; Weinstain, R., et al., Chem Commun (Camb), 2010. 46(4): p.
553-5], to this point, most polymer-based theranostic nanomedicines
studies utilize an `always ON` theranostic systems. In these
systems, a fluorescent signal is obtained from the background and
desired site at once, resulting in low TBR.
[0010] Among methods used to obtain a selective Turn-ON mechanism,
Forster resonance energy transfer (FRET) is the most common and
efficient. Using FRET technique to monitor drug release, two types
of fluorophores are incorporated into the core of drug-carrying
nanoparticles and serve as energy donors and acceptors. In this
process, following excitation of the donor, the acceptor will
absorb the emission energy of the donor and will turn off the
fluorescent signal. The donor and the acceptor are required to have
overlapping emission and absorbance spectra, as well as close
proximity between them. A FRET-based probe is turned-ON upon
distance that results in the diffusion of the donor fluorophore
away from the acceptor, and generation of a measurable fluorescent
signal [Lee et al. 2010 supra; Johansson, M. K., et al., Journal of
the American Chemical Society, 2002. 124(24): p. 6950-6956]. This
process includes two approaches, fluorophore-fluorophore
(self-quenching) and fluorophore-quencher activation. The donor is
always a fluorophore, however the acceptor can be either a
quencher--a dye with no native fluorescence (FRET) or a second
fluorophore (self-quenching) [Redy, O., et al., Org Biomol Chem,
2012. 10(4): p. 710-5].
[0011] In the fluorophore-fluorophore (self-quenching) approach,
excited fluorophores of similar type absorb the energy from each
other that would otherwise have led to an emitted photon, thus
reducing the fluorescence of the entire compound. This can occur
when the excitation and emission peaks overlap or when the Stokes
shift is small, like in the case of Cy5. Hence, the fluorophore can
serve as a quencher and adsorb the excitation energy. Under these
circumstances the emitted energy from one fluorophore is absorbed
by another fluorophore (self-quenching) [Melancon, M. P., et al.,
Pharm Res, 2007. 24(6): p. 1217-24].
[0012] Self-quenching involving only fluorophores may still yield
weak fluorescence even in the quenched state. A second alternative
to fluorophore-fluorophore quenching, is to use a
fluorophore-quencher combinations in which the quencher is
non-fluorescent and plays as the acceptor, whereas the donor is a
fluorophore. When a FRET fluorophore-quencher process occurs, the
excited fluorophore can transfer its emission energy to the nearby
quencher [Redy, O., et al., Org Biomol Chem, 2012. 10(4): p.
710-5].
[0013] Optical imaging in the near-infrared (NIR) range enables
detection of molecular activity in vivo due to high penetration of
NIR photons through organic tissues and low auto-fluorescence
background. Cyanine dyes are widely employed as fluorescence labels
for NIR imaging, since they are compounds with large extinction
coefficient and relatively high quantum yield.
[0014] In order to generate a Turn-ON system for a cyanine
molecule, a FRET (fluorescence resonance energy transfer) approach
is usually applied. In such approach, the cyanine dye and a
quencher are attached through a specific linker to obtain a
quenched fluorophore. A linker, which is cleaved by a specific
enzyme, separates the fluorophore from the quencher and thus,
turn-ON its fluorescence signal. Exemplary such FRET-based probes
are described in Redy, O., et al., Org Biomol Chem, 2012. 10(4): p.
710-5, which is incorporated by reference as if fully set forth
herein. An alternative approach, to turn OFF and ON a fluorophore,
could be achieved by disrupting the pull-push conjugated
.pi.-electron system of the dye. Such a concept, referred to as
Internal Charge Transfer (ICT) probe, is described in WO
2012/123916, which is incorporated by reference as if fully set
forth herein, and in Kisin-Finfer E., et al., 1; 24(11):2453-8;
Bioorg Med Chem Lett. 2014, which is also incorporated by reference
as if fully set forth herein.
[0015] Additional background art includes Jones et al. Langmuir,
2001, 17 (9), pp 2568-2571; U.S. Patent Application Publication No.
20120122734; Theodora Krasia-Christoforou and Theoni K. Georgiou,
J. Mater. Chem. B, 2013, 1, 3002-3025; Morton et al., Biomaterials.
2014 April; 35(11): 3489-3496; and Luk and Zhang, Appl. Mater.
Interfaces 2014, 6, 21859-21873.
SUMMARY OF THE INVENTION
[0016] Although polymeric nanocarriers conjugated to low molecular
weight drugs greatly improve their efficacy and toxicity profile,
these nanocarriers lack information concerning drug-release time
and location. Combining therapeutic and diagnostic modalities on
the same delivery system, thereby forming theranostic (therapy and
diagnostic) nanomedicine, enables simultaneous monitor and
treatment of angiogenesis-dependent diseases, like cancer.
Information obtained from theranostic nanomedicines allows tuning
therapy dose, while monitoring diseased tissue and delivery
kinetics. This, from a clinical prospective, may increase early
detection of disease and decrease drug under-dosing or over-dosing,
resulting in a more personalized treatment.
[0017] The present inventors have now designed various theranostic
systems, which are based on a polymeric system in which a
fluorogenic moiety is attached to a portion of the backbone units
composing the polymeric backbone of a polymeric moiety, wherein the
fluorogenic moiety is attached to the backbone units via a
cleavable linking such that upon cleavage of the linking moiety, a
fluorescent moiety is generated, and a detectable signal can be
measured.
[0018] According to an aspect of some embodiments of the present
invention there is provided a polymeric system comprising a first
polymeric moiety comprising a polymeric backbone composed of a
plurality of backbone units and having attached to at least a
portion of the backbone units a fluorogenic moiety, the fluorogenic
moiety being attached to the backbone units via a first cleavable
linking moiety such that upon cleavage of the linking moiety, a
fluorescent signal is generated, the system further comprising a
therapeutically active agent, such that: (i) the fluorogenic moiety
is attached to one portion of the backbone units and the
therapeutically active agent is attached to another portion of the
backbone units; (ii) the therapeutically active agent forms a part
of the fluorogenic moiety; (iii) the therapeutically active agent
is attached to the first cleavable linking moiety; or (iv) the
system further comprises a second polymeric moiety comprising a
second polymeric backbone composed of a plurality of backbone units
and having attached to at least a portion of the backbone units a
therapeutically active agent.
[0019] According to some of any of the embodiments described
herein, upon the cleavage, a fluorescent moiety is generated.
[0020] According to some of any of the embodiments described
herein, the fluorescent moiety emits UV-vis light.
[0021] According to some of any of the embodiments described
herein, the fluorescent moiety emits near infrared light.
[0022] According to some of any of the embodiments described
herein, the fluorescent moiety is or comprises a cyanine dye.
[0023] According to some of any of the embodiments described
herein, the first cleavable linking moiety is a first biocleavable
linking moiety.
[0024] According to some of any of the embodiments described
herein, the first cleavable linking moiety is an
enzymatically-cleavable linking moiety.
[0025] According to some of any of the embodiments described
herein, the first polymeric moiety further comprises a quenching
agent.
[0026] According to some of any of the embodiments described
herein, the fluorogenic agent is attached to one portion of the
backbone units and the quenching agent is attached to another
portion of the backbone units.
[0027] According to some of any of the embodiments described
herein, the quenching agent forms a part of the fluorogenic
moiety.
[0028] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by, or comprises a
moiety represented by, formula II:
##STR00001##
wherein:
[0029] Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heterocylic moiety;
[0030] R.sub.1 is hydrogen, a substituted or unsubstituted alkyl or
a substituted or unsubstituted cycloalkyl;
[0031] n is an integer of from 1 to 10; and
[0032] R' and R'' are each independently hydrogen, a substituted or
unsubstituted alkyl and a substituted or unsubstituted cycloalkyl,
or, alternatively, R' and R'' form together an aryl.
[0033] According to some of any of the embodiments described
herein, Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heteroaryl.
[0034] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by, or comprises a
moiety represented by, formula IIA or IIB, as depicted herein.
[0035] According to some of any of the embodiments described
herein, the fluorogenic moiety comprises a fluorescent moiety
linked by the first cleavable linking moiety or by a degradable
spacer to the quenching agent.
[0036] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by a formula selected
from Formula IIIA, IIIB, IIIC, and IIID, as depicted herein.
[0037] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by Formula IV, as
depicted herein.
[0038] According to some of any of the embodiments described
herein, the fluorogenic moiety is attached to one portion of the
backbone units and the therapeutically active agent is attached to
another portion of the backbone units.
[0039] According to some of any of the embodiments described
herein, the system further comprises a second polymeric moiety
comprising a second polymeric backbone composed of a plurality of
backbone units and having attached to at least a portion of the
backbone units a therapeutically active agent.
[0040] According to some of any of the embodiments described
herein, the therapeutically active agent is attached to the
backbone units via a second cleavable linking moiety.
[0041] According to some of any of the embodiments described
herein, the second linking moiety is a biocleavable linking
moiety.
[0042] According to some of any of the embodiments described
herein, the second linking moiety is an enzymatically-cleavable
linking moiety.
[0043] According to some of any of the embodiments described
herein, the first and second cleavable linking moieties are the
same or are cleavable by the same mechanism (e.g., the same
enzyme).
[0044] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, or is attached to the first cleavable linking
moiety, and wherein upon the cleavage, the therapeutically active
agent is released. According to some embodiments, upon the
cleavage, a fluorescent moiety is generated.
[0045] According to some of any of the embodiments described
herein, the fluorescent moiety is or comprises a cyanine dye.
[0046] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, and the fluorogenic moiety is represented by
Formula VIA, VIB, VIC, or VID, as depicted herein.
[0047] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, and the fluorogenic moiety is represented by
Formula IIIA, IIIB, IIIC or IIID, and wherein the therapeutically
active agent is attached to one of the spacers or to the cleavable
linking moiety.
[0048] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, and the fluorogenic moiety is represented by
Formula IV, wherein the therapeutically active is attached to the
donor moiety or to the cleavable linking moiety.
[0049] According to some of any of the embodiments described
herein, the backbone units in the first polymeric backbone and/or
in the second polymeric backbone, if present, form a polymeric
backbone of HPMA co-polymer.
[0050] According to some of any of the embodiments described
herein, the backbone units in the first polymeric backbone and/or
in the second polymeric backbone, if present, form a polymeric
backbone of a PGA polymer.
[0051] According to an aspect of some embodiments of the present
invention there is provided a polymeric conjugate comprising a
polymeric backbone composed of a plurality of backbone units and
having attached to at least a portion of the backbone units a
fluorogenic moiety, the fluorogenic moiety being attached to the
portion of backbone units via a cleavable linking moiety such that
upon cleavage of the linking moiety, a fluorescent moiety is
generated, wherein the fluorescent moiety is a cyanine dye.
[0052] According to some of any of the embodiments described
herein, the polymeric conjugate further comprises a quenching agent
attached to the polymeric backbone.
[0053] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by, or comprises a
moiety represented by, formula II, as depicted herein.
[0054] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by, or comprises a
moiety represented by, formula IIA or IIB, as depicted herein.
[0055] According to some of any of the embodiments described
herein, the fluorogenic moiety comprises a fluorescent moiety
linked by a cleavable linking moiety and/or a degradable spacer to
a quenching agent.
[0056] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by a formula selected
from Formula IIIA, IIIB, IIIC and IIID as depicted herein.
[0057] According to some of any of the embodiments described
herein, the fluorogenic moiety is represented by Formula IV, as
depicted herein.
[0058] According to some of any of the embodiments described
herein, the polymeric conjugate further comprises a therapeutically
active agent attached to the cleavable linking moiety or forming a
part of the fluorogenic moiety, such that upon the cleavage, the
therapeutically active agent is released.
[0059] According to an aspect of some embodiments of the present
invention there is provided a polymeric system comprising a
fluorogenic cyanine moiety covalently attached via a cleavable
linking moiety to a quenching agent, such that upon cleavage of the
linking moiety, a fluorescent cyanine moiety is generated, the
system further comprising a polymeric moiety attached to the
fluorogenic cyanine moiety.
[0060] According to some of any of the embodiments described
herein, the polymeric system is represented by a formula selected
from Formula VA or VB, as depicted herein.
[0061] According to some of any of the embodiments described
herein, the cyanine moiety is attached to the polymeric moiety via
a spacer.
[0062] According to some of any of the embodiments described
herein, the polymeric system further comprises a therapeutically
active agent, wherein:
[0063] (i) the therapeutically active agent is attached to the
cleavable linking moiety;
[0064] (ii) the therapeutically active agent is attached to the
degradable spacer; or
[0065] (iii) the therapeutically active agent is attached to a
second polymeric moiety.
[0066] According to an aspect of some embodiments of the present
invention there is provided a polymeric system as described in any
one of the embodiments described herein, where the system comprises
a therapeutically active agent, for use in the treatment and
diagnosis of a medical condition treatable by the therapeutically
active agent, or for use in the preparation of a medicament for
treating the medical condition.
[0067] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition, the method comprising administering to a subject in need
thereof a polymeric system as described herein, which comprises a
therapeutically active agent that is usable in treating the medical
condition.
[0068] According to some of any of the embodiments described
herein, the medical condition is cancer.
[0069] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0070] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0071] Some embodiments of the invention are herein described, by
way of example only, with reference to the accompanying drawings.
With specific reference now to the drawings in detail, it is
stressed that the particulars shown are by way of example and for
purposes of illustrative discussion of embodiments of the
invention. In this regard, the description taken with the drawings
makes apparent to those skilled in the art how embodiments of the
invention may be practiced.
[0072] In the drawings:
[0073] FIGS. 1A-1E present the chemical structure and cleavage
mechanism by Cathepsin B of an exemplary HPMA copolymer-Cy5 (FIG.
1A); an exemplary HPMA copolymer-PTX and a release mechanism of PTX
therefrom by cathepsin B (FIG. 1B); an exemplary HPMA
copolymer-PTX-FK and a release mechanism of PTX therefrom by
cathepsin B (FIG. 1C); an exemplary HPMA copolymer-Cy5-PTX (FIG.
1D), and an exemplary HPMA copolymer-Cy5-PTX-FK, according to some
embodiments of the present invention.
[0074] FIG. 2 is a scheme depicting a two-step synthesis of
HPMA-GFLG-en 10% copolymer-Cy5 conjugate, carried out by activation
of Cy5 with NHS group, followed by coupling with HPMA
copolymer.
[0075] FIG. 3 is a scheme depicting a synthesis of HPMA
copolymer-PTX conjugate, carried out by activation of PTX with
PNp-C1, followed by conjugation of HPMA to PTX, by mixing activated
PTX with HPMA-GFLG-en 10 mol % copolymer.
[0076] FIG. 4 is a scheme depicting a synthesis of HPMA
copolymer-PTX-FK conjugate, carried out by forming a Phe-Lys-PABC
linker and conjugating the linker to PTX, followed by coupling the
PTX-Phe-Lys with HPMA copolymer.
[0077] FIG. 5 is a scheme depicting a synthesis of HPMA
copolymer-Cy5-PTX conjugate, carried out by conjugating of Cy5 to
HPMA copolymer, and activation of PTX with 4-Nitrophenyl, followed
by its conjugation to the HPMA copolymer-Cy5 so as to generate HPMA
copolymer-Cy5-PTX.
[0078] FIG. 6 is a scheme depicting a synthesis of HPMA
copolymer-Cy5-PTX-FK conjugate, carried out by conjugating Cy5 to
HPMA copolymer, forming a FK-PABC linker and conjugating the linker
to PTX, followed by coupling the PTX-Phe-Lys to the HPMA
copolymer-Cy5-PTX-FK so as to generate HPMA copolymer-Cy5-PTX-FK;
DCM=dichloromethane, DMF=N,N-dimethylformamide, TFA=trifluoroacetic
acid.
[0079] FIGS. 7A-7D present a scheme depicting a synthesis of a PGA
polymer, carried out by hexylamine-initiated polymerization (FIG.
7B) of the N-carboxyanhydride (NCA) of y-benzyl-L-glutamate (FIG.
7A), followed by deprotection (FIG. 7C); and of a PGA-PTX
conjugate, carried out by activation of PGA with CDI coupling
reagent supported by DMAP as a catalyst in basic environment and
conjugation of PGA to PTX, by mixing activated polymer with PTX
(FIG. 7D).
[0080] FIGS. 8A-8B present schemes depicting a synthesis of
PGA-PTX-Cy5 conjugate, carried out by removing the BOC protecting
group of a Cy5-NH.sub.2-BOC (FIG. 8A); and conjugating the obtained
Cy5-NH.sub.2 to a PGA-PTX conjugate, by mixing an activated PGA
with the Cy5-NH.sub.2 (FIG. 8B).
[0081] FIGS. 9A-9B present graphs showing PTX release kinetics from
HPMA copolymer-PTX-FK conjugate upon incubation in the absence
(diamonds) or presence (squares) of cathepsin B [1 Unit/ml] in
phosphate buffer (pH 6) (FIG. 9A); and PTX release kinetics from
HPMA copolymer-PTX conjugate upon incubation in the presence of
cathepsin B [1 Unit/ml] (diamonds) (FIG. 9B).
[0082] FIGS. 10A-10D present graphs showing the anti-proliferative
activity of PTX and HPMA copolymer-PTX conjugate in murine 4T1
cells (FIG. 10A) and in HUVEC cells (FIG. 10B), upon incubation for
96 hours; the anti-proliferative activity of HPMA copolymer-PTX-FK
conjugate in human MDA-MB-231 mammary adenocarcinoma cells, upon
incubation for 72 hours (FIG. 10C), and the IC50 values obtained in
these assays (FIG. 10D).
[0083] FIGS. 11A-11C present comparative plots showing the
self-quenching capability of an HPMA copolymer-Cy5 conjugate (3.8
mol % loading) (blank diamonds) and of free Cy5 (squares) (FIG.
11A); Comparative plots showing the changes in fluorescence
intensity (.lamda..sub.Ex=600 nm, .lamda..sub.Em=670 nm) emitted
upon incubation of HPMA copolymer-Cy5 conjugate [0.01 mM] in the
presence (blank diamonds) of cathepsin B [1 Units/ml] in Phosphate
buffer (pH 6) and in the absence of cathepsin B in PBS (pH 7.4)
(squares), with data acquired throughout 160 hours following enzyme
addition at 37.degree. C. (FIG. 11B); and a bar graph showing the
in vitro degradation of HPMA copolymer-Cy5 (gray bars) in cultured
MDA-MB-231 cells, compared to non-treated cells (white bars), as
measured by activation of a fluorescence signal (The data represent
mean SD (n=3); *p<0.05, **p<0.01) (FIG. 11C).
[0084] FIGS. 12A-12B present graphs showing quantification of the
flourescence signal following intra-tumoral injection of free Cy5
[0.1 mM; 30 .mu.l] (blank diamonds) and equivalent dose of HPMA
copolymer-Cy5 conjugate (squares) into subcutaneous 4T1 mammary
adenocarcinoma (FIG. 12A) and images showing that the fluorescence
signal of HPMA copolymer-Cy5 conjugate is maintained 8 hours
following injection, while free Cy5 exhibits 80% bleach already
within 3 hours [Images were acquired and quantified using CRI
Maestro.TM. imaging system; Filter set: excitation--635 nm,
emission cutoff--675 nm] (FIG. 12B).
[0085] FIGS. 13A-13C present an image (FIG. 13A) and a bar graph
(FIG. 13B) showing the fluorescent signal and tumor/background
ratio of HPMA copolymer-Cy5 conjugate in a 4T1 tumor, upon
administering the conjugate (10 .mu.M; 200 .mu.l) via the tail vein
of mice, as monitored using CRI Maestro.TM. imaging system; and a
bar graph (FIG. 13C) showing the Cy5-fluorescent spectrum (composed
images of unmixed multispectral cubes) in resected organs of mice
bearing 4T1 tumors treated with HPMA copolymer-SQ-Cy5 conjugate (10
.mu.M; 200 .mu.l), demonstrating greater intensity of
Cy5-fluorescence spectrum in tumor tissue, liver and kidneys
compared with other organs.
[0086] FIGS. 14A-14C present graphs showing the emitted
fluorescence intensity (.lamda..sub.Ex=650 nm) by HPMA
copolymer-SQ-Cy5-PTX conjugate (FIG. 14A) and HPMA
copolymer-SQ-Cy5-PTX-FK conjugate (FIG. 14B) as measured using
SpectraMax.RTM. M5.sup.e plate reader, upon incubation of the
conjugates [0.01 mM] in the presence or absence of cathepsin B [1
Units/ml] in Phosphate buffer (pH 6); Data was acquired throughout
48 hours following enzyme addition at 37.degree. C.; and
comparative plots showing the emission of HPMA-PTX-Cy5 conjugate
loaded with 5.38 mol % Cy5 and HPMA-PTX-FK-Cy5 loaded with 1.95 mol
% Cy5 compared to free Cy5 emission (.lamda..sub.ex=650 nm,
.lamda..sub.em=670 nm) with a similar equivalent concentration of
Cy5 (13-19 .mu.M)(FIG. 14C).
[0087] FIGS. 15A-15D present comparative plots showing: the
absorption spectrum of PGA-PTX-Cy5 (red) compared to a free Cy5
(blue) (FIG. 15A); the emission spectrum (.lamda..sub.ex=650 nm,
.lamda..sub.em=670 nm) of PGA-PTX-Cy5 conjugate loaded with 4 mol %
Cy5 (red), PGA-PTX-Cy5 loaded with 7.5 mol % Cy5 (green) and of
free Cy5 (blue) (FIG. 15B); the emitted fluorescence
(.lamda..sub.ex=650 nm, .lamda..sub.em=670 nm) following enzymatic
release of Cy5 from the PGA-PTX-SQ-Cy5 conjugate upon incubation in
the presence (black) and in the absence (dashed gray) of cathepsin
B enzyme [1 Units/ml] as a function of time (FIG. 15C); and the PTX
release kinetics from PGA-PTX-Cy5 conjugate upon incubation in the
presence of cathepsin B enzyme [1 Units/ml] as a function of time
(FIG. 15D).
[0088] FIGS. 16A-16D present comparative plots showing the
anti-proliferative activity of free PTX, PGA-PTX and PGA-PTX-Cy5
conjugate, free PTX, PGA-PTX and PGA-PTX-Cy5 in human MDA-MB-231
mammary adenocarcinoma cell line (FIG. 16A), murine 4T1 cell line
(FIG. 16B) and human WM239A melanoma cell line (FIG. 16C), upon
incubating the cells with the tested agent for 72 hours; and the
IC50 values obtained in these assays (FIG. 16D) [Data represents
mean.+-.SD. The X-axis is presented at a logarithmic scale].
[0089] FIG. 17 presents a bar graph demonstrating the inhibition of
the migration of HUVECs by a PGA-PTX-Cy5 conjugate, compared to
free PTX, PGA-PTX, PGA-PTX-Cy5 and control (non-treated HUVECs).
Migration was normalized to percent migration with 100%
representing migration control [Data represents mean.+-.SD, (***
p<0.005)].
[0090] FIGS. 18A-18B present representative images showing the
effect of free PTX, PGA-PTX, PGA-PTX-Cy5, PGA and Cy5-amine,
compared to control (untreated), on capillary-like tube structures
formation of HUVEC, following incubation (FIG. 18A); and a bar
graph showing a quantitative analysis of the mean length of
capillary tubes following incubation (Data represents mean
displayed as % of control .+-.SD; * p<0.05; ** p<0.01; ***
p<0.005).
[0091] FIGS. 19A-19B present schemes depicting the Chemical
syntheses of MA-Gly-Gly-diamine-Boc monomer (FIG. 19A) and
MA-Gly-Phe-Leu-Gly-PABA monomer (FIG. 19B);
NHS=N-hydroxy-succinimide, DCC=dicyclohexyl carbodiimide,
DMF=N,N-dimethylformamide, NMM=N-methylmorpholine,
THF=tetrahydrofuran.
[0092] FIG. 20 is a scheme depicting a two-step synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-PTX, carried out by RAFT
polymerization of the copolymer precursor, followed by coupling
with PTX, as an exemplary drug and Cy5, as an exemplary fluorogenic
moiety.
[0093] FIG. 21 is a scheme depicting a two-step synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-PTX, carried out by RAFT
polymerization of the copolymer precursor, followed by coupling
with PTX, as an exemplary drug, and FITC, as an exemplary
fluorescent moiety.
[0094] FIGS. 22A-22B present schemes depicting the syntheses of
exemplary drug and dye dipeptide-PABC moieties: Boc-NH-LG-PABC-PTX,
Boc-NH-LG-PABC-Cy5 and Boc-NH-LG-PABC-FITC (FIG. 22A), and
ivDde-NH--FK-PABC-PTX and ivDde-NH--FK-PABC-Cy5 (FIG. 22B), useful
for further conjugation to HPMA copolymer-dipeptide-ONp
(Gly-Gly-ONp).
[0095] FIG. 23 presents an illustration of the general design and
mode of action of a FRET-based turn-ON system.
[0096] FIG. 24 is a scheme depicting a two-step synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX, carried out by
RAFT polymerization of the copolymer precursor, followed by
coupling with the drug (PTX), dye (Cy5) and finally the
quencher.
[0097] FIG. 25 is a scheme depicting a two-step synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX, carried out by RAFT
polymerization of the copolymer precursor, followed by coupling
with the drug (PTX), dye (FITC) and finally the quencher, DR1.
[0098] FIG. 26 is a scheme depicting a FRET-based theranostic
system in which a quencher-amine is coupled to a COOH
end-functionalized HPMA copolymer-PTX-Cy5 conjugate, providing a
conjugate with one quencher molecule per polymeric chain.
[0099] FIG. 27 is a scheme depicting a FRET-based theranostic
system in which a DR1-amine is coupled to a COOH end-functionalized
HPMA copolymer-PTX-FITC conjugate, providing a conjugate with one
quencher molecule per polymeric chain.
[0100] FIG. 28 is a scheme depicting the synthesis of a HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-PTX-Cy5-quencher conjugate by RAFT
polymerization of a copolymer precursor
HPMA-Gly-Phe-ONp/Gly-Gly-diamine-Boc, followed by coupling to the
precursor amine-Leu-Gly-PABC-PTX, amine-Leu-Gly-PABC-Cy5 and
quencher-COOH.
[0101] FIG. 29 is a scheme depicting the synthesis of a HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-PTX-FITC-DR1 conjugate by RAFT
polymerization of a copolymer precursor
HPMA-Gly-Phe-ONp/Gly-Gly-diamine-Boc, followed by coupling to the
precursor amine-Leu-Gly-PABC-PTX, amine-Leu-Gly-PABC-FITC and
DR1-amine.
[0102] FIG. 30 is a scheme depicting the synthesis of a FRET-based
PGA-Cy5-Quencher conjugate. Coupling of PGA to the Cy5-NH.sub.2 is
carried out by mixing a CDI activated polymer and the fluorophore,
followed by the coupling of PGA-Cy5 conjugate to a deprotected
Quencher-NH.sub.2.
[0103] FIGS. 31A-31B present a scheme depicting the structure and
chemical synthesis of a FRET-based probe-polymer conjugate based on
Cy5 conjugated to PEG, a latent central linker conjugated to
phenyl-boronic ester as a triggering substrate for hydrogen
peroxide, and a quencher (FIG. 31A), and the generation of a
fluorescent signal upon contact with hydrogen peroxide (FIG.
32B).
[0104] FIGS. 32A-32B present comparative plots (FIG. 32A) showing
the NIR fluorescence (.lamda..sub.ex=630 nm, .lamda..sub.em=670 nm)
emitted upon incubation of the Cy5-PEG conjugate [30 .mu.M] in the
presence or absence of hydrogen peroxide (5 equivalents) in 0.1 M
PBS, pH 7.4, monitored by RP-HPLC; gradient: 10-90% ACN in 0.1% TFA
in water; and images acquired using CRI Maestro.TM. Imaging system
(FIG. 32B).
[0105] FIG. 33 presents an image acquired using CRI Maestro.TM.
Imaging system (.lamda..sub.ex=630 nm, .lamda..sub.em=670 nm) of
SCID mice bearing -U-87 MG tumors, 2 minutes after injection i.v.
of 200 .mu.l of a 1 .mu.M solution of a PEG-Cy5 conjugate via the
tail vein.
[0106] FIG. 34 presents the chemical structure and a schematic
illustration of the activation mechanism of a FRET-based cathepsin
B fluorescent probe with the cyanine dye Cy5.
[0107] FIG. 35 is a scheme depicting a synthetic pathway for
preparing a FRET-based cathepsin B-activated fluorescent probe with
the cyanine dye Cy5.
[0108] FIG. 36 presents comparative plots showing the NIR
fluorescence (.lamda..sub.ex=620 nm, .lamda..sub.em=670 nm) emitted
upon incubation of a FRET-based cathepsin B fluorescent probe with
the cyanine dye Cy5 [25 .mu.M, 10% DMSO] in the presence (red) or
absence (blue) of cathepsin B [1.4 U/ml] in activity buffer
(pH=6.0) solution.
[0109] FIGS. 37A-37B present comparative images showing the NIR
fluorescence turn-ON response of a FRET-based cathepsin B
fluorescent probe with the cyanine dye Cy5 upon reaction with
cathepsin B (solutions in activity buffer of pH 6.0). FIG. 37A
presents images of the probe [0.01 mM] in the presence and in the
absence of cathepsin B [10 U/ml] (1 minute after enzyme's addition)
(most and second left vials, respectively), and of and Cy5 [0.01
mM] under the same conditions (third and fourth from the left,
respectively. FIG. 37B presents images of the probe [0.01 mM] in
the presence (4 hours after addition) and in the absence of
cathepsin B [10 U/ml]. Images were taken by CRI Maestro.TM. Imaging
system. Filter set: excitation at 635 nm, emission cut-off filter
of 675 nm.
[0110] FIG. 38 presents a quantification of time-dependent
fluorescence signal upon intratumoral injection of a FRET-based
cathepsin B fluorescent probe into cathepsin B-overexpressing 4T1
mammary adenocarcinoma [50 .mu.l; 0.01 mM]. Images were acquired
and quantified using non-invasive intravital CRI Maestro.TM.
imaging system. Filter set: excitation at 635 nm, emission cut-off
filter of 675 nm.
[0111] FIG. 39 is a scheme depicting the synthesis of HPMA
copolymer-Gly-Gly-Phe-Lys-PABC-PTX-Cy5-Quencher by RAFT
polymerization of copolymer precursor HPMA-Gly-Gly-ONp followed by
coupling to amine-Phe-Lys-PABC-PTX, amine-Phe-Lys-PABC-Cy5-Quencher
as an example for FRET-based fluorescent Turn-On moiety.
[0112] FIG. 40 presents a schematic illustration of a general
design and mode of action of an ICT-based fluorescent probe.
[0113] FIG. 41 is a scheme depicting the synthesis of HPMA
copolymer-Gly-Gly-Phe-Lys-PABC-PTX-QCy7 by RAFT polymerization of
copolymer precursor HPMA-Gly-Gly-ONp followed by coupling to
amine-Phe-Lys-PABC-PTX, amine-Phe-Lys-PABC-QCy7 as an example for
ICT-based fluorescent Turn-On moiety.
[0114] FIG. 42 is a schematic illustration presenting an activation
mechanism of QCy7-based probe by cathepsin B to release free
Camptothecin drug and produce a fluorescent turn-ON response.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0115] The present invention, in some embodiments thereof, relates
to therapy and diagnosis (theranostic) and, more particularly, but
not exclusively, to polymeric systems in which a labeling agent or
a labeling agent and a therapeutically active agents are attached
to a polymeric backbone, to probes useful for inclusion in such
polymeric systems, and to uses thereof in diagnostic and
theranostic applications.
[0116] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0117] The present invention, in some embodiments thereof, relates
to therapy and diagnosis (theranostic) and, more particularly, but
not exclusively, to polymeric systems in which a labeling moiety
(e.g., a fluorescent or fluorogenic moiety) or a labeling moiety
and a therapeutically active agents are attached to a polymeric
backbone, and to uses thereof in diagnostic and theranostic
applications.
[0118] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0119] The present inventors have now devised and successfully
practiced two theranostic systems, which permit simultaneous drug
release and imaging ability: (i) a polymeric system composed of two
separate polymeric moieties, one designed to release a
therapeutically active agent and one designed to generate a
fluorescent signal; and (ii) a combined polymeric system in which a
fluorogenic moiety and therapeutically active agent are attached to
a single polymeric backbone.
[0120] In some embodiments, the diagnostic system is composed of an
efficient high-loading, FRET-based (self-quenched (SQ) or paired)
"Turn-ON" system with a NIR fluorescent cyanine dye or an analog
thereof. In some embodiments, the therapeutic system includes a
therapeutically active agent, such as an anti-cancer agent (e.g.,
paclitaxel; PTX).
[0121] In some embodiments, the polymers are water soluble,
non-toxic, biocompatible and stable polymers (e.g., HPMA, PEG or
the biodegradable PGA).
[0122] In some embodiments, the cyanine dye and/or a
therapeutically active agent are conjugated to the polymeric
backbone in a manner enabling their site-specific cleavage, for
example, by a tumor-specific enzyme such as cathepsin B.
[0123] According to an aspect of some embodiments of the present
invention there is provided a polymeric system comprising a first
polymeric moiety which comprises a first polymeric backbone
composed of a plurality of backbone units and having attached to at
least a portion of the backbone units a fluorogenic moiety, the
fluorogenic moiety being attached to the backbone units via a first
cleavable linking moiety such that upon cleavage of the linking
moiety, a fluorescent signal is generated. The first polymeric
moiety described herein represents the diagnostic part of a
theranostic system. The first polymeric moiety described herein is
a polymeric conjugate in which a fluorogenic moiety is conjugated
to the first polymeric backbone.
[0124] According to some of any of the embodiments of the present
invention, the polymeric system further comprises a therapeutically
active agent.
[0125] In some embodiments, the polymeric system comprises a second
polymeric moiety which comprises a second polymeric backbone
composed of a plurality of backbone units and having attached to at
least a portion of the backbone units a therapeutically active
agent. This second polymeric moiety represents the therapeutic part
of a theranostic system. The second polymeric moiety described
herein is a polymeric conjugate in which a therapeutically active
agent is conjugated to the second polymeric backbone. The second
polymeric backbone can be the same or different from the first
polymeric backbone. In some of these embodiments, the
therapeutically active agent is attached to the backbone units via
a second cleavable linking moiety, which can be the same as or
different from the first cleavable linking moiety.
[0126] In some embodiments, the therapeutically active agent is
attached to a portion of the backbone units of the first polymeric
backbone, such that the fluorogenic moiety is attached, via the
cleavable linking moiety, to one portion of the backbone units, and
the therapeutically active agent is attached to another portion of
the backbone units. Such a polymeric system represents a single
polymeric theranostic system. In some of these embodiments, the
therapeutically active agent is attached to the backbone units via
a second cleavable linking moiety, which can be the same as or
different from the first cleavable linking moiety. Such a system
can be regarded as a polymeric system which comprises two polymeric
moieties or polymeric conjugates, each comprising a polymeric
backbone, namely, a first and a second polymeric backbone as
described herein, whereby the second polymeric backbone forms a
part of the first polymeric backbone, resulting in a polymeric
backbone in which the fluorogenic moiety is attached, via the
cleavable linking moiety, to one portion of the backbone units, and
the therapeutically active agent is attached to another portion of
the backbone units.
[0127] In some embodiments, the therapeutically active agent forms
a part of the fluorogenic moiety, such that upon the cleavage of
the first cleavable linking moiety, the therapeutically active
agent is released and a fluorescent signal is generated. Such a
polymeric system represents a single polymer theranostic
system.
[0128] In some embodiments, the therapeutically active agent and
the fluorogenic moiety are both attached to the first cleavable
linking moiety, for example, by means of a spacer, as described
herein, such that upon cleavage of the first cleavable linking
moiety, the therapeutically active is released and a fluorescent
signal is generated. Such a polymeric system represents a single
polymer theranostic system.
[0129] Thus, in some embodiments of the present invention the
polymeric system can comprise two (or more) polymeric conjugates,
each comprising a polymeric backbone, which can be the same or
different. One of the polymeric conjugates, referred to herein as a
first polymeric moiety, comprises a fluorogenic moiety attached to
a portion of the backbone units of a first polymeric backbone.
Another polymeric conjugate, referred to herein as a second
polymeric moiety, comprises a therapeutically active agent attached
to a portion of the backbone unit of the second polymeric
backbone.
[0130] In other embodiments of the present invention, the polymeric
system comprises one polymeric conjugate, referred to herein as a
first polymeric moiety, in which both the fluorogenic moiety and
the therapeutically active agent are attached to the same polymeric
backbone, each being attached to a portion of the backbone units in
the polymeric backbone. In these embodiments, the second polymeric
backbone forms a part of the first polymeric backbone, such that
the conjugate comprises one polymeric backbone.
[0131] According to some embodiments of the invention, the first
and the second polymeric backbones are not covalently associated
therebetween, such that the system comprises two separate polymeric
conjugates (polymeric moieties).
[0132] According to some embodiments of the invention, the second
polymeric backbone forms a part of the first polymeric backbone,
such that the polymeric system comprises a polymeric backbone
comprising a plurality of backbone units having the fluorogenic
moiety attached to one portion of the backbone units and having the
therapeutically active agent attached to another portion of the
backbone units, such that the system comprises one polymeric
conjugate or moiety as described herein.
[0133] In some of any of the embodiments described herein, the
fluorogenic moiety is attached to the first cleavable linking
moiety via a spacer. In some embodiments, the first cleavable
linking moiety is attached to the respective portion of backbone
units of the first polymeric backbone via a spacer.
[0134] In some of any of the embodiments described herein, the
therapeutically active agent is attached to the second cleavable
linking moiety via a spacer. In some embodiments, the second
cleavable linking moiety, if present, is attached to the respective
portion of backbone units of the polymeric backbone via a
spacer.
[0135] In some of the embodiments described herein, when the
therapeutically active agent forms a part of the fluorogenic
moiety, the therapeutically active agent is attached to the
fluorogenic moiety via a spacer.
[0136] In some of any of these embodiments, the spacer is a
degradable spacer, as described herein.
[0137] In some of any of the embodiments described herein, the
first polymeric moiety, which comprises the fluorogenic moiety,
further comprises a quenching agent, as described herein.
[0138] In some embodiments, the quenching agent is attached to a
portion of the backbone units of the first polymeric backbone, such
that the fluorogenic moiety is attached to one portion of the
backbone units of the first polymeric backbone and the quenching
agent is attached to another portion of the backbone units of the
first polymeric backbone.
[0139] In some embodiments, the quenching agent is attached to a
terminus of the first polymeric backbone, that is, the quenching
agent is attached to a terminal backbone unit of the first
polymeric backbone.
[0140] The quenching agent can be attached to the backbone unit(s)
via a linking moiety, or via a spacer, which can be degradable or
non-degradable.
[0141] In some embodiments, the quenching agent forms a part of the
fluorogenic moiety. In some of these embodiments, the quenching
agent is attached to a fluorescent moiety via a spacer.
[0142] The polymeric conjugates described herein can be used each
separately or in any combination thereof.
[0143] The polymer:
[0144] As used herein, the term "polymer" or "polymeric moiety"
describes a substance composed of a plurality of repeating
structural units (backbone units) covalently connected to one
another and forming a polymeric backbone. The term "polymer" as
used herein encompasses organic and inorganic polymers and further
encompasses one or more of a homopolymer, a copolymer or a mixture
thereof (a blend). The term "homopolymer" as used herein describes
a polymer that is made up of one type of monomeric units and hence
is composed of homogenic backbone units. The term "copolymer" as
used herein describes a polymer that is made up of more than one
type of monomeric units and hence is composed of heterogenic
backbone units. The heterogenic backbone units can differ from one
another by the pendant groups thereof.
[0145] The term "polymer" or "polymeric moiety" is used herein to
describe the polymeric backbone to which the agents/moieties
described herein are attached.
[0146] The polymer comprises a polymeric backbone which is
comprised of backbone units whereby one or more of the
therapeutically active and the fluorogenic moiety, and optionally
other agents and/or moieties as described herein, are attached to
at least a portion of these backbone units. Some or all of these
backbone units are typically functionalized prior to conjugation,
so as to have a reactive group for attaching the therapeutically
active agent and/or the fluorogenic moiety and/or other agents or
moieties. Those backbone units that are not functionalized and/or
do not participate in the conjugation of the therapeutically active
agent and/or the fluorogenic moiety and/or other agents or
moieties, are referred to herein as "free" backbone units.
[0147] Polymers which are suitable for use in the context of the
present embodiments are biocompatible, non-immunogenic and
non-toxic. The polymers serve as carriers that enable targeting to
and delivery into tumor tissue, possibly due to the EPR effect.
[0148] The polymer may be a biostable polymer, a biodegradable
polymer or a combination thereof. The term "biostable", as used in
this context of embodiments of the invention, describes a compound
or a polymer that remains intact under physiological conditions
(e.g., is not degraded in vivo).
[0149] The term "biodegradable" describes a substance which can
decompose under physiological and/or environmental conditions into
breakdown products. Such physiological and/or environmental
conditions include, for example, hydrolysis (decomposition via
hydrolytic cleavage), enzymatic catalysis (enzymatic degradation),
and mechanical interactions. This term typically refers to
substances that decompose under these conditions such that 50
weight percents of the substance decompose within a time period
shorter than one year.
[0150] The term "biodegradable" as used in the context of
embodiments of the invention, also encompasses the term
"bioresorbable", which describes a substance that decomposes under
physiological conditions to break down products that undergo
bioresorption into the host-organism, namely, become metabolites of
the biochemical systems of the host-organism.
[0151] The polymer can be water-soluble or water-insoluble. In some
embodiments, the polymer is water soluble at room temperature.
[0152] The polymer can further be a charged polymer or a
non-charged polymer. Charged polymers can be cationic polymers,
having positively charged groups and a positive net charge at a
physiological pH; or anionic polymers, having negatively charged
groups and a negative net charge at a physiological pH. Non-charged
polymers can have positively charged and negatively charged group
with a neutral net charge at physiological pH, or can be
non-charged.
[0153] In some embodiments, the polymer has an average molecular
weight in the range of 100 Da to 800 kDa. In some embodiments, the
polymer has an average molecular weight lower than 60 kDa. In some
embodiments, the polymer's average molecular weight range is 15 to
40 kDa.
[0154] Polymeric substances that have a molecular weight higher
than 10 kDa typically exhibit an EPR effect, as described herein,
while polymeric substances that have a molecular weight of 100 kDa
and higher have relatively long half-lives in plasma and an
inefficient renal clearance. Accordingly, a molecular weight of a
polymeric conjugate can be determined while considering the
half-life in plasma, the renal clearance, and the accumulation in
the tumor of the conjugate.
[0155] The molecular weight of the polymer can be controlled, at
least to some extent, by the degree of polymerization (or
co-polymerization).
[0156] The polymer used in the context of embodiments of the
invention can be a synthetic polymer or a naturally-occurring
polymer. In some embodiments, the polymer is a synthetic
polymer.
[0157] The polymeric backbone of a polymeric conjugate as described
herein may be derived from, or correspond to, a polymeric backbone
of polymers such as, for example, polyacrylates, polyvinyls,
polyamides, polyurethanes, polyimines, polysaccharides,
polypeptides, polycarboxylates, and mixtures thereof.
[0158] Exemplary polymeric backbones which are suitable for use in
the context of the present embodiments are polymeric backbones
which correspond to the polymeric backbones of polymers such as,
but are not limited to, polyglutamic acid (PGA), a
poly(hydroxyalkylmethaacrylamide) (HPMA), a polylactic acid (PLA),
a polylactic-co-glycolic acid (PLGA), a
poly(D,L-lactide-co-glycolide) (PLA/PLGA), a polyamidoamine
(PAMAM), a polyethylenimine (PEI), dextran, pollulan, a water
soluble polyamino acid, and a polyethylenglycol (PEG).
[0159] These polymers can be of any molecular weight, as described
herein, and preferably have a molecular weight within the range of
10 to 60 kDa, or of 10 to 40 kDa.
[0160] It is to be understood that the polymers as discussed herein
describe those polymers that are formed from homogenic or
heterogenic, non-functionalized monomeric units, and that the
polymeric backbone constituting the polymeric conjugates disclosed
herein corresponds to such polymers by being comprised of the same
monomeric units, while some of these monomeric backbone units have
moieties attached thereto, as described herein. Thus, the polymeric
backbone of a polymeric conjugate is similar to that of the
polymers described herein, and differs from the polymers by having
the above-described agents attached to at least some of the
backbone units therein.
[0161] In some of any of the embodiments described herein, the
polymeric backbone of a polymeric moiety or conjugate corresponds
to (as described herein), or is derived from (as described herein),
a polymeric backbone of a poly(hydroxyalkylmethaacrylamide) or a
copolymer thereof. Such a polymeric backbone comprises
methacrylamide backbone units having attached thereto either
2-hydroxypropyl groups or such 2-hydroxypropyl groups that have
been modified by attaching thereto (directly or indirectly) the
moieties described herein (e.g., therapeutically active agent(s)
and/or fluorogenic moiety).
[0162] Poly(hydroxyalkylmethacrylamide) (HPMA) polymers are a class
of water-soluble synthetic polymeric carriers that have been
extensively characterized as biocompatible, non-immunogenic and
non-toxic. One advantage of HPMA polymers over other water-soluble
polymers is that they may be tailored through relatively simple
chemical modifications, in order to regulate their respective drug
and targeting moiety content. Further, the molecular weight and
charge of these polymers may be manipulated so as to allow renal
clearance and excretion from the body, or to alter biodistribution
while allowing tumor targeting.
[0163] In some of any of the embodiments described herein, the
polymeric backbone is derived from, or corresponds to, polyglutamic
acid (PGA). PGA is a polymer composed of units of naturally
occurring L-glutamic acid linked together through amide bonds. The
pendant free .gamma.-carboxyl group in each repeating unit of
L-glutamic acid is negatively charged at a neutral pH, which
renders the polymer water-soluble. The carboxyl groups also provide
functionality for drug attachment. PGA is biodegradable and
FDA-approved.
[0164] Cysteine proteases, particularly cathepsin B, play key roles
in the lysosomal degradation of PGA to its nontoxic basic
components, L-glutamic acid, D-glutamic acid and D,L-glutamic acid.
The cellular uptake of negatively charged polymers can be hindered
due to electrostatic repulsion forces between the polymers and the
rather negatively charged surface of the cells. Although PGA is no
exception to this rule, it does not diminish the EPR effect and the
accumulation and retention of PGA-drug conjugates in solid tumors.
Specific receptor-mediated interactions of PGA-drug conjugates
containing targeting ligands may also increase the rate of polymer
uptake into the target cells.
[0165] As used herein, "a polyglutamic acid" or "polyglutamic acid
polymer" encompasses poly(L-glutamic acid), poly(D-glutamic acid),
poly(D,L-glutamic acid), poly(L-gamma glutamic acid), poly(D-gamma
glutamic acid) and poly(D,L-gamma glutamic acid).
[0166] PGA is usually prepared from
poly(.gamma.-benzyl-L-glutamate) by removing the benzyl protecting
group with the use of hydrogen bromide. A sequential copolymer of
protected PGA may be synthesized by peptide coupling reactions. For
the preparation of high-molecular-weight homopolymers and block or
random copolymers of protected PGA, tri-ethylamine-initiated
polymerization of the N-carboxyanhydride (NCA) of
.gamma.-benzyl-L-glutamate is used.
[0167] Water-soluble copolymers such as N-2-hydroxypropyl
methacrylamide (HPMA) copolymer and polyglutamic acid (PGA) are
biocompatible, non-immunogenic and non-toxic carriers that enable
specific delivery into tumor tissue (Satchi-Fainaro et al. Nat Med
2004; 10: 255-261). These macromolecules do not diffuse through
normal blood vessels but rather accumulate selectively in the tumor
site because of the EPR effect. This phenomenon of passive
diffusion through the hyperpermeable neovasculature and
localization in the tumor interstitium is observed in many solid
tumors for macromolecular agents and lipids.
[0168] For any of the polymeric moieties or conjugates described
herein, the plurality of the backbone units forming the polymeric
backbone in the conjugate comprises two or more different portions
of backbone units that differ from one another by the presence
and/or nature of the moiety or agent attached thereto. For example,
one portion of the backbone units are "free" backbone units, and
one portion of the backbone units have a fluorogenic moiety
attached thereto. In another example, a third portion of the
backbone units have a therapeutically active agent attached
thereto, or a quenching agent attached thereto.
[0169] The different backbone units that have a moiety or agent
attached thereto can be randomly dispersed within the polymeric
backbone.
[0170] Thus, in some embodiments, a polymeric backbone as described
herein is formed of a plurality of backbone monomeric units, which
are covalently linked to one another so as to form the polymeric
backbone. The backbone units are therefore such that, if not having
certain moieties attached thereto, as described herein, form a
polymeric backbone of a polymer. The plurality of backbone units as
described herein, and the polymeric backbone comprised thereof, are
therefore also defined herein as derived from, or corresponding to,
the polymeric backbone of such a polymer. The plurality of backbone
units as described herein, and the polymeric backbone comprised
thereof, therefore correspond to, or are derived from, a polymer,
whereby one or more moieties or agents, as described herein, are
attached to one or more portions of the backbone units. Since once
the one or more moieties are attached to one or more portions of
the backbone units forming the polymeric backbone, the backbone
units forming the polymeric backbone are not identical to one
another, as is the case of an "intact" polymer, and hence the
polymeric conjugate is actually a copolymer, or has a copolymeric
backbone, which is comprised of two or more types of backbone
units. The phrase "polymeric backbone" as used herein therefore
describes a "copolymeric backbone" comprised of at least two
different types of backbone units.
[0171] It is to be noted that portions of the backbone units differ
from one another by the presence and type of the moiety or agent
that are attached to the backbone unit, but maintain the chemical
structure of the portion of the backbone unit that forms the
polymeric backbone. In analogy to a peptide, where the portions of
the backbone units differ from one another by the side chain of the
amino acid, the portions of the backbone units differ from one
another by the presence and/or nature of the pendant group
thereof.
[0172] In some of any of the embodiments described herein, a
polymeric conjugate or moiety as described herein comprises a
polymeric (or copolymeric) backbone formed from a plurality of
backbone units, and the plurality of backbone units comprise one or
more of the following backbone units:
[0173] -A.sub.1-, which represents a backbone unit within the
polymeric backbone, or, in other words, a backbone unit of the
polymer from which the polymeric backbone is derived, and is "free"
of moieties that attached thereto;
[0174] -A.sub.2-, which represents a backbone unit of the polymer
from which the polymeric backbone is derived (a backbone unit
within the polymeric backbone), having a fluorogenic moiety (F), as
described herein, attached thereto via a cleavable linking moiety,
as described in further detail hereinafter;
[0175] -A.sub.3-, which represents a backbone unit of the polymer
from which the polymeric backbone is derived (a backbone unit
within the polymeric backbone), having a therapeutically active
agent (D), as described herein attached thereto, optionally via a
cleavable linking moiety, as described in further detail
hereinafter;
[0176] -A.sub.4-, which represents a backbone unit of the polymer
from which the polymeric backbone is derived (a backbone unit
within the polymeric backbone), having a quenching agent attached
thereto; and optionally
[0177] -A.sub.5-, which represents a backbone unit of the polymer
from which the polymeric backbone is derived (a backbone unit
within the polymeric backbone), having a functional/reactive group
attached thereto. Such backbone units can be present in a polymeric
moiety as described herein, in cases where a polymer comprising a
plurality of functionalized backbone units is used for forming a
polymeric conjugate as described herein, whereby not all the
functionalized backbone units participate in the conjugation
reaction to form one or more of A.sub.2, A.sub.3 or A.sub.4 as
described herein. Such backbone units can be regarded as "free"
backbone units to the extent that they do not contain a moiety or
agent as described herein conjugated thereto, yet they contain a
reactive/functional pendant group, which is denoted herein as
R.
[0178] The backbone units can be arranged within the polymeric
backbone in any order, such that each of the backbone units can be
a terminal backbone unit, which is attached to one other backbone
unit, or is attached to two other backbone units, which can be the
same or different.
[0179] In some of any of the embodiments of the present invention,
a polymeric moiety or conjugate as described herein comprises at
least backbone units A.sub.2 as described herein, and optionally
also backbone units A.sub.1, and further optionally also backbone
units A.sub.4 and A.sub.5. Such a polymeric moiety represents a
diagnostic part of a theranostic system, and in some embodiments, a
polymeric system comprising such a polymeric moiety, further
comprises a second polymeric moiety.
[0180] In some of these embodiments, the second polymeric moiety
comprises backbone units A.sub.3 as described herein, and
optionally also backbone units A.sub.1, and further optionally also
backbone units A.sub.5.
[0181] In some of any of the embodiments of the present invention,
a polymeric moiety or conjugate as described herein comprises
backbone units A.sub.1, A.sub.2 and A.sub.3 as described herein,
and optionally also backbone units A.sub.4 and A.sub.5.
[0182] In some of any of the embodiments of the present invention,
the moiety or agent attached to the backbone units can be
conjugated or attached directly to pendant group of the backbone
units, or indirectly, via a spacer or a linker, as described
herein.
[0183] In some embodiments, the plurality of backbone units forming
the polymeric backbone comprises the following portions of backbone
units:
[0184] -(A.sub.1)w-;
[0185] -(A.sub.2-F)x-;
[0186] -(A.sub.3-D)y-; and
[0187] -(A.sub.4-Q)s,
[0188] and optionally -(A.sub.5-R)z
[0189] wherein:
[0190] A.sub.1 is a backbone unit within the polymeric backbone, as
described herein;
[0191] A.sub.2-F is a backbone unit within the polymeric backbone
having attached thereto, via a cleavable linking moiety, a
fluorogenic moiety F, as described herein;
[0192] A.sub.3-D is a backbone unit within the polymeric backbone
having attached thereto a therapeutically active agent D, as
described herein;
[0193] A.sub.4-Q is a backbone unit within the polymeric backbone
having attached thereto a quenching agent (Q), as described
herein;
[0194] and A.sub.5 in a functionalized backbone unit within the
polymeric backbone, as described herein, wherein R is said reactive
or functional group.
[0195] The backbone units can further comprise second and third
linking moieties, and/or spacers, through which the agents or
moieties are attached, as described in further detail
hereinafter.
[0196] Herein, the phrases "loading onto the polymer", or simply
"load", are used to describe the amount of an agent or moiety that
is attached to the polymeric backbone of the conjugates described
herein, and is represented herein by the mol percent (mol %) of the
backbone units having the agent or moiety attached thereto, as
defined hereinafter.
[0197] Herein "mol percent" represents the number of moles of
backbone units having the agent or moiety attached thereto, as
defined hereinafter, per 1 mol of the polymeric backbone,
multiplied by 100, and hence represents the number of moles of an
attached moiety or agent per 1 mol of the polymer, multiplied by
100.
[0198] The % loading can be measured by methods well known by those
skilled in the art, some of which are described hereinbelow under
the Materials and Methods of the Examples section that follows.
[0199] The mol percent of each of the backbone units is represented
herein by "w", "x", y", "s", and "z", respectively. "x", "y" and
"s" represent the loading of the respective moieties.
[0200] In some of any of the embodiments described herein, a load
of a therapeutically active agent, when present within the
polymeric moiety, denoted herein also as "y", ranges from 0.1 to
100 mol percent, or from 0.1 to 20 mol percent, and can be, for
example, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20, and even higher values, including any
subranges and values therebetween.
[0201] In some of any of the embodiments described herein, the load
of the fluorogenic moiety, denoted also as "x" herein, ranges from
0.1 to 100 mol percent, or from 0.1 to 20 mol percent, or from 1 to
20 mol percent, and can be, for example, 0.1, 0.5, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, and even
higher values, including any subranges and values therebetween.
[0202] In some of any of the embodiments described herein, the load
of the quenching agent, if present within separate backbone units,
denoted also as "s" herein, ranges from 0.1 to 50 mol percent, or
from 0.1 to 20 mol percent, or from 1 to 20 mol percent, and can
be, for example, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 and 20, and higher values, including any
subranges and values therebetween.
[0203] According to some embodiments of the invention, w is an
integer having a value such that x/(x+y+w+s+z) multiplied by 100 is
in the range of from 70 to 99.9; y is an integer having a value
such that y/(x+y+w+s+z) multiplied by 100 is in the range of from
0.01 to 20, as described herein; and x is an integer having a value
such that w/(x+y+w+z+x) multiplied by 100 is in the range of from
0.01 to 20, as described herein.
[0204] For example w/(x+y+w+z+x) multiplied by 100 may be 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9; y/(x+y+w+z+s)
multiplied by 100 may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14 or 15; x/(x+y+w+z+s) multiplied by 100 may be 0.01,
0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;
s/(x+y+w+z+s) multiplied by 100 may be 0.01, 0.02, 0.03, 0.04,
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
[0205] It would be appreciated that x, y, s, z and w can be
controlled as desired by selecting the mol ratio of the respective
monomeric units used for forming the polymeric conjugate, as
discussed hereinbelow.
[0206] Any of the polymeric systems described herein, in some
embodiments, have a large enough hydrodynamic diameter. The term
"large enough" is used herein to describe a polymeric moiety having
a hydrodynamic diameter which leads to an increase in the ratio of
polymer accumulated in tumor tissue as compared to other tissues.
The determination of the optimal ratio is well within the
capability of those skilled in the art. For example, the ratio may
be 1.1, 2, 3, 4, 5 etc. In some embodiments, the hydrodynamic
diameter is in the range of from 15 nm to 200 nm. In some
embodiments, the hydrodynamic diameter is in the range of from 50
nm to 150 nm. In some embodiments the hydrodynamic diameter is in
the range of from 70 nm to 90 nm. In yet another embodiment the
hydrodynamic diameter is 95 nm. The hydrodynamic diameter can be
measured by methods known in the art.
[0207] The polymeric moieties described hereinabove may be
administered or otherwise utilized in this and other aspects of the
present invention, either as is, or as a pharmaceutically
acceptable salt, enantiomer, diastereomer, solvate, hydrate or a
prodrug thereof.
[0208] The phrase "pharmaceutically acceptable salt" refers to a
charged species of the parent compound and its counter ion, which
is typically used to modify the solubility characteristics of the
parent compound and/or to reduce any significant irritation to an
organism by the parent compound, while not abrogating the
biological activity and properties of the administered compound.
The neutral forms of the compounds are preferably regenerated by
contacting the salt with a base or acid and isolating the parent
compound in a conventional manner. The parent form of the compound
differs from the various salt forms in certain physical properties,
such as solubility in polar solvents, but otherwise the salts are
equivalent to the parent form of the compound for the purposes of
the present invention.
[0209] The phrase "pharmaceutically acceptable salts" is meant to
encompass salts of the moieties and/or polymeric backbone which are
prepared with relatively nontoxic acids or bases, depending on the
particular substituents found on the compounds described herein.
When polymeric moieties of the present embodiments contain
relatively acidic functionalities, base addition salts can be
obtained by contacting the neutral (i.e., non-ionized) form of such
conjugates with a sufficient amount of the desired base, either
neat or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition salts include sodium, potassium, calcium,
ammonium, organic amino, or magnesium salt, or a similar salt. When
polymeric moieties of the present invention contain relatively
basic functionalities, acid addition salts can be obtained by
contacting the neutral form of such polymeric moieties with a
sufficient amount of the desired acid, either neat or in a suitable
inert solvent. Examples of pharmaceutically acceptable acid
addition salts include those derived from inorganic acids like
hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,
phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, for
example, Berge et al., "Pharmaceutical Salts", Journal of
Pharmaceutical Science, 1977, 66, 1-19). Polymeric moieties of the
present embodiments may contain both basic and acidic
functionalities that allow the conjugates to be converted into
either base or acid addition salts.
[0210] The neutral forms of the polymeric moieties are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent polymeric moiety in a conventional manner. The
parent form of the polymeric moiety differs from the various salt
forms in certain physical properties, such as solubility in polar
solvents, but otherwise the salts are equivalent to the parent form
of the polymeric moiety for the purposes of the present
invention.
[0211] The Fluorogenic Moiety:
[0212] As generally stated in the art, the term "fluorogenic"
encompasses a state or condition of having the capability to be
fluorescent (i.e. to absorb and emit light, as defined hereinbelow)
following a chemical, biochemical or physical occurrence or event.
Thus, a "fluorogenic moiety" or a "fluorogenic compound" describes
a non-fluorescent moiety or compound or a weakly fluorescent moiety
or compound that becomes more fluorescent (e.g., by at least 10%,
at least 20%, at least 30%, at least 40%, at least 50%, at least
60%, at least 70%, at least 80%, at least 90 5, at least 100% (at
least 2-fold), optionally at least 3-fold, optionally at least
4-fold more fluorescent, and optionally at 10-fold or higher more
fluorescent) upon the occurrence of a chemical, biochemical or
physical event.
[0213] As used herein, a "chemical event" describes an event that
involves a change in the chemical structure of a compound,
including, but not limited to, bond cleavage, bond formation,
protonation, deprotonation, oxidation, reduction, and more.
[0214] In some of any of the embodiments described herein, the
chemical event is bond cleavage.
[0215] The phrase "fluorogenic moiety" as used herein therefore
describes a moiety which changes its fluorescence upon a chemical
event, and is therefore regarded, and in also referred to herein
interchangeably, as a chemically-activatable fluorogenic moiety, as
a probe, as a chemically-activatable probe or as a Turn-ON probe.
The fluorogenic moieties described herein throughout are also
referred to in the context of the fluorescent (dye) compounds or
moieties generated upon said activation.
[0216] The phrase "Turn-ON" is used herein to describe a
fluorogenic moiety, which upon a chemical event, becomes
fluorescent, as defined herein. In the fluorogenic moiety,
fluorescence is OFF, yet, upon being subjected to a chemical event,
fluorescence turns ON (and a fluorescent signal is generated). The
chemical event comprises a cleavage of a linking moiety, as
described herein.
[0217] The phrase "fluorescent" refers to a compound or moiety that
emits light upon return to the base state from a singlet
excitation. The fluorescent compounds or moieties disclosed herein
are also referred to herein throughout as dye compounds or dye
molecules or as fluorophores or as fluorchromes.
[0218] In some embodiments, the emitted light is a near infrared
light (near IR or NIR), being in the range of from about 700 nm to
about 1400 nm. In some embodiments, the emitted light has emission
maxima at a wavelength that is suitable for biological applications
(e.g., in vivo applications), which ranges from 650 nm to 900 nm,
and in some embodiments, from 700 nm to 800 nm.
[0219] Thus, a fluorogenic moiety as disclosed herein does not
exhibit fluorescence and hence does not emit light at a near
infrared range, for example, a light having a wavelength in the
range of from about 650 nm to about 900 nm, and is designed such
that it is capable of exhibiting fluorescence and thus of emitting
light at such a near infrared range upon said cleavage.
[0220] In some embodiments, the emitted light is a UV-vis light.
Thus, a fluorogenic moiety as disclosed herein does not exhibit
fluorescence and hence does not emit light at a UV-vis range, and
is designed such that it is capable of exhibiting fluorescence and
thus of emitting light at such a UV-vis range upon said
cleavage.
[0221] In some of any of the embodiments described herein, a
fluorogenic moiety comprises a fluorescent moiety or compound,
which is attached to said cleavable linking moiety (optionally via
a spacer such as a degradable spacer), yet, the fluorescence of the
moiety is quenched and hence is OFF. Once the linking moiety is
cleaved, and the fluorescent moiety or compound is released from
the polymeric moiety, and diffuses away therefrom, quenching is no
longer effected, the fluorescence of the moiety turns ON, and a
fluorescent signal is generated. A polymeric moiety or system
comprising such a fluorogenic moiety is also referred to herein as
a FRET system, or FRET-based system, as is discussed in detail
hereinafter.
[0222] In some of these embodiments, the quenching of the
fluorescence is self-quenching (SQ), and is effected by having at
least two fluorescent moieties per a polymeric backbone. Once the
linking moiety is cleaved, and the fluorescent moieties or
compounds are released from the polymeric moiety, and diffuse away
therefrom, self-quenching is no longer effected, the fluorescence
of the moieties turns ON, and a fluorescent signal is
generated.
[0223] In some of these embodiments, the quenching of the
fluorecsence is effected by means of a quenching agent.
[0224] In some embodiments, the quenching agent is attached to one
or more backbone units of the first polymeric backbone, and the
fluorescent moiety is attached to other, one or more, backbone
units of the polymeric backbone units, such that the fluorescence
of the fluorescent moiety is quenched and is OFF. Once the linking
moiety is cleaved, and the fluorescent moiety or compound is
released from the polymeric moiety, and diffuses away therefrom,
quenching is no longer effected, the fluorescence of the moiety
turns ON, and a fluorescent signal is generated.
[0225] In other embodiments, the quenching agent forms a part of
the fluorogenic moiety. In these embodiments, the fluorogenic
moiety comprises a quenching agent attached to a fluorescent
moiety, optionally via a spacer such as a degradable spacer, such
that upon the cleavage of the first linking moiety, the fluorescent
moiety is released, quenching is no longer effected, the
fluorescence of the fluorescent moiety turns ON and a fluorescent
signal is generated.
[0226] In some of any of the embodiments described herein, the
fluorogenic moiety comprises a compound or a moiety which is
non-fluorescent (i.e., does not absorb and emit light) or which has
weak fluorescence (e.g., have a quantum yield lower by at least
2-fold than a quantum yield of a corresponding fluorescent molecule
with the strong fluorescence), when attached to the polymeric
backbone, and which becomes more fluorescent, as defined herein,
upon a chemical event, due a change (rearrangement) in the
structure of the compound or moiety. In some of these embodiments,
the change in the structure of the compound or moiety involves
relocalization of .pi.-electrons, as a result of the chemical event
(cleavage of the linking moiety), which may generate, for example,
a conjugated .pi.-electron system, which accounts for fluorescence.
A polymeric moiety or system comprising such a fluorogenic moiety
is also referred to herein as an ICT system, or ICT-based system,
as is discussed in detail hereinafter.
[0227] In some of any of the embodiments described herein, the
fluorogenic moiety has a cyanine-like structure and the fluorescent
moiety or compound is a cyanine dye or a cyanine-like dye.
[0228] As used herein, the phrase "cyanine-like structure"
describes a molecule that has two nitrogen-containing moieties
which are joined by a polymethine-containing chain (e.g., a
carbomethine chain). One or both nitrogens can be a part of a
nitrogen-containing heteroaromatic moiety, or, alternatively, be a
secondary or tertiary ammonium.
[0229] In some embodiments, the polymethine-containing chain
comprises 2 carbon atoms and the cyanine-like structure is of a Cy2
type cyanine structure, as this term is widely recognized in the
art.
[0230] In some embodiments, the carbomethine-containing chain
comprises 3 carbon atoms and the cyanine-like structure is of a Cy3
type cyanine structure.
[0231] In some embodiments, the carbomethine-containing chain
comprises 5 carbon atoms and the cyanine-like structure is of a Cy5
type cyanine structure.
[0232] In some embodiments, the carbomethine-containing chain
comprises 7 carbon atoms and the cyanine-like structure is of a Cy7
type cyanine structure.
[0233] In some embodiments, the carbomethine-containing chain
comprises 5 or 7 carbon atoms.
[0234] Thus, in some embodiments, the fluorogenic moiety as
described herein is a modified cyanine dye, including any of the
known cyanine dyes, which is modified by the means used to attach
it to the polymeric backbone.
[0235] In some embodiments, a cyanine-like fluorogenic moiety as
described herein can be regarded as comprising the same basic
chemical arrangement as cyanine dyes, yet, because of its
attachment to the polymeric backbone in sufficient amount and in
close proximity, the fluorogenic moiety is spectroscopically
silenced in the NIR range before activation by said cleavage, due
to self-quenching.
[0236] In some embodiments, a cyanine-like fluorogenic moiety as
described herein can be regarded as comprising the same basic
chemical arrangement as cyanine dyes, yet, because of its
attachment to the polymeric backbone in close proximity to a
quenching agent, the fluorogenic moiety is spectroscopically
silenced in the NIR range before activation by said cleavage, due
to quenching.
[0237] In some embodiments, a cyanine-like fluorogenic moiety as
described herein has a chemical arrangement which is different from
cyanine dyes (e.g., a delocalized .pi.-electrons system), and hence
the fluorogenic moiety is spectroscopically silenced in the NIR
range before activation by said cleavage.
[0238] Fluorogenic moieties which have a modified cyanine structure
are also referred to herein as cyanine-based fluorogenic moieties,
and the fluorescent moieties or compounds generated upon said
cleavage are referred to herein as cyanine dyes. Exemplary such
moieties are described in further detail hereinafter.
[0239] Other fluorogenic or fluorescent moieties are also
contemplated.
[0240] In embodiments of the present invention where the
fluorogenic moiety comprises a fluorescent moiety or compound
attached to the cleavable linker or moiety, the fluorescent moiety
can be selected from the myriad of known fluorescent moieties. FITC
is a non-limiting example. Other examples are listed in "Methods in
Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic
Acid Probes: Designs and Protocols", Edited by: V. V. Didenko
.COPYRGT. Humana Press Inc., Totowa, N.J., Chapter 2, page 17, by
Mary Katherine Johans son, which is incorporated by reference as if
fully set forth herein.
[0241] The Quenching Agent:
[0242] Herein, the phrase "quenching agent" is also referred to
interchangeably as "quencher", and describes a moiety or compound
which is capable of decreasing the fluorescence intensity of
another moiety or compound, as described herein. The decrease in
fluorescence intensity by quenching can result from processes such
as excited state reactions, energy transfer, complex-formation and
the like.
[0243] A quencher is selected in accordance with a selected
fluorescent moiety or compound, namely, as capable of, for example,
absorbing energy emitted from the fluorescent moiety or compound,
interacting with the fluorescent moiety or compound which it is in
its excited state, etc.
[0244] In some of any of the embodiments described herein, the
quencher is selected suitable for FRET, which is based on
dipole-dipole interactions between the transition dipoles of the
fluorescent moiety (which acts as a donor) and the quencher (which
acts as an acceptor). A suitable quencher should be positioned at a
distance of distances up to 100 .ANG. from the donor, should have a
suitable orientation of the dipole moment relative to the donor,
and should have a spectral overlap with the donor.
[0245] Those skilled in the art would readily recognize which
quenching agent or dye is suitable for use for quenching the
fluorescence of a selected fluorescent moiety or moiety.
[0246] Exemplary quenching agent-fluorescent moiety pairs include,
but are not limited to, two cyanine dyes (which can be the same,
for SQ, or different, for pair FRET) (NIR), FITC and DR1 (visible),
Fluorescein & Cy5 (visible), Cy5 & IR783 (NIR), Cy7 &
IR783 (NIR), Cy3 & BHQ2 (visible), Cy5 & BHQ2 (NIR).
Additional pairs are listed in "Methods in Molecular Biology, vol.
335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and
Protocols", Edited by: V. V. Didenko .COPYRGT. Humana Press Inc.,
Totowa, N.J., Chapter 2, page 17, by Mary Katherine Johansson,
which is incorporated by reference as if fully set forth
herein.
[0247] The Linking Moiety:
[0248] Herein throughout, the phrase "linking moiety" is also
referred to herein as "linker".
[0249] In any of the embodiments described herein, the fluorogenic
moiety is attached to the polymeric backbone (to at least a portion
of the backbone units composing the first polymeric backbone), via
a linking moiety, which is a cleavable linking moiety (referred to
herein as a first cleavable linking moiety).
[0250] In some of any of the embodiments described herein, the
therapeutically active agent is attached to a polymeric backbone
(e.g., to at least a portion of the first or second polymeric
backbone) via a linking moiety, preferably, a cleavable linking
moiety, referred to herein as a second cleavable linking moiety.
The linker linking the therapeutically active agent to the
polymeric backbone, if present, and the linker linking the
fluorogenic moiety to the polymeric backbone may be the same or
different. In some embodiments, the linker is the same and in some
embodiments, the linker or either the same or is cleavable under
the same conditions (e.g., by the same enzyme).
[0251] The linker described herein refers to a chemical moiety that
serves to couple the fluorogenic moiety and/or the therapeutically
active agent to the polymeric backbone (to the respective portion
of backbone units).
[0252] The phrase "cleavable linking moiety" or "cleavable linker"
describes a chemical moiety which can undergo a bond cleavage upon
a chemical event, as described herein.
[0253] In some embodiments, the linker is a biodegradable or
biocleavable linker.
[0254] The phrases "biodegradable linker" and "biocleavable linker"
as used herein, describe a linker that is capable of being
degraded, or cleaved (undergo bond cleavage), when exposed to
certain physiological conditions. Such physiological conditions can
be, for example, pH, a presence of a certain enzyme, a presence of
chemical substance (e.g., analyte) and the like.
[0255] In some embodiments, the linker is designed as being
cleavable at conditions characterizing the desired bodily site
(e.g., by certain enzymes, chemical substances or pH), as detailed
hereinbelow.
[0256] According to some embodiments, the biodegradable linker is a
pH-sensitive linker, a hydrolysable linker or an
enzymatically-cleavable linker.
[0257] In some embodiments, the linker is capable of being cleaved
by pre-selected cellular enzymes, for instance, those found in
osteoblasts, osteoclasts, lysosomes of cancerous cells or
proliferating endothelial cells, or in tumor tissues.
[0258] Alternatively, an acid hydrolysable linker could comprise an
ester or amide linkage.
[0259] In some embodiments the biodegradable linker is an
enzymatically cleavable linker. Such a linker is typically designed
so as to include a chemical moiety, typically, but not exclusively,
an amino acid sequence that is recognized by a pre-selected enzyme.
Such an amino acid sequence is often referred to in the art as a
"recognition motif". A polymeric conjugate comprising such a linker
typically remains substantially intact in the absence of the
pre-selected enzyme in its environment, and hence does not cleave
or degrade so as to the release the moiety attached thereto via the
linker until contacted with the enzyme.
[0260] In some embodiments, the enzymatically cleavable linker is
cleaved by an enzyme which is overexpressed in tumor tissues. A
polymeric conjugate comprising such a linker ensures, for example,
that a substantial amount of a conjugated moiety is released from
the conjugate only at the tumor tissue.
[0261] Exemplary such enzymes include, but are not limited to,
Cathepsins (cysteine proteases) such as Cathepsin B, Cathepsin K,
Cathepsin D, Cathepsin H, Cathepsin L, and Cathepsin S, legumain,
matrix metalloproteinases such as MMP-2 and MMP-9, as well as MMP1,
MMP3, MMP7, MMP13 and MMP14, KLK6 (kallikrein-related peptidase-6
which encodes a trypsin-like serine peptidase), PIM
serine/threonine kinases such as PIM 1, PIM 2 and PIM 3, histone
deacetylases (HDAC) such as HDAC1, HDAC2, HDAC3, HDAC6 AND
kdac8.
[0262] Suitable linking moieties having a Cathepsin K cleavable
site include amino acid sequences such as, but not limited to,
-[Asn-Glu-Val-Ala]- and -[Lys-Lys]-.
[0263] Suitable linking moieties having cathepsin-B cleavable sites
include amino acid sequences such as, but are not limited to,
-[Gly-Phe-Lys]-, -[Cit-Val]-, -[Arg]-, -[Arg-Arg]-, -[Val-Arg]-,
-[Phe-Lys]-, -[Phe-Arg]-, [Gly-Phe-Leu-Gly], -[Gly-Phe-Ala-Leu]-
and -[Ala-Leu-Ala-Leu]-, -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-,
-[Gly-Phe-Leu-Gly-Phe-Lys]-, -[(Glu).sub.6-(Asp).sub.8]- and
combinations thereof.
[0264] In some embodiments the linking moiety comprises the amino
acid sequences -[Gly-Phe-Lys]-, -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-,
-[Gly-Leu-Phe-Gly]-, -[Gly-Phe-Leu-Gly]-, -[Phe-Lys]- and
-[Gly-Phe-Leu-Gly-Phe-Lys]-. In some embodiments, the trigger unit
consists of these amino acid sequences or a combination thereof.
Suitable linking moieties having cathepsin-D cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Gly-Pro-Ile-Cys(Et)-Phe-Phe-Arg-Leu]-.
[0265] Suitable linking moieties having cathepsin-K cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Gly-Gly-Pro-Nle]-.
[0266] Suitable linking moieties having cathepsin-L cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Phe-Arg]-.
[0267] Suitable linking moieties having Legumain cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Ala-Ala-Asn]-, -[Asn-Glu-Val-Ala]- and
-[(Glu).sub.6-(Asp).sub.8]-, and any combination thereof.
[0268] Suitable linking moieties having MMP cleavable sites include
an amino acid sequence such as, but are not limited to,
-[Cys-Gly-Leu-Asp-Asp]-, -[Gly-Pro-Leu-Gly-Val]-,
-[Gly-Pro-Leu-Gly-Ala-Gly]-, -[Cys-Asp-Gly-Arg]-,
-[Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys]- and
-[Pro-Leu-Gly-Met-Thr-Ser]-, and any combination thereof. In some
embodiments, the linking moieties have only a part of the
above-described amino acid sequences. In some embodiments, the
linking moiety consists of 3 amino acids of the above-described
sequences.
[0269] Suitable linking moieties having MMP-2 and MMP-9 cleavable
sites include an amino acid sequence such as, but are not limited
to, -[Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln]-,
-[Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln]-,
-[Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln]-, -[Pro-Leu-Gly-Val-Arg]-,
[Pro-Leu-Gly-Leu-Tyr-Leu]-, -[Pro-Leu-Gly-Leu-Tyr-Ala-Leu]-,
-[Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln]-,
-[Gly-Pro-Leu-Gly-Leu-Trp-Ala-Gln]-,
-[Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys]-,
-[His-Pro-Val-Gly-Leu-Leu-Ala-Arg]-,
-[Gly-Gly-Pro-Leu-Gly-Leu-Trp-Ala-Gly-Gly]-,
-[Ala-Ala-Ala-Pro-Leu-Gly-Leu-Trp-Ala]- and combinations thereof.
In some embodiments, the linking moieties have only a part of the
above-described amino acid sequences. In some embodiments, the
linking moiety consists of 3 amino acids of the above-described
sequences.
[0270] Suitable linking moieties having MMP-7 cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Gly-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly-Cys]- and
-[Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser]- and combinations thereof. In
some embodiments, the linking moieties have only a part of the
above-described amino acid sequences. In some embodiments, the
linking moiety consists of 3 amino acids of the above-described
sequences.
[0271] Suitable linking moieties having MMP-13 cleavable sites
include an amino acid sequence such as, but are not limited to,
-[Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-Gly-Lys]-. In some embodiments,
the linking moieties have only a part of the above-described amino
acid sequence. In some embodiments, the linking moiety consists of
3 amino acids of the above-described sequence.
[0272] Suitable linking moieties having KLK6 cleavable sites
include amino acid sequences such as, but are not limited to,
-[Gly-Ala-Arg-Arg-Arg-Gly]-, -[Trp-Ala-Arg-Arg-Ser]-,
-[Trp-Ala-Arg-Lys -Arg]-, -[Leu-Arg-Lys -Arg-Trp]-, -[Ala-Lys
-Arg-Arg-Gly]-, abd -[Trp-Lys-Lys-Lys-Arg]. In some embodiments,
the linking moieties have only a part of the above-described amino
acid sequences. In some embodiments, the linking moiety consists of
3 amino acids of the above-described sequences. Suitable linking
moieties having PIM cleavable sites include an amino acid sequence
such as, but are not limited to,
-[(Arg/Lys).sub.3-AA.sub.1-[Ser/Thr-AA.sub.2]-, with AA.sub.1 and
AA.sub.2 being independently any amino acid residue except basic or
large hydrophobic residues. An exemplary amino acid sequence
include:
-[Ala-Arg-Lys-Arg-Arg-Arg-His-Pro-Ser-Gly-Pro-Pro-Thr-Ala]-.
[0273] Suitable linking moieties having HDAC cleavable sites
include acetylated Lysine.
[0274] Suitable linking moieties having caspase cleavable sites
include an amino acid sequence such as, but not limited to,
-[Asn-Glu-Val-Ala]-, -[(Glu).sub.6-(Asp).sub.8]-,
-[Asp-Glu-Val-Asp]-, and [Asp-Glu-Val-Asp-Ala-Pro-Lys]-.
[0275] In some embodiments, the linker is a Cathepsin B-cleavable
linker.
[0276] Cathepsin B is a lysosomal enzyme over-expressed in both
epithelial and endothelial tumor cells. Suitable exemplary linkers
having cathepsin-B cleavable sites include amino acid sequences
such as, but are not limited to, -[Gly-Phe-Leu-Gly]-,
-[Gly-Phe-Ala-Leu]-, -[Ala-Leu-Ala-Leu]-, -[Gly-Leu-Gly]-,
-[Gly-Phe-Gly]-, -[Gly-Phe-Leu-Gly-Phe-Lys]-, -[Cit-Val]-, -[Arg]-,
-[Arg-Arg]-, -[Phe-Lys]-, -[Val-Arg]-, -[Phe-Arg]-,
-[6-Glu-8-Asp]-, and any combination thereof.
[0277] In some embodiments the enzymatically cleavable linker is
cleaved by cathepsin K.
[0278] Cathepsin K is a lysosomal cysteine protease involved in
bone remodeling and resorption and is predominantly expressed in
osteoclasts. Its expression is stimulated by inflammatory cytokines
that are released after tissue injury and in bone neoplasms [Pan et
al. 2006, J Drug Target 14:425-435; Husmann et al. 2008, Mol
Carcinog 47: 66-73; Segal et al. PUS One 2009, 4(4):e5233].
[0279] A non-limiting example of a linker having cathepsin K
cleavable sites is -[Gly-Gly-Pro-Nle]-.
[0280] In some embodiments the linker comprises the amino acid
sequences -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-, -[Gly-Leu-Phe-Gly]-,
-[Gly-Phe-Leu-Gly]-, -[Phe-Lys]-, -[Gly-Phe-Leu-Gly-Phe-Lys]- and
-[Gly-Gly-Pro-Nle]-. In some embodiments, the linker consists of
these amino acid sequences or a combination thereof.
[0281] Other suitable linkers include, but are not limited to,
alkyl chains; alkyl chains optionally substituted with one or more
substituents and in which one or more carbon atoms are optionally
interrupted by nitrogen, oxygen and/or sulfur heteroatom. Other
suitable linkers include amino acids and/or oligopeptides.
[0282] Such alkyl chains and/or oligopeptides can optionally be
functionalized so as allow their covalent binding to the moieties
linked thereby (e.g., the polymeric backbone units and the
fluorogenic moiety, the polymeric backbone units and the
therapeutically active agent). Such a functionalization may include
incorporation or generation of reactive groups that participate in
such covalent bindings, as detailed hereinunder.
[0283] In some embodiment, the linker is a biodegradable
oligopeptide which contains, for example, from 2 to 10 amino acid
residues.
[0284] An oligopeptide linker which contains the pre-selected amino
acid sequence (recognition motif) can also be constructed such that
the recognition motif is repeated several times within the linker,
to thereby enhance the selective release of the attached agent or
moiety. Various recognition motifs of the same or different enzymes
can also be incorporated within the linker.
[0285] Similarly, the linker may comprise multiple pH sensitive
bonds or moieties. Linkers comprising such multiple cleavable sites
can enhance the selective release of the therapeutically active
agent at the desired bodily site, thereby reducing adverse side
effects, and further enhance the relative concentration of the
released drug at the bodily site when it exhibits its activity.
[0286] In some embodiments of the present invention, the
fluorogenic moiety and/or the therapeutically active agent(s)
is/are bound directly to the polymeric backbone units, whereby the
bond linking these moieties to the polymeric backbone is
biodegradable, for example, a hydrolysable bond, an
enzymatically-cleavable bond or a pH-sensitive bond. Such a bond
can be formed upon functionalizing the polymeric backbone units,
the fluorogenic moiety and/or the therapeutically active agent, so
as to include compatible reactive groups, as defined herein, for
forming the required bond. Such a bond is also referred to herein
as a cleavable or biocleavable linker.
[0287] According to some embodiments, the biodegradable linker is a
pH-sensitive linker or an enzymatically-cleavable linker.
[0288] A pH-sensitive linker comprises a chemical moiety that is
cleaved or degraded only when subjected to a certain pH condition,
such as acidic pH (e.g., lower than 7), neutral pH (6.5-7) or basic
pH (higher than 7).
[0289] Such a linker may, for example, be designed to undergo
hydrolysis under acidic or basic conditions, and thus, the
conjugate remains intact and does not release the agents or
moieties attached to the polymeric backbone in the body, until it
reaches a physiological environment where a pH is either acidic or
basic, respectively.
[0290] Exemplary pH-sensitive linkers include, but are not limited
to, a hydrazone bond, ester (including orthoester) bond, amide bond
of cis-aconytil residue, a trityl group, acetals, ketals, Gly-ester
and a -[Gly-Phe-Gly]- moiety.
[0291] The peptide linker may also include a peptide sequence which
serves to increase the length of the linker. Longer peptides may be
advantageous due to a more efficient steric interaction of the
linker with the cleaving enzyme due to enhanced accessibility.
[0292] In some embodiments, the linker is
-[Gly-Phe-Leu-Gly-Phe-Lys]-. Such a linker comprises two
"recognition motifs" of Cathepsin B, and a cleavage thereof so as
to release the moiety attached thereto is effected only in the
presence of high enzyme concentration. This feature enhances the
selective release of the attached moiety at a site where the enzyme
is over-expressed.
[0293] In some embodiments, the linker is -[Gly-Phe-Leu-Gly]-.
[0294] In some embodiments, the linker is or comprises
-[Phe-Lys]-.
[0295] In some of any of the embodiments described herein, the
first and second cleavable linking moieties, if present, are the
same or are cleavable by the same chemical event.
[0296] A spacer:
[0297] In some of any of the embodiments described herein, the
fluorogenic moiety is attached to the first cleavable linker via a
spacer.
[0298] In some of any of the embodiments described herein the first
cleavable linker is attached to the respective backbone units via a
spacer.
[0299] In some of any of the embodiments described herein, the
therapeutically active agent is linked to the respective polymeric
backbone units and/or to the second cleavable linker via a spacer.
The spacers can be the same or different.
[0300] In some of the embodiments described herein, the quenching
agent, if present, is linked to the respective polymeric backbone
units via a spacer.
[0301] In some of the embodiments described herein, when the
quenching agent forms a part of the fluorogenic moiety, it is
linked to the fluorescent moiety via a spacer.
[0302] In some of the embodiments described herein, when the
therapeutically active agent forms a part of the fluorogenic
moiety, it is linked to the fluorescent moiety via a spacer.
[0303] The term "spacer" as used herein describes a chemical moiety
that is covalently attached to, and interposed between, two other
moieties, or a moiety/agent and a polymeric backbone unit, thereby
forming a bridge-like.
[0304] In some cases, a spacer is utilized for enabling a more
efficient and simpler attachment of the fluorogenic moiety and/or
therapeutically active agent and/or quenching agent to the
polymeric backbone units or linker or to one another, in terms of
steric considerations (e.g., renders the site of the polymeric
backbone to which coupling is effected less hindered) or chemical
reactivity considerations (adds a compatible reactive group to
facilitate coupling). In some cases, the spacer may contribute to
the performance of the resulting polymeric conjugate. For example,
the spacer may render an enzymatically cleavable linker less
sterically hindered and hence more susceptible to enzymatic
interactions.
[0305] In some embodiments, the spacer facilitates the attachment
of the moiety or agent to the polymeric backbone units or the
linker. This may be effected by imparting a reactive group to the
moiety to be attached, which is chemically compatible with
functional groups in the polymeric backbone units and/or the linker
attached to the polymeric backbone, and/or by modifying the
solubility of the moiety to be attached to the polymer, so as to
facilitate the reaction between the polymer (or co-polymer) and the
moiety.
[0306] Suitable spacers include, but are not limited to, alkylene
chains, optionally substituted by one or more substituents and
which are optionally interrupted by one or more nitrogen, oxygen
and/or sulfur heteroatom.
[0307] Other suitable spacers include amino acids and amino acid
sequences, optionally functionalized with one or more reactive
groups for being coupled to the respective portion or moiety of the
polymeric conjugate.
[0308] In some embodiments, a spacer has the formula
G-(CH.sub.2)n-K, wherein n is an integer from 1 to 10; and G and K
are each a reactive group such as, for example, NH, O or S. In some
embodiments, G and K are each NH and n is 2.
[0309] An exemplary spacer is --[NH--(CH.sub.2).sub.mNH.sub.2]--
wherein "m" stands for an integer ranging from 1-10. Preferably m
is 2.
[0310] In some embodiments, the spacer is or comprises an amino
acid sequence, optionally an inert amino acid sequence (namely,
does not affect the affinity or selectivity of the polymeric
conjugate). Such a spacer can be utilized for elongating or
functionalizing the linker.
[0311] Exemplary such sequences include, for example,
-[Gly-Gly-].
[0312] In some embodiments, the spacer is a degradable spacer,
which is capable of undergoing degradation reactions so as to
release an agent attached thereto. In some embodiments, the spacer
is biodegradable, as defined herein.
[0313] In some embodiments the spacer is a substituted or
unsubstituted aryl group and substituted or unsubstituted
heteroaryl group whereby the substituents can be carbonate,
C-amido, N-amido and amine, whereby the spacer may be linked to the
desired agent or moiety or to the polymeric backbone units either
directly, through the aromatic group or alternatively, via one or
more of the substituents.
[0314] In some embodiments, the spacer is a degradable spacer
selected such that it undergoes a spontaneous degradation once it
is cleaved from the polymeric conjugate. Such spacers are also
referred to herein as self-immolative spacers.
[0315] Such a spacer can be, for example, attached to a
biodegradable linker at one end and to another moiety or agent
(e.g., the fluorogenic moiety) at another end, such that once the
biodegradable linker is cleaved, so as to release the spacer and
the moiety attached thereto, the spacer undergoes a spontaneous
degradation so as to release the moiety attached thereto.
[0316] Exemplary spacers that can undergo such a spontaneous
degradation include, but are not limited, chemical moieties that
can undergo a spontaneous 1,4-, 1,6-, 1,8-, etc. elimination, via a
cascade of immolative electronic reactions. Such chemical groups
are known in the art, or, otherwise, can be devised by those
skilled in the art.
[0317] In an exemplary embodiment, the spacer is such that can
undergo a spontaneous 1,6-benzyl elimination. An example of such a
spacer is p-aminobenzyl carbonate (PABC).
[0318] In some embodiments, the spacer comprises one or more of the
exemplary spacers described herein.
[0319] In some embodiments, a spacer is used to connect 3 moieties
to one another, or to connect 2 moieties to a cleavable linking
moiety or to respective polymeric backbones.
[0320] For example, a spacer can connect a fluorescent moiety and a
quenching agent and/or a therapeutically active agent to one
another, so as to form a fluorogenic moiety, and to connect the
fluorogenic moiety to a cleavable linking moiety. Such a spacer can
include a bi- or tri-functional moiety (e.g., an aryl moiety as
described herein), which is also referred to herein as a branching
spacer. Such a spacer can combine also spacers which are attached
to the branching spacer, and connect the moieties/agents to the
branching spacer unit.
[0321] Exemplary such multi-functional spacers are shown, for
example, in FIGS. 31A-B, 34, 35, 39 and 42.
[0322] The spacer may be varied in length and in composition. A
spacer as defined herein encompasses also any combination of the
spacers described herein.
[0323] The following describes exemplary polymeric conjugates and
polymeric systems comprising same according to some embodiments of
the present invention.
[0324] The First Polymeric Moiety:
[0325] According to an aspect of some embodiments of the present
invention there is provided a polymeric conjugate, also referred to
herein as a first polymeric moiety or a first polymeric conjugate,
which comprises a (first) polymeric backbone composed of a
plurality of backbone units and having attached to at least a
portion of the backbone units a fluorogenic moiety, as described
herein, the fluorogenic moiety being attached to the portion of
backbone units via a cleavable linking moiety such that upon
cleavage of the linking moiety, a fluorescent signal is generated
(e.g., upon release and/or generation of a fluorescent moiety).
[0326] According to some embodiments of this aspect of the
invention, the fluorescent moiety emits near infrared light.
[0327] According to some embodiments of this aspect of the
invention, the fluorescent moiety is a cyanine dye.
[0328] According to some of any of the embodiments of the
invention, the first cleavable linking moiety is a first
biocleavable linking moiety, as described herein.
[0329] In some of any of the embodiments described herein, the
fluorogenic moiety is such that when it is attached to the
polymeric backbone via the first cleavable linking moiety, it does
not emit light, whereby when the first linking moiety is cleaved,
the generated fluorescent moiety emits light (e.g., near infrared
light), thus featuring a Turn-ON mechanism, as described
herein.
[0330] According to some of any of the embodiments described
herein, the fluorogenic moiety is attached to the cleavable linking
moiety via a spacer, as described in any of the respective
embodiments.
[0331] According to some embodiments, the spacer is selected
degradable such that it allows releasing or generating the
fluorescent moiety, as described herein, upon cleavage of the
linking moiety.
[0332] According to some embodiments, the spacer is selected
degradable such that it allows generating the fluorescent signal
upon cleavage of the linking moiety.
[0333] Spacers usable in the context of a fluorogenic moiety can be
selected to act via ICT or FRET mechanism, as described herein.
[0334] In some of any of the embodiments described herein, the
first polymeric moiety further comprises a quenching agent, either
attached to one or more polymeric backbone units of the first
polymeric backbone or forming a part of the fluorogenic moiety, as
described herein.
[0335] In some of any of the embodiments described herein, the
fluorogenic moiety is a fluorescent moiety, which is attached
directly or via a spacer (e.g., a degradable or self-immolative
spacer as described herein) to the cleavable linking moiety.
[0336] In some of any of the embodiments described herein, the
first polymeric moiety is a homo-FRET system, and is devoid of a
quenching agent.
[0337] In some of these embodiments, a loading of the fluorescent
moiety is such that allows self-quenching, namely, the loading
results in a distance between the fluorescent moieties attached to
the backbone units which is up to 100 angstroms.
[0338] In some of these embodiments, the loading of the fluorescent
moiety is at least 1 mol %, preferably at least 2 mol %, at least 3
mol % or at least 4 mol % and/or ranges from 1 to 10 mol %.
[0339] In some of any of the embodiments described herein, the
first polymeric moiety is a pair-FRET system, which further
comprises a quenching agent, as described herein.
[0340] In some of these embodiments, the quenching agent is
attached to the respective backbone units via a spacer, for
example, a non-degradable spacer, such as, for a non-limiting
example, a spacer that comprises a -[Gly-Gly]- moiety.
[0341] In some of any of the embodiments described herein, the
quenching agent is attached to a terminal backbone unit of the
polymeric backbone. In some of these embodiments, the polymeric
backbone is functionalized or is designed so as to feature a
reactive group, or a spacer featuring a reactive group, for
attaching the quenching agent. An exemplary such group can be
generated while synthesizing a HPMA copolymer by RAFT
polymerization, as exemplified in the Examples section that
follows.
[0342] A first polymeric moiety as described herein can be
represented by Formula IA:
##STR00002##
[0343] wherein:
[0344] A.sub.1, A.sub.2 and A.sub.4 are polymeric backbone units as
described herein;
[0345] L.sub.2 is the first cleavable lining moiety, as described
herein,
[0346] S.sub.2 is a first spacer, linking the fluorogenic moiety to
L.sub.2, as described herein, or is absent;
[0347] L.sub.4 is a third linking moiety, linking the quenching
agent to the backbone units, and which can be cleavable or
non-cleavable, or is absent;
[0348] S.sub.4 is a third spacer linking the quenching agent to the
linking moiety, or is absent;
[0349] F is a fluorogenic moiety as described in any one of the
respective embodiments herein;
[0350] Q is a quenching agent as described in any one of the
respective embodiments herein;
[0351] w is an integer having a value such that w/(x+s+w)
multiplied by 100 is in the range of from 0 to 99.9;
[0352] x is an integer having a value such that x/(x+s+w)
multiplied by 100 is in the range of from 0.1 to 100; and
[0353] s is an integer having a value such that s/(x+s+w)
multiplied by 100 is in the range of from 0 to 99.9.
[0354] Each [A.sub.2-L.sub.2-S.sub.2-F] independently represents a
backbone unit having attached thereto the fluorogenic moiety;
and
[0355] Each [A.sub.4-L.sub.4-S.sub.4-Q] independently represents a
backbone unit having attached thereto the quenching agent.
[0356] Optionally, the polymeric moiety further comprises backbone
units -[A.sub.5]z- as described herein, wherein z is an integer
having a value such that z/(x+s+w+z) multiplied by 100 is in the
range of from 0 to 99.9.
[0357] When z is other than 0, w, x and s in Formula I are divided
by "x+w+z+s" instead of "x+s+w".
[0358] In some of any of the embodiments described herein: [0359] w
is an integer having a value such that w/(x+s+w) multiplied by 100
is in the range of from 0.1 to 99.9, or from 10 to 99.9, or from 20
to 99.9, or from 30 to 999, or from 40 to 99.9, or from 50 to 99.9,
or from 60 to 99.9, or from 70 to 99.9, or as further described
herein; [0360] x is an integer having a value such that x/(x+s+w)
multiplied by 100 is in the range of from 0.1 to 99.9, or from 0.1
to 20, or from 0.1 to 15, or from 1 to 15, or from 2 to 15, or from
3 to 15 or from 4 to 15, as is further described herein; and [0361]
s is an integer having a value such that s/(x+s+w) multiplied by
100 is in the range of from 0 to 100, or from 0 to 20, or from 0 to
15, or from 0 to 1, as is further described herein.
[0362] In some embodiments, s is 0, and the polymeric moiety is a
homo-FRET system, as described herein.
[0363] In some embodiments, s is a positive integer and is such
that a single molecule of a quenching agent is attached to the
polymeric backbone. In some of these embodiments, A.sub.4 is a
terminal backbone unit in the polymeric backbone.
[0364] In some embodiments, s is a positive integer and is in a
ratio to x which is in a range of from 20:1 to 1:20, or from
10:1:10, or from 5:1 to 1:5, or from 2:1 to 1:2, or is 1:1,
including any subratios therebetween.
[0365] In some of these embodiments, each of the backbone units
A.sub.1, A.sub.2 and A.sub.4 can be a terminal unit, attached to
one other unit, or is attached to two other units, which can be the
same of different.
[0366] In some of any of the embodiments described herein, the
fluorogenic moiety is, or comprises a fluorescent moiety, as
described herein.
[0367] The fluorescent moiety is also referred to herein as F*.
[0368] In some of any of the embodiments described herein, the
fluorogenic moiety is, or comprises a fluorescent moiety which is,
or comprises, a cyanine dye, or a cyanine-like moiety, as described
herein.
[0369] In cyanine-like dye molecules, one nitrogen is positively
charged (e.g., in a form of an ammonium ion) and one nitrogen atom
is neutral (e.g., in a form of an amine) and thus has a lone pair
of electrons. The positive charge in cyanine-like dyes therefore
resonates between the two nitrogen atoms via the polymethine
chain.
[0370] In some of these embodiments, the fluorogenic moiety is
represented by, or comprises, a moiety represented by, formula
II:
##STR00003##
wherein:
[0371] Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heterocylic moiety;
[0372] R.sub.1 is hydrogen, a substituted or unsubstituted alkyl or
a substituted or unsubstituted cycloalkyl;
[0373] n is an integer of from 1 to 10; and
[0374] R' and R'' are each independently hydrogen, a substituted or
unsubstituted alkyl and a substituted or unsubstituted cycloalkyl,
or, alternatively, R' and R'' form together an aryl.
[0375] In cyanine-like fluorescent moiety, two heterocylic moieties
are linked therebetween via a substituted or unsubstituted
polymethine chain, such that one heterocylic moiety acts as a donor
moiety (Z.sub.2) and one acts as an acceptor moiety (Z.sub.1).
[0376] The phrase "polymethine chain" describes a chain of methine
groups (e.g., --CH.dbd.CH-- groups) each can independently be
substituted, as long as the substituent does not interfere with the
optical properties of the disclosed moiety, as defined herein.
[0377] The polymethine-containing moiety forms a chain that can
comprise from 2 to 13 carbon atoms, preferably from 2 to 7 carbon
atoms.
[0378] Exemplary heterocyclic moieties suitable for inclusion in
the fluorogenic compounds disclosed herein as donor moieties
include, but are not limited to, imidazoline ring, imidazole ring,
benzimidazole ring, .alpha.-naphthoimidazole ring,
.beta.-naphthoimidazole ring, indole ring, isoindole ring,
indolenine ring, isoindolenine ring, benzindolenine ring,
pyridinoindolenine ring, oxazoline ring, oxazole ring, isoxazole
ring, benzoxazole ring, pyridinooxazole ring, .alpha.-naphthoxazole
ring, .beta.-naphthoxazole ring, selenazoline ring, selenazole
ring, benzoselenazole ring, .alpha.-naphthselenazole ring,
.beta.-naphthselenazole ring, thiazoline ring, thiazole ring,
isothiazole ring, benzothiazole ring, .alpha.-naphthothiazole ring,
.beta.-naphthothiazole ring, tellulazoline ring, tellulazole ring,
benzotellulazole ring, .alpha.-naphthotellulazole ring,
.beta.-naphthotellulazole ring, isoquinoline ring, isopyrrole ring,
imidaquinoxaline ring, indandione ring, indazole ring, indoline
ring, oxadiazole ring, carbazole ring, xanthene ring, quinazoline
ring, quinoxaline ring, thiodiazole ring, thiooxazolidone ring,
tetrazole ring, triazine ring, naphthyridine ring, piperazine ring,
pyrazine ring, pyrazole ring, pyrazoline ring, pyrazolidine ring,
pyrozolone ring, pyridine ring, pyridazine ring, pyrimidine ring,
pyrylium ring, pyrrolidine ring, pyrroline ring, pyrrole ring,
phenazine ring, phenanthridine ring, phthalazine ring, furazan
ring, benzoxazine ring, morpholine ring, and rhodanine ring.
[0379] Acceptor moieties can be an ammonium form of any of the
foregoing.
[0380] In some embodiments, Z.sub.1 and Z.sub.2 are each
independently a substituted or unsubstituted heteroaryl, whereby
the acceptor moiety is positively charged.
[0381] In some embodiments, one of acceptor and donor moieties
comprises a pyridine ring and the other is an indolenine ring, as
defined herein.
[0382] In some embodiments, the donor moiety is an indolenine ring,
and the acceptor moiety is an ammonium form thereof.
[0383] The phrase "indolenine ring" describes a ring having an
aromatic portion having fused thereto a 5-membered aromatic.
[0384] In some embodiments, the fluorogenic moiety is, or
comprises, a moiety represented by Formula IIA:
##STR00004##
wherein:
[0385] Y.sub.1 and Y.sub.2 are each independently a substituted or
unsubstituted aromatic moiety; and
[0386] X.sub.1 and X.sub.2 are each independently selected from the
group consisting of CR.sub.3R.sub.4, NR.sub.3, and S, wherein
R.sub.3 and R.sub.4 are each independently selected from the group
consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic,
heteroaryl, alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy,
thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide,
carboxy, sulfonyl, sulfoxy, sulfinyl, sulfonamide, and a
saccharide.
[0387] Whenever the carbon, nitrogen or sulfur representing X in
the above formula, are substituted, the substituents can be
independently an alkyl, cycloalkyl, alkyl, cycloalkyl, aryl,
heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy,
thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo,
trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl,
sulfonamide, and a saccharide.
[0388] In some embodiments, Y.sub.1 and Y.sub.2 are each a
substituted or unsubstituted phenyl.
[0389] In some embodiments, X.sub.1 and X.sub.2 are each
independently CR.sub.3R.sub.4.
[0390] In some embodiments, R.sub.3 and R.sub.4 are each an
alkyl.
[0391] In some embodiments, R.sub.1 is hydrogen or alkyl.
[0392] In some embodiments, the fluorogenic moiety is represented
by, or comprises, a moiety represented by, Formula IIB:
##STR00005##
wherein:
[0393] g and k are each independently an integer of from 0 to 5;
and
[0394] R.sub.2 and R.sub.3 are each independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl,
heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy,
thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo,
trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl,
sulfonamide, and a saccharide.
[0395] In some embodiments, R.sub.1 is hydrogen or a substituted or
unsubstituted alkyl. In some embodiments, one or both R.sub.2 and
R.sub.3 is an alkyl substituted by a sulfonyl or sulfinyl.
[0396] It is to be noted that fluorogenic compounds in which one or
more of the indolenine-like rings is replaced by any of the
acceptor or donor moieties described herein, for example, any of
the ammonium acceptor moieties described herein (e.g., a pyridinium
moiety), are also contemplated.
[0397] In some of any of the embodiments described herein, the
quenching agent forms a part of the fluorogenic moiety.
[0398] In some of these embodiments, s is 0.
[0399] In some embodiments, a FRET-based modular system is used as
a fluorogenic moiety, in which both the fluorescent moiety and the
quenching agent are linked to one another, and/or to the first
cleavable linking moiety.
[0400] In some embodiments, such a system can be generally
represented, according to some embodiments of the invention, by
formulae III or III*:
##STR00006##
[0401] wherein F* is a fluorescent moiety, as described herein; Q
is the quenching agent;
[0402] S' and S''' (if present) are each independently a spacer,
preferably one or more of which is degradable, or absent; and
[0403] S'' is a multifunctional spacer, as described herein, for
example, a self-immolative spacer, which connects the fluorogenic
moiety to the first cleavable moiety, or to an additional spacer
which is connected to the cleavable linking moiety.
[0404] In some embodiments, at least S'' in Formula III is a
degradable spacer, e.g., a self-immolative spacer, as described
herein, which, upon cleavage of the linking moiety, degrades so as
to no longer have the quenching agent linked to the fluorescent
moiety. As a result, a fluorescent signal is generated.
[0405] In some embodiments, at least S'' and S' in Formula III* is
a degradable spacer, e.g., a self-immolative spacer, as described
herein, which, upon cleavage of the linking moiety, degrades so as
to no longer have the fluorescent moiety linked to the quenching
agent and to the polymeric moiety. As a result, a fluorescent
signal is generated.
[0406] In some of any of the embodiments described herein, the
fluorescent moiety is a cyanine-like structure or moiety, as
described herein, and the fluorogenic moiety can represented by one
or more of the following formulae:
##STR00007##
[0407] wherein:
[0408] Z.sub.1, Z.sub.2, R', R'' and n are as described herein for
Formula II, and can form any of the cyanine structures depicted and
described herein in Formulae IIA and IIB; and
[0409] R.sub.1 and R.sub.2, if present, are each independently
hydrogen, a substituted or unsubstituted alkyl or a substituted or
unsubstituted cycloalkyl;
[0410] In some embodiments, Z.sub.1 and Z.sub.2 are each
independently a substituted or unsubstituted heterocylic moiety, as
described herein.
[0411] Exemplary such system is presented in FIG. 39.
[0412] Herein throughout, the curled line indicates an attachment
point to the first cleavable linking moiety, either directly, or
via spacer (e.g., degradable spacer, as described herein).
[0413] In some of any of the embodiments described herein, an ICT
modular system is used as a fluorogenic moiety. In these
embodiments, the fluorogenic moiety is a modified fluorescent
moiety which exhibits reduced fluorescence, as described herein,
due to an alteration in its chemical structure. Upon cleavage of
the linking moiety, the fluorogenic undergoes rearrangement and a
fluorescent moiety is generated. Thus, a fluorescent signal is
generated.
[0414] In some of these embodiments, s is 0.
[0415] In some of these embodiments, a cyanine-like fluorogenic
moiety as described herein has a chemical arrangement which is
different from cyanine dyes (e.g., a delocalized .pi.-electrons
system), and hence the fluorogenic moiety is spectroscopically
silenced in the NIR range before activation by said cleavage.
[0416] In some embodiments, such a fluorogenic moiety is
represented by Formula IV:
##STR00008##
[0417] wherein:
[0418] Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heterocylic moiety, as described in any one of the
embodiments herein for an acceptor moiety of a cyanine-like
structure;
[0419] R.sub.1 and R.sub.2 are each independently hydrogen, a
substituted or unsubstituted alkyl or a substituted or
unsubstituted cycloalkyl;
[0420] m and n are each independently an integer of from 0 to
4;
[0421] R' and R'' are each independently hydrogen, a substituted or
unsubstituted alkyl and a substituted or unsubstituted cycloalkyl,
or, alternatively, R' and R'' form together an aryl, as described
herein; and
[0422] A is a donor-containing moiety, which, when attached to
A.sub.2 via said first cleavable linking moiety, and optionally
also via a spacer (e.g., a degradable spacer) interferes with a
conjugation of .pi. electrons between Z.sub.1 and Z.sub.2, and upon
cleavage, participates in said conjugation of .pi. electrons.
[0423] The curled line denotes an attachment point.
[0424] As shown in Formula IV, both Z.sub.1 and Z.sub.2 moieties
are acceptor moieties, and the donor moiety A is inactivated by its
linkage to the linking moiety. Thus, the conjugation of .pi.
electrons in the moiety is disrupted. Once the linking moiety is
cleaved, the donor-containing moiety functions as a donor, and a
conjugated .pi. electron system is generated, resulting in a
fluorescent moiety and generation of a fluorescent signal.
[0425] Unlike cyanine dyes, in the modified cyanine structures
disclosed herein, the presence of a donor moiety interferes with
the resonance (the delocalization of .pi. electrons) between the
two nitrogen atoms, and both nitrogen atoms are positively charged
(e.g., in a form of an ammonium ion). As such, there is no
delocalization of .pi.-electrons (no resonating electrons) between
the nitrogen-containing moieties.
[0426] Thus, in some embodiments, the fluorogenic moiety disclosed
herein has a cyanine-like structure, modified so as to include two
positively charged nitrogen (e.g., ammonium)-containing moieties
(instead of two nitrogen-containing moieties with one positive
charge resonating therebetween) and a donor moiety that forms a
conjugated .pi.-electron system with the two ammonium-containing
moieties, whereby the donor-moiety interferes with the
delocalization of the .pi.-electrons system of a non-modified
cyanine-like molecule, by restricting delocalization of
.pi.-electrons to portions of the molecule that do not involve the
nitrogen-containing moieties, and thus reduces or abolishes the
delocalization of the positive charge that is present in
non-modified cyanine-like molecules.
[0427] Such a fluorogenic moiety is designed such that upon the
cleavage of the first linking moiety, delocalization of the
positive charge is restored.
[0428] Thus, the fluorogenic moieties described in these
embodiments follow a design in which the inclusion of the donor
moiety results in delocalization of .pi. electrons through a
smaller portion of the molecule (smaller number of overlapping
p-orbitals), as compared to non-modified cyanine structures, and
hence the moiety is incapable of interacting with light so as to
emit NIR light.
[0429] The fluorogenic moiety disclosed herein, however, further
follows a design in which upon the cleavage of the linking moiety,
rearrangement of the donor moiety occurs and results in a structure
in which .pi. electrons are relocalized such that one of the
ammonium-containing moieties becomes an amine-containing moiety,
and thus a resonating positive charge between two
nitrogen-containing moieties, as in cyanine dyes, is restored. The
.pi. electrons relocalization thus results in a moiety that has
spectroscopic behavior similar to cyanine dyes, and is thus capable
of emitting NIR light.
[0430] Accordingly, the cyanine-based fluorogenic moieties
described in these embodiments are designed after known cyanine
dyes, by having two nitrogen-containing moieties and a
carbomethine-containing chain linking therebetween, yet differ from
cyanine dyes by the presence of two positively charged (e.g.,
ammonium) nitrogen-containing moieties (instead of one positively
charged nitrogen-containing moiety), and further by the presence of
a donor moiety as described herein. Fluorogenic moieties which are
equivalent to such fluorogenic moieties, but in which the
donor-containing moiety is attached to cleavable moiety Y are
disclosed in WO 2012/123916. Each of these moieties can be used in
these embodiments, upon the modification explained herein.
[0431] An exemplary such a system is presented in FIG. 41.
[0432] Any of the fluorogenic moieties described herein can be
attached to a terminal or non-terminal backbone unit of the first
polymeric backbone.
[0433] In some of any of the embodiments described herein, the
backbone units form a polymeric backbone of a HPMA copolymer, as
described herein.
[0434] In some of these embodiments, the polymeric moiety can be
represented by Formula IA:
##STR00009##
[0435] wherein the variables are as defined herein, and R
represents a reactive group, as described herein.
[0436] In some of these embodiments, x and s are other than 0.
[0437] In some of these embodiments, the backbone units containing
the quenching agent is a terminal backbone unit, and the quenching
agent Q is attached to the backbone unit via a spacer
(S.sub.4).
[0438] In some of any of the embodiments described herein, the
backbone units form a polymeric backbone of a PGA polymer, as
described herein.
[0439] In some of these embodiments, the polymeric moiety can be
represented by Formula IB:
##STR00010##
wherein the variables are as defined herein.
[0440] In some of these embodiments, x and s are other than 0.
[0441] In some of any of the embodiments described herein, the
backbone units form a polymeric backbone of a PEG polymer, as
described herein. In some of these embodiments, the quenching agent
forms a part of the fluorogenic moiety, and the fluorogenic moiety
is attached to a terminal backbone unit of the polymer.
[0442] A Second Polymeric Moiety:
[0443] According to some of any of the embodiments described
herein, the fluorogenic moiety is attached to one portion of the
backbone units and the therapeutically active agent is attached to
another portion of the backbone units.
[0444] According to some of any of the embodiments described
herein, the system further comprises a second polymeric moiety
comprising a second polymeric backbone composed of a plurality of
backbone units and having attached to at least a portion of the
backbone units a therapeutically active agent.
[0445] According to some of any of the embodiments described
herein, the therapeutically active agent is attached to the
backbone units via a second cleavable linking moiety.
[0446] According to some of any of the embodiments described
herein, the second linking moiety is a biocleavable linking
moiety.
[0447] According to some of any of the embodiments described
herein, the second linking moiety is an enzymatically-cleavable
linking moiety.
[0448] According to some of any of the embodiments described
herein, the first and second cleavable linking moieties are the
same or are cleavable by the same mechanism (e.g., the same
enzyme).
[0449] In some embodiments, the therapeutically active is attached
to the backbone units or to the linking moiety, if present, via a
spacer, as described in any one of the respective embodiments.
[0450] In some embodiments, the backbone units in the second
polymeric backbone form a HPMA copolymer backbone.
[0451] In some embodiments, the backbone units in the second
polymeric backbone form a PGA polymer backbone.
[0452] The backbone units if the second polymeric backbone can be
the same or different from the backbone units of first polymeric
backbone, and are preferably the same.
[0453] A System with a Single Polymeric Backbone:
[0454] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, or is attached to the first cleavable linking
moiety, such that upon the cleavage of the first linking moiety, as
described herein, the therapeutically active agent is released.
According to some embodiments, upon the cleavage, a fluorescent
moiety is generated, as described herein.
[0455] According to some of any of the embodiments described
herein, the fluorescent moiety is or comprises a cyanine dye, as
described herein.
[0456] According to some of any of the embodiments described
herein, the therapeutically active agent is attached to the first
linking moiety, preferably via a degradable spacer, as described
herein.
[0457] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, and the fluorogenic moiety is represented by
Formula VIA, VIB, VIC, or VID, as depicted herein.
##STR00011##
wherein:
[0458] Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heterocylic moiety, as described herein;
[0459] R.sub.1 and R.sub.2 are each independently hydrogen, a
substituted or unsubstituted alkyl or a substituted or
unsubstituted cycloalkyl;
[0460] n is an integer of from 1 to 10;
[0461] R' and R'' are each independently hydrogen, a substituted or
unsubstituted alkyl and a substituted or unsubstituted cycloalkyl,
or, alternatively, R' and R'' form together an aryl;
[0462] S', S'' and S' are each independently a degradable spacer,
or absent, as described herein for Formulae IIIA, IIIB, IIIC and
IIID; and
[0463] D is the therapeutically active agent,
[0464] wherein the curled line indicates an attachment point.
[0465] According to some of any of the embodiments described
herein, both the therapeutically active agent and the quenching
agent form a part of the fluorogenic moiety, and the fluorogenic
moiety is represented by Formula IIIA, IIIB, IIIC or IIID, and
wherein the therapeutically active agent is attached to one of the
spacers shown therein or to the cleavable linking moiety.
[0466] According to some of any of the embodiments described
herein, the therapeutically active agent forms a part of the
fluorogenic moiety, and the fluorogenic moiety is represented by
Formula IV, wherein the therapeutically active is attached to the
donor moiety or to the cleavable linking moiety.
[0467] A polymeric system according to some of these embodiments
can be represented by Formula I:
##STR00012##
[0468] wherein:
[0469] D is a therapeutically active agent, as described
herein;
[0470] F is a fluorogenic moiety as described in any one of its
respective embodiments; Q is a quenching agent, as described in any
one of its respective embodiments;
[0471] L.sub.2 is said first linking moiety;
[0472] L.sub.3 is said second linking moiety or absent;
[0473] L.sub.4 is a linking moiety linking the quenching agent, as
described herein, or absent;
[0474] each of S.sub.2, S.sub.3 and S.sub.4 is independently a
spacer, as described in any one of its respective embodiments, or
absent;
[0475] w is an integer having a value such that w/(x+y+w+s)
multiplied by 100 is in the range of from 0 to 99.9;
[0476] x is an integer having a value such that x/(x+y+w+s)
multiplied by 100 is in the range of from 0.1 to 100;
[0477] y is an integer having a value such that y/(x+y+w+s)
multiplied by 100 is in the range of from 0 to 100; and
[0478] s is an integer having a value such that s/(x+y+w+s)
multiplied by 100 is in the range of from 0 to 99.9.
[0479] A.sub.1, A.sub.2, A.sub.3 and A.sub.4 are each backbone
units covalently linked to one another and forming a polymeric
backbone,
[0480] such that each [A.sub.3-L.sub.3-S.sub.3-D] independently
represents a backbone unit having attached thereto said
therapeutically active agent;
[0481] each [A.sub.2-L.sub.2-S.sub.2-F] independently represents a
backbone unit having attached thereto said fluorogenic moiety;
and
[0482] each [A.sub.4-L.sub.4-S.sub.4-Q] independently represents a
backbone unit having attached thereto said quenching agent.
[0483] According to some embodiments, A.sub.4 is a terminal
backbone unit, as described herein.
[0484] According to some embodiments, the quenching agent forms a
part of the fluorogenic moiety, as described herein, in which case,
"s" is 0.
[0485] According to some embodiments, the therapeutically active
agent forms a part of the fluorogenic moiety, as described herein,
in which case, "y" is 0.
[0486] According to some embodiments, the polymeric system further
comprises backbone units A.sub.5 as described herein, and the mol
percent is defined accordingly, as shown herein for Formula IA.
[0487] According to some of any of the embodiments described
herein, the backbone units form a polymeric backbone of HPMA
co-polymer.
[0488] In some of these embodiments, the polymeric system is
represented by the Formula 1A as described herein, and further
comprises suitable backbone units comprising the therapeutically
active agent, as described herein.
[0489] According to some of any of the embodiments described
herein, the backbone units form a polymeric backbone of a PGA
polymer.
[0490] In some of these embodiments, the polymeric system is
represented by the Formula 1B as described herein, and further
comprises suitable backbone units comprising the therapeutically
active agent, as described herein.
[0491] Processes of Preparing a Polymeric System:
[0492] According to an aspect of some embodiments of the present
invention there is provided a process of preparing a polymeric
system which comprises a first and a second polymeric moieties, as
described herein, the process comprising:
[0493] conjugating the fluorogenic compound to a first polymeric
backbone in which at least a portion of the backbone units have the
first cleavable linking moiety attached thereto and terminate with
a first reactive group, thereby preparing the first polymeric
backbone; and
[0494] conjugating the therapeutically active agent to a second
polymeric backbone in which at least a portion of the backbone
units terminate with a second reactive group, thereby preparing the
second polymeric backbone.
[0495] According to some embodiments, the therapeutically active
agent is attached to the second polymeric backbone via a cleavable
linking moiety, the process comprising conjugating the
therapeutically active agent to a second polymeric backbone in
which the portion of the backbone units have the second cleavable
linking attached thereto, thereby preparing the second polymeric
backbone.
[0496] According to some embodiments, conjugating the
therapeutically active agent and/or to the fluorogenic moiety to
the backbone units comprises attaching a spacer to the
therapeutically active agent and/or to the fluorogenic moiety, and
conjugating the spacer to the backbone units.
[0497] According to an aspect of some embodiments of the present
invention there is provided a process of preparing a polymeric
system which comprises a single polymeric moiety, the process
comprising:
[0498] polymerizing a first plurality of monomers, at least one
portion of the monomers have the first linking moiety attached to
and terminate with a first reactive group for reacting with the
fluorogenic moiety or with a fluorogenic moiety conjugated to a
spacer, to thereby form the first polymeric backbone in which a
portion of the backbone units terminate with the first reactive
group;
[0499] polymerizing a second plurality of monomers, at least a
portion of the monomers terminate with a second reactive group for
reacting with the therapeutically active agent or with the
therapeutically active agent conjugated to a spacer, to thereby
form the second polymeric backbone in which a portion of the
backbone units terminate with the second reactive group; and
[0500] attaching the fluorogenic moiety to the first reactive group
and the therapeutically active agent to the second reactive
group,
[0501] thereby providing the polymeric system.
[0502] The copolymerization of the various monomers can be effected
by any polymerization method known in the art, using suitable
polymerization initiators and optionally chain transfer agents.
Such suitable polymerization initiators and chain transfer agents
can be readily identified by a person skilled in the art.
[0503] Using the RAFT approach enables to perform the
copolymerization at room temperature.
[0504] The "reversible addition-fragmentation chain transfer"
(RAFT) polymerization technique typically involves the use of
thiocarbonylthio compounds, such as dithioesters, dithiocarbamates,
trithiocarbonates, and xanthates in order to mediate the
polymerization via a reversible chain-transfer process. This allows
access to polymers with low polydispersity and high
functionality.
[0505] In some embodiments, the reactive groups can be protected
prior to the respective conjugation thereto. In such cases, the
process further comprises deprotecting the reactive group prior to
the respective conjugation.
[0506] This allows a regio-controlled conjugation of, for example,
the anti-angiogenesis agent to those backbone units that comprises
a biodegradable linker.
[0507] According to some embodiments, the polymerizing or
co-polymerizing is performed via RAFT polymerization.
[0508] Exemplary processes are described in detail in the Examples
section that follows. These processes can be utilized with any of
the fluorogenic moieties, therapeutically active agents and
quenching agents as described herein.
[0509] The exemplary processes described in the Examples section
that follows and accompanying figures, can be manipulated as
desired to suit the selected cleavable linking moiety or moieties,
therapeutically active agent and fluorogenic/fluorescent moiety
(and the selected Turn-ON mechanism).
[0510] Additional Polymeric Systems:
[0511] According to an aspect of some embodiments of the present
invention there is provided a polymeric system which comprises a
fluorogenic cyanine moiety covalently attached via a cleavable
linking moiety to a quenching agent, such that upon cleavage of the
linking moiety, a fluorescent cyanine moiety is generated. In some
embodiments, the system further comprising a polymeric moiety
attached to the fluorogenic cyanine moiety.
[0512] According to some of any of the embodiments described
herein, the polymeric system is represented by a formula selected
from Formula VA or VB:
##STR00013##
[0513] wherein:
[0514] Z.sub.1 and Z.sub.2 are each independently a substituted or
unsubstituted heterocylic moiety, as described herein;
[0515] R.sub.1 and R.sub.2 are each independently a polymeric
moiety;
[0516] n is an integer of from 1 to 10;
[0517] R' and R'' are each independently hydrogen, a substituted or
unsubstituted alkyl and a substituted or unsubstituted cycloalkyl,
or, alternatively, R' and R'' form together an aryl;
[0518] S.sub.5 and S'.sub.5 are each independently a degradable
spacer, as described herein, or absent;
[0519] L.sub.5 is the cleavable linking moiety; and
[0520] Q is the quenching agent.
[0521] The cleavable moiety can be any of the cleavable moieties
described herein.
[0522] According to some of any of the embodiments described
herein, the cyanine moiety is attached to the polymeric moiety via
a spacer, preferably a degradable spacer as described herein.
[0523] In some embodiments, the fluorogenic moiety described herein
is a FRET-based system, as described herein, preferably a pair-FRET
system.
[0524] Modular FRET systems previously described in the art (e.g.,
Redy et al. supra), can be conjugated according to these
embodiments, via a degradable spacer, to a polymeric moiety.
[0525] In some of these embodiments, the polymeric moiety is PEG.
Other polymeric moieties, for example, as described herein, are
contemplated.
[0526] According to some of any of the embodiments described
herein, the polymeric system further comprises a therapeutically
active agent, wherein:
[0527] (i) the therapeutically active agent is attached to the
cleavable linking moiety, such that upon its cleavage, the
therapeutically active agent is released;
[0528] (ii) the therapeutically active agent is attached to the
degradable spacer, such that upon its cleavage, the therapeutically
active agent is released; or
[0529] (iii) the therapeutically active agent is attached to a
second polymeric moiety, for forming a combined polymeric system,
similarly to any of the other embodiments described herein.
[0530] Applications:
[0531] According to an aspect of some embodiments of the present
invention there is provided a polymeric system as described in any
one of the embodiments described herein, where the system comprises
a therapeutically active agent, for use in the treatment and
diagnosis of a medical condition treatable by the therapeutically
active agent, or for use in the preparation of a medicament for
treating the medical condition.
[0532] According to an aspect of some embodiments of the present
invention there is provided a method of treating a medical
condition, the method comprising administering to a subject in need
thereof a polymeric system as described herein, which comprises a
therapeutically active agent that is usable in treating the medical
condition.
[0533] According to some of any of the embodiments described
herein, the medical condition is cancer.
[0534] According to some embodiments of the invention, the
therapeutically active agent is an anti-cancer agent.
[0535] In some of these embodiments, the therapeutically active
agent is an anti-tumor agent (an anti-cancer agent, an
anti-proliferative agent, an anti-angiogenesis agent, a
chemotherapeutic agent), such as paclitaxel (PTX), as exemplified
herein.
[0536] According to an aspect of some embodiments of the present
invention there is provided a polymeric system according to any one
of the embodiments described herein, for use in the treatment and
diagnosis of a medical condition treatable by the therapeutically
active agent.
[0537] According to an aspect of some embodiments of the present
invention there is provided a method of treating and monitoring a
medical condition treatable by the therapeutically active
agent.
[0538] According to an aspect of some embodiments of the present
invention there is provided a method of treating and monitoring a
medical condition treatable by the therapeutically active agent, by
administering the polymeric system as described herein to a subject
in need of such treatment.
[0539] According to an aspect of some embodiments of the present
invention there is provided a use of a polymeric system as
described herein for preparing a medicament for treating and
monitoring a medical condition treatable by the therapeutically
active agent.
[0540] According to some embodiments of the invention, the medical
condition is cancer, the therapeutically active agent is an
anti-tumor agent and the cleavable linking moiety/moieties are
cleavable by an enzyme expressed or overexpressed in tumor
tissues.
[0541] According to some embodiments of the present invention,
treatment and monitoring or diagnosis are performed simultaneously,
and thus may allow real-time monitoring and evaluation of the
treatment.
[0542] The terms "cancer" and "tumor" are used interchangeably
herein to describe a class of diseases in which a group of cells
display uncontrolled growth (division beyond the normal limits).
The term "cancer" encompasses malignant and benign tumors as well
as disease conditions evolving from primary or secondary tumors.
The term "malignant tumor" describes a tumor which is not
self-limited in its growth, is capable of invading into adjacent
tissues, and may be capable of spreading to distant tissues
(metastasizing). The term "benign tumor" describes a tumor which is
not malignant (i.e. does not grow in an unlimited, aggressive
manner, does not invade surrounding tissues, and does not
metastasize). The term "primary tumor" describes a tumor that is at
the original site where it first arose. The term "secondary tumor"
describes a tumor that has spread from its original (primary) site
of growth to another site, close to or distant from the primary
site.
[0543] Non-limiting examples of therapeutically active agents that
can be efficiently incorporated in the herein described polymeric
systems include amino containing chemotherapeutic agents such as
daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin,
anthracycline, mitomycin C, mitomycin A, 9-amino camptothecin,
aminopertin, antinomycin, N.sup.8-acetyl spermidine,
1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, bleomycin,
tallysomucin, and derivatives thereof; hydroxy containing
chemotherapeutic agents such as etoposide, camptothecin,
irinotecaan, topotecan, 9-amino camptothecin, paclitaxel,
docetaxel, esperamycin, 1,8-dihydroxy-bicyclo [7.3.1]
trideca-4-ene-2,6-diyne-13-one, anguidine, morpholino-doxorubicin,
vincristine and vinblastine, and derivatives thereof, sulfhydril
containing chemotherapeutic agents and carboxyl containing
chemotherapeutic agents. Any other anti-cancer agents are also
contemplated.
[0544] Other therapeutically active agents that can be beneficially
incorporated in the herein described polymeric systems include, for
example, antihistamines, anesthetics, analgesics, anti-fungal
agents, antibiotics, anti-inflammatory agents, vitamins and
anti-infectious agents.
[0545] It is expected that during the life of a patent maturing
from this application many relevant cyanine dyes, fluorogenic
moieties, therapeutically active agent and/or polymers will be
developed and the scope of the terms cyanine dye, cyanine-like
structure, polymeric backbone and therapeutically active agent, is
intended to include all such new technologies a priori.
[0546] General:
[0547] The term "alkyl" describes a saturated aliphatic hydrocarbon
including straight chain and branched chain groups. Preferably, the
alkyl group has 1 to 20 carbon atoms. Whenever a numerical range;
e.g., "1-20", is stated herein, it means that the group, in this
case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3
carbon atoms, etc., up to and including 20 carbon atoms. In some
embodiments, the alkyl group has 1-10 carbon atoms. In some
embodiments, the alkyl group has 1-4 carbon atoms. Exemplary alkyl
groups include, but are not limited to methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl, octadecyl
and nonadecyl.
[0548] The term "cycloalkyl" describes an all-carbon monocyclic or
fused ring (i.e., rings which share an adjacent pair of carbon
atoms) group wherein one of more of the rings does not have a
completely conjugated pi-electron system. Examples, without
limitation, of cycloalkyl groups are cyclopropane, cyclobutane,
cyclopentane, cyclopentene, cyclohexane, cyclohexadiene,
cycloheptane, cycloheptatriene, and adamantane.
[0549] The term "aryl" describes an all-carbon monocyclic or
fused-ring polycyclic (i.e., rings which share adjacent pairs of
carbon atoms) groups having a completely conjugated pi-electron
system. Examples, without limitation, of aryl groups are phenyl,
naphthalenyl and anthracenyl. The aryl group may be substituted or
unsubstituted.
[0550] The term "heteroaryl" describes a monocyclic or fused ring
(i.e., rings which share an adjacent pair of atoms) group having in
the ring(s) one or more atoms, such as, for example, nitrogen,
oxygen and sulfur and, in addition, having a completely conjugated
pi-electron system. Examples, without limitation, of heteroaryl
groups include pyrrole, furane, thiophene, imidazole, oxazole,
thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline
and purine.
[0551] The term "heteroalicyclic" describes a monocyclic or fused
ring group having in the ring(s) one or more atoms such as
nitrogen, oxygen and sulfur. The rings may also have one or more
double bonds. However, the rings do not have a completely
conjugated pi-electron system.
[0552] The term "hydroxy" describes an --OH group.
[0553] The term "alkoxy" describes both an --O-alkyl and an
--O-cycloalkyl group, as defined herein.
[0554] The term "thiol" describes a --SH group.
[0555] The term "thioalkoxy" describes both an --S-alkyl group, and
an --S-cycloalkyl group, as defined herein.
[0556] The term "cyano" describes a --C.ident.N group.
[0557] The term "carbonyl" describes a --C(.dbd.O)--R' group, where
R' is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through
a ring carbon) or heteroalicyclic (bonded through a ring carbon) as
defined herein.
[0558] The term "thiocarbonyl" describes a --C(.dbd.S)--R' group,
where R' is as defined herein.
[0559] The term "O-carbamyl" describes an --OC(.dbd.O)--NR'R''
group, where R' is as defined herein and R'' is hydrogen, alkyl,
cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) or
heteroalicyclic (bonded through a ring carbon) as defined
herein.
[0560] The term "N-carbamyl" describes an R'OC(.dbd.O)--NR''--
group, where R' and R'' are as defined herein.
[0561] The term "O-thiocarbamyl" describes an --OC(.dbd.S)--NR'R''
group, where R' and R'' are as defined herein.
[0562] The term "N-thiocarbamyl" describes an R''OC(.dbd.S)NR'--
group, where R' and R'' are as defined herein.
[0563] The term "C-amido" describes a --C(.dbd.O)--NR'R'' group,
where R' and R'' are as defined herein.
[0564] The term "N-amido" describes an R'C(.dbd.O)--NR'' group,
where R' and R'' are as defined herein.
[0565] The term "C-carboxy" describes a --C(.dbd.O)--O--R' groups,
where R' is as defined herein.
[0566] The term "O-carboxy" describes an R'C(.dbd.O)--O-- group,
where R' is as defined herein.
[0567] The term "nitro" group describes an --NO.sub.2 group.
[0568] The term "amino" group describes an --NH.sub.2 group.
[0569] The term "sulfonyl" group describes an --S(.dbd.O).sub.2--R'
group, where R' is as defined herein.
[0570] The term "halogen" or "halo" describes fluoro, chloro, bromo
or iodo atom.
[0571] Herein, the phrase "therapeutically active agent" is also
referred to herein as "drug".
[0572] The polymeric moieties described herein may possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0573] As used herein, the term "enantiomer" describes a
stereoisomer of a compound that is superposable with respect to its
counterpart only by a complete inversion/reflection (mirror image)
of each other. Enantiomers are said to have "handedness" since they
refer to each other like the right and left hand. Enantiomers have
identical chemical and physical properties except when present in
an environment which by itself has handedness, such as all living
systems.
[0574] The polymeric moieties described herein can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention.
[0575] The term "solvate" refers to a complex of variable
stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on),
which is formed by a solute (the conjugate described herein) and a
solvent, whereby the solvent does not interfere with the biological
activity of the solute. Suitable solvents include, for example,
ethanol, acetic acid and the like.
[0576] The term "hydrate" refers to a solvate, as defined
hereinabove, where the solvent is water.
[0577] As used herein, a "reactive group" describes a chemical
group that is capable of reacting with another group so as to form
a chemical bond, typically a covalent bond. Optionally, an ionic or
coordinative bond is formed.
[0578] A reactive group is termed as such if being chemically
compatible with a reactive group of an agent or moiety that should
be desirably attached thereto. For example, a carboxylic group is a
reactive group suitable for conjugating an agent or a moiety that
terminates with an amine group, and vice versa.
[0579] A reactive group can be inherently present in the monomeric
units forming the backbone units, or be generated therewithin by
terms of chemical modifications of the chemical groups thereon or
by means of attaching to these chemical groups a spacer or a linker
that terminates with the desired reactive group.
[0580] The term "subject" (alternatively referred to herein as
"patient") as used herein refers to an animal, preferably a mammal,
most preferably a human, who has been the object of treatment,
observation or experiment.
[0581] In any of the methods and uses described herein, any of the
polymeric moieties described herein can be provided to an
individual either per se, or as part of a pharmaceutical
composition where it is mixed with a pharmaceutically acceptable
carrier.
[0582] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the polymeric moieties described
herein (as active ingredient), or physiologically acceptable salts
or prodrugs thereof, with other chemical components including but
not limited to physiologically suitable carriers, excipients,
lubricants, buffering agents, antibacterial agents, bulking agents
(e.g. mannitol), antioxidants (e.g., ascorbic acid or sodium
bisulfite), anti-inflammatory agents, anti-viral agents,
chemotherapeutic agents, anti-histamines and the like. The purpose
of a pharmaceutical composition is to facilitate administration of
a compound to a subject. The term "active ingredient" refers to a
compound, which is accountable for a biological effect.
[0583] The terms "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably
used refer to a carrier or a diluent that does not cause
significant irritation to an organism and does not abrogate the
biological activity and properties of the administered
compound.
[0584] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of a drug. Examples, without limitation, of
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0585] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0586] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen. The dosage may vary depending upon the dosage form employed
and the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition (see e.g., Fingl et
al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.
1).
[0587] The pharmaceutical composition may be formulated for
administration in either one or more of routes depending on whether
local or systemic treatment or administration is of choice, and on
the area to be treated. Administration may be done orally, by
inhalation, or parenterally, for example by intravenous drip or
intraperitoneal, subcutaneous, intramuscular or intravenous
injection, or topically (including ophtalmically, vaginally,
rectally, intranasally).
[0588] Formulations for topical administration may include but are
not limited to lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable.
[0589] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
sachets, pills, caplets, capsules or tablets. Thickeners, diluents,
flavorings, dispersing aids, emulsifiers or binders may be
desirable.
[0590] Formulations for parenteral administration may include, but
are not limited to, sterile solutions which may also contain
buffers, diluents and other suitable additives. Slow release
compositions are envisaged for treatment.
[0591] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0592] The pharmaceutical composition may further comprise
additional pharmaceutically active or inactive agents such as, but
not limited to, an anti-bacterial agent, an antioxidant, a
buffering agent, a bulking agent, a surfactant, an
anti-inflammatory agent, an anti-viral agent, a chemotherapeutic
agent and an anti-histamine.
[0593] According to an embodiment of the present invention, the
pharmaceutical composition described hereinabove is packaged in a
packaging material and identified in print, in or on the packaging
material, for use in the treatment and/or monitoring of a disease
or disorder or medical condition as described herein.
[0594] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accommodated by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert.
[0595] In any of the methods, uses and compositions described
herein, the polymeric systems described herein can be utilized in
combination with additional therapeutically active agents. Such
additional agents include, as non-limiting examples,
chemotherapeutic agents, anti-angiogensis agents, hormones, growth
factors, antibiotics, anti-microbial agents, anti-depressants,
immunostimulants.
[0596] As used herein the term "about" refers to .+-.10%
[0597] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0598] The term "consisting of" means "including and limited
to".
[0599] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0600] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0601] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0602] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0603] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0604] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical
symptoms of a condition or substantially preventing the appearance
of clinical or aesthetical symptoms of a condition.
[0605] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0606] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0607] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
MATERIALS AND METHODS
[0608] Materials:
[0609] HPMA copolymer-Gly-Phe-Leu-Gly-ONp incorporating 10 mol % of
the methacryloyl-Gly-Phe-Leu-Gly-p-nitrophenol ester monomer units
and HPMA copolymer-Gly-Phe-Leu-Gly-ethylenediamine (HPMA-GFLG-en)
incorporating 10 mol % of the
methacryloyl-Gly-Phe-Leu-Gly-ethylenediamine were obtained from
Polymer Laboratories (Church Stretton, U.K.). The HPMA copolymers
have a molecular weight of 31,600 Da and a polydispersity of
1.66.
[0610] PTX was purchased from Petrus Chemicals and Materials 1986
(LTD) (China).
[0611] Dulbecco's modified Eagle's medium (DMEM) and PBS, RPMI
1640, fetal bovine serum (FBS), penicillin, streptomycin, nystatin,
1-glutamine, Hepes buffer, sodium pyruvate, and fibronectin were
purchased from Biological Industries Ltd. (Kibbutz Beit Haemek,
Israel).
[0612] EGM-2 medium was purchased from Cambrex, USA and endothelial
cells growth supplement (ECGS) from Zotal (Israel).
[0613] All other chemical reagents, including salts and solvents,
were purchased from Sigma-Aldrich (Rehovot, Israel).
[0614] All reactions requiring anhydrous conditions were performed
under Argon or N.sub.2 atmosphere. Chemicals and solvents were
either AR grade or purified by standard techniques.
[0615] Thin layer chromatography (TLC): silica gel plates Merck 60
F.sub.254: compounds were visualized by irradiation with UV
light.
[0616] Flash chromatography (FC): silica gel Merck 60 (particle
size 0.040-0.063 mm), eluent given in parentheses.
[0617] High pressure liquid chromatography (HPLC): C18 5u,
250.times.4.6 mm, eluent given in parentheses.
[0618] Preparative HPLC: C18 5u, 250.times.21 mm, eluent given in
parentheses.
[0619] .sup.1H-NMR spectra were measured using Bruker Avance
operated at 400 MHz as mentioned. .sup.13C-NMR spectra were
measured using Bruker Avance operated at 400 MHz as mentioned.
[0620] Absorption and fluorescence spectra were recorded on
Spectramax-M2 fluorescent spectrometer using quartz cuvettes or
quartz 96-wells plate reader.
[0621] Some Abbreviations:
[0622] ACN--Acetonitrile, DCM--Dichloromethane,
DMAP--4-Dimethylaminopyridine, DMF--N,N'-Dimethylformamide,
EtOAc--Ethylacetate, Hex--n-Hexanes, MeOH--Methanol,
THF--Tetrahydrofurane, TFA--Trifluoroacetic acid,
Et.sub.3N--Triethylamine, EtOH--Ethyl alcohol, NaOAc--Sodium
acetate, Ac.sub.2O--Acetic anhydride,
DCC--N,N'-Dicyclohexylcarbodiimide, AcOH--Acetic acid.
[0623] Dynamic Light Scattering (DLS) Analysis and Surface Charge
Measurements:
[0624] The mean hydrodynamic diameter of the HPMA copolymer-PTX
conjugate and the zeta-potential measurements were performed using
a ZetaSizer Nano ZS instrument with an integrated 4Mw He--Ne laser
(.lamda.=532 nm; Malvern Instruments Ltd., Malvern, Worcestershire,
UK). HPMA copolymer-PTX sample were prepared by dissolving 1 mg of
polymer conjugate in 1 ml of 15.5 mM phosphate buffer, pH=7.4. The
polymer solution was vortexed and then filtered through 0.2 .mu.M
filter. All measurements were performed at 25.degree. C. using
polystyrol/polystyrene (10.times.4.times.45) mm cell for DLS
analysis and folded capillary cell (DTS 1070) for zeta-potential
measurements.
[0625] Cathepsin B Activity Assays:
[0626] PTX and Cy5 enzymatically-directed release from the
conjugates was studied in vitro, upon incubation at 37.degree. C.
with Cathepsin B (1 unit/ml) in freshly prepared activity phosphate
buffer (0.1 M, pH=6.0), containing 0.05 M NaCl, 1 mM
Ethylenediaminetetraacetic acid (EDTA) and 5 mM reduced glutathione
(GSH). As a control, conjugates were incubated in the absence of
Cathepsin B in activity phosphate buffer (0.1 M, pH=6.0),
containing 0.05 M NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA)
and 5 mM reduced glutathione (GSH), and/or in Dulbecco's PBS (pH
7.4).
[0627] Release of Cy5 from HPMA Copolymer-Cy5 Conjugate:
[0628] Free Cy5 release was monitored by measuring the change in
the fluorescence intensity at sequential time points. The
fluorescence measurements were carried out at excitation
wavelengths of 600 nm using SpectraMax M5.sup.e multi-detection
reader. Samples (50 .mu.l) were collected every 24 hours (up to 105
hours) and immediately analyzed.
[0629] Release of PTX from HPMA Copolymer-PTX-FK Conjugate:
[0630] The free PTX release was monitored by reversed phase (RP)
HPLC. UltiMate.RTM. 3000 Nano LC systems (Dionex) was used,
equipped with 3000 pump, VWD-3000 UV-Vis detector and
Chromeleon.RTM. 6.80 software. The column in use was Phenomenex
Jupiter 5.mu. 250.times.4.60 mm C-18 300A. Chromatographic
conditions were: flow: 1.0 ml/min, gradient: 20% to 100% solution B
in 20 minutes (sol. A--0.1% TFA in water; sol. B--0.1% TFA in
acetonitrile (MeCN)).
[0631] Samples (100 .mu.l) were collected every 24 hours, until a
plateau was observed (up to 50 hours). For PTX extraction, sodium
carbonate buffer solution (0.2 M, pH=9.6) was added to each sample,
followed by ethyl acetate. Samples were vigorously vortex and
centrifuged. The organic layer was carefully removed and
evaporated. The residue was dissolved in MeCN and analyzed.
[0632] Release of PTX from HPMA Copolymer-PTX Conjugate:
[0633] The free PTX release was monitored by reversed phase (RP)
HPLC. UltiMate.RTM. 3000 Nano LC systems (Dionex) was used,
equipped with 3000 pump, VWD-3000 UV-Vis detector and
Chromeleon.RTM. 6.80 software. The column in use was Phenomenex
Jupiter 5.mu. 250.times.4.60 mm C-18 300A. Chromatographic
conditions were: flow: 1.0 ml/min, gradient: 20% to 100% solution B
in 20 minutes (sol. A--0.1% TFA in water; sol. B--0.1% TFA in
acetonitrile (MeCN)).
[0634] Samples (100 .mu.l) were collected every 24 hours, until a
plateau was observed (up to 50 hours). For PTX extraction, sodium
carbonate buffer solution (0.2 M, pH=9.6) was added to each sample,
followed by ethyl acetate. Samples were vigorously vortex and
centrifuged. The organic layer was carefully removed and
evaporated. The residue was dissolved in MeCN and analyzed.
[0635] Release of Cy5 from PGA-PTX-Cy5 Conjugate:
[0636] Cy5 release was monitored by measuring the change in the
fluorescence intensity at sequential time points. The fluorescence
measurements were carried out at excitation wavelengths of 650 nm
using SpectraMax M5.sup.e multi-detection reader. Samples (50
.mu.l) were collected every 24 hours (up to 160 hours) and
immediately analyzed.
[0637] Cell Cultures:
[0638] MDA-MB-231 human mammary adenocarcinoma cell line, 4T1
murine mammary adenocarcinoma cell line and WA239A human melanoma
cell line were purchased from the American Type Culture Collection
(ATCC, Manassas, Va., USA).
[0639] MDA-MB-231 cells were cultured in DMEM supplemented with 10%
FBS, 100 .mu.g/mL penicillin, 100 .mu.t/mL streptomycin, 12.5
.mu.l/mL nystatin and 2 mM L-glutamine.
[0640] 4T1 cells were cultured in RPMI 1640 supplemented with 10%
FBS, 100 mg/mL Penicillin, 100 .mu.l/mL Streptomycin, 12.5 .mu.l/mL
Nystatin, and 2 mM L-glutamine, 1 mM Sodium pyruvate, 10 mM HEPES
buffer and 2.5 g/L D-glucose.
[0641] WM239A cells were cultured in RPMI 1640 supplemented with
10% FBS, 100 mg/ml Penicillin, 100 .mu.l/ml Streptomycin, 12.5
.mu.l/mg Nystatin, and 2 mM L-glutamine.
[0642] Cells were grown at 37.degree. C.; 5% CO.sub.2.
[0643] Human umbilical vein endothelial cells (HUVEC) were
purchased from Lonza, Switzerland and were cultured in EGM-2 medium
(Lonza, Switzerland). Cells were grown at 37.degree. C.; 5%
CO.sub.2.
[0644] For the study of in vitro degradation of HPMA copolymer-Cy5
conjugate, MDA-MB-231 cells (30,000 cells/ml) were seeded to
24-well culture plates with DMEM supplemented with 10% FBS, 100
.mu.g/mL penicillin, 100 U/mL streptomycin, 12.5 U/mL nystatin and
2 mM L-glutamine. 24 hours later, HPMA-copolymer-Cy5 (3.8%) at a
final concentration of 10 .mu.M eq. Cy5 was added. At 0.5, 24 and
48 hours after the addition of the substrate, DMEM was replaced
with PBS and the degradation of the conjugate was monitored using
SpectraMax M5.sup.e multi-detection reader. Non-treated MDA-MB-231
cells were used as a control.
[0645] Cell Viability Assay:
[0646] For the study of HPMA copolymer-PTX conjugate antitumor
activity:
[0647] 4T1 cells (3,000 cells/well), HUVEC (10,000 cells/well) and
MDA-MB-231 cells (10,000 cells/well) were plated onto 24-well
culture plates in RPMI supplemented with 2% FBS, EBM-2 supplemented
with 5% FBS or in DMEM supplemented with 10% FBS respectively, and
incubated for 24 hours (37.degree. C.; 5% CO2). The medium was then
replaced with RPMI 1640 supplemented with 10% FBS, EGM-2 or DMEM
supplemented with 10% FBS. Cells were exposed to PTX and PTX
bound-conjugates at serial dilutions, at equivalent dose of the
free PTX. Number of viable cells was counted by a Z1 Coulter
Counter.RTM. Cell and Particle Counter (Beckman Coulter.RTM.)
following 96 hours of incubation.
[0648] For the study of PGA-PTX-Cy5 conjugate antitumor
activity:
[0649] 4T1 cells (8,000 cells/wells), WM239A cells (15,000
cells/well) and MDA-MB-231 cells (10,000/well) were plated onto
24-well culture in RPMI supplemented with 10% FBS, or in DMEM
supplemented with 10% FBS respectively, and incubated for 24 hours
(37.degree. C.; 5% CO.sub.2). The medium was then replaced with
RPMI 1640 supplemented with 10% FBS or DMEM supplemented with 10%
FBS.
[0650] Cells were exposed to PTX and PTX-bound conjugates at serial
dilutions, at equivalent dose of free PTX. Number of viable cells
was counted by a Z1 Coulter Counter.RTM. Cell and Particle Counter
(Beckman Coulter.RTM.) following 72 hours of incubation.
[0651] Animals and Tumor Cell Inoculation:
[0652] 4T1 cells (3.times.10.sup.6) were injected subcutaneously
(s.c.) into the flank of female BALB/C mice aged 6-8 weeks). Tumor
volume was calculated using the standard formula:
length.times.width.sup.2.times.0.52.
[0653] Intravital Non-invasive Imaging of Cy5 cathepsin B-dependent
release: BALB/c mice bearing subcutaneous 4T1 tumors (about 100
mm.sup.3) were injected intra-tumorally with HPMA copolymer-Cy5
(0.1 mM; 30 .mu.l) or with equivalent dose of free Cy5. Fluorescent
signal within tumor was assessed at different time points 30 hours
following injection using non-invasive imaging system CRI
Maestro.TM. Multispectral image-cube were acquired through 650-800
nm spectral range in 10 nm steps using excitation (635 nm longpass)
and emission (675 nm longpass) filter set. Mice auto-fluorescence
and undesired background signals were eliminated by spectral
analysis and linear unmixing algorithm.
[0654] Body Distribution of HPMA Copolymer-SQ-Cy5:
[0655] BALB/c mice bearing sub-cutaneous 4T1 tumors (about 300
mm.sup.3) were injected intravenously (i.v.) with HPMA
copolymer-SQ-Cy5 (10 .mu.M; 200 .mu.l). Accumulation of the
conjugate in the tumor and organs was assessed at different time
points for 12 hours. At termination, tumors and organs were excised
and imaged. Organs were imaged using non-invasive imaging system
CRI Maestro.TM. (filter set--Ex/Em 635/675). Mice auto-fluorescence
and undesired background signals were eliminated by spectral
analysis and linear unmixing algorithm. Time dependent tumor
contrast profile was determined by the ratio between fluorescence
intensities of tumors and those of normal skin.
[0656] Statistical Methods:
[0657] Data is expressed as mean.+-.standard deviation (s.d.) for
in vitro assays or .+-.standard error of the mean (s.e.m.) for in
vivo. Statistical significance was determined using an unpaired
t-test. All statistical tests were two-sided. All experiments were
performed in triplicates and repeated at least three times.
Example I
[0658] Chemical syntheses of HPMA copolymer conjugates Synthesis of
HPMA Copolymer-Cy5 conjugate:
[0659] The structure of an exemplary HPMA copolymer-Cy5 conjugate
is depicted in FIG. 1A. An exemplary synthesis of a HPMA
Copolymer-Cy5 conjugate is depicted in FIG. 2.
[0660] Cy5-COOH was synthesized as previously described [Redy, O.,
et al., Org. Biomol. Chem., 2012. 10(4): p. 710-5].
[0661] Cy5-COOH fluorophore was conjugated with HPMA
copolymer-GFLG-en in two-step synthesis, as follows. First,
Cy5-COOH (15.1 mg, 0.023 mmol) was dissolved in 0.7 mL anhydrous
N,N-Dimethylformamide (DMF). N-Hydroxysuccinimide (NHS) (5.3 mg,
0.046 mmol) and N,N'-dicyclohexylcarbodiimide (DCC) (9.5 mg, 0.046
mmol) were added in order to activate the free carboxylic group of
the fluorophore, for further coupling to the HPMA copolymer. The
reaction mixture was stirred at room temperature (rT) in dark for
12 hours. Then, HPMA-GFLG-en copolymer (21.1 mg, 0.114 mmol) was
dissolved in 0.5 mL anhydrous DMF and added to the reaction
mixture. Following the reaction by High Pressure Liquid
Chromatography (HPLC) (UltiMate.RTM. 3000 Nano LC systems, Dionex),
the precipitate was washed with acetone and dried under vacuum.
[0662] Purification of the conjugate by size exclusion
chromatography (SEC) was performed using AKTA/FPLC system
(Pharmacia/GE Healthcare), HiTrap Desalting columns (Sephadex G-25
Superfine) in DDW, flow rate 1.0 ml/min; UV detection.
[0663] In order to remove all excess of free fluorophore, the
residue was dissolved in water and dialyzed for 1 day at 4.degree.
C. (MWCO 6-8 kDa) against DI water. The conjugate was isolated by
freeze-drying.
[0664] Cy5 loading was determined using SpectraMax M5.sup.e
multi-detection reader. The absorbance of conjugated Cy5 was
measured and compared to that of free Cy5.
[0665] Quenching efficiency was expressed as a percentage of the
fluorescence intensity of the HPMA copolymer-Cy5 conjugate
(.lamda..sub.Em=670 nm) compared with the emission of the free Cy5
at the equivalent concentration, as shown in Example 5 below and
FIG. 11A.
[0666] Synthesis of HPMA Copolymer-PTX Conjugate:
[0667] The structure of an exemplary HPMA copolymer-PTX conjugate
is depicted in FIG. 1B. Paclitaxel (PTX) was conjugated with HPMA
copolymer-GFLG-en in two-step synthesis, as depicted in FIG. 3.
First, PTX was activated using 4-Nitrophenyl chloroformate (PNP-Cl)
in order to form PTX-ONp. PTX (107.2 mg, 0.125 mmol) was dissolved
in 1 ml pre-distilled Tetrahydrofuran (THF) and was stirred at
-30.degree. C. Triethylamine (Et.sub.3N) (140 .mu.l, 1.0 mmol) and
a grain of 4-Dimethylaminopyridine (DMAP) were dissolved in the dry
solvent and added to the reaction mixture. PNP-Cl (151.2 mg, 0.750
mmol) was dissolved in another 1 ml of THF and added to the
reaction. The reaction was followed by Thin Layer Chromatography
(TLC) and quenched with 1M HCl at -30.degree. C. The product was
extracted from the aqueous media using ethyl acetate and purified
by silica gel column.
[0668] Then, PTX-ONp was conjugated to the HPMA-GFLG-en copolymer
in the presence of Et.sub.3N and Nitrogen atmosphere. The PTX
content of the HPMA copolymer-PTX conjugate was determined by HPLC
analysis, at .lamda.=270 nm, against a calibration curve for free
PTX.
[0669] Synthesis of HPMA Copolymer-PTX-FK Conjugate:
[0670] The structure of an exemplary HPMA copolymer-PTX-FK
conjugate is depicted in FIG. 1C. The synthesis of HPMA
copolymer-PTX-FK conjugate is illustrated in FIG. 4.
[0671] The conjugation of PTX with HPMA copolymer was performed as
previously described [Duncan, R., et al., J Control Release, 2001.
74(1-3): p. 135-46]. Briefly,
[0672] PTX (168.5 mg, 0.197 mmol) was first attached to an FK-PABC
linker (147.5 mg, 0.197 mmol) and the obtained FK-PABC-PTX was
conjugated to HPMA copolymer-GFLG-ONp. L-Boc-Phe-ONp was conjugated
to L-Lys (alloc)-OH to afford Compound 2. Amidation with
4-aminobenzyl alcohol (PABA) afforded Compound 3, and was followed
by activation with p-nitrophenol to afford Compound 4, which was
then reacted with PTX to afford Compound 5. Deprotection of the Boc
group afforded the free amine, which was then conjugated with HPMA
copolymer-GFLG-ONp, as depicted in FIG. 4. Finally, deprotection of
the alloc group of the amine residue of Lys afforded the desired
HPMA copolymer-PTX-FK, the structure of which is depicted as the
final product in FIG. 4 and in FIG. 1C.
[0673] The PTX-FK content of the HPMA copolymer-PTX-FK conjugate
was determined by HPLC analysis. The PTX-FK content was determined
against a calibration curve for free PTX-FK.
[0674] Preparation of Compound 2:
[0675] L-Boc-Phe-ONp (104.3 mg, 0.27 mmol) was dissolved in 2 mL
DMF. Then commercially available L-Lys(alloc)-OH (62 mg, 0.27 mmol)
and Et.sub.3N (100 .mu.L) were added. The reaction mixture was
stirred for 12 hours and was monitored by TLC (AcOH:MeOH:EtOAc
0.5:10:89.5). Upon completion of the reaction the solvent was
removed under reduced pressure and the crude product was purified
using column chromatography on silica gel (AcOH:MeOH:EtOAc
0.5:10:89.5) to give compound 2 (107 mg, 83%) as a white solid
(FIG. 4).
[0676] Preparation of Compound 3:
[0677] Compound 2 (832.1 mg, 1.74 mmol) was dissolved in dry THF
and the solution was cooled to -15.degree. C. Then NMM (0.19 mL,
1.74 mmol) and isobutyl chloroformate (0.27 mL, 2.09 mmol) were
added. The reaction was stirred for 20 minutes and a solution of
4-aminobenzyl alcohol (321.85 mg, 2.61 mmol) in dry THF was added.
The reaction mixture was stirred for 2 hours and was monitored by
TLC (EtOAc 100%). Upon completion of the reaction, the solvent was
removed under reduced pressure and the crude product was purified
using column chromatography on silica gel (EtOAc 100%) to give
compound 3 (835 mg, 82%) as a yellow solid (FIG. 4).
[0678] Preparation of Compound 4:
[0679] Compound 3 (353.6 mg, 0.60 mmol) was dissolved in dry THF
and the solution was cooled to 0.degree. C. Then DIPEA (0.42 mL,
2.42 mmol), PNP-chloroformate (367 mg, 1.82 mmol) and a catalytic
amount of pyridine were added. The reaction was stirred for 2 hours
and monitored by TLC (EtOAc:Hex 3:1). Upon completion of the
reaction, the solvent was removed under reduced pressure. The crude
product was diluted with EtOAc and washed with saturated
NH.sub.4Cl. The organic layer was dried over magnesium sulfate and
the solvent was removed under reduced pressure. The crude product
was purified using column chromatography on silica gel (EtOAc:Hex
3:1) to give compound 4 (453.2 mg, 79%) as a white solid (FIG.
4).
[0680] Preparation of Compound 5:
[0681] Compound 4 (360.3 mg, 0.48 mmol) was dissolved in dry DCM.
Then PTX (494.06 mg, 0.57 mmol) and DMAP (70.61 mg, 0.57 mmol) were
added. The reaction mixture was stirred for 8 hours and monitored
by TLC (EtOAc 100%). Upon completion of the reaction, the solvent
was removed under reduced pressure and the crude product was
purified using column chromatography on silica gel (EtOAc 100%) to
give compound 5 (662 mg, 94%) as a white solid (FIG. 4).
[0682] Preparation of HPMA Copolymer-PTX-FK (Alloc):
[0683] Compound 5 (12 mg, 7.57 .mu.mol) was dissolved in 0.5 mL TFA
and stirred for 2 minutes at 0.degree. C. The excess of acid was
removed under reduced pressure and the crude amine salt was
dissolved in 0.5 mL DMF. HPMA copolymer (26.3 mg, ONp=8.32 .mu.mol)
was added, followed by the addition of Et.sub.3N (3 .mu.L). The
reaction mixture was stirred for 12 hours and the solvent was
removed under reduced pressure.
[0684] Free PTX, FK and ONp were removed by FPLC using XK26/70
column with Sephadex LH.sub.2O column (MeOH 100%, 1 mL/1 minute) to
give the alloc protected HPMA copolymer-PTX-FK as a white solid (20
mg) (FIG. 4).
[0685] Preparation of HPMA Copolymer-PTX-FK:
[0686] Alloc protected HPMA copolymer-PTX-FK (30 mg, alloc=max. 9.9
.mu.mol) was dissolved in DMF (1 mL). Then acetic acid (2.71 .mu.L,
47.4 .mu.mol), Bu.sub.3SnH (30.6 .mu.L, 113 .mu.mol) and a
catalytic amount of Pd(PPh.sub.3).sub.4 were added. The reaction
mixture was stirred for 2 hours and was concentrated under reduced
pressure, followed by addition of 10 mL of acetone. The precipitate
was filtered out and washed with acetone several times. The crude
product was purified by HPLC using XK26/70 column with Sephadex
LH.sub.2O (MeOH 100%, 1 mL/1 minute) to give HPMA copolymer-PTX-FK
(20 mg) as a white solid (FIGS. 1D and 4).
[0687] Synthesis of HPMA Copolymer-PTX-Cy5 Conjugate:
[0688] The structure of exemplary HPMA copolymer-PTX-Cy5 is
depicted in FIG. 1D. The synthesis of the HPMA copolymer-PTX-Cy5
conjugate is depicted in FIG. 5.
[0689] Cy5-COOH was synthesized as described hereinabove. Next,
Cy5-COOH fluorophore was conjugated with HPMA copolymer-GFLG-en in
two-step synthesis. First, Cy5-COOH (15.1 mg, 0.023 mmol) was
dissolved in 0.7 mL anhydrous N,N-Dimethylformamide (DMF).
N-Hydroxysuccinimide (NHS) (5.3 mg, 0.046 mmol) and
N,N'-dicyclohexylcarbodiimide (DCC) (9.5 mg, 0.046 mmol) were added
in order to activate the free carboxylic group of the fluorophore,
for further coupling to the HPMA copolymer. The reaction mixture
was stirred at room temperature (RT) in dark for 12 hours. Then,
HPMA-GFLG-en copolymer (21.1 mg, 0.114 mmol) was dissolved in 0.5
mL anhydrous DMF and added to the reaction mixture. At reaction
termination, PTX-ONp, prepared as described hereinabove, was added
to the reaction round bottom flask. Following the reaction
completion (as monitored by High Pressure Liquid Chromatography
(HPLC) (UltiMate.RTM. 3000 Nano LC systems, Dionex), the
precipitate was washed with acetone and dried under vacuum.
[0690] Free Cy5 and PTX-ONp were removed by FPLC using XK26/70
column with Sephadex LH20 column (MeOH 100%, 1 mL/1 min); UV
detection.
[0691] The conjugate was isolated by freeze-drying.
[0692] Cy5 loading was determined using SpectraMax M5.sup.e
multi-detection reader. The absorbance of conjugated Cy5 was
measured and compared to that of free Cy5.
[0693] The PTX content of the HPMA copolymer-PTX-Cy5 conjugate was
determined by HPLC analysis. The PTX content was determined against
a calibration curve for free PTX.
[0694] Quenching efficiency was expressed as a percentage of the
fluorescence intensity of the HPMA copolymer-Cy5 conjugate
(.lamda..sub.Em=670 nm) compared with the emission of the free Cy5
at the equivalent concentration, as is detailed hereinunder.
[0695] Synthesis of HPMA Copolymer-PTX-FK-Cy5 Conjugate:
[0696] The structure of an exemplary HPMA copolymer-PTX-FK-Cy5 is
shown in FIG. 1E. The synthesis of the HPMA copolymer-PTX-FK-Cy5 is
illustrated in FIG. 6.
[0697] Previously synthesized L-Boc-Phe-ONp was reacted with
L-Lys(alloc)-OH, as described hereinabove, to give dipeptide
compound 2. The latter was conjugated with 4-aminobenzyl alcohol as
described hereinabove to generate alcohol compound 3. Activation of
alcohol compound 3 with p-nitrophenyl chloroformate as described
hereinabove afforded carbonate compound 4, which was reacted with
PTX as described hereinabove to yield compound 5. Deprotection of
Boc-Cy5 with TFA, followed by conjugation with HPMA
copolymer-Gly-Phe-Leu-Gly-ONp gave compound 6. Deprotection of
Boc-Phe-Lys(alloc)-PABC-PTX 5 with TFA, followed by conjugation
with HPMA copolymer-Cy5 6 gave compound 7. Both Gly-Phe-Leu-Gly and
Phe-Lys cathepsin B-cleavable peptides were used in order to
provide convenient conjugation chemistry, longer spacer and higher
probability of cleavage. Deprotection of the alloc residue of 7
afforded the desired conjugate 1 (see, FIG. 1E).
[0698] Preparation of Compound 6:
[0699] Cy5 (20 mg, 25.41 .mu.mol) was dissolved in 0.5 mL TFA and
the solution was stirred for 5 minutes at 0.degree. C. The excess
of acid was removed under reduced pressure and the crude amine salt
was dissolved in 0.5 mL DMF. HPMA copolymer-Gly-Phe-Leu-Gly-ONp (24
mg, ONp=12.7 .mu.mol) was added followed by the addition of
Et.sub.3N (5 .mu.L). The reaction mixture was stirred for 12 hours
and the solvent was removed under reduced pressure. The crude
product was used for the next step without further purification
(FIG. 6).
[0700] Preparation of Compound 7:
[0701] Compound 5 (12 mg, 7.57 .mu.mol) was dissolved in 0.5 mL TFA
and the solution was stirred for 2 minutes at 0.degree. C. The
excess of acid was removed under reduced pressure and the crude
amine salt was dissolved in 0.5 mL DMF. Compound 6 (26.3 mg,
ONp=8.32 .mu.mol) was added followed by the addition of Et.sub.3N
(3 .mu.L). The reaction mixture was stirred for 12 hours and the
solvent was removed under reduced pressure.
[0702] Free amine (Cy5 and PTX FK), ONp and Cy5 were removed by
FPLC using XK26/70 column with Sephadex LH20 column (MeOH 100%, 1
mL/1 min) to give compound 7 as a white solid (20 mg) (FIG. 6).
[0703] Preparation of Compound I:
[0704] Compound 7 (30 mg, alloc=max. 9.9 .mu.mol) was dissolved in
DMF (1 mL). Then acetic acid (2.71 .mu.L, 47.4 .mu.mol),
Bu.sub.3SnH (30.6 .mu.L, 113 .mu.mol) and a catalytic amount of
Pd(PPh.sub.3).sub.4 were added. The reaction mixture was stirred
for 2 hours and was concentrated under reduced pressure, followed
by addition of 10 mL of acetone. The precipitate was filtered out
and washed with acetone several times. The crude product was
purified by HPLC using XK26/70 column with Sephadex LH.sub.2O (MeOH
100%, 1 mL/1 min) to give compound 1 (20 mg) as a white solid (FIG.
1E).
Example 2
Chemical Synthesis of PGA-PTX-Cy5 Conjugate
[0705] The synthesis of PGA-PTX-Cy5 conjugate is depicted in FIGS.
7A-D and 8A-B.
[0706] PGA was synthesized via the N-carboxyanydride (NCA)
polymerization of glutamic acid, as shown in FIGS. 7A-C. The
synthesized PGA was dissolved in anhydrous N,N-Dimethylformamide
(DMF) and mixed with carbonyldiimidazole (CDI) coupling reagent in
order to activate the free polymer's carboxyl groups. The reaction
mixture was stirred at room temperature for 4 hours in basic
environment. Then PTX was dissolved in anhydrous DMF and added to
the reaction mixture to obtain the PGA-PTX conjugate, through
formation of an ester bond. The reaction mixture was stirred
overnight at 4.degree. C. The reaction, shown in FIG. 7D, was
followed by High Pressure Liquid Chromatography (HPLC)
(UltiMate.RTM. 3000 Nano LC systems, Dionex). At the end of the
reaction the precipitate was washed with acetone:chloroform (1:4)
solution and dried under vacuum.
[0707] The drug loading (x, FIG. 7D) on a polymer was determined
using a High Pressure Liquid Chromatography (HPLC) (UltiMate.RTM.
3000 Nano LC systems, Dionex).
[0708] The obtained PGA-PTX conjugate was dissolved in an anhydrous
DMF and mixed again with carbonyldiimidazole (CDI) coupling reagent
in order to activate the unoccupied polymer's carboxyl groups. The
reaction mixture was stirred at room temperature for 4 hours. The
solution was removed to a round-bottom flask containing
Cy5-NH.sub.2, which was treated with Trifluoroacetic Acid (TFA) to
remove a protecting group (Boc) from it (See, FIG. 8A). The
reaction was stirred overnight at room temperature in basic
environment. The reaction, shown in FIG. 8B, was followed by High
Pressure Liquid Chromatography (HPLC) (UltiMate.RTM. 3000 Nano LC
systems, Dionex). Upon completion of the reaction the precipitate
was washed with acetone:chloroform (4:1) mixture and dried under
vacuum.
[0709] In order to remove the excess of free fluorophore, the
residue was dissolved in NaHCO.sub.2 0.2M buffer and dialyzed for 1
day at 4.degree. C. (MWCO 6-8 kDa) against DI water. The final
purification of the conjugate by size exclusion chromatography
(SEC) was performed using AKTA/FPLC system (Pharmacia/GE
Healthcare), HiTrap Desalting columns (Sephadex G-25 Superfine) in
DDW, flow rate 1.0 ml/min; UV detection.
[0710] Cy5 loading (y in FIG. 8B) was determined using SpectraMax
M5.sup.e multi-detection reader. The absorbance of conjugated Cy5
was measured and compared to that of free Cy5. Quenching efficiency
was expressed as a percentage of the fluorescence intensity of the
PGA-PTX-Cy5 conjugate (.lamda..sub.Em=670 nm) compared with the
emission of the free Cy5 at the equivalent concentration.
[0711] Preparation of PGA: The starting PGA was synthesized via the
N-carboxyanhydride (NCA) polymerization of glutamic acid. First,
NCA glutamate was prepared as described in FIG. 7A with a proposed
mechanism. H-Glu(OBzl)-OH was used as a starting material, in which
the .gamma.COOH is protected with O-benzyl (OBzl). Then, hexylamine
(denoted as R.sub.2--NH.sub.2) initiated polymerization of the NCA
of .gamma.-benzyl-L-glutamate (FIG. 7B).
[0712] PGA was characterized by GPC, zetasizer and .sup.1H-NMR.
Following deprotection of the OBzl protecting group in TFA/HBr/AcOH
mixture, the carboxyl group becomes available for coupling to PTX
and Cy5.
[0713] Preparation of PGA-PTX:
[0714] The anti-cancer drug PTX was conjugated to the PGA polymeric
backbone via its carboxyl groups, as depicted in FIG. 7D. First,
PGA functional groups were activated by carbonyldiimidazole (CDI)
coupling in anhydrous DMF, and then PTX was added to an activated
reaction mixture to obtain the PGA-PTX conjugate, through a
formation of an ester bond.
[0715] As PGA is a substrate of a lysosomal enzyme cathepsin B,
upon endocytosis of the conjugate to the cells it should be
cleaved, and result in release of free PTX in the tumor
interstitium.
[0716] Preparation of PGA-PTX-Cy5:
[0717] Cy5, in a sufficient amount, was conjugated to the polymeric
backbone of PGA, to achieve a fluorophore self-quenching. The Cy5
is attached to the PGA via an amide bond. The polymeric backbone
cleavage by the cathepsin B enzyme should release the fluorophore
from the conjugate, thus removing the self-quenching and activating
the fluorescence.
[0718] PGA-PTX-Cy5 conjugate was synthesized as described in FIGS.
8A-B. First, the unoccupied carboxylic groups of PGA were activated
with CDI coupling agent, supported by DMAP as a catalyst in a basic
environment and then a Cy5-NH.sub.2, after the protecting group
(Boc) removal (FIG. 8A), was mixed with the activated PGA-PTX
polymeric conjugate, to form an amide bond and obtain the desired
conjugate (FIG. 8B).
Example 3
Characterization and Activity of HPMA Copolymer-PTX Conjugate
[0719] Cathepsin B-Mediated Degradation and Release:
[0720] Cathepsin B-mediated degradation and release of PTX from
HPMA copolymer-PTX over time is described in FIG. 9B. PTX
concentration was increased as a function of time and expressed by
area under the curve (AUC), and represents a satisfactory
efficiency of cathepsin B activity. Release kinetics of PTX release
show a complete release from HPMA copolymer after approximately 80
hours.
[0721] Dynamic Light Scattering and Zeta Potential of HPMA
Copolymer-PTX Conjugate:
[0722] The hydrodynamic diameter size distribution and
zeta-potential of HPMA copolymer-PTX conjugate was determined using
a ZetaSizer analyzer. Table 1 presents physico-chemical
characterization of HPMA copolymer-PTX conjugate.
TABLE-US-00001 TABLE 1 Total PTX Size Zeta Loading Conjugate Mw
(kDa).sup.a (nm).sup.b Potential (mV).sup.b (mol %).sup.c HPMA-
37.70 11.99 3.69 4.0 PTX .sup.atheoretical value, .sup.bdetermined
by Zetasizer in 10% PBS (1 mg/mL), .sup.cdetermined by analytical
HPLC at .lamda. = 270 nm.
[0723] The mean hydrodynamic diameter was 11.99 nm and the zeta
potential value was 3.69 mV (Table 1). As expected, HPMA
copolymer-PTX has a neutral charge and its size is in the nano
range, which enables its targeting to the tumor via the EPR
effect.
[0724] HPMA Copolymer-PTX Conjugate Inhibits the Proliferation of
4T1 Mammary Adenocarcinoma and MDA-MB-231 Cancer Cell Lines:
[0725] The mitotic inhibitor PTX is a potent cytotoxic agent
approved as first line therapy for breast cancer. The cytotoxic
effect of the conjugate was evaluated on murine 4T1 mammary
adenocarcinoma cells. The obtained data are presented in FIG. 10A.
As shown therein, the proliferation of 4T1 cells was inhibited by
HPMA copolymer-PTX conjugate with an IC50 of about 15 .mu.M (see,
FIGS. 10A and 10D).
[0726] HPMA copolymer alone served as control and was inert at all
the concentrations tested (data not shown), in agreement with
previously published data [Duncan et al., 2001, supra]. IC50 for
free PTX was about 35 nM (see, FIG. 10D). The difference in
IC.sub.50 between the free drug and the conjugate can be attributed
to the slow release kinetics of the drug from the carrier.
[0727] These results are in accordance with previous reports
showing the cytotoxic effect of an analogous HPMA copolymer-PTX
conjugate on breast cancer cells [Miller, K., et al., Angew Chem
Int Ed Engl, 2009. 48(16): p. 2949-54; Miller, K., et al., Mol
Pharm, 2011. 8(4): p. 1052-62].
[0728] HPMA Copolymer-PTX Exhibits Anti-Angiogenic Effect In
Vitro:
[0729] The anti-angiogenic effect of PTX on endothelial cells was
previously demonstrated [Miller et al., 2009 and 2011, supra;
Clementi, C., et al., Mol Pharm, 2011. 8(4): p. 1063-72]. These
studies have demonstrated inhibitory effect of PTX on different
stages of the angiogenic cascade-proliferation, migration and
formation of tube-like structures. Since endothelial cells that
construct the tumor vasculature, also overexpress cathepsin B, the
inhibitory effect of the herein described cathepsin B-dependent
delivery system on HUVEC proliferation was evaluated.
[0730] The obtained results are presented in FIGS. 10B and 10D, and
indeed show that the HPMA copolymer-PTX conjugate exhibited
cytotoxic effect on HUVEC proliferation with an IC.sub.50 of about
90 nM compared to about 2 nM of the free drug. Similarly to the
aforementioned experiments performed on 4T1 cells, HPMA copolymer
alone served as control and was nontoxic at all the concentrations
tested (data not shown).
[0731] The results demonstrate that PTX maintained its cytotoxic
activity in vitro upon conjugation to HPMA copolymer and that a
cathepsin B-dependent release mechanism is efficient for active
targeting of HPMA copolymer-PTX to breast cancer and its
vasculature overexpressing the enzyme in vivo.
Example 4
Characterization and Activity of HPMA Copolymer-PTX-FK
Conjugate
[0732] The chemical structure of HPMA copolymer-PTX-FK conjugate is
presented in FIG. 1D. PTX was conjugated to HPMA copolymer through
a Gly-Phe-Leu-Gly (GFLG) linker and an addition of Phe-Lys-PABC
linker respectively, both cleavable by cathepsin B enzyme.
[0733] The conjugation to the HPMA copolymer was a two-step
procedure in which PTX was first attached to the FK-PABC linker and
then conjugated to HPMA copolymer-GFLG-ONp (FIG. 4).
[0734] The resulting conjugate was water-soluble, PTX-FK loading
was 1.12 mol % (2.02 PTX molecules per polymeric chain).
[0735] Cathepsin B-mediated degradation and release of PTX from
HPMA copolymer-PTX-FK over time is described in FIG. 9A. PTX
concentration was increased as a function of time and expressed by
area under the curve (AUC), and presents a satisfactory efficiency
of cathepsin B activity. Release kinetics of PTX show a complete
release from HPMA copolymer after approximately 50 hours.
[0736] HPMA Copolymer-PTX-FK Conjugate Inhibits the Proliferation
of MDA-MB-231 Human Mammary Adenocarcinoma Cancer Cell Line:
[0737] The cytotoxic effect of the conjugate was evaluated on human
MDA-MB-231 mammary adenocarcinoma cells. The obtained data is
presented in FIGS. 10C and 10D. As shown therein, the proliferation
of MDA-MB-231 cells was inhibited by HPMA copolymer-PTX-FK
conjugate with an IC50 of 100 nM. HPMA copolymer alone served as
control and was inert at all the concentrations tested (data not
shown), in agreement with previously published data.
[0738] IC.sub.50 for free PTX was about 0.5 nM. The difference in
IC.sub.50 between the free drug and the conjugate can be attributed
to the slow release kinetics of the drug from the carrier. These
results are in accordance with previous reports showing the
cytotoxic effect of an analogous HPMA copolymer-PTX conjugate on
breast cancer cells [Miller et al. supra].
Example 5
Characterization and Activity of HPMA Copolymer-SQ-Cy5
Conjugate
[0739] The chemical structure of the HPMA-copolymer-GFLG-en-Cy5
conjugate, also referred to herein as HPMA copolymer-SQ-Cy5
conjugate (SQ=self-quenching), or simply as HPMA copolymer-Cy5, is
presented in FIG. 1A.
[0740] HPMA-copolymer-GFLG-en-Cy5 conjugate was synthesized with
3.8 mol % loading (7.5 dye molecules per polymeric chain) and its
fluorescence spectrum was characterized, in order to evaluate both
the self-quenching of the conjugated fluorophore and its
biodegradability by cathepsin B.
[0741] As shown in FIG. 11A, HPMA copolymer-SQ-Cy5 conjugate
exhibits significant self-quenching; the fluorescent signal of the
HPMA copolymer-SQ-Cy5 conjugate was reduced compared to an
equivalent concentration of free Cy5. At the signal's linear range,
the two linear trend lines slopes of free Cy5 and HPMA
copolymer-Cy5 were compared and a reduction of 54% therebetween was
obtained. In addition, after the signal reached saturation, a
reduction of about 80% was observed.
[0742] In order to evaluate the increase in fluorescence intensity
due to enzymatic cleavage, HPMA copolymer-SQ-Cy5 was incubated in
the presence of cathepsin B and release of Cy5 was assessed by
fluorescence signal. The results are presented in FIG. 11B, and
show that the measured fluorescence intensity was dramatically
increased over time and plateaued after about 100 hours. In the
absence of the enzyme, there was no increase in fluorescent
signal.
[0743] A HPMA copolymer-SQ-Cy5, with loading of 3.8 mol % Cy5,
exhibited satisfactory self-quenching properties and activation by
cathepsin B. Under physiological conditions, the conjugate is
relatively optically silent in its quenched state (i.e., turn-OFF),
and becomes highly fluorescent after enzymatic cleavage of the GFLG
linker by cathepsin B. It is postulated that the loading of the
fluorophore affects the optimal performance of the conjugate. At
low fluorophore loading, only limited quenching may occur, whereby
at high fluorophore loading, the Turn-ON may not occur as the
enzyme may not reach its target site [Melancon, M. P., et al.,
Pharm Res, 2007. 24(6): p. 1217-24].
[0744] In Vitro Turn-on Capacity on HPMA-SQ-Cy5 Conjugate:
[0745] As shown in FIG. 11C, incubation of HPMA copolymer-GFLG-Cy5
in cultured MDA-MB-231 cells resulted in significantly higher
fluorescence signal intensity than that observed in culture
non-treated MDA-MB-231 cells during a period of 0.5-48 hours.
[0746] In Vivo Characterization of HPMA Copolymer-SQ-Cy5
Cathepsin-Dependent Release:
[0747] As mentioned above, overproduction of cathepsin B in vivo is
associated with breast carcinoma, both tumor cell population and
tumor endothelium. Thus, the ability of the probe conjugate to
exhibit Turn-ON properties, and to image endogenously produced
cathepsin B in a murine model of 4T1 breast adenocarcinoma tumors
was evaluated.
[0748] Mice bearing approximately 100 mm.sup.3 tumors were injected
intra-tumorally with 0.1 mM free Cy5 and equivalent Cy5 dose of
HPMA copolymer-SQ-Cy5. Injected mice were imaged using CRI
Maestro.TM. non-invasive fluorescence imaging systems over time for
approximately 8 hours.
[0749] The obtained data is presented in FIGS. 12A and 12B. The
initial fluorescent signal of HPMA copolymer-SQ-Cy5 is
significantly lower than that of free Cy5, which exhibits the
self-quenching properties of high-loaded Cy5. In addition, FIG. 12A
clearly shows an increase of 1.8-fold change in fluorescence signal
within 1 hour of injection. Interestingly, HPMA copolymer-SQ-Cy5
exhibited improved biocompatibility for in vivo florescence
imaging. While the free Cy5 bleached almost completely about 3
hours following injection, although lower, the fluorescent signal
of the conjugated Cy5 retained for long period of time (FIG. 12A).
Consequently, HPMA copolymer-SQ-Cy5 may represent a suitable
approach for in vivo imaging of endogenous cathepsin B in tumor, to
indicate on drug release in real time and for tumor monitoring over
time.
[0750] HPMA Copolymer-SQ-Cy5 Exhibit Improved Pharmacokinetics
Profile in Mice:
[0751] To assess whether HPMA copolymer-PTX exhibits preferable
accumulation and release at the tumor site once injected
systemically, HPMA copolymer-SQ-Cy5 was administered into mice and
its pharmacokinetics profile was utilized to deduce on HPMA
copolymer-PTX pharmacokinetics profile.
[0752] Mice bearing about 300 mm.sup.3 4 T1 tumors were
administered via the tail vein with HPMA copolymer-Cy5 (10 .mu.M;
200 .mu.l). It was hypothesized that conjugation will result in
half-life prolongation and tumor specific accumulation and
release.
[0753] As shown in FIG. 13A, and in accordance with this
hypothesis, HPMA copolymer-SQ-Cy5 demonstrated accumulation in the
tumor. Mice were imaged over time and fluorescent signal in the
tumor was measured. As described in FIG. 13B, at the first 3 hours
following administration, HPMA copolymer-Cy5 exhibited no
preferable accumulation at the tumor. However, after 4 hours
increased fluorescent signal in the tumor was measured.
[0754] Next, healthy organs (heart, lungs, liver, spleen and
kidneys) and tumors were resected from mice injected with the
conjugate at different time points and the fluorescent intensity
was evaluated. The obtained data is presented in FIG. 13C and show
that increased fluorescent signal was measured within tumors 12
hours following administration. HPMA copolymer-SQ-Cy5 was hardly
detectable in the heart and spleen. Interestingly, increased
fluorescent signal was also measured in the liver and kidneys.
However, since the fluorescent signal in these organs did not
increase over time, it can be concluded that Cy5 was not released
from HPMA copolymer, hence, PTX will not be released and
fluorescent as well. After several hours, the signal decreased in
these organs and it was only increased over time within tumors. To
conclude, HPMA copolymer-SQ-Cy5 exhibited preferable accumulation
in the tumor, liver and kidneys, but Cy5 was released, presumably
by enzymatic cleavage, only within tumor cells expressing cathepsin
B.
Example 6
Characterization and Activity of HPMA Copolymer-PTX-Cy5 and HPMA
Copolymer-PTX-FK-Cy5 Conjugates
[0755] In HPMA copolymer-PTX-Cy5 conjugate, also referred to herein
as HPMA copolymer-SQ-Cy5-PTX, both Cy5 and PTX were conjugated to
HPMA copolymer through a Gly-Phe-Leu-Gly (GFLG) linker, cleavable
by cathepsin B enzyme. For HPMA copolymer-PTX-FK-Cy5, also referred
to herein as HPMA copolymer-SQ-Cy5-PTX-FK, both Cy5 and PTX were
conjugated to HPMA copolymer through a Gly-Phe-Leu-Gly (GFLG)
linker and an addition of Phe-Lys-PABC linker, respectively,
cleavable by cathepsin B enzyme. Characterization of HPMA
copolymer-SQ-Cy5-PTX and HPMA copolymer-SQ-Cy5-PTX-FK conjugates
show an increase in fluorescence following incubation with
cathepsin B, as presented in FIGS. 14A-B. FIG. 14C presents
comparative plots showing that fluorescence intensity
(.lamda..sub.Ex=650 nm) decreased with increasing load of Cy5.
Example 7
Characterization and Activity of PGA-PTX-Cy5 Conjugate
[0756] Characterization:
[0757] To characterize the conjugate, its absorption and
fluorescence were evaluated. Cy5 loading on the conjugate was
calculated by spectroscopy analysis.
[0758] In addition, these conjugate are substrate for the enzyme
cathepsin B. When mixing the conjugate with the enzyme (in a
suitable buffer with a low pH), the enzyme should cleave the
conjugate and release the attached moieties. The enzymatic reaction
was followed using HPLC and spectrophotometer. The absorption
spectrum of the PGA-PTX-Cy5 conjugate relative to the absorption
spectrum of the free Cy5 is presented in FIG. 15A. As shown in the
emission spectrum (FIG. 15B), a fluorescent signal observed for the
conjugates of 4 mol % and 7.5 mol % Cy5 loading is significantly
lower relative to a signal emitted from an unconjugated Cy5. The
fluorescence intensity decreases as Cy5 loading on a polymer
increases.
[0759] Cy5 release from the conjugate was also monitored by
measuring the change in the fluorescence intensity at sequential
time points. The fluorescence measurements were carried out at
excitation wavelengths of 650 nm using SpectraMax M5.sup.e
multi-detection reader. Samples (50 .mu.l) were collected every 24
hours (up to 160 hours) and immediately analyzed. The incubation of
the PGA-PTX-Cy5 conjugate with cathepsin B enzyme, showed an
increase in emitted fluorescent signal as a function of time. In
the absence of cathepsin B, almost no increase in fluorescence was
observed (FIG. 15C). This data shows a self-quenching ability of
PGA-PTX-Cy5 conjugate, since while the conjugate is intact the
fluorescent signal is significantly silent, while when the Cy5 is
released from the PGA polymeric backbone in the presence of
cathepsin B, there is no longer self-quenching effect and the
fluorescent signal increases.
[0760] Release of PTX from the conjugate incubated with cathepsin B
enzyme over time was monitored by reversed phase (RP) HPLC.
UltiMate.RTM. 3000 Nano LC systems (Dionex), equipped with 3000
pump, VWD-3000 UV-Vis detector and Chromeleon.RTM. 6.80 software.
The column in use was Phenomenex Jupiter 5.mu. 250.times.4.60 mm
C-18 300A. Chromatographic conditions were: flow: 1.0 ml/min,
gradient: 20% to 100% solution B in 20 minutes (sol. A--0.1% TFA in
water; sol. B--0.1% TFA in acetonitrile (MeCN)). Samples (50 .mu.l)
were collected simultaneously with samples for Cy5 release
determination, every 24 hours (up to 160 hours). To each sample 150
.mu.l of methanol added and immediately analyzed. The AUC of a PTX
peak was increase over the time as shown in FIG. 15D.
[0761] Anti-Proliferative Activity:
[0762] The cytotoxic effect of the conjugate was evaluated on human
MDA-MB-231 mammary adenocarcinoma cells, on murine 4T1
adenocarcinoma cells and on human WM239A melanoma cells. As shown
in FIGS. 16A-D, the proliferation of all cell lines was inhibited
by PGA-PTX-Cy5 conjugate with an IC.sub.50 of about 40 nM for
MDA-MB-231 cells, an IC.sub.50 of about 650 nM for 4T1 cells and
with IC.sub.50 of about 80 nM for WM239A cells.
[0763] Free Paclitaxel and PGA-PTX polymeric conjugate served as
controls. IC.sub.50 for free PTX was about 2 nM in case of
MDA-MB-231 cells, about 60 nM in case of 4T1 cells and about 5 nM
in case of WM239A cells (FIG. 16D). The difference in IC50 between
the free drug and the conjugate can be attributed to the slow
release kinetics of the drug from the carrier. These results are in
accordance with previous reports showing the cytotoxic effect of an
analogous PGA-PTX conjugate on breast cancer cells.
[0764] PGA-PTX-Cy5 conjugate inhibits the migration of HUVEC: The
migration of HUVEC in the presence of PGA-PTX-Cy5 conjugate was
evaluated using the scratch assay. The method is based on the
observation that, upon creation of a new artificial gap, so called
"scratch", on a confluent cell monolayer, the cells on the edge of
the newly created gap will move toward the opening to close the
"scratch" until new cell-cell contacts are established again.
Following 24 hours incubation of HUVEC in 6 wells plate (500,000
cells per well) the cells were treated with the conjugate and the
different controls (such as: PTX, PGA-PTX and no treatment). At
time zero (t=0) images were taken by phase-contrast microscope in a
reference point. Following another 12 h of incubation, images of
the reference point were taken again. The samples were analyzed
quantitatively by ImageJ software. The PGA-PTX-Cy5 conjugate, at
PTX-equivalent concentrations of 20 nM inhibited efficiently the
migration of HUVEC by 36% of gap closure (FIG. 17). As expected,
PTX alone, which is known to be anti-angiogenic at low doses,
showed higher inhibitory effect of 20% of gap closure.
[0765] The effect of PGA-PTX-Cy5 conjugate on the ability of HUVECs
to form capillary-like tube structures on matrigel was also
evaluated. As previously reported, such an assay can emulate the
capability of endothelial cells to form vascular networks in vivo.
HUVEC were incubated in the presence of PGA-PTX-Cy5 conjugate, free
PTX, free Cy5, and PGA, and in absence of treatment, for 8 hours,
pictured and quantitatively analyzed. The obtained data is
presented in FIGS. 18A-B. As shown therein, PGA-PTX-Cy5 at PTX load
equivalent of 20 nM inhibited the tubular structures formation by
about 40%, compared to untreated cells used as negative
control.
Example 8
Syntheses of HPMA Copolymer Conjugates by RAFT Polymerization
[0766] Reversible addition-fragmentation chain transfer (RAFT)
polymerization is a versatile controlled/"living" free radical
polymerization technique resulting in predetermined molecular
weight with narrow polydispersity. This technique enables the
theoretical calculation of molecular weight of the polymers by the
ratio of monomer concentration to chain transfer agent
concentration and the conversion of the polymerization. Additional
advantage is the ease of manufacturing since the synthesis is
carried out in a one-pot reaction.
[0767] Functional monomers were therefore designed and synthesized
for RAFT polymerization of HPMA copolymer conjugates for
theranostics. This design of RAFT synthesized copolymer conjugates
benefits from controlled polymerization and a lower polydispersity
that may improve its biodistribution and accumulation at the tumor
site, and further, the higher amount of the activatable diagnostic
moiety can lead to an increased signal emitted upon the probe
activation to a Turn-ON state.
[0768] Preparation of Functional Monomers:
[0769] The syntheses of N--(N-Boc-ethylenediamine)
methacryloylglycylglycylamide (MA-Gly-Gly-diamine-Boc) and
methacryloylglycylphenylalanylleucylglycyl-p-aminophenylcarbonate
p-nitrophenyl ester (MA-Gly-Phe-Leu-Gly-PABC-ONp; MA-GFLG-PABA;
MA-Gly-Phe-Leu-Gly-PABA) are shown in FIGS. 19A and 19B,
respectively.
[0770] Preparation of MA-Gly-Phe-Leu-Gly-OH:
[0771] MA-Gly-Phe-Leu-Gly-OH was synthesized by solid phase peptide
synthesis (SPPS) and manual Fmoc/tBu strategy using 2 grams of
2-chlorotrityl chloride beads with 80% of loading leading to a
yield of 0.88 grams, 95%.
[0772] Preparation of MA-Gly-Phe-Leu-Gly PABA:
[0773] MA-GFLG-OH (400 mg, 0.815 mmol) was dissolved in dry THF and
the solution was cooled to -15.degree. C. Then NMM (90 .mu.L, 0.815
mmol) and isobutyl chloroformate (128 .mu.L, 0.978 mmol) were
added. The reaction was stirred for 20 minutes and a solution of
4-aminobenzyl alcohol (151 mg, 1.22 mmol) in dry THF was added. The
reaction mixture was stirred for 12 hours and was monitored by TLC
(EtOAc 100%). Upon completion of the reaction, the solvent was
removed under reduced pressure and the crude product was purified
by using column chromatography on silica gel (1-8% MeOH in EtOAc)
to give MA-Gly-Phe-Leu-Gly (262 mg, 53%) as a yellow solid. See,
FIG. 19B.
[0774] Preparation of MA-Gly-Gly-diamine-Boc:
[0775] MA-Gly-Gly-OH was synthesized as described in Rejmanova et
al. (1977) Makromol Chem 178, 2159-2168, followed by amination that
was carried out as follows: MA-Gly-Gly-OH (200 mg, 0.864 mmol), DCC
(196.2 mg, 0.951 mmol), NHS (99.5, 10 mmol) were stirred in DMF for
1 hour, then N-(tert-butoxycarbonyl) (Boc)-ethylenediamine (138.5
mg, 0.864 mmol) was added, and the reaction mixture was stirred for
24 hours at room temperature. The product was obtained by
precipitation in ethyl ether. See, FIG. 19A.
[0776] RAFT Polymerization:
[0777] Exemplary synthetic schemes utilizing the above-described
functional monomers are presented in FIGS. 20 and 21. A RAFT
synthesized HPMA copolymer precursor is obtained, bearing
functional groups (ONp and/or NH.sub.2-Boc) for facile conjugation
of a drug (e.g., paclitaxel) and a fluorescent agent (e.g., Cy5 or
FITC).
[0778] FIG. 20 presents the chemical structure, two-step synthesis
and cleavage mechanism by Cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-PTX, an exemplary self-quenching
(homo-FRET) based theranostic system synthesized by RAFT
polymerization.
[0779] FIG. 21 presents the chemical structure, two-step synthesis
and cleavage mechanism by Cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-PTX, an exemplary
self-quenching (homo FRET) based theranostic system synthesized by
RAFT polymerization.
[0780] FIG. 22A presents exemplary synthetic schemes for the
preparation of Boc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-Cy5 and
Boc-NH-LG-PABC-FITC, useful for conjugating PTX, Cy5 and FITC,
respectively, as non-limiting examples of a drug and dyes, to the
HPMA copolymer precursor prepared by RAFT polymerization as
described hereinabove.
[0781] FIG. 22A presents an exemplary synthetic scheme of drug and
dye dipeptide-PABC moieties (ivDde-NH--FK-PABC-PTX and
ivDde-NH--FK-PABC-Cy5 respectively) useful for conjugation to HPMA
copolymer-dipeptide-ONp (Gly-Gly-ONp).
[0782] The Cy5 is presented herein as a representative example and
can be replaced with other fluorogenic or fluorescent dye moieties
or probes such as QCy7 and FRET probes as described herein.
[0783] The PTX can also be replaced by other therapeutically active
agents as described herein.
Example 9
FRET-Based Polymeric Systems
[0784] Forster Resonance Energy transfer (FRET)-based probes are
typically composed of a fluorescent dye attached through a
cleavable linker to a quencher, as depicted in FIG. 23. Under such
circumstances, the excited fluorophore transfers its excitation
energy to the nearby quencher-chromophore in a non-radiative manner
through long range dipole-dipole interactions. Cleavage of the
linker moiety (e.g., by an analyte or enzyme of interest), results
in diffusion of the fluorescent dye away from the quencher and
thereby in generation of a measurable fluorescent signal.
[0785] In some exemplary polymeric FRET-based systems according to
the present embodiments, a fluorescent dye is attached to the
polymeric backbone units through a cleavable linker. In such cases,
upon cleavage of the linker, the fluorescent dye molecules diffuse
away from one another, thus generating a measurable fluorescent
signal. These systems are referred to herein as self-quenching (SQ)
or homo-FRET based systems. When a therapeutically active agent
(drug) is also attached to the polymeric backbone, the system is
theranostic. Exemplary such systems are described hereinabove and
are shown in FIGS. 1A-E, 2, 5, 6, 8B, 20 and 21.
[0786] In other exemplary polymeric FRET-based systems, the
fluorescent dye is attached to the polymeric backbone units via a
cleavable linker, and a quencher is also attached to the polymeric
backbone. The quencher can be attached to some of the polymeric
backbone units via a linker (preferably a non-cleavable linker). In
some embodiments, the fluorescent dye is attached to a portion of
the backbone units and the quencher is attached to another portion
of backbone units of the polymeric backbone (referred to herein
also as FRET mode I). In some embodiments, the quencher is attached
to the end of the polymeric backbone (referred to herein also as
FRET mode II). Exemplary such systems are described hereinafter and
in FIGS. 24-30.
[0787] In other exemplary systems, a moiety composed of a
fluorescent dye (as a fluorogenic moiety) and a quencher, linked to
one another via a linker, is utilized. This moiety can be attached
to polymeric backbone units, preferably via a cleavable linker,
whereby the system is designed such that upon cleavage of the
linker, the fluorescent dye diffuses away from the quencher and a
measurable fluorescent signal is generated (referred to herein as
FRET mode III). Exemplary such moieties are described hereinafter
and are shown in FIGS. 34 and 35, and an exemplary polymeric system
comprising such a moiety is shown in FIG. 39. Alternatively, such a
moiety can be attached to the end of the polymeric backbone
(referred to herein as FRET mode IV). A polymeric system comprising
such a moiety is shown in FIGS. 31A-B.
[0788] The present inventors have designed exemplary FRET-based
polymeric systems and moieties to be incorporated in such systems
as follows.
[0789] FRET-Based Systems with HPMA Copolymer Conjugates (FRET
Modes I and II):
[0790] HPMA copolymer conjugates having a drug (e.g., PTX) and a
dye (e.g., a fluorogenic moiety such as Cy5 or FITC) attached to
the HPMA backbone units, and further comprising a quencher attached
to the polymeric backbone are prepared by RAFT polymerization as
described in Example 8 hereinabove, and the quencher is attached to
the backbone by one of the following approaches: (i) through
coupling chemistry of quencher-COOH to a linker of Gly-Gly-NH.sub.2
(see, FIGS. 24, 25, 28 and 29; FRET mode I); or (ii) by coupling
chemistry of quencher-NH.sub.2 to a COOH end-functionalized
HPMA-copolymer chain, which results from the rational design of
RAFT agent with functional carboxylic acid end group (see, FIGS. 26
and 27; FRET mode II).
[0791] FIG. 24 presents the chemical structure, two-step synthesis
and cleavage mechanism by cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX as an illustration
for a FRET-based theranostic system synthesized by RAFT
polymerization (FRET mode I). The functional monomers and
preparation thereof, for synthesizing HPMA copolymer precursor are
described in Example 8 hereinabove and in FIGS. 19A and 19B. The
functionalized PTX and Cy5 moieties and the preparation thereof are
presented in FIGS. 22A-B.
[0792] FIG. 25 presents the chemical structure, two-step synthesis
and cleavage mechanism by cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX as an illustration for
a FRET-based theranostic system synthesized by RAFT polymerization
(FRET mode I). The functional monomers and preparation thereof, for
synthesizing HPMA copolymer precursor are described in Example 8
hereinabove and in FIGS. 19A and 19B. The functionalized PTX and
FITC moieties and the preparation thereof are presented in FIGS.
22A-B.
[0793] FIG. 26 presents the chemical structure, two-step synthesis
and cleavage mechanism by Cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX as an illustration
for a FRET-based theranostic system synthesized by RAFT
polymerization. The quencher-amine is coupled to the COOH
end-functionalized HPMA copolymer-PTX-Cy5 conjugate, providing one
quencher molecule per polymeric chain (FRET mode II). The
functional monomers and preparation thereof, for synthesizing HPMA
copolymer precursor are described in Example 8 hereinabove and in
FIGS. 19A and 19B. The functionalized PTX and Cy5 moieties and the
preparation thereof are presented in FIGS. 22A-B.
[0794] FIG. 27 presents the chemical structure, two-step synthesis
and cleavage mechanism by Cathepsin B of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX as an illustration for
a FRET-based theranostic system synthesized by RAFT polymerization.
The quencher, DR1-amine is coupled to the COOH end-functionalized
HPMA copolymer-PTX-FITC conjugate, providing one quencher molecule
per polymeric chain (FRET mode II). The functional monomers and
preparation thereof, for synthesizing HPMA copolymer precursor are
described in Example 8 hereinabove and in FIGS. 19A and 19B. The
functionalized PTX and FITC moieties and the preparation thereof
are presented in FIGS. 22A-B.
[0795] FIG. 28 is a scheme depicting a synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX conjugate (FRET
mode I), effected by conjugating Boc-NH-LG-PABC-PTX,
Boc-NH-LG-PABC-Cy5 and a quencher to HPMA copolymer precursor
bearing Gly-Phe-ONp/Gly-Gly-diamine Boc linkers, and addition of
LG-PABC linker to PTX and Cy5 followed by their addition to the
resulting conjugate HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX. The functional
monomers and preparation thereof, for synthesizing HPMA copolymer
precursor are described in Example 8 hereinabove and in FIGS. 19A
and 19B. The functionalized PTX and Cy5 moieties and the
preparation thereof are presented in FIGS. 22A-B.
[0796] FIG. 29 is a scheme depicting a synthesis of HPMA
copolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX conjugate (FRET mode
I), effected by conjugating Boc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-FITC
and DR1 to HPMA copolymer precursor bearing
Gly-Phe-ONp/Gly-Gly-diamine Boc linkers, and addition of LG-PABC
linker to PTX and FITC followed by their addition to the resulting
conjugate HPMA copolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX. The
functional monomers and preparation thereof, for synthesizing HPMA
copolymer precursor are described in Example 8 hereinabove and in
FIGS. 19A and 19B. The functionalized PTX and FITC moieties and the
preparation thereof are presented in FIGS. 22A-B.
[0797] FRET-Based Systems with PGA Conjugates (FRET Mode I):
[0798] A PGA-Cy5 conjugate was prepared according to the procedure
described hereinabove (see, Example 2 and FIGS. 8A-B). A Quencher
was thereafter conjugated to the polymeric backbone upon activating
the unoccupied carboxylic groups of PGA with CDI coupling agent in
a basic environment, as depicted in FIG. 30.
[0799] PGA was synthesized via the N-carboxyanydride (NCA)
polymerization of glutamic acid, as shown in FIGS. 7A-D. The
obtained polymer was dissolved in anhydrous N,N-Dimethylformamide
(DMF) and mixed with carbonyldiimidazole (CDI) coupling reagent in
order to activate the free polymer's carboxyl groups. The reaction
mixture was stirred at room temperature for 4 hours in basic
environment. The activated polymers was then added to Cy5-NH.sub.2
(see, FIG. 8A), and the reaction mixture was stirred overnight at
room temperature in basic environment. The reaction was followed by
High Pressure Liquid Chromatography (HPLC) (UltiMate.RTM. 3000 Nano
LC systems, Dionex). At the end of the reaction, the precipitate
was washed with acetone:chloroform (4:1) solution and dried under
vacuum. In order to remove excess of free fluorophore, the dried
residue was dissolved in NaHCO.sub.2 0.2M buffer and dialyzed for 1
day at 4.degree. C. (MWCO 6-8 kDa) against DI water. The obtained
conjugate was purified by size exclusion chromatography (SEC)
performed using AKTA/FPLC system (Pharmacia/GE Healthcare), HiTrap
Desalting columns (Sephadex G-25 Superfine) in DDW, flow rate 1.0
ml/min; UV detection. Cy5 loading was determined using SpectraMax
M5.sup.e multi-detection reader. The absorbance of conjugated Cy5
was measured and compared to that of free Cy5.
[0800] The obtained PGA-Cy5 conjugate was dissolved in an anhydrous
DMF and mixed again with carbonyldiimidazole (CDI) coupling reagent
in order to reactivate the unoccupied polymer's carboxyl groups.
The reaction mixture was stirred at room temperature for 4 hours.
The solution was removed to a round bottom flask containing
Quencher-NH.sub.2, and the reaction mixture was stirred overnight
at room temperature in basic environment and was monitored by High
Pressure Liquid Chromatography (HPLC) (UltiMate.RTM. 3000 Nano LC
systems, Dionex). Once the reaction was completed, the precipitate
was washed with acetone:chloroform (4:1) mixture and dried under
vacuum. In order to remove the excess of free Quencher, the dried
residue was dissolved in NaHCO.sub.2 0.2M buffer and dialyzed for 1
day at 4.degree. C. (MWCO 6-8 kDa) against DI water. The final
purification of the conjugate by size exclusion chromatography
(SEC) was performed using AKTA/FPLC system (Pharmacia/GE
Healthcare), HiTrap Desalting columns (Sephadex G-25 Superfine) in
DDW, flow rate 1.0 ml/min; UV detection. Quencher loading was
determined using SpectraMax M5.sup.e multi-detection reader.
[0801] FRET-based systems with PEG conjugates (FRET mode IV): A
FRET-based probe-polymer conjugate for use as the diagnostic
component in a theranostic system was designed, using Cy5 as a
fluorophore, conjugated to the end (terminus) of polyethylene
glycol (PEG) as the polymeric nanocarrier. The PEG-Cy5 conjugate
was further conjugated via an analyte-cleavable linker to a
quencher, to provide a PEG-Cy5-Q conjugate, forcing a Turn-OFF
fluorescent state on the probe. An exemplary conjugate was designed
to undergo specific activation by hydrogen peroxide, which is
overproduced by various tumors, and therefore can be used as
analyte for selective activation. Activation turns on a
fluorescence signal through separation of the quencher from PEG-Cy5
conjugate, as depicted in FIG. 31B.
[0802] The PEG-Cy5-Q conjugate was synthesized as depicted in FIG.
31A. In brief, A FRET-based probe, actuvatable by hydrogen
peroxide, was prepared as described in Redy et al., 2012 (supra),
and was dissolved in a minimal amount of DMF. HBTU (4 equivalents)
and DIPEA (15 equivalents) were added and the mixture was stirred
for 30 minutes. Monofunctional PEG amine (13 kDa) was dissolved in
DMF, heated to 50.degree. C. and then was added to the mixture. The
reaction was monitored using RP-HPLC. Upon completion, the obtained
polymeric conjugate was purified using preparative HPLC.
[0803] The emitted fluorescence signal in the presence and absence
of hydrogen peroxide was measured by.
[0804] The emitted fluorescence signal in the presence and absence
of hydrogen peroxide was determined by incubation with and without
hydrogen peroxide in PBS pH 7.4, while monitoring the emission
using a spectrofluorometer (.lamda..sub.ex-630 nm,
.lamda..sub.em-670 nm). The results are presented in FIGS. 32A-B,
and show the emission from the probe as a function of time after
addition of hydrogen peroxide. A significant increase of the
emitted fluorescence was observed within minutes after addition of
hydrogen peroxide, whereas no change in fluorescence was observed
in the absence of hydrogen peroxide. The conjugate exhibits stable
Turn-OFF properties in the absence of hydrogen peroxide for several
hours. Upon incubation with hydrogen peroxide, a gradual increase
of the fluorescence signal was observed (i.e. Turn-ON) that reached
saturation within approximately 3 hours. As shown in FIG. 32B, the
signal was 10 times higher than the background as measured by HPLC
and CRI Maestro.TM. imaging system.
[0805] The in vivo activation was measured following intravenous
injection into tumor-bearing mice. Mice bearing U-87 MG tumors were
injected with the PEG-Cy5-Q conjugate into the tail vein and the
emitted fluorescence was monitored immediately following injection.
At two minutes post injection, a strong fluorescence signal was
observed in the tumor alone, as shown in FIG. 33, and retained for
more than 6 hours (data not shown). These results indicate the
selective activation of the conjugate at the tumor site.
[0806] Conjugating a therapeutically active agent to a conjugate as
described herein provides a FRET-based PEG theranostic system (FRET
mode IV).
[0807] Cathepsin B-Cleavable FRET-Based Fluorogenic Moiety (for Use
in FRET Modes III and IV):
[0808] An exemplary FRET-based fluorogenic moiety (probe) which can
be attached to a polymeric backbone by any of the approaches
described herein, has been designed and synthesized.
[0809] The chemical structure and the activation mechanism of such
a FRET-based probe is presented in FIG. 34. The probe is composed
of Cy5 fluorescent dye attached through a cathepsin B substrate to
a quencher dye. The selected cathepsin B substrate was the
dipeptide Phe-Lys. The NH.sub.2-terminus of the dipeptide was link