U.S. patent application number 14/970660 was filed with the patent office on 2016-04-07 for targeted polymeric conjugates and uses thereof.
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., Universita degli Studi di Padova. Invention is credited to Gianfranco PASUT, Ronit SATCHI-FAINARO.
Application Number | 20160095838 14/970660 |
Document ID | / |
Family ID | 46208110 |
Filed Date | 2016-04-07 |
United States Patent
Application |
20160095838 |
Kind Code |
A1 |
SATCHI-FAINARO; Ronit ; et
al. |
April 7, 2016 |
TARGETED POLYMERIC CONJUGATES AND USES THEREOF
Abstract
Polymeric conjugates comprising a polymeric backbone having
attached thereto a bone targeting moiety and a therapeutically
active agent, wherein the bone targeting moiety is attached to one
end of the polymeric backbone via a branching unit such that a
molar ratio of the bone targeting moiety to the polymer is at least
2:1, are disclosed. Pharmaceutical compositions containing these
conjugates and uses thereof in the treatment of bone related
disorders are also disclosed.
Inventors: |
SATCHI-FAINARO; Ronit;
(Tel-Aviv, IL) ; PASUT; Gianfranco; (Padova,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Universita degli Studi di Padova
Ramot at Tel-Aviv University Ltd. |
Padova
Tel-Aviv |
|
IT
IL |
|
|
Assignee: |
Ramot at Tel-Aviv University
Ltd.
Tel-Aviv
IL
Universita degli Studi di Padova
Padova
IT
|
Family ID: |
46208110 |
Appl. No.: |
14/970660 |
Filed: |
December 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14117043 |
Nov 12, 2013 |
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PCT/IB2012/052338 |
May 10, 2012 |
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14970660 |
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61484991 |
May 11, 2011 |
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Current U.S.
Class: |
424/9.6 ;
514/1.3; 514/100; 530/329; 549/220 |
Current CPC
Class: |
A61K 47/641 20170801;
A61K 49/0008 20130101; A61K 49/0054 20130101; G01N 33/574 20130101;
A61K 47/56 20170801; A61K 47/24 20130101; G01N 33/5091 20130101;
A61P 35/00 20180101; A61K 47/548 20170801; A61K 31/337 20130101;
A61K 47/60 20170801 |
International
Class: |
A61K 31/337 20060101
A61K031/337; A61K 49/00 20060101 A61K049/00; A61K 47/48 20060101
A61K047/48 |
Claims
1. A conjugate comprising a polymeric backbone having attached
thereto a therapeutically active agent and a bone targeting moiety,
said therapeutically active agent being attached to one end of said
polymeric backbone and said bone targeting moiety being attached to
another end of said polymeric backbone via a branching unit, said
branching unit being arranged in a dendritic structure composed of
a cascade of branching moieties, wherein a molar ratio of said bone
targeting moiety to said polymer and is at least 2:1.
2. The conjugate of claim 1, being represented by the general
Formula I:
D-L.sub.1-[B*]-P-[B.sub.1].sub.m.sup.0-[B.sub.2].sub.m.sup.1-[B.sub.3].su-
b.m.sup.2 . . . [Bg-L.sub.2].sub.m.sup.g-1-[T].sub.m.sup.g Formula
I wherein: D is said therapeutically active agent; P is said
polymeric backbone; T is said bone targeting moiety; B* is a
branching unit or is absent; L.sub.1 is a linking moiety, linking
said therapeutically active agent to said one end of said polymeric
backbone; L.sub.2 is a second linking moiety, linking said
targeting moiety to said another end of said polymer, via said
branching unit, or is absent; B.sub.1, B.sub.2, B.sub.3 . . . Bg
are each independently a branching moiety, wherein B.sub.1,
B.sub.2, B.sub.3 . . . Bg together form said branching unit having
said dendritic structure; m is an integer that equals 2, 3, 4, 5 or
6, representing the ramification number of said dendritic
structure; and g is an integer that ranges from 1 to 20,
representing the number of generations of said dendritic
structure.
3. The conjugate of claim 1, wherein said branching unit comprises
at least one trifunctional moiety which comprises at least 3
functional groups, each of said functional groups being
independently selected from the group consisting of an amine, a
carboxylate, a thiocarboxylate, hydroxy, thiol, carbamate,
thiocarbamate, sulfonate, sulfinate, sulfonamide, phosphonate,
phosphinate, phosphoryl, urea and thiourea.
4. The conjugate of claim 3, wherein said trifunctional moiety is
selected from the group consisting of glutamic acid, beta-glutamic
acid, amino adipic acid aspartic acid, lysine, and
3-hydroxy-2-amine propanol.
5. The conjugate of claim 1, wherein said therapeutically active
agent is useful in treating a bone-related disease or disorder.
6. The conjugate of claim 1, wherein said therapeutically active
agent is paclitaxel (PTX).
7. The conjugate of claim 1, wherein said therapeutically active
agent is attached to said polymeric backbone via a biocleavable
linking moiety.
8. The conjugate of claim 7, wherein said biocleavable linking
moiety is selected from the group consisting of a
hydrolytically-cleavable linking moiety, a pH-sensitive linking
moiety and an enzymatically-cleavable linking moiety.
9. The conjugate of claim 7, wherein said biocleavable moiety is a
hydrolytically-cleavable linking moiety.
10. The conjugate of claim 9, wherein said hydrolytically-cleavable
linking moiety comprises an ester bond.
11. The conjugate of claim 10, wherein said
hydrolytically-cleavable linking moiety is derived from succinic
acid.
12. The conjugate of claim 8, wherein said enzymatically-cleavable
linking moiety is cleaved by an enzyme that is overexpressed in a
diseased bone tissue.
13. The conjugate of claim 1, wherein said polymeric backbone is
derived from a poly(alkylene glycol).
14. The conjugate of claim 13, wherein said polymeric backbone is
derived from poly(ethylene glycol) (PEG).
15. The conjugate of claim 1, wherein said bone targeting moiety is
a bisphosphonate moiety.
16. The conjugate of claim 15, wherein said bisphosphonate is
alendronate.
17. The conjugate of claim 1, wherein said polymer is a
poly(ethylene glycol), said bone targeting moiety is alendronate,
and said therapeutically active agent is paclitaxel.
18. The conjugate of claim 17, wherein said branching unit
comprises at least 3 beta-glutamic acid moieties arranged in said
dendritic structure.
19. The conjugate of claim 18, wherein said paclitaxel is attached
to said terminus backbone unit via a hydrolytically-cleavable
linking moiety.
20. The conjugate of claim 19, wherein said
hydrolytically-cleavable linking moiety comprises an ester
bond.
21. The conjugate of claim 20, having the structure: ##STR00004##
wherein n is an integer that ranges from 10 to 1000.
22. The conjugate of claim 1, further comprising a labeling agent
attached thereto.
23. A pharmaceutical composition comprising, as an active
ingredient, the conjugate of claim 1 and a pharmaceutically
acceptable carrier.
24. The pharmaceutical composition of claim 23, being packaged in a
packaging material and identified in print, in or on said packaging
material, for use in the treatment of a bone related disease or
disorder.
25. The pharmaceutical composition of claim 24, wherein said
conjugate comprises a labeling agent, the composition being
packaged in a packaging material and identified in print, in or on
said packaging material, for use in monitoring a bone related
disease or disorder.
26. A method of treating a bone related disease or disorder in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of the conjugate of
claim 1.
27. A method of monitoring a bone related disease or disorder in a
subject, the method comprising: administering to the subject the
conjugate of claim 22; and employing an imaging technique for
monitoring a distribution of the conjugate within the body or a
portion thereof.
28. A conjugate comprising a polymeric backbone having attached to
one end thereof a bisphophonate moiety, said bisphophonate being
attached to said polymeric backbone via a branching unit, said
branching unit being arranged in a dendritic structure composed of
a cascade of branching moieties, wherein a mol ratio of said
bisphosphonate to said polymer is at least 2:1.
29. The conjugate of claim 28, wherein said polymeric backbone is
derived from a poly(alkylene glycol).
30. A conjugate comprising polymeric backbone having attached
thereto a therapeutically active agent, said therapeutically active
agent being attached to one end of said polymeric backbone, wherein
said polymeric backbone further comprises a reactive group attached
to another end of said polymeric backbone via a branching unit,
said branching unit being arranged in a dendritic structure
composed of a cascade of branching moieties, wherein a molar ratio
of said functional group to said polymer and is at least 2:1,
wherein said therapeutically active agent is useful in the
treatment of a bone related disease or disorder.
31. A process of preparing the conjugate of claim 1, the process
comprising: attaching said therapeutically active agent to a
onjugate comprising said polymeric backbone having attached to one
end thereof said bisphophonate moiety, said bisphophonate being
attached to said polymeric backbone via said branching unit,
wherein a mol ratio of said bisphosphonate to said polymer is at
least 2:1, thereby preparing the conjugate.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/117,043 filed on Nov. 12, 2013, which is a
National Phase of PCT Patent Application No. PCT/IB2012/052338
having International Filing Date of May 10, 2012, which claims the
benefit of priority of U.S. Provisional Patent Application No.
61/484,991 filed on May 11, 2011. The contents of the above
applications are all incorporated by reference as if fully set
forth herein in their entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 64646SequenceListing.txt, created
on Dec. 14, 2015, comprising 4,185 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to polymeric conjugates and their use in therapy and/or diagnosis
and, more particularly, but not exclusively, to bone-targeted
polymeric conjugates and to uses thereof in treating and/or
monitoring bone-related diseases and disorders.
[0004] A limiting factor for the success of cancer chemotherapy
lies in the accumulation of the therapeutic agents in tumors.
Difficulties are encountered in the administration of sufficient
quantities of chemotherapeutic agents which provide the in vivo
concentration of the chemotherapeutic agent required to afford an
effective killing of cancer cells.
[0005] The accumulation of chemotherapeutic agents in tumors depend
on several factors including the size, surface characteristics and
circulation half-life of the chemotherapeutic agents, as well as
the degree of angiogenesis in the tumors.
[0006] Polymer-anticancer drug conjugates have been investigated,
as therapies against cancer aimed at addressing the relevant
limitations of current protocols using low molecular weight drugs.
The coupling of anti-cancer agents with water-soluble polymers has
been demonstrated to improve both the safety profile and antitumor
efficacy due to, for example, possible avoidance of toxic
formulations; and to contribute to improved biodistribution and
pharmacokinetics, which results from their restricted distribution
and the enhanced permeability and retention (EPR) effect, which
promotes passive targeting to solid tumors.
[0007] An example of the increased activity yet reduced toxicity
obtained by conjugation of anti-tumor drugs to water-soluble
polymers is presented in U.S. Pat. No. 6,884,817.
[0008] Recent studies have been directed to either synthesizing
targeted conjugates [Allen, T. M. Nat. Rev. Cancer 2002; 2:
750-763; Brumlik et al. Expert Opin. Drug Delivery 2008; 5: 87-103;
Segal et al. PLoS ONE 2009; 4: e5233; Canal et al. J. Controlled
Release 2010, 146: 388-399] or polymers bearing two anticancer
drugs for combination therapy [Vicent et al. Angew. Chem., Int. Ed.
Engl. 2005; 44 (26); 4061-4066; Pasut, et al. J. Med. Chem. 2009;
52; 6499-6502. Greco, F.; Vicent et al. J. Adv. Drug Delivery Rev.
2009; 61: 1203-1213.]. Satchi-Fainaro et al. disclose targeted
conjugates, in which paclitaxel (PTX) and alendronate (ALN) are
coupled to HPMA copolymer [Miller et al. Angew. Chem., Int. Ed.
Engl. 2009; 48: 2949-2954.]. An exemplary such conjugate was shown
to exhibit increased anticancer and anti-angiogenic activity with
respect to the free drugs and, remarkably, reduced toxicity. Other
studies in this regard are described in Segal et al. [PLoS ONE
2009; 4: e5233]; and Wang et al. [Mol. Pharmaceutics 2006; 3:
717-25].
[0009] WO 2004/062588 teaches water soluble polymeric conjugate for
bone targeted drug delivery. The polymeric drug delivery systems
taught in this application are based on hydroxypropyl methacrylate
(HPMA) conjugates of bone-targeting agents, such as alendronate and
D-Asp.sub.8, together with a chemotherapeutic agent (e.g.,
tetracycline).
[0010] WO 2009/141823 teaches polymeric conjugates comprising a
plurality of polymeric backbones (e.g., derived from HPMA) having
attached thereto a bone-targeting moiety such as alendronate and an
anti-angiogenesis agent such as paclitaxel or TNP-470.
[0011] WO 2009/141826 teaches conjugates of a polymer (e.g., PGA)
having attached thereto an angiogenesis targeting moiety and a
therapeutically-active agent such as an anti-cancer agent or
anti-angiogenesis agent.
[0012] WO 2009/141827 teaches conjugates of hydroxypropyl
methacrylamide (HPMA)-derived copolymers having attached thereto
anti-angiogenesis agents such as TNP-470 and a high load of a
bone-targeting moiety such as alendronate (ALN).
[0013] PTX is a potent anticancer drug, used for the treatment of
several cancers, however, it is associated with severe side effects
due to both its scarce tumor selectivity and the formulation in
Cremophor EL. In recent years, it has become evident that
paclitaxel at low doses has antiangiogenic properties (Wang, et al.
Anticancer Drugs 2003; 14: 13-19).
[0014] A HPMA copolymer conjugate of paclitaxel has been described
by Meerum Terwogt et al. [PNU166945; Anticancer drugs 2001; 12:
315-323]. This conjugate was aimed at improving drug solubility and
providing controlled release of paclitaxel.
[0015] Bisphosphonates, such as alendronate (ALN), are molecules
used to treat osteoporosis, bone metastases and to prevent bone
fractures. These compounds exhibit an exceptionally high affinity
to the bone-mineral hydroxyapatite, and therefore are known to be
used also as a targeting moiety [Uludag, H. Curr Pharm Des 2002; 8:
1929-1944].
[0016] Alendronate is considered potent for the treatment of bone
related diseases and cancer-associated hypercalcemia. It was shown
to have antitumor effect in several in vivo cancer models through
several different mechanisms [Tuomela et al. 2008, BMC Cancer 8:81;
Molinuevo et al. 2007, Eur J Pharmacol 562:28-33; Hashimoto et al.
2005, Cancer Res 65: 540-545]. In addition, alendronate was found
to have anti-angiogenic activity through (i) suppression of
VEGF-induced Rho activation in an ovarian cancer model [Hashimoto
et al. 2007, Biochem Biphys Res Commun 354: 478-484], (ii)
inhibition of farnesyl pyrophosphate synthase, in the mevalonate
pathway [Russell R G 2007, Pediatrics 119 Suppl 2: S150-162]; and
(iii) regulation of cellular level of MMP-2 expression in
osteosarcoma cell lines [Cheng et al. 2004, Pediatr Blood Cancer
42; 410-415].
[0017] Poly(ethylene glycol) (PEG) is a polymer approved for human
use. While it is known to be non-biodegradable, it is readily
excretable after administration into living organisms. High
excretion is typically observed for polymers having a molecular
weight lower than 40 kDa or for polymers having a hydrodynamic
diameter of less than 100 nm. In vitro studies showed that its
presence in aqueous solutions has shown no deleterious effect on
protein conformation or activities of enzymes. Covalent attachment
of PEG to biologically active compounds is described, for example,
in Yamaoka et al. [1994, J Pharm Sci 83; 601-606].
[0018] However, the potential of PEG as a carrier of low molecular
weight drugs (small molecules) has been limited by its intrinsic
low loading, owing to the polymer's chemical structure. In fact,
only the end chain groups (at the termini) of PEG can be modified
and exploited for drug coupling.
[0019] Wang et al. [in Bioconj. Chem., 2003, 14, 853-859] teach
bone-targeted drug delivery systems based on water-soluble polymers
such as PEG and HPMA, have attached thereto bone targeting moieties
such as alendronate and Asp.sub.8, and FITC as a model drug for
detection purposes.
[0020] Katsumi et al. [in J. Pharma. Sci., 2011, 100, 3783-3792]
also teach PEG-conjugated alendronate, and its effect in treating
osteoporosis.
[0021] Pasut et al. [in J. Bioactive and Comp. Polym., 2005, 20,
213] discloses PEG-epirubicin conjugates with high drug loading,
having dendrimeric (dendritic) structures based on adipic acid or
beta-glutamic acid branching units.
[0022] Pasut et al. [in J. Med. Chem. 2009; 52 (20), 6499-6502]
reported on the synthesis, characterization, and biological
performance of PEG conjugates carrying epirubicin (EPI) and one or
more nitric oxide (NO) molecules per PEG.
[0023] Bioconjugates of poly(ethylene glycol), gemcitabine (an
antitumor agent), and a targeting moiety, differing in the drug
loading, have also been reported [Pasut et al, J. Control Release.
2008; 127(3): 239-48].
[0024] Canal et al. [in J. Controlled Release 2010; 146: 388-399]
disclosed a series of PEG-epirubicin conjugates with different
folic acid contents per polymeric chain. A dendron structure was
synthesized at one end of the PEG chain with the aim of increasing
the number of folic acid molecules.
[0025] Choe et al. [in J. Controlled Release 2002; 79: 41-53]
reported on a study of various N-amino PEG-prodrugs of ara-C. In an
LX-1 solid lung tumor model, some of the PEG prodrugs exhibited
superior activity to ara-C when compared on a molar basis. However,
the degree of loading ara-C onto PEG was limited by the high
viscosity of the obtained solutions.
[0026] Choe et al. [in J. Controlled Release 2002; 79: 55-70]
described the synthesis of branched PEG (40,000) acids which had
been achieved using aspartic acid (Asp) and AspAsp dendrons.
Conjugation of these dendritic acids with cytosine arabinoside
(ara-C) was achieved by the use of spacers that allowed a greater
separation of the branches to accommodate several large ara-C
molecules in proximity to each other.
[0027] Berna et al. [in Biomacromolecules 2006, 7:146-153]
synthesized novel monodisperse PEG-dendrons with amino or
carboxylic terminal groups. The PEG-based dendrons were prepared
using monodisperse Fmoc-amino PEG propionic acid as a monomer, and
cadaverine, tris(2-aminoethyl)amine or lysine as the branching
moieties.
[0028] Other combinations of dendritic structures and drugs, or
other biologically active molecules, are disclosed, for example, in
U.S. Pat. Nos. 5,714,166, 6,417,339 and 6,632,889; and in U.S.
patent applications having Publication Nos. 2003/064050 and
2003/023968.
[0029] Bone metastases are one of the most common complications
related to advanced malignancies, particularly in the three leading
cancers; breast cancer, prostate cancer and lung cancer. Bone
metastases from breast cancer are typically osteolytic, involving
the mobilization of osteoclasts that cause pathological bone
resorption, with intense pain, bone fractures, nerve compression,
and hypercalcemia. The development and osteolytic nature of these
lesions are based on complex interactions between cancer cells and
bone marrow stroma in a cycle of bone destruction and tumor
expansion. The complexity of cellular interactions and molecular
components implicated in bone metastasis has hindered a mechanistic
elucidation of key biological features of this process, in
particular the basis for long-term survival of metastatic cells in
the bone marrow.
[0030] Chemotherapeutic agents, hormonal deprivation and
bisphosphonates are the common treatments for advanced metastatic
disease. However, with time, the disease progresses to a phase when
the standard therapy fails to control the malignancy and further
progresses to a highly chemotherapy-resistant state.
SUMMARY OF THE INVENTION
[0031] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric
backbone having attached thereto a therapeutically active agent and
a bone targeting moiety, the therapeutically active agent being
attached to one end of the polymeric backbone and the bone
targeting moiety being attached to another end of the polymeric
backbone via a branching unit, wherein a molar ratio of the bone
targeting moiety to the polymer and is at least 2:1.
[0032] According to some embodiments of the present invention, the
branching unit has a dendritic structure.
[0033] According to some embodiments of the present invention, the
branching unit comprises at least one trifunctional moiety which
comprises at least 3 functional groups, each of the functional
groups being independently selected from the group consisting of an
amine, a carboxylate, a thiocarboxylate, hydroxy, thiol, carbamate,
thiocarbamate, sulfonate, sulfinate, sulfonamide, phosphonate,
phosphinate, phosphoryl, urea and thiourea.
[0034] According to some embodiments of the present invention, the
trifunctional moiety is selected from the group consisting of
glutamic acid, beta-glutamic acid, amino adipic acid aspartic acid,
lysine, and 3-hydroxy-2-amine propanol.
[0035] According to some embodiments of the present invention, the
branching unit has a dendritic structure and the conjugate is
represented by the general Formula I:
D-L.sub.1-[B*]-P-[B.sub.1].sub.m.sup.0-[B.sub.2].sub.m.sup.1-[B.sub.3].s-
ub.m.sup.2 . . . [Bg-L.sub.2].sub.m.sup.g-1-[T].sub.m.sup.g Formula
I
[0036] wherein:
[0037] D is the therapeutically active agent;
[0038] P is the polymeric backbone;
[0039] T is the bone targeting moiety;
[0040] B* is a branching unit or is absent;
[0041] L.sub.1 is a linking moiety, linking the therapeutically
active agent to the one end of the polymeric backbone;
[0042] L.sub.2 is a second linking moiety, linking the targeting
moiety to the another end of the polymer, via the branching unit,
or is absent;
[0043] B.sub.1, B.sub.2, B.sub.3 . . . Bg are each independently a
branching moiety, wherein B.sub.1, B.sub.2, B.sub.3 . . . Bg
together form the branching unit having the dendritic
structure;
[0044] m is an integer that equals 2, 3, 4, 5 or 6, representing
the ramification number of the dendritic structure; and
[0045] g is an integer that ranges from 1 to 20, representing the
number of generations of the dendritic structure.
[0046] According to some embodiments of the present invention, the
therapeutically active agent is selected from the group consisting
of an anti-angiogenesis agent and an anti-cancer agent.
[0047] According to some embodiments of the present invention, the
therapeutically active agent is useful in treating a bone-related
disease or disorder.
[0048] According to some embodiments of the present invention, the
therapeutically active agent is selected from the group consisting
of paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY
12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic
acid, dimethylxanthenone acetic acid, endostatin, IM-862,
marimastat, a matrix metalloproteinase, penicillamine, PTK787/ZK
222584, RPI.4610, squalamine lactate, SU5416, thalidomide, TNP-470,
combretastatin, tamoxifen, COL-3, neovastat, BMS-275291, SU6668,
anti-VEGF antibody, Medi-522 (Vitaxin II), CAI, Interleukin-12,
IM862, Amilloride, Angiostatin.RTM.Protein, Angiostatin K1-3,
Angiostatin K1-5, Captopril, DL-alpha-Difluoromethylornithine,
DL-alpha-Difluoromethylornithine HCl, His-Tag.RTM.
Endostatin.TM.Protein, Fumagillin, Herbimycin A,
4-Hydroxyphenylretinamide, Juglone, Laminin, Laminin Hexapeptide,
Laminin Pentapeptide, Lavendustin A, Medroxyprogesterone,
Medroxyprogesterone Acetate, Minocycline, Minocycline HCl,
Placental Ribonuclease Inhibitor, Suramin, Sodium Salt Suramin,
Human Platelet Thrombospondin, Neutrophil Granulocyte, a monoclonal
antibodies directed against specific proangiogenic factors and/or
their receptors, a tyrosine kinase inhibitor of multiple
proangiogenic growth factor receptors, an inhibitor of mTOR, an
interferon, IL-12, EMD121974 (Cilengitide), Vitaxin; Squalamin, a
COX-2 inhibitor, a PDGFR inhibitor, NM3 and 2-ME2.
[0049] According to some embodiments of the present invention, the
therapeutically active agent is paclitaxel (PTX).
[0050] According to some embodiments of the present invention, the
therapeutically active agent is attached to the polymeric backbone
via a biocleavable linking moiety.
[0051] According to some embodiments of the present invention, the
biocleavable linking moiety is selected from the group consisting
of a hydrolytically-cleavable linking moiety, a pH-sensitive
linking moiety and an enzymatically-cleavable linking moiety.
[0052] According to some embodiments of the present invention, the
biocleavable moiety is a hydrolytically-cleavable linking
moiety.
[0053] According to some embodiments of the present invention, the
hydrolytically-cleavable linking moiety comprises an ester
bond.
[0054] According to some embodiments of the present invention,
hydrolytically-cleavable linking moiety is derived from succinic
acid.
[0055] According to some embodiments of the present invention, the
enzymatically-cleavable linking moiety is cleaved by an enzyme that
is overexpressed in a diseased bone tissue.
[0056] According to some embodiments of the present invention, the
enzyme is an extracellular enzyme.
[0057] According to some embodiments of the present invention, the
enzymatically-cleavable linking moiety is cleaved by an enzyme
selected from the group consisting of Cathepsin K, Cathepsin D,
Cathepsin H, Cathepsin L, legumain, MMP-2 and MMP-9.
[0058] According to some embodiments of the present invention, the
polymeric backbone is derived from a polymer selected from the
group consisting of a poly(alkylene glycol), a
poly(2-alkyl-2-oxazoline), and a copolymer comprising a
poly(alkylene glycol) and/or a poly(2-alkyl-2-oxazoline).
[0059] According to some embodiments of the present invention, the
polymeric backbone is derived from a poly(alkylene glycol).
[0060] According to some embodiments of the present invention, the
polymeric backbone is derived from poly(ethylene glycol) (PEG).
[0061] According to some embodiments of the present invention, the
bone targeting moiety is a bisphosphonate moiety.
[0062] According to some embodiments of the present invention, the
bisphosphonate moiety is selected from a group consisting of
alendronate, cimadronate, clodronate, tiludronate, etidronate,
ibandronate, neridronate, olpadronate, risedonate, piridronate,
pamidronate and zoledronate.
[0063] According to some embodiments of the present invention, the
bisphosphonate is alendronate.
[0064] According to some embodiments of the present invention, the
polymer is a poly(ethylene glycol), the bone targeting moiety is
alendronate, and the therapeutically active agent is
paclitaxel.
[0065] According to some of these embodiments of the present
invention, the branching unit has a dendritic structure and
comprises at least 3 beta-glutamic acid moieties arranged in the
dendritic structure.
[0066] According to some embodiments of the present invention, the
paclitaxel is attached to the terminus backbone unit via a
hydrolytically-cleavable linking moiety.
[0067] According to some embodiments of the present invention, the
hydrolytically-cleavable linking moiety comprises an ester
bond.
[0068] According to some embodiments of the present invention, the
conjugate further comprises a labeling agent attached thereto.
[0069] According to some embodiments of the present invention, the
labeling agent is selected from the group consisting of a
fluorescent agent, a radioactive agent, a magnetic agent, a
chromophore, a bioluminescent agent, a chemiluminescent agent, a
phosphorescent agent and a heavy metal cluster.
[0070] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition
comprising, as an active ingredient, any of the conjugates as
described herein and a pharmaceutically acceptable carrier.
[0071] According to some embodiments of the present invention, the
pharmaceutical composition is packaged in a packaging material and
identified in print, in or on the packaging material, for use in
the treatment of a bone related disease or disorder.
[0072] According to some embodiments of the present invention, the
conjugate comprises a labeling agent, and the composition is
packaged in a packaging material and identified in print, in or on
the packaging material, for use in monitoring a bone related
disease or disorder.
[0073] According to an aspect of some embodiments of the present
invention there is provided a method of treating a bone related
disease or disorder in a subject in need thereof, the method
comprising administering to the subject a therapeutically effective
amount of the conjugate as described herein.
[0074] According to an aspect of some embodiments of the present
invention there is provided a method of monitoring a bone related
disease or disorder in a subject, the method comprising:
[0075] administering to the subject a conjugate as described
herein, which further comprises a labeling agent; and employing an
imaging technique for monitoring a distribution of the conjugate
within the body or a portion thereof.
[0076] According to an aspect of some embodiments of the present
invention there is provided a use of the conjugate as described
herein as a medicament.
[0077] According to an aspect of some embodiments of the present
invention there is provided a use of the conjugate as described
herein in the manufacture of a medicament for treating a
bone-related disease or disorder.
[0078] According to an aspect of some embodiments of the present
invention there is provided a use of the conjugate as described
herein, which further comprises a labeling agent, as a diagnostic
agent.
[0079] According to an aspect of some embodiments of the present
invention there is provided a use of the conjugate as described
herein, which further comprises a labeling agent, in the
manufacture of a diagnostic agent for monitoring a bone related
disease or disorder.
[0080] According to an aspect of some embodiments of the present
invention there is provided a conjugate as described herein,
identified for use in the treatment of a bone related disease or
disorder.
[0081] According to an aspect of some embodiments of the present
invention there is provided a conjugate as described herein, which
further comprises a labeling agent, identified for use in
monitoring of a bone related disease or disorder.
[0082] According to some embodiments of the present invention, the
disease or disorder is associated with angiogenesis.
[0083] According to some embodiments of the present invention, the
disease or disorder is selected from the group consisting of bone
metastases and bone cancer.
[0084] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric
backbone having attached to one end thereof a bisphosphonate
moiety, the bisphosphonate being attached to the polymeric backbone
via a branching unit, wherein a mol ratio of the bisphosphonate to
the polymer is at least 2:1.
[0085] According to some embodiments of the present invention, the
polymeric backbone is derived from a poly(alkylene glycol).
[0086] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising polymeric
backbone having attached thereto a therapeutically active agent,
the therapeutically active agent being attached to one end of the
polymeric backbone, wherein the polymeric backbone further
comprises a reactive group attached to another end of the polymeric
backbone via a branching unit, wherein a molar ratio of the
functional group to the polymer and is at least 2:1.
[0087] According to some embodiments of the present invention, the
reactive group is useful for attaching to the conjugate a targeting
moiety.
[0088] According to an aspect of some embodiments of the present
invention there is provided a process of preparing a conjugate as
described herein, the process comprising:
[0089] providing a conjugate comprising a polymeric backbone having
attached to one end thereof a bisphosphonate moiety, wherein the
bisphosphonate is being attached to the polymeric backbone via a
branching unit, as described herein;
[0090] providing the therapeutically active agent; and
[0091] attaching the therapeutically active agent to the conjugate
comprising a bisphosphonate moiety described immediately
hereinabove, thereby preparing the conjugate.
[0092] 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
[0093] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0094] 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.
[0095] In the drawings:
[0096] FIGS. 1A-1C present the chemical structures of the exemplary
conjugates, according to some embodiments of the present invention,
PEG-ALN (Compound 1, FIG. 1A), PTX-PEG (Compound 2, FIG. 1B), and
PEG-PTX-ALN (Compound 3, FIG. 1C); Non-labeled conjugates are
represented by chemical structures in which X is --C(.dbd.O)-- and
FITC-labeled conjugates are represented by chemical structures in
which X is a lysine residue coupled to FITC.
[0097] FIG. 2 presents a schematic illustration of an exemplary
synthetic pathway for preparing PTX-PEG-ALN conjugate (Compound 3)
according to some embodiments of the present invention.
[0098] FIG. 3 presents a schematic illustration of an exemplary
synthetic pathway for preparing FITC labeled-PTX-PEG-ALN conjugate
(FITC labeled-Compound 3) according to some embodiments of the
present invention.
[0099] FIGS. 4A-4B are bar graphs presenting the mean hydrodynamic
diameter of PTX-PEG (Compound 2; FIG. 3A) and PTX-PEG-ALN (Compound
3; FIG. 3B), as determined by a real time particle analyzer
(NanoSight LM20.TM.).
[0100] FIGS. 5A-5B present comparative plots demonstrating the
stability of the exemplary PTX-PEG-ALN conjugate, expressed as % of
the conjugate out of the initial amount of the conjugate, following
incubation in plasma (diamonds), at pH 7.4 (squares) and at pH 5
(triangles), as monitored by RP-HPLC, at the indicated time points
(FIG. 5A); and the stability of the exemplary PTX-PEG-ALN conjugate
following incubation at pH 7.4 (blank squares) and at pH 5 (black
squares) and of the exemplary PTX-PEG conjugate following
incubation at pH 7.4 (blank triangles) and at pH 5 (black
triangles), expressed by the average micelles' size, as monitored
by dynamic light scattering (Malvern Nano-S) (FIG. 5B).
[0101] FIG. 6 presents plots demonstrating the binding kinetics of
the exemplary conjugates PEG-ALN and PTX-PEG-ALN to the bone
mineral HA, following incubation for 0, 2, 5, 10, and 60 minutes,
as analyzed by FPLC.
[0102] FIG. 7 presents plots demonstrating the effect of
PTX-PEG-ALN (black squares), PEG (black diamonds), PEI (black
circles), PTX vehicle (1:1:8 ethanol/Cremophor EL/saline, black
diamonds), and a combination of PTX plus ALN as free drugs (blank
squares) on hemolysis of rat red blood cells upon incubation for 1
hours at serial concentrations. Results are presented as % of
hemoglobin release. Due to similar values, some symbols
overlap.
[0103] FIGS. 8A-8B present comparative plots demonstrating the
effect of various concentrations of PEG (black diamonds), the
PEG-ALN conjugate as described herein (black circles), the PTX-PEG
conjugate as described herein (black triangles), free PTX (blank
triangles) and free ALN (blank circles) at equivalent
concentrations (FIG. 8A) and of PEG (black diamonds), the
PTX-PEG-ALN conjugate as described herein (black squares) and a
combination of PTX and ALN as free drugs at equivalent
concentrations (FIG. 8B), on PC3 cells, upon incubation for 72
hours. Data represent mean.+-.s.d. X-axis is presented at a
logarithmic scale.
[0104] FIG. 9 is a bar graph demonstrating a quantitative analysis
of the effect of a combination of PTX and ALN as free drugs, free
PTX, free ALN, PEG, a PTX-PEG-ALN conjugate as described herein, a
PTX-PEG conjugate as described herein and a PEG-ALN conjugate as
described herein, on the migration of PC3 cells, presented as % of
migrated cells compared with control, untreated cells, following 2
hours incubation. Data represent mean.+-.s.d.
[0105] FIGS. 10A-10B present comparative plots demonstrating the
effect of various concentrations of a combination of PTX and ALN as
free drugs (blank squares), free PTX (blank triangles), free ALN
(blank circles), and equivalent concentrations of PEG (black
diamonds), PTX-PEG-ALN (black squares), PTX-PEG (black triangles)
and PEG-ALN (black circles) conjugates on the proliferation of
murine 4T1 (FIG. 10A) and human MDA-MB-231 (FIG. 10B) mammary
adenocarcinoma cell lines, following incubation for 72 hours. Data
represent mean.+-.s.d. X-axis is presented at a logarithmic
scale.
[0106] FIGS. 11A-11B present comparative plots demonstrating the
effect of various concentrations of a combination of PTX and ALN as
free drugs (blank squares), free PTX (blank triangles), free ALN
(blank circles), and equivalent concentrations of PEG (black
diamonds), PTX-PEG-ALN (black squares), PTX-PEG (black triangles)
and PEG-ALN (black circles) conjugates on the proliferation of
HUVEC (FIG. 11A), wherein the X-axis is presented at a logarithmic
scale (FIG. 11A); and the effect of these treatments on the
migration of HUVEC towards the chemoattractant VEGF, wherein
migration was normalized to percent migration with 100%
representing migration to VEGF alone (FIG. 11B). The quantitative
analysis of the number of migrated cells is presented.
[0107] FIGS. 12A-12B present representative images of
capillary-like tube structures of HUVEC seeded on Matrigel.RTM.
following treatment with a combination of PTX and ALN as free
drugs, free PTX, free ALN, and equivalent concentrations of PEG,
PTX-PEG-ALN, PTX-PEG and PEG-ALN conjugates (FIG. 12A; scale bar
represents 100 .mu.m); and a bar graph showing the effect of these
treatments on the ability of HUVEC to form capillary-like tube
structures as a quantitative analysis of the mean length of the
tubes (FIG. 12B). Data represents mean.+-.s.d. * P<0.05, **
P<0.01.
[0108] FIGS. 13A-13B are bar graphs showing the biodistribution of
FITC labeled-PEG (PEG Dendron; black), PTX-PEG (grey), PTX-PEG-ALN
(white), and PEG-ALN (strips) conjugates, following intravenous
injection to SCID mice bearing MDA-MB-231 human mammary tumors in
the tibia, measured using the fluorescence non-invasive imaging
system (CRI.TM. Maestro), and presenting semi-quantitative time
dependent tumor accumulation profile of FITC-labeled conjugates in
vivo, assessed as tumor/background (normal skin) ratios of
florescence intensities of representative regions of interest (FIG.
13A), and ex vivo fluorescence intensities of tumors and organs
resected 8 hours post treatment (FIG. 13B). Data represent
mean.+-.s.e.m.
[0109] FIG. 14 presents comparative plots showing the
pharmacokinetic profile of PTX in 1:1:8 Ethanol:Cremophor EL:Saline
(blank triangles), of PTX-PEG conjugate in PBS pH=6 (black
triangles), and of PTX-PEG-ALN conjugate in PBS pH=6 (black
squares) (dose: 10 mg/Kg PTX equiv., n=10 animals per group) in
female Balb/C mice. Each point is the mean of PTX serum level in
animals (*=p<0.05 of PTX-PEG vs PTX; **=p<0.05 of PTX-PEG-ALN
vs PTX). Y-axis is presented at a logarithmic scale.
[0110] FIGS. 15A-15D present comparative plots showing the effect
of intravenous administration of 15 mg/kg free PTX (blank
triangles), 35 mg/kg free ALN (blank circles), a combination of ALN
and PTX as free drugs (blank squares), of the PTX-PEG (black
triangles), PEG-ALN (black circles), PTX-PEG-ALN (black squares)
conjugates at equivalent concentrations and of saline (black
diamonds) or PTX-vehicle (blank diamonds) as controls, every other
day, to mice bearing 4T1-mCherry tumors in the tibia, as measured
by intravital non-invasive fluorescence imaging of the tumors (FIG.
15A; scale bar represents 15 mm); the corresponding fluorescence
images of 4T1-mCherry tumors in the tibia (FIG. 15B); comparative
plots showing the percent body weight change from initial weight in
mice following the indicated treatments at equivalent dose of the
free drugs (FIG. 15C); and images showing the H&E histology
staining of tumor sections of the saline control and the various
treatment groups (FIG. 15D). * P<0.05 value of mice treated with
PEG conjugates was analyzed against saline treated mice, P value of
free PTX was analyzed against control mice treated with
PTX-vehicle. Data represent mean.+-.s.e.m. (n=6 mice per
group).
[0111] FIGS. 16A-16B are bar graphs presenting WBC counts from
blood samples collected on day 11 (FIG. 16A) and Micro Vessels
Density (MVD) analysis assessed by vascular marker CD34 staining
(FIG. 16B) in mice bearing 4T1-mCherry adenocarcinoma of the
mammary in the tibia and treated by intravenous administration
every other day of 15 mg/kg free PTX, and the conjugates PTX-PEG,
PEG-ALN, PTX-PEG-ALN at equivalent concentrations and with saline
or PTX-vehicle as controls. * P<0.05 value of mice treated with
PEG conjugates was analyzed against saline treated mice, P value of
free PTX was analyzed against control mice treated with
PTX-vehicle. Data represent mean.+-.s.e.m. (n=6 mice per
group).
[0112] FIGS. 17A-17B are bar graphs presenting the effect of
intravenous administration, every other day, of 15 mg/kg free PTX,
and of the PTX-PEG, PEG-ALN, PTX-PEG-ALN conjugates at equivalent
PTX concentrations and of saline or PTX-vehicle controls, on the
apoptotic CEC counts (FIG. 17A) and viable CEP levels (FIG. 17B) in
mice bearing 4T1-mCherry tumors in the tibia, as measured using
flow cytometry analysis performed on blood samples taken on day 11
of treatment. The calculation of the number of apoptotic CEC and
viable CEP in peripheral blood was based on the WBC of each mouse.
*P<0.05 value of mice treated with PEG-PTX-ALN conjugate was
analyzed against saline control mice. Data represent mean.+-.s.e.m.
(n=6 mice per group).
[0113] FIGS. 18A-18D present comparative plots demonstrating the
anti-tumor effect of intravenous administration, every other day,
of a combination of 35 mg/kg free ALN and 15 mg/kg free PTX (blank
squares), a combination of 17.5 mg/kg free ALN and 7.5 mg/kg free
PTX (black triangles), and of the PTX-PEG-ALN conjugate (black
squares) (dose: 15 mg/kg PTX and 35 mg/kg ALN equiv.), and of
saline (black diamonds) or PTX-vehicle (blank diamonds) controls,
as measured by intravital non-invasive fluorescence imaging of
MDA-MB-231-mCherry tumors in the tibia (FIG. 18A), with the inset
showing the obtained fluorescence images of the MDA-MB-231-mCherry
tumors in the tibia on a scale bar of 15 mm; comparative plots
showing the percent body weight change from initial weight in each
of the tested groups (FIG. 18B); images showing H&E staining of
tumor sections of the MDA-MB-231-mCherry-labeled tumors in the
tibia in each of the tested groups (FIG. 18C); and images showing
H&E staining of ossea medulla tumor sections of the saline
control and PTX-PEG-ALN-treated mice (FIG. 18D). Data represent
mean.+-.s.e.m. (n=6 mice per group). *P<0.05 value of mice
treated with PTX-PEG-ALN conjugate was analyzed against saline
treated mice, P value of combination of free PTX plus ALN was
analyzed against control mice treated with PTX-vehicle.
[0114] FIGS. 19A-19B are bar graphs presenting WBC counts from
blood samples collected on day 20 (FIG. 16A) and Micro Vessels
Density (MVD) analysis assessed by vascular marker CD34 staining
(FIG. 16B) in mice bearing MDA-MB-231-mCherry-labeled in the tibia
and treated by intravenous administration every other day with a
combination of 15 mg/kg free PTX and 35 mg/kg free ALN, a
combination of 17.5 mg/kg free ALN and 7.5 mg/kg free PTX as free
drugs, the PTX-PEG-ALN conjugate at equivalent concentrations and
with saline or PTX-vehicle as controls. *P<0.05 value of mice
treated with PTX-PEG-ALN conjugate was analyzed against saline
treated mice, P value of a combination of free PTX and free ALN was
analyzed against control mice treated with PTX-vehicle. Data
represent mean.+-.s.e.m. (n=6 mice per group).
[0115] FIGS. 20A-20B are bar graphs presenting the effect of
intravenous administration, every other day, of a combination of 15
mg/kg free PTX and 35 mg/kg free ALN, of the PTX-PEG-ALN conjugate
(dose: 15 mg/kg PTX and 35 mg/kg ALN equiv.), and of saline as
control, on the apoptotic CEC counts (FIG. 20A) and viable CEP
levels (FIG. 20B) in mice bearing MDA-MB-231-mCherry tumors in the
tibia, as measured using flow cytometry analysis performed on blood
sample taken on day 20 of treatment. The calculation of the number
of apoptotic CEC and viable CEP in peripheral blood was based on
the WBC of each mouse. *P<0.05 value of mice treated with
PEG-PTX-ALN conjugate was analyzed against saline control mice.
Data represent mean.+-.s.e.m. (n=6 mice per group).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0116] The present invention, in some embodiments thereof, relates
to chemical conjugates and their use in therapy and/or diagnosis
and, more particularly, but not exclusively, to bone-targeted
polymeric conjugates and to uses thereof in monitoring and/or
treating bone-related diseases and disorders.
[0117] 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.
[0118] As discussed hereinabove, currently known agents for
treating bone related cancer and other angiogenesis-related bone
conditions, at doses where therapeutic activity is achieved, are
characterized by high toxicity, which limits their use.
[0119] While reducing the present invention to practice, the
present inventors have devised and successfully prepared and
practiced novel conjugates, based on a heterobifunctional
PEG-dendrimer (also referred to herein as PEG-dendron) such as, for
example, NH.sub.2-PEG-.beta.Glu-(.beta.Glu).sub.2-(COOH).sub.4,
having attached thereto a bone targeting moiety and a
therapeutically active agent. The present inventors have
demonstrated that such conjugates can be obtained with a high
degree of homogeneity, and a great control over the active agents'
ratio, which can be pre-determined by defining the dendritic
structure of the polymer.
[0120] The devised heterobifunctional PEG-dendrimer allows the
subdivision of targeting and therapeutic functions by linking the
therapeutic agent and the targeting agent at the two different end
chains of the polymer. This design may lead to the obtainment of
amphiphillic conjugates, in cases of a hydrophobic therapeutically
active agent and a hydrophilic targeting moiety. The spatial
separation of the active agents (therapeutically active agent and
targeting moiety), besides offering the possibility to form
self-assembled micelles, maintains all of the molecules of a
hydrophilic targeting moiety exposed to the water, and thereby
promptly available for binding to the desired target (e.g., a bone
mineral).
[0121] The devised heterobifunctional PEG-dendrimer further allows
obtaining a conjugate with a high degree of homogeneity, as it
offers a great control over the ratio of the conjugated moieties
and on the chemical structure of the conjugate. The optimal
therapeutically active moiety/targeting moiety ratio can be
selected by controllably growing the dendrimer structure. In
addition, a high loading of a bone targeting moiety such as ALN,
which has been reported to account for both rapid and elevated
targeting to bone tumors and enhanced anti-angiogenic activity
(see, for example, WO 2009/141827, can be achieved.
[0122] As demonstrated in the Examples section that follows, an
exemplary PTX-PEG-ALN conjugate (see, FIG. 1, Compound 3) has been
successfully synthesized and was shown to target bone neoplasms,
possible by dual-targeting; through ALN (active mechanism), and by
exploiting the EPR effect (passive mechanism). The building blocks
of the conjugate (succinic acid, PEG and .beta.-Glutamic acid) are
all non-toxic, and no hemolytic activity was found to be exhibited
by the conjugate (as opposed to the commercial solubilizing vehicle
for PTX that contains Cremophor EL).
[0123] While devising exemplary polymeric conjugates, the present
inventors have considered the high affinity of the conjugates to
extracellular bone matrix, which can affect the conjugate
internalization into cancer cells and consequently slow the rate of
PTX release, in cases where such a release is designed to be
lysosomotropic drug release (e.g., by using cathepsin-cleavable
linking moieties which are susceptible to intracellular
cathepsins). To this effect, exemplary conjugates were designed so
as to exhibit a faster drug release and/or drug release in the
surroundings of bone metastasis, where the conjugate will fast
accumulate. This can be achieved either by linking the drug to the
polymer through an ester or other hydrolytically-cleavable linkage,
which releases the drug at physiological pH by simple hydrolysis,
or via a linker that is cleavable by extracellular enzymes (e.g.,
enzymes which are present in extracellular matrix surrounding the
bone).
[0124] Indeed, as demonstrated in the Examples section that
follows, it has been shown for exemplary conjugates that the drug
(PTX) is released by a hydrolytically-based mechanism without a
significant contribution of esterases.
[0125] It has further been demonstrated the fast drug release at
physiological pH affected also the stability of conjugate's
micelles, which at such pH were stable up to about three hours. The
pharmacokinetic profiles of exemplary conjugates in mice models
showed marked half-lives increase with respect to free PTX
solubilized in Cremophor EL (about 5 and 6 times longer,
respectively), whereby the cytotoxicity of PTX was comparable to
that of free PTX, thereby indicating that conjugating the drug does
not reduce its therapeutic activity, yet results in reduced side
effects compared to known PTX formulations.
[0126] As further demonstrated in the Examples section that
follows, the effect of exemplary conjugates in inhibiting
proliferation, capillary-like tube formation, and migration of
endothelial cells suggested that these conjugates possess
anti-angiogenic properties.
[0127] Biodistribution analysis demonstrated preferred accumulation
in tumors in all FITC-labeled conjugates tested following 8 hours
of injection, possibly as a result of the EPR effect. Exemplary
conjugates were found to explicitly accumulate in the kidneys, due
to renal excretion.
[0128] Exemplary conjugates according to some embodiments of the
present invention showed substantial antitumor effects in both
murine syngeneic and human xenograft mouse models tested.
Additionally, the superiority of such conjugates was further
evidenced by enhanced safety compared to the free drugs, without
hindering bone-targeting affinity.
[0129] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric
backbone having attached thereto a therapeutically active agent and
a bone targeting moiety. In some embodiments, the therapeutically
active agent and the bone targeting moiety are spatially separated
by the polymeric backbone. In some embodiments, the therapeutically
active agent is attached to one end of the polymeric backbone
(e.g., to a terminus backbone unit at one end of the polymeric
backbone) and the bone targeting moiety is attached to another end
of the polymeric backbone (e.g., to a terminus backbone unit at
another end of the polymeric backbone). In some embodiments, the
bone targeting moiety is attached to the polymeric backbone (e.g.,
to a terminus backbone unit of the polymeric backbone) via a
branching unit, such that a mol ratio of the bone targeting moiety
to said polymer and is at least 2:1.
[0130] The conjugates described herein can also be referred to as
polymeric conjugates.
[0131] The Polymeric Backbone:
[0132] As used herein, the phrase "polymeric backbone" describes a
plurality of backbone units that are covalently linked to one
another. The backbone units and hence the polymeric backbone are
those present in a polymer from which the conjugate is derived
from.
[0133] By "derived from" it is meant that the polymeric backbone is
the same as the polymer from which it is derived, except for having
the moieties as described herein conjugated thereto (optionally via
the branching unit, linking moiety and/or spacer, as described
herein).
[0134] As used herein, the term "polymer" describes a substance,
preferably an organic substance, but alternatively an inorganic
substance, composed of a plurality of repeating structural units
(referred to interchangeably as backbone units or monomeric units)
covalently connected to one another and forming the polymeric
backbone of the polymer. 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.
[0135] For the sake of simplicity, the terms "polymer" and
"polymeric backbone" as used hereinthoroughout interchangeably,
relate to both homopolymers, copolymers and mixtures thereof.
[0136] In some embodiments, the polymeric conjugates described
herein are composed of a polymeric backbone, formed from a
plurality of backbone units that are covalently linked to one
another. The therapeutically active agent and the bone targeting
moiety are each attached, directly or indirectly, to a different
end of the polymeric backbone, e.g., to a different terminus
backbone unit of the polymeric backbone. Thus, in some embodiments,
the therapeutically active agent and the bone targeting moiety are
spatially separated from one another by the polymeric backbone.
[0137] By "terminus backbone unit" it is meant a backbone unit at
the end of the polymeric chain, which is attached only to one other
backbone unit of the polymer (and not to two backbone units, as do
all other backbone units in the polymeric backbone). In some
embodiments, the targeting moiety and the therapeutically active
agent are each attached to the terminus backbone unit either
directly or indirectly, while utilizing a functional group that
forms a part of the terminus backbone unit, or which is generated
within, or attached to, the terminus backbone unit, in order to
facilitate the attachment.
[0138] A polymeric conjugate as described herein therefore
comprises, in some embodiments, a polymeric backbone which
comprises a plurality of backbone units being the same (in case of
a homopolymer) or different (in case of a copolymer), wherein all
of these backbone units, except for the terminus backbone units at
each end of the backbone, are non-functionalized, namely, do not
have any of a therapeutically active agent, a labeling agent and/or
a targeting moiety attached thereto and do not bear any functional
group that can be utilizing for attaching a therapeutically active
agent, a labeling agent and/or a targeting moiety, either directly
or indirectly, thereto.
[0139] 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 specific
delivery into tumor tissue, possibly due to the EPR effect
discussed herein.
[0140] The polymer may be a biostable polymer, a biodegradable
polymer or a combination thereof (in case of a copolymer).
[0141] 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, and hence is non-biodegradable or non-biocleavable).
[0142] 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.
[0143] 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.
[0144] In some embodiments, the polymer is a biostable polymer, as
defined herein. Such polymers may allow designing the polymeric
conjugate so as to selectively release the therapeutically active
agent at the desired bodily site (e.g., a bone tissue), as
biodegradation of the polymer before it reaches the desired site is
avoided.
[0145] The polymers can be water-soluble or water-insoluble. In
some embodiments, the polymers are water soluble at room
temperature.
[0146] In some embodiments, the polymer is an amphiphillic
polymer.
[0147] The polymers can further be charged polymers or non-charged
polymers. 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 no charge
at all.
[0148] 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 10 to
60 kDa, or 15 to 60 kDa, or 10 to 40 kDa or 15 to 40 kDa. Any
intermediate range or value is also contemplated.
[0149] 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.
[0150] The molecular weight of the polymer can be controlled, at
least to some extent, by the degree of polymerization (or
co-polymerization). Optionally, commercially available polymers,
which have a desired molecular weight, are utilized.
[0151] 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.
[0152] Exemplary polymers which are suitable for use in the context
of the present embodiments include, but are not limited to,
poly(alkylene glycol)s, poly(2-alkyl-2-oxazoline)s, dextran, water
soluble polyamino acids, a polyglutamic acid (PGA), a polylactic
acid (PLA), a polylactic-co-glycolic acid (PLGA), a
poly(D,L-lactide-co-glycolide) (PLA/PLGA), a
poly(hydroxyalkylmethaacrylamide), a polyglycerol, a polyamidoamine
(PAMAM), and a polyethylenimine (PEI).
[0153] In some embodiments, the polymer is an amphiphilic,
biostable, biocompatible and immunogenic polymer. Exemplary such
polymers include, but are not limited to, a poly(alkylene glycol),
a poly(2-alkyl-2-oxazoline), and a copolymer comprising a
poly(alkylene glycol) and/or a poly(2-alkyl-2-oxazoline).
[0154] In some embodiments, a suitable polymer for use in the
context of the present embodiments can be represented by the
following general Formula:
ZWx-{[(CRxRy)k]-W}n-[(CRxRy)k]-WyL
[0155] wherein:
[0156] k is an integer from 1 to 6, preferably from 2 to 4, more
preferably being 2 or 3, representing the number of carbon atoms
(the length) in each backbone unit;
[0157] n is an integer from 100 to 1000, representing the number of
backbone units in the polymer, and is preferably selected or
determined in accordance with a desired molecular weight of the
polymer, as outlined hereinabove;
[0158] Z and L are each independently a group at an end of the
polymer, and can be hydrogen, alkyl, cycloalkyl, aryl, alkoxy,
thioalkoxy, aryloxy, thioaryloxy, carbonyl, thiocarbonyl, and can
also be selected from the group consisting of carbamate,
thiocarbamate, guanyl, guanidine, hydrazine, hydrazide, and the
like;
[0159] W, Wx and Wy are each independently a heteroatom-containing
group selected from the group consisting of oxygen, sulfur, NRw,
PRw, and SiRwRz, and is preferably selected from the group
consisting of oxygen, sulfur or NRw; and
[0160] Rz, Ry, Rw and Rz are each independently selected from the
group consisting of hydrogen, alkyl, cycloalkyl, aryl, alkoxy,
thioalkoxy, aryloxy, thioaryloxy, carbonyl, thiocarbonyl, and can
also be selected from the group consisting of oxo, carbamate,
thiocarbamate, guanyl, guanidine, hydrazine, hydrazide, as long as
the substituent(s) do not interfere with the binding of, and/or are
reactive with, the bone targeting moiety and/or the therapeutically
active moiety.
[0161] The group [(CRxRy)k]-W represents the repeating backbone
unit in the polymer. In some embodiments, all of the backbone units
are the same. Optionally, the backbone units are different from one
another by the heteroatom W, the RxRy substituents, the value k, or
both.
[0162] In exemplary embodiments, at least 50% of the backbone units
are identical, e.g., they comprise the same heteroatoms and the
same k values as one another, and may further by identically
substituted. Optionally, at least 70%, optionally at least 90%, and
optionally 100% of the backbone units are identical.
[0163] The group WxZ and WyL represent the functional groups at the
ends of the polymer. In cases where W, Wx and WY are the same and Z
and L are each hydrogen, the polymer is considered as being
functionalized by the intrinsic functionalities of the repeating
units (e.g., it terminated by groups such as --OH, NHR, and the
like. In some embodiments, the polymer can be modified so as to
include groups that are not derived from the repeating units, for
example, by replacing an and functional hydroxy group which is
present when each of W, Wx and Wy is hydrogen, by an amine or any
other group.
[0164] In some embodiments, in any of the above-described
embodiments of a polymer, W is O, such that the polymer is a
poly(alkylene glycol).
[0165] The phrase "poly(alkylene glycol)", as used herein,
encompasses a family of polyether polymers which share the
following general formula: --O--[(CRxRy)k-O-]n-, wherein k
represents the number of methylene groups present in each alkylene
glycol unit, and n represents the number of repeating units, and
therefore represents the size or length of the polymer. Optionally,
k varies among the units of the poly(alkylene glycol) chain. For
example, a poly(alkylene glycol) chain may comprise both ethylene
glycol (k=2) and propylene glycol (k=3) units linked together.
[0166] When Rx and Ry are each hydrogen and m is 2, the polymer is
a poly(ethylene glycol) (PEG).
[0167] The WxZ and WyL groups can each be hydroxy (OH) groups, or
can be modified such that the PEG is modified so as to include
other functional groups at one or both ends thereof.
[0168] In some embodiments, W in NRw, and Rw is a carbonyl, as
defined herein, such that the polymer is a
poly(2-alkyl-2-oxazoline) or an analog thereof. By "analog" of a
poly(2-alkyl-2-oxazoline), it is meant that the carbonyl can be
replaced by a thiocarbonyl and/or the alkyl can be replaced by a
cycloalkyl or aryl.
[0169] The WxZ and WyL groups can each be NHRw groups, as defined
herein for these embodiments, or can be modified such that the
poly(2-alkyl-2-oxazoline) is modified so as to include other
functional groups at one or both ends thereof.
[0170] These polymers can be of any molecular weight, as described
herein.
[0171] These polymers can also form a part of a copolymer, which
further comprise backbone units of another polymer. The backbone
units of another polymer can be interdispersed between the backbone
units as described herein, or a chain of backbone units of one type
of a polymer can be linked at one or both ends to a chain of
backbone units of another polymer.
[0172] An exemplary suitable copolymer of a poly(alkylene glycol)
is a copolymer of PEG and PGA.
[0173] It is to be noted that the description provided herein for
the term "polymer" refers to those polymers from which the
polymeric backbone unit in the conjugates described herein is
derived. However, in the herein described conjugates, the terminus
backbone units in each end of the polymeric backbone is utilized
for attaching thereto the bone targeting moiety and the
therapeutically active agent, and thus these terminus backbone
units are derivatized so as to have these moieties attached
thereto, either directly or indirectly, and hence possess at least
some different structural properties.
[0174] In some embodiments, the WxZ and WyL groups is the Formula
described hereinabove, or any other reactive group at the polymer's
ends, is utilized for attaching, either directly or indirectly, the
therapeutically active moiety or the bone targeting moiety.
[0175] In some embodiments, a functional group at one end of the
polymeric backbone is used per se for attaching the bone targeting
moiety and/or the group at the other end of the polymer is used per
se for attaching the therapeutically active agent.
[0176] In some embodiments, one or both end groups are modified so
as to facilitate the attachment of the therapeutically active
moiety and/or the bone targeting moiety, and/or to include a
desirable linking moiety, as is further detailed herein.
[0177] The Therapeutically Active Agent:
[0178] As used herein, a "therapeutically active agent" encompasses
any agent that is capable of exhibiting a beneficial therapeutic
effect, such as treating or preventing a medical condition, a
disease or a disorder, as defined herein. The terms
"therapeutically active agent" and "drug" are used herein
interchangeably.
[0179] In some embodiments, the therapeutically active agent
attached to one end of the polymeric backbone (e.g., one of the
terminus backbone units), is such that exhibits a therapeutic
effect in the environment of a bone tissue. Thus, in some
embodiments, the therapeutically active agent is such that is
suitable for treating a bone-related disease or disorder, as
defined hereinafter.
[0180] In some embodiments, the therapeutically active agent is an
anti-angiogenesis agent.
[0181] The phrase "anti-angiogenesis agent", which is also referred
to herein, interchangeably as "anti-angiogenic agent" or
"angiogenesis inhibitor", describes an agent having the ability to
(a) inhibit endothelial cell proliferation or migration, (b) kill
proliferating endothelial cells, and/or (c) inhibit the formation
of new blood vessels in a tissue (e.g., a tumor tissue).
[0182] Exemplary anti-angiogenesis agents that are suitable for use
in the context of embodiments of the invention include, but are not
limited to, paclitaxel, 2-methoxyestradiol, prinomastat,
batimastat, BAY 12-9566, carboxyamidotriazole, CC-1088,
dextromethorphan acetic acid, dimethylxanthenone acetic acid,
endostatin, IM-862, marimastat, a matrix metalloproteinase,
penicillamine, PTK787/ZK 222584, RPI.4610, squalamine lactate,
SU5416, thalidomide, TNP-470, combretastatin, tamoxifen, COL-3,
neovastat, BMS-275291, SU6668, anti-VEGF antibody, Medi-522
(Vitaxin II), CAI, Interleukin-12, IM862, Amilloride,
Angiostatin.RTM.Protein, Angiostatin K1-3, Angiostatin K1-5,
Captopril, DL-alpha-Difluoromethylornithine,
DL-alpha-Difluoromethylornithine HCl, His-Tag.RTM.
Endostatin.TM.Protein, Fumagillin, Herbimycin A,
4-Hydroxyphenylretinamide, Juglone, Laminin, Laminin Hexapeptide,
Laminin Pentapeptide, Lavendustin A, Medroxyprogesterone,
Medroxyprogesterone Acetate, Minocycline, Minocycline HCl,
Placental Ribonuclease Inhibitor, Suramin, Sodium Salt Suramin,
Human Platelet Thrombospondin, Neutrophil Granulocyte, monoclonal
antibodies directed against specific proangiogenic factors and/or
their receptors (e.g. Avastin, Erbitux, Vectibix, Herceptin); small
molecule tyrosine kinase inhibitors of multiple proangiogenic
growth factor receptors (e.g. Tarceva, Nexavar, Sutent, Iressa);
inhibitors of mTOR (mammalian target of rapamycin) (e.g. Torisel);
interferon alpha, beta and gamma; IL-12; matrix metalloproteinases
(MMP) inhibitors (e.g. COL3, Marimastat, Batimastat); EMD121974
(Cilengitide); Vitaxin; Squalamin; COX-2 inhibitors; PDGFR
inhibitors (e.g., Gleevec); NM3 and 2-ME2.
[0183] As used herein, the term "COX-2 inhibitor" refers to a
non-steroidal drug that relatively inhibits the enzyme COX-2 in
preference to COX-1. Preferred examples of COX-2 inhibitors
include, but are no limited to, celecoxib, parecoxib, rofecoxib,
valdecoxib, meloxicam, and etoricoxib.
[0184] In some embodiments, the anti-angiogenesis agents is
selected from the group consisting of TNP-470, Paclitaxel,
monoclonal antibodies directed against specific proangiogenic
factors and/or their receptors (e.g. Avastin, Erbitux, Vectibix,
Herceptin); small molecule tyrosine kinase inhibitors of multiple
proangiogenic growth factor receptors (e.g. Tarceva, Nexavar,
Sutent, Iressa); inhibitors of mTOR (mammalian target of rapamycin)
(e.g. Torisel); interferon alpha, beta and gamma; IL-12; matrix
metalloproteinases (MMP) inhibitors (e.g. COL3, Marimastat,
Batimastat); EMD121974 (Cilengitide); Vitaxin; Squalamin; COX-2
inhibitors; PDGFR inhibitors (e.g., Gleevec); NM3; and 2-ME2.
[0185] In some embodiments, the anti-angiogenesis agent is
Paclitaxel.
[0186] The microtubule-interfering agent Paclitaxel is a drug
commonly used for the treatment of advanced metastatic breast
cancer. However, it is neurotoxic, it causes hematological toxicity
and many breast tumors develop resistance thereto. It has been
recently shown that Paclitaxel at ultra low doses inhibits
angiogenesis. However, Paclitaxel is poorly soluble and the
excipients Cremophor EL or ethanol used today to solubilize its
commercial form, cause hypersensitivity reactions.
[0187] Alternatively, the therapeutically active agent can be an
anti-cancer agent (anti-neoplastic agent) that acts via other
mechanism of action (namely, not via inhibition of angiogenesis).
Such agents include, but are not limited to, alkylating agents,
antimetabolites and antitumor antibiotics, as these are known for
any person skilled in the art.
[0188] The Linking Moiety:
[0189] Since the conjugates as described herein are aimed at
releasing the therapeutically active agent at the diseased bodily
site, in some embodiments, the therapeutically active agent is
attached to the polymer via a biocleavable moiety. The biocleavable
moiety can be a biocleavable bond or a biocleavable linking
group.
[0190] In some embodiments, the therapeutically active agent is
attached to the terminus backbone unit of the polymeric backbone
directly via a bond, preferably via a biocleavable bond, and
preferably via a hydrolytically-cleavable bond, as defined
herein.
[0191] In some embodiments, the therapeutically active agent is
linked to the end of the polymeric backbone (e.g., to a terminus
backbone unit of the polymeric backbone) directly, or indirectly
(e.g., via a spacer), through a linking moiety (also referred to
herein as a linker, a linker group a linker moiety or a linking
group), whereby, in some embodiments, the direct/indirect linkage
is designed as being cleavable at conditions characterizing the
desired bodily site, as detailed hereinbelow.
[0192] The linking moiety linking the therapeutically active agent
to the polymer is also referred to herein as a first linking
moiety, and, in the representative Formula I hereinbelow, is
represented as L.sub.1.
[0193] The linking moiety described herein refers to a chemical
moiety that serves to couple the therapeutically active agent to
the polymeric backbone while not adversely affecting the
therapeutic effect of agent.
[0194] In some embodiments, the linking moiety is a biodegradable
(or biocleavable) linking moiety.
[0195] The phrase "biodegradable linking moiety", as used herein,
describes a linker that is capable of being degraded, or cleaved,
when exposed to physiological conditions. Such physiological
conditions can be, for example, an aqueous environment, pH, a
certain enzyme, and the like.
[0196] Accordingly, according to some embodiments, the
biodegradable linker is a hydrolytically-cleavable linking moiety,
a pH-sensitive linker or an enzymatically-cleavable linker.
[0197] In some embodiments, the biodegradable linking moiety is a
hydrolytically-cleavable linking moiety.
[0198] As used herein, the phrase "hydrolytically-cleavable linking
moiety or bond" describes a linking moiety or bond that can be
cleaved by hydrolysis in an aqueous environment such as a
physiological medium. This phrase does not encompass linking
moieties or bonds that can be cleaved at a certain pH or by enzymes
in a physiological medium, but rather encompasses linking moieties
or bonds that can be cleaved via hydrolysis once contacting an
aqueous medium such as a physiological medium, at or about the
physiological pH (e.g., pH 7).
[0199] A hydrolytically-cleavable linking moiety or bond is
advantageous as it allows fast release of the therapeutically
active agent, once the polymeric conjugate contacts a
physiological, aqueous medium. In case of conjugates comprising a
high load of bone-targeting moieties as described herein, a
hydrolytically-cleavable linking moiety or bond is even more
advantageous since the presence of the bone targeting moiety leads
to fast accumulation at the extracellular bone matrix and slows the
internalization of the polymeric conjugate into the cell. Thus, a
linking moiety that allows releasing the therapeutically active
agent at conditions present at the extracellular bone matrix,
namely, via simple hydrolysis at the matrix's pH, would lead to
fast and efficient release of the therapeutically active agent at
the desired bodily site.
[0200] In some embodiments, the hydrolytically-cleavable moiety
comprises or consists of one or more hydrolytically-cleavable
bond(s). Examples of such hydrolytically-cleavable moieties include
one or more hydrolytically-cleavable bond(s) such as, but not
limited to, an ester bond, an imine bond, a hydrazone bond, a ketal
bond, an acetal bond and a carbonate bond.
[0201] In some embodiments, the hydrolytically-cleavable moiety
comprises a hydrolytically-cleavable bond such as an ester and thus
can be derived, for example, from a carboxylic acid or an alcohol,
which is attached to the end of the polymeric backbone (e.g., to
the terminus backbone unit).
[0202] In some embodiments, the hydrolytically-cleavable moiety is
formed upon coupling the therapeutically active agent to the end of
the polymeric backbone (e.g., the terminus backbone unit of the
polymer) and is defined by the functional groups which are present
or generated in the therapeutically active agent and at the end of
the polymeric backbone (e.g., within the terminus backbone
unit.
[0203] For example, when a hydrolytically-cleavable moiety
comprises an ester bond, such a moiety can be formed between a
hydroxyl functional group that is present or generated (e.g., by
means of a spacer or by chemical modification) in the
therapeutically active agent and a carboxylic group that is present
or is generated (e.g., by means of a linking moiety and/or a spacer
and/or chemical modification) at the end of the polymeric backbone
(e.g., within the terminus backbone unit of the polymeric
backbone).
[0204] Alternatively, for example, when a hydrolytically-cleavable
moiety comprises an ester bond, such a moiety can be formed between
a carboxylate functional group that is present or generated (e.g.,
by means of a spacer or chemical modification) in the
therapeutically active agent and a hydroxy group that is present or
is generated (e.g., by means of a linking moiety and/or a spacer
and/or a chemical modification) at the end of the polymeric
backbone (e.g., within the terminus backbone unit of the polymeric
backbone.
[0205] Further alternatively, for example, when a
hydrolytically-cleavable moiety comprises an imine bond, such a
moiety can be formed between an aldehyde functional group and an
amine functional group, one present or generated (e.g., by means of
a spacer) in the therapeutically active agent and one present or is
generated (e.g., by means of a linking moiety and/or a spacer) in
the terminus backbone unit of the polymeric backbone.
[0206] Similarly, a hydrazone can be formed from amide and
hydrazine.
[0207] A person skilled in the art would readily recognize how to
devise a conjugate in which the therapeutically active agent is
attached to the end of the polymeric backbone (e.g., to the
terminus backbone unit of the polymeric backbone) via a
hydrolytically-cleavable moiety, based on the functional groups
that are intrinsically present at the terminus of the polymer and
in the therapeutically active agent.
[0208] In one example, a carboxylate is generated on a
therapeutically active agent by attaching to a free hydroxyl group
on the drug to a carboxylic acid, to thereby generate a
hydrolytically-cleavable ester bond. The carboxylic acid, in these
embodiments, represents a hydrolytically-cleavable linking moiety,
and can be attached to the terminus backbone unit via and
additional ester bond and/or any other bond, via a functional group
at the other end thereof. Exemplary such bifunctional carboxylic
acids include, but are not limited to, dicarboxylic acids such as
succinic acid, malonic acid, oxalic acid, glutaric acid, adipic
acid, sebacic acid, phthalic acid, and the like. Such an esterified
bifunctional carboxylic acid is an exemplary
hydrolytically-cleavable linking moiety.
[0209] 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).
[0210] 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 attached
to the polymer in the body, until its reaches a physiological
environment where a pH is either acidic or basic, respectively.
[0211] Exemplary pH-sensitive linking moieties 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]- (SEQ ID NO:1) moiety.
[0212] In some embodiments, the biodegradable linking moiety is an
enzymatically-cleavable linking moiety.
[0213] 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 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 therapeutically active agent attached thereto
until contacted with the enzyme.
[0214] In some embodiments, the enzymatically-cleavable linker is
cleaved by an enzyme which is expressed in tumor tissues. In some
embodiments, the enzymatically-cleavable linker is cleaved by an
enzyme which is overexpressed in tumor tissues. A conjugate
comprising such a linker ensures, for example, that a substantial
amount of the conjugated therapeutically active agent is released
from the conjugate only at the tumor tissue, thus reducing the side
effects associated with non-selective administration of the drug
and further enhancing the concentration of the drug at the tumor
site.
[0215] 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 a nitrogen, oxygen and/or sulfur heteroatom.
[0216] Other suitable linkers include amino acids and/or
oligopeptides.
[0217] 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 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.
[0218] In some embodiment, the linker is a biodegradable
oligopeptide which contains, for example, from 2 to 10 amino acid
residues.
[0219] In some embodiments the enzymatically-cleavable linker is
cleavable by pre-selected cellular enzymes, for instance, those
found in osteoblasts, osteoclasts, lysosomes of cancerous cells or
proliferating endothelial cells.
[0220] Non-limiting examples of such enzymes include, but are not
limited to, Cathepsin K, Cathepsin D, Cathepsin H, Cathepsin L, and
legumain, Cathepsin B, MMP-2 and MMP-9.
[0221] Since the conjugates described herein can be designed so as
to be targeted to bone minerals, and are hence not internalized
into the cells, in some embodiments, the pre-selected enzyme is
such that is present in the extracellular matrix, outside the
cells. Exemplary such enzymes include, but are not limited to,
Cathepsin K, Cathepsin D, Cathepsin H, Cathepsin L, MMP-2 and
MMP-9.
[0222] Legumain and cathepsin B are expressed mainly in the
lysosome (intracellularly), yet some amount is secreted to the
extracellular matrix.
[0223] In some embodiments, the linker is cleavable by Cathepsin
K.
[0224] 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].
[0225] A non-limiting example of a linker having Cathepsin K
cleavable sites is a linker which comprises the amino acid sequence
-[Gly-Gly-Pro-Nle]- (SEQ ID NO:2).
[0226] Non-limiting examples of linkers having Cathepsin D
cleavable sites are those which comprise or consist of the amino
acid sequences -[Gly-Thr-Gln-Phe-Phe]- (SEQ ID NO:3) and
-[Gly-Ser-Thr-Phe-Phe]- (SEQ ID NO:4).
[0227] A non-limiting example of a linker having Cathepsin H
cleavable sites is a linker which comprises or consists of the
amino acid sequence -[Leu-Gly]- (SEQ ID NO:5).
[0228] A non-limiting example of a linker having Cathepsin L
cleavable sites is a linker which comprises or consists of the
amino acid sequence -[Ala-Phe-Arg-Ser-Ala-Ala-Gln]- (SEQ ID
NO:6).
[0229] A non-limiting example of a linker having legumain cleavable
sites is a linker which comprises or consists of the amino acid
sequence -[Ala-Ala-Asn]- (SEQ ID NO:7).
[0230] Non-limiting examples of linkers having MMP-2 and MMP-9
cleavable sites are those which comprise or consist of the amino
acid sequences -[His-Pro-Val-Gly-Leu-Leu-Ala-Arg]- (SEQ ID NO:8),
-[Pro-Val-Ser-Leu-Ser-Tyr]- (SEQ ID NO:9), and
-[Gly-Pro-Val-Gly-Leu-Ile-Gly-Lys]- (SEQ ID NO:10).
[0231] Non-limiting examples of linkers having Cathepsin B
cleavable sites are those which comprise or consist of the amino
acid sequences -[Arg]-, -[Cit-Val]- (SEQ ID NO:11), -[Arg-Arg]-
(SEQ ID NO:12), -[Phe-Lys]- (SEQ ID NO:13), [Gly-Phe-Leu-Gly] (SEQ
ID NO:14), -[Gly-Phe-Ala-Leu]- (SEQ ID NO:15),
-[Ala-Leu-Ala-Leu]-(SEQ ID NO:16), -[Gly-Leu-Gly]- (SEQ ID NO:17),
-[Gly-Phe-Gly]- (SEQ ID NO:18), -[Gly-Phe-Leu-Gly-Phe-Lys]-(SEQ ID
NO:19) and combinations thereof.
[0232] 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.
Various recognition motifs of the same or different enzymes can
also be incorporated within the linker. 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
where it exhibits its activity.
[0233] In some embodiments the therapeutically active agent is
linked to the polymeric backbone and/or to the linking via a
spacer, as described herein.
[0234] In some embodiments, the therapeutically active agent is
attached to the first linking moiety or to the polymeric backbone
via a branching unit, as defined herein and is represented by the
variable "B*" in Formula I hereinafter.
[0235] In these embodiments, the load of the therapeutically active
agent can be increased as the branching unit allows attaching more
than one mol of the therapeutically active agent (e.g., 2 or 3
mols) per mol of the polymer.
[0236] The branching unit can be attached to the polymeric backbone
either directly or via a spacer as defined herein.
[0237] The branching unit can further be attached to the polymeric
backbone via a linking moiety and/or a spacer, as described
herein.
[0238] The branching unit can be attached to the polymeric backbone
either directly or via a spacer, and the linking moiety can be
attached to the branching unit, thus linking the therapeutically
active agent to the polymer via the branching unit, as described
hereinbelow for the bone targeting moiety.
[0239] The therapeutically active agent that is attached to each
branch of the branching unit can be the same or different.
[0240] The Bone Targeting Moiety:
[0241] As used herein throughout, the phrase "bone targeting
moiety" describes a moiety that is capable of preferentially
accumulating in hard tissues (i.e. bone tissues) rather than any
other organ or tissue, after administration in vivo.
[0242] In some embodiments, a bone targeting moiety is
characterized by a strong affinity to bone minerals (e.g, to
hydroxyapettite).
[0243] In some embodiments, the bone targeting moiety is a
bisphosphonate.
[0244] Bisphosphonates (BPs) such as alendronate are compounds with
a chemical structure similar to that of inorganic pyrophosphate
(PPi), an endogenous regulator of bone mineralization. The
pharmacokinetic profile of bisphosphonates, which exhibit a strong
affinity to bone mineral under physiological conditions, their low
toxicity and anti-angiogenic activity (typically exhibited at
relatively high concentration thereof) are advantageous for
targeting to tumors confined to bony tissues.
[0245] Accordingly, in some embodiments, the bone targeting moiety
described herein is a compound which comprises at least two
phosphonate (--P(.dbd.O)(OH).sub.2) groups, and optionally other
functional groups.
[0246] Exemplary compounds have the following general formula:
##STR00001##
[0247] or a pharmaceutically acceptable salt thereof, as defined
herein,
[0248] wherein R.sub.1 and R.sub.2 are each independently selected
from the group consisting of hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heteroalicyclic, halo, hydroxy, thiol,
alkoxy, thioalkoxy, aryloxy, and thioaryloxy, as defined
hereinbelow.
[0249] In some embodiments, at least one of R.sub.1 and R.sub.2 is
an alkyl, cycloalkyl, aryl, heteroaryl or heteroalicyclic,
optionally substituted as defined herein.
[0250] In some embodiments, the alkyl, cycloalkyl, aryl, heteroaryl
or heteroalicyclic is substituted by a reactive group such as
amine, hydroxy, thiol, halo, carboxylate, and the like, as defined
herein, which enables its conjugation to compatible reactive groups
(functional groups) of the branching unit.
[0251] In some embodiments, at least one of R.sub.1 and R.sub.2 is
hydroxy and the other one is an alkyl, cycloalkyl, aryl, heteroaryl
or heteroalicyclic, as described herein.
[0252] In some embodiments, R.sub.1 is hydroxy and R.sub.2 is an
alkyl terminating with an amino group. The alkyl can have from 1 to
6 carbon atoms in its backbone chain.
[0253] In some embodiments, the bisphosphonate is an
amino-bisphosphonate, which comprises an amino group in one or both
R.sub.1 and R.sub.2 substituents.
[0254] Exemplary bisphosphonate bone targeting moieties that are
suitable for use in the context of embodiments of the invention
include, but are not limited to, alendronate, cimadronate,
clodronate, tiludronate, etidronate, ibandronate, neridronate,
olpadronate, risedronate, piridronate, pamidronate and
zoledronate.
[0255] In some embodiments the bone targeting moiety is alendronate
(4-amino-1-hydroxybutylidene) bisphosphonic acid):
##STR00002##
[0256] Herein, the terms "alendronate" and "bisphosphonate"
encompass any pharmaceutically acceptable salts, solvates and/or
hydrates thereof, as defined hereinafter.
[0257] As described herein, the molar ratio of the bone targeting
moiety to the polymer is at least 2:1, and is determined by the
nature of the branching unit and the number of functional groups in
the branching unit to which the targeting moiety can be
attached.
[0258] Highly-branched branching units, such as those arranged in a
dendritic structure as described herein, allow the attachment of 3,
4 or more molecules of the targeting moiety to a single polymeric
backbone, such that the mol ratio of the bone targeting moiety to
the polymer is 3:1, 4:1 and even higher, namely, 6:1, 8:1, 9:1,
10:1, 16:1, and can be even higher.
[0259] Accordingly, the load of the bone targeting moiety, in terms
of weight percents of the total weight of the conjugate, can be
high.
[0260] In some embodiments, a load of the bone targeting moiety is
at least 3 weight percent, or at least 5 weight percents or at
least 7 weight percents, or at least 10 weight percents. For
example, a load of alendronate as an exemplary bone targeting
moiety can be 5, 6, 7, 8, 9, 10, 11 or 12 weight percent and even
higher.
[0261] The high load of a bone targeting moiety can be efficiently
utilized for delivering a therapeutically active agent to a bone
tissue.
[0262] The bone targeting moiety can be attached to the branching
unit directly, via a bond, or via a linking moiety (L.sub.2 in
Formula I) and/or a spacer. The linking moiety can be used to
facilitate the attachment of the bone targeting moiety to the
branching unit. Thus, for example, in cases where the branching
unit has functional groups that are chemically incompatible with
the functional groups of the bone targeting moiety, a linking
moiety and/or a spacer can be attached to the branching unit or to
the bone targeting moiety so as to enable attachment of the bone
targeting moiety to the polymer via the branching unit.
[0263] Optionally, the branching unit is attached to the terminus
backbone unit of the polymeric backbone via a linking moiety or a
spacer, for the same reasons as applied herein for the branching
unit.
[0264] The linkage of the bone targeting moiety to the branching
unit and of the branching unit to the polymer, whether being a bond
or via a linking moiety and/or a spacer, can be biocleavable or
biostable (non-biocleavable).
[0265] By "biocleavable" it is meant that the bond or linking
moiety can be cleaved under physiological conditions, for example,
hydrolytically, enzymatically, or at a physiological pH, as is
described in further detail hereinafter.
[0266] By "biostable" it is meant that the bond or linking moiety
cannot be cleaved under physiological conditions.
[0267] In some embodiments, the bone targeting moiety is attached
to the polymer via biostable linkages, that is, both the linkage
between the bone targeting moiety and the branching unit is
biostable and the linkage between the branching unit and the
polymer is biostable. This may avoid release of the bone targeting
moiety before it reaches its target, and thus improves the
targeting of the conjugate and avoids adverse effects (e.g.,
cytotoxicity) which may be caused by free bone targeting moieties
when present in non-diseased tissues or in tissues other than bone
tissues.
[0268] Further optionally, the bone targeting moiety can be
attached to the branching unit via a spacer. The spacer can be used
to avoid spatial interactions and/or steric hindrance which can be
imparted by attaching two or more targeting moieties to the
branching units. The spacer may allow attachment of two or more
bulky targeting moieties to the branching unit. Further optionally,
the branching unit can be attached to the polymeric backbone via a
spacer, as described herein.
[0269] The Branching Unit:
[0270] As discussed hereinabove, the bone targeting agent is
attached to the polymeric backbone via a branching unit. The
branching unit is utilized for generating more than one functional
group (or reactive group) at the polymer's end, that can be used
for attaching the bone targeting moiety to the polymer and thus is
selected so as to provide a desired mol ratio of the bone targeting
moiety and the polymer, which is 2:1 or more.
[0271] Herein, the phrase "branching unit" describes a chemical
moiety which can be regarded as a spacer or a linking moiety for
attaching one moiety to two or more other moieties via the same
position of the first moiety. That is, the branching moiety is a
chemical moiety that, when attached to a single position, group or
atom of a substance, creates two or more functional groups that are
linked to this single position, group or atom, and thus "branches"
a single functionality into two or more functionalities.
[0272] In some embodiments, the branching unit is derived from a
chemical moiety that has one functional group for attaching,
directly or indirectly, a terminus backbone unit of the polymer,
and two or more additional functionalities, each comprising a
reactive group for attaching the bone targeting moiety.
[0273] Thus, in some embodiments, a branching unit is derived from
a trifunctional moiety that comprises 3 or more functional groups,
as described hereinabove.
[0274] It is to be noted that for any of the embodiments described
herein for the conjugates, moieties, units and/or polymers, the
moieties, units and/or polymers within the conjugate can be derived
from the described conjugates, moieties, units and/or polymers, and
that "derived from" is used to describe the moiety, unit or
polymeric backbone after being conjugated to another moiety and/or
unit, whereby upon conjugation, the functional moieties which were
present in the polymer, unit or moiety, are already interacted with
the conjugated unit, moiety or polymeric backbone.
[0275] In some embodiments, the branching unit is derived from a
chemical moiety that comprises at least one trifunctional moiety.
Such a trifunctional moiety comprises at least 3 functional groups,
and optionally 4, 5, 6 or more functional groups, in which one
functional group is utilized for attaching to the terminus backbone
unit of the polymer and two or more other functional groups are
utilized for attaching to the bone targeting moiety. The 3 or more
functional groups can be the same or different. Exemplary such
functional groups include, but are not limited to, amine,
carboxylate, thiocarboxylate, hydroxy, thiol, carbamate,
thiocarbamate, sulfonate, sulfinate, sulfonamide, phosphonate,
phosphinate, phosphoryl, urea and thiourea. In some embodiments,
each of the 3 functional groups is independently an amine, a
hydroxyl, a thiol or a carboxylate, as there terms are defined
herein.
[0276] Exemplary trifunctional moieties include, but are not
limited to, glutamic acid, beta-glutamic acid, amino adipic acid
aspartic acid, lysine, 3-hydroxy-2-amine propanol, and any other
amino acid that has a carboxylate-containing side chain, or an
amino-containing side-chain, or a hydroxyl-containing side chain,
or a thiol-containing side-chain or a combination of two or more of
the above-described functional moieties in addition to the
intrinsic amine and carboxylate groups of an amino acid.
[0277] In some embodiments, any of the branching units as described
herein comprises a trifunctional moiety as described herein,
arranged in a dendritic structure.
[0278] By "dendritic structure" it is meant that a perfectly
cascade-branched, highly defined, structure which generally
comprises a core, a number of generations of ramifications (also
known and referred to herein as "branches" or "branching moieties")
and an external surface. The generations of ramifications are
composed of repeating structural units, which radially extend
outwardly from the core. The external surface of a dendritic
structure of an Nth generation is, in general, composed of the
terminal functional groups (also known and referred to herein as
"end groups") of the Nth (final) generation. A first generation
dendritic structure has one branching moiety and the number of end
groups will depend on the number of ramifications of the branching
moiety. A second generation dendritic structure has additional two
branching moieties, and the number of end groups will depend on the
number of ramifications of the branching moiety and will be raised
accordingly.
[0279] Conjugates in which the branching unit is arranged in a
dendritic structure can be represented by the general Formula
I:
D-L.sub.1-[B*]-P-[B.sub.1].sub.m.sup.0-[B.sub.2].sub.m.sup.1-[B.sub.3].s-
ub.m.sup.2 . . . [Bg-L.sub.2].sub.m.sup.g-1-[T].sub.m.sup.g Formula
I
[0280] wherein:
[0281] D is a therapeutically active agent, as defined herein;
[0282] P is a polymer as defined herein, or a polymeric backbone
derived from a polymer as defined herein;
[0283] T is a bone targeting moiety, as defined herein;
[0284] B* is a branching unit, as defined herein, through which the
therapeutically active agent is attached to the polymeric backbone,
or is absent;
[0285] L.sub.1 is a first linking moiety, linking the
therapeutically active agent to the terminus backbone unit of the
polymer, and can optionally be absent, as further discussed
herein;
[0286] L.sub.2 is a second linking moiety, linking the bone
targeting moiety to the other terminus backbone unit of said
polymer, via the branching unit, or is absent;
[0287] B.sub.1, B.sub.2, B.sub.3 . . . Bg are each independently a
branching moiety, wherein B.sub.1, B.sub.2, B.sub.3 . . . Bg
together form a branching unit having a dendritic structure, as
described herein;
[0288] m is an integer that equals 2, 3, 4, 5 or 6, representing
the ramification number of the dendritic structure, and is
preferably, 2, 3 or 4; and
[0289] g is an integer that ranges from 1 to 20, representing the
number of generations of the dendritic structure, and preferably
ranges from 1 to 10, or from 1 to 6, or is 1, 2, 3 or 4.
[0290] In some embodiments, g is 2.
[0291] The dendritic structure is thus composed of a cascade of
branching moieties, wherein the number of branching moieties in
each generation equals m.sup.g-1. Thus, for example, when g=1, the
number of branching moieties is one)(m.degree., and the number of
the bone targeting moieties attached to the polymer via the
branching unit equals to the ramification number of branching
moiety. When g=1, the branching unit consists of a single branching
moiety. When m=2, there are 2 branching units attached to the
polymeric backbone.
[0292] When g=2, the number of branching moieties is the second
generation is (m.sup.1), and the number of the bone targeting
moieties attached to the polymer via the branching unit is a
(mathematic) power of the ramification number of branching moiety.
When g=2, the branching unit consists of m.sup.1+1 branching
moieties. When m=2, there are 4 branching units attached to the
polymeric backbone.
[0293] The branching moieties composing the branching unit in a
dendritic structure can be any of the moieties described herein as
suitable for a branching unit. Two or more types of branching
moieties can be used within a branching unit, although preferably,
the same branching moieties compose the dendritic branching
unit.
[0294] The Spacer:
[0295] The term "spacer" as used herein describes a chemical moiety
that is covalently attached to, and interposed between, the
polymeric backbone and the branching unit, the branching unit and
the bone targeting moiety, the branching unit and a linking moiety
or the polymeric backbone and a linking moiety, thereby forming a
bridge-like structure between the spaced moieties.
[0296] The term "spacer" as used herein also describes such a
chemical moiety that is covalently attached to, and interposed
between, the polymeric backbone and the therapeutically active
agent, the polymeric backbone and the linking moiety through which
the therapeutically active agent is attached to the polymer, the
linking moiety and the therapeutically active agent, or any of
these moieties and a branching unit, if present at the end of the
polymer where the therapeutically active agent is attached, thereby
forming a bridge-like structure between the spaced moieties.
[0297] 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.
[0298] Other suitable spacers include amino acids and amino acid
sequences, optionally functionalized with one or more reactive
groups for being coupled to the polymeric backbone/bone targeting
moiety/branching unit/linking moiety/therapeutically active
agent.
[0299] In some embodiments, the 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, carboxylate,
amide, carbonyl, S and the like.
[0300] In some embodiments, the spacer is an amino acid sequence,
optionally an inert amino acid sequence (namely, does not affect
the affinity or selectivity of the conjugate).
[0301] In some cases, a spacer is utilized for enabling a more
efficient and simpler attachment of spaced moieties, in terms of
steric considerations (renders the site of attachment less
hindered) or chemical reactivity considerations (adds a compatible
reactive group to the site of attachment). In some cases, the
spacer may contribute to the performance of the resulting
conjugate. For example, the spacer may render an
enzymatically-cleavable linking moiety less sterically hindered and
hence more susceptible to enzymatic interactions.
[0302] In some embodiments, the spacer is a degradable spacer,
which is capable of undergoing degradation reactions so as to
release the agent attached thereto. In some embodiments, the spacer
is biodegradable, as defined herein.
[0303] The spacer can be, for example, a substituted or
unsubstituted cycloalkyl group, a substituted or unsubstituted
heteroalicyclic group, a substituted or unsubstituted aryl group
and a substituted or unsubstituted heteroaryl group; wherein the
substituents can be, for example, hydroxy, alkoxy, thiohydroxy,
thioalkoxy, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy,
O-carboxy, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,
sulfinyl, sulfonyl, C-amido, N-amido, amino and NRaRb wherein Ra
and Rb are each independently hydrogen, alkyl, cycloalkyl, aryl,
carbonyl, sulfonyl, trihalomethysulfonyl and, when combined, a
five- or six-member heteroalicyclic ring, whereby the spacer may be
linked to the therapeutically active agent/bone targeting
moiety/linker/polymer either directly, through the cyclic group or
alternatively, via one or more of the substituents.
[0304] In some embodiments, the spacer facilitates the attachment
of the therapeutically active agent or the linking moiety to the
polymeric backbone, or the attachment of the bone targeting moiety
to the branching unit or of the branching unit to the polymeric
backbone. This may be effected by imparting a reactive group to one
or both moieties to be coupled to one another and/or by modifying
the solubility of one of the moieties, so as to facilitate its
reaction with another moiety.
[0305] For example, in some cases the polymer is a water-soluble
polymer while the therapeutically active agent is hydrophobic, and
hence has a limited solubility in aqueous solutions or in polar
organic solvents. In such cases, a spacer can be attached to the
therapeutically active agent so as to enhance the water solubility
thereof and to facilitate the conjugation thereof to the polymer in
an aqueous solution or a protic or polar organic solvent.
[0306] A spacer may also be used in order to attach other agents
(e.g., a labeling agent, as described hereinbelow) to the
conjugate.
[0307] The spacer may be varied in length and in composition,
depending on steric consideration and may be used to space the
therapeutically active agent and/or bone targeting moiety form the
polymeric backbone.
[0308] Any of the above-described spacers and linking moieties can
be utilized for any of the branching units described herein, having
a dendritic structure or not.
[0309] Exemplary Polymeric Conjugates:
[0310] In some embodiments, the polymeric conjugate can be in a
form of micelles, formed sue to the hydrophilic nature of bone
targeting moieties (such as alendronate), an amphiphillic polymer
and a hydrophobic nature which is common for anti-cancer and/or
anti-angiogenesis agents, as discussed in further detail in the
Examples section that follows.
[0311] As discussed hereinabove, the present inventors have
conjugated alendronate to a PEG polymeric backbone together with
Paclitaxel and the bone targeting capacity of the obtained
polymeric conjugate was demonstrated by the enhanced binding of the
conjugate to hydroxyapatite (as a modal mimicking bone tissue). The
beneficial therapeutic activity of the conjugate in the treatment
of a mouse model of bone cancer metastasis was also
demonstrated.
[0312] According to some embodiments, the conjugate comprises any
of the polymers described herein (or polymeric backbones derived
therefrom), the bone targeting moiety is alendronate, and the
therapeutically active agent is paclitaxel.
[0313] According to some embodiments, the conjugate comprises a
polymeric backbones derived from poly(ethylene glycol), the bone
targeting moiety is alendronate, and the therapeutically active
agent is paclitaxel.
[0314] In some of these embodiments, the branching unit has a
dendritic structure as defined herein and, in some of these
embodiments, the branching unit comprises at least 3 beta-glutamic
acid moieties arranged in a dendritic structure, as described
herein.
[0315] In some of these embodiments, the paclitaxel is attached to
the terminus backbone unit via a hydrolytically-cleavable linking
moiety such as an ester-containing moiety (a carboxylate).
[0316] In some of these embodiments, the ester-containing moiety is
derived from a bifunctional carboxylic acid such as succinic
acid.
[0317] In some embodiments, the chemical structure has the
structure:
##STR00003##
[0318] wherein n is an integer that ranges from 10 to 1000.
[0319] The chemical structure of an exemplary such conjugate is
depicted in FIG. 1 (as Compound 3).
[0320] Labeled Conjugates:
[0321] Any of the conjugates as described herein may further
comprise a labeling agent attached thereto. The labeling agent can
be attached to any one of the linking moieties, spacers, branching
moieties or units, as described herein.
[0322] In some embodiments, the labeling agent is attached to a
spacer, as described herein, and the spacer bridges between two of
the therapeutically active agent, the first linking moiety and
terminus backbone unit the polymer.
[0323] In some embodiments, the labeling agent is attached to a
spacer, as described herein, and the spacer bridges between two of
the bone targeting moiety, the branching unit and terminus backbone
unit the polymer.
[0324] The attachment of a labeling agent to the conjugate, enables
utilizing these conjugates for monitoring bone related disease or
disorders, for example, monitoring the therapeutic effect exhibited
by the conjugate described herein.
[0325] As used herein, the phrase "labeling agent" describes a
detectable moiety or a probe. Exemplary labeling agents which are
suitable for use in the context of these embodiments include, but
are not limited to, a fluorescent agent, a radioactive agent, a
magnetic agent, a chromophore, a bioluminescent agent, a
chemiluminescent agent, a phosphorescent agent and a heavy metal
cluster.
[0326] The phrase "radioactive agent" describes a substance (i.e.
radionuclide or radioisotope) which loses energy (decays) by
emitting ionizing particles and radiation. When the substance
decays, its presence can be determined by detecting the radiation
emitted by it. For these purposes, a particularly useful type of
radioactive decay is positron emission. Exemplary radioactive
agents include .sup.99mTc, .sup.18F, .sup.131I and .sup.125I.
[0327] The term "magnetic agent" describes a substance which is
attracted to an externally applied magnetic field. These substances
are commonly used as contrast media in order to improve the
visibility of internal body structures in Magnetic resonance
imaging (MRI). The most commonly used compounds for contrast
enhancement are gadolinium-based. MRI contrast agents alter the
relaxation times of tissues and body cavities where they are
present, which depending on the image weighting can give a higher
or lower signal.
[0328] As used herein, the term "chromophore" describes a chemical
moiety that, when attached to another molecule, renders the latter
colored and thus visible when various spectrophotometric
measurements are applied.
[0329] The term "bioluminescent agent" describes a substance which
emits light by a biochemical process.
[0330] The term "chemiluminescent agent" describes a substance
which emits light as the result of a chemical reaction.
[0331] The phrase "fluorescent agent" refers to a compound that
emits light at a specific wavelength during exposure to radiation
from an external source.
[0332] The phrase "phosphorescent agent" refers to a compound
emitting light without appreciable heat or external excitation as
by slow oxidation of phosphorous.
[0333] A heavy metal cluster can be for example a cluster of gold
atoms used, for example, for labeling in electron microscopy
techniques.
[0334] In some embodiments, the labeling agent is a fluorescent
agent such as FITC. An exemplary FITC labeled-conjugate as
described herein is depicted in FIG. 1 (FITC labeled-Compound
3).
[0335] As discussed hereinabove, the tumor vasculature possesses an
enhanced capacity for the uptake of macromolecules and colloidal
drug carriers having a high molecular weight and large hydrodynamic
diameter due to the EPR effect. Therefore, a conjugate as described
herein, having a large enough hydrodynamic diameter is beneficial.
The term "large enough" is used herein to describe a conjugate
having a hydrodynamic diameter which leads to an increase in the
ratio of conjugate accumulated in the 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 300 nm. In
some embodiments, the hydrodynamic diameter is in the range of from
50 nm to 250 nm. In some embodiments the hydrodynamic diameter is
in the range of from 100 nm to 250 nm. In yet another embodiment
the hydrodynamic diameter is in the range of 150 nm to 200 nm. The
hydrodynamic diameter can be measured as described below under the
Materials and Methods of the Example section which follows
hereinbelow.
[0336] Chemical Forms of the Conjugates:
[0337] The conjugates described hereinabove may be administered or
otherwise utilized either as is, or as a pharmaceutically
acceptable salt, solvate, hydrate or a prodrug thereof.
[0338] 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.
[0339] The phrase "pharmaceutically acceptable salts" is meant to
encompass salts of the moieties and/or conjugates which are
prepared with relatively nontoxic acids or bases, depending on the
particular substituents found on the compounds described herein.
When conjugates of the present invention 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
conjugates of the present invention contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such conjugates 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). Certain specific
conjugates of the present invention contain both basic and acidic
functionalities that allow the conjugates to be converted into
either base or acid addition salts.
[0340] The neutral forms of the conjugates are preferably
regenerated by contacting the salt with a base or acid and
isolating the parent conjugate in a conventional manner. The parent
form of the conjugate 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
conjugate for the purposes of the present invention.
[0341] In an example, a pharmaceutically acceptable salt of
alendronate is utilized. An exemplary such salt is sodium
alendronate. An alendronate-containing conjugate can therefore
comprise a sodium salt of alendronate.
[0342] The term "prodrug" refers to an agent, which is converted
into the active compound (the active parent drug) in vivo. Prodrugs
are typically useful for facilitating the administration of the
parent drug. The prodrug may also have improved solubility as
compared with the parent drug in pharmaceutical compositions.
Prodrugs are also often used to achieve a sustained release of the
active compound in vivo.
[0343] The conjugates described herein may possess asymmetric
carbon atoms (optical centers) or double bonds; the racemates,
enantiomers, diastereomers, geometric isomers and individual
isomers are encompassed within the scope of the present
invention.
[0344] 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.
[0345] The conjugates 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.
[0346] 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.
[0347] The term "hydrate" refers to a solvate, as defined
hereinabove, where the solvent is water.
[0348] Certain conjugates of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0349] Uses:
[0350] As discussed hereinabove, the conjugates described herein
comprise a bone targeting moiety which enables the targeting of the
conjugate to bone and bone related (osteoid) structures. Due to the
therapeutic activity of the conjugates, they can be efficiently
used for treating bone related disease and disorders.
[0351] Hence, according to another aspect of some embodiments of
the present invention there are provided methods of treating a bone
related disease or disorder in a subject in need thereof. These
methods are effected by administering to the subject a
therapeutically effective amount of any of the conjugates described
herein.
[0352] Accordingly, according to another aspect of some embodiments
of the present invention there are provided uses of any of the
conjugates described herein as a medicament. In some embodiments,
the medicament is for treating a bone-related disease or
disorder.
[0353] According to another aspect of some embodiments of the
present invention, the conjugates described herein are identified
for use in the treatment of a bone related disease or disorder.
[0354] 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.
[0355] The phrase a "bone related disease or disorder" describes a
disease or disorder wherein bone formation, deposition, or
resorption is abnormal, especially those characterized by excessive
angiogenesis. The phrase "bone related disease or disorder"
encompasses disease and disorders occurring in bodily sites other
than bone which evolved from a bone related disease or disorder
such as, for example, metastasis of bone cancer in another organ.
Bone-related diseases and disorders include, but are not limited
to, bone cancer and bone cancer metastases, osteopenia due to bone
metastases, periodontal disease, periarticular erosions in
rheumatoid arthritis, Paget's disease, malignant hypercalcemia,
osteolytic lesions produced by bone metastasis, bone abnormalities
caused by cancer therapeutics and hyperostosis.
[0356] 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. When the
treatable disease is bone cancer, the term would encompass any
inhibition of tumor growth or metastasis, or any attempt to
inhibit, slow or abrogate tumor growth or metastasis.
[0357] It is noted herein that by targeting a therapeutically
active agent via the methodologies described herein, the toxicity
of the therapeutically active agent is substantially reduced, due
to the conjugate selectivity towards bone tissues. Consequently,
besides the use of the conjugates described herein in a clinically
evident disease, optionally in combination with other drugs, these
conjugates may potentially be used as a long term-prophylactic for
individuals who are at risk for relapse due to residual dormant
cancers.
[0358] 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.
[0359] As demonstrated in the Examples section that follows, an
exemplary conjugate, according to some embodiments described
herein, inhibited angiogenesis as well as cell proliferation and
therefore can be utilized for the treatment of bone related disease
and disorders characterized by pathologically excessive
angiogenesis wherein the inhibition of angiogenesis and/or cell
proliferation is beneficial.
[0360] Hence, in some embodiments the bone related disease or
disorder is associated with angiogenesis.
[0361] Tumor growth and metastasis are particularly dependent on
the degree of angiogenesis. Tumor angiogenesis is the proliferation
of a network of blood vessels that penetrate into cancerous tumors
in order to supply nutrients and oxygen and remove waste products,
thus leading to tumor growth. Tumor angiogenesis involves hormonal
stimulation and activation of oncogenes, expression of angiogenic
growth factors, extravasation of plasma protein, deposition of a
provisional extracellular matrix (ECM), degradation of ECM, and
migration, proliferation and elongation of endothelial capillaries.
Inhibition of further vascular expansion has therefore been the
focus of active research for cancer therapy.
[0362] Hence, in some embodiments the bone related disease or
disorder is selected from the group consisting of bone cancer
metastases and bone cancer.
[0363] 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.
[0364] The term "bone cancer" describes tumors that arise from the
tissues of the bone. The term "bone cancer", as used herein,
further encompasses tumors in tissues located in proximity to bone
structures and associated with bone such as cartilage, bone cavity
and bone marrow. The term "Bone cancer" further encompasses cancer
which evolved from bone cells (i.e. primary tumor) as well as
cancer cells which have "breaken away", "leaked", or "spilled" from
a primary tumor located in bone, entered the lymphatic and/or blood
vessels, circulated through the lymphatic system and/or
bloodstream, settled down and proliferated within normal tissues
elsewhere in the body thereby creating a secondary tumor. For
example, metastases originating from osteosarcoma can be frequently
found in the lungs and in other organs. These lesions produce an
osteoid and therefore can be targeted similarly with compounds with
high affinity to bone mineral, hydroxyapatite, such as alendronate,
and other bisphosphonates as well as oligoaspartates.
[0365] Bone cancer is found most often in the bones of the arms and
legs, but it can occur in any bone.
[0366] Bone cancers are also known as sarcomas. There are several
types of sarcomas of bone, depending upon the kind of bone tissue
where the tumor developed.
[0367] Exemplary types of bone cancers that are treatable according
to embodiments of the invention include, but are not limited to,
osteosarcoma, Ewing's sarcoma, chondrosarcoma, fibrosarcoma,
malignant giant cell tumor, and chordoma.
[0368] Osteosarcoma is the most common type of primary bone cancer
and classified as a malignant mesenchymal neoplasm in which the
tumor directly produces defective osteoid (immature bone). It is a
highly vascular and extremely destructive malignancy that most
commonly arises in the metaphyseal ends of long bones. Several
strategies were proposed, such as immune-based therapy,
tumor-suppressor or suicide gene therapy, or anticancer drugs that
are not commonly used in osteosarcoma [Quan et al. Cancer
Metastasis Rev 2006; 10: 707-713]. However, still one-third of
patients die from this devastating cancer, and for those with
unresectable disease there are no curative systemic therapies.
[0369] The term "bone metastases" describes cancer evolving form a
primary tumor located in bodily site other than bone but
metastasizing to the bone (i.e. a secondary tumor). Cancers that
commonly metastasize, or spread, to the bones include breast
cancer, lung cancer, thyroid cancer, prostate cancer, some brain
cancers and cancers of the kidney.
[0370] For example, prostate cancer is the most common cancer of
males in industrialized countries and the second leading cause of
male cancer mortality. Prostate cancer predominantly metastasizes
to bone, but other organ sites are affected including the lung,
liver, and adrenal gland. Bone metastases incidence in patients
with advanced metastatic disease is approximately 70%. Bone
metastases are associated with considerable skeletal morbidity,
including severe bone pain, pathologic fracture, spinal cord or
nerve root compressions, and hypercalcemia of malignancy.
[0371] As discussed hereinabove, the conjugates described herein
may be further utilized for monitoring bone related disease or
disorders. In such a case the conjugate further comprises a
labeling agent, as defined herein for easy detection of the
conjugate in the body of the patient, using well known imaging
techniques. For example, in the case of the bone related disease or
disorder being bone cancer the detection of the conjugate, as
assessed by the level of labeling agent signal, can serve to detect
bone cancer metastases in bodily sites other than bone.
[0372] Hence, according to another aspect of some embodiments of
the present invention there are provided methods of monitoring a
bone related disease or disorder in a subject. The method according
to these embodiments of the invention is effected by administering
to the subject any of the conjugates described herein, having a
labeling agent attached to the polymer, as described herein, and
employing an imaging technique for monitoring a distribution of the
conjugate within the body or a portion thereof.
[0373] Accordingly, according to another aspect of some embodiments
of the present invention there are provided uses of any of the
conjugates described herein, having a labeling agent as described
herein, as diagnostic agents and/or in the manufacture of a
diagnostic agent for monitoring a bone related disease or
disorder.
[0374] According to another aspect of some embodiments of the
present invention, each of the conjugates described herein, which
comprises a labeling agent, is identified for use as a diagnostic
agent, for monitoring a bone related disease or disorder.
[0375] Suitable imaging techniques include but are not limited to
positron emission tomography (PET), gamma-scintigraphy, magnetic
resonance imaging (MRI), functional magnetic resonance imaging
(FMRI), magnetoencephalography (MEG), single photon emission
computerized tomography (SPECT) computed axial tomography (CAT)
scans, ultrasound, fluoroscopy and conventional X-ray imaging. The
choice of an appropriate imaging technique depends on the nature of
the labeling agent, and is within the skill in the art. For
example, if the labeling agent comprises Gd ions, then the
appropriate imaging technique is MRI; if the labeling agent
comprises radionuclides, an appropriate imaging technique is
gamma-scintigraphy; if the labeling agent comprises an ultrasound
agent, ultrasound is the appropriate imaging technique, etc.
[0376] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising, as an active
ingredient, any of the conjugates described herein and a
pharmaceutically acceptable carrier.
[0377] Accordingly, in any of the methods and uses described
herein, any of the conjugates 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.
[0378] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the conjugates 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.
[0379] 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.
[0380] 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.
[0381] 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.
[0382] 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).
[0383] 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).
[0384] 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.
[0385] 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.
[0386] 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.
[0387] 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.
[0388] 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.
[0389] 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 of a bone related disease or
disorder, as described herein.
[0390] According to another embodiment of the present invention,
the pharmaceutical composition is packaged in a packaging material
and identified in print, in or on the packaging material, for use
in monitoring a bone related disease or disorder, as described
herein.
[0391] 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.
[0392] In any of the methods, uses and compositions described
herein, the conjugates 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, and any other agent that may enhance the
therapeutic effect of the conjugate and/or the well-being of the
treated subject.
Syntheses and Intermediates
[0393] In the course of devising a synthetic pathway for preparing
the conjugates as described herein, the present inventors have
successfully prepared representative intermediate structures that
are useful for preparing the conjugates as described herein and/or
for evaluating the biological activity of the conjugates as
described herein.
[0394] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric
backbone having attached to an end thereof (e.g., to a terminus
backbone unit thereof) a bisphosphonate moiety, said bisphosphonate
being attached to said terminus backbone via a branching unit,
wherein a mol ratio of said bisphosphonate to said polymer is at
least 2:1.
[0395] Embodiments as described herein for the polymer, the
branching unit and the bone targeting moiety in the context of
bisphosphonates are all contemplated in the herein described
embodiments of this aspect.
[0396] In some embodiments, the branching unit has a dendrite
structure.
[0397] In some embodiments, such a conjugate can be represented by
Formula II as follows:
A-P-[B.sub.1].sub.m.sup.0-[B.sub.2].sub.m.sup.1-[B.sub.3].sub.m.sup.2
. . . [Bg-L.sub.2].sub.m.sup.g-1-[T*].sub.m.sup.g Formula II
[0398] wherein:
[0399] P is said polymeric backbone;
[0400] T* is a bisphosphonate bone targeting moiety as described
herein;
[0401] A is an end group of the polymeric backbone, and can be a
functional group intrinsic to the polymer, per se, or protected, or
any other functional group generated at the end of the polymer, as
described hereinabove;
[0402] L.sub.2 is a linking moiety, linking said targeting moiety
to one end of the polymeric backbone (e.g., to a terminus backbone
unit of the polymer) via the branching unit, as described herein,
or is absent;
[0403] B.sub.1, B.sub.2, B.sub.3 . . . Bg are each independently a
branching moiety, wherein B.sub.1, B.sub.2, B.sub.3 . . . Bg
together form a branching unit having a dendritic structure, as
described herein;
[0404] m is an integer that equals 2, 3, 4, 5 or 6, representing
the ramification number of said dendritic structure; and
[0405] g is an integer that ranges from 1 to 20, representing the
number of generations of said dendritic structure.
[0406] In some embodiments, the polymer is a poly(alkylene
glycol).
[0407] In some embodiments, the bisphosphonate is alendronate.
[0408] In some embodiments, the polymer is a poly(alkylene glycol),
and the bisphosphonate is alendronate.
[0409] According to an aspect of some embodiments of the present
invention there is provided a conjugate comprising a polymeric
backbone having attached thereto a therapeutically active agent,
the therapeutically active agent being attached to one end of the
polymeric backbone (e.g., to a terminus backbone unit at one end of
the polymer) wherein the polymer further comprises a reactive group
attached to a another end of the polymeric backbone 9 e.g., to
terminus backbone unit at another end of the polymer) via a
branching unit, as described herein, wherein a mol ratio of said
functional group to said polymer and is at least 2:1.
[0410] In some embodiments, the reactive group is useful for
attaching a targeting moiety, e.g., a bone targeting moiety, to the
conjugate.
[0411] Embodiments as described herein for the polymer, the
branching unit, the linking moiety and the therapeutically active
agent are all contemplated in the herein described embodiments of
this aspect.
[0412] In some embodiments, the branching unit has a dendrite
structure.
[0413] In some embodiments, such a conjugate can be represented by
Formula III as follows:
D-L.sub.1-[B*]-P-[B.sub.1].sub.m.sup.0-[B.sub.2].sub.m.sup.1-[B.sub.3].s-
ub.m.sup.2 . . . [Bg-L.sub.2].sub.m.sup.g-1-[R].sub.m.sup.g Formula
I
[0414] wherein:
[0415] D is a therapeutically active agent as described herein;
[0416] P is a polymeric backbone as described herein;
[0417] R is a reactive group;
[0418] B* is a branching unit or is absent;
[0419] L.sub.1 is a linking moiety, linking the therapeutically
active agent to the end of the polymeric backbone, as described
herein;
[0420] L.sub.2 is a second linking moiety, linking the reactive
group to the other end of the polymeric backbone, via the branching
unit, or is absent;
[0421] B.sub.1, B.sub.2, B.sub.3 . . . Bg are each independently a
branching moiety, wherein B.sub.1, B.sub.2, B.sub.3 . . . Bg
together form a branching unit having a dendritic structure, as
described herein;
[0422] m is an integer that equals 2, 3, 4, 5 or 6, representing
the ramification number of the dendritic structure; and
[0423] g is an integer that ranges from 1 to 20, representing the
number of generations of the dendritic structure.
[0424] In some embodiments, the first linking moiety is a
hydrolytically-cleavable moiety as described herein.
[0425] In some embodiments, the polymeric backbone is derived from
a ply(alkylene glycol), as described herein, and in some
embodiments, it is derived from PEG.
[0426] The reaction group can be, for example, hydroxy, amine,
carboxylate, halide, sulfate, sulfonate, and the like.
[0427] In some embodiments, the reactive group is carboxylate.
[0428] According to an aspect of the some embodiments of the
present invention there is provided a process of preparing a
polymeric conjugate as described herein.
[0429] In some embodiments, the process is effected by reacting a
conjugate comprising a polymeric backbone having a bone targeting
moiety attached to one end thereof via a branching moiety, with a
therapeutically active agent.
[0430] In some embodiments, the conjugate and the therapeutically
active agent are selected so as to generate a
hydrolytically-cleavable linking moiety, as described herein. In
some of these embodiments, the process further comprises, prior to
the reacting, attaching such a linking moiety to the
therapeutically active agent, whereby the reacting in performed by
forming a bond between the conjugate and the linking moiety.
[0431] Conditions, optional protecting groups, optional activating
groups and the like, can be selected by one of skill in the art so
as to efficiently perform the reaction while considering the
chemical structures of the reactants.
[0432] In some embodiments, the reacting conjugate has general
Formula III, as described herein.
[0433] Providing such a conjugate can be performed by sequentially
and controllably growing at one end of a polymeric backbone (e.g.,
protected at the other end thereof) a dendritic branching unit, by
sequential attachment of the branching moieties, as described
herein.
[0434] An exemplary process as described herein is described in the
Examples section that follows, and in FIG. 2.
[0435] In some embodiments, a process of preparing the polymeric
conjugates as described herein is effected by reacting a conjugate
of a polymeric backbone and a therapeutically active agent as
described herein, which terminates by two or more reactive groups,
with a bone targeting moiety or moieties.
[0436] General:
[0437] As used herein the term "about" refers to .+-.10%.
[0438] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0439] The term "consisting of" means "including and limited
to".
[0440] 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.
[0441] The word "exemplary" is used herein to mean "serving as an
example, instance or illustration". Any embodiment described as
"exemplary" is not necessarily to be construed as preferred or
advantageous over other embodiments and/or to exclude the
incorporation of features from other embodiments.
[0442] The word "optionally" is used herein to mean "is provided in
some embodiments and not provided in other embodiments". Any
particular embodiment of the invention may include a plurality of
"optional" features unless such features conflict.
[0443] 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.
[0444] 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.
[0445] 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.
[0446] 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.
[0447] 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.
[0448] As used herein throughout, the term "alkyl" refers to 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 implies 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. More preferably, the alkyl is a
medium size alkyl having 1 to 10 carbon atoms. Most preferably,
unless otherwise indicated, the alkyl is a lower alkyl having 1 to
4 carbon atoms. The alkyl group may be unsubstituted or
substituted, as long as the substituent does not interfere with the
performance and/or intended use of the compound. When substituted,
the substituent group can be, for example, cycloalkyl, alkenyl,
alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy,
aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl,
cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl,
thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl,
O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy,
O-carboxy, sulfonamido, and amino, as these terms are defined
herein.
[0449] A "cycloalkyl" group refers to 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. A cycloalkyl group
may be unsubstituted or substituted, as long as the substituent
does not interfere with the performance and/or intended use of the
compound. When substituted, the substituent group can be, for
example, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy,
thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide,
phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea,
thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,
C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as
these terms are defined herein.
[0450] An "alkenyl" group refers to an alkyl group which consists
of at least two carbon atoms and at least one carbon-carbon double
bond.
[0451] An "alkynyl" group refers to an alkyl group which consists
of at least two carbon atoms and at least one carbon-carbon triple
bond.
[0452] An "aryl" group refers to 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 unsubstituted
or substituted, as long as the substituent does not interfere with
the performance and/or intended use of the compound. When
substituted, the substituent group can be, for example, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,
halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl,
phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these
terms are defined herein.
[0453] A "heteroaryl" group refers to 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, indole, indolenine,
quinoline, isoquinoline and purine. The heteroaryl group may be
unsubstituted or substituted, as long as the substituent does not
interfere with the performance and/or intended use of the compound.
When substituted, the substituent group can be, for example, alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic,
halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy,
thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl,
phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea,
O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,
N-amido, C-carboxy, O-carboxy, sulfonamido, and amino, as these
terms are defined herein.
[0454] A "heteroalicyclic" group refers to 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. The heteroalicyclic may be
unsubstituted or substituted, as long as the substituent does not
interfere with the performance and/or intended use of the compound.
When substituted, the substituted group can be, for example, lone
pair electrons, alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy,
thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano,
nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl,
urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl,
N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy,
sulfonamido, and amino, as these terms are defined herein.
Representative examples are piperidine, piperazine,
tetrahydrofuran, tetrahydropyran, morpholine and the like.
[0455] A "hydroxy" group refers to an --OH group.
[0456] An "azide" group refers to a --N.dbd.N.sup.+.dbd.N.sup.-
group.
[0457] An "alkoxy" group refers to both an --O-alkyl and an
--O-cycloalkyl group, as defined herein.
[0458] An "aryloxy" group refers to both an --O-aryl and an
--O-heteroaryl group, as defined herein.
[0459] A "thiohydroxy" or "thiol" group refers to a --SH group.
[0460] A "thioalkoxy" group refers to both an --S-alkyl group, and
an --S-cycloalkyl group, as defined herein.
[0461] A "thioaryloxy" group refers to both an --S-aryl and an
--S-heteroaryl group, as defined herein.
[0462] A "carbonyl" group refers to a --C(.dbd.O)--R' group, where
R' is defined as hereinabove.
[0463] A "thiocarbonyl" group refers to a --C(.dbd.S)--R' group,
where R' is as defined herein.
[0464] A "C-carboxy" group refers to a --C(.dbd.O)--O--R' groups,
where R' is as defined herein.
[0465] An "O-carboxy" group refers to an R'C(.dbd.O)--O-- group,
where R' is as defined herein.
[0466] An "oxo" group refers to a .dbd.O group.
[0467] A "carboxylate" or "carboxyl" encompasses both C-carboxy and
O-carboxy groups, as defined herein.
[0468] A "carboxylic acid" group refers to a C-carboxy group in
which R' is hydrogen. A "thiocarboxy" or "thiocarboxylate" group
refers to both --C(.dbd.S)--O--R' and --O--C(.dbd.S)R' groups.
[0469] An "ester" refers to a C-carboxy group wherein R' is not
hydrogen. An ester bond refers to a --O--C(.dbd.O)-- bond.
[0470] A "halo" group refers to fluorine, chlorine, bromine or
iodine.
[0471] A "sulfinyl" group refers to an --S(.dbd.O)--R' group, where
R' is as defined herein.
[0472] A "sulfonyl" group refers to an --S(.dbd.O).sub.2--R' group,
where R' is as defined herein.
[0473] A "sulfonate" group refers to an --S(.dbd.O).sub.2--O--R'
group, where R' is as defined herein.
[0474] A "sulfate" group refers to an --O--S(.dbd.O).sub.2--O--R'
group, where R' is as defined as herein.
[0475] A "sulfonamide" or "sulfonamido" group encompasses both
S-sulfonamido and N-sulfonamido groups, as defined herein.
[0476] An "S-sulfonamido" group refers to a
--S(.dbd.O).sub.2--NR'R'' group, with each of R' and R'' as defined
herein.
[0477] An "N-sulfonamido" group refers to an
R'S(.dbd.O).sub.2--NR'' group, where each of R' and R'' is as
defined herein.
[0478] An "O-carbamyl" group refers to an --OC(.dbd.O)--NR'R''
group, where each of R' and R'' is as defined herein.
[0479] An "N-carbamyl" group refers to an R'OC(.dbd.O)--NR''--
group, where each of R' and R'' is as defined herein.
[0480] A "carbamyl" or "carbamate" group encompasses O-carbamyl and
N-carbamyl groups.
[0481] A carbamate bond describes a --O--C(.dbd.O)--NR'-- bond,
where R' is as described herein.
[0482] An "O-thiocarbamyl" group refers to an --OC(.dbd.S)--NR'R''
group, where each of R' and R'' is as defined herein.
[0483] An "N-thiocarbamyl" group refers to an R'OC(.dbd.S)NR''--
group, where each of R' and R'' is as defined herein.
[0484] A "thiocarbamyl" or "thiocarbamate" group encompasses
0-thiocarbamyl and N-thiocarbamyl groups.
[0485] A thiocarbamate bond describes a --O--C(.dbd.S)--NR'-- bond,
where R' is as described herein.
[0486] A "C-amido" group refers to a --C(.dbd.O)--NR'R'' group,
where each of R' and R'' is as defined herein.
[0487] An "N-amido" group refers to an R'C(.dbd.O)--NR''-- group,
where each of R' and R'' is as defined herein.
[0488] An "amide" group encompasses both C-amido and N-amido
groups.
[0489] An amide bond describes a --NR'--C(.dbd.O)-- bond, where R'
is as defined herein.
[0490] A "urea" group refers to an --N(R')--C(.dbd.O)--NR''R'''
group, where each of R' and R'' is as defined herein, and R''' is
defined as R' and R'' are defined herein.
[0491] A "nitro" group refers to an --NO.sub.2 group.
[0492] A "cyano" group refers to a --C.ident.N group.
[0493] The term "phosphonyl" or "phosphonate" describes a
--P(.dbd.O)(OR')(OR'') group, with R' and R'' as defined
hereinabove.
[0494] The term "phosphate" describes an --O--P(.dbd.O)(OR')(OR'')
group, with each of R' and R'' as defined hereinabove.
[0495] A "phosphoric acid" is a phosphate group is which each of R
is hydrogen.
[0496] The term "phosphinyl" describes a --PR'R'' group, with each
of R' and R'' as defined hereinabove.
[0497] The term "thiourea" describes a --N(R')--C(.dbd.S)--NR''--
group, with each of R' and R'' as defined hereinabove.
[0498] 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.
[0499] 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
[0500] Reference is now made to the following examples which,
together with the above descriptions, illustrate the invention in a
non limiting fashion.
Example 1
Chemical Syntheses and Characterization
Materials and Experimental Methods
[0501] Materials:
[0502] All reactions requiring anhydrous conditions were performed
under an Ar or N.sub.2 atmosphere. Chemicals and solvents were
either A.R. grade or purified by standard techniques.
[0503] Paclitaxel (PTX) was obtained from Indena (Milan, IT) or
from Alcon Biosciences Ltd. (Mumbai, India; Petrus Chemicals and
Materials Ltd., Israel).
[0504] Alendronate (ALN) was purchased from Alcon Biosciences Ltd.
(Mumbai, India; Petrus Chemicals and Materials Ltd., Israel).
[0505] Boc-NH-PEG5 kDa-NHS and Lys(cFmoc)-OH was obtained from Iris
Biotech GmbH (Marktredwitz, Germany).
[0506] N-Hydroxysuccinimide (NHS), N,N-Dicyclohexylcarbodiimmide
(DCC), succinic anhydride, .beta.-glutamic acid (.beta.-Glu),
silica gel (SiO.sub.2), sodium sulfate anhydrous
(Na.sub.2SO.sub.4), triethylamine (TEA), trifluoroacetic acid
(TFA), 2,4,6-trinitrobenzenesulfonic acid (TNBS),
dimethylsulfoxide-d.sub.6 and D.sub.2O were purchased from
Sigma-Aldrich.
[0507] Glycil-glycine (Gly-Gly) was obtained from Merck (Darmstadt,
Germany).
[0508] All other chemical reagents, including salts and solvents
were purchased from Sigma-Aldrich.
[0509] Instrumental Data:
[0510] Thin layer chromatography (TLC) was performed using silica
gel plates Merck 60 F.sub.254; compounds were visualized by
irradiation with UV light and/or by treatment with a solution of
phosphomolybdic acid (20% wt. in ethanol), followed by heating.
[0511] .sup.1H NMR measurements were performed using Bruker AMX 200
or 400 instrument. The chemical shifts are expressed in 6 relative
to TMS (6=0 ppm) and the coupling constants J in Hz. The spectra
were recorded in CDCl.sub.3, as a solvent at room temperature,
unless otherwise indicated.
[0512] Determination of Free and Total PTX Contents in the
Conjugates:
[0513] The amount of PTX in the conjugates was evaluated by reverse
phase HPLC using an Agilent 300-Extend C18 (4.6.times.250 mm; 5
.mu.m) column, with the UV detector settled at 227 nm. The eluents
A and B were H.sub.2O and CH.sub.3OH, respectively. The elution was
performed by the following gradient: from 5% B to 50% B in 5
minutes, from 50% B to 80% B in 14 minutes, from 80% B to 100% B in
5 minutes, and from 100% B to 5% B in 5 minutes, at a flow rate of
1 mL/minute.
[0514] The total drug content was evaluated by RP-HPLC following
the release of PTX from the conjugates. 3 mg of conjugate were
dissolved in 1 mL of MeOH. Following the addition of 2% (v/v) of
NaOH 0.2 N, the solution was incubated at 50.degree. C. for 2
hours. The drug was then extracted by ethyl acetate. The organic
phase was evaporated and the residue was solubilized in methanol.
The elution was performed as reported above. The amount of PTX was
calculated using PTX calibration curve obtained using the same
method. The standard error for this analysis, calculated using
solutions of PTX at known concentrations, is .+-.1.89%.
[0515] Determination of ALN Content Bound to PEG:
[0516] The formation of chromophoric complex between ALN and
Fe.sup.3+ ions in perchloric acid solution was used to determine
the ALN content by spectrophotometry [as described in Kuljanin et
al. J. Pharm. Biomed. Anal. 2002, 28, 1215-1220]. Briefly,
conjugates (2.5, 5 and 10 mg) were dissolved in a mixture of 0.1 mL
of 4 mM FeCl.sub.3 and 0.8 mL of 0.2 M perchloric acid
(HClO.sub.4). The content of ALN in the conjugates was determined
against a calibration graph of serial dilutions of 0-3 mM ALN.
Sample absorbance was measured spectrophotometrically at
.lamda.=300 nm.
[0517] Dynamic Light Scattering (DLS) of Conjugates:
[0518] The mean hydrodynamic diameter of the conjugates was
evaluated using a real time particle analyzer (NanoSight LM20.TM.).
PTX-PEG and PTX-PEG-ALN (5 mg/mL) were injected into the chamber,
allowed to equilibrate for 30 seconds and analyzed by a
Nanoparticle Tracking Analysis (NTA) software.
[0519] Conjugates' Stability in Buffer Solution at Different pH
Values and in Plasma:
[0520] Each conjugate (3 mg/mL) was incubated at 37.degree. C. for
48 hours in PBS at pH 5 and 7.4 to evaluate the drug release.
Samples of 50 .mu.L were withdrawn at predetermined times and
analyzed by RP-HPLC using the conditions reported above, evaluating
the decrease of the conjugate peak in the chromatographic
profile.
[0521] The tested conjugates were also incubated at 37.degree. C.
for 48 hours in mouse plasma, obtained after centrifugation of
blood sample at 2000.times.g for 10 minutes. Samples of 60 .mu.L
were withdrawn at predetermined times and 60 .mu.L of CH.sub.3CN
were added to achieve plasma protein precipitation. Samples were
centrifuged at 15000.times.g and the supernatant was withdrawn and
analyzed by RP-HPLC using the conditions reported above.
[0522] The stability of the conjugates was also evaluated by
dynamic light scattering. Solution of each conjugate (7 mg/mL) in
PBS pH 5 and 7.4 were prepared and immediately extruded with manual
extruder (Liposofast Avestin) at 200 nm and analyzed using a light
scattering instrument (Malvern Nano-S, Worcestershire, United
Kingdom). The instrument was settled at 37.degree. C., the detector
position was at 173.degree. and the analysis was performed every 20
minutes (the first measurement was performed after 5 minutes of
equilibration) for 4 hours and, after storage in similar
conditions, the sample was analyzed at 24 hours.
Chemical Syntheses
Synthesis of PEG-ALN, PEG-PTX and PTX-PEG-ALN Conjugates
[0523] The chemical structures of PEG-ALN (Compound 1) PEG-PTX
(Compound 2) and PTX-PEG-ALN (Compound 3), exemplary conjugates
according to some embodiments of the present invention, are
depicted in FIGS. 1A, 1B and 1C, respectively, wherein X is
--C(.dbd.O)--.
[0524] An exemplary synthetic pathway for preparing PTX-PEG-ALN
(Compound 3) is depicted in FIG. 2.
[0525] The synthesis of PTX-PEG (Compound 2) was performed in three
main steps: synthesis of SPTX, synthesis of PEG-dendrimer and
binding of SPTX to PEG-dendrimer (see, FIG. 2).
[0526] The PEG-dendrimer was built at carboxylic activated terminus
of commercial Boc-NH-PEG-NHS using .beta.-Glutamic acid (.beta.Glu)
as symmetric bicarboxylic branching unit.
[0527] PEG-ALN was obtained by firstly linking the ALN targeting
residues to the PEG dendrimer carboxylic group and then by removing
the Boc protecting group.
[0528] The coupling of SPTX to PEG-ALN yielded PTX-PEG-ALN.
[0529] Preparation of 2'-succinyl-paclitaxel (SPTX):
[0530] To 1 gram (1.17 mmol) of paclitaxel, dissolved in 30 mL of
anhydrous pyridine, 585 mg (5.85 mmol) of succinic anhydride were
added. The reaction was stirred at room temperature for 48 hours.
The SPTX was purified by chromatography on a SiO.sub.2 column
(30.times.2.5 cm) eluted with a chloroform-methanol mixture (97:3
to 90:10) and determined by TLC (Rf 0.5 in chloroform-methanol
90:10).
[0531] SPTX was characterized by .sup.1H-NMR spectroscopy, showing
the characteristic signals of PTX together with those of the
succinic spacer, as follows.
[0532] .sup.1H-NMR of SPTX (CDCl.sub.3): .delta.=1.15 (s, 3H, C16),
1.24 (s, 3H, C17), 1.68 (s, 3H, C18), 1.79 (s, 3H, C19), 2.24 (s,
3H, C31), 2.38 (s, 3H, C29), 2.5-2.7 (m, 4H, --CH2-CH2- succinic
spacer), 4.9 (d, 1H, C5), 5.66 (d, 1H, C2'), 6.27 (s, 1H, C10),
7.25 (s, 3'-Ph), 7.4 (m, 3'-NBz), 7.5 (m 2-OBz), 7.75 (d, 3'NBz),
8.1 (d, 2-OBz) ppm.
[0533] Preparation of Boc-NH-PEG-.beta.Glu-(COOH).sub.2 (Compound
4):
[0534] Boc-NH-PEG-NHS (MW 4928 Da; 3.5 grams; 0.71 mmol) was added
to .beta.-glutamic acid (.beta.Glu; 313 mg; 2.13 mmol), dissolved
in 150 mL of 0.1 M borate buffer/CH.sub.3CN (3:2) mixture having pH
8.0. The reaction mixture was let to proceed for 5 hours under
stirring. The reaction mixture pH was thereafter adjusted to about
4.5 with 0.2N HCl and the excess of .beta.Glu was removed by
extractions with CHCl.sub.3 (6.times.300 mL). The organic phase was
dried over anhydrous Na.sub.2SO.sub.4, concentrated under vacuum
and dropped into 1 L of cold diethyl ether under stirring. After 1
hour at -20.degree. C., the precipitate was filtered and dried
under vacuum, to thereby afford Compound 4 (3.345 grams, 95%
yield). The absence of free .beta.Glu in the conjugate was verified
by TNBS test according to Snyder and Sabocinsky assay [Anal.
Biochem. 1975, 64, 284-288].
[0535] Preparation of Boc-NH-PEG-.beta.Glu-(NHS).sub.2 (Compound
5):
[0536] Compound 4 (3.33 grams; 0.67 mmol) was dissolved in 100 mL
of anhydrous CH.sub.2Cl.sub.2, and NHS (469 mg; 4.07 mmol) and DCC
(1.114 gram; 5.4 mmol) were added. The reaction mixture was stirred
at room temperature overnight, and was thereafter filtered and
dropped into 1 liter of cold diethyl ether. After 1 hour at
-20.degree. C., the precipitate was filtered and dried under vacuum
to afford Compound 5 (3.1 mg, 89.5% yield). The degree of
activation was 91%, determined on the basis of the amino group
modification of an equimolar solution of Gly-Gly as reported
elsewhere [see, Pasut et al., 2005, supra].
[0537] Preparation of
Boc-NH-PEG-.beta.Glu-(.beta.Glu).sub.2-(COOH).sub.4 (Compound 6;
PEG-dendrimer):
[0538] .beta.Glu (532 mg; 3.6 mmol) was dissolved in 200 mL of 0.1
M borate buffer/CH.sub.3CN (3:2) mixture at pH 8.0, and Compound 5
(3.09 mg; 0.6 mmol) was added to the solution. The reaction mixture
was treated as described hereinabove for preparing Compound 4 and
the product was similarly purified so as to afford Compound 6 (2.9
grams, 92% yield).
[0539] Preparation of
Boc-NH-PEG-.beta.Glu-(.beta.Glu).sub.2-(NHS).sub.4 (Compound
7):
[0540] Compound 6 (1.7 gram; 0.32 mmol) was with NHS and DCC as
described hereinabove for preparing Compound 5, so as to afford
Compound 7 (1.52 gram, 89% yield). The degree of activation was
81%.
[0541] Preparation of
Boc-NH-PEG-.beta.Glu-(.beta.Glu).sub.2-(ALN).sub.4 (Compound
8):
[0542] ALN (802 mg; 2.46 mmol) was dissolved in 0.1 M borate buffer
at pH 8.0, Compound 7 (1.45 gram; 0.25 mmol) was added and the
reaction mixture was stirred for 5 hours at room temperature. The
product was purified as described hereinabove for Compound 4, to
thereby afford Compound 8 (1.3 gram, 83% yield).
[0543] Preparation of PEG-ALN (Compound 1):
[0544] Compound 8 (1.2 gram) was dissolved in 4 mL of a mixture of
CH.sub.2CH.sub.2/CF.sub.3COOH/H.sub.2O (55.4:45.4:0.1 volume ratio)
and the reaction mixture was stirred at room temperature for 3
hours, and was thereafter evaporated to remove TFA and the
solvents. The obtained oil was dissolved in CH.sub.2Cl.sub.2 and
the solution was dropped into 400 mL of diethyl ether. The
precipitate was filtered and dried under vacuum to afford the
PEG-ALN conjugate Compound 1 (1.1 grams, 91% yield).
[0545] Preparation of
H.sub.2N-PEG-.beta.Glu-(.beta.Glu.sub.2-(COOH).sub.4 (Compound 9;
PEG-dendron):
[0546] Compound 6 (1.2 gram) was dissolved in 4 mL of a mixture of
CH.sub.2CH.sub.2/CF.sub.3COOH/H.sub.2O (55.4:45.4:0.1 volume ratio)
and the reaction mixture was stirred at room temperature for 3
hours to remove the protecting group t-Boc, and was thereafter
evaporated to remove TFA and the solvents. The obtained oil was
dissolved in CH.sub.2Cl.sub.2 and the solution was dropped into 400
mL of diethyl ether. The precipitate was filtered and dried under
vacuum to afford Compound 9 (1.17 grams, 97% yield).
[0547] Preparation of Compound 2 (PEG-PTX):
[0548] SPTX (190 mg; 0.2 mmol) was dissolved in anhydrous DMF (5
mL), and a solution of HOBT (40.5 mg; 0.3 mmol), EDC (40.2 mg; 0.22
mmol) in anhydrous DMF (2 mL) was added. The reaction mixture was
stirred for 5 hours at room temperature and then 530 mg of Compound
9, dissolved in 5 mL of DMF, were added and the obtained mixture
was allowed to react 24 hours under stirring at room temperature.
The reaction mixture was thereafter reduced to small volume (about
5 mL) under vacuum and the product was purified from excess of SPTX
by gel-filtration chromatography using Sephadex LH-20 resin eluted
with DMF. The fractions containing Compound 2 were collected in a
round bottom flask and DMF was evaporated under vacuum. The residue
was dissolved in 5 mL of anhydrous CH.sub.2Cl.sub.2 and the
solution was dropped into 500 mL of cold diethyl ether under
stirring. After 1 hour at -20.degree. C., the precipitate was
filtered and dried under vacuum, to thereby afford the PEG-PTX
conjugate (Compound 2; 425 mg; 69.2% yield.
[0549] Preparation of Compound 3 (PTX-PEG-ALN):
[0550] SPTX (190 mg; 0.2 mmol) was dissolved in anhydrous DMF (5
mL), and a solution of HOBT (40.5 mg; 0.3 mmol) and EDC (40.2 mg;
0.22 mmol), in anhydrous DMF (2 mL), was added. The reaction
mixture was stirred for 5 hours at room temperature and then 650 mg
of Compound 1 (PEG-ALN), dissolved in 5 mL DMF, were added and the
obtained mixture was allowed to react 24 hours under stirring at
room temperature. The reaction mixture was reduced to small volume
(about 5 mL) under vacuum and the product was purified from the
excess SPTX by gel-filtration chromatography using Sephadex LH-20
resin eluted with DMF. The fractions containing Compound 3 were
collected in a round bottom flask and DMF was evaporated under
vacuum. The residue was dissolved in 5 mL of anhydrous
CH.sub.2Cl.sub.2 and the solution was dropped into 500 mL of cold
diethyl ether under stirring. After 1 hour at -20.degree. C., the
precipitate was filtered and dried under vacuum, to thereby afford
the PTX-PEG-ALN conjugate (Compound 3; 550 grams; 74.7% yield).
Synthesis of FITC Labeled PTX-PEG, PEG-ALN and PTX-PEG-ALN
Conjugates
[0551] The chemical structures of FITC labeled-PEG-ALN (FITC
labeled-Compound 1) FITC labeled-PEG-PTX (FITC labeled-Compound 2)
and FITC labeled-PTX-PEG-ALN (FITC labeled-Compound 3), exemplary
FITC labeled-conjugates according to some embodiments of the
present invention, are depicted in FIGS. 1A, 1B and 1C,
respectively, wherein X is the structure stands for FITC coupled to
a lysine residue.
[0552] An exemplary synthetic pathway for preparing PTX-PEG-ALN
(FITC labeled-Compound 3) is depicted in FIG. 3.
[0553] The syntheses of FITC labeled-conjugates (FITC
labeled-Compounds 1, 2 and 3) was performed by exploiting the same
chemical strategy used for the preparation of non-labeled
conjugates, as described hereinabove, and attachment of FITC was
performed by incorporating a Lys residue and exploiting the
.epsilon. amino group of Lys for coupling with FITC (see, FIG.
3).
[0554] Preparation of Boc-NH-PEG-L-Lys(.epsilon.Fmoc)-OH (Compound
13):
[0555] L-Lys(.epsilon.Fmoc)-OH (313 mg; 0.67 mmol) was dissolved in
50 mL of H.sub.2O/CH.sub.3CN (3:2) mixture having pH=8,
Boc-NH-PEG-NHS (MW 4928 Da; 1.1 gram; 0.2 mmol) was added and the
reaction mixture was let to proceed for 5 hours under stirring at
room temperature. The pH was thereafter adjusted to about 4.5 by
addition of 0.2N HCl and Compound 13 was purified from the excess
of L-Lys(cFmoc)-OH by extractions with CHCl.sub.3 (5.times.80 mL).
The organic phase was dried over anhydrous Na.sub.2SO.sub.4,
concentrated under vacuum and was precipitated from 500 mL of
diethyl ether. The product was recovered by filtration and dried
under vacuum to thereby afford the Boc-NH-PEG-L-Lys(cFmoc)-OH
(Compound 13; 1.0 grams; 83.0% yield). The product was
characterized by the Snaider Sobociski assay as described
hereinabove, to verify the absence of free L-Lys(cFmoc)-OH.
[0556] .sup.1H-NMR (d.sub.6-DMSO): .delta.=1.3 (s, 9H, Boc),
3.4-3.6 (s, 422H, CH.sub.2 PEG), 7.2-7.5 (m, 8H, Fmoc group), 7.7
(d, 4H, Fmoc group), 7.9 (d, 4H, Fmoc group) ppm.
[0557] Preparation of Boc-NH-PEG-L-Lys(.epsilon.Fmoc)-NHS (Compound
14):
[0558] Compound 13 (800 mg) was activated by reacting it with NHS
and DCC, as described hereinabove for Compounds 5 and 7, to afford
Compound 14 (94% yield).
[0559] Preparation of
Boc-NH-PEG-L-Lys(.epsilon.Fmoc)-.beta.Glu-(.beta.Glu).sub.2-(COOH).sub.4
(Compound 15; FITC labeled-PEG dendron):
[0560] Compound 14 was reacted with .beta.Glu as described
hereinabove for Compounds 4 and 6, to afford Compound 15 (89.1%
yield).
[0561] Preparation of
Boc-PEG-L-Lys(.epsilon.NH.sub.2)-.beta.Glu-(.beta.Glu).sub.2-(COOH).sub.4
(Compound 16):
[0562] Compound 15 (530 mg) was dissolved in 10 mL of a mixture of
DMF and 20% (v/v) piperidine, and the solution was stirred at room
temperature for 15 minutes to remove the Fmoc protecting group. The
reaction mixture was then evaporated to remove the solvent, the
residue was dissolved in 5 mL of CH.sub.2Cl.sub.2 and the solution
was dropped into 300 mL of diethyl ether. The precipitate was
filtered and dried under vacuum to afford Compound 16 (92.7%
yield).
[0563] Preparation of
Boc-PEG-L-Lys(.epsilon.FITC)-.beta.Glu-(.beta.Glu.sub.2-(COOH).sub.4
(Compound 17):
[0564] FITC (38.9 mg) was dissolved in DMF (10 mL), and Compound 16
(470 mg) and Et.sub.3N (11 .mu.L) were added. The reaction mixture
was stirred at room temperature for 5 hours and excess of FITC was
thereafter removed by extensive dialysis vs. 0.1 M phosphate buffer
pH=8.0 using a membrane with cut-off 3500 Da. The last step of
dialysis was performed overnight with H.sub.2O mQ to eliminate the
phosphate salts. The product was then lyophilized to thereby afford
Compound 17 (86.4% yield). Compound 17 was analyzed by RP-HPLC to
verify the absence of free FITC.
[0565] Preparation of
Boc-PEG-L-Lys(.epsilon.FITC)-.beta.Glu-(.beta.Glu.sub.2-(ALN).sub.4
(Compound 18):
[0566] Compound 17 (244 mg) was activated with NHS/DCC, as
described hereinabove for Compound 7 and the NHS-activated product
was thereafter coupled to ALN, as described hereinabove for
Compound 8, to afford Compound 18 (79.2% yield).
[0567] Preparation of FITC labeled-PEG-ALN (FITC labeled-Compound
1):
[0568] The Boc protecting group was removed from Compound 18 by TFA
hydrolysis procedure as described hereinabove for the preparation
of Compound 1, to thereby afford FITC labeled-Compound 1 (90.4%
yield).
[0569] Preparation of FITC labeled-PTX-PEG (FITC labeled-Compound
2):
[0570] Compound 17 was reacted to remove the Boc protecting group
as described hereinabove for Compound 9, and the obtained product
was reacted with SPTX, as described hereinabove for Compound 2, to
thereby afford FITC labeled-Compound 2 (65.9% yield).
[0571] Preparation of FITC labeled-PTX-PEG-ALN (FITC
labeled-Compound 3):
[0572] FITC-labeled Compound 1 was coupled to SPTX, as described
hereinabove for Compound 3, to thereby afford FITC labeled-Compound
3 (62.8% yield).
Physicochemical Properties of the Conjugates
[0573] The content of ALN in the PTX-PEG-ALN (Compound 3) and
PEG-ALN (Compound 1) non-labeled and labeled conjugates was
determined spectrophotometrically via the chromophoric complex
formed between ALN and Fe.sup.3+ ions in perchloric acid, and
against a calibration graph of ALN, as described in the Methods
section hereinabove.
[0574] The content of free PTX in the PTX-PEG-ALN and PTX-PEG
conjugates was determined directly by RP-HPLC analysis of the
conjugates when dissolved in DMSO. Free PTX impurity in all
conjugates was below 0.6% (w/w).
[0575] Determination of the total PTX amount in the conjugates was
performed by RP-HPLC after hydrolysis of the conjugates to release
the linked drug.
[0576] The content of FITC in the labeled conjugates was measured
spectrophotometrically using .epsilon.64185 M.sup.-1 cm.sup.-1 in
PBS Ph=8.
[0577] The hydrodynamic diameter and size distribution of
PTX-PEG-ALN and of PTX-PEG conjugates were evaluated using laser
light scattering microscopy with Nanoparticle Tracking Analysis
(NTA) technology (NanoSight LM20.TM., Salisbury, UK). The obtained
data is presented in FIGS. 4A and 4B. As shown therein, the mean
hydrodynamic diameter of both PTX-PEG-ALN and PTX-PEG conjugates in
PBS pH 7.4 was about 190 nm.
[0578] The physicochemical properties of PEG conjugates and the
FITC labeled conjugates are presented in table 1.
TABLE-US-00001 TABLE 1 % PTX Micelles Molecular loading % ALN
loading size Product Weight wt/wt wt/wt (nm) PEG 4667 Da -- -- --
PEG-ALN 5913 Da -- 11.9% -- PTX- 5620 Da 6% -- 190 nm PEG
PTX-PEG-ALN 6850 Da 4.68% 11% 200 nm FITC-PEG-ALN 6430 Da -- 7.2%
-- FITC-PEG-PTX 6137 Da 4.26 -- -- FITC-PEG-PTX- 7367 Da 3.6% 6.9%
-- ALN
[0579] Evaluation of the Stability of the Conjugates at Different
pH Values and at Plasma:
[0580] The stability of the exemplary PTX-PEG-ALN conjugate,
Compound 3, was evaluated in buffer solutions at physiological pH
(7.4), at lysosomal pH (5), and in mice plasma, upon incubation at
37.degree. C. for 48 hours, and degradation of the conjugates was
monitored by RP-HPLC. The obtained data is presented in FIG. 5A. At
pH 7.4 and in plasma, about 50% of the PTX-PEG-ALN conjugate was
degraded within the first 1 hour, and the remaining conjugate was
degraded within 24 hours. Similar results were found with
PTX-PEG.
[0581] The stability of the conjugates micelles at 37.degree. C.
during 48 hours was also monitored by dynamic light scattering, and
the results are presented in FIG. 5B. As shown therein, the
micelles stability was in line with the kinetic of PTX release. The
micelles of the PTX-PEG-ALN and PTX-PEG conjugates preserved the
same size for up to 24 hours when incubated at pH 5, whereas at pH
7.4 the same micelles were stable for 3 hours, after which the size
of the samples starts to increase owing to the release of PTX from
the conjugates. Th released PTX is insoluble in the aqueous buffer
and precipitates, thus forming a suspension and destabilizing the
system.
Example 2
In Vitro Studies
Materials and Experimental Methods
[0582] Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, Fetal
bovine serum (FBS), Penicillin, Streptomycin, Nystatin,
L-glutamine, Hepes buffer, sodium pyruvate, and fibronectin were
obtained from Biological Industries Ltd. (Kibbutz Beit Haemek,
Israel).
[0583] EGM-2 medium was purchased from Cambrex (Walkersville, Md.,
U.S.A).
[0584] Matrigel.RTM. matrix was purchased from BD Biosciences,
USA.
[0585] Peroxidase Block was purchased from Merck, Germany.
[0586] Human umbilical vein endothelial cells (HUVEC) were obtained
from Cambrex (Walkersville, Md., U.S.A).
[0587] Hydroxyapatite Binding Assay:
[0588] PEG, PEG-ALN and PTX-PEG-ALN conjugates were dissolved in
phosphate buffered saline (PBS), pH 7.4 (5 mg/mL). The conjugate
solution (600 .mu.L) was incubated with hydroxyapatite (HA) powder
(30 mg), in 600 .mu.L PBS, pH 7.4. NH.sub.2-PEG-(COOH).sub.4
(H.sub.2N-PEG-.beta.Glu-(.beta.Glu).sub.2-(COOH).sub.4;
PEG-dendron, Compound 9, denoted as PEG) was used as control.
Incubated samples were centrifuged at 7000 RPM for 3 minutes and a
sample from the upper layer (100 .mu.L) was collected after 0, 2,
5, 10 and 60 minutes. Fast protein liquid chromatography (FPLC,
AKTA.TM. Purifier.RTM., Amersham Biosciences) analysis using
HiTrap.TM. desalting column (Amersham.RTM.) was used for detection
of unbound conjugates in the samples (FPLC conditions: AKTA.TM.
Purifier.RTM., mobile phase 100% DDW, 2 mL/minute, .lamda.=215 nm).
HA-binding kinetic analysis of the conjugates was performed using
the Unicorn.RTM. AKTA.TM. software. Areas under the curve (AUC)
were calculated from chromatographs at each time point. AUC of each
HA-incubated conjugate chromatogram was normalized to percent AUC
of conjugate sample in the absence of HA.
[0589] Cell Culture:
[0590] PC3 human prostate adenocarcinoma MDA-MB-231 and MDA-MB-231
cells were cultured in DMEM supplemented with 10% Fetal Bovine
Serum (FBS), 100 .mu.g/mL Penicillin, 100 U/mL Streptomycin, 12.5
U/mL Nystatin and 2 mM L-glutamine.
[0591] 4T1 cells were cultured in RPMI 1640 supplemented with 10%
FBS, 100 .mu.g/mL Penicillin, 100 U/mL Streptomycin, 12.5 U/mL
Nystatin, 2 mM L-glutamine, 10 mM Hepes buffer, and 1 mM sodium
pyruvate.
[0592] Human umbilical vein endothelial cells (HUVEC) were grown
according to the manufacturer's protocol in EGM-2 medium
(Cambrex).
[0593] Cells were grown at 37.degree. C.; 5% CO.sub.2.
[0594] Cell Viability Assays:
[0595] PC3 cells were plated onto 96 well plate (5.times.10.sup.3
cells/well) in DMEM supplemented with 5% FBS and incubated for 24 h
(37.degree. C.; 5% CO.sub.2). Following 24 hours of incubation,
medium was replaced with DMEM containing 10% FBS. Cells were
exposed to the combination of PTX and ALN, each drug alone, and
with PEG, PEG-ALN, PEG-PTX, PTX-PEG-ALN conjugates at serial
concentrations for 72 hours. Following incubation, PC3 cells were
counted by MTT.
[0596] HUVECs were plated onto 24-well plate (1.5.times.10.sup.4
cells/well) in growth factors reduced media, (EBM-2, Cambrex, USA)
supplemented with 5% FBS. Following 24 hours of incubation
(37.degree. C.; 5% CO.sub.2), medium was replaced with EGM-2
(Cambrex, USA). 4T1 and MDA-MB-231 cells were plated onto 96 well
plate (5.times.10.sup.3 cells/well) in DMEM supplemented with 5%
FBS and incubated for 24 hours (37.degree. C.; 5% CO.sub.2). The
medium was thereafter replaced with DMEM containing 10% FBS and the
cells were challenged with a combination of free PTX and ALN, with
each free drug alone, and with PEG, and the exemplary PEG-ALN,
PTX-PEG, PTX-PEG-ALN conjugates, at serial concentrations, for up
to 72 hours. Following incubation, HUVEC were counted by Coulter
Counter.
[0597] 4T1 and MDA-MB-231 cells viability was measured by Thiazolyl
Blue Tetrazolium Blue (MTT) (Sigma-Aldrich, Israel) as follows: a
30 .mu.l solution of 2 mg/mL MTT was added to wells containing
cells grown at 100 .mu.l medium. Following 5 hours incubation, the
medium was replaced with dimethyl sulfoxide (DMSO) until blue color
was developed. Viability was measured spectrophotometrically at 560
nm.
[0598] Migration assay: Cell migration assay was performed using
modified 8 .mu.m Boyden chambers Transwells.RTM. (Costar Inc., USA)
coated with 10 .mu.g/mL fibronectin (Biological industries, Beit
Haemek, Israel). PC3 (15.times.10.sup.4 cells/100 .mu.L) were
challenged with a combination of free PTX (10 nM) and ALN (46 nM),
each free drug alone, and with PEG, PEG-ALN, PTX-PEG, PTX-PEG-ALN
conjugates, at equivalent PTX and ALN concentrations, and were
added to the upper chamber of the transwells for 2 hours incubation
prior to migration towards DMEM containing 10% FBS. Following
incubation, cells were allowed to migrate to the underside of the
chamber for 4 hours in the presence or absence of 10% FBS in the
lower chamber. Cells were then fixed and stained (Hema 3 Stain
System; Fisher Diagnostics, USA). The stained migrated cells were
imaged using Nikon TE2000E inverted microscope integrated with
Nikon DS5 cooled CCD camera by 10.times. objective, brightfield
illumination. Migrated cells from the captured images per membrane
were counted using NIH image software. Migration was normalized to
percent migration, with 100% representing migration to medium
containing FBS.
[0599] Capillary-Like Tube Formation Assay:
[0600] The surface of 24-well plates was coated with Matrigel.RTM.
matrix (50 .mu.L/well) (BD Biosciences, USA) on ice and
polymerization was thereafter effected at 37.degree. C. for 30
minutes. HUVEC (3.times.10.sup.4) were challenged with a
combination of free PTX (5 nM) and ALN (23 nM), with each free drug
alone, and with PEG, PEG-ALN, PTX-PEG and PTX-PEG-ALN conjugates,
at equivalent concentrations, and were thereafter seeded on coated
plates in the presence of complete EGM-2 medium. After 8 hours of
incubation (37.degree. C.; 5% CO.sub.2), wells were imaged using
Nikon TE2000E inverted microscope integrated with Nikon DS5 cooled
CCD camera by 4.times. objective, brightfield technique.
[0601] Red Blood Cells (RBC) Lysis Assay:
[0602] Rat RBC solution (2% w/w) was incubated with serial
dilutions of a combination of free PTX and free ALN, PEG, and a
PTX-PEG-ALN conjugate as described herein at equivalent PTX and ALN
concentrations, for 1 hour at 37.degree. C.
[0603] Negative controls were PBS and Dextran (MW of about 70000
Da) while positive controls were 1% w/v solution of Triton X100
(100% lysis) and poly(ethylenimine) (PEI). Following
centrifugation, the supernatant was drawn off and its absorbance
measured at 550 nm using a microplate reader (Genios, TECAN). The
results were expressed as percent of hemoglobin released relative
to the positive control (Triton X100).
[0604] Statistical Methods:
[0605] In vitro data is expressed as mean.+-.standard deviation
(s.d.). Statistical significance was determined using an unpaired
t-test. P<0.05 was considered statistically significant. All
statistical tests were two-sided.
Results
Binding of the Conjugates to Hydroxyapatite (HA)
[0606] The bisphosphonate ALN, known as bone targeting moiety with
strong bone affinity, was chosen as the bone targeting moiety. The
binding capacity of the exemplary ALN-containing conjugates to bone
mineral was evaluated. Hydroxyapatite was used as a model mimicking
bone tissue. An in vitro HA binding assay and FPLC analysis using
HiTrap.TM. desalting column was performed, as described
hereinabove. As shown in FIG. 6, following 5 minutes incubation,
80% or 90% of PTX-PEG-ALN or PEG-ALN conjugates, respectively, were
bound to HA and reached a plateau, indicating the high binding
capacity of the conjugates to bone minerals.
[0607] Biocompatibility of PTX-PEG-ALN Conjugate:
[0608] The biocompatibility of PTX-PEG-ALN was evaluated using rat
red blood cell (RBC) hemolysis assay. Rat RBC solution was
incubated with serial concentrations of a combination of PTX and
ALN, PEG, and the PTX-PEG-ALN conjugate at equivalent PTX and ALN
concentrations, a PTX vehicle (1:1:8 Ethanol:Cremophor EL:Saline),
and polyethylene imine (PEI) which served as control for hemolysis.
The obtained data is presented in FIG. 7 and shows that the
PTX-PEG-ALN conjugate (black squares) did not exhibit detectable
RBC hemolysis at all tested concentrations up to 5 mg/mL (the
estimated blood concentration after in vivo administrations is
about 0.5 mg/mL). PTX vehicle cytotoxicity is known on normal
non-proliferating cells, and indeed, a RBC hemolysis of about 8%
was observed in RBCs incubated with PTX vehicle (black diamonds).
About 5% hemolysis was observed in RBCs incubated with the
combination of PTX plus ALN as free drugs (blank squares) at the
highest equivalent to the conjugate concentration of 5 mg/mL. This
hemolysis observed is probably caused by the Cremophor EL vehicle
in which these drugs were dissolved.
[0609] Anti-Proliferative Effect of PC3 Cells:
[0610] The taxane PTX is a potent cytotoxic agent approved as first
line of therapy for metastatic breast cancer, and it is being
tested in the clinic in combination with other chemotherapeutic
agents for the treatment of metastatic prostate cancer. To evaluate
whether PTX retained its cytotoxic activity following conjugation
with PEG polymer, a proliferation assay of PC3 human prostate
adenocarcinoma cells was performed. The obtained data is presented
in FIGS. 8A and 8B. PEG-.beta.-Glutamic acid Dendron (denoted as
PEG) served as control and was found to be non-toxic at any of the
concentrations tested. The proliferation of PC3 cells was similarly
inhibited by PTX-PEG and PTX-PEG-ALN conjugates, by free PTX alone
and by a combination of free PTX and free ALN, all exhibiting an
IC.sub.50 of 25-60 nM, and indicating the PTX maintains its potent
cytotoxicity when conjugates to PEG.
[0611] ALN alone was found to be toxic only at the highest
concentration tested of 10 .mu.M, however ALN bound to PEG at
equivalent concentration was not toxic at any of the concentrations
tested.
[0612] Effect on the Migration of PC3 Human Prostate Adenocarcinoma
Cells:
[0613] The effect of the exemplary PTX-PEG, PEG-ALN and PTX-PEG-ALN
conjugates on the ability of PC3 cells to migrate towards FBS was
evaluated compared to the free drugs alone, at equivalent
concentrations, and to PEG-.beta.-Glutamic acid dendron, denoted as
PEG. The results are presented in FIG. 9 and show that migration of
PC3 incubated with both PTX-PEG and PTX-PEG-ALN conjugates and the
combination of free PTX plus ALN towards PBS was similarly
inhibited by about 70%.
[0614] Anti-Proliferative Effect of Murine 4T1 and Human MDA-MB-231
Adenocarcinoma of the Mammary Cell Lines:
[0615] To evaluate whether PTX and ALN retained their cytotoxic
activity following conjugation with PEG polymer, a proliferation
assay of 4T1 and MDA-MB-231 cells was performed. The results are
presented in FIG. 10A (for 4T1 cells) and FIG. 10B (MDA-MB-231
cells). As shown in FIG. 10A, proliferation of 4T1 cells was
inhibited in a similar manner by all PTX-containing formulations,
exhibiting an IC.sub.50 of about 10 nM, and in a similar manner
also for a combination of PTX and ALN as free drugs and for
PTX-PEG-ALN conjugate, exhibiting an IC.sub.50 of about 20 nM.
[0616] As shown in FIG. 10B, the proliferation of MDA-MB-231 cells
was also inhibited by all PTX-containing formulations, exhibiting
an IC.sub.50 of about 1 nM, and in a similar manner also for a
combination of PTX and ALN as free drugs and for PTX-PEG-ALN
conjugate, exhibiting an IC.sub.50 of about 10 nM.
[0617] The PEG-.beta.-Glutamic acid dendron, denoted as PEG, served
as control and was non-toxic at all the concentrations tested. ALN
alone was found to be toxic only at the highest concentration
tested of 10 .mu.M, however ALN bound to PEG at equivalent
concentration was not toxic at all the concentrations tested.
[0618] Anti-Angiogenic Properties:
[0619] To assess whether similarly to PTX, the conjugates described
herein possess anti-angiogenic properties, endothelial cell
proliferation, capillary-like tube formation and migration assays
were carried out on human umbilical vein endothelial cells (HUVEC).
FIG. 11A presents the effect of various concentrations of a
combination of free PTX plus ALN (blank squares), PTX (blank
triangles), ALN (blank circles), and equivalent concentrations of
PEG (black diamonds), PTX-PEG-ALN (black squares), PTX-PEG (black
triangles) and PEG-ALN (black circles) conjugates on the
proliferation of HUVEC. X-axis is presented at a logarithmic scale.
As shown in FIG. 11A, the proliferation of HUVEC was inhibited
similarly by all PTX-containing formulations, exhibiting an
IC.sub.50 of about 2 nM, and in a similar manner also for a
combination of PTX and ALN as free drugs and for PTX-PEG-ALN
conjugate, exhibiting an IC.sub.50 of about 4 nM.
[0620] FIG. 11B presents the effect of a combination of PTX and ALN
as free drugs, PTX and ALN each alone, and equivalent
concentrations of PEG, PTX-PEG-ALN, PTX-PEG and PEG-ALN conjugates
on the ability of HUVEC to migrate towards VEGF. As shown in FIG.
11B, the migration of HUVEC incubated with both PTX-PEG and
PTX-PEG-ALN conjugates and the combination of free PTX and ALN
towards VEGF was inhibited by about 80%.
[0621] Having shown that free and conjugated PTX and ALN possess
anti-angiogenic potential by inhibiting the proliferation and
migration of HUVEC, the effect of the conjugates on the ability of
HUVEC to form capillary-like tube structures on Matrigel.RTM. was
measured as being indicative of an additional crucial step in the
angiogenic cascade of events.
[0622] FIG. 12A presents representative images of capillary-like
tube structures of HUVEC seeded on Matrigel.RTM. following the
indicated treatment (scale bar represents 100 .mu.m), demonstrating
the inhibition of capillary-like tube formation by all
PTX-containing formulations (with and without ALN).
[0623] In terms of capillary length, as shown in FIG. 12B, the
combination of PTX and ALN as free drugs inhibited the formation of
tubular structures of HUVEC by about %. Both PTX-PEG and
PTX-PEG-ALN conjugates at PTX-equivalent concentrations inhibited
the formation of the tubular structures of HUVEC by about 50%.
[0624] The concentrations of treatments used in both migration and
capillary-like tube formation assays on HUVEC were tested and found
as non-cytotoxic at the indicated incubation times, but rather
specifically inhibited the ability to migrate and form
capillary-like tubes.
[0625] Overall, the in vitro studies conducted showed that PTX,
when bound with PEG, exhibited similar cytotoxicity to various cell
lines, compared with free PTX, suggesting that PTX can be released
from the conjugates and achieve similar tumor cells killing
efficacy. Inhibition of proliferation, capillary-like tube
formation, and migration of endothelial cells revealed that both
PTX-PEG and PTX-PEG-ALN conjugates possesses anti-angiogenic
properties and are as potent as the free drugs at equivalent
concentrations. The improved binding capacity to HA demonstrated
the combined targeting effect exhibited by ALN-containing
conjugates.
Example 3
In Vivo Studies
Materials and Experimental Methods
[0626] Materials:
[0627] All monoclonal antibodies were purchased from BD Biosciences
and used for flow cytometry analysis in accordance with the
manufacturer's protocols.
[0628] Primary rat anti-murine CD34 antibody (MEC 14.7) was from
Abcam, (Cambridge, Mass.). Rabbit anti-rat antibody, anti-rabbit
horseradish peroxidase-conjugated antibody (ABC detection kit) and
ImmPACT.TM. DAB diluent kit were from Vector Laboratories
(Burlingame, Calif., USA).
[0629] pEGFPLuc plasmid was from Clontech (Mountain View, Calif.,
USA). Nuclear staining was from Procount, BD Pharmingen (San Jose,
Calif., USA).
[0630] 7-aminoactinomycin D (7AAD) was from Chemicon (Billerica,
Mass.).
[0631] Dextran (MW of about 70000) and all other chemical reagents,
including salts and solvents were purchased from Sigma-Aldrich,
Israel.
[0632] PC3 human prostate adenocarcinoma, MDA-MB-231 human mammary
adenocarcinoma and 4T1 murine mammary adenocancinoma cell lines
were purchased from the American Type Culture Collection
(ATCC).
[0633] Balb/C mice were obtained from Harlan.
[0634] SCID mice were obtained from Harlan.
[0635] Human embryonic kidney 293T (HEK 293T) cells were obtained
from ATCC.
[0636] All other reagents and solvents were obtained from known
vendors.
[0637] Pharmacokinetic Studies in Mice:
[0638] Pharmacokinetics of PTX, PTX-PEG and PTX-PEG-ALN were
determined in 30 female Balb/C mice (23-25 grams). The mice were
randomly divided in three groups of 10 animals. 150 .mu.L of PTX in
1:1:8 Ethanol:Cremophor EL:Saline, PTX-PEG conjugate as described
herein in PBS pH 6 or PTX-PEG-ALN conjugate as described herein in
PBS pH 6 (dose: 10 mg/Kg PTX equiv.) were administered via tail
vein to mice anaesthetized with 5% isoflurane gas (mixed with
O.sub.2 in enclosed cages). At predetermined times, two blood
samples (150 .mu.L) were withdrawn from the retro-orbital
plexus/sinus of two animals, with a heparinized capillary, and then
centrifuged at 1,500 g for 15 minutes. To 50 .mu.L of plasma, 350
.mu.L of CH.sub.3CN was added for protein precipitation and the
resulting mixture was centrifuged at 20,000 g for 5 minutes. A 300
.mu.L aliquot of the supernatant was collected and freeze-dried.
The residue was dissolved in 50 .mu.L of CH.sub.3OH and analyzed by
RP-HPLC under the condition reported hereinabove. For PTX-PEG and
PTX-PEG-ALN conjugates, the residues after freeze-drying were also
hydrolyzed by incubation with a solution of 2% NaOH 2N as reported
above.
[0639] Generation of mCherry-Infected Human MDA-MB-231 and Murine
4T1 Mammary Adenocarcinoma Cell Lines:
[0640] mCherry was subcloned from pART7-mCherry (provided by A.
Avni from Tel Aviv University), into pQCXIP (Clontech). Human
embryonic kidney 293T (HEK 293T) cells were co-transfected with
pQC-mCherry and the compatible packaging plasmids (pMD.G.VSVG and
pGag-pol.gpt). Forty eight (48) hours following transfection, the
pQC-mCherry retroviral particles containing supernatant was
collected. 4T1 and MDA-MB-231 cells were infected with the
retroviral particles media, and 48 hours following the infection,
mCherry positive cells were selected by puromycin resistance.
[0641] Evaluation of Antitumor Activity of PTX-PEG-ALN
Conjugate:
[0642] A syngeneic mouse model of mammary adenocarcinoma was
established by injecting Balb/c female mice with 100 .mu.l of
4.times.10.sup.5 mCherry-labeled 4T1 cells intra-tibia. Therapy was
initiated one day following tumor cells inoculation. Mice were
randomly divided into 9 groups (n=6 mice/group) and intravenously
(i.v.) injected with 100 .mu.l PTX (15 mg/kg), ALN (35 mg/kg), a
combination of PTX and ALN as free drugs, PEG, and the PTX-PEG,
PEG-ALN, or PTX-PEG-ALN conjugates at equivalent concentrations.
Mice injected intravenously with commercial the PTX vehicle 1:1:8
Ethanol:Cremophor EL:Saline or saline were used as controls.
[0643] A xenograft mouse model was established by injecting female
SCID mice with 100 .mu.l of 1.times.10.sup.5 mCherry-labeled
MDA-MB-231 cells intra-tibia.
[0644] Therapy was initiated 10 days following tumor inoculation,
when most mice had fluorescent signals indicating tumors uptake.
Mice were divided into 5 groups (n=6 mice/group) and the mean
fluorescence intensity was approximately equivalent for all groups.
These groups were randomly assigned and received intravenuous
(i.v.) injections of 100 .mu.l PTX (15 mg/kg) plus ALN (35 mg/kg),
PTX (7.5 mg/kg) plus ALN (17.5 mg/kg), PTX-PEG-ALN conjugate at
equivalent concentration, and controls of PTX-vehicle, or saline.
All treatments for both mouse models were injected i.v. via the
tail vain, every other day, 5 injections. Tumor progression was
monitored by CRI.TM. Maestro non-invasive intravital imaging
system. At termination, tibias were removed and analyzed, as
described hereinbelow. Data is expressed as mean.+-.SEM.
[0645] Body Distribution of FITC Labeled PEG, PTX-PEG, PEG-ALN and
PTX-PEG-ALN Conjugates:
[0646] SCID mice bearing MDA-MB-231 tumors in the tibia were
injected i.v. (intravenously) with FITC-labeled PEG, and the
FITC-labeled PTX-PEG, PEG-ALN and PTX-PEG-ALN conjugates.
Accumulation of the conjugates in the tumor was assessed at
different time points (0, 2, 4, 6, and 8 hours) by measuring
fluorescence intensity signal. At termination (after 8 hours),
tumors, organs and bones were excised and imaged. Organs were
imaged using non-invasive imaging system (CRI Maestro.TM.)
Fluorescence was determined using defined regions of interest (ROI)
measurements on tumors and other tissues. Time dependent tumor
contrast profile was determined by the ratio between fluorescence
intensities of tumors and those of normal skin. Data were expressed
as mean.+-.standard deviation (s.d.) (n=3).
[0647] Measurement of Circulating Endothelial Cells (CEC) and
Circulating Endothelial Progenitor (CEP) by Flow Cytometry:
[0648] Blood was obtained from anaesthetized mice by retro-orbital
sinus bleeding. CEC and CEP were quantitated using flow cytometry,
as described in Shaked et al. [in Cancer Cell 2005; 7: 101-111].
Briefly, 24 hours after treatment, blood was collected in tubes
containing EDTA to avoid clotting. Monoclonal antibodies were used
to detect CEC and CEP population with the following antigenic
phenotypes: CD13+/VEGFR2+/CD45-/dim. CEP population was also
CD117+. Nuclear staining was used in some experiments to exclude
platelets or cellular debris. 7-Aminoactinomycin D (7AAD) was used
to distinguish apoptotic and dead cells from viable cells. After
red cell lysis, cell suspensions were analyzed and at least 200,000
cells per sample were acquired. Analyses were considered
informative when an adequate number of events (i.e. >50,
typically 50-150) was collected in the CEC and CEP enumeration gate
in untreated control animals.
[0649] Percentages of stained cells were determined and compared
with appropriate negative controls. Positive staining was defined
as being greater than non-specific background staining.
[0650] Flow cytometry studies were performed on Cyan ADP flow
cytometer (Beckman Coulter) and analyzed with Summit (Beckman
Coulter) software. Data is expressed as mean.+-.standard error of
the mean (SEM).
[0651] White Blood Cell (WBC) Counts:
[0652] Blood was obtained from anaesthetized mice by retro-orbital
sinus bleeding. Twenty four hours after treatment, blood was
collected in tubes containing 0.1 M EDTA to avoid clotting. Samples
were counted no longer than five minutes after blood was drawn from
mice. Ten .mu.l of blood samples were mixed with 90 .mu.l of track
solution (1% acetic acid in DDW), and cells were counted by a Z1
Coulter.RTM. Particle Counter (Beckman Coulter.TM.). Data is
expressed as mean.+-.s.e.m.
[0653] Immunohistochemistry:
[0654] Immunohistochemistry of tumors in the tibia was performed
using samples fixed with 4% paraformaldehyde, following
decalcification in EDTA and paraffin embedding by the standard
procedure. Paraffin sections of 4 .mu.m were de-paraffinized,
rehydrated, and stained by hematoxylin and eosin (H&E). For
CD34 staining, slides were deparaffinized and pre-treated with 10
mM citrate buffer, pH=6.0, for 20 minutes in a steam pressure
cooker (Decloaking Chamber, BioCare Medical, Walnut Creek,
Calif.).
[0655] All further steps were performed at room temperature in a
hydrated chamber. Slides were covered with Peroxidase Block (Merck,
Germany) for 10 minutes to quench endogenous peroxidase activity,
followed by incubation with 2% of horse serum in 50 mM Tris-HCl, pH
7.4, for 30 minutes to block non-specific binding sites. Primary
rat anti-murine CD34 antibody (MEC 14.7 1:50 dilution; Abcam,
Cambridge, Mass.) was applied in 1% rabbit serum albumin in
Tris-HCl, pH 7.4 at 4.degree. overnight. Slides were washed in 50
mM TrisHCl, pH 7.4 and rabbit anti-rat antibody (1:750 dilution;
Vector Laboratories, Calif., USA) was applied for 30 minutes.
Following further washing, immunoperoxidase staining was developed
using HistoMark TrueBlue peroxidase system (KPL, USA) per the
manufacturer instructions and counterstained with safranin.
Microvessel density (MVD) was calculated as previously described
[Weidner et al., N Engl J Med 1991; 324: 1-8].
[0656] Statistics:
[0657] In vivo data is expressed as mean.+-.s.e.m. Statistical
significance was determined using an unpaired t-test. P<0.05 was
considered statistically significant. All statistical tests were
two-sided.
Results
Pharmacokinetic Studies
[0658] Pharmacokinetics of PTX dissolved in 1:1:8 Ethanol:Cremophor
EL:Saline, and of the exemplary conjugates PTX-PEG and PTX-PEG-ALN
were determined in mice. The serum levels of PTX were evaluated by
RP-HPLC and the obtained data are presented in FIG. 14. As shown in
FIG. 14, after administration of free PTX, high levels of the drug
were recorded, however at 5 minutes post-injection, the PTX
concentration decreased dramatically, and it was not detectable at
60 minutes. On the contrary, the two conjugates showed a marked
half-life prolongation, with detectable levels of PTX after 3 hours
for PTX-PEG and after 24 hours for PTX-PEG-ALN. In particular,
elimination half-lives (T.sub.1/2.beta.) were 15.1 minutes, 77.9
minutes and 85.5 minutes for PTX, PTX-PEG and PTX-PEG-ALN,
respectively.
[0659] Table 2 below summarizes the pharmacokinetic parameters
obtained in these studies, and clearly demonstrate the prolonged
blood circulation of both the PTX-PEG and PTX-PEG-ALN
conjugate.
TABLE-US-00002 TABLE 2 PTX-PEG- Parameter PTX PTX-PEG ALN
T.sub.1/2.alpha. 1.3 .+-. 0.3 7.5 .+-. 0.6 18.9 .+-. 0.8
T.sub.1/2.beta. 15.1 .+-. 4.8 77.9 .+-. 4.0 85.5 .+-. 19.8
AUC.sup.0-.infin. (.mu.L min/mL) 31.2 .+-. 9.2 407.3 .+-. 70.7
948.4 .+-. 119.2 Clearance (mL/min) 7.4 .+-. 1.5 0.56 .+-. 0.08
0.24 .+-. 0.03 Vd (mL) 160.9 .+-. 2.1 63.4 .+-. 6.2 29.9 .+-.
5.5
[0660] As shown in Table 2, the elimination half-lives
(T.sub.1/2.beta.) of PTX-PEG and PTX-PEG-ALN were 77.9 minutes and
85.5 minutes, respectively, which is a marked prolongation with
respect to the 15.1 minutes of free PTX. Consequently, also the
area under the curve (AUC) of PTX-PEG and PTX-PEG-ALN was increased
resulting in 13-fold and 30-fold larger values than the AUC value
of free PTX, respectively.
[0661] In Vivo Tumor Accumulation and Body Distribution:
[0662] Non-invasive fluorescence imaging technology was utilized to
monitor the real-time distribution, and tumor accumulation of
FITC-labeled PEG, PTX-PEG, PEG-ALN and PTX-PEG-ALN conjugates. Mice
bearing MDA-MB-231-mCherry breast cancer tumors in the tibia were
injected i.v. with FITC-labeled conjugates. Immediately following
administration of the conjugates, mice became entirely fluorescent.
A semi-quantitative time-dependent tumor/background contrast
profile was derived from the average fluorescence intensities of
equal areas within tumor and normal skin regions and is presented
in FIG. 13A. As shown therein, the tested FITC-labeled conjugates
accumulated gradually and preferentially at tumor sites. At 8 hours
post injection, tumors and major organs were excised for ex vivo
imaging to determine tissue distribution, as presented in FIG. 13B.
As shown therein, for all of thye tested conjugates, apart for
tumors, uptake was predominant in kidney tissues due to renal
excretion. Preferred accumulation in bones was observed in the
PEG-ALN and PTX-PEG-ALN conjugates, indicating that ALN retained
its binding capacity to bone mineral.
[0663] Anti-Tumor Efficacy and Toxicity on Syngeneic 4T1-m Cherry
Murine Mammary Adenocarcinoma in the Tibia:
[0664] The antitumor effect of PTX-PEG-ALN conjugate following
intravenous injection was evaluated on syngeneic mCherry-labeled
4T1 murine mammary adenocarcinomas in the tibia. Mice were treated
with MTD of PTX and equivalent concentrations of the conjugates.
Tumor growth was monitored non-invasively using fluorescence
imaging system (CRI.TM. Maestro).
[0665] As presented in FIGS. 15A-D, a significant tumor growth
inhibition was recorded in mice treated with both PTX-PEG and
PTX-PEG-ALN conjugates. On day 15, when mice were euthanized,
PTX-PEG-ALN and PTX-PEG conjugates inhibited tumor growth by 48%
and 37%, respectively, as compared with saline treated mice (see,
FIG. 15A). Treatment with ALN was very toxic and caused severe body
weight loss and mortality within 2 injections both in free
ALN-treated mice and in mice treated with the combination of free
ALN plus PTX (see, FIG. 15C). Therefore, in these groups, tumor
progression could not be determined. In contrast to free ALN, mice
treated with PEG-ALN conjugate did not lose weight. Further body
weight loss was not recorded in any of the other treatment groups
(see, FIG. 15C).
[0666] Representative histology sections of H & E staining
through the tibia demonstrated that most of the PEG conjugates
treated-mice had intact cortical and trabecular bone (FIG. 15D).
However, in the control groups and in mice treated with PTX, tumor
filled the bone marrow space and destroyed both trabecular and
cortical bone. Increased percent necrosis was observed in control
groups of mice due to the larger size of tumors incorporating a
necrotic hypoxic core, as compared with smaller tumors and
decreased percent necrosis observed in all treated groups of
mice.
[0667] As shown in FIG. 16A, WBC counts in mice treated with the
tested conjugates or with PTX-vehicle were all at the normal range
and similar to those of control mice injected with saline. Only in
mice treated with free PTX, a significant decrease in the WBC
counts was recorded. Due to the severe toxicity effect of free ALN
and the combination of free ALN and PTX, that caused mortality
prior to day 11, no data concerning the WBC counts could be
obtained from these mice groups.
[0668] Immunohistochemical analysis of paraffin-embedded sections
of CD34 staining is presented in FIG. 16B and show a significant
reduction, of about 50%, in micro-vessel density (MVD) in mice
treated with PTX, PTX-PEG, and PTX-PEG-ALN there was PTX-PEG-ALN
conjugate, as compared to the saline-treated control group.
[0669] Following the in vitro results demonstrating the
anti-angiogenic activity of the conjugates described herein, the
effect of various treatments on CEC and CEP populations in blood
circulation in mice bearing the 4T1-mCherry adenocarcinomas in the
tibia was tested.
[0670] Viable CEP have been shown to correlate with angiogenesis. A
substantial increase in the number of viable CEP was observed in
peripheral blood of mice 24 hours after they were treated with
paclitaxel chemotherapy. Such cells were found in large numbers in
treated tumor sites, and thus may account for the induction in
angiogenesis and tumor re-growth following therapy. In addition,
apoptotic CEC are likely to represent an indirect marker of vessel
damage and/or turnover and remodeling.
[0671] Using multi-parametric flow cytometry, both apoptotic CEC
and viable CEP populations were analyzed, and the obtained data is
presented in FIGS. 17A and 17B. As shown in FIG. 17A, in all
treatments there was no difference in apoptotic CEC counts in the
blood. However, in mice treated with PTX-PEG-ALN conjugate there
was a significant increase in the apoptotic CEC counts in the
blood. As shown in FIG. 17B, an increase in viable CEP following
PTX therapy, as opposed to PTX-PEG-ALN or PTX-PEG therapy was
observed. These results provide further support for the
anti-angiogenic activity exhibited by the PTX-PEG-ALN conjugaes as
described herein.
[0672] Anti-Tumor Efficacy and Toxicity on a Xenograft Model of
MDA-MB-231-mCherry Mammary Adenocarcinoma in the Tibia:
[0673] The antitumor effect of exemplary conjugates as described
herein, following intravenous injection was also evaluated on a
xenograft mouse model of mCherry-labeled MDA-MB-231 mammary
adenocarcinomas in the tibia. The results are presented in FIGS.
18A-D and show that mice treated with the PTX-PEG-ALN conjugate
exhibited superior antitumor efficacy, 50% inhibition in tumor
growth, as compared to saline control mice (see, FIG. 18A).
[0674] As shown in FIG. 18B, the PTX-PEG-ALN conjugate did not
induce body weight loss. However, combination of MTD (minimal
therapeutic dose) of free PTX and ALN was very toxic and induced
mortality within one treatment. Treatment with half dose of the
combination of free PTX and ALN was also very toxic and caused
severe body weight loss that almost reached 20% decrease, but was
recovered after treatment withdrawal (FIG. 18B).
[0675] As shown in FIG. 18C, representative H&E staining of
paraffin-embedded sections of MDA-MB-231-mCherry tumors in the
tibia demonstrated similarly to the H&E from mCherry-4T1 tumors
that in control mice, tumor filled the bone marrow, destroyed bone,
and penetrated into soft tissues and the proximal joint. In
contrast, mice treated with PTX-PEG-ALN conjugate had intact
cortical and trabecular bone. In control mice, the tumor diffusely
invaded bone marrow with destruction of bone trabeculae. When
treated with the herein disclosed conjugate, the bones looked
normal with slight irregularity of the trabecular outlines.
[0676] As shown in FIG. 18D, an overall increased percent necrosis
due to larger size of tumors with hypoxic core was observed in
control groups of mice, as compared with smaller tumors and
decreased overall percent necrosis observed in mice treated with
PTX-PEG-ALN conjugate. On the contrary, a significant necrotic core
in the ossea medulla was observed only in mice treated with the
conjugate, whereas in control mice tumor within the ossea medulla
was viable with no necrosis observed.
[0677] WBC counts of mice treated with the conjugate or combination
of the free drugs were comparable to control mice treated with
saline or PTX-vehicle (see, FIG. 19A).
[0678] As shown in FIG. 19B, immunohistochemical analysis of
paraffin-embedded sections of CD34 staining revealed that in mice
treated with the combination of free PTX plus ALN, and the
PTX-PEG-ALN conjugate, there was a significant reduction in the
micro-vessel density (MVD) of about 73% and 54% in PTX-PEG-ALN
conjugate and the combination of free PTX plus ALN,
respectively.
[0679] FIGS. 20A and 20B present the effect of the various
treatments on the blood levels of CEC and CEP in this mice model.
It is shown therein that, as opposed to the 4T1, in MDA-MB-231
mouse model, there was a substantial increase in apoptotic CEC only
in the PTX-vehicle-treated group, when compared to the other groups
(see, FIG. 20A). In addition, similar to 4T1 tumor model, in
MDA-MB-231 tumors, a decrease in viable CEP following therapy with
the PTX-PEG-ALN conjugate, as opposed to the other treatments, was
observed, suggesting that the conjugate possesses anti-angiogenic
effect (see, FIG. 20B).
[0680] In summary, the data obtained in the in vivo studies
conducted show that an exemplary conjugate according to some
embodiments of the present invention, the PTX-PEG-ALN conjugate as
described herein, showed substantial antitumor effects in both
murine syngeneic and human xenograft mouse models. The superiority
of the conjugate is evident in its safety compared to the free
drugs. In both mouse models treatment with the combination of free
PTX plus ALN caused mortality within the first injection. Even
treatment with free PTX plus ALN at half-dose was very toxic and
caused a reduction of 20% in body weight, whereas treatment with
the PTX-PEG-ALN conjugate did not. Also, in contrast to free ALN,
mice treated with PEG-ALN conjugate did not lose weight, suggesting
that the conjugation with PEG increased the safety of ALN without
hindering its bone-targeting affinity. Without being bound by any
particular theory, it is assumed that while free ALN diffuses
through the blood vessels and affects normal healthy tissues
besides the bones, and causes toxicity, the conjugate is targeted
only to the bones.
[0681] WBC levels in mice treated with the exemplary PTX-PEG-ALN
conjugate were comparable to those in control mice, whereas mice
treated with free PTX, displayed a significant decrease in WBC
levels. These results indicate that the conjugation of PTX with PEG
and ALN decreases the toxic effect of PTX on the bone marrow.
[0682] H&E staining for both 4T1-mCherry and mCherry-MDA-MB-231
models showed intact bone in mice treated with the exemplary
PTX-PEG-ALN conjugate. However, in control mice, bones were
destroyed, and tumor penetrated into the proximal soft tissues and
the proximal joint. Although overall percent necrosis was increased
in control treated mice, as compared to mice treated with the
exemplary PTX-PEG-ALN conjugate, a specific larger necrotic area
was observed in the ossea medulla of conjugate-treated mice. These
findings suggest that the exemplary PTX-PEG-ALN conjugate, as
designed, is targeted into bone, and is active in the bones.
H&E staining of PEG-ALN-treated mice showed more preserved
bones, as compared with saline-treated control mice.
[0683] Immunohistochemical CD34 staining carried out on 4T1 and
MDA-MB-231 tumor sections showed that PTX-PEG-ALN conjugate is
directed against tumor endothelial cells and inhibits angiogenesis,
suggesting that the antitumor effect caused by the conjugates as
described herein is mediated by impairing the blood supply to the
tumor. These data is in corroboration with the in vitro
anti-angiogenic activity presented by exemplary conjugates and the
in vivo evaluation of the angiogenic cellular markers, apoptotic
CEC, and viable CEP.
[0684] Overall, these studies demonstrate the superior efficacy and
reduced toxicity exhibited by the conjugates as described herein,
particularly as compared to free PTX.
[0685] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0686] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
1913PRTArtificial sequenceExemplary pH-sensitive linking moiety
1Gly Phe Gly 1 24PRTArtificial sequenceAn example of a linker
having a Cathepsin K cleavable site 2Gly Gly Pro Xaa 1
35PRTArtificial sequenceAn example of a linker having a Cathepsin D
cleavable site 3Gly Thr Gln Phe Phe 1 5 45PRTArtificial sequenceAn
example of a linker having a Cathepsin D cleavable site 4Gly Ser
Thr Phe Phe 1 5 52PRTArtificial sequenceAn example of a linker
having a Cathepsin H cleavable site 5Leu Gly 1 67PRTArtificial
sequenceAn example of a linker having a Cathepsin L cleavable site
6Ala Phe Arg Ser Ala Ala Gln 1 5 73PRTArtificial sequenceAn example
of a linker having a legumain cleavable site 7Ala Ala Asn 1
88PRTArtificial sequenceAn example of a linker having an MMP-2 and
an MMP-9 cleavable site 8His Pro Val Gly Leu Leu Ala Arg 1 5
96PRTArtificial sequenceAn example of a linker having an MMP-2 and
an MMP-9 cleavable site 9Pro Val Ser Leu Ser Tyr 1 5
108PRTArtificial sequenceAn example of a linker having an MMP-2 and
an MMP-9 cleavable site 10Gly Pro Val Gly Leu Ile Gly Lys 1 5
112PRTArtificial sequenceAn example of a linker having a Cathepsin
B cleavable site 11Xaa Val 1 122PRTArtificial sequenceAn example of
a linker having a Cathepsin B cleavable site 12Arg Arg 1
132PRTArtificial sequenceAn example of a linker having a Cathepsin
B cleavable site 13Phe Lys 1 144PRTArtificial sequenceAn example of
a linker having a Cathepsin B cleavable site 14Gly Phe Leu Gly 1
154PRTArtificial sequenceAn example of a linker having a Cathepsin
B cleavable site 15Gly Phe Ala Leu 1 164PRTArtificial sequenceAn
example of a linker having a Cathepsin B cleavable site 16Ala Leu
Ala Leu 1 173PRTArtificial sequenceAn example of a linker having a
Cathepsin B cleavable site 17Gly Leu Gly 1 183PRTArtificial
sequenceAn example of a linker having a Cathepsin B cleavable site
18Gly Phe Gly 1 196PRTArtificial sequenceAn example of a linker
having a Cathepsin B cleavable site 19Gly Phe Leu Gly Phe Lys 1
5
* * * * *