Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Therapeutic Uses of Peroxometallic Compounds
FIELD OF THE INVENTION
This invention relates to the use of peroxometallic compounds, such as
potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(phen)],
potassium bisperoxo(pyridine-2-carboxylato)oxovanadate [bpV(pic)] and
potassium bisperoxo(2,2'-bipyridyl) oxovanadate [bpV(bipy)], for preventing
angiogenesis, restenosis and the production of endothelins, and as adjuvants
for vaccination. The compounds of the present invention are also suitable as
antitumorigenic agents; for example, experimental results reveal that they are
efficacious in the treatment of breast cancer and prostate cancer.
BACKGROUND OF THE INVENTION
A) PEROXOVANADIUM COMPOUNDS:
Synthetic peroxovanadium (pV) compounds are structurally versatile
molecules which are potent inhibitors of phosphotyrosyl phosphatases
(PTPs) (1 ). These compounds contain one oxo ligand, one or two peroxo
groups, one ancillary ligand, all coordinated to vanadium. They are stable in
aqueous solution at physiological pH when shielded from light. pVs are
cytostatic agents, unlike many other antitumor molecules. Their mode of
. action lies in the modulation of the activity of cellular transduction
pathways
involved in the progression of pathological conditions. Their effects are
transitory and disappear within a few days after administration(2).
Phosphotyrosine phosphatases (PTPs) are enzymes which remove
phosphates from tyrosine residues of proteins. They are involved in several
cell functions regulating proliferation, differentiation and metabolism. Their
number is estimated at about 100 in the human genome (3). These enzymes
function by engulfing in their catalytic site phosphates located on the
tyrosine
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residues of target proteins. The mechanisms underlying the inhibition of
PTPs and the specificity of these peroxo-anionic compounds have been
characterized. The inhibiting potential of PTPs by pVs is a 100 to a 1000
times more powerful than that of oxovanadate (1 ). When compared to known
inhibitors of PTPs such as orthovanadate, molybdate, tungstate and zinc, the
increased inhibiting potential of pV can be explained by the presence of the
peroxide groups, which have the ability to irreversibly oxidize an essential
conserved cysteine residue located in the catalytic domain of practically all
PTPs (4).
The possibility of manipulating the ancillary ligands of pVs is important in
regulating potency and specificity (1 ). The ancillary ligands are more or
less
hydrophilic or hydrophobic and provide the molecule with a specific mode of
action and distribution for the different PTPs. These ligands allow for the
specific targeting of certain PTPs.
B) ANGIOGENESIS:
Almost all tissues and organs develop a vascular network which provides
cells with nutrients and oxygen and enables the elimination of metabolic
waste. Once formed, the vascular network is a stable system with a slow rate
of cellular turn over (5) and yet, the endothelium can become one of the most
rapidly proliferating of all cell types when stimulated (6). Indeed, the
formation of new blood vessels can cause serious physiological
complications. For example, while cornea and cartilage are avascular in
healthy situations, several diseases involving these tissues are complicated
by the massive arrival of blood vessels. Eye angiogenic diseases include
neovascular glaucoma, retrolental fibroplasia, macular degeneration and
neovascularization of corneal grafts. Joint angiogenic diseases include
rheumatoid arthritis and arthrosis. Psoriasis, a chronic condition of the
skin,
also exhibits hypervascularization at the surface of the skin. Finally, solid
tumor growth is critically dependent upon the formation of new blood vessels
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to progress locally and spread all over the body (7,8). The maintenance of
existing blood vessels also requires the regulation of cell replication and/or
differentiation. For example, acute and chronic pathological processes, such
as atherosclerosis, post-angioplastic restenosis and hypertension, involve the
proliferation of different cellular components of mature blood vessels
(endothelial cells, smooth muscle cells, myocytes and fibroblasts).
Several factors, including certain peptides and proteins, can induce a
vascular response in vivo. They are endogenous substances, such as
EGF/TGF-a (Epidermal Growth FactorlTransforming Growth Factor-alpha),
TGF-~3 (Transforming Growth Factor-beta), TNF-a (Tumor Necrosis Factor-
alpha), angiogenin, prostaglandine E2, and monobutyrine (9-16). However,
these factors have almost no mitogenic effect on endothelial cells in culture
(TGF-a, EGF, angiogenine, prostaglandine E2, monobutyrine) and,
paradoxically, inhibit their growth (TGF-~, TNF-a) (10-17). Their angiogenic
action is thus indirect, depending for the most part upon the inflammatory
response (17). Inflammatory cells produce some factors, such as aFGF
(acidic Fibroblast Growth Factor), bFGF (basic Fibroblast Growth Factor),
PDGF (Platelet-Derived Growth Factor), and VEGF (Vascular Endothelial
Growth Factor), which are capable of stimulating the proliferation of
endothelial cells in vitro and angiogenesis in vivo (17-27).
The angiogenic process, as currently understood, can be summarized as
follows: a cell activated by a mutation, lack of oxygen, etc, releases
angiogenic molecules (28-33) that attract inflammatory and endothelial cells
and promote their proliferation. Following the binding of leukocytes to
vascular endothelial cells, the latter reorganize the protein arrangements on
their membranes to activate the angiogenic process (34, 35). During
migration to the target tissue, inflammatory cells release substances that
intensify the angiogenic effect (36, 37). Activated vascular endothelial cells
respond to the angiogenic signal by secreting proteases which digest blood
vessel walls to enable migration toward the target tissue (38-40). Several
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protein fragments produced by the digestion of the blood-vessel walls
intensify the proliferative and migratory activity of the endothelial cells
(41-
43). Finally, the endothelial cells rearrange their adhesive membrane proteins
to generate the formation of capillary tubes.
S
Angiogenesis is thus a complex process consisting of several critical cellular
events (44-46), among which the following may be readily identified:
~ binding of leukocytes to endothelial cells and induction;
~ migration of inflammatory cells to the target tissue;
~ regression of the pericytes of the existing vascular system;
~ dissolution of blood-vessel walls by proteases;
~ endothelial cell migration;
~ endothelial cell proliferation;
~ endothelial cell differentiation and arrangement into a tubular shape;
1 S ~ formation of the capillary network;
~ anastomosis; and
~ initiation of blood flow.
Agents that are known to induce the proliferation of endothelial cells include
sodium orthovanadate (47). The mechanism by which this agent induces an
invasive phenotype in capillary endothelial cells is not clearly understood.
However, the effects of vanadate on cultured cells are similar, in many
respects, to those elicited by PMA (48), bFGF (49), and certain retroviral
transforming proteins, which act by inducing tyrosine-specific protein
phosphorylation (50-53). Consistent with these observations is the finding
that both phorbol ester and various growth factors including bFGF, VEGF
and PDGF stimulate the phosphorylation of cellular proteins on tyrosine
residues whereas vanadate, perhaps by inhibiting tyrosine phosphatase(s),
produces a marked increase in tyrosine phosphorylation.
pVs were expected to promote endothelial cell proliferation, like vanadate.
On the contrary, these agents have been shown to inhibit the proliferation of
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several cell types (2). In some cases, the cells are arrested at G2/M. The
simplest hypothesis to explain this restriction is that PTPs controlling cell
mitosis are targets for bpV(phen) and pbV (pic) inhibitors. The cdc25 protein
was proposed as one candidate target for pV compounds (2).
5
International Patent Publication WO 95/19177 teaches the use of vanadate
compounds for the treatment of proliferative disorders, metastasis and drug-
resistant tumors. Although these vanadate compounds are stated to be anti-
proliferative and anti-collagenolytic, no indication of any anti-angiogenic
activity has been ascribed to them. This publication further shows that an
anti-tumor effect is observed at dosages of vanadate higher than 5 mM. It is
admitted that a concentration of vanadate compound of 1.3 mM or lower has
no apparent anti-tumor effect.
Montesano et al. (47) teach, on the contrary, that vanadate compounds
cause endothelial cells to proliferate. Hence, their findings would indicate
that
these compounds are pro-angiogenic and not anti-angiogenic.
US Patent 5,716,981 (Hunter et al.) mentions the use of vanadium
compounds, namely oxovanadate, orthovanadate and vanadyl compounds,
in anti-angiogenic applications. However, this reference provides no
experimental evidence to substantiate the anti-angiogenic effects of these
compounds. The compounds are stated to be possibly equivalent to
Paclitaxel (an anti-angiogenic compound described in detail). Since the
remaining body of the prior art suggests that vanadium compounds are pro-
angiogenic, there is no enabling teaching in this patent to validate the use
of
these compounds as anti-angiogenics.
pVs are more powerful anti-tumor molecules than oxo compounds, such as
vanadate. The direct antiproliferative activity of pVs on transformed cells is
known (2). At concentrations found to be ineffective for vanadate
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compounds in International Patent Publication WO 95119177, pV compounds
are efficacious anti-tumor compounds.
Although the anti-tumor activity ascribed to cytotoxicity of vanadium
S compounds is known, their anti-angiogenic activity has not heretofore been
disclosed in relation to either oxo or peroxo derivatives thereof.
C) ENDOTHELINS:
Endothelins (ETs), a family of three isopeptides, acting as important
regulators of the physiological state of mature blood vessels. They are the
most potent vasoconstrictors identified to date. In addition, they are known
to
stimulate the proliferation of endothelial cells, smooth muscles, myocytes and
fibroblasts, as well as the synthesis of various growth factors, including
1S VEGF (54, 55).
ETs are also considered to be angiogenic factors involved in tumor
development (56). Most tumor cells synthesize and secrete ETs (57-61 ).
Patients affected by different cancers show elevated blood concentrations of
ETs (62-64). A reduction in the expression of ET receptor type B (RETg)
decreases the growth of tumor cells incubated in the presence of ETs (64).
As indicated above, ETs promote proliferation and migration of endothelial
cells (65). Expression of ET ligands and of ET receptors (RETA and RETg)
was observed in the endothelial cells of a plurality of tumors (66, 67). ETs
act
2S in an autocrine fashion, promoting local angiogenesis (67). ETs are further
involved in hemodynamic changes that go along with metastatic
development. For example, the ratio of arterial hepatic blood flow to portal
vein blood flow is abnormally high in patients having hepatic metastasis from
colorectal tumors (69). This high blood flow ratio is due to the presence of a
humoral mediator as demonstrated in vivo (70).
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A tumoral vascular bed has no innervation and consequently does not
respond to vasoconstrictive drugs. However, these drugs decrease normal
hepatic blood flow and increase blood flow in tumors. Since ETs are potent
vasoconstrictive and angiogenic factors involved in vascular remodeling and
tumor development, they may be responsible for these altered
hemodynamics. An ET inhibitor may therefore be a valuable tool for
controlling intratumor blood flow and for influencing the growth and
degeneration of tumors.
. D) ANGIOPLASTY:
ETs are also known to stimulate the production of extracellular matrices by
endothelial cells (71, 72). That is frequently observed upon vascular
reconstruction or angioplasty (73, 74). This effect is particularly
deleterious
following vascular trauma due to restenosis. In 30-50% of patients,
restenosis is characterized by the reexpansion of atherosclerotic lesions. The
causes of this vascular disorder are due to a local vascular blockage caused
by cell proliferation, cell migration and extracellular matrix production.
Recently, ETs have been shown to be major players in vascular remodelling,
particularly in such conditions as long term atherosclerosis, cardiac
hypertrophy (congestive heart failure), hypertension (pulmonary and other),
renal problems and certain systemic dysfunctions (75-79). ETs are also
strongly involved in coronary and brain vasospasm leading to ischemia and a
reduced rate of survival (80, 81 ). Thus, because of the potent
vasoconstrictor
and nitogenic activities of ETs, inhibitors of ET production would be expected
to be generally useful as anti-hypertensive and anti-proliferative agents, and
particularly useful prior to, during and after vascular surgery.
E) IMMUNE RESPONSE:
To develop an effective cell-mediated immune response, infected
macrophages (MO) must be able to induce T lymphocyte activation in a
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specific manner. In turn, T cells, by secreting several lymphokines, can
activate M0 for cytocidal functions (82, 84). Several investigators have
demonstrated the importance of cytokines in the control of Leishmania
infection (85-90). Thus, the integrity of M0-T lymphocyte interactions is a
S primordial step for the immune response development, and MQl participation
is crucial to its initiation and support. Tyrosine phosphorylation is a common
event in the initiation of cell proliferation, and its role in signal
transduction
regulating cellular functions of nonproliferative haematopoietic cells is also
well documented (91-97). We have recently demonstrated the importance of
PTPs in NO regulation (98). Correlation between M0 PTP inhibition and an
enhancement of NO production was supported by an increase in M0 PTK
activity and tyrosyl residue hyperphosphorylation (78).
It has been demonstrated that the modulation of protein tyrosyl
1 S phosphorylation states will, in many cases, result in better responses
from
some cells to extracellular stimuli (98, 99-101 ). PTP can play a crucial role
in
the negative regulation of signal transduction culminating in T-lymphocyte
activation. Prior studies have shown that pervanadate (a mixture of vanadate
and hydrogen peroxide) stimulates transcription of the c-fos gene and
accumulation of its mRNA as well as the expression of CD69 antigen and
CD25 (101 ). Also, it was demonstrated that bpV(phen) could induce nucleus
NF-kB translocation in T lymphocyte (102) and increase MO WOS mRNA
expression (98). Rat kupffer cells in culture stimulated with platelet-
activating
factor and vanadate have shown a time- and concentration-dependent
increase in phosphotyrosine in several proteins and of Prostaglandin E2
generation (99). As mentioned earlier, we recently demonstrated that M0
pretreated with bpV(phen) were more responsive to IFNy stimulation as
reflected by the greater amount of NO produced in comparison to Vanadate-
treated and untreated cells (98). Although there is evidence that suggests
that bpV(phen) can be a powerful activator of some immune functions, the
capacity of either oxo or peroxo derivatives to induce cytokines (e.g. IFN(,
IL-
12 and IL-1) and chemokines (e.g. RANTES, MIP-1a,a, MIP-2, IP-10, MCP-
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1 ) recognized for their pivotal role in inflammatory response and the
development of an effective immune response toward a specific antigen, and
thus acting as an adjuvant, has not heretofore been disclosed.
SUMMARY OF THE INVENTION
The present invention provides peroxometallic compounds, such as (pVs),
that are useful against angiogenesis, restenosis and endothelin production,
and as immunomodulators. They have also been found to be suitable for
preventing further growth of established tumors. These molecules comprise
a transition metal (such as vanadium, molybdenum, tungsten) and one oxo or
peroxo groups. Molecules comprising peroxo groups are the more potent
anti-angiogenic molecules. Preferably, the molecules also contain an
ancillary ligand, which includes any molecule capable of binding the
transition
metal atom (usually, through bonds involving oxygen and nitrogen).
Phenanthroline, picolinic acid, bipyridine, oxalic acid, 4,7-dimethyl-
phenanthroline and peptides are examples of such ligands.
All these molecules can be used to inhibit the formation of new blood vessels
and/or control systemic and local levels of endothelins (ETs) in the
reparation
of existing blood vessels.
The molecules containing peroxo anions are more powerful than their oxo
counterparts. Therefore, the former can be used at much lower
concentrations to reduce the toxicity that results from overexposure to
transition metals (2).
Oxo transition metal complexes include oxo complexes such as vanadate,
tungstate, molybdate, and vanadyl complexes, such as the following:
methavanadate (V03-), orthovanadate (V043-), salts thereof, vanadyl
compounds (V02+) like vanadyl acetyl acetonate and vanadyl sulfate.
Similar complexes exist for other transition metals. Other suitable tungsten
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and molybdenum complexes include hydroxo derivatives derived from
glycerol, tartaric acid and sugars, for example. The peroxo transition metal
complexes include any oxidizing agent capable of combining with the
transition metal. As such, the preferred peroxides comprise the following: t-
5 butylhydroperoxide, benzoyl peroxide, m-chloroperoxibenzoic acid, cumene
hydroperoxide, peracetic acid, hydroperoxiloneic acid, ethyl peroxide,
pyridine peroxide and hydrogen peroxide.
The general structure of the compounds of the present invention is the
10 following:
Y
Z T Z'
L L'
wherein: T is a transition metal selected from the group consisting of
vanadium, molybdenum, tungsten;
Y is oxygen or hydroxyl;
Z and Z' are independently selected from oxygen and peroxide and at least
one of them is peroxide; and
L and L' are any group which can donate an electron pair.
In a preferred embodiment, the transition metal T is vanadium, Y is oxygen, Z
and Z' are peroxide and L and L' are the nitrogen atoms of 1,10-
phenanthroline.
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In another preferred embodiment, the transition metal T is vanadium, Y is
oxygen, Z and Z' are peroxide and L and L' are nitrogen or oxygen atoms of
picolinic acid.
S In yet another preferred embodiment, the transition metal T is vanadium, Y
is
oxygen, Z and Z' are peroxide and L and L' are nitrogen atoms of 2,2'-
bipyridine.
The above pV compounds are potent anti-angiogenics, since they inhibit
endothelial cell proliferation. They further inhibit neovascularization and
the
production of endothelins.
Moreover, administration of the pV compound bpV(bipy) in the rat model
revealed a significant reduction in the degree of post-angioplastic vascular
remodeling of the carotid artery. Additionally, the pV compound bpV(phen)
was found to be a potent immunomodulator based on its capacity to induce
cytokine and chemokine gene expression, to enhance cellular recruitment in
response to an agonist and consequently act as an adjuvant in the context of
vaccination.
In one specific embodiment, the present invention relates to the inhibiting
action on tumor growth of bpV(phen), demonstrating efficiency in vivo. In
addition it is shown that bpV(phen) has also the capacity to inhibit the
migration of tumor cells in vitro.
Other objects, advantages and features of the present invention will become
apparent upon reading the following non-restrictive description of preferred
embodiments thereof, with references to the accompanying drawings.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENT
INVENTION
This invention will now be described by referring to specific embodiments and
the appended Figures, which purpose is to illustrate the invention rather than
to limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Peroxovanadium compounds inhibit the proliferation of endothelial
cells. Human umbilical vein endothelial cells (HUVECs) were extracted with
collagenase-controlled digestion. Pure HUVECs were used before the fourth
passage (trypsin-EDTA at each passage). The cells were analyzed for their
capacity to incorporate di-acetyl LDL and to be labelled with factor VIII-
related antigen. Endothelial cells were plated at a density of 2500 cells/cm2
in a sterile plate coated with gelatin. Cells were cultured in complete medium
(M199: heparin (90mg/ml) or L-glutamine (2mM), bicarbonate, FBS (20%)
and ECGS (100mg/ml) for 24 hours to ensure cell adhesion. Then, cells
were washed 3 times with PBS and culture medium was added according to
experimental conditions. The last PBS wash was considered as time t=0..
Cell proliferation was evaluated with the amount of DNA present in the petri
dishes. Each experiment was performed in triplicate. The culture medium
was changed daily. After 96 hours in culture, cells were lysed with Na-
Citrate-SDS solution and incubated with Hoescht 33358. Samples were read
at 365 nm with a spectrofluorometer. The results show a dose-response
inhibition of endothelial cell proliferation with the pV compounds. The
approximate IC5o is 2mM for bpV(phen) and 3.5 mM for bpV(pic).
Figure 2: A and B show an anti-angiogenic response to bpV(phen),
orthovanadate (Van) and protamine (Prot) using a vascularization test on the
vitelline membrane of chick embryos. Each point represents a minimum of 15
embryos (15 to 28). In Figure 2A), neovascularization was assessed using
N/+ scoring system; the proportion of embryos showing anti-angiogenesis
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increased with dosage, bpV(phen) being the most potent agent. In Figure 2B,
neovascularization was assessed using the 1-2-3 scoring system; the
angiogenic score decreased with dosage, bpV(phen) being the most potent
agent.
Figure 3: Micrographs of the cell migration from aortic rings embedded in
fibrin (x12). Compared to the sample control (A), cell migration was impaired
in the presence of doses of bpV(phen) (1, 3.5 NM; B, C) with a strong
inhibition at 3.5 NM (C). In the presence of bpV(pic) (3.5, 10, 25 NM ; D, E,
F),
inhibition was seen at 10-25NM. In the presence of orthovanadate, partial
inhibition was reached at 25 NM (G) with no effect at 3.5 NM (H). The
migration of well-defined microvessels in the presence of bpV(pic) is evident
at 10 NM, while in the control sample very thin cell strands were frequently
observed.
Figure 4: Quantification of the cell migration from aortic rings in the
presence
of orthovanadate (plain bars); bpV(phen) (light gray bars); and bpV(pic) (dark
gray bars). The control appears as an empty bar. Means and standard errors
of the means are presented. The Student-Newman-Keuls method was used
for statistical analysis (p value=0.001 )
Figure 5: bpV(phen) inhibits angiogenesis in vivo in the s.c. Matrigel assay.
Graph showing the results of an experiment expressed as the number of
cells that migrated in three microscopic fields for each s.c. Matrigel plug.
Fields were equidistant from the edge of the plug. (1 ) Four mice that did not
receive any treatment after the Matrigel plug implantation. (2) Four mice that
received daily administration of 10 Ng bpV(phen) during the 7 days post-
implantation. (3) Four mice that received daily administration of 50 Ng
bpV(phen) during the 7 days post-implantation. (4) Four mice that received
daily administration of 100 Ng bpV(phen) during the 7 days post-implantation.
(5) Four mice that received daily administration of PBS during the 7 days
post-implantation.
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Figure 6: Neutrophils, eosinophils and MQ~ recruited in air pouch exudates of
bpV(phen)-treated BALB/c mice in response to L. major promastigotes intra-
pouch injection. Air pouch exudates were collected from animals injected
with endotoxin-free PBS, LPS (20 ~g/ml), L. major and bpV(phen) (500 nM
S i.p., 2 hour) + L. major, 6 hr post-inoculation. Each time point represents
the
mean ~ SD (n= 4 mice) of 2 experiments similarly performed. Differences
observed for specific leukocytes population recruited were significant (p <
0.01, Student's t test) over their respective control.
Figure 7: In vivo Leishmania-induced pro-inflammatory mediator generations
in pV-treated mice. (A) Nitrate level determination in air pouch exudates
collected 6 hr post-inoculation of endotoxin-free PBS, L. major and
bpV(phen) followed by L. major has been performed. Values are the mean ~
SEM of 2 experiments performed independently. (B) Evaluation of pro-
inflammatory cytokines (IL-6 and IL-1(3) and chemokines (MCP-1 and MIP-2)
secreted in air pouch exudates collected as described above were measured
by ELISA as described in more detail below. Values are the mean ~ SEM of
2 experiments performed independently. Levels of pro-inflammatory
molecules measured in exudate supernatants were significantly augmented
(p < 0.01, Student's t test) in response to L. major infection and
significantly
further increased by bpV(phen) treatment over their respective control. IL-6,
IL-1[3, MCP-1 and MIP-2 secretion were respectively 12.8-; 2-, 8- and 6.8-
time more secreted in L. major -injected bpV(phen)-treated animals
compared to L. major alone. PBS control was equal to background.
Figure 8: In vivo bpV(phen)-induced cytokine gene expression in spleen of
BALB/c mice. Expression of Th1 (IFN-(, IL-2, IL-12) and Th2 (IL-4 and IL-10)
cytokines were monitored in spleen of BALB/c mice in response to bpV(phen)
over a 24-hr period. Expression of cytokine genes has been monitored using
a multi-probe RNase protection assay as described in more detail below.
Results obtained are representative of 3 experiments performed
independently.
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Figure 9: Effect of bpV(phen) on chemokine mRNA expression in murine
B10R macrophages. (A) Cells were incubated for 2 h with or without various
concentrations of bpV(phen) (1-50 pM). Total RNA was isolated and
analyzed by RNase protection. (B) Kinetic expression of chemokine genes in
5 murine B10R macrophages. Cells were incubated for increasing period of
time (1-4 h) with or without bpV(phen) at 10 NM. Following extraction of total
RNA, RNase protection assay was performed. Free RNA probe is shown in
the far left lane. The fold increase was calculated from the densitometry of
the autoradiogam.
Figure 10: Use of bpV(phen) as an adjuvant in Leishmania vaccination trial.
BALB/c mice were inoculated with soluble Leishmania major antigen (SLA;
100 Ng) with or without daily injection of bpV(phen) (500 NM) for 5 days. Two
weeks later animals received a second set of treatment as above. Control
animals received either PBS or bpV(phen) alone without any SLA. At 4
weeks post-vaccination, animals were challenged with 5 million Leishmania
major stationary phase promastigotes injected in the right hind footpad.
Progression of infection was followed by measuring footpad thickness with a
caliper on a weekly basis. Differences measured for reduced footpad
thickness were significant (p < 0.01, Student's f test, n=5-10 animals per
group) over their respective controls.
Figure 11: Illustration of the antitumor activity of bpV(phen) in vitro using
PC3 prostate cancer cells. Collagen gels containing the PC3 cells were
prepared according to the method of Esdale and Bard (1972). Briefly, stock
collagen solution (3.5mg/ml in acetic acid 0.02N; Rat tail) was added to a
mixture composed of culture medium (5x), fetal bovine serum (FBS),
bicarbonate (0.26M), and was neutralized with 0.1 N NaOH. A cell suspension
(1.42 x 106 cells/ml) was mixed into the collagen-medium mixture to obtain a
final concentration of 2.5x105 cells/ml.
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A DMEM medium, having a normal concentration of glucose and without
phenol red, was used. After gelification (within 1 h) of the collagen mixture
containing the cells, the gels were removed from their culture wells (mould)
and interwoven into a receptor hole prepared in a fibrin gel, as previously
described (103). The fibrin gel was made from a 0.3 % fibrinogen solution in
Hank's balanced salt solution. Fibrin was allowed to polymerize with thrombin
(stock solution at 1.75mg/ml) at a ratio of 1:003 v/v fibrin to thrombin. The
collagen-fibrin complexes were then covered with serum-supplemented
medium according to the cell types. An inhibitor of plasminogen activator
(Trasylol, Parke Davies) was added into the medium at 10 pl/ml (100UIml).
Cell behavior was periodically monitored over 15 days of culture.
In this model, collagen is believed to mimic the tumor stroma, and fibrin is
well recognized as the primary matrix for cancer cell expansion and migration
(104). bpV(phen) was used at 2, 5 and 10 ~M; the compound was renewed
daily over a 2 week period. Phase contrast microscopic observations and
micrographs were taken after 15 days, and samples were prepared for
histological sections. Histological sections were stained with periodic acid
Shiff stain to enhance the matrix contrast.
Figure 12: Illustration of the antitumor activity of bpV(phen) in vitro using
ZR-
75 breast cancer cells. Experiments were done as described for the figure
11 except that DMEM medium having a high glucose concentration and
without phenol red was used. 10% FBS was used instead of 5% FBS. The
media was supplemented with 109 M estradiol (final concentration).
Figure 13: Figures 13A and 13B illustrates the antitumor activity of
bpV(phen) in vivo.
Figure 14: In vitro antitumor activity of lymphocytes pretreated with
bpV(phen). PC-3 cancer cells embedded in a collagen gel, grew as a
"primary tumor". Some cells migrated from the primary tumor toward the
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fibrin gel, forming front edges that can be quantified. Small clumps of cells
progressively appear in the fibrin gel and we assume that these cell
extension are representative of the invasive potential of the cancer cells. In
the control panel (left), PC-3 cells migrated slightly from the primary tumor
and formed extensive secondary tumors in the fibrin gel. In the presence of
bpV(phen)-treated lymphocytes no secondary tumor was observed and there
was no migration front. In addition, the primary tumors appeared less dense
than in the control.
Definitions
In order to provide a clear and consistent understanding of terms used in the
present description, a number of definitions are provided hereinbelow.
Unless defined otherwise, the scientific and technological terms and
nomenclature used herein have the same meaning as commonly understood
by a person of ordinary skill to which this invention pertains.
For the purposes of the present application, the term "animal" is meant to
signify human beings, primates, domestic animals (such as horses, cows,
pigs, goats, sheep, cats, dogs, guinea pigs, mice, birds, fish etc.).
From the specification and appended claims, the term "therapeutic agent"
should be taken in a broad sense so as to also include a combination of at
least two such therapeutic agents.
For administration to humans, the prescribing medical professional will
ultimately determine the appropriate form and dosage for a given patient, and
this can be expected to vary according to the chosen therapeutic regimen,
the response and condition of the patient as well as the severity of the
disease.
CA 02386759 2002-05-17
18
Compositions within the scope of the present invention should contain the
active agent (e.g. compound) in an amount effective to achieve the desired
therapeutic effect while avoiding adverse side effects. Typically, the
compounds of the present invention can be administered to mammals (e.g.
S humans) in doses ranging from 0.001 to 50 mg per kg of body weight per day
of the mammal which is treated. Pharmaceutically acceptable preparations
and salts of the active agent are within the scope of the present invention
and
are well known in the art (Remington's Pharmaceutical Science, 16th Ed.,
Mack Ed.). The dosage will be adapted by the clinician in accordance with
conventional factors such as the extent of the disease and different
parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be
administered to the mammal.
A) PEROXOVANADIUM COMPOUNDS and ANGIOGENESIS
1. Peroxovanadium compounds inhibit the proliferation of endothelial
cells and the formation of tubular structures
Human umbilical vein endothelial cells (HUVECs) were extracted with
collagenase-controlled digestion, as previously described (105). Pure
HUVECs were used before the fourth passage (trypsin-EDTA at each
passage). The cells were analyzed for their capacity to incorporate di-acetyl
LDL and to be labelled with factor VIII-related antigen.
Endothelial cells were plated at a density of 2500 cells/cm2 in a sterile
plate
coated with gelatin. Cells were cultured in complete medium (M199: heparin
(90 ~g/ml) or L-glutamine (2mM), bicarbonate, FBS (20%) and ECGS
(100 ~g/ml)) for 24 hours to ensure cell adhesion. Then, cells were washed 3
times with PBS and culture medium was added according to experimental
conditions. The last PBS wash was considered as time t=0.
Cell proliferation was evaluated with the amount of DNA present in the petri
dishes. Each experiment was performed in triplicate. The culture medium
CA 02386759 2002-05-17
19
was changed daily. After 96 hours in culture, cells were lysed with Na-Citrate-
SDS solution and incubated with Hoescht 33358. Samples were read at 365
nm with a spectrofluorometer.
S The results show a dose-response inhibition of endothelial cell
proliferation
with the pV compounds (Figure 1 ). The approximate IC5o is 2 ~M for
bpV(phen) and 3.5 pM for bpV(pic).
Alternatively, HUVECs cultured in a fibrin matrix can form 3-dimensional
tubular-like structures in the presence of serum (103). This assay was
performed in the presence of either bpV(phen), bpV(pic) or vanadate to
assess the influence of each one of these compounds on HUVEC
differentiation and organization (Table 1 ). HUVECs were seeded on the
bottom of gelatin-coated wells at high density to provide a confluent
monolayer at 48 hours.
Then, 50 000 HUVECs/ml were embedded in fibrinogen solution prior to
polymerization. The fibrin matrix was covered with culture medium containing
the molecule to be tested, while culture medium without any of these
molecules served as controls. Cell behavior was observed periodically by
phase contrast microscopy. After 21 days in culture in the presence of 1 ~M
bpV(phen), cord-like structures (or cords), tube-like structures (or tubes)
and
stellate structures (or Stell.struct.) were observed. At higher doses (2 and
3.5
uM), fragmented cord-like structures were apparent. In the presence of
bpV(pic), cords and tubes were observed at 1, 2 and 3.5 pM. In the presence
of orthovanadate, cords were still apparent at 10 ~M, and at 25 pM dead
cells were sparsely distributed. These results suggest that pV compounds
interfere with endothelial cell organization and terminal differentiation.
Furthermore, the nature of the ancillary ligand is important since bpV(phen)
is
more potent in inhibiting tube formation than bpV(pic).
CA 02386759 2002-05-17
The results show that vanadate has an anti-angiogenic effect inasmuch as
the stellate structures are affected (organization and terminal
differentiation).
Vanadate is at least 3 and 5 times less potent than bpV(pic) and bpV(phen),
respectively. The anti-angiogenic effect observed with vanadate is in
5 contradiction to the teachings of Montesano (47).
Indeed, it was observed that at low doses, all the tested vanadium
compounds seemed to be pro-angiogenic, while at higher doses (i.e., at
almost double the dose), the same compounds were clearly anti-angiogenic.
10 The type of radical L, L' greatly affects the potency of the vanadium
compounds; for example, phenanthroline was found to be twice as potent as
picolinic acid.
CA 02386759 2002-05-17
21
TABLE I: Effects of bpV(phen)i, bpVi(pic) and Vanadate on HUVEC
Differentiation and Orgianization
Doses Stell.
(uM) cords tubes struct.
bpV 0 +++ +++ +++
(phen)
+++ +++ +++
++ + _
3.5 - - -
bpV (pic)0 +++ +++ +++
+++ +++ +++
2 +++ +++ +++
3.5 ++ + _
van 0 +++ +++ +++
2.5 +++ +++ +++
10 +++ +++
25 t 1'
t Dead cells
CA 02386759 2002-05-17
22
2- Rat aortic ringassay
Rat aorta rings were embedded in fibrin matrix as previously described (107).
Thoracic aortas excised from adult rats were rinsed in Hank's balanced salt
solution (HBSS), and cleansed of periadventitial. One-mm long aortic rings
were sectioned and rinsed in culture medium. Each of the aortic rings were
then embedded in fibrin gel matrix. Migration of microvessels in fibrin gel
was
quantified by measuring the distance from the external surface of rat aortic
rings towards the migrating vessels. Measurements were performed at day
by digitizing morphometry with a NIH image analysis system (Fig. 3 and
10 4).
3- Peroxovanadium compounds inhibit neovascularization ex ovo
a. Definition of the test-s ski tem:
The normal development of a chick embryo involves the formation of an
external vascular system which is located in the vitelline membrane and
15 which carries nutrients from the vitellus (yolk of the egg) to the
developing
embryo. When placed onto the vitelline membrane, anti-angiogenic
substances can inhibit blood vessel development in the vitelline membrane.
To facilitate access to the vitelline membrane, chick embryos were
transferred to a sterile culture box (Petri dish) and placed in a humidity-
and
temperature-controlled incubator. Embryos could then develop in this ex ovo
condition for several days.
An aliquot of the tested compound was mixed with a methylcellulose solution
and allowed to air-dry into thin discs. Methylcellulose forms an inert matrix
from which the tested compound can diffuse slowly. Methylcellulose discs
containing the tested compound were placed on the external border of the
vascular perimeter of the vitelline membrane where the angiogenic process
was still active.
The effects of the discs containing the tested compound on proximal vascular
development were, in all cases, compared with those of discs containing
control buffer. The discs were placed on the embryos' vitelline membrane on
CA 02386759 2002-05-17
23
Day 0 or Day 1 of the ex ovo growth process; at this point, only beginnings of
the main blood vessels are invading the vitellus. The embryos were then put
in culture conditions until vascularization was assessed (approximately 24 h).
Control buffer- and compound-containing discs were in all cases added
simultaneously on the vitelline membrane of the same embryo. Both discs
were arranged in a symmetrical fashion with respect to the cephalo-caudal
axis of the embryo in order to minimize inter-individual variations when
comparing the tested compounds with controls.
b. Anti-angriogienic activity:
Embryonic vascularization tests (EVTs) were performed using different
concentrations of protamine (5 to 20 fig) as a positive control or a tested
compound (0.001 to 10 fig). After one day in culture, the level of
vascularization in the area covered by the discs was graded by a pair of
scientists in the usual blind fashion. To facilitate the location of the
discs,
black O-rings were placed around them just after their placement on the
vitelline membrane. The evaluation scale for the EVTs was based on two
different scoring systems.
Assessment of blood vessel formation:
Blood vessel formation was assessed in a blind fashion. The areas of the
vitelline membranes that lay beneath the methylcellulose discs were scored
for the degree of vascularization, using two scoring systems ("N/+" or " 1-2-
3"). The following selection criteria were used:
NI+ system:
A score of "N" for "Normal" was attributed when all the following conditions
were met:
Blood vessels in the evaluated area grew along their path with no abnormal
deviation. Collateral branching density was normal and the growth path of the
lateral branches was also normal.
CA 02386759 2002-05-17
24
A score of "+" was attributed when at least one of the following conditions
was met:
~ Major blood vessels grew across the evaluated area but their paths were
clearly affected (winding).
~ Major blood vessels grew across the evaluated area but collateral
branching density was clearly diminished.
~ Major blood vessels penetrated the evaluated area but their growth path
rapidly deviated.
~ A kink was observed in the blood vessel.
~ Major blood vessels penetrated the evaluated area but were stunted. No
growth was observed beyond that point.
~ A drastic deviation in the growth path occurred when major blood vessels
reached the proximal ridge of the disc.
1-2-3 grading scale system:
A score of 3 was attributed when the following conditions were met:
~ Blood vessels, present in the evaluated area, grew along their paths with
no abnormal deviation. Collateral branching density was normal and the
growth paths of the lateral branches were also normal.
A score of 2 was attributed when at least one of the following conditions was
met:
~ Major blood vessels grew across the evaluated area but their paths were
clearly affected (winding).
~ Major blood vessels grew across the evaluated area but collateral
branching density was clearly diminished.
A score of 1 was attributed when at least one of the following conditions was
met:
CA 02386759 2002-05-17
~ Major blood vessels penetrated the evaluated area but their growth paths
were rapidly deviated.
~ A kink was observed in the blood vessels.
~ Major blood vessels penetrated the evaluated area but were stunted. No
5 growth was observed beyond that point.
~ A drastic deviation in the growth paths occurred when major blood
vessels reached the proximal ridge of the disc.
A score of 3 meant that blood vessel development was normal whereas a
10 score of 1 indicated the greatest degree of angiostatic activity.
CA 02386759 2002-05-17
O
_N
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M r- t~ 00 Cfl d'
M N N ~ - N N
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CA 02386759 2002-05-17
O N ~ d" ~' O O
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tfj ~ t' N N CEO T
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CA 02386759 2002-05-17
28
A dose-response inhibition was obtained with protamine, bpV(phen), and
orthovanadate (Figure 2). The approximate IC5o is 0.1 pg for bpV(phen),
0.6 ~g for orthovanadate, and 11 ~g for protamine (regardless of the scoring
system). A summary of the experimental data collected is shown in Table II.
The results show that bpV(phen) is a potent inhibitor of the angiogenic
process. Prior art on the subject (vanadate) taught the opposite, namely that
vanadium compounds promote angiogenesis.
4- In vivo Matrigel slug assay
This assay was performed as previously described (107). Briefly, Matrigel
(liquid at 4°C) was mixed with 200 ng/ml ECGS and injected s.c. into
four
C57B1/6N female mice (1 ml/mice). After injection, the Matrigel polymerized
to form a plug. After 7 days, the animals were sacrified, and the Matrigel
plugs were removed and fixed in 10% neutral-buffered formalin solution
(Sigma Chemical Co.) and embedded in paraffin. Histological sections were
stained with Masson's trichrome and angiogenesis was scored by counting
the number of cells that migrated in three microscopic fields for each s.c.
Matrigel plug.
Experiments were performed to determine whether bpV(phen) inhibits vessel
formation in vivo. Animals were divided into five groups (4 mice/group): two
control groups that received no treatment or daily i.p. PBS injection,
respectively, and three groups that received daily administration of various
doses (10, 50 and 100 pg) of bpV(phen), from day 1 to day 7 (intraperitoneal
daily administration of 100 NI solution). The results show that bpV(phen) is a
potent angiogenesis inhibitor in vivo (Fig. 5). Daily administration of 10
pg/mice or higher doses decreased by half the number of endothelial cells
having invaded Matrigel plugs.
CA 02386759 2002-05-17
29
The angiogenic potential of pV compounds was tested using in vifro, ex ovo,
and in vitro systems of analysis. The results show that in contrast to what
might have been expected from the teaching of the prior art, pV compounds
are potent inhibitors of the angiogenic process (see below), and this includes
high doses of vanadate.
B) PEROXOVANADIUM COMPOUNDS and THE PRODUCTION OF
ENDOTHELINS
The data presented in Table III show the effect of bpV(phen) on the plasmatic
levels of ETs. In this study, rats were injected intraperitoneally with
bpV(phen) (0.5 mg/100g b.w.) 16 hours before the administration of either
insulin or vehicle. Two minutes following insulin administration (1.5 ~g/100g
b.w, or vehicle), the plasma levels of ETs were determined. Insulin
administration induced a strong increase of seric ET concentration (54). This
increase was completely abolished by bpV(phen). In addition, bpV(phen)
decreased the insulin-stimulated levels of plasmatic ETs below control levels.
These results suggest that bpV(phen) inhibits the insulin-induced release of
ETs that subsequently can lead to diabetic complications like vasculopathy
and nephropathy.
TABLE 111: Effect of bpV i(phen) on Endothelin Plasma Levels
Plasma endothelins
(pg/ml)
Control 113.41 10.91
Insulin 253.10
Insulin, bpV(phen) 77.68 3.08
ETs (ET-1, ET-2, ET-3) were measured by RIA (Amersham kit RPA 555)
after lyophilization and extraction on C2 columns (500 mg). Results are mean
~SEM, p<0.0209, control (n=4) vs Insulin, pV (n=3).
CA 02386759 2002-05-17
C) PEROXOVANADIUM COMPOUNDS and RESTENOSIS
The data presented in Table IV show the effect of the pV compound
bpV(bipy) on restenosis. Male Sprague-Dawley rats (325-350g) were
5 anaesthetized with Rogarsetic, a mixture of ketamine hydrochloride and
xylazine hydrochloride. The animals were divided into two groups of 14-31
rats that underwent balloon angioplasty of the left carotid artery, with or
without treatment (108). The treated group received bpV (bipy) (10 mg I kg I
i.p. b.i.d.) for 14 days, starting on the day of the angioplasty. Every animal
in
10 each group underwent balloon angioplasty of the left external carotid
whereas the contra-lateral carotid served as a control for each animal. The
left carotid artery balloon angioplasty was performed under aseptic
conditions. The left external carotid was isolated and an arteriotomy was
performed. A 2F Fogarty arterial embolectomy catheter was then inserted in
15 the left common carotid and positioned near the aortic arch. The balloon
was
then inflated and retracted with a twisting motion to its insertion point.
This
procedure was repeated twice and the external carotid was ligated. There
were no differences in the lumina (L), intima (I), media (M), the ratio of I/M
and calculated value of (M - L / P) as a measure of neointimal proliferation
20 (NIP) between normal, non-ballooned carotid artery of treated and non-
treated rats, serving as controls (data not shown). Treatment caused a 32%
decreased in NIP and the I/M ratio, a 21 % reduction in the thickness of the I
and a 28% increase in the opening of the L. Thus, treatment with bpV (bipy)
revealed a significant reduction in the degree of post-angioplastic vascular
25 remodeling of the carotid artery in the rat model.
The above results illustrate the antiangiogenic and anti-restenosis potential
of
vanadium compounds. The administration of pV compounds could be used to
control the progression of several angiogeno-dependent conditions as well as
30 post-angioplastic vascular remodeling. Since vanadates are anti-tumor
compounds, the present pV compounds should function as anti-tumor
therapeutic agents, and part of this activity should be due to an anti-
CA 02386759 2002-05-17
31
angiogenic effect. Pharmaceutical compositions would comprise potent pV
compounds capable of achieving an extracellular concentration of about 0.1
to 100 ~M, preferably 2-40 ~M. In cats, a dose of 20 moles per kilogram
was successful in reversing the endothelin increase after insulin
administration. Moreover, pV compounds, which as previously stated are
more potent and provoke less side effects than the vanadium "oxo"
compounds, can be administered to achieve the desired dosage. For
example, see in vivo effects on blood glucose levels have been reported (1,
3).
TABLE IV: Effect of bpV (bipY) on Restenosis
NIP I M I / L
mm mm2 M mm2
Control 0.064 0.138 0.090 1.546 0.090
bpV(bipy)0.045 0.112 0.104 1.086 0.112
p < x 0.002 0.04 0.005 0.003 0.003
Abbreviations: I, intima; M, media; NIP, neointimal proliferation; L, lumina.
D) PEROXOVANADIUM COMPOUNDS and THE IMMUNE RESPONSE
The present invention provides pV compounds as novel immunomodulator
and adjuvant molecules. The pV compound bpV(phen) can be used as an
immunomodulator in vitro (macrophage; chemokine genes expression) and in
vivo (murine model; cytokine genes expression, cytokine and chemokine
generations and cellular recruitment in response to various stimulation), and
as an adjuvant to maximize the protective effect of known Leishmania
antigen based vaccine. The above bpV(phen) compound is a potent
immunomodulator based on its capacity to induce cytokine (e.g. IL-12, IFN(,
IL-1~3) and chemokine (e.g. RANTES, MIP-1a,(3, MIP-2, IP-10, MCP-1) gene
CA 02386759 2002-05-17
32
expression to enhance cellular recruitment in response to an agonist and
consequently act as an adjuvant in the context of vaccination.
1-bpVl~phen) treatment enhance pro-inflammatory molecules secretion
S and polymorphonuclear recruitment in response to Leishmania
infection
With the use of a murine air pouch model, we investigated early inflammatory
events that occur following L. major skin injection in bpV(phen)-treated
animals (Figure 6). As shown in Figure 6A, both L. major and LPS (positive
control) could induce significant leukocyte recruitment in air pouch exudates
as measured 6 hours post-injection. Of interest, bpV(phen)-treated BALB/c
mice, in response to L. major promastigotes (107 parasites/ml) intra-pouch
injection, manifested a five times greater recruitment of cells within the
same
time period. In addition, differential counts performed on Diff-Quick-stained
cytospin preparations from these cells (Fig. 6B) revealed that --70% of
recruited leukocytes were neutrophils (18% eosinophils and 12%
macrophages), whereas in untreated animals receiving L. major
promastigotes ~48% of recruited cells were neutrophils with the rest of the
cell population consisting of eosinophils (26%) and macrophages (26%). This
data revealed that bpV(phen) is a good modulator of the early inflammatory
response and could have a major impact on the progression of L. major
infection since the dramatic increase in inflammatory cell recruitment
consisted mainly of neutrophils, already recognized for their importance to
restrain Leishmania pathogenesis (109).
In parallel to this experiment, in vivo generation of Leishmania-induced NO
and pro-inflammatory mediators in bpV(phen)-treated mice were measured in
air pouch exudates (Figure 7).
The present set of data reinforce the idea that bpV(phen) is an excellent NO
modulator as revealed by its enhanced generation in response to L. major
infection (Fig. 7A). This elevated NO generation not only further increased
CA 02386759 2002-05-17
33
the microbicidal activity observed in bpV(phen)-treated animals, but also
partly explained the strong inflammatory response observed in bpV(phen)-
treated animals since NO has been recognized as an important mediator of
inflammation and regulator of neutrophil migration (110, 111 ). In addition,
bpV(phen)-treatment has shown elevation of some pro-inflammatory
cytokines (IL-6 and IL-1~i) and chemokines (MCP-1 and MIP-2) measured in
air pouch exudates of animals inoculated with L. major promastigotes (Fig.
7B). MIP-2 chemokine is recognized as a specific chemoattractant for
neutrophils (112) whereas MCP-1 has been shown to be a powerful
monocyte/macrophage recruiter to sites of inflammation. Additionally, the
pro-inflammatory cytokines IL-1(3 and IL-6 are well known chemokine
modulators playing a pivotal role in the development of inflammation (113)
including the regulation of MCP-1 and MIP-2 expression (114).
1S 2- Up-regulation of IL-12, IL-2 and IFN~~ mRNA expression in spleen of
bpV(phenJl-treated animals
As shown in Figure 8, in vivo bpV(phen)-induced cytokine gene expression-
monitored with the use of a multi-probe RNAse protection assay-in spleen of
naive mice was significantly up-regulated for up to 24 hr (i.e. IL-12, IL-10,
IL-
2 and IFN() in comparison to animals injected with PBS. Our observation
provides evidence that the PTP inhibitor bpV(phen) is a powerful
immunomodulator favoring the activation of protective Th1 type cytokines
recognized to modulate NO generation in vivo resulting in control over
cutaneous leishmaniasis (115, 116). Additionally, whereas little is known
concerning the role of PTPs on the regulation of cytokines, the present
observation revealed that different signaling events being or not under the
control of PTPs conduct to cytokine genes regulation. Overall, these last
experiments clearly demonstrated the capacity of bpV(phen) to up-regulate
several protective inflammatory and immunological functions of the host in
response to a pathogen.
CA 02386759 2002-05-17
34
3- Effect of bpV(phen) on chemokine gene expression
Various concentrations of bpV(phen) were added to B10R macrophages for 4
h. mRNA expression was measured by RNase protection assays. RANTES,
S MIP-1a/~i MIP-2, IP-10 and MCP-1 chemokine mRNA levels increased in
response to bpV(phen) (Fig. 9A). MCP-1, MIP-2, MIP-1~3 and RANTES were
induced by the addition of bpV(phen) in a dose-dependent manner whereas
IP-10 and MIP-1a were expressed at markedly lower concentrations (10 pM).
Thus, bpV(phen) used at 10 ~M seems to be the most effective dose for the
induction of chemokine gene expression. In addition, we noted that their
expression was were differently modulated by bpV(phen) treatment,
suggesting that different type of PTPs must play a specific role in the
regulation of various chemokines.
To determine the kinetics of chemokine expression following pV compound
treatment, B10R macrophages were incubated with 10 DM bpV(phen) for
increasing time intervals. Total RNA was extracted from macrophages at
various time-points and subjected to RNase protection assay. There was a
transient induction of MIP-1a, MIP-2, IP-10, and MCP-1 chemokines mRNA,
reaching maximum levels at 4 h and rapidly declining afterward (Fig. 9B),
whereas for MIP-1 ~3, the optimal expression was achieved at 8h post
treatment. However, the expression of RANTES mRNA increased in a time
dependent manner over 24 h (Fig. 9B). Thus, conditions for the induction of
chemokine expression in response to bpV(phen) seems to vary with the
different chemokines.
These results clearly showed that bpV(phen) had a stronger effect on some
chemokines than on others. Evaluation of mRNA expression by densitometry
analysis (data not shown) demonstrated a greater induction of MIP-2>MIP-
1a>MCP-1 in presence of bpV(phen) in comparison to MIP-1~3 >RANTES>IP-
10.
CA 02386759 2002-05-17
We report that modulation of host PTPs by bpV(phen) was effective in
inducing RANTES, MIP-1a, MIP-1~i MIP-2, IP-10 and MCP-1 chemokine
gene expression in B10R murine macrophages. However, its action varied
according to each chemokine, demonstrating a more potent effect on specific
5 chemokines. Indeed, RNase protection assays have shown a greater
induction of MIP-2> MIP-1a> MCP-1 by adding bpV(phen) compared to MIP-
1 ~i >RANTES>IP-10. Thus, our results designate PTPs as important negative
regulators of the signaling process implicated in the chemokine production
when they are activated. This is consistent with several studies where PTPs
10 negatively regulate many cellular signaling such as B-cell receptor (BCR)
(117) and erythropoietin receptor (118).
4- Use of bpVi(phen) as adjuvant in the context of Leishmania vaccine
trial
The adjuvant potential of bpV(phen) was tested in vivo in the context of
Leishmania infection and resulted in complete protection against infectious
challenge.
In the past, use of total and soluble Leishmania antigen (SLA) for vaccination
has been reported and permitted some levels of protection against infectious
challenges (85). Using a murine model, we have tested whether bpV(phen)-
modulated cytokine and chemokine generation (as reported above) in
combination with SLA administration could lead to protection against
Leishmania infection. Mice have received PBS and bpV(phen) as controls,
and SLA (100 fig) with or without daily bpV(phen) injection (500 nM, 5 days).
All injections were done intra-peritoneally. Two weeks later, all groups were
inoculated similarly with their respective treatment. Finally, 4 weeks post-
vaccination, all animals were challenged with infectious Leishmania (5x106 L.
major intradermally injected in the right hind footpad) and progression of
infection followed over a period of 4 weeks post-infection (Fig. 10).
Protection
mediated by SLA+bpV(phen) has been successful since no significant
CA 02386759 2002-05-17
36
footpad inflammation and skin lesion development was observed in
comparison to all the other experimental groups.
E) PEROXOVANADIUM COMPOUNDS AND THE INHIBITION OF
TUMOR GROWTH
Methods
'- Cells
ZR-75: hormone-dependent cancer (ductal carcinoma) with oestrogen
receptors (ATCC, USA is depository)
PC-3: adenocarcinoma (grade IV) with bone metastasis (ATCC, USA is
depository).
A) ZR-75-1 human breast cancer: ZR-75-1 human breast cancer cells
obtained from the American Tissue Culture Collection (Rockville, MD) were
cultured in phenol red-free RPMI 1640. The cells were supplemented with
2mM L-glutamine, 1 mM Na-pyruvate, 100 IU penicillin/ml, 100~,g
streptomycin/ml, and 10% (v/v) fetal bovine serum and incubated under a
humidified atmosphere comprised of 95% air arid 5% COZ at 37°C.
Female homozygous HSD nu/nu athymic mice (50 days old) were obtained
from Harlan Sprague Dawley Inc. (Indianapolis, IN). Five mice were housed
per vinyl cage, which was equipped with air filter lids and kept in laminar
air
flow hoods under pathogen-limiting conditions. The photoperiod was
composed of a period of 14h of light and a period of 10h of darkness. Cages,
bedding, and food (Agway Pro-Lab R-M-H diet #4018) were autoclaved prior
to use. Water was acidified to pH of 2.8, autoclaved, and provided ad libitum.
Bilateral ovariectomy (OVX) was performed on all animals one week prior to
cell inoculation, under 2.5% isoflurane anesthesia mixed with oxygen.
Simultaneously, an oestrogen (E2) implant was inserted subcutaneously to
CA 02386759 2002-05-17
37
stimulate initital tumor growth and appearance. E2 implants were prepared in
1-cm long silastic tubing (inside diameter, 0.062 inch; outside diameter,
0.095
inch) containing 0.5 cm of estradiol/cholesterol diluted at a ratio of 1:10
(w:w).
One week after the ovariectomy, 2.0 x 106 ZR-75-1 cells, in their logarithmic
growth phase, were harvested with 0.083% pancreatin/0.3 mM EDTA and
inoculated s.c. in 0.1 ml of RPMI 1640 culture medium containing 30% of
Matrigel, from each flank of each animal through a 2.5-cm-long 20-gauge
needle. Four weeks after ZR-75-1 cell inoculation, the E2 implants were
replaced in all animals by estrone-containing implants (E~: cholesterol; 1:25
w:w) (104). Treatments consisting of increased doses of bpV(phen) versus
control were started 5 weeks after cell inoculation. Mice bearing tumors of an
average area of 15 mm2 were randomly assigned to 3 groups, each group
containing more than 15 mice. OVX animals first received the most potent
natural estrogen, to initiate cell proliferation and the development of
tumors.
Thereafter, the E2 implants were replaced by E~ implants as a model for post-
menopausal women in which E~ is the main circulating estrogen that is
converted into E2 in peripheral tissues.
On day 0 of the experiment (5 weeks after inoculation), the E~-releasing
implants were removed from the animals in Group 1 only. All mice received a
daily administration of bpV(phen) over a period of 42 days (i.p., 1001, in a 2
blind manner). Groups 1 and 2 received PBS, Group 3 received 2.5 mg/Kg
bpV(phen).
B) Human prostate adenocarcinoma (PC-3) in the athymic mice: Male
Balb/c nude (nu/nu) were purchased at 4-6 weeks of age from Charles Rivers
Inc. Mice were housed under pathogen free conditions and maintained on a
12-h light/12-h dark cycle with food and water supplied ad libitum. The
hormono-independent PC3 human tumor cells were from the American
Tissue Cuture Collection. Cells were grown in DMEM in the presence of 5
foetal bovine serum. Cells were collected at confluence, included in a matrix
CA 02386759 2002-05-17
38
(1.0 X 106 cells /ml; 30 % Matrigel). An equal volume of the tumor cell
suspension was injected s.c. in the right flank of each mouse. After 5 days, a
palpable tumor of approximately 5X5 mm was detected in the inoculated
animals. Mice with palpable tumors were divided into five groups (18
mice/group) for the treatment study. All mice in each treatment group had
tumor of similar size at the start of treatment. For administration to mice,
bpV(phen) was dissolved and diluted in phosphate buffered saline (PBS) at
pH 7.4. A Smg/Kg dose of bpV(phen) was administed daily by i.p. for 39
days. Taxol was used as a positive control at the dose of 20mg/Kg and
injected i.p. at every three days. A control group of 10 animals was injected
with PBS. The injection volume was kept constant at 100 pl/g body weight.
The mice were weighed three times during the experimental period to assess
toxicity of the treatment, and the tumors were measured twice weekly using
calipers. Tumor volume was calculated from the two-dimensional caliper
measurements using the following formula: tumor volume = length X (width)2
X 0.53.
The treatment period was completed after 39 days, when the PBS treated
group of mice had large tumors, requiring that the animals be sacrificed
according to the Animal Care Procedures. On the final day of the study, the
mice were sacrificed by carbon dioxyde inhalation. The s.c. tumors was
removed and weighed.
Statistical analysis: Tumor growth curves are presented in terms of
treatment group means and SEs. Statistical significance of treatment effect
was assessed by repeated measures ANOVA after applying a power
transformation to equalize residual variances and linearize the tumor growth
curves.
CA 02386759 2002-05-17
39
Results
1. Progression of tumor cells in vitro
The cells embedded in the collagen gel, grew as a "primary tumor". Some
cells migrated from the primary tumor towards the fibrin gel, forming front
edges. Small clumps of cells were observed in the fibrin gel; in this model
they represent "secondary tumors". Their extension and numbers are
representative of the invasive potential of the cancer cells.
PC-3: In control experiments, PC-3 cells migrated slightly from the primary
tumor and formed extensive secondary tumors in the fibrin gel. In the
presence of 2pM bpV(phen), a decrease in the size of the secondary tumor
was observed and the migration front from the primary tumor was similar to
1 S that seen in the control gels. In the presence of 5 and 10 uM bpV(phen),
there was no migration front and there were no secondary tumors in the fibrin
gel. In addition, the primary tumors appeared clearer than in the control
(Figure 11 ).
ZR-75-1: In control experiments ZR-75-1 cells migrated into the fibrin as
small spheroidal secondary tumors, with a limited and sparsely visible
migration front. The presence of 2pM bpV(phen) restricted the growth of the
primary tumor. At 5 and 10~M bpV(phen) there were no secondary tumors
and the primary tumor had a lower cell density (Figure 12).
2. Inhibition of tumor progression in vivo
A- ZR-75-1 human breast cancer:
The tumor size in the control group, having not received E~ replacement
therapy, did not increase. The tumor size in the animals in the other control
groups having received E~ replacement therapy was found to have increased
CA 02386759 2002-05-17
significantly (p<0.05) from 15 to 26 mm2 on day 42. The daily administration
of bpV(phen) do not resulted in increase of tumor size (p<0.05). The results
show that bpV(phen) has the capacity to inhibit the progression of tumors in
vivo (Figure 13A).
5
B- Human prostate adenocarcinoma (PC-3) in the athymic mice'
Daily administration of bpV(phen) caused a significant (p<0.001 ) 59
suppression of the final tumor compared with PBS-treated control animals
10 (Figure 13B ). No death were observed among the vehicle-treated controls or
bpV(phen), and, on average these mice gained 1.5 and 1.7 grams in body
weight respectively, relative to their weight at the initiation of the
treatment.
15 F) THE USE OF INHIBITORS OF PROTEIN TYROSINE PHOSPHATASES
(PTP) FOR ANTI-TUMOR IMMUNOTHERAPY
Lymphocytes with anti-tumor activity can be isolated from patients and grown
in vitro for use in cell-tranfer therapies (119). The incubation of immune
cells
20 with the PTP inhibitor bpV(phen) augments their activation state (120).
Therefore, the re-administration of bpV(phen)-activated immune cells to
cancer patients may enhance the immune response towards tumor cells. The
results described below demonstrate the efficacy of a method in which a
peroxometallic compound (bpV(phen) is used ex vivo on autologous immune
25 cells in order to stimulate the potency of these cells and once returned
into
blood circulation of cancer patients fight invasion malignant cells.
Method
30 An in vitro cancer invasion system that has been previously designed was
used. Briefly, prostate cancer cells (PC-3; American Type Culture Collection,
Rockville MD) were grown in DMEM medium with 5 % fetal bovine serum, 2
mM L-glutamine, and antibiotics. They were, incubated under a humidified
atmosphere of 95 % air/5% C02 at 37~C. Collagen gels containing the PC-3
CA 02386759 2002-05-17
41
cells were prepared according the method of Esdale and Bard (121 ). The
cell-embedded collagen gels were laid down onto a layer of fibrin gel, and
anchored by a second layer of fibrin gel. The top of the collagen gel was not
fully covered with fibrin gel in order to allow direct contacts between the
cancer cells in collagen and the splenocytes. The latter were directly seeded
onto the top layer of the collagen and fibrin gel. Prior to the molding of the
cancer invasion system, leucocytes were isolated from spleen of either
healthy mice or mice bearing PC-3 tumors. They were treated in vitro with
bpV(phen) (25 NM) for 24 hr. Thereafter, treated cells were washed, counted
and seeded (106 cells per gel) on the cancer cells-embedded gels. Untreated
leukocytes seeded on the top of the cancer invasion system (same
concentration) were used as a control experiment. Medium was renewed
periodically. During the whole experiment, most leucocytes remained on the
top of the gels, and have a normal morphology. Cell behavior was
periodically observed for 7 days of culture, then recorded (by photography).
Results
Clumps of PC3 cells progressively appeared in the fibrin gel representing the
invasive potential of the cancer cells. In the control 3D culture system, PC-3
cells migrated slightly from the primary site and formed extensive secondary
tumors in the fibrin gel as described in previous studies (122, 123). In
contrast to this, in the presence of bpV(phen)-treated leucocytes, neither
secondary tumors nor migration front was observed. In addition, the primary
tumors appeared less dense than in the control, indicating a smaller number
of growing cells (Figure 14).
Conclusion
The above describes a method consisting in the ex vivo autologous activation
by bpV(phen) of leucocytes and their potential to trigger a cellular immune
response against cancer cells. This may prove to be beneficial for several ex
vivo technologies. For example: alone or in combination with interleukins and
CA 02386759 2002-05-17
42
chemokines that trigger immune response to specific antigens or mutated cell
types (124).
Although the present invention has been described by way of preferred
embodiments thereof, these embodiments can be modified at will, within the
scope of the appended claims, without departing from the spirit and nature of
the subject invention.
CA 02386759 2002-05-17
43
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