Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION THERAPY FOR TREATING
PROLIFERATIVE DISEASES
Field of the Invention
The present invention relates generally to new chemical combinations and
methods for
their use in the treatment of proliferative diseases and in particular cancer.
Background of the Invention
Cancer is typically treated with either chemotherapy and/or radiation therapy.
While often
effective to destroy a significant amount of tumour cells, such therapies
often leave behind
a number of tumour cells that are resistant to the treatment. These resistant
cells can
proliferate to form new tumours that are then resistant to treatment. As a
result, the
constant use of known combinations of chemotherapeutic drugs has given rise to
multidrug
resistant ('MDR') tumour cells.
The mode of proliferative diseases, such as tumours, is multi-factorial. For
instance,
research over the last forty years has led to the realisation that cytotoxic
agents (or anti-
proliferative agents) includes anti-metabolic agents which intefere with
microtubule
formulation, alkylating agents which are able to cross-link DNA, platinum
based agents
which are able to interfere with DNA alkylation by blocking DNA replication,
antitumour
antibiotic agents, topoisomerase inhibitors, etc. In the treatment of such
diseases drugs
with different mechanisms may be combined (i.e, combination therapies) with
beneficial
effects including the effective treatment of MDR tumour cells and to minimise
side effects
such as undesireable cytotoxicity. The difficulty here is though that not all
known
antiproliferative agents provide useful or beneficial effects in combination
and accordingly
research in many laboratories is presently focused on developing new and
useful anti-
proliferative combination partners.
It has now been found that a combination comprising a compound selected from a
small
CA 02686587 2009-11-27
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class of particularly substituted benzofuran tubulin polymerisation inhibitors
and at least
one other anti-proliferative agent selected from a group of specific agents is
particularly
useful in treating proliferative diseases and in particular cancer.
Summary of the Invention
The present invention provides a pharmaceutical combination comprising: (a) a
compound
of formula (I) or a salt, solvate or prodrug thereof;
O
H3C0 CH3
H3C0
0
\
(I)
H3C0
OH
and (b) at least one other anti-proliferative agent selected from alkylating
agents,
antitumour antibiotics, antimetabolites, natural alkaloids and inhibitors of
protein tyrosine
kinases and/or serine/threonine kinases, for simultaneous, separate or
sequential use in the
treatment of a proliferative disease.
The present invention also provides use of (a) a compound of formula (I) or a
pharmaceutically acceptable salt, solvate or prodrug thereof;
o
H3c0 cH3
H3C0 410
0
1401\ CH3 (I)
H3c0
OH
in combination with (b) at least one other anti-proliferative agent selected
from alkylating
CA 02686587 2009-11-27
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agents, antitumour antibiotics, antimetabolites, natural alkaloids and
inhibitors of protein
tyrosine kinases and/or serine/threonine kinases, for treating a proliferative
disease.
The present invention also provides the use of: (a) a compound of formula (I)
or a
pharmaceutically acceptable salt, solvate or prodrug thereof:
O
H3C0 CH3
H3C0 41)
0
\ CH3 (I)
0
H3C0
OH
and (b) at least one other anti-proliferative agent selected from alkylating
agents,
antitumour antibiotics, antimetabolites, natural alkaloids and inhibitors of
protein tyrosine
kinases and/or serine/threonine kinases,
in the manufacture of a medicament for the treatment of a proliferative
disease.
The present invention further provides a pharmaceutical composition comprising
(a) a
compound of formula (I) or a pharmaceutically acceptable salt, solvate or
prodrug thereof:
O
H3C0 CH3
H3C0
0
1111111\ pfi
1.3 (I)
0
H3C0
Off
and (b) at least one other anti-proliferative agent selected from alkylating
agents,
antitumour antibiotics, antimetabolites, natural alkaloids and inhibitors of
protein tyrosine
kinases and/or serine/threonine kinases.
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The present invention further provides a kit comprising:
(a) an amount of a compound of formula (I) or a pharmaceutically acceptable
salt, solvate or prodrug thereof:
O
H3co CH3
H3CO
(I)
0\CH
H300
OH
(b) an amount of at least one other anti-proliferative agent selected from
alkylating agents, antitumour antibiotics, antimetabolites, natural alkaloids
and inhibitors of protein tyrosine kinases and/or serine/threonine kinases;
and
(c) instructions for use of (a) and (b) in combination for treating a
proliferative
disease.
Surprisingly it has been found that the effects in treating proliferative
diseases with a
combination which comprises: (a) a compound of formula (I) or a
pharmaceutically
acceptable salt, solvate or prodrug thereof:
O
H3c0 cH3
H3co
(I)
CH3
H3C0
OH
and (b) at least one other anti-proliferative agent selected from alkylating
agents,
antitumour antibiotics, antimetabolites, natural alkaloids and inhibitors of
protein tyrosine
kinases and/or serine/threonine kinases,
CA 02686587 2009-11-27
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is greater than the effects that can be achieved with either (a) or (b) alone.
That is, the
present combinations have been found to possess a supra additive or
synergistic effect or
provides beneficial additive effects in anti-cancer therapy.
Brief Description of the Figures
Figure 1 Table of combination index values. Range of combination
index =
synergy (<0.1-0.90), additive (0.90-1.10), and no additive benefit
(antagonism) (1.10->10).
Figure 2 Graph showing tumour growth over 23 days calculated
relative to
average tumour volume (mm3). denotes
cisplatin treatment
day. 1-0. denotes Example 2 treatment day.
Figure 3 Graph showing % survival of mice vs days of treatment.
Figure 4 Graph showing tumour growth ratio (Day X / Day 1) vs days
of
treatment for combination with Doxorubicin or 5-Fluorouracil.
Figure 5 depicts a graph of A perfusion control against an amount
of
compound (mg/kg) in relation to comparative levels of vascular
shutdown (reduction in tumour perfusion) between CA4P and
compound Example 2 of the present invention.
Figure 6 depicts a graph of Tumour Volume ratio (Day*/Day 1) against
time
(Days) in relation to tumour growth inhibition of compound
example 2 in Balb/c nu/nu mice bearing MDA-MB-231 orthotopic
breast solid tumours.
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Description of the Preferred Embodiments
Throughout this specification and the claims which follow, unless the context
requires
otherwise, the word "comprise", and variations such as "comprises" and
"comprising", will
be understood to imply the inclusion of a stated integer or step or group of
integers or steps
but not the exclusion of any other integer or step or group of integers or
steps.
The reference in this specification to any prior publication (or information
derived from it),
or to any matter which is known, is not, and should not be taken as an
acknowledgment or
admission or any form of suggestion that that prior publication (or
information derived
from it) or known matter forms part of the common general knowledge in the
field of
endeavour to which this specification relates.
Combination partner (a); compound offormula (I)
Compound of formula (I) (2-Methy1-7-hydroxy-3-(3,4,5-trimethoxybenzoy1)-6-
methoxybenzofuran) can be prepared by the synthetic methodology described in
PCT/AU2007/000101 (WO 07/087684).
The compound of formula (I) is observed to be potent tubulin polymerisation
inhibitor
(TPI). TPI compounds are important in the treatment of cancers primarily as a
result of
their capacity to selectively shut down blood flow through a tumour. Compounds
that
inhibit tumour blood flow are generally referred to as vascular disrupting
agents (VDAs)
(Tozer, G. M.; Kanthou, C.; Baguley, B. C. Nature Rev., Vol. 5, 2005, 423).
TPIs are
VDAs because they inhibit a certain cell signalling pathway associated with
microtubules,
leading to interference in the regulation of the cytoskeleton of the
endothelial cells that line
the blood vessels of the tumour. As a result, these usually flat cells become
more rounded,
ultimately occluding blood flow through the vessels. The selectivity
associated with these
agents results from the fact that tumour vasculature is weaker and more prone
to collapse
than normal vasculature. Nonetheless, a number of the dose limiting toxicities
associated
with VDAs are due to a reduction in blood flow in healthy tissues. An
important aspect of
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the compound of formula (I) is the combination of the specific C-6 and C-7
substituents
together with the C-2 methyl group which appears to confer greater potency and
selectivity
when compared to other structurally related TN compounds. In this compound
selectivity
is not simply reliant on the predisposition of tumour vasculature towards
collapse when
challenged with the VDA but on a capacity of the VDA to distinguish between
tumour
endothelial cells and normal endothelial cells. Normal endothelial cells,
found in healthy
tissues, are in a "quiescent" state and tumour endothelial cells are in an
"activated" state.
Most VDAs do not distinguish between these two states, for example,
Combretastatin A4
is equally potent against quiescent and activated endothelial cells. However,
the
compound of formula (I) shows high potency towards tumour endothelial cells
(activated)
over normal endothelial cells (quiescent).
It will be appreciated that the compound of formula (I) can be administered to
a subject as
a pharmaceutically acceptable salt thereof Suitable pharmaceutically
acceptable salts
include, but are not limited to salts of pharmaceutically acceptable inorganic
acids such as
hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and
hydrobromic
acids, or salts of pharmaceutically acceptable organic acids such as acetic,
propionic,
butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic,
mucic, gluconic,
benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic,
benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic,
stearic, palmitic,
oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
Base salts include, but are not limited to, those formed with pharmaceutically
acceptable
cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and
alkylammonium. In particular, the present invention includes within its scope
cationic
salts eg sodium or potassium salts, or alkyl esters (eg methyl, ethyl) of the
phosphate
group.
The term "pro-drug" is used in its
broadest sense and encompasses those derivatives that are converted in vivo to
the
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compound of formula (I). Such derivatives would readily occur to those skilled
in the art,
and include, for example, compounds where the free hydroxy group at the C-7
position is
converted into an ester, such as an acetate or phosphate ester. Procedures for
esterifying,
cg. acylating, the compound of formula (I) are well known in the art and may
include
treatment of the compound with an appropriate carboxylic acid, anhydride or
chloride in
the presence of a suitable catalyst or base. A particularly preferred prodrug
is a disodium
phosphate ester. The disodium phosphate ester of the compound of the invention
may be
useful in increasing the solubility of the compound. This, for instance, may
allow for
delivery of the compound in a benign vehicle like saline. The disodium
phosphate ester
may be prepared in accordance with the methodology described in Pettit, G. R.,
et al,
Anticancer Drug Des., 1995, 10, 299. Other texts which generally describe
prodrugs (and
the preparation thereof) include: Design of Prodrugs, 1985, H. Bundgaard
(Elsevier); The
Practice of Medicinal Chemistry, 1996, Camille G. Wermuth et al., Chapter 31
(Academic
Press); and A Textbook of Drug Design and Development, 1991, Bundgaard et al.,
Chapter
5, (Harwood Academic Publishers).
The compound of formula (I) (or a prodrug thereof) may be in crystalline form
either as
the free compound or as a solvate (e.g. hydrate) and it is intended that both
forms are
within the scope of the present invention. Methods of solvation are generally
known
within the art.
Combination partner (b); the anti-proliferative agent
The "at least one other anti-proliferative agent" or combination partner (b)
may include any
chemotherapeutic agent that can be used in the treatment of proliferative
diseases selected
from known anti-proliferative alkylating agents, antitumour antibiotics,
antimetabolites,
natural alkaloids and inhibitors of protein tyrosine kinases and/or
serine/threonine kinases.
Such agents include:
(i) alkylating agents, such as cis-platinum(II)-diaminedichloride (platinol
or
cisplatin); oxaliplatin (Eloxatin or Oxaliplatin Medac); and carboplatin
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(Paraplatin);
(ii) antitumour antibiotics, including those selected from the group
comprising
anthracyclines, such as doxorubicin (Adriamycin, Rubex);
(iii) antimetabolites, including folic acid analogues such as pyrimidine
analogues
such as 5-fluorouracil (Fluoruracil, 5-FU), gemcitabine (Gemzar), or histone
deacetylase inhibitors (HDI) for instance, Vorinostat (rINN);
(iv) natural alkaloids, including paclitaxel (Taxol);
(v) inhibitors of protein tyrosine kinases and/or serine/threonine
kinases
including Sorafenib (Nexavar), Erlotinib (Tarceva), Dasatanib (BMS-354825 or
Sprycel).
Preferred anti-proliferative agents include:
carboplatin, cisplatin, doxorubicin, 5-FU, gemcitabine, paclitaxel,
oxaliplatin, sorafenib,
tarceva, dasatanib, and vorinostat.
These above anti-proliferative agents are known in the art and their synthesis
would also
be known to those skilled in the art.
As used herein the term "proliferative disease" broadly encompasses any
neoplastic disease
including those which are potentially malignant (pre-cancerous) or malignant
(cancerous).
The term therefore encompasses the treatment of tumours.
Accordingly, the term "tumour" is used generally to define any malignant
cancerous or
pre-cancerous cell growth, and may include leukemias, and carcinomas such as
melanomas, colon, lung, ovarian, skin, breast, pancrease, pharynx, brain
prostate, CNS,
and renal cancers, as well as other cancers.
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In a preferred embodiment the combination may be used in the treatment of
tumours and in
particular in the following tumours: breast carcinoma, brain glioblastoma,
colorectal
carcinoma, lung carcinoma, ovary carcinoma, pancreas carcinoma, prostate
carcinoma,
renal cell carcinoma, and pharynx squamous cell carcinoma.
Thus the present invention also provides a method of treating tumours
comprising the
administration of an effective amount of (a) a compound of formula (I) or a
pharmaceutically acceptable salt, solvate or prodrug thereof and (b) at least
one other anti-
proliferative agent.
An "effective amount" is intended to mean that the amount of each combination
partner,
when administered to a mammal (in particular a human) in need of such
treatment, is
sufficient to effect treatment for a particular proliferative disease. Thus,
for example, a
therapeutically effective amount of a compound of the formula (I) (or a
pharmaceutically
acceptable salt, solvate, or prodrug thereof) is a quantity sufficient to
synergise or
potentiate the activity of the at least one anti-proliferative agent such that
a targeted disease
state is reduced or alleviated.
Accordingly, the terms "treatment" or "treating" as used herein are intended
to include at
least partially attaining the desired effect, or delaying the onset of, or
inhibiting the
progression of, or halting or reversing altogether the onset or progression of
the particular
disease (e.g., tumour) being treated.
Without wanting to be bound by any particular theory it is believed that the
tumour
vascular disruption effect caused by compound of formula (I) is transient (at
least in some
tumours) with tumour re-vascularisation occurring after around 48 hrs. It is
believed that
the synergistic or additive effect proposed by the present combination is (at
least in respect
of some of the combination partners for (a)) the inhibition or delay of this
re-
vascularisation event and tumour recovery from the initial disruption with
combination
partner (a).
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In some embodiments the following combinations of (a) and (b) have found to be
particularly synergistic in the treatment of particular tumours:
(a) (b) Treatment of
1. Compound of formula (I) or + carboplatin breast carcinoma
prodrug thereof
2. Compound of formula (I) or + carboplatin pancrease carcinoma
prodrug thereof
3. Compound of formula (I) or + carboplatin renal cell carcinoma
prodrug thereof
4. Compound of formula (I) or + cisplatin breast carcinoma
prodrug thereof
5. Compound of formula (I) or + cisplatin brain glioblastoma
prodrug thereof
6. Compound of formula (I) or -I- cisplatin colorectal carcinoma
prodrug thereof
7. Compound of formula (I) or + cisplatin lung carcinoma
prodrug thereof
S. Compound of formula (I) or + cisplatin pancrease carcinoma
prodrug thereof
9. Compound of formula (I) or + doxorubicin colorectal carcinoma
prodrug thereof
10. Compound of formula (I) or + doxorubicin pancrease carcinoma
prodrug thereof
11. Compound of formula (I) or + 5-FU breast carcinoma
prodrug thereof
12. Compound of formula (I) or + 5-FU brain glioblastoma
prodrug thereof
13. Compound of formula (I) or + 5-FU colorectal carcinoma
prodrug thereof
14. Compound of formula (I) or + 5-FU lung carcinoma
prodrug thereof
15. Compound of formula (I) or + 5-FU ovary carcinoma
prodrug thereof
16. Compound of formula (1) or + 5-FU pancrease carcinoma
prodrug thereof
17. Compound of formula (I) or + 5-FU prostate carcinoma
prodrug thereof
18. Compound of formula (I) or + 5-FU renal cell carcinoma
prodrug thereof
19. Compound of formula (I) or + gemcitabine lung carcinoma
prodrug thereof
20. Compound of formula (I) or + oxaliplatin colorectal carcinoma
prodrug thereof
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(a) (b) Treatment of
21. Compound of formula (I) or + sorafenib brain glioblastoma
prodrug thereof
22. Compound of formula (I) or + tarceva colorectal carcinoma
prodrug thereof
23. Compound of formula (I) or + tarceva pharynx squamous cell
prodrug thereof
carcinoma
24. Compound of formula (I) or + tarceva prostate carcinoma
prodrug thereof
25. Compound of formula (I) or + dasatanib colorectal carcinoma
prodrug thereof
26. Compound of formula (I) or + dasatanib ovary carcinoma
prodrug thereof
27. Compound of formula (I) or + dasatanib pharynx squamous cell
prodrug thereof
carcinoma
28. Compound of formula (I) or + dasatanib prostate carcinoma
prodrug thereof
29. Compound of formula (I) or + vorinostat prostate carcinoma
prodrug thereof
30. Compound of formula (I) or + vorinostat lung carcinoma
prodrug thereof
Clinical studies such as open-label, dose escalation studies in patients with
proliferative
diseases may include studies to prove the synergism of the active ingredients
of the
combination. The beneficial and/or synergistic effects can be determined
directly through
the results of these studies which are known as such to a person skilled in
the art. These
studies are also able to compare the effects of a monotherapy using the active
ingredients
and a combination of the invention. Preferably, the dose of combination
partner (a) may
be escalated until the Maximum Tolerated Dosage (MTD) is reached, and agent
(b) is
administered as a fixed dose. Alternatively, combination partner (a) is
administered in a
fixed dose and the dose of agent (b) is escalated. Each patient may receive
doses of agent
(a) either daily or intermittent. The efficacy of the treatment can be
determined in such
studies, e.g., after 6, 12, 18 or 24 weeks by evaluation of symptom scores
every 6 weeks.
In another study agent (b) would be given at recommended dose and combination
partner
(a) progressively escalated from 4.2, 8.4, 12.6 and 16.0 mg/m2. If no dose
limiting
toxicities were identified agent (a) could be used at the highest dose in a
further clinical
CA 02686587 2009-11-27
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study.
The administration of the pharmaceutical combination of the present invention
may result
not only in a beneficial effect, e.g., a synergistic therapeutic effect, for
instance, with
regard to alleviating, delaying progression of or inhibiting the symptoms, but
also in
further surprising beneficial effects. Such other effects may include fewer
side effects, an
improved quality of life or a decreased morbidity, compared with a monotherapy
applying
only one of the pharmaceutically active ingredients used in the combination of
the present
invention.
A further benefit of the invention is that lower doses of the active
ingredients of the
combination can be used. The dosages need not only be smaller but may also be
applied
less frequently, which may diminish the incidence or severity of side effects.
The term "administration" relates to the co-administration of the combination
partners to a
single patient, and is intended to include treatment regimens in which the
agents are not
necessarily administered by the same route of administration or at the same
time.
Accordingly, combination partners (a) and (b) may be administered together,
one after the
other or separately in one combined unit dosage form or in two separate unit
dosage forms.
The unit dosage form may also be a fixed combination such as a pharmaceutical
composition which comprises both a compound of formula (I) (or a salt, solvate
or prodrug
thereof) and at least one other anti-proliferative agent selected from
alkylating agents,
antitumour antibiotics, antimetabolites, natural alkaloids and inhibitors of
protein tyrosine
kinases and/or serine/threonine kinases.
In particular, a therapeutically effective amount of each of the combination
partners of the
combination of the invention may be administered simultaneously or
sequentially and in
any order, and the combination partners may be administered separately or as a
fixed
combination.
For example, the method of preventing or treating proliferative diseases
according to the
CA 02686587 2009-11-27
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invention may comprise: (i) administration of the first agent (a) in free or
pharmaceutically
acceptable salt form; and (ii) administration of an agent (b) in free or
pharmaceutically
acceptable salt form, simultaneously or sequentially in any order, in jointly
therapeutically
effective amounts, preferably in synergistically effective amounts, e.g., in
daily or
intermittently dosages corresponding to the amounts described herein. The
individual
combination partners of the combination of the invention may be administered
separately
at different times during the course of therapy or concurrently in divided or
single (e.g.,
fixed) combination forms. Furthermore, the term administering also encompasses
the use
of a pro-drug of a combination partner that convert in vivo to the combination
partner as
such. The instant invention is therefore to be understood as embracing all
such regimens of
simultaneous or alternating treatment and the term "administering" is to be
interpreted
accordingly.
The effective dosage of each of the combination partners employed in the
combination of
the invention may vary depending on the particular compound or pharmaceutical
composition employed, the mode of administration, the condition being treated,
the
severity of the condition being treated. Thus, the dosage regimen of the
combination of the
invention is selected in accordance with a variety of factors including the
route of
administration and the renal and hepatic function of the patient. A physician
of ordinary
skill can readily determine and prescribe the effective amount of the single
active
ingredients required to treat the disease state.
Daily dosages for combination parties (a) and (b) will, of course, vary
depending on a
variety of factors, e.g., the compound chosen, the particular disease to be
treated and the
desired effect. In general, however, satisfactory results may be achieved on
administration
of agent (a) at daily dosage rates of about 0.03 to 5 mg/kg per day,
particularly 0.1 to 5
mg/kg per day, e.g. 0.1 to 2.5 mg/kg per day, as a single dose or in divided
doses.
Combination partner (a) and agent (b) may be administered by any conventional
route, in
particular enterally, e.g., orally, e.g., in the form of tablets, capsules,
drink solutions or
parenterally, e.g., in the form of injectable solutions or suspensions.
Suitable unit dosage
forms for oral administration comprise from about 0.02 to 50 mg active
ingredient, usually
CA 02686587 2009-11-27
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0.1 to 30 mg, e.g. combination partner (a) or (b), together with one or more
pharmaceutically acceptable diluents or carriers therefore.
Combination partner (b) may be administered to a human in a daily dosage range
of 0.5 to
1000 mg. Suitable unit dosage forms for oral administration comprise from
about 0.1 to
500 mg active ingredient, together with one or more pharmaceutically
acceptable diluents
or carriers therefore.
The pharmaceutical compositions for separate administration of combination
partner (a)
and agent (b) or for the administration in a fixed combination (i.e., a
composition
comprising both (a) and (b)) according to the invention, may be prepared in a
manner
known in the art and are those suitable for enteral, such as oral or rectal,
and parenteral
administration to mammals (warm-blooded animals), particularly humans,
comprising a
therapeutically effective amount of at least one pharmacologically active
combination
partner alone, e.g., as indicated above, or in combination with one or more
pharmaceutically acceptable carriers or diluents, especially suitable for
enteral or
parenteral application.
Suitable pharmaceutical compositions contain, e.g., from about 0.1% to about
99.9%,
preferably from about 1 % to about 60%, of the active ingredient(s).
The composition may contain any suitable carriers, diluents or excipients.
These include
all conventional solvents, dispersion media, fillers, solid carriers,
coatings, antifungal and
antibacterial agents, dermal penetration agents, surfactants, isotonic and
absorption agents
and the like. It will be understood that the compositions of the invention may
also include
other supplementary physiologically active agents.
The carrier must be pharmaceutically "acceptable" in the sense of being
compatible with
the other ingredients of the composition and not injurious to the subject.
Compositions
include those suitable for oral, rectal, nasal, topical (including buccal and
sublingual),
vaginal or parental (including subcutaneous, intramuscular, intravenous and
intradermal)
CA 02686587 2009-11-27
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administration. The compositions may conveniently be presented in unit dosage
form and
may be prepared by any methods well known in the art of pharmacy. Such methods
include the step of bringing into association the active ingredient with the
carrier which
constitutes one or more accessory ingredients. In general, the compositions
are prepared
by uniformly and intimately bringing into association the active ingredient
with liquid
carriers or finely divided solid carriers or both, and then if necessary
shaping the product.
Compositions of the present invention suitable for oral administration may be
presented as
discrete units such as capsules, sachets or tablets each containing a
predetermined amount
of the active ingredient; as a powder or granules; as a solution or a
suspension in an
aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a
water-in-oil
liquid emulsion. The active ingredient may also be presented as a bolus,
electuary or
paste.
A tablet may be made by compression or moulding, optionally with one or more
accessory
ingredients. Compressed tablets may be prepared by compressing in a suitable
machine
the active ingredient in a free-flowing form such as a powder or granules,
optionally mixed
with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium
starch glycolate,
cross-linked polyvinyl pyn-olidone, cross-linked sodium carboxymethyl
cellulose) surface-
active or dispersing agent. Moulded tablets may be made by moulding in a
suitable
machine a mixture of the powdered compound moistened with an inert liquid
diluent. The
tablets may optionally be coated or scored and may be formulated so as to
provide slow or
controlled release of the active ingredient therein using, for example,
hydroxypropylmethyl
cellulose in varying proportions to provide the desired release profile.
Tablets may
optionally be provided with an enteric coating, to provide release in parts of
the gut other
than the stomach.
Compositions suitable for topical administration in the mouth include lozenges
comprising
the active ingredient in a flavoured base, usually sucrose and acacia or
tragacanth gum;
pastilles comprising the active ingredient in an inert basis such as gelatine
and glycerin, or
CA 02686587 2009-11-27
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sucrose and acacia gum; and mouthwashes comprising the active ingredient in a
suitable
liquid carrier.
Compositions suitable for topical administration to the skin may comprise the
compounds
dissolved or suspended in any suitable carrier or base and may be in the form
of lotions,
gel, creams, pastes, ointments and the like. Suitable carriers include mineral
oil, propylene
glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan
monostearate,
polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and
water. Transdermal patches may also be used to administer the compounds of the
invention.
Compositions for rectal administration may be presented as a suppository with
a suitable
base comprising, for example, cocoa butter, glycerin, gelatine or polyethylene
glycol.
Compositions suitable for vaginal administration may be presented as
pessaries, tampons,
creams, gels, pastes, foams or spray formulations containing in addition to
the active
ingredient such carriers as are known in the art to be appropriate.
Compositions suitable for parenteral administration include aqueous and non-
aqueous
isotonic sterile injection solutions which may contain anti-oxidants, buffers,
bactericides
and solutes which render the composition isotonic with the blood of the
intended recipient;
and aqueous and non-aqueous sterile suspensions which may include suspending
agents
and thickening agents. The compositions may be presented in unit-dose or multi-
dose
sealed containers, for example, ampoules and vials, and may be stored in a
freeze-dried
(lyophilised) condition requiring only the addition of the sterile liquid
carrier, for example
water for injections, immediately prior to use. Extemporaneous injection
solutions and
suspensions may be prepared from sterile powders, granules and tablets of the
kind
previously described.
Preferred unit dosage compositions are those containing a daily dose or unit,
daily sub-
dose, as herein above described, or an appropriate fraction thereof, of the
active ingredient.
CA 02686587 2009-11-27
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It should be understood that in addition to the active ingredients
particularly mentioned
above, the compositions of this invention may include other agents
conventional in the art
having regard to the type of composition in question, for example, those
suitable for oral
administration may include such further agents as binders, sweeteners,
thickeners,
flavouring agents disintegrating agents, coating agents, preservatives,
lubricants and/or
time delay agents. Suitable sweeteners include sucrose, lactose, glucose,
aspartame or
saccharine. Suitable
disintegrating agents include cornstarch, methylcellulose,
polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable
flavouring
agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry
flavouring.
Suitable coating agents include polymers or copolymers of acrylic acid and/or
methacrylic
acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
Suitable
preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic
acid, methyl
paraben, propyl paraben or sodium bisulphite. Suitable lubricants include
magnesium
stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time
delay agents
include glyceryl monostearate or glyceryl distearate.
The present invention also relates to a kit of parts comprising
(i) an amount of a compound of formula (I) or a pharmaceutically acceptable
salt,
solvate or prodrug thereof in a first unit dosage form; and
(ii) an amount of at least one other anti-proliferative selected from
allcylating
agents, antitumour antibiotics, antimetabolites, natural alkaloids and
inhibitors
of protein tyrosine kinases and/or serine/threonine kinases, in each case,
where
appropriate, a pharmaceutically acceptable salt thereof, in the form of one,
two
or three more separate unit dosage forms, optionally with instructions for use
of
(i) and (ii) components in combination for treating a proliferative disease.
Preferably the kit comprises a first unit dosage form of (i) and a second unit
dosage form
of one other anti-proliferative agent (ii).
The invention furthermore relates to a commercial package comprising the
combination
CA 02686587 2016-02-26
- 19 -
according to the present invention together with instructions for
simultaneous, separate or
sequential use.
In an embodiment, the (commercial) product is a commercial package comprising
as active
ingredients the combination according to the present invention (in the form of
two or three
or more separate units of the components as described above), together with
instructions
for simultaneous, separate or sequential use, of any combination thereof, in
the treatment
of the disease states as mentioned herein.
Those skilled in the art will appreciate that the invention described herein
is
susceptible to variations and modifications other than those specifically
described.
The scope of the claims should not be limited by the preferred embodiments and
examples, but should be given the broadest interpretation consistent with the
description as a whole. The invention also includes all of the steps,
features,
compositions and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations of any two or more
of
said steps or features.
Certain embodiments of the invention will now be described with reference to
the
following examples which are intended for the purpose of illustration only and
are not
intended to limit the scope of the generality hereinbefore described.
Examples
Synthetic Protocols
Preparation of 2-Bromo-7-acetoxy-3-(3,4,5-trimethoxybenzoy1)-6-
methoxybenzofuran.
O
Me0 Me
Me0
0
=
\ Br
Me0 0
OAc
CA 02686587 2009-11-27
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Step 1: 2-t-Butyldimethylsily1-3-(t-butyldimethylsilyloxymethylene)-6-methoxy-
7-
isopropoxybenzofuran (Larock coupling).
A suspension of 2-isopropoxy-3-methoxy-5-iodophenol (4.41 mmol), 1-(tert-
butyldimethylsily1)-3-(tert-butyldimethylsilyloxy)propyne (1.5 g, 5.28 mmol),
lithium
chloride (189 mg, 4.45 mmol) and sodium carbonate (2.34 g, 22.08 mmol) in dry
dimethylformamide (5 mL) at 100 C was deoxygenated 4 times by evacuation and
backfilling with nitrogen. Palladium acetate (135 mg, 0.60 mmol) was added and
the
reaction vessel was degassed twice with nitrogen. The reaction mixture was
then stirred at
this temperature for 4 hours (tic) and the solvent was removed by distillation
under
vacuum. The residue was dissolved in ethyl acetate (75 mL), stirred well,
filtered and
treated with triethylamine (5 mL). The solution was concentrated onto silica
gel (10 g) and
purified by flash chromatography (silica gel, eluent = hexane/diethyl
ether/triethylamine;
95:5:1%) to afforded the title compound as a yellow oil (1.45 g, 96 %); 1H NMR
(300
MHz, CDC13) 6 7.24(d, 1H, J = 8.45 Hz), 6.88(d, 1H, J = 8.47 Hz), 4.80(s, 2H,
CH2),
4.73(m, 1H), 3.88(s, 3H, OMe), 1.36(d, 6H, J= 6.17 Hz), 0.94(s, 9H), 0.92(s,
9H), 0.35(s,
6H), 0.12(s, 6H).
Step 2: 24-Butyldimethylsily1-3-formy1-6-methoxy-7-isopropoxybenzofuran
To a solution of 2-t-butyldimethy Isily1-3-(t-butyldimethylsilyloxymethylene)-
6-methoxy-
7-isopropoxybenzofuran (2.69 mmol) in methanol (100 mL) was added concentrated
hydrochloric acid (200 viL) and the reaction was stirred for 30 minutes
(monitored by tic),
quenched with triethylamine (2 mL) and the solvent removed by distillation
under vacuum.
The residue was dissolved in dichloromethane (20 mL), washed with water (10
mL), dried
over magnesium sulfate, concentrated under vacuum and co-distilled with
toluene (20 mL).
The crude product was dissolved in dry dichloromethane (4 mL) and added to a
stirred
solution of Collins reagent (chromium trioxide (1.01 g), pyridine (1.65 mL) in
dry
dichloromethane (30 mL)). The suspension was stirred for 10 minutes, filtered
and the
residue washed with diethyl ether (20 mL). The filtrate was concentrated onto
silica (10 g)
and purified by flash chromatography (silica gel, eluent = hexane/diethyl-
ether/triethylamine (90:9:1) to afford the title compound as a light yellow
oil (503 mg,
48%); 1H NMR (300 MHz, CDC13) 6 10.25(s, 1H, CHO), 7.79(d, 1H, J= 8.45 Hz),
6.98(d,
CA 02686587 2009-11-27
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1H, J = 8.46 Hz), 4.65(m, 1H), 3.89(s, 3H, OMe), 1.35(d, 6H, J = 6.17 Hz),
0.97(s, 9H),
0.45(s, 6H).
Step 3: 2-t-
Butyldimethylsily1-3-(3,4,5-trimethoxybenzoy1)-6-methoxy-7-
isopropoxybenzofuran
To a stirred solution of 3,4,5-trimethoxyiodobenzene (377 mg, 1.27 mmol) in
dry
tetrahydrofuran (1 mL) at -78 C under nitrogen was added n-butyllithium (795
L, 1.59
mmol, 2M solution in cyclohexane) and the reaction mixture was stirred at this
temperature for 40 minutes. After this time a solution of 2-t-
butyldimethylsily1-3-formy1-6-
methoxy-7-isoproxybenzofuran (1.07 mmol) in dry tetrahydrofuran (1 mL) was
added to
the reaction dropwise via syringe pipette. The reaction mixture was stirred at
-60 C for 20
minutes and then allowed to warm to 0 C, stirred for 10 minutes, quenched with
saturated
ammonium chloride solution (2 mL) and diluted with ethyl acetate (20 mL). The
organic
layer was washed with water (10 mL), dried over magnesium sulfate and the
solvent was
removed under vacuum to give a residue that was co-distilled with toluene. The
crude
product (908 mg) was dissolved in dry tetrahydrofuran (10 mL) and treated with
2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (900 mg, 1.59 mmol) was added. The
reaction
mixture was stirred at room temperature for 16 hours (monitored by tic) and
then loaded
onto silica (10 g) and purified by flash chromatography (silica gel, eluent =
hexane/diethyl
ether/triethylamine, 90:9:1) to afford the title compound as a light yellow
oil (498 mg,
69%); NMR (300
MHz, CDC13) 6 7.14(s, 2H, benzoyl Hs), 6.81(d, 1H, J= 8.64 Hz),
6.77(d, 1H, J = 8.64 Hz) 4.74(m, 1H), 3.93(s, 3H, OMe), 3.86(s, 3H, OMe),
3.78(s, 6H, 2 x
OMe), 1.39(d, 6H, J = 6.14 Hz), 1.01(s, 9H), 0.26(s, 6H).
Step 4: 2-(tert-butyldimethylsilyloxy)-7-acetoxy-3-(3,4,5-trimethoxybenzoy1)-6-
methoxybenzofuran
To a stirred solution of 2-(t-butyldimethylsilyloxy)-7-isopropoxy-3-(3,4,5-
trimethoxybenzoy1)-6-methoxy-benzofuran (160 mg, 0.31 mmol) in dry DCM (2 mL)
at
room temperature under nitrogen was added solid aluminium trichloride (83 mg,
0.62
mmol) and the reaction mixture was stirred for 15 minutes (monitored by tic).
The
reaction was quenched with a saturated solution of ammonium chloride,
extracted with
CA 02686587 2009-11-27
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dichloromethane and dried over magnesium sulfate. The solvent was removed by
distillation and residue was dried by azeotropic removal of water with
toluene. The crude
product was dissolved in pyridine (2 mL), acetic anhydride (1 mL) was added
and reaction
mixture was stirred for 2 hours at room temperature. The solvent was distilled
under
vacuum and the residue was loaded onto silica gel (1 g) and purified by column
chromatography (silica gel, eluent, hexane:diethyl-ether; 80:20) (134 mg,
84%); 1H NMR
(300 MHz, CDC13) 6 7.14(s, 2H, benzoyl Hs), 6.98(d, 1H, J = 8.72 Hz), 6.85(d,
1H, J =
8.72 Hz), 3.93(s, 3H, OMe), 3.86(s, 3H, OMe), 3.80(s, 6H, 2 x OMe), 2.41(s,
3H), 0.99(s,
9H), 0.25(s, 6H).
Step 5: 2-Bromo-7-acetoxy-3-(3,4,5-trimethoxybenzoy1)-6-methoxybenzofuran
To a stirred solution of 2-t-butyldimethylsily1-7-acetoxy-3-(3,4,5-
trimethoxybenzoy1)-6-
methoxybenzofuran (120 mg, 0.44 mmol) in 1,2-dichloroethane (1 mL) at room
temperature under nitrogen was added bromine (12 ul, 0.44 mmol) dropwise and
the
reaction mixture was stirred at this temperature for 10 minutes. After this
time the reaction
was quenched with saturated sodium thiosulfate solution, extracted with ethyl
acetate (20
mL), dried over magnesium sulfate and the solvent removed by distillation
under vacuum.
The crude product was purified by silica gel column chromatography (eluent =
Hexane:diethyl ether; 8:2 - 7:3) to afford the title compound as a colourless
crystalline
solid (91 mg, 81%); 11-1 NMR (300 MHz, CDC13) 6 7.40(d, 1H, J= 8.70 Hz),
7.14(s, 2H,
benzoyl-Hs), 6.98(d, 1H, J= 8.75 Hz), 3.94(s, 3H, OMe), 3.89(s, 3H, OMe),
3.86(s, 6H, 2
x OMe), 2.43(s, 3H); 13C NMR (75 MHz, CDC13) 6 187.95(C0), 167.71, 152.75,
149.54,
147.49, 142.59, 131.92, 131.80, 123.91, 121.84, 119.89, 117.72, 109.89,
106.92, 60.69,
56.61, 56.00, 20.09.
Example 1
Preparation of 2-Methy1-7-hydroxy-3-(3,4,5-trimethoxybenzoy1)-6-
methoxybenzofuran
CA 02686587 2009-11-27
- 23 -
O
Me0 Me
Me0
0
Me0
OH
Preparation A
To a stirred solution of 2-Bromo-7-acetoxy-3-(3,4,5-trimethoxybenzoy1)-6-
methoxybenzofuran (20 mg, 0.042 mmol), methyl-boronic acid (40 mg, 0.67 mmol),
in 1,4-
dioxane (2 mL) at 90 C was added tetrakis-triphenylphosphine palladium (11
mg, 0.01
mmol) followed by the addition of a solution of sodium bicarbonate (40 mg,
0.48 mmol) in
distilled water (0.5 mL). The reaction mixture turned red after 5 minutes.
After 2 hours
(tic) the reaction mixture was brought to room temperature and was added
saturated
ammonium chloride (2 mL) and diluted with dichloromethane (20 mL). The organic
layer
was separated and washed with water, dried over magnesium sulfate and the
solvent was
removed by distillation under vacuum. The residue was purified by PTLC (eluent
=
Dichloromethane/Methanol, 1:1) to give the title compound (acetate cleaved
during
reaction) as a fluffy white solid; (3 mg, 19%).
Preparation B (Negishi Coupling)
To a stirred solution of zinc-bromide (592 mg, 2.63 mmol) in dry THF(1.5 mL)
at 0 C was
added the solution of methyl lithium (1.6 M solution in diethyl-ether, 2,6 mL,
4.15 mmol)
and the reaction mixture was stirred for 2 hours. Solid 2-bromo-7-acetoxy-3-
(3,4,5-
trimethoxybenzoy1)-6-methoxy-benzofuran (300 mg, 0.63 mmol) was added and the
ether
was removed under vacuum and to the rest suspension was added
dichlorobis(triphenylphosphine)palladium catalyst (21 mg) and catalytic amount
of copper
(I) iodide. The reaction mixture was stirred at room temperature for 36 hours
(monitored
by tic), quenched with saturated ammonium chloride solution and extracted with
dichloromethane (10 mL), dried over magnesium sulfate and solvent distilled
under
CA 02686587 2009-11-27
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vacuum and the product was purified by silica gel column (eluent =
hexane/ethyl acetate;
8:2). The product was crystallized in methanol (106 mg, 46%); 11-1 NMR (300
MHz,
CDC13) 6 7.09(s, 2H, benzoyl Hs), 6.93(d, 1H, J = 8.54 Hz), 6.83(d, 1H, J =
8.56 Hz),
5.70(bs, 1H, OH), 3.93(s, 3H, OMe), 3.92(s, 3H, OMe), 3.83(s, 6H, 2 x OMe),
2.54(s, 3H,
2-Me)
Example 2
Preparation of Disodium 6-methoxy-2-
methy1-3-(3,4,5-
trimethoxybenzoyl)benzofuran-7-y1 phosphate
Me0 OMe
Me0
0
101
Me0
0, /0Na
(/), ONa
Step 1: Dibenzyl 6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyDbenzofuran-7-y1
phosphate:
To a mixture of 0.081 g (0.22 mmol) of (7-hydroxy-6-methoxy-2-methylbenzofuran-
3-
yl)(3,4,5-trimethoxyphenyl)methanone, 0.086 g (0.261 mmol) of carbon
tetrabromide and
0.063 ml (0.283 mmol) of dibenzylphosphite in 2.5 ml of anhydrous acetonitrile
0.046 ml
of anhydrous triethylamine was added dropwise at 0 C under nitrogen
atmosphere. The
resulting mixture was stirred for 2h at room temperature, then diluted to 20
ml with ethyl
acetate, washed with water brine, dried over anhydrous magnesium sulfate,
filtered off and
evaporated to dryness under reduced pressure. The residue was purified by
flash column
chromatography (dichloromethane/ ethyl acetate, 9:1) to give the title
compound as a
colorless foam (0.13g, 94%); 11-1 NMR (CDC13) 6 2.42 (s, 3H, Me-2); 3.83 (s,
1H, OMe);
3.93 (s, 3H, OMe); 5.33 (m, 4H, CH2Ph); 6.89 (d, CH aromatic, J= 8.7 Hz); 7.21
(dd, 1H,
CH aromatic, J= 8.72 Hz; J= 1.2 Hz); 7.08 (s, 2H, CH aromatic); 7.29 ¨ 7.43
(m, 10 H,
CH aromatic).
CA 02686587 2009-11-27
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Step 2: Disodium 6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyDbenzofuran-7-y1
phosphate:
To a stirred solution of 0.122 g (0.193 mmol) of the product from Step 1 in 1
ml of
anhydrous acetonitrile 0.075 ml (0.58 mmol) of bromotrimethylsilane was added
at ¨5 C
under nitrogen atmosphere. The resulting mixture was stirred for 1 h at 0 C,
then
evaporated to dryness in vacuo. The residue was diluted to 5 ml with anhydrous
methanol
and pH of the solution was brought up about 10 by the addition of sodium
methoxide.
After evaporation of the resulting mixture under reduced pressure the solid
residue was
washed with anhydrous isopropanol (4 x 1.5 ml) and anhydrous ethanol (3 x 1.5
ml) and
dried under vacuum to give 0.062 g (65 % yield) of title compound as an
colorless solid;
IH NMR (D20) 6 2.37 (s, 3H, Me-2); 3.76 (s, 6H, OMe); 3.79 (s, 3H, OMe); 3.82
(s, 3H,
OMe); 4.66 (s, H20); 6,93 (d, 1H, CH aromatic, J= 8.6 Hz); 7.04 (d, 1H, CH
aromatic, J
= 8.6 Hz); 7.10 (s, 2H, CH aromatic).
Biological data
(A) (i) In Vitro Studies for combination partner (a)
Table 1: In Vitro Data for Compounds: These are the results for growth
inhibition
studies of compounds using the Sulforhodamine B (SRB) or Systmex cell counting
(CC)
assays. IC50 is the concentration required to inhibit net cell growth by 50%.
Entries 1-4
are provided for comparison, entry 5 is a compound of the invention
(combination partner
(a)).
Entry Example/ Structure Cancer HUVECs`
Comparator cell Tun): IC50, nM
line': Norm: IC50,
IC50, nM
nM
CA 02686587 2009-11-27
- 26 -
Entry Example/ Structure Cancer HUVECs`
Comparator cell Turn: IC50, nM
line': Norm: ICso,
1C5o, nM
nM
1. Comparator OMe 5 Turn: 1-10
A is OMe Norm: 1-10
OMe
1 si OH
OMe
2. Comparator OMe 5 Turn: 1-10
B is OMe Norm: 1-10
OMe ONa
I L-ONa
S
0
OMe
3. Comparator Me0 OMe 55 Tum: 10-100
C Norm: 10-100
Example 5
Me0 .
0
Me0
4. Comparator Me0 OMe 500 Turn: 100-1000
D Norm: 100-1000
Example 3 Me0 4It
o
0 \ Br
Me0 0
5. Example 1 Me0 OMe 2.0 Turn: 0.1-1
Norm: 10-100
Me0 4Ik
0
el \ Me
Me0
OH
CA 02686587 2009-11-27
- 27 -
a
Unless otherwise stated the cancer cell line is MCF-7. For description of
method of
MCF-7 inhibition see: Verdier-Pinard, P etal. Mol. Pharmacol 1998, 53, 62-76
Human umbilical vein endothelial cells (HUVECs) tumour type activated
endothelial cells (Turn) and normal quiescent type endothelial cells (Norm).
General Description of Biological Experiments:
Proliferation Assay ¨ quiescent endothelium: Human umbilical vein endothelial
cells
(CC-2519, Clonetics) were plated at 15000 cells/well in EBM2 (CC-3156,
Clonetics) +
0.5%FBS (CC-4101A, Clonetics) + GA-1000 (CC-4381A, Clonetics) in a 96 well
plate in
triplicate. Cells were cultured overnight at 37 C 5% CO2. Medium was
subsequently
replaced with fresh medium including the compound or negative control. Cells
were
cultured for a period of 48hrs. An MTT assay was performed to measure changes
in cell
numbers. Briefly, 20111 of MTT reagent was added to cells containing 100u1 of
EBM2 +
0.5%FBS and incubated at 37 C for 2 hours. Absorbance was measured at 492nm.
Proliferation Assay ¨ activated endothelium: Human umbilical vein endothelial
cells
(CC-2519, Clonetics) were plated at 2500 cells/well in EGM2 (CC-3162,
Clonetics) in a
96 well plate in triplicate. Cells were cultured overnight at 37 C 5% CO2.
Medium was
subsequently replaced with fresh medium including the compound or negative
control.
Cells were cultured for a period of 48hrs. An MTT assay was performed to
measure
changes in cell numbers. Briefly, 201.11 of MTT reagent was added to cells
containing
100111 of EGM2 and incubated at 37 C for 2 hours. Absorbance was measured at
492nm.
(ii) In Vivo Studies for combination partner (a)
Vascular Disruption Assay: Female athymic BALB/c-nu/nu mice (nude mice) were
used
for this study. Mice were between 6-8 weeks old and were purchased from the
Animal
Resource Centre, Perth, Western Australia and allowed to acclimatize for a
couple of days.
All the animals were housed under pathogen-free conditions and cared for in
accordance
with Flinders University of South Australia and NH&MRC guidelines and the
Australian
CA 02686587 2009-11-27
- 28 -
Code of Practice for the care and use of animals for scientific purposes. The
human breast
cancer MDA MB 231 was grown as orthotopic xenografts in the mammary fat pad of
nude
mice. Each mouse was injected with 2 x 106 cells in 501.d Dulbecco's PBS
subcutaneously
just above the mammary fat pad, below the right forward limb. Tumours were
selected for
treatment when they reached a diameter of 100-150 mm3 (3 weeks after
implantation). The
test compound (Example 2) was dissolved in saline solution and injected
intravenously at
concentrations ranging from 150 mg/kg ¨ 1 mg/kg in a total volume of 400u1.
Tumour
bearing animals were injected intravenously with 10mg/kg Hoechst 33342, 24
hours after
the injection of the test compound. Animals were euthanised I minute after the
Hoechst
33342 injection. Tumours were recovered for histochemical analysis. Tumour
perfusion
analysis was performed by assessing the amount of Hoechst 33342 staining
across an entire
tumour cross-section. 10 micron sections of frozen tumour biopsies were viewed
under an
ultraviolet light filter. Using a 4x objective lens, 8-bit monochromatic
images were
captured in succession, representing the total area of the tumour section.
Composite
images of the total tumour section were generated by overlaying common areas
of the
monochromatic images. Hematoxylin and Eosin-Y staining of the same tumour
section
was performed to identify non-tumour regions. Non-tumour regions were mapped
on
Hoechst 33342 composite images and excluded from the quantitation analysis.
Quantitation was performed by measuring the pixel area of Hoechst 33342
staining and the
total pixel area of the tumour region. Perfusion was expressed as a percentage
of Hoechst
33342 stained area to total tumour area (see Figure 5).
Tumour Growth Inhibition: Balb/c nu/nu mice bearing MDA-MB-231 solid
orthotopic
tumours were treated with compound Example 2 at 40mg/kg. Animals were i.v.
dosed with
a total of two cycles of Example 2 treatment. Each cycle was dosing on days
and 8
followed by a three week no-dosing period. Tumour growth represented as a
ratio to initial
tumour volume is shown over a total of 72-days.
Tumour growth as well as animal health were monitored for up to 72 days post-
day 1 of
treatment. The results seen in this experiment (see Figure 6) clearly show
tumour growth
inhibition in animals treated with two cycles of Example 2. Significant
differences in
CA 02686587 2009-11-27
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tumour growth between Example 2 treated (n=64) and vehicle treated (n=20)
animals were
observed as early as day 4 (p<0.001; unpaired t-test; Prism analysis) through
to Day 70.
(B) (i) In vitro studies for combinations of (a) and (b)
Combination Index Values
Human cancer cell lines (list cell lines: MDA-MB-231, U87, HT29, Calu-6, SKOV-
3,
BxPC-3, DU145, 786-0, Lovo, FaDu) were used to evaluate combination treatments
in
vitro. Analysis was based on measurements of in vitro cell proliferation in
the presence of
the compounds under evaluation. Cells were seeded at an average of 500-2000
cells/well in
96 well plates allowed to adhere overnight before addition of the test
compounds. Cell
proliferation was assessed after 48-72hrs of culture in the presence of the
test substances.
Cells were treated with a combination of Example 1 and the compound under
evaluation, or
with each of these agents alone. Proliferation measurements were carried out
by a
tetrazolium-based colorimetric assay (MTS). Metabolically active cells were
measured
using CellTiter 96 Aqueous One Solution (Promega Corp. Madison WI, USA)
according
to the manufacturers instructions and absorbance readings taken at 492nm.
Absorbance
readings for each compound concentration were normalized to corresponding
vehicle
control cultures. A sigmoidal dose response curve was fitted to the data, and
the
concentration at which proliferation decreased by 50% was calculated using
Graph Pad
Prism 4 software (San Diego, USA). ED50 data derived from these evaluations
were
analyzed using the quantitative software CaluSyn to determine the combination
index (CI)
(Chou 2006 Theoretical Basis, Experimental Design and Computerised Simulation
of
Synergism and Antagonism in Drug Combination Studies, Pharmacological Reviews
58
(3): 621-681). Based on their CI, compounds were classified as antagonistic,
additive or
synergistic combination partners for Example 1. The results are tabulated in
Figure 1.
CalcuSyn is a dose effect analyzer program for single and multiple drugs. It
uses the
Median Effect method to quantify the effects of drug combinations to determine
whether
they give greater effects together that expected from a simple summation of
their
individual effects. Data was processed for individual drugs and for constant
ratio
CA 02686587 2009-11-27
- 30 -
combinations. The program automatically graphs the data and produces reports
of
summary statistics for all drugs and detailed analysis of drug interactions
including the
Combination Index and EDx (effective dose).
Cell culture and cell lines. Cancer cell lines included Calu-6, DU145, U87-MG,
BxPc-3,
HT29, Sk-OV-3, 786-0, LoVo (ATCC, Manassas, VA, USA), FaDu (kind gift from
Peter
Mac, Melbourne, AU) and MDA-MB-231 (kind gift from WCH, Adelaide, AU).
Calu-6, DU145 and U87-MG cells were cultured in MEM media (Gibco0) with 10%
FCS,
2mM penicillin-streptomycin-glutamine (Gibco0), 10mM Hepes buffered solution
(Gibco0), 1mM sodium pyruvate solution (Gibco0) and 0.1mM non-essential amino
acids
solution (Gibco0). FaDu cells were cultured in RPMI 1640 media (Gibco0) with
10%
FCS and 10mM Hepes buffered solution. Bx-PC-3 cells were cultured in RPMI 1640
media with 10% FCS, 2mM penicillin-streptomycin-glutamine and 1mM sodium
pyruvate
solution. MDA-MB-231 cells were cultured in RPMI 1640 media with 10% FCS, 2mM
penicillin-streptomycin-glutamine and 10mM Hepes buffered solution. HT29 cells
were
cultured in MEM media with 10% FCS and 2mM penicillin-streptomycin-glutamine.
SK-
OV-3 cells were cultured in DMEM/F12 (Gibco0) media with 10% FCS, 2mM
penicillin-
streptomycin-glutamine and 10mM Hepes buffered solution. 786-0 cells were
cultured in
in RPMI 1640 media with 10% FCS, 2mM penicillin-streptomycin-glutamine, 10mM
Hepes buffered solution and 1mM sodium pyruvate solution and 0.1mM non-
essential
amino acids solution. Lovo cells were cultured in F 12K (Gibco0) media with
10% FCS,
2mM penicillin-streptomycin-glutamine and 10mM Hepes buffered solution.
All cells were cultured in a humidified incubator at 37 C with 5% CO2.
(B) (ii) In vivo studies for combinations of (a) and (b)
(a) Example 2 treatment in combination with Cisplatin (combination partner
(b))
Balb/c nu/nu mice were inoculated with 2 x 106 Calu-6 cells (human lung
carcinoma line)
subcutaneously. Once mean tumour size had reached 150mm3 mice were treated
with
cisplatin on day 1 at 4mg/kg, weekly treatment of Example 2 at 10mg/kg
starting on day 2
CA 02686587 2009-11-27
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or a combination of both drugs (n=10/group). Tumour volume measurement were
taken
throughout the treatment period and statistical analysis performed on complete
groups.
Survival was assessed throughout the trial and is shown below. Average weight
loss in
combined treatment group did not exceed acceptable levels. The results are
illustrated in
Figure 2 and Figure 3.
(b) Example 2 treatment in combination with 5-Fluorouracil or Doxorubicin
(combination partner (b))
Balb/c nuinu mice were inoculated with 2 x 106 MDA-MB-231 cells (human breast
carcinoma line) subcutaneously. Once mean tumour size had reached 50-100mm3
mice
were treated with Example 2 (10mg/kg) as a single i.v. dose every 96 hrs
followed on the
next day by either a saline injection or injection of the chemotherapeutics
Doxorubicin
(5mg/kg) or 5-Fluorouracil (50mg/kg). Tumour volume measurements were taken
throughout the treatment period and are expressed as a ratio relative to
starting volume on
day 1. Results are illustrated in Figure 4.
