Note: Descriptions are shown in the official language in which they were submitted.
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COMBINATION PREPARATION OF A BIOLOGICAL RESPONSE MODIDIER AND AN ANTICANCER
AGENT AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to anticancer biological response modifier
combinations,
pharmaceutical compositions comprising the same, and the use thereof in the
treatment of cancer.
BACKGROUND OF THE INVENTION
There are a number of therapies directed towards the treatment of cancer,
including
chemotherapeutic drugs, radiation, gene therapy and antisense
oligonucleotides. One drawback to
current therapies is the toxicity associated with most treatments. Moreover,
oftentimes large
dosages must be administered over an extended period of time in order to
attain therapeutic benefit.
Thus, a need remains for more effective treatments.
A bile extract has been prepared that is known to be able to modify the
biological response
of cells of the immune system. The production and characterization of this
bile-derived Biological
Response Modifier (BD-BRM) has been described in International Patent
Application Serial No.
PCT/CA94/00494, published February 16, 1995 as WO 95/07089, International
Patent
Application Serial No. PCT/CA96/00152, published September 19, 1996 as WO
96128175 and
U.S. Patent No. 6,280,774. The use of this immunomodulatory composition as an
anti-viral has
been described in International Patent Application Serial No. PCT/CA98/00494,
published
2 0 November 26, 1998 as WO 98/52585. These applications are herein
incorporated by reference in
their entirety.
The BD-BRM composition is composed of small molecular weight components of
less than
3000 daltons, and has one or more of the following properties:
a) is extracted from bile of animals;
2 5 b) is capable of stimulating monocytes and/or macrophages in vitro and/or
in vivo;
c) is capable of modulating tumor necrosis factor production and/or release;
1
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d) contains no measurable level of IL-la, IL-Iii, TNF, IL-6, IL-8, IL-4, GM-
CSF or IFN-'y;
e) shows no cytotoxicity to human peripheral blood mononuclear cells or
lymphocytes; and
f) is not an endotoxin.
The bile-derived biologic response modifier (BD-BRM) is a composition that has
been
hypothesized to exert anti-tumour activity via the activation of macrophages,
with subsequent
enhancement of cell-mediated i'rnmune response to tumours. Its precise
mechanism of action
remains unknown.
The cumulative results of studies with BD-BRM revealed following:
(1) BD-BRM does not directly stimulate lymphocytes to synthesize DNA or
undergo
blastogenesis and cell division. BD-BRM does not directly stimulate the
development of
lymphocyte-mediated cytotoxicity.
(2) BD-BRM can stimulate normal peripheral blood monocytes to express
cytocidal activity in
a dose-dependent manner. The activity elicited by BD-BRM is equal to or
greater than the
activity produced in response to more conventional macrophage activators that
are currently
under investigation in cancer patients including: Gamma Interferon;
Granulocyte-Monocyte
Colony Stimulating Factor; Monocyte Colony Stimulating Factor; and Interleukin-
12.
(3) BD-BRM can stimulate both the peripheral blood monocytes and regional,
tumour-
associated macrophages from cancer patients to express significant cytocidal
activity. This
included peritoneal macrophages from women with gynaecological malignancies
and
2 0 alveolar macrophages from patients with lung cancer. BD-BRM has been found
to
stimulate macrophages from cancer patients to kill autologous and heterologous
tumour cells
obtained from surgical specimens of patients. Of potentially greater
importance is the
finding that BD-BRM can often stimulate cancer patient macrophages that are
unresponsive
to stimulation with conventional activators such as gamma interferon +
endotoxin.
2
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(4) The hypersecretion of prostaglandins, both by macrophages and by tumor
cells from cancer
patients has been shown to be a principal cause of the immunosuppression seen
in patients
with advanced malignant disease. One determinant of the biological activity of
different
macrophage activators in cancer patients PBMs, therefore, is the sensitivity
of the activator
to arachidonic~acid metabolism and the secretion by the cell of
prostaglandins: The
development of macrophage cytocidal function in response to BD-BRM was found
to be
insensitive to the inhibitory effects of prostaglandins. This is considered
important
therapeutically because the effectiveness of many other biological activators
is limited by
prostaglandins.
(5) BD-BRM can stimulate cytocidal function in macrophages obtained from
cancer patients
(including pancreatic cancer) who are undergoing cytotoxic therapy. Of note is
the fact that
BD-BRM was more effective in stimulating tumouricidal function than
conventional
activators such as gamma interferon plus endotoxin.
(6) BD-BRM can also stimulate cytocidal function in macrophages obtained from
patients with
Kaposi's sarcoma even at very late stages of the disease. Thus, the action of
BD-BRM
appears to be independent of the need for collaboration with other immune cell
types
including helper T-lymphocytes.
(7) The macrophage cytocidal function that develops in response to BD-BRM may
be
associated with the expression of TNFa by the macrophages. However, other
mechanisms
2 0 for cytotoxicity may also be involved. The BD-BRM composition from bovine
sources
promotes the release of TNF from human peripheral blood mononuclear cells and
from the
pre-monocyte cell line U-937 in what appears to be physiological quantities.
Because TNF
is known to initiate a cascade of inflammatory and antitumor cytokine effects,
the
composition could exert its antineoplastic effect by stimulating human
leukocytes to release
2 5 TNF (and possibly other cytokines).
(8) Demonstrates anti-tumour activity in a mouse tumour (plasrnacytoma) model.
3
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(9) Exhibits no toxicity in animals at doses up to 125 X the doses used
inhuman toxicity studies
with no LD5° yet reached in toxicity studies.
(10) Induces the phenomenon of apoptosis in some continuous cell lines.
(k) Is non-cytotoxic to human PBMNs and lymphocytes. The survival of human
peripheral
blood mononuclear cells (PBMNs) and lymphocytes is not affected by BD-BRM.
The central hypothesis guiding investigations of the BD-BRM composition is
that the
therapeutic efficacy of a powerful biological stimulator can depend on its
ability to elicit suitable
modulation of the immune system, such as by activating macrophages and/or
monocytes to produce
certain cytokines or promote activity to seek and remove or destroy disease-
causing viruses or cells
negatively affected by such viral infections. Such function could be generated
by direct stimulation
of resident immune cells in tumour microenvironments. Alternatively, this
function could be
generated by stimulation of circulating immune cells if those cells were then
able to home in on
tumour sites and to function in that environment.
This background information is provided for the purpose of making known
information
believed by the applicant to be of possible relevance to the present
invention. No admission is
necessarily intended, nor should be construed, that any of the preceding
information constitutes
prior art against the present invention. Publications referred to throughout
the specification are
hereby incorporated by reference in their entireties in this application.
2 0 SUMMARY OF THE INVENTION
An object of the present invention is to provide anticancer biological
response modifier
combinations. In accordance with an aspect of the present invention, there is
provided a
combination comprising: (i) a composition comprising small molecular weight
components of less
than 3000 daltons, and having the following properties: is extracted from bile
of animals; is capable
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of stimulating monocytes and/or macrophages in vitro and/or in vivo; is
capable of modulating
tumor necrosis factor production and/or release; contains no measurable level
of IL-la, IL-lei,
TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; is not cytotoxic to human
peripheral blood
mononuclear cells; is not an endotoxin; and (ii) one or more anticancer
agent(s), wherein said
combination has therapeutic synergy or improves the therapeutic index in the
treatment of cancer
over the composition or the anticancer agents) alone. Another aspect of the
present invention
provides the use of this combination in the manufacture of a medicament or a
pharmaceutical kit.
In accordance with another aspect of the invention, there is provided a
pharmaceutical kit
comprising: (i) a dosage unit of a composition and a pharmaceutically
acceptable carrier wherein the
composition comprises small molecular weight components of less than 3000
daltons, and has the
following properties: is extracted from bile of animals; is capable of
stimulating monocytes and/or
macrophages in vitro and/or ih vivo; is capable of modulating tumor necrosis
factor production
and/or release; contains no measurable level of IL-la, IL-1~, TNF, IL-6, IL-8,
IL-4, GM-CSF or
IFN-gamma; is not cytotoxic to human peripheral blood mononuclear cells; is
not an endotoxin; and
(ii) a dosage unit of one or more chemotherapeutic drugs) and a
pharmaceutically acceptable
carrier, (i) and (ii) being provided in amounts that are effective, in
combination, for killing tumour o~
metastatic cells:
In accordance with another aspect of the invention, there is provided a
pharmaceutical
composition comprising: (i) a composition comprising small molecular weight
components of less
2 0 than 3000 daltons, and having the following properties: is extracted from
bile of animals; is capable
of stimulating monocytes and/or macrophages ih vitro and/or in vivo; is
capable of modulating
tumor necrosis factor production and/or release; contains no measurable level
of IL-1 a, IL-1 (3,
TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; is not cytotoxic to human
peripheral blood
mononuclear cells; is not an endotoxin; (ii) one or more chemotherapeutic
drug(s); and (iii) a
2 5 pharmaceutically acceptable carrier; wherein said pharmaceutical
composition has therapeutic
synergy or improves the therapeutic index in the treatment of cancer over the
composition or the
chemotherapeutic drugs) alone.
In accordance with another aspect of the invention, there is provided a
combination for use
in the treatment of cancer, comprising: (i) a composition comprising small
molecular weight
3 0 components of less than 3000 daltons, and having the following properties:
is extracted from bile of
5
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WO 02/38164 PCT/CA01/01558
animals; is capable of stimulating monocytes and/or macrophages ire vitro
and/or in vivo; is
capable of modulating tumor necrosis factor production and/or release;
contains no measurable
level of IL-1 a, IL-1 /3, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; is not
cytotoxic to human
peripheral blood mononuclear cells; is not an endotoxin; and (ii) one or more
anticancer agent(s),
wherein said combination has therapeutic synergy or improves the therapeutic
index in the treatment
of cancer over the composition or the anticancer agents) alone.
In accordance with another aspect of the invention, there is provided a method
for treating
cancer, comprising the step of administering a therapeutically effective
amount of a combination
comprising: (i) a composition comprising small molecular weight components of
Iess than 3000
daltons, and having .the following properties: is extracted from bile of
animals; is capable of
stimulating monocytes and/or macrophages ih vitro and/or in vivo; is capable
of modulating tumor
necrosis factor production and/or release; contains no measurable level of IL-
la, IL-1(3, TNF, IL-
6, IL-8, IL-4, GM-CSF or IFN-gamma; is not cytotoxic to human peripheral blood
mononuclear
cells; is not an endotoxin; and (ii) one or more anticancer agent(s), wherein
said combination has
therapeutic synergy or improves the therapeutic index in the treatment of
cancer over the
composition or the anticancer agents) alone.
BRIEF DESCRIPTION OF THE DRAWINGS
Further details of the invention are described below with the help of the
examples illustrated in the
accompanying drawings in which:
2 0 Figure 1 is a graph showing dose response of the composition of the
invention in stimulating
peripheral blood monocyte function.
Figure 2 illustrates the growth of Human Pancreatic Adenocarcinoma (BxPC-3) in
CD-1 Nude
Mice.
Figure 3 illustrates the weight of Human Pancreatic Adenocarcinoma (BxPC-3) in
CD-1 Nude
2 5 Mice.
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Figure 4 illustrates the growth of Human Pancreatic Carcinoma (SU.86.86.) in
CD-1 Nude Mice.
Figure 5 illustrates the weight of Human Pancreatic Carcinoma (SU.86.86.) in
CD-1 Nude Mice.
Figure 6 illustrates the growth of Human Melanoma(A2058) in CD-1 Nude Mice.
Figure 7 illustrates the weight of Human Melanoma(A2058) in CD-1 Nude Mice.
Figure 8 illustrates the growth of Human Melanoma(C8161) in CD-1 Nude Mice.
Figure 9 illustrates the weight of Human Melanoma(C8161) in CD-1 Nude Mice.
Figurel0 illustrates the growth of Human Breast Adenocarcinoma(MDA-MB-231) in
CD-1 Nude
Mice.
Figurell illustrates the weight of Human Breast Adenocarcinoma (MDA-MB-231) in
CD-1 Nude
Mice.
Figure 12 illustrates the growth of Human Breast Adenocarcinoma (MDA-MB-231)
in CD-1
Nude Mice.
Figure 13 illustrates the weight of Human Breast Adenocarcinoma (MDA-MB-231)
in CD-1
Nude Mice.
Figure 14 illustrates the growth of Human Prostate Carcinoma (PC-3) in SCID
Mice.
Figure 15 illustrates the weight of Human Prostate Carcinoma (PC-3) in SCID
Mice.
Figure 16 illustrates the growth of Human Pancreatic Carcinoma (BxPC-3) in CD-
1 Nude Mice.
7
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WO 02/38164 PCT/CA01/01558
Figure 17 illustrates the weight of Human Pancreatic Carcinoma (SU.86.86) in
CD-1 Nude Mice.
Figure 18 illustrates the growth of Human Prostate Carcinoma (DU145) in SCID
Mice.
Figure 19 illustrates the weight of Human Prostate Carcinoma (DU145) in SCID
Mice.
Figure 20 illustrates the growth of Human Ovary Adenocarcinoma (SK-OV-3) in CD-
1 Nude
Mice.
Figure 21 illustrates the growth of Human Ovary Adenocarcinoma (SK-OV-3) in CD-
1 Nude
Mice.
Figure 22 illustrates the growth of Human Lung Adenocarcinoma (H460) in CD-1
Nude Mice.
Figure 23 illustrates the weight of Human Lung Adenocarcinoma (H460) in CD-1
Nude Mice.
Figure 24 illustrates the growth of Human Small Cell Lung Carcinoma (H209) in
SLID Mice.
Figure 25 illustrates the weight of Human Small Cell Lung Carcinoma (H209) in
SLID Mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides anticancer biological response modifier (BD-
BRM)
combinations. The combination comprises (i) a composition comprising small
molecular weight
components of less than 3000 daltons, and having the following properties: is
extracted from bile of
animals; is capable of stimulating monocytes and/or macrophages ih vitro
and/or i~a vivo; is
capable of modulating tumor necrosis factor production and/or release;
contains no measurable
level of IL-la, IL-lei, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma; is not
cytotoxic to human
2 0 peripheral blood mononuclear cells; is not an endotoxin; and (ii) one or
more anticancer agent(s),
wherein the BD-BRM combination has therapeutic synergy or improves the
therapeutic index in the
CA 02428145 2003-05-07
WO 02/38164 PCT/CA01/01558
treatment of cancer over the composition or the anticancer agents) alone. The
present invention
further provides the use of the combination in the manufacture of a medicament
or a~ pharmaceutical
kit and in the treatment of cancer.
Components of the Combination
BD-BRM composition
Experimental evidence to date indicates that the unique immunomodulatory
properties of
BD-BRM activity are associated with low molecular weight material derived from
bile. The BD-
BRM composition of the present invention comprises small molecular weight
components of less
than 3000 daltons, and having at least one of the following properties:
a) is extracted from the bile of animals;
b) is capable of stimulating or activating monocytes and/or macrophages in
vitro and/or in vivo;
c) is capable of modulating tumor necrosis factor production and/or release;
d) contains no measurable level of IL-Ia, IL-I ~, TNF, IL-6, IL-8, IL-4, GN-
CSF or IFN-y;
e) shows no cytotoxicity to human peripheral blood mononuclear cells or
lymphocytes; and
f) is not an endotoxin.
As mentioned above, the production and characterization of the BD-BRM
composition has
been described in preceding patent applications, and is also summarized in
Example 1.. The
composition can be produced in a consistently reproducible form using the
method as generally .
described above with demonstrated identity, potency and purity from batch to
batch. Identity and
2 0 purity are determined using reverse-phase high pressure liquid
chromatography. (See Example 1).
The compositions have a consistently reproducible pattern on reverse-phase
HPLC. The
composition may be used in a concentrated form. The composition may also be
lyophilized. The
composition may be used without further modification by simply packaging it in
vials and sterilizing
2 5 The compositions are also characterized by the properties hereinbefore
mentioned, for
example their ability to stimulate monocytes and macrophages in vitro a~zd ih
vivo, etc. The
compositions activate PBMNs to release TNF in vitro as measured by the
Monocyte/Macrophage
Activation Assay (TNF-Release) as described in Example 2.
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Anticancer Ag-e,_ nts
This invention provides for a BD-BRM composition in combination with one or
more other
anticancer agents. An "anticancer agent" is any compound, composition or
treatment that prevents
or delays the growth and/or metastasis of cancer cells. Such anticancer agents
include but are not
limited to chemotherapeutic drug treatment, radiation, gene therapy, hormonal
manipulation,
immunotherapy and antisense oligonucleotide therapy. It is to be understood
that anticancer agents
for use in the this invention also include novel compounds or treatments
developed in the future that
can be used to generate therapeutic combinations as described herein.
Examples of candidate anti-cancer compounds that may be useful in the
combinations of
this invention are: antisense sequences, Drugs for Promyelocytic Leukemia:
Tretinoin (Vesanoid~);
Drugs for Chronic Myeloid Leukemia: Low-dose Interferon (IFN)-alpha; Drugs
Used in Gastric
Cancer: Antibiotics, Antineoplastics; Acute Lymphoblastic Leukemia:
Pegaspargase (Oncaspar~),
Rhone-poulenc Rorer, L-asparaginase, Il-2; Drugs for Colon Cancer: Edatrexate
or 10-ethyl-10-
deaza-aminopterin or 10-edam, 5-fluorouracil (5-FU) and Levamisole, Methyl-
ccnu (Methyl-
chloroethyl-cyclohexyl-nitrosourea), Fluorodeoxyuridine (Fudr), Vincristine;
Drugs for Esophageal
Cancer: Porfimer Sodium (Photofrin~), Quadra Logic Technologies, or Treatment
with a
Neodymium:yag (Nd:yag~) Laser; Drugs Used in Colorectal Cancer: Irinotecan
(Camptosar~),
Pharmacia & Upjohn, Topotecan (Hycamtin~), Loperamide (Imodium~), 5-
fluorouracil (5-FU);
Drugs For Advanced Head and Neck Cancers: Docetaxel (Taxotere~); Drugs for Non-
hodgkin's
2 0 Lymphoma: Rituximab, Etoposide; Drugs for Non-small-cell Lung Cancer: A
Vinca Alkaloid,
Vinorelbine Tartrate (Navelbine~), Wellcome, Paitaxel, (Taxol~), Docetaxel
(Taxotere~),
Topotecan, Irinotecan, Gemcitabine; Drugs for Ovarian Cancer: Docetaxel
(Taxotere~),
Gemcitabine, (Gemzar~), Irinotecan (CamptosarC~), Paclitaxel (Taxol~),
Topotecan (Hycamtin~),
Amifostine (Ethyol~), Us Bioscience (For Reducing the Cumulative Renal
Toxicity Associated witl
2 5 Repeated Cisplatin Therapy in Patients with Advanced Ovarian Cancer);
Drugs to Prevent
Melanoma (Sun Screens): 2-ethylhexyl-p-methoxy-cinnamate (2-ehmc), Octyl- N-
dimethyl-p-
aminobenzoate (O-paba), Benzophenone-3 (Bp-3); Drugs for Prostate Cancer:
Flutamide
(Eulexin~), Finasteride (Proscar~), Terazosin (Hytrin~), Doxazosin (Cardura~),
Goserelin
Acetate (Zoladex~), Liarozole, Nilutamide (Nilandron~), Mitoxantrone
(Novantrone~),
30 Prednisone (Deltasone~); Drugs fox Pancreatic Cancer:
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WO 02/38164 PCT/CA01/01558
Gemcitabine (Gemzar~), 5-fluorouracil; Drugs for Advanced Renal Cancer:
Interleukin-2
(Proleukin~), Chiron Corp.; Additional Anti-neoplastic Drugs: Porfimer Sodium,
Axcan,
Dacarbazine, Faulding, Etoposide, Faulding, Procarbazine HCI, Sigma-tau,
Rituximab, Roche,
Paclitaxel (Taxol~), Bristol-myers Squibb, Trastuzumab (Herceptin~), Roche,
Temozolomide
(Temodal~), Schering; Alkylating Agents Used in Combination Therapy for
Different Cancers:
Cyclophosphamide, Cisplatin, Melphalan.
Antisense Compounds
The specificity and sensitivity of antisense compounds makes them useful in
diagnostics,
therapeutics, prophylaxis, as research reagents and in kits. In the context of
the present invention,
the terms "antisense compound" and "antisense oligonucleotide" each refer to
an oligomer or
polymer of ribonucleic acid (RNA), or deoxyribonucleic acid (DNA), or mimetics
thereof. These
terms also include chimeric antisense compounds, which are antisense compounds
that contain two
or more chemically distinct regions, each made up of at least one monomer
unit. In accordance with
the present invention, the terms "antisense compound" and "antisense
oligonucleotide" further
include oligonucleotides composed of naturally occurring nucleobases, sugars
and covalent
internucleoside (backbone) linkages, as well as oligonucleotides comprising
non-naturally-occurring
moieties that function similarly. Such modified or substituted
oligonucleotides are well known to
2 0 workers skilled in the art and often preferred over native forms because
of desirable properties such
as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid
target and increased
stability in the presence of nucleases. The antisense compounds in accordance
with the present
invention comprise from about 7 to about 50 nucleobases, or from about 7 to
about 30.
Alternatively, the antisense compounds comprise a mixture of short oligomers
which will bind to the
target nucleic acid in tandem (i.e. they are complementary to sequences that
are adjacent to one
another in the target nucleic acid).
Examples of antisense compounds useful in the present invention include
oligonucleotides
containing modified backbones or non-natural internucleoside linkages. In
accordance with the
present invention, oligonucleotides having modified backbones include those
that retain a
3 0 phosphorus atom in the backbone and those that do not have a phosphorus
atom in the backbone.
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WO 02/38164 PCT/CA01/01558
For the purposes of the present invention, and as sometimes referenced in the
art, modified
oligonucleotides that do not have a phosphorus atom in their internucleoside
backbone can also be
considered to be oligonucleosides.
The antisense compounds used in accordance with this invention may be
conveniently and
routinely made through the well-known technique of solid phase synthesis.
Equipment for such
synthesis is sold by several vendors including, for example, Applied
Biosystems (Foster City, CA).
Any other means for such synthesis known in the art may be additionally or
alternatively employed.
Similar techniques using phosphorothioates and alkylated derivatives have been
employed to
produce oligonucleotides.
Antisense oligonucleotides have been successfully employed as therapeutic
moieties in the
treatment of disease states such as cancer. Antisense compounds exert their
effects by specifically
modulating expression of a gene implicated in a specific disease state. Thus,
the present invention
contemplates the therapeutic administration of an effective amount of a
combination of the BD-
BRM composition of the present invention and an appropriate antisense compound
to a mammal
suspected of having a disease or disorder Which can be treated by specifically
modulating gene
expression. The present invention further contemplates the prophylactic use of
a combination of the
BD-BRM composition and an antisense compound in the prevention of a cancer
which is related to
over- or under-expression of a specific gene.
2 0 Pharmaceutical Compositions
The combinations of the present invention may be converted using customary
methods into
pharmaceutical compositions. The pharmaceutical composition contain the
combination of the
invention either alone or together with other active or inactive substances.
Such pharmaceutical
compositions can be for oral, topical, rectal, parenteral, local, inhalant, or
intracerebral use. They
2 5 are therefore in solid or semisolid form, for example pills, tablets,
creams, gelatin capsules, capsules,
suppositories, soft gelatin capsules, gels, membranes, and tubelets. For
parenteral and intracerebral
uses, those forms for intramuscular or subcutaneous administration can be
used, or forms for
infusion or intravenous or intracerebral injection can be used, and can
therefore be prepared as
solutions of the combinations or as powders of the combinations to be mixed
with one or more
12
CA 02428145 2003-05-07
WO 02/38164 PCT/CA01/01558
pharmaceutically acceptable excipients or diluents, suitable for the aforesaid
uses and with an
osmolarity that is compatible with the physiological fluids. For local use,
those preparations in the
form of creams or ointments for topical use or in the form of sprays maybe
considered; for inhalant
uses, preparations in the form of sprays, fox example nose sprays, may be
considered. Preferably,
the BD-BRM composition of the combination is administered intramuscularly.
The pharmaceutical compositions can be prepared by per se known methods for
the
preparation of pharmaceutically acceptable compositions which can be
administered to patients,
and such that an effective quantity of the active substance is combined in a
mixture with a
pharmaceutically acceptable vehicle. Suitable vehicles are described, for
example, in Remin tg on's
Pharmaceutical Sciences (Nack Publishing Company, Easton, Pa., USA 1985).
On this basis, the pharmaceutical compositions include, albeit not
exclusively, the
combination of the invention in association with one or more pharmaceutically
acceptable vehicles
or diluents, and are contained in buffered solutions with a suitable pH and
iso-osmotic with the
physiological fluids.
The compositions and agents of the invention are intended for administration
to humans or
animals.
The dosage requirements of the pharmaceutical.compositions according to the
present
invention will vary with the particular combinations employed, the route of
administration and the
particular cancer and cancer patient being treated. Treatment will generally
be initiated with small
2 0 dosages less than the optimum dose of the compound. Thereafter the dosage
is increased until the
optimum effect under the circumstances is reached. In general, the
pharmaceutical compositions
according to the present invention are most administered at a concentration
that will generally afford
effective results without causing any harmful or deleterious side effects. The
compounds can be
administered either as a single unit dose, or if desired, the dosage can be
divided into convenient
2 5 subunits administered at suitable times throughout the day. The amount of
the pharmaceutical
composition that will be effective in treatment can be determined by standard
clinical techniques,
known to a worker skilled in the art [for example, see Remzngto~'s Phay-
maceutical Sciefaces,
18t" Edition, Mack Publishing Co., Easton, PA (1990)].
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WO 02/38164 PCT/CA01/01558
Therapeutic Activity of the Combination
The combination of the present invention has a net anticancer effect that is
greater than the
anticancer effect of the individual components of the combination when
administered alone. The
anticancer effect is increased without a concomitant increased toxic effect.
Without being limited b;
mechanism, by combining one or more anticancer agents with a BD-BRM
composition it is possiblf
to:
(i) increase the therapeutic effect of the anticancer agent(s);
(ii) increase the therapeutic effect of the BD-BRM composition;
(iii) decrease or delay the toxicity phenomena associated with the anticancer
agent(s); and/or
(iv) decrease or delay the toxicity phenomena associated with the BD-BRM
composition,
in comparison to treatment with the individual components of the combination.
In one embodiment the combination of the present invention provides an
improved efficacy,
over treatment using the components of the combination alone, that may be
demonstrated by
determination of the therapeutic synergy.
A combination manifests therapeutic synergy if it is therapeutically superior
to one or other
of the constituents used at its optimum dose [T. H. Corbett et al., (1982)
Cancer Ti~eatmerat
Reports, 66, 1187 ]. To demonstrate the efficacy of a combination, it may be
necessary to
compare the maximum tolerated dose of the combination with the maximum
tolerated dose of each
of the separate constituents in the study in question. This efficacy may be
quantified using technique
2 0 and equations commonly known to workers skilled in the art. [T. H. Corbett
et al. , (1977) Cancer,
40, 2660.2680; F. M. Schabel et al., (1979) Cancer Drug Development, Part B,
Methods in
Cancer Research, 17, 3-51, New York, Academic Press Inc.].
The combination, used at its own maximum tolerated dose, in which each of the
constituent
will be present at a dose generally not exceeding its maximum tolerated dose,
will manifest
2 5 therapeutic synergy when the efficacy of the combination is greater than
the efficacy of the best
constituent when it is administered alone.
In another embodiment the combination of the present invention improves the
therapeutic
index in the treatment of cancer over that of the BD-BRM composition or the
anticancer agents)
when administered to a patient alone.
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A median effective dose (EDSO) of a drug is the dose required to produce a
specified effect
in 50% of the population. Similarly, the median lethal dose (LDSQ) of a drug,
as determined in
preclinical studies, is the dose that has a lethal effect on 50% of
experimental animals. The ratio of
the LDSO to the EDSO can be used as an indication of the therapeutic index.
Alternatively the
therapeutic index can be determined based on doses that produce a therapeutic
effect and doses
that produce a toxic effect (e.g. ED9o and LDIO, respectively). During
clinical studies, the dose, or
the concentration (e.g. solution, blood, serum, plasma), of a drug required to
produce toxic effects
can be~compared to the concentration required for the therapeutic effects in
the population to
evaluate the clinical therapeutic index. Methods of clinical studies to
evaluate the clinical therapeuti
index are well known to workers skilled in the art.
In one embodiment the combination of the present invention provides an
improved
therapeutic index, in comparison to that of the individual components of the
combination when
administered alone, by decreasing the observed LDSO of at least one of the one
or more anticancer
agents in the combination.
In a related embodiment the combination of the present invention provides an
improved
therapeutic index, in comparison to that of the individual components of the
combination when
administered alone, by increasing the observed EDSO of at least one of the one
or more anticancer
agents in the combination. In a further embodiment the combination of the
present invention
provides an improved therapeutic index, in comparison to that of the
individual components of the
2 0 combination when administered alone, by increasing the observed EDSO of
the bile-derived
biological response modifier.
In another embodiment the efficacy of a combination according to the present
invention may
also be characterized by adding the actions of each constituent.
In order to prepare a combination according to the present invention one first
selects one or
2 5 more candidate anticancer agents) and measure its efficacy in a model of a
cancer of interest, as
would be well understood by one skilled in the art. The next step may be to
perform a routine
analysis to compare the efficacy of the one or more anticancer agents) alone
to the efficacy of the
one or more anticancer agents) in combination with varying amounts of the BD-
BRM composition.
Successful candidates for use in the combinations of the present invention
will be those that
CA 02428145 2003-05-07
WO 02/38164 PCT/CA01/01558
demonstrate a therapeutic synergy with the BD-BRM or that improve the
therapeutic index in
comparison to the therapeutic index of the candidate agent(s).
The efficacy of the combinations of the present invention may be determined
experimentally
using standard techniques using cancer models well known to workers skilled in
the art. Such
cancer models allow the activity of combinations to be tested ih vitro and in
vivo in relation to the
cancer of interest. Exemplary methods of testing activity are described in the
Examples provided
herein, although, it should be understood that these methods are not intended
to limit the present
invention.
One example of a method for studying the efficacy of the combinations on solid
tumors in
vivo involves the use of subject animals, generally mice, that are
subcutaneously grafted bilaterally
with 30 to 60 mg of a tumor fragment on day 0. The animals bearing tumors axe
mixed before being
subjected to the various treatments and controls. In the case of treatment of
advanced tumors,
tumors are allowed to develop to the desired size, animals having
insufficiently developed tumors
being eliminated. The selected animals are distributed at random to undergo
the treatments and
controls. Animals not bearing tumors may also be subjected to the same
treatments as the
tumor-bearing animals in order to be able to dissociate the toxic effect from
the specific effect on
the tumor. Chemotherapy generally begins from 3 to 22 days after grafting,
depending on the type
of tumor, and the animals are observed every day. The different animal groups
are weighed 3 or 4
times a week until the maximum weight loss is attained, and the groups are
then weighed at Ieast
2 0 once a week until the end of the trial.
The tumors are measured 2 or 3 times a week until the tumor reaches
approximately 2 g, or
until the animal dies if this occurs before the tumor reaches 2 g. The animals
are autopsied when
sacrificed. The antitumour activity is determined in accordance with various
recorded parameters.
For a study of the combinations on leukaemias, the animals are grafted with a
particular
2 5 number of cells, and the antitumour activity is determined by the increase
in the survival time of the
treated mice relative to the controls.
Administration of the Combination
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The uses and methods of the present invention comprise administering to a
subject in need
thereof an effective amount of a BD-BRM composition in combination with one or
more anticancer
agents to a subject. As used herein, combination components are said to be
administered in
combination when the two or more components are administered simultaneously or
are
administered independently in a fashion such that the components will act at
the same time.
Components administered independently can, for example, be administered
separately (in
time) or concurrently. Separately in time means at least minutes apart, and
potentially hours, days or
weeks apart. The period of time elapsing between the administration of the
components of the
combination of the invention can be determined by a worker of skill in the
art, and will be
dependent upon, for example, the age, health, and weight of the recipient,
nature of the
combination treatment, side effects associated with the administration of
other components) of the
combination, frequency of administration(s), and the nature of the effect
desired. Components of
the combinations of the invention may also be administered independently with
respect to location
and, where applicable, route of administration.
In another embodiment, an effective amount of a therapeutic composition
comprising a BD-
BRM composition and one or more anticancer agents, and a pharmaceutically
acceptable carrier is
administered to a subject. The combination or the pharmaceutical composition
of the invention can
be administered before during or after other anticancer treatment(s), or
treatments for other
diseases or conditions. For example a drug to treat adverse side effects of
the anticancer
2 0 treatments) can be administered concurrently with a combination of the
invention or a
pharmaceutical composition of the invention.
As indicated above the components of the combination of the present invention
may be
administered separately, concurrently, or simultaneously. In the case of
separate administration the
BD-BRM composition may be administered before, during or after administration
of the anticancer
2 5 agent(s). Furthermore, it would be readily apparent to a worker skilled in
the art that the route of
administration of each component of the combination is selected in order to
maximize the
therapeutic benefit of the component and it is not necessary that each
component be delivered via
the same route. The BD-BRM composition andlor the anticancer agents) of the
combination may
be administered via a single dose or via continuous perfusion.
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The agents, compounds and compositions of this invention can be utilised ire
vivo, ordinarily
in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and
mice, or i~z vitr°o to
treat cancer or cancer cells.
Cancers
As used herein, "cancer" refers to all types of cancer or neoplasm or
malignant tumors
found in mammals, including carcinomas and sarcomas. Examples of cancers are
cancer of the
brain, breast, cervix, colon, head and neck, kidney, lung, non-small cell
lung, melanoma,
mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
The term "leukemia" refers broadly to progressive, malignant diseases of the
blood-forming
organs and is generally characterized by a distorted proliferation and
development of leukocytes
and their precursors in the blood and bone marrow. Leukemia is generally
clinically classified on the
basis of (1) the duration and character of the disease--acute or chronic; (2)
the type of cell
involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and
(3) the increase or
non-increase in the number of abnormal cells in the blood--leukemic or
aleukemic (subleukemic) .
Leukemia includes, for example, acute nonlymphocytic leukemia, chronic
lymphocytic leukemia,
acute granulocytic leukemia, chronic granulocytic leukemia, acute
promyelocytic leukemia, adult
T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic
leukemia, blast cell
leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis,
embryonal leukemia,
eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic
leukemia, hemocytoblastic
2 0 leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic
leukemia, leukopenic leukemia,
lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous
leukemia,
lymphoid leukemia,.lymphosarcoma cell leukemia, mast cell leukemia,
megakaryocytic leukemia,
micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia,
rnyelocytic leukemia,
myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia,
plasma cell leukemia,
2 5 plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia,
Schilling's leukemia, stem cell
leukemia, subleukemic leukemia, and undifferentiated cell leukemia.
The term "sarcoma" generally refers to a tumor which is made up of a substance
like the
embryonic connective tissue and is generally composed of closely ,packed cells
embedded in a
fibrillar or homogeneous substance. Sarcomas include chondrosarcoma,
fibrosarcoma,
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lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma,
adipose
sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,
botryoid sarcoma,
chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma,
endometrial
sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic
sarcoma, giant cell
sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple
pigmented hemorrhagic
sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of
T-cells, Jensen's
sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,
malignant
mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,
serocystic
sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
The term "melanoma" is taken to mean a tumor arising from the melanocytic
system of the
skin and other organs. Melanomas include, for example, acral-lentiginous
melanoma, amelanotic
melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-
Passey
melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma,
nodular melanoma
subungal melanoma, and superficial spreading melanoma.
The term "carcinoma" refers to a malignant new growth made up of epithelial
cells tending
to infiltrate the surrounding tissues and give rise to metastases. Exemplary
carcinomas include, for
example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid
cystic carcinoma,
carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma,
alveolar cell
carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma,
basosquamous cell
2 0 carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic
carcinoma,
cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma,
colloid carcinoma,
comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en
cuirasse, carcinoma
cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,
carcinoma durum,
embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma
epitheliale
2 5 adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,
gelatiniform carcinoma,
gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,
glandular carcinoma,
granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma,
hepatocellular carcinoma,
Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile
embryonal
carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial
carcinoma, Krompecher's
3 0 carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular
carcinoma, carcinoma
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lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma
medullare, medullary
carcinoma, melanotic carcinoma, carcinoma mope, mucinous carcinoma, carcinoma
muciparum,
carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous
carcinoma,
carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma
ossificans,
osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive
carcinoma, prickle cell
carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell
carcinoma,
carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma
scroti,
signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma, spheroidal
cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous
carcinoma, squamous cell
carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma
telangiectodes, transitional cell
carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and
carcinoma
villosum.
Additional cancers include, for example, Hodgkin's Disease, Non-Hodgkin's
Lymphoma,
multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer,
rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors,
primary brain tumors,
stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant
carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer, lymphomas,
thyroid cancer,
neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant
hypercalcemia, cervical
cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer.
2 0 Pharmaceutical Kits
The present invention additionally provides for therapeutic kits containing
(i) a dosage unit of a
composition and a pharmaceutically acceptable carrier wherein the composition
comprises small
molecular weight components of less than 3000 daltons, and has the following
properties: is
extracted from bile of animals; is capable of stimulating monocytes and/or
macrophages i~ vitro
2 5 and/or in vivo; is capable of modulating tumor necrosis factor production
and/or release; contains
no measurable level of IL-la, IL-lei, TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-
gamma; is not
cytotoxic to human peripheral blood mononuclear cells; is not an endotoxin;
and (ii) dosage unit of
one or more chemotherapeutic drugs) and a pharmaceutically acceptable carrier,
said (i) and (ii)
CA 02428145 2003-05-07
WO 02/38164 PCT/CA01/01558
being provided in amounts that are effective, in combination,.for selectively
killing tumor or
metastatic cells.
As used herein, a "dosage unit" is a pharmaceutical composition or formulation
comprising
at least one active ingredient and optionally one or more inactive
ingredient(s). The dosage unit can
be unitary, such as a single pill or liquid, containing all of the desired
active ingredients and the
inactive ingredients necessary and desired for making a dosage suitable for
administration (e.g.,
tabletting compounds such as binders, fillers, and the like); the dosage unit
can consist of a number
of different dosage forms (e.g., pill (s) and/or liquid(s)) designed to be
taken simultaneously as a
dosage unit. '
The contents of the kit can be lyophilized and the kit can additionally
contain a suitable
solvent for reconstitution of the lyophilized components. Individual
components of the kit would be
packaged in separate containers and, associated with such containers, can be a
notice in the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or sale fox
human administration.
EXAMPLES
A worker skilled in the art can produce BD-BRM compositions, and assay BD-BRM
compositions for activities such as ifa vitro and/or in vivo monocyte and/or
macrophage
stimulation, modulation of tumor necrosis factor production and/or release,
content of IL-la, IL-lei,
2 0 TNF, IL-6, IL-8, IL-4, GM-CSF or IFN-gamma and endotoxin and cytotoxicity
to human
peripheral blood mononuclear cells, using the methods described in
International Patent Application
Serial No. PCT/CA94/00494, published February 16, 1995 as WO 95/07089.
Example 1: In Vivo Evaluation Of Efficacy Of BD-BRM In The Treatment Of Human
Pancreatic Adenocarcinoma In Cd-1 Nude Mice
2 5 The mouse xenograft model of neoplasia was used in these studies to
demonstrate the effect
of treatment with a BD-BRM composition on tumor growth in mice. For
comparison, separate
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groups of mice were treated with saline (control), a conventional
chemotherapeutic drug or
concurrently with a combination of a BD-BRM composition and a chemotherapeutic
drug.
A human carcinoma cell line was grown as monolayer culture in Minimum
essential medium
(a-MEM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential
amino acid,
1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 ~,g/ml streptomycin and 0.25
~.g/ml
amphotericin B and 2mM L-alanyl-1-glutamine at 37°C in an atmosphere of
5% COz in air. The
tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment.
The cells were
harvested from subconfluent logarithmically growing culture by treatment with
trypsin-EDTA and
counted for tumor inoculation. The cell lines used in the experiments herein
are listed hereafter,
though any carcinoma cell line capable of tumor formation upon inoculation
could be used:
pancreatic adenocarcinoma (BxPC-3) (a gemcitabine-resistant cell line)
melanoma (A2058)
melanoma(C8161)
breast adenocarcinoma(MDA-MB-231)
prostate carcinoma (PC-3)
ovary adenocarcinoma (SK-OV-3)
large cell lung adenocarcinoma (H460)
small cell lung carcinoma (H209).
Tun2or hZbculatioh: An acclimation period of at least 7 days was allowed
between receipt of the
2 0 immunocomprornised animal and its inoculation. Typically CD-1 or SCID mice
were used. When
the female mice were 6-9 (most typically 6-7) weeks of age, each mouse was
subcutaneously
injected in the right flank with 3-10 million human carcinoma cells in 0.1 ml
of PBS. Inoculated
animals were divided into equal sized treatment groups of 9-20 (typically
about 10) mice each and
treated daily with saline (0.2 ml/mouse/day, i.p.), BD-BRM (0.2 ml/mouse/day,
i.p.), a
2 5 chemotherapeutic drug, or concurrently with BD-BRM (0.2 ml/mouse/day,
i.p.) and a
chemotherapeutic drug. The drug doses used in the experiments herein are
listed hereafter, though
any chemotherapeutic drugs) or other anticancer agents) could be used:
gemcitabine (100 mg/kg in 0.1 ml saline/mouse/3 day, i.v.)
dacarbazine (DTIC) (80 mg/kg in 0.1 ml saline/mouse/day, i.p.)
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taxol (10 mg/kg/week, i.v.)
5-fluorouracil °
taxotere
cisplatin
mitoxanthrone (i.v.)
Tumour sizes were measured every other day' in two dimensions using a caliper,
and the
volume was expressed in mm3 using the formula: V = 0.5 a x b2, where a and b
are the long and
short diameters of the tumor, respectively. Mean tumor volumes calculated from
each measurement
were then plotted in a standard graph to compare the anti-tumor efficacy of
drug treatments to that
of control. A day after the last treatment, tumors were excised from the
animals and their weights
were measured. The data are displayed as a tumour growth curve, and a bar
graph showing mean
tumor weights.
Mouse
xenograft
experiments
with
BD-BRM
compositions
and
BD-BRM
combinations
FigureHuman Mouse combination# mice with total
# carcinomacell linestraindrug eXpt tumor regression
BRM: 4 (of 9)
2, pancreatic CD-1 gemcitabine-
3
4, pancreaticSU.86.86 CD-1 gemcitabinegemcitabine
5
6, melanomaA2058 CD-1 dacarbazinedacarbazine
7
2 8, melanomaC8161 CD-1 - dacarbazinecomb: 5 (of 10)
0 9
10, breast MDA-MB-231CD-1 Taxol Taxol
11
12, breast MDA-MB-231CD-1 Taxol Taxol BRM: 2; comb:
13 5 (of 10)
14, prostatePC-3 SCID mitoxantrone
15
16 pancreaticBxPC-3 CD-1 5-fluorouracil5-fluorouracilcomb: (5 of 10)
2 17 pancreaticSU.86.86 CD-1 5-fluorouracil5-fluorouracil
5
18, prostateDU145 SCID mitoxantrone-
19
ovarianSK-OV-3 CD-1 cisplatincisplatin
21 ovarianSK-OV-3 CD-1 taxol taxol
lung,
large
22, cell H460 CD-1 taxotere taxotere
23
24 lun H209 SCID - -
25 small
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The results of the mouse xenograft experiments outlined in the table above are
shown in
Figures 2-25. BD-BRM treatments always resulted in significant delay of tumor
growth compared
to saline control. Where a chemotherapeutic drug treatment group was included,
the delay in tumor
growth achieved with BD-BRM was typically superior to the inhibitory effects
observed with the
chemotherapeutic drug. As indicated in the above table, total regression of
the tumor was also
observed in some of the animals, when the animals were treated with a BRM
composition alone or
with a combination of the BD-BRM composition and a chemotherapeutic drug was
used. In the
remaining animals treated with a combination, significantly enhanced antitumor
effects were
observed.
The efficacy of the combinations of the invention can also be determined
experimentally
using other protocols to study animal models grafted with cancerous cells. The
animals subjected t<
the experiment, can be grafted with a tumor fragment, and the graft may be
placed subcutaneously.
In the case of the treatment of advanced tumors, tumors are allowed to develop
to the desired size,
animals having insufficiently developed tumors being eliminated. Animals not
bearing tumors may
also be subjected to the same treatments as the tumor-bearing animals in order
to b able to
dissociate the toxic effect from the specific effect on the tumor. Treatment
generally begins 3 days
to 4 weeks after grafting, depending on the type of tumor, and the animal are
observed and animal
weight change recorded, and the tumors measured regularly, for example daily,
or 2 or 3 times per
week until the tumor reaches a defined size (e.g. 2 g in a mouse), or until
the animal dies if this
2 0 occurs before hte tumor reaches 2 g. The animals are autopsied when
sacrificed. To study
leukemia, cancerous cells can be injected intravenously. Antitumor activity is
determined by the
increase in the survival time of the treated animals relative to the controls.
The efficacy of the
treatment with the combination of the invention is assessed in terms of
changes in the mean surviva
time of the animal. Alternative methods of assessing efficacy, and therapeutic
synergy, can also be
2 5 used.
These animal models are recognized in the art to be predictive tests for
anticancer effects in
humans.
From the foregoing, it will be appreciated that, although specific embodiments
of the invention havf
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WO 02/38164 PCT/CA01/01558
been described herein for purposes of illustration, various modifications may
be made without
deviating from the spirit and scope of the invention. Accordingly, the
invention is not limited excep
as by the appended claims.
