Note: Descriptions are shown in the official language in which they were submitted.
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BIOLOGICAL RESPONSE MODIFIER COMPOSITION AND USES
THEREOF
FIELD OF THE INVENTION
The present invention relates to a synthetic biological response modifier
composition,
pharmaceutical compositions comprising the same and the uses thereof, alone
and in
combination with other drugs in the treatment of various disorders.
BACKGROUND OF THE INVENTION
Numerous therapies exist that are directed towards the treatment of cancer,
viral disorders,
and many other diseases including chemotherapeutic drugs, radiation, gene
therapy and
antisense oligonucleotides. ,One drawback to current therapies is the toxic
side effects
associated with the pharmaceuticals utilized to treat different human
diseases. 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
therapeutic treatments for
human disease.
Therapies are continuously being developed for the prophylaxis and treatment
of cancer and
infectious diseases, such as Acquired Immunodefieiency Syndrome (AIDS). Some
of these
therapies attempt to use the immune system therapeutically. One approach is
based on the
antigen specific elements of the immune system, namely antibodies and T cells.
For example,
research has been aimed at developing vaccines against foreign agents, or
against certain
endogenous chemical messengers, such as interleukins, to suppress antibody
reactions. A
second approach is based on the isolation, cloning, expression and production
of peptides and
proteins from the non-antigen specifc parts of the immune system. For example,
proteins,
such as cytokines, which comprise the interleukins produced by white blood
cells, and
interferons which stimulate lymphocytes and scavengers cells that digest
foreign antigens,
offer possibilities for therapies.
The treatment of many human diseases could be greatly enhanced if the early
immune
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response to a foreign antigenic molecule, substance, cell or organism could be
augmented so
that the immune response of the affected individual is enhanced. Strategies
which have been
suggested to augment the immune response include vaccines specific for disease-
associated
antigens; the use of monoclonal antibodies against antigens on the surface of
cells or viruses
and superantigens.
Relatively recently, the role of the physiologically active polypeptide, known
as tumor
necrosis factor ("TNF") has been studied, particularly with respect to its
ability to induce
necrosis of tumors, with no effect upon the normal tissues of the living body.
The amino acid
sequence of TNF, as well as the base sequence of the DNA coding for TNF has
been
disclosed in U.S. Patent.No. 4,879,226.
Because TNF has been shown to have a role in controlling the immune system of
mammals in
response to a variety of different diseases, any agent that can stimulate the
production or
bioavailability of TNF in vivo has potential utility as a treatment for
various human diseases,
including cancer, HIV, heart disease and others. Additionally, any agent that
can stimulate
human monocytes and macrophages to produce TNF ih vitro, is useful as a means
for
providing a source of TNF for therapeutic administration, as well as for
analytical and
diagnostic purposes.
The mechanism of action of TNFa appears to be derived from accumulating
evidence which
indicates that TNFa is a regulatory cytokine with pleiotrophic biological
activities. These .
activities include: inhibition of lipoprotein lipase synthesis, activation of
polymorphonuclear
leukocytes, inhibition of cell growth or stimulation of cell growth, cytotoxic
action on certain
transformed cell lines, antiviral activity, stimulation of bone resorption,
stimulation of
collagenase and prostaglandin E2 production and immunoregulatory actions
including
activation of T-cells, B-cells, monocytes, thymocytes and stimulation of the
cell-surface
expression of major histocompatibility complex class I and class II molecules.
Unfortunately, treatments with high dosages of TNF alone have been associated
with such
side effects as hypotension, leukocytosis, fever, chills, neurotoxicity,
nausea and vomiting.
TNF therapy has therefore played an important role in the field of cancer
therapy, however
excessive or unregulated TNF production has been implicated in exacerbating a
number of
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disease states. These include rheumatoid arthritis, rheumatoid spondylitis,
osteoaithritis,
gouty arthritis and o~'~r arthritic conditions, sepsis, septic shock, gram
negative sepsis, toxic
shock syndrome, adult resipratory stress syndrome, cerebral malaria, chronic
pulmonary
inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption
disease, reperfusion
injury, graft v. host reaction, allograft rejections, fever and myalgias due
to infection, such as
influenza, cachexia secondary to infection or malignancy, cachexia secondary
to AIDS, keloid
formation, scar tissue formation, Crohn's disease, ulcerative colitis and
pyresis plus a number
of autoimmune diseases such as multiple sclerosis, autoimmune diabetes and
systemic lupus
erythematosis.
Cytokines, specifically TNF, have been implicated in the activation of T-cell
mediated HIV
protein expression and/or virus replication by playing a role in maintaining T-
lymphocyte
activation. ,Therefore, extensive research has been directed towards
interfering with cytokine
production, notably TNF, in a HIV-infected individual. The therapeutic aim
being to limit the
maintenance of T-cell activation, thereby reducing the progression of HIV
infectivity to
previously uninfected cells, thereby resulting in a slowing or elimination of
the progression of
immune dysfunction caused by HIV infection. Hence there is mounting evidence
supporting
the use of inhibitors of cytokines, particularly TNF, (U.5. Patent Nos.
5,563,143 and
5,506,340) in the treatment of AIDS.
Numerous clinical trials have also been carried out in patients with Kaposi's
sarcoma with
immune modulators such as Interferona (J.AIDS., 1:111-118; 1988). This drug
has been
licensed in Canada for the treatment of Kaposi's sarcoma. Interferon has been
shown to have
antitumor and antiretroviral effects. Response rates to treatment with IFN are
ixutially high
(Known, et al., Recomb. Leucocyte A IFN in Kaposi=s sarcoma, N.Y. Acad. Sci.,
437:431-
43 8, 1984). However prolonged responses are not frequent, possibly because of
the
emergence of anti-IFN antibodies (Autavelli, et al., J.LD. 163:882-885, 1991).
Patients
invariably require chemotherapy or radiotherapy to control tumor growth. Both
IFN and
chemotherapy have substantial toxic side effects on bone marrow resulting in
the termination
of therapy (Fischl, M.A., Am. J. Med., April 10, 1991).
Both TNF and IFN individually possess antiviral activity, making them
potential candidates
in the treatment of viral infections and tumors. However, serious side effects
have been
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observed in the treatment with therapeutically valuable doses' of TNF and IFN
which have
limited their clinical usefulness.
Infectious diseases, such as those caused by viruses can only succeed by
avoiding or defeating
the body's immune system. The immune system mounts or elicits either or both
non-specific
immune responses and specific immune response factors to fight such pathogens.
Non-specific immune responses are focused on cytokine production, principally
by
macrophages, and serve as a prelude to specific antibody responses. The
inflammatory
cytokines include TNF-a and mediate an acute response directed to the injury
or infection
sites, which is manifested by an increased blood supply. The pathogenic
bacteria or viruses
are engulfed by neutrophils and macrophages in an attempt to contain the
infection to a small
tissue space. Macrophages, therefore, play a key role in the defense against
infectious
diseases as follows: (1) processing and presentation of antigens to
lymphocytes so that
antibody-mediated and cell-mediated immune responses can occur; (2) secretion
of cytokines
central to immune response; and (3) destruction of antibody-coated bacteria,
tumor cells or
host cells.
Macrophages can ingest and kill a wide variety of pathogens, such as bacteria,
fungi, and
protozoa (parasites). This ability is augmented when the macrophages are
activated. Secreted
products of activated macrophages are more diverse than those from any other
immune cell.
These regulate both pro- and anti-inflammatory effects arid regulate other
cell types. These
products include TNF-a, IL-1 [3, IL-6, hydrolytic enzymes, and products of
oxidative
metabolism Bacteria that are eliminated primarily through this cell-mediated
immune process
include tuberculosis and other related mycobacterial infections, such as
atypical
mycobacterial infections seen in up to 50% of AIDS patients, and anthrax, a
potential
bacteriological warfare agent. Fungal infections are common problems in
immunosuppressed
patients, such as those afflicted with AIDS or organ transplant patients.
Therefore a need remains for a synthetic BRM (Syn-BRM) that alone or in
combination with
other pharmaceuticals modulates the mammalian immune system in a manner that
effectively
treats human disease.
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SUMMARY OF THE INVENTION
The invention provides a synthetic biological response modifier (Syn-BRM)
composition
In accordance with an aspect of the present invention there is provided a
synthetic biological
response modifier composition comprising 57 ~ 20 mg/L 3-hydroxybutyric acid,
125 ~ 44
mg/L lactic acid, 155 ~ 54 mg/L acetic acid, 1.4 ~ 0.5 mglL creatine, 22 ~ 8
mg/L creatinine,
2.5 ~ 0.9 mg/L carnitine, 6.8 ~ 2.4 mg/L taurine, 20 ~ 7 mg/L choline, 815 ~
285 mg/L urea,
wherein said composition: (a) stimulates or activates monocytes and
macrophages; and/or (b)
modulates tumor necrosis factor production and/or release.
In accordance with another aspect of the present invention there is provided a
synthetic
biological response modifier composition comprising 57 ~ 20 mg/L 3-
hydroxybutyric acid,
125 ~ 44 mg/L lactic acid, 155 ~ 54 mg/L acetic acid, 1.4 ~ 0.5 mg/L creatine,
22 ~ 8 mg/L
creatinine, 2.5 ~ 0.9 mg/L carnitine, 6.8 ~ 2.4 mg/L taurine, 20 ~ 7 mg/L
choline, 815 ~ 285
mg/L urea, 40 ~ 14 mg/L formic acid, wherein said composition stimulates or
activates
monocytes and macrophages; and/or modulates tumor necrosis factor production
and/or.
release.
In accordance with another aspect of invention there is provided a synthetic
biological
response modifier composition comprising 57 ~ 20 mg/L 3-hydroxybutyric acid,
125 ~ 44
mg/L lactic acid, 155 ~ 54 mg/L acetic acid, 1.4 ~ 0.5 mg/L creatine, 22 ~ 8
mg/L creatinine,
2.5 ~ 0.9 mg/L carnitine, 6.8 ~ 2.4 mg/L taurine, 20 ~ 7 mg/L choline, 815 t
285 mg/L urea,
40 ~ 14 mg/L and the relative proportions of the components in the composition
remains
approximately the same.
In accordance with a further aspect of the invention there is provided a
combination
comprising: a composition of the invention and 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.
The invention also encompasses pharmaceutical compositions comprising the
synthetic
biologic response modifier compositions of the invention.
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The present invention encompasses a method of treating a mammal (or patient)
comprising
the step of administering to said mammal an effective amount of a synthetic
biological
response modifier composition, either alone or in combination with one or more
anti-cancer
drugs and/or anti-viral drugs and/or antisense sequences, as well as to
pharmaceutical
compositions and kits comprising a combination of the synthetic biological
response modifier
composition and anticancer drugs, anti-virals and/or antisense sequences.
These and other aspects of the present invention will become evident upon
reference to the
following detailed description and attached drawings.
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:
Figure 1 presents results of an ih vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human pancreatic carcinoma (BxPC-3)
in CD-1
nude mice. Figure 1A demonstrates the effect on tumor size and Figure 1B
demonstrates the
effect on tumor weight.
Figure 2 presents results of an i~ vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human pancreatic carcinoma (BxPC-3)
in CD-1
nude mice, compared to a natural BRM. Figure 2A demonstrates the effect on
tumor size and
Figure 2B demonstrates the effect on tumor weight.
Figure 3 presents results of an ih vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human melanoma (C8161) in CD-1 nude
mice,
compared to a natural BRM. Figure 3A demonstrates the effect on tumor size and
Figure 3B
demonstrates the effect on tumor weight.
Figure 4 presents results of an in vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human breast adenocarcinorna (MDA-
MB-231)
in CD-1 nude mice, compared to a natural BRM. Figure 4A demonstrates the
effect on tumor
size and Figure 4B demonstrates the effect on tumor weight.
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Figure 5 presents results of an i~ vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human pancreatic carcinoma (BxPC-3)
in CD-1
nude mice, compared to a natural BRM. Figure SA demonstrates the effect on
tumor size and
Figure SB demonstrates the effect on tumor weight.
Figure 6 presents results of an ih vivo demonstration of the effect of two
embodiments of a
synthetic BRM composition on the tumors of human breast adenocarcinoma (MDA-MB-
231)
in CD-1 nude mice, compared to a natural BRM. ' Figure 6A demonstrates the
effect on tumor
size and Figure 6B demonstrates the effect on tumor weight.
Figure 7A presents results of an i~ vivo demonstration of the effect of a
synthetic BRM
composition on the weight of human pancreatic carcinoma tumors (BxPC-3) in CD-
1 nude
mice, compared to a natural BRM.
Figure 7B demonstrates the effect of a synthetic BRM composition on TNF-a
release.
Figure 8 shows the effect of syn-BRM on NIA cell infiltration into tumors in
mice harboring
Human Melanoma (C8161) xenografts.
Figure 9 illustrates a 2D COSY spectrum of Nat-BRM with various J-coupling
connections of
known compounds labeled in the spectrum.
Figure 10 illustrates a 2D NOESY spectrum of Nat-BRM with various NOE
connections of
known compounds labeled in the spectrum.
Figure 11 illustrates a 1D NMR spectrum of Nat-BRM with added formic acid (18A
+ Formic
acid) and Nat-BRM (18A) alone.
Figure 14 illustrates a 1 D proton NMR spectrum of Nat-BRM with chemical shift
assignments for identified compounds at, a) 8.1-8.8 ppm, b) 2.7-4.4 ppm, and
c) 0.8-2.6 ppm.
Figure 16 compares the percentage of inhibition of tumor weight by Syn-BRM
(Syn-BRM#4)
and various batches of Nat-BRM in human carcinoma models. Inhibition of tumor
weight by
Nat-BRM was calculated using the formula [(C-T)/C] x 100, where C represents
mean tumor
weight of saline-treated mice and T represents that of Nat-BRM-treated mice.
Each bar
represents the calculated percentage of tumor weight inhibition (mean ~ SE)
for the treatment
groups. The total number of experiments performed per treatment group is
indicated on the
right.
Figure 17 illustrates the effects of Nat-BRM treatment on numbers of tumor
infiltrating NK
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cells. (A) Mice harboring C8161 human melanoma xenografts were treated with
saline or
Nat-BRM and isolated.tumors were subjected to FACS analyses using macrophage-
and NK
cell-specific antibodies. Increased NK infiltration (6.45% to 10.55%) to
tumors isolated from
mice treated with Nat-BRM was observed. (B) Mice harboring Capan-1 human
pancreatic
carcinoma xenografts were treated with saline or Nat-BRM and isolated tumors
were
subjected to flow cytometric analyses using macrophage- and NK cell-specific
antibodies.
Increased NK infiltration (4.49% to 9.70%) to tumors isolated from mice
treated with Nat-
BRM was observed.
Figure 18 illustrates the effects of macrophage depletion on isz vivo anti-
tumor efficacy of
Nat-BRM. (A) the effects on tumor size and (B) the effects on tumor weight.
Figure 19 illustrates the effects of Nat-BRM on NK cell infiltration to tumors
in macrophage
depleted mice. CD-1 nude mice harboring C8161 human melanoma xenografts were
depleted
of macrophages using C12MDP and treated with saline control or Nat-BRM. NK
infiltration
is significantly compromised in tumors isolated from macrophage-depleted mice,
suggesting
an important role of macrophages in Nat-BRM-mediated NK infiltration.
Figure 20 illustrates the growth of Human Pancreatic Adenocarcinoma (BxPC-3)
in CD-1
Nude Mice.
Figure 21 illustrates the weight of Human Pancreatic Adenocarcinoma (BxPC-3)
in CD-1
Nude Mice.
Figure 22 illustrates the growth of Human Pancreatic Carcinoma (SU.86.86.) in
CD-1 Nude
Mice.
Figure 23 illustrates the weight of Human Pancreatic Carcinoma (SU.86.86.) in
CD-1 Nude
Mice.
Figure 24 illustrates the growth of Human Melanoma(A2058) in CD-1 Nude Mice.
Figure 25 illustrates the weight of Human Melanoma(A2058) in CD-1 Nude, Mice.
Figure 26 illustrates the growth of Human Melanoma(C8161) in CD-1 Nude Mice.
Figure 27 illustrates the weight of Human Melanoma(C8161) in CD-1 Nude Mice.
Figure28 illustrates the growth of Human Breast Adenocarcinoma(MDA-MB-231) in
CD-1
Nude Mice.
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Figure 29 illustrates the weight of Human Breast Adenocarcinoma (MDA-MB-231 )
in CD-1
Nude Mice.
Figure 30 illustrates the growth of Human Breast Adenocarcinoma (MDA-MB-231)
in CD-1
Nude Mice.
Figure 31 illustrates the weight of Human Breast Adenocarcinoma (MDA-MB-231 )
in CD-1
Nude Mice.
Figure 32 illustrates the growth of Human Prostate Carcinoma (PC-3) in SCID
Mice.
Figure 33 illustrates the weight of Human Prostate Carcinoma (PC-3) in SCID
Mice.
Figure 34 illustrates the growth of Human Pancreatic Carcinoma (BxPC-3) in CD-
1 Nude
Mice.
Fi-gore 35 illustrates the weight of Human Pancreatic Carcinoma (SU.86.86) in
CD-1 Nude
Mice.
Figure 36 illustrates the growth of Human Prostate Carcinoma (DU145) in SCID
Mice.
Figure 37 illustrates the weight of Human Prostate Carcinoma (DU145) in SCID
Mice.
Figure 38 illustrates the growth of Human Ovary Adenocarcinoma (SK-OV-3) in CD-
l Nude
Mice.
Figure 39 illustrates the growth of Human Ovary Adenocarcinoma (SK-OV-3) in CD-
1 Nude
Mice.
Figure 40 illustrates the growth of Human Lung Adenocarcinoma (H460) in CD-1
Nude
Mice.
Figure 41 illustrates the weight of Human Lung Adenocarcinoma (H460) in CD-1
Nude
Mice.
Figure 42 illustrates the growth of Human Small Cell Lung Carcinoma (H209) in
SCID Mice.
Figure 43 illustrates the weight of Human Small Cell Lung Carcinoma (H209) in
SCID Mice.
Figure 44 illustrates the growth of human melanoma (C8161) in CD-1 Nude Mice.
Human
melanoma cells were injected into the right flanks of CD-1 nude mice to induce
tumor
growth. Treatments with saline, defined osmolarity solutions, Nat-BRM and Syn-
BRM were
started several days following tumor cell inoculation when the tumor had
reached a palpable
size. Tumor volume was measured by calipers on the indicated days.
Table 1: A synthetic biologic response modifier composition (Syn-BRM #1) of
the present
invention.
Table 2: A synthetic biologic response modifier composition (Syn-BRM #2) of
the present
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invention.
Table 3: Illustrates further embodiments of the synthetic biologic response
modifier
composition (Table 3A: Syn-BRM #3 and Table 3B: Syn-BRM#4) of the present
invention.
Where present, formic acid may be present in the concentration range of 40-107
mg/L.
Table 4: Shows proton chemical shift assignments from NMR spectra of Nat-BRM.
b= peak
overlapped with other compound peaks. n = not observed in the spectra due to
low
concentrations.
Table 6: Summary of Inorganic components detected in Nat-BRM.
Table 7: Summary of Nat-BRM in vivo anti-tumour activity assessed in mice
harbouring
human cancer.xenoplants. (N/A) not done; (+) significant tumor growth
suppression
(P<0.05); (-) no tumor growth suppression; (+l-) variable in different
experiments; ( + )
suppression of tumor growth statistically greater than either treatment alone
(P<0.05); ( -)
:.
suppression of tumor growth not greater than either treatment alone; ( +/- )
variable in
different experiments; (~) tumor suppression greater than either treatment
alone but not
statistically different.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is a synthetic biologic response modifier (Syn-BRM)
comprising
components in the following amounts: 57 + 20 mg/L 3-hydroxybutyric acid, 125 +
44 mg/L
lactic acid, 155 + 54 mg/L acetic acid, 1.4 ~ 0.5 mg/L creatine, 22 + 8 mg/L
creatinine, 2.5 +
0.9 mg/L carnitine, 6.8 + 2.4 mg/L taurine, 20 + 7 mg/L choline, 815 + 285
mg/L urea. The
compositions may additionally comprise 40 + 14 mg/L of formic acid. The Syn-
BRM been
shown to modify biological response ih vivo. In one example, the Syn-BRM
demonstrates
anti-cancer activity and enhances cell-mediated immune response to tumours.
In an embodiment of the invention the compositions may additionally comprise
inorganic
components including sodium, phoshphate and chloride (as described in the
tables and
examples) in amounts (+ 35%) of 14900 mg/L of NaCI, 1390 mg/L of Na2HP04 and
780
mg/L of NaHZP04.
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The compositions of the present invention can be made according to known and
standard
methods in the, art. For example, commercially available, optionally
pharmaceutical grade
compounds or components can be mixed and solubilized with sterile water and/or
using
appropriate buffers. One skilled in the art would appreciate how to adjust,
for example, the
osmolarity and pH of the compositions so that the compositions are
physiologically
acceptable.
For example, the pH of the composition may be adjusted to physiological pH,
i.e. 7.4-7.5,
using hydrochloric acid (1%) solution and sodium hydroxide (1% solution), and
a buffered
solution maybe obtained using dibasic and monobasic sodium phosphate salts as
buffers,
using conventional methods. In an embodiment of the invention the pH is 7Ø
In an embodiment of the invention an osmolarity of about 650 mOsm, but may be
as high as
850 mOsm.
The composition of the invention can modulate tumor necrosis factor (TNF)
production
and/or release. In one embodiment a composition of the invention, promotes the
release of
TNF from human peripheral blood mononuclear cells and from the pre-monocyte
cell line U-
937. As TNF is known to initiate a cascade of inflammatory and antitumor
cytokine effects,
one embodiment stimulates human leukocytes to release TNF and other cytokines
including
those listed in Example 22.
Once prepared, therefore, an embodiment of the composition it can be tested
for its ability to
stimulate TNF release, for example, using methodologies as taught in Example
8. The
compositions activate PBMNs to release TNF in vitro as measured by the
Monocyte/Macrophage Activation Assay (TNF-Release).
The efficacy of the compositions 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 in 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.
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One example of a method for studying the efficacy of the compositions on solid
tumors ih
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 are
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 least 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.
The composition may be used without further modification by simply packaging
it in vials
and sterilizing it. A preferred sterilization method is to subject the
composition to.three
sterilization cycles by autoclaving followed by incubation. The composition
may also be used
in a concentrated form. This solution is then tested for biological activity.
The composition
may also be lyophilized.
In other embodiments of the invention the compositions may be used in
conjunction with
known anticancer therapies or agents to enhance the effect of said therapies.
In one specific
embodiment the anticancer agent is a chemotherapeutic agent. One skilled in
the art would
appreciate how to test relative amounts of the compositions of the invention
with anticancer
agents (eg. chemotherapeutics) and/or anticancer therapies (eg. radiation
therapy).
Anticancer Agents
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This invention provides for the use of BRM in combination with other anti-
cancer'
compounds and antisense sequences. Examples of axlti-cancer compounds are:
antisense
sequences; Bleomycin; Docetaxel (Taxotere); Doxorubicin; Edatrexate;
Etoposide;
Finasteride (Proscar); Flutamide (Eulexin); Gemcitabine (Gemzar); Goserelin
Acetate
(Zoladex); Granisetron (Kytril); Irinotecan (Gampto/Camptosar); Leuprolide
(Viadur);
Methotrexate; Ondansetron (Zofran); Paclitaxel (Taxol); Pegaspargase
(Oncaspar);
Pilocarpine Hydrochloride (Salagen); Porfimer Sodium (Photofrin); Interleukin-
2 {Proleukin);
Rituximab (Rituxan); Topotecan (Hycamtin); Trastuzumab (Herceptin); Tretinoin
(Retin-a);
Triapine; Vincristine; Vinorelbine Tartrate (Navelbine); Drugs Used in Breast
Cancer such
as: Capecitabine (Xeloda); Cyclophosphamide (Cytoxan); Docetaxel (Taxotere);
Doxorubicin
Injection (Adriamycin); Doxorubicin, Liposomal-entrapped (Doxil); Epirubicin
(Ellence);
Exemestane ( Aromasin ); Raloxifene (Evista); Tamoxifen (Nolvadex);
Trastuzumab
(Herceptin); Goserelin Acetate (Zoladex), Zeneca; Drugs Used for Kaposi's
Sarcoma such as: ,
Liposomal-entrapped Doxorubicin (Doxil); Liposomal Daunorubicin (Daunoxome);
Drugs
for Promyelocytic Leukemia; Tretinoin (Vesanoid); Drugs for Chronic Myeloid
Leukemia
such as low-dose IFN-alpha; Drugs Used in Gastric Cancer; Antibiotics;
Antineoplastics;
Acute Lymphoblastic Leukemia; Pegaspargase (Oncaspar); L-asparaginase; IL-2;
Drugs for
Colon Cancer such as Edatrexate or 10-ethyl-10-deaza-aminopterin or I O-edam;
5-
fluorouracil (5-fu) and Levamisole; Methyl-ccnu (Methyl-chloroethyl-cyclohexyl-
nitrosourea); Fluorodeoxyuridine (Fudr); Vincristine; Drugs for Esophageal
Cancer such as:
Porfimer Sodium (Photofrin) or Treatment with a Neodymium:yag (Nd:yag) Laser;
Drugs
Used in Colorectal Cancer such as: Irinotecan (Camptosar); Topotecan
(Hycamtin);
Loperamide (Imodium); 5-fluorouracil (5-fu); Drugs for Advanced Head and Neck
Cancers
such as Docetaxel (Taxotere); Drugs for Non-hodgkin's Lymphoma such as
Rituximab;
Etoposide; Drugs for Non-Small-Cell Lung Cancer such as a Vinca Alkaloid,
Vinorelbine
Tartrate (Navelbine): Paitaxel, (Taxol); Docetaxel (Taxotere); Topotecan;
Irinotecan;
Gemcitabine; Drugs for Ovarian Cancer: Docetaxel (Taxotere); Gemcitabine
(Gemzar);
Irinotecan (Camptosar); Paclitaxel {Taxol); Topotecan (Hycamtin); Amifostine
(Ethyol), (For
Reducing the Cumulative Renal Toxicity Associated with Repeated Cisplatin
Therapy in
Patients with Advanced Ovarian Cancer); Drugs to Prevent Melanoma such as: 2-
ethylhexyl-
p-methoxy-cinnamate (2-ehmc); Octyl- N-dimethyl-p-aminobenzoate {O-paba);
Benzophenone-3 (Bp-3); Drugs for Prostate Cancer such as: Flutamide (Eulexin);
Finasteride
(Proscar); Terazosin (Hytrin); Doxazosin (Cardura); Goserelin Acetate
(Zoladex); Liarozole;
13
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Nilutamide (Nilandron); Mitoxantrone (Novantrone); Prednisone (Deltasone);
Drugs for
Pancreatic Cancer such as Gemcitabine (Gemzar); 5-fluorouracil; Drugs for
Advanced Renal
Cancer such as Interleukin-2 (Proleukin); Other Anti-neoplastic Drugs such as
Porfimer
Sodium, Axcan; Dacarbazine; Etoposide; Procarbazine HCI; Rituximab; Paclitaxel
(Taxol),
Trastuzumab (Herceptin); Temozolomide (Temodal); Alkylating Agents Used in
Combination Therapy for Different Cancers such as Cyclophosphamide; Cisplatin;
Melphalan; Therapies used for treatment of Cancer such as Photodynamics and
photosensitizing agents; Surgery; Cryotherapy; Chemotherapy; Biotherapy;
immunotherapy
(similar to biotherapy); Angiogenesis inhibitors; and hormone replacement
therapy.
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 compowlds, 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 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).
14
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
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 phosphorus atom in the backbone and those that do not have
a phosphorus
atom in the backbone. 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 Syn-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 Syn-BRM composition
and an
antisense compound in the prevention of a cancer which is related to over- or
under-
expression of a specific gene. .
Pharmaceutical Compositions
The compositions of the invention may be converted using customary methods
into
pharmaceutical compositions. The pharmaceutical composition containing the
composition
of the invention either alone or together with other active substances. Such
pharmaceutical
compositions can be for oral, topical, rectal, parenteral, local, inhalant, or
intracerebral use.
They 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 admin-
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
istration can be used, or forms for infusion or intravenous or intracerebral
injection can be
used, and can therefore be prepared as solutions of the compositions or as
powders of the
active compositions to be mixed with one or more pharmaceutically acceptable
excipients or
diluents, suitable for the aforesaid uses and with an osmolaxity 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 may be considered; for inhalant uses,
preparations in the
form of sprays, for example nose sprays, may be considered. Preferably, the
composition is
administered intramuscularly.
The pharmaceutical compositions can be prepared by peg 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~,ton's Pharmaceutical Sciences (Hack Publishing Company, Easton, Pa.,
USA 195).
On this basis, the pharmaceutical compositions include, albeit not
exclusively, the
composition 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 are indicated as therapeutic agents either alone or in
conjunction with other
therapeutic agents or other forms of treatment. For example, other antiviral
compounds,
including but not limited to; 3TC, interferon, ganciclovir, famciclovir,
rimantadine, foscarnet
sodium, zidovudine, amantadine hydrochloride, valacyclovir, ribavirin,
acyclovir, may be
used in combination with the composition of the present invention. The
compositions and
agents of the invention are intended for administration to humans or animals.
It will be appreciated by medical practitioners that it may be necessary to
deviate from the
amounts recormnended and, in particular, to do so as a function of the body
weight and
condition of the mammal to be treated, the particular disease to be treated,
the nature of the
administration route and the therapy desired. In addition, the type of mammal
and its
individual behavior towards the medicine or the nature of its formulation and
the time or
interval at which it is administered may also indicate use of amounts
different from those
16
CA 02451674 2003-12-17
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mentioned. Thus it may suffice, in some cases, to manage with less than the
above-
mentioned minimum amounts while in other cases the upper limit mentioned must
be
exceeded. Where major amounts are administered, it may be advisable to divide
these into
several administrations over the course of the day.
The present invention comprises a composition and it's use either alone or in
combination
with other drugs or therapies, wherein the composition shows no cytotoxicity
to human
peripheral blood mononuclear cells, and has at least one of the following
properties:
(a) is capable of stimulating monocytes and/or macrophages in vitro or in vivo
to produce
one or more cytokines; and/or
(b) is capable.of stimulating monocytes and/or macrophages to produce tumor
necrosis
factor in vitro and/or in vivo;
In a preferred embodiment of the composition, the composition stimulates tumor
necrosis
factor production in vitro or in vivo, and most preferably in humans.
Therapeutic Activity of the Combination
The combinations of the present invention will have a net anticancer effect
that is
greater than the anticancer effect of the individual components of the
combination when
admiiustered alone. Without being limited by mechanism, by combining one or
more
anticancer agents with a Syn-BRM composition it is possible to:
(i)increase the therapeutic effect of the anticancer agent(s);
(ii)increase the therapeutic effect of the Syn-BRM composition;
(iii)decrease or delay the toxicity phenomena associated with the a~iticancer
agent(s); and/or
(iv)decrease or delay the toxicity phenomena associated with the Syn-B1RM
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~eatmetzt Reports, 66, 1187 ]. To demonstrate the efficacy of a combination,
it may be
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CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
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 techniques and equations commonly known to workers skilled
in the art.
[T. H. Corbett et al., (1977) Caacer~, 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
constituents will be present at a dose generally not exceeding its maximum
tolerated dose,
will manifest 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 Syn-BRM
composition or the
anticancer agents) when administered to a patient alone.
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 (LDSO) 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 for other proportions
of the treated
population. Examples include, but are not limited to EDX, where x = 90, 95 or
99 and LDy,
where y = 10, 5 or 1 respectively (note that the values of x and y need not
add up to 100). It is
well known in the art that the acceptability of therapeutic index value (for
any given EDx/LDy
ratio) varies depending on the severity of the disease and availability of
other more
efficacious and/or less toxic treatment options. 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 a clinical
therapeutic 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, for example, the observed LDy of at
least one of the
one or more anticancer agents in the combination.
In a related embodiment the combination of the present uivention provides an
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CA 02451674 2003-12-17
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improved therapeutic index, in comparison to that of the individual components
of the
combination when administered alone, by increasing the observed EDX 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 combination when administered alone, by
increasing the
observed EDX of the Syn-BRM.
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 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 Syn-BRM composition. Successful candidates for use in the
combinations of
the present invention will be those that demonstrate a therapeutic synergy
with the Syn-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 ih
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 ih 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 are mixed before being subjected to the various treatments and
controls. In the case of
treatment of advanced tumors, tumors axe 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
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CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
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 least 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 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
The uses and methods of the present invention comprise administering to a
subject in
need thereof an effective amount of a Syn-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 Syn-BRM composition and one or more anticancer agents, and a
pharmaceutically
acceptable carrier is administered to a subject. The combination or the
pharmaceutical
compositiori 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 treatments) can be administered
concurrently with a
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
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 Syn-BRM composition may be administered before, during or
after
administration of the anticancer 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 Syn-
BRM
composition and/or the anticancer agents) of the combination may be
administered via a
single dose or via continuous perfusion.
The agents, compounds and compositions of this invention can be utilised in
vivo,
ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs,
cats, rats and mice,
or in vitro 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 leukemia, histiocytic
leukemia, stem cell
leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic
leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia,
lymphosarcoma
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cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic
leukemia,
monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid
granulocytic
leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia,
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,
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, I~upffer
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 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
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carcinoma, epiermoid carcinoma, carcinoma epitheliale 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
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular
carcinoma, carcinoma
lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma
medullare,
medullary carcinoma, melanotic carcinoma, carcinoma molle, 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.
Pharmaceutical Kits
The present invention additionally provides for therapeutic kits containing
(i) a dosage
unit of a composition and a pharmaceutically acceptable carrier; and (ii)
dosage unit of one or
more chemotherapeutic drugs) and a pharmaceutically acceptable carrier, said
(i) and (ii)
being provided in amounts.that are effective, in combination, for selectively
killing tumor or
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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 munber 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 for human administration.
EXAMPLES
EXAMPLE 1: SYNTHETIC BRM COMPOSITIONS
A synthetic BRM composition was prepared by combining the compounds in the
approximate
amounts as shown in Table 1. This composition is referred to herein as
Synthetic BRM #1.
A synthetic BRM composition was prepared by combining the compounds listed in
Table 2 in
the approximate amounts as indicated. These compositions are referred,to
herein as Synthetic
BRM #2.
A Syn-BRM composition was prepared according to Table 3b (Synthetic BRM #4)
The above compositions are biological response modifier compositions each
comprising the
components: 3-hydroxybutyric acid, lactic acid, acetic acid, creatine,
creatinine, carnitine,
taurine, choline, urea. Synthetic B1ZM #4 additionally comprises formic acid.
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These Syn-BRM compositions are shown to have activity similar to Nat-BRM. The
compositions are compared to naturally occurring BRM.compositions, isolated
from animal
bile, referred to herein as Nat-BRM. These are also described using batch
numbers such as #
311 or #313, or alternatively as BD-BRM.
EXAMPLE 2: IN VIVO DEMONSTRATION OF EFFICACY OF SYN-BRM
COMPOSITION IN THE TREATMENT OF HUMAN PANCREATIC CARCINOMA
IN CD-1 NUDE MICE ,
This experiment demonstrates and compares the ability of two synthetic BRM
compositions:
Syn-BRM#1 and Syn-BRM#1, to inhibit the growth of human pancreatic carcinoma
(BxPC-
3) in CD-1 nude mice.
Human pancreatic carcinoma cell line (BxPC-3) were 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
p.g/ml
streptomycin, 0.25 ~.g/ml amphotericin B and 2mM L-alanyl-1-glutamine at 37
°C in an
atmosphere of S% 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 tiypsin-EDTA and counted for tumor inoculation.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected at the right flank with 3 x
106 BxPC-3
human pancreatic carcinoma cells in 0.1 ml of PBS to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
demonstration.
Group 1: Saline Control (0.2 mllmouse/day, i.p., n=10)
Group 2: Syn-BRM #1 (0.2 ml/mouse/day, i.p., n=10)
Group 3: Syn-BRM #2 (0.2 ml/mouse/day, i.p., n=10)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
cured by the treatment with synthetic BRM compositions and to demonstrate how
the anti-
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
tumor effects of different BRM compositions compare with each other. Tumor
sizes were
measured every other day from day 19 after the tumor cell inoculation 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.
The treatments
were terminated at day 68. 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. One day after the last treatment, tumors were excised from the
animals and tumor
weights were measured. A standard bar graph was used to demonstrate the
differences in
tumor weights with each bar representing mean tumor weight calculated from 10
animals.
The purpose of this study was to demonstrate efficacy of Syn-BRM compositions
based on
the components. Two embodiments of synthetic BRM=s showed biological responses
that
were similar to the natural BRM (Nat-BRM)'with respect to TNF-a, release. The
in vivo nude
mouse xenograft model of BxPC-3 human pancreatic carcinoma has been tested
using these
two synthetic BRM compositions.
The results of the tumor growth curve and tumor weight measurements are shown
in the
Figure 1 (A) and (B). As illustrated, treatment with each of the two synthetic
BRM
compositions resulted in significant delay of tumor growth compared to saline
control.
EXAMPLE 3: IN VIVO DEMONSTRATION OF EFFICACY OF SYN-BRM IN THE
TREATMENT OF HUMAN PANCREATIC CARCINOMA IN CD-1 NUDE MICE
This experiment demonstrates and compares the ability of two synthetic BRM
compositions
and a natural BRM (batch #311) to inhibit the growth of human pancreatic
carcinoma (BxPC-
3) in CD-1 nude mice.
Human pancreatic carcinoma cell line (BxPC-3) 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
p.g/ml
streptomycin, 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
26
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WO 02/102363 PCT/CA02/00932
trypsin-EDTA treatment. The cells were harvested from subconfluent
logarithmically growing
culture by treatment with trypsin-EDTA and counted for tumor inoculation.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected at the right flank with 3 x
106 BxPC-3
human pancreatic carcinoma cells in 0.1 ml of PBS to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
experiment.
Group 1: Saline Control (0.2 ml/mouse/day, i.p., n=10)
Group 2: Natural BRM (batch #311) (0.2m1/mouse/day, i.p., n=~10)
Group 3: Synthetic BRM #1 (0.2 ml/mouse/day, i.p., n=10)
Group 4: Synthetic BRM #2 (0.2 ml/rnouse/day, i.p., n=10)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
cured by the treatment with natural BRM or synthetic BRM compositions and to
see how the
anti-tumor effects of different synthetic BRM compositions compare with those
of natural
BRM. Tumor sizes were measured every other day from day 23 after the tumor
cell .
inoculation 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. The treatments were terminated at day 58. 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. One day after the last treatment, tumors
were excised from
the animals and tumor weights were measured. A standard bar graph was used.to
demonstrate
the differences in tumor weights with each bar representing mean tumor weight
calculated
from 10 animals.
The purpose of this study was to demonstrate the efficacy of synthetic BRM
based on the
components in Table 1. Two embodiments of a synthetic BRM used in this study
showed
biological responses that were similar to the natural BRM with respect to TNF-
a release. The
in vivo nude mouse xenograft model of BxPC-3 pancreatic carcinoma has been
tested using
these two synthetic BRM compositions and their antitumor efficacy has been
compaxed with
that of natural BRM.
27
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
The results of the tumor growth curve and tumor weight measurements~are shown
in Figure 2
(A) and (B). As illustrated, treatment with each of the two synthetic BRM
compositions
resulted in significant delay of tumor growth compared to saline control. The
delay in tumor
growth achieved with both synthetic BRM compositions was as effective as that
observed
with the natural BRM.
EXAMPLE 4: IN VIVO DEMONSTRATION OF EFFICACY OF SYNTHETIC BRM
IN THE TREATMENT OF HUMAN MELANOMA IN CD-1 NUDE MICE
This experiment demonstrates and compares the ability of two embodiments of a
synthetic
BRM composition and a natural BRM (batch #311 ), to inhibit the growth of
human
melanoma (08161) in CD-1 nude mice.
Human melanoma cell line (08161) 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, 0.25
~,g/ml amphotericin B and 2mM L-alanyl-1-glutamine at 37 °C in an
atmosphere of 5% COa
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.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected with 5 x 106 08161 human
melanoma
cells in 0.1 ml of PBS at the right flank of the mice to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
experiment.
Group 1: Saline Control (0.2 ml/mouse/day, i.p., n=10)
Group 2: Natural BRM (batch #311) (0.2m1/mouse/day, i.p., n=10)
Group 3: Synthetic BRM #1 (0.2 ml/mouse/day, i.p., n=10)
Group 4: Synthetic BRM #2 (0.2 ml/mouse/day, i.p., n=10)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
28
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
cured by the treatment with either the natural BRM or the synthetic BRM
compositions and to
see how the anti-tumor effects of different synthetic BRM compositions compare
with those
of natural BRM. Tumor sizes were measured every other day from day 5 after the
tumor cell
inoculation 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. The treatments were terminated at day 38. 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. One day after the last treatment, tumors
were excised from
the animals and tumor weights were measured. A standard bar graph was used to
demonstrate
the differences in tumor weights with each bar representing mean tumor weight
calculated
from 10 animals.
The purpose of this study was to demonstrate the efficacy of a synthetic BRM
based on the
components presented in Table 1. Two embodiments of a synthetic BRM were used
in this
study showed biological responses that were similar to the natural BRM with
respect to TNF-
oc release. The ih vivo nude mouse xenograft model of 08161 melanoma has been
tested using
these two synthetic BRM compositions and their antitumor efficacy has been
compared with
that of natural BRM. .
The results of the tumor growth curve and tumor weight measurements are shown
in Figure 3
(A).and (B). As illustrated, treatment with each of the two synthetic BRM
compositions
resulted in significant delay of tumor growth compaxed to saline control. The
delay in tumor
growth achieved with synthetic BRM #1 and #2.compositions was as effective as
that
observed with natural BRM.
EXAMPLE 5; IN VIVO DEMONSTRATION OF EFFICACY OF SYNTHETIC BRM
IN THE TREATMENT OF HUMAN BREAST ADENOCARCINOMA IN CD-1 NUDE
MICE
This experiment demonstrates and compares the ability of two synthetic BRM
compositions
and a natural BRM (batch #311), to inhibit the growth of human breast
adenocarcinoma
(MDA-MB-231) in CD-1 nude mice.
29
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WO 02/102363 PCT/CA02/00932
Human breast adenocarcinoma cell line (MDA-MB-231) was grown as monolayer
culture in
Minimum essential medium (oc-MEM) supplemented with 10% fetal bovine serum
(FBS), 0.1
mM non-essential amino acid, 1.0 mM sodium pyruvate, 100 Ulml penicillin, 100
p.g/ml
streptomycin, 0.25 pg/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.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected at the right flank with 1 x
107 MDA-MB-
231 human breast adenocarcinoma cells in 0.1 ml of PBS to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
experiment.
Group 1: Saline Control (0.2 ml/mouse/day, i.p., n=8)
Group 2: Natural BRM (batch #311) (0.2m1/mouse/day, i.p., n=8)
Group 3: Synthetic BRM #1 (0.2 ml/mouse/day, i.p., n=8)
Group 4: Synthetic BRM #2 (0.2 ml/mouse/day, i.p., n=8)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
cured by the treatment with either the natural BRM or the synthetic BRM
compositions and to
see how the anti-tumor effects of different synthetic BRM compositions compare
with those
of natural BRM. Tumor sizes were measured every other day from day 4 after the
tumor cell
inoculation 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. The treatments were terminated at day 29. 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. One day after the last treatment, tumors
were excised from
the animals and tumor weights were measured. A standard bar graph was used to
demonstrate
the differences in tumor weights with each bar representing mean tumor weight
calculated
from 8 animals.
The purpose of this study was to demonstrate the efficacy of a synthetic BRM
based on the
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
components presented in Table 1. Two embodiments of a synthetic BRM used in
this study
showed biological responses that were similar to the natural BRM with respect
to TNF-a.
release. The ivc vivo nude mouse xenograft model of MDA-MB-231 breast
adenocarcinoma
has been tested using these two synthetic BRM compositions and their antitumor
efficacy has
been compared with that of natural BRIM.
The results of the tumor growth curve and tumor weight measurements are shown
in Figure 4
(A) and (B). As illustrated, treatment with each ofthe two synthetic
BRM~compositions
resulted in significant delay of tumor growth compared to saline control. The
delay in tumor
growth achieved with synthetic BR.M #1 was as effective as that observed with
natural BRM.
EXAMPLE 6: IN VIVO DEMONSTRATION OF EFFICACY OF A SYNTHETIC
BRM COMPOSITION IN THE TREATMENT OF HUMAN PANCREATIC
CARCINOMA IN CD-1 NUDE MICE
This experiment demonstrates and compares the ability of two embodiments of a
synthetic
BRM composition and a natural BRM (batch #313), to inhibit the growth of human
pancreatic carcinoma (BxPC-3) in CD-1 nude mice.
Human pancreatic carcinoma cell line (BxPC-3) was grown as monolayer culture
in
Minimum essential medium (oc-MEM) supplemented with 10% fetal bovine serum
(FBS), 0.1
mM non-essential amino acid, 1.0 mM sodium pyruvate, 100 LT/ml penicillin, 100
p,g/ml
streptomycin, 0.25 ~,g/ml amphotericin B and 2mM L-alanyl-1-glutamine at 37
°C in an
atmosphere of 5% C02 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.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected at the right flank with 3 x
106 BxPC-3
human pancreatic carcinoma cells in 0.1 ml of PBS to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
experiment.
3I
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
Group I: Saline Control (0.2 mI/mouse/day, i.p., n=10)
Group 2: Natural BRM (batch #313) (0.2m1/mouselday, i.p., n=10)
Group 3: Synthetic BRM #1 (0.2 ml/mouselday,~i.p., n=10)
Group 4: Synthetic BRM #2 (0.2 ml/mouse/day, i.p., n=10)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
cured by the treatment with natural BRM or synthetic BRM compositions and to
demonstrate
how the anti-tumor effects of different synthetic BRM compositions compare
with those of
natural BRM. Tumor sizes were measured every other day from day 17 after the
tumor cell
inoculation 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. The treatments were terminated at day 64. 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. One day after the last treatment, tumors
were excised from
the animals and tumor weights were measured. A standard bar graph was used to
demonstrate
the differences in tumor weights with each bar representing mean tumor weight
calculated
from 10 animals.
The purpose of this study was to demonstrate the efficacy. of a synthetic BRM
based on the
components in Table 1. Two embodiments of a synthetic BRM composition used in
this study
showed biological responses that were similar to the natural BRM with respect
to TNF-a
release. The in vivo nude mouse xenograft model of BxPC-3 pancreatic carcinoma
has been
tested using these two embodiments of a synthetic BRM composition and their
antitumor
efficacy has been compared with that of natural BRM.
The results of the tumor growth curve and tumor weight measurements are shown
in Figures
(A) and (B). As illustrated, treatment with each of the two synthetic BRM
compositions
resulted in significant delay of tumor growth compared to saline control. The
delay in tumor
growth achieved with synthetic BRM #2 was as effective as that observed with
the natural
BRM.
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EXAMPLE 7: IN VIVO DEMONSTRATION OF EFFICACY OF A SYNTHETIC
BRM COMPOSITION IN THE TREATMENT OF HUMAN BREAST
ADENOCARCINOMA IN CD-1 NUDE MICE
This experiment demonstrates and compares the ability of two embodiments of a
synthetic
BRM composition and a natural BRM (batch #313), to inhibit the growth of human
breast
adenocarcinoma (MDA-MB-231) in GD-1 nude mice.
Human breast adenocarcinoma cell line (MDA-MB-231) was grown asmonolayer
culture in
Minimum essential medium (a-MEM) supplemented with 10% fetal bovine serum
(FBS), 0.1
mM non-essential amino acid, 1.0 rnM sodium pyruvate, 100 U/ml penicillin, 100
~.g/ml
streptomycin, 0.25 pg/ml amphotericin B and 2mM L-alanyl-1-glutamine at 37
°C in an
atmosphere of S% 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.
An acclimation period of at least 7 days was allowed between animal receipt
and
commencement of tumor inoculation. When the female CD-1 mice were 6-7 weeks of
age
(20-25 g), each mouse was subcutaneously injected at the right flank with 1 x
10' MDA-MB-
231 human breast adenocaxcinoma cells in 0.1 ml of PBS to induce tumor growth.
The following treatment (or control) conditions were evaluated for this
experiment.
Group 1: Saline Control (0.2 ml/mouse/day, i.p., n=10)
Group 2: Natural BRM (batch #313) (0.2m1/mouse/day, i.p., n=10)
Group 3: Synthetic BRM #1 (0.2 ml/mouse/day, i.p., n=10)
Group 4: Synthetic BRM #2 (0.2 ml/mouse/day, i.p., n=10)
The major endpoint was to demonstrate that tumor growth could be delayed or
mice could be
cured by the treatment with either the natural BRM or the synthetic BRM
compositions and to
see how the anti-tumor effects of different synthetic BRM compositions compare
with those
of natural BRM. Tumor sizes were measured every other day from day 4 after the
tumor cell
inoculation 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,
33
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
respectively. The treatments were terminated at day 21. Meaai 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. One day after the last treatment, tumors
were excised from
the animals and tumor weights were measured. A standard bar graph was used to
demonstrate
the differences in tumor weights with each bar representing mean tumor weight
calculated
from 8 animals.
The purpose of this study was to demonstrate the efficacy of a synthetic BRM
composition
based on the components presented in Table 1. Two embodiments of a synthetic
BRM
composition used in this study showed biological responses that were similar
to the natural
BRM with respect to TNF-a release. The in vivo nude mouse xenograft model of
MDA-MB-
231 breast adenocarcinoma has been tested using these two synthetic BRM
compositions and
their antitumor efficacy has been compared with that of natural BRM.
The results of the tumor growth curve and tumor weight measurements are shown
in Figures
6 (A) and (B). As illustrated, treatment with each of the two synthetic BRM
compositions
resulted in significant delay of tumor growth compared to saline control. The
delay in tumor
growth achieved with synthetic BRM #2 was as effective as that observed with
natural BRM
EXAMPLE 8: THE BIOLOGICAL ACTIVITY OF THE BRM COMPOSITION
Studies are conducted to demonstrate the effect of the composition on cytokine
release from
peripheral blood mononuclear cells (PBMN) and/or U937 cells which is a stable
line of pre-
monocyte cells (American Type Culture Collection (ATCC), Rockville, Maryland).
These
studies provide the basis for a standardized test for quantitatively
evaluating the potency of a
given batch of a synthetic BRM composition to evaluate the ability of the
synthetic BRM
composition to stimulate TNF-a production in the PBMN or U937 cells.
Whole blood is drawn from 5 healthy human subjects. into heparinized
Vacutainer tubes
(Beckton Dickinson, Canada). PBMNs were isolated by gradient centrifugation on
Ficoll-
Hypaque (Pharmacia). The PBMNs are washed twice with phosphate-buffered saline
(PBS),
counted and resuspended in RPMI 1640 culture medium (Gibco Labs) at a
concentration of
106 cells/0.5 ml. These cells are cultured in 24-well, flat-bottomed tissue
culture plates
34
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
(Falcon, Becton, Dickinson). A 0.5 ml aliquot of the PBMN suspension is added
to each
well, which contains 50 ng lipopolysaccharide (LPS) (from E. coli), 10 p,1
fetal calf serum and
10-300 p,1 of the composition of the invention (see Example 1). If necessary,
the
hyperosmolar effect of the composition is neutralized by adding distilled
water to the culture
wells at a volume equivalent to 10% of the volume of composition used. The
total volume is
then made up to 1 ml/well with RPMI. PBS is used as a control. The cells are
cultured for 2,
6, 24, 48 and 72 hrs at 37°C in a humidified 5% COZ incubator. At the
end of each
incubation period, the cells are harvested and cell-free culture fluids are
obtained by
centrifugation at 9000 rpm for 10 mins. The samples are then stored for up to
2 weeks at -
70°C until immunoassays, such as ELISA, are conducted to quantify the
cytokines present.
Cytokine synthesis in the supernatants are measured after stimulating human
PBMN with the
BRM composition at volumes of 100 and 200 w1 per weh.
Cytokine synthesis in the supernatants is measured at 24 hrs at 37°C
after stimulating PBMNs
with the BRM composition and LPS (or LPS alone as positive control), using
volumes of 100
p.1 of the BRM composition per well. TNF is measured by a TNF-a ELISA kit
(Endogen,
Inc.), which detects a minimum level of 5 pg/ml of the cytokine. The other
ELISA
immunoassay kits that are used includ: IL-la (Endogen, Inc.); GM-CSF (Endogen,
Inc.);
RFN-a (Endogen, Inc.); IL-2 (Advanced Magnetics, Inc.); IL-6 (Advanced
Magnetics, Inc.);
IL-1 (Advanced Magnetics, Inc.); IL-4 (R&D Systems); and IL-8 (R&D Systems).
The
results indicate that TNF is the major, cytokine present in the supernatants,
along with smaller
amounts of IL-1 (3 and GM-CSF. For example, a 40 p,1 dose of the BRM
composition of
stimulates the production and release of T'NF-a, GM-CSF, and IL-1(3.
Different deletion batches of the BRM composition are examined for their
effect on LPS-
induced release of TNF.
The PBMN-TNF assay as described above is standardized using 100 p1 of the BRM
composition and 50 ng of LPS. PBMNs from 3 different human subjects are
obtained as
described above and used the same day. The results of each of the three assays
(using
individual subject cells) are averaged to compensate for variations in
response between
different subjects. The analysis involves determining the amount of TNF-a
released in RPMI
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
media alone and in the presence of 50 ng LPS. The TNF-a released in the
presence of 100 p1
of the BRM composition in combination with 50 ng LPS is also determined. The
TNF-a
released in media is subtracted from the LPS value to obtain the TNF-a
released in the
presence of LPS alone. The media and LPS values are subtracted from the
combined
composition and LPS value to obtain the TNF-a released in the presence of the
composition
alone (reported in pg/ml). Accordingly, the TNF release assay serves to
quantify the potency
of the BRM composition.
The BRM composition can stimulate release of TNF-a from U937 cells, which are
originally
derived from a patient with histocytic lymphoma and display many
characteristics of
monocytes. U937 cells can be obtained from the ATCC. They are routinely
maintained in
RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat-
inactivated
fetal calf serum (FCS, GIBCO), 2 mM L-glutamine (ICN Biomedical Inc, Costa
Mesa, CA),
and 10 ~g/ml Gentamycin Sulfate (SIGMA, Mississauga, Ontario, Canada) at
37°C, 5% CO2.
Passage of the U937 cells is performed every 3-4 days and seeding is at an
initial
concentration of 5 x 105 cells/ml. The U937 cells can be stimulated to
differentiate to
monocytes by exposure to phorbol 12-myristate 13-acetate (PMA; Sigma Chemical
Co., St.
Louis, MO). The resulting monocytes have the capacity to release TNF upon
stimulation,
such as with the composition of Example 1, alone or in combination with LPS.
PMA is first dissolved in dimethyl sulfoxide (DMSO, SIGMA) at a concentration
of 10 mM
and then diluted 1000-fold with PBS to a stock solution concentration of 10
~,M and stored at
-20°C. U937 cell suspensions are centrifuged at 350 x g for 10 mins at
room temperature and
reconstituted in fresh complete RPMI-1640 medium at a concentration of 2 x 106
cells/ml.
Cell viability is determined by trypan blue exclusion and is routinely greater
than 95%. PMA
is further diluted 500-fold with complete culture media to a concentration of
20 nM.
Aliquots of 0.5 ml of U937 cells (106 cell/ml) are cultured in the presence or
absence of 0.5
ml of PMA (20 nM) in 24-well, flat-bottom tissue culture plates (Becton
Dickinson, Lincoln
Park, NJ) and incubated for 72 hrs at 37°C, 5% C02. The final
concentrations per well are 5
x 105 cells and 10 nM PMA.
36 .
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
After 72 hrs of incubation, 120 p1 of media are removed and replaced by 100 p1
of the
composition of Example 1 and 10 ~1 of sterile deionized distilled water, in
the presence or
absence of 10 p,1 of LPS (5 ng/~l). After 24 hrs of incubation, any cells and
particulate matter
are pelleted by centrifugation at 350 x g for 10 min and the resulting
supernatants are stored
at -200C until they are assayed for TNF-a. BRM samples are tested on two
separate occa-
sions.
Two-site sandwich ELISAs are performed to quantify TNF-a in the U937 cell
culture
supernatants using TNF-a ELISA kits purchased from Endogen, Inc. (Cedarlane
Laboratories,
Hornby, Ontario). The protocol recommended by the manufacturer is used.
Briefly, 100.~p1 of
TNF-a standards and test samples are added to antihuman TNF-a pre-coated 96-
well plates
and incubated at 37°C, 5% COZ for 3, hrs. After extensive washing with
washing buffer, 100
p,1 of antihuman TNF-a conjugated to alkaline phosphatase is added to plates
and incubated at
37°C, 5% COZ for 2 hrs. After incubation, the plates are washed as
described above and 100
p,1 of premixed TMB substrate is added to each well and the enzymatic color
reaction is
allowed to develop at room temperature in the dark for 30 min. Then 100 ~,l of
stop solution
is added to each well to stop the reaction and the plates are read using an
SLT Lab Instrument
ELISA reader at 450 nm. The detection limit of the assay is 5 pg/ml.
TNF values for U937 cells are determined as described for PBMN cells.
EXAMPLE 9: EFFECTS OF THE BRM COMPOSITION ON T AND B LYMPHO-
CYTES IN CULTURE
The growth of human lymphocytes is examined under carefully controlled
conditions in the
presence and absence of the BRM composition. Standard concentrations of
lymphocytes are
incubated in wells containing various concentrations of the composition. When
normal T and
B human lymphocytes.are incubated with the composition in concentrations
similar to those
that are used clinically, there are no adverse effects as judged by trypan
blue dye exclusion.
Accordingly, the composition of the invention are non-toxic to normal T and B
lymphocytes
in culture.
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The effect of the composition on the survival of human PBMN is also examined.
PBMNs are
incubated for 24 and 48 hrs in plastic microwell plates with various volumes
of the composi-
tion and tissue culture medium. At the end of this period, the number'of
surviving cells is
estimated by trypan blue dye exclusion.
The number of surviving cells fell at 24 and again at 48 hours in the presence
or absence of
the composition is indicative of the cytotoxicity to human PBMN.
The ability of the composition to stimulate lymphocytes can be evaluated in
the following 3
indicator systems: 1) stimulation of lymphocyte DNA synthesis; 2) induction of
lymphocyte-
mediated.cytotoxic function; and 3) induction of monocyte/macrophage-mediated
cytotoxic
function. These tests were chosen for the screen because they measure
~immunological
functions that have been shown to be associated with different clinical
parameters in patients
with malignant disease. These indicators of immune function also can be
modulated in
cancer patients treated with different biological response modifying agents,
such as IFN or IL- .
2.
EXAMPLE 10: PHARMACODYNAMIC STUDIES IN MICE WITH THE BRM
COMPOSITION
Peritoneal macrophages are harvested from C57BL/6 mice 72 hours after
intraperitoneal
injection of 1.5 ml of 4% protease peptone. The macrophages are then
stimulated i~ vity~o
with medium alone, 50 ng LPS, or BRM. Measurements of the stimulation are
performed
with respect to TNF (by ELISA) and NO (by spectrophotometric assay using the
Greiss
reagent) levels in duplicate experiments.
Ih vitro synergy of BRM with LPS for TNF-a release can similarly be addressed.
Peritoneal
macrophages were harvested from C57BL16 mice after the same aforementioned
treatment.
The macrophages were then stimulated with 50 ng LPS alone or LPS with
different dilutions
of Nat-BRM. As above, TNF was determined via ELTSA. LPS alone induces about
TNF-a
release from mouse peritoneal macrophages in vitro.
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Ih vitro synergy of BRM with LPS for nitric oxide (NO) is addressed in the
same procedure as
above, except NO is determined in the supernatant of the treated macrophages.
As above, the
assay for NO is spectrophotometric and uses a Greiss reagent. LPS causes some
release of
NO. BRM in synergy with LPS induces a marked increase in NO production. Nat-
BRM by
itself does not induce release of NO by macrophages.
In vitro synergy of BRM with IFN-y for TNF-a release is studied using the same
peritoneal
mouse macrophages derived from C57BL/6 mice treated as above. Peritoneal mouse
macro-
phages exhibit a baseline release of TNF-a after 24 hours of in vitro culture.
The same
macrophages stimulated with either LPS or IFN-y release almost 3000 pg/ml of
TNF-a.
In vitro synergy of BRM with IFN-y for NO release is studied, using the same
peritoneal
mouse macrophages derived from C57BL16 mice treated as above. LPS and IFN-y
alone
each enhanced NO production.
In vivo production of TNF-a over 72 hours is studied on macrophages harvested
from
C57BL/6 mice that, prior to harvest, were treated with nothing, injected
intraperitoneally 72
hours previously with 1.5 ml of 4% protease peptone, or injected
intraperitoneally 72, 48, or
24 hours previously with 1.0 ml Nat-BRM diluted 1:10 in PBS. The macrophage
monolayers
are treated i~ vitro for 24 hours with IFN-'y (50 ~l/rnl), LPS only (5 ng/ml),
or the combina-
tion thereof. TNF and NO are determined as recited above.
The release of TNF-a from macrophages is examined in the absence of a stimulus
or with
IFN-~y, LPS, or LPS/IFN-'y after 24 hrs irk vitro culture. When harvested
macrophages are
exposed to IFN-y at 24 and 48 hrs prior to testing, they show a small increase
in production of
TNF-a. By contrast, harvested macrophages stimulated with LPS at 24 and 48
hrs, but not 72
hrs prior to testing, show enhanced release of TNF-a. Likewise, there is a
synergistic effect of
LPS and IFN-y on harvested macrophages that were stimulated 24 and 48 hrs but
not 72 hrs
before testing.
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EXAMPLE 11: THE BRM COMPOSITION HAS TNF-A RELEASING ACTIVITY
This Example demonstrates, in summary, the following: (1) the composition has
TNF-a
releasing activity and the TNF-a releasing activity is not related to any
contamination with
endotoxin; (2) priming of macrophages enhances the ability of the composition
to stimulate
release of TNF-a; and (3) the hyperosmolarity of the composition is not
responsible for TNF
a releasing activity.
To test whether an endotoxin effect is associated with the biological activity
noted above for
the composition of Example 1, further composition experiments are performed
with
polymyxin added to the reactants. Polymyxin inhibits the action of endotoxin
on leukocytes
The osmolarity of different batches is determined using standard methods. The
effect of the
hyperosmolarity of the composition on TNF-a releasing activity is also
studied.
EXAMPLE 12: ACTIVATION OF MONOCYTES AND MACROPHAGES WITH
THE BRM COMPOSITION
BRM compositions will activate normal monocytes to demonstrate cytotoxicity
towards the
Chang hepatoma cell line, which is used to measure monocyte toxicity, and that
the
monocytes and macrophages from cancer patients (e.g., those afflicted with
cancers of the
cervix, ovaries, ear/nose/throat, and endometrium/uterus, and chronic
myelogenous leukemia)
which have been stimulated by the composition to attack and destroy tumor
cells derived
from the same patient.
The monocyte tumoricidal function is tested in the presence of the composition
of the
invention and the basic procedure for these experiments is outlined below.
This procedure
has been named the "Monocyte/Macrophage Cytotoxicity Assay to Cell Lines and
Autologous Tumor Cells," or "Cytotoxicity Assay" for short.
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The method requires isolation of monocytes/macrophages, which is accomplished
as follows:
Venous blood is collected aseptically in heparinized Vacutainer tubes. Sterile
preservative-
free heparin is added to a final concentration of 20 units/ml. The blood is
diluted 3:1 in
Hanks balanced salt solution (HBSS), layered onto lymphocyte separation medium
and
centrifuged to obtain a band of peripheral blood mononuclear cells (PBMNs).
After centrifu-
gation, the mononuclear cell layer is recovered from the interface, washed
twice in medium
(medium is Roswell Park Memorial Institute [RPMI] 1640 media supplemented with
10%
heat-inactivated fetal bovine serum, 50 unitslml penicillin, and 50 ~,g/ml
streptomycin) and
monocytes are enumerated by latex ingestion. Monocytes are isolated by
adherence in 96-well
plastic plates (for 2 hours at 37°C, followed by two cycles of washing
with medium).
Adherent cells are estimated to be greater than 90% monocytes: Wells
containing adherent
cells are incubated overnight in the presence of Nat-BRM (1:10-1;200 final
dilution). Then,
adherent cells are washed to remove Nat-BRM and incubated overnight with tumor
cells. The
tumor cells are maintained in medium in which endotoxin concentration is
guaranteed by the
manufacturer to be low and is non-stimulatory in the assay.
For studies using a standaxd cell line, SICr (chromium) labelled Chang
hepatoma cells are
used because this cell line is insensitive to natural killer cell
cytotoxicity. These hepatoma
target tumor cells are added to adherent cell monolayers at effectoraarget
(E:T) cell ratios of
20:1 to 15:1. This E:T ratio is used because it falls well into the plateau
range on a curve
prepared by varying the E:T ratio from 5:1 to 30:1. After 24 hours,
supernatants are collected
and 5lCr release is quantitated as a representation of cytotoxicity (i.e. cell
lysis). The percent
specific cytotoxicity is calculated as:
specific release = T - S x 100
In the equation above, E = CPM released from target cells in the presence of
effector cells; S
= CPM released from target cells in the absence of effector cells; T = CPM
released from
target cells after treatment with 2% sodium dodecyl sulfate.
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For studies using autologous tumor cells, these cells are obtained from
surgical biopsies,
labelled with S~C, and used in the same way as the hepatoma cells described
above.
Preparation, of peritoneal and alveolar macrophages is done by the methods
described in
Braun et al., Cancer Research, 53 3362-3365 (1993).
Using this protocol, the composition is found to cause monocytes from healthy
donors to
exert cytotoxicity toward the Chang hepatoma cell line. Subsequently, whether
monocytes
and macrophages from a cancer patient could be stimulated by the composition
to attack and
destroy their own particular tumor is investigated. Using similar protocols as
described for
the standard cell line (Chang hepatoma cells), monocytes and/or peritoneal
macrophages from
cancer patients are isolated. Peritoneal macrophages are isolated from
peritoneal fluids
collected at the time of laparoscopy. '
EXAMPLE 13: MONOCYTE/MACROPHAGE STUDIES WITH SYN-BRM
COMPOSITION
A number of comparative studies aimed at determining the dose response
characteristics of
the composition in stimulating monocyte/macrophage tumoricidal function are
performed as
well as testing different batches of the composition. The main emphasis of the
studies is to
demonstrate the capacity of the composition to simulate tumoricidal function
in monocytes
and macrophages from different anatomical sites of cancer patients. For these
investigations,
the following can be relied upon: (1) peripheral blood monocytes from cancer
patients and
control subjects; (2) alveolar macrophages from lung cancer patients and
control patients with
non-malignant lung diseases; and (3) peritoneal macrophages from patients with
gynecological malignancies.
Dose response studies with different batches of the composition, all prepared
in accordance
with Example 1, are completed. These studies rely on peripheral blood
monocytes to test the
stimulatory activities of different doses and different batches of the
composition. Each batch
42
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of the composition is tested without dilution (neat), a 1:10 dilution and a
1:50 dilution of
material.
Tumoricidal function in peripheral blood monocytes is also evaluated. -Tests
are performed
on 4 peripheral blood monocyte samples from control subjects. . These tests
utilize an optimal
stimulating concentration of the composition (eg. 1:10 dilution) and an
optimal stimulating
concentration of IFN-~ plus LPS. The target cells in these studies are a
cultured, NK-
insensitive cell line, namely the Chang Hepatoma.
A test is also performed on 1 monocyte sample from a patient with cervical
cancer. This test
is important because the patient's own tumor cells are available to be used as
target cells in
the assay. As before, this test utilized an optimal stimulating concentration
of the
composition (eg., 1:10, dilution) and an optimal stimulating concentration of
IFN-y plus LPS.
Also, the effector/target cell ratio is reduced to 15/1 to conserve patient
tumor cells.
In the peripheral blood monocytes from control subjects, the composition
stimulates
monocyte tumoricidal function against the Chang Hepatoma cells at a level
equal to or greater
than the level elicited by an optimal stimulating concentration of IFN-y +
LPS. In the
peripheral blood monocytes from a patient with cervical cancer, the
composition stimulates
tumoricidal function against the patient's own tumor cells at a level which
exceeds that
elicited by IFN-y plus LPS.
Tumoricidal function in peritoneal macrophages from patients with
gynecological
malignancies can be tested. These tests are performed on peritoneal macrophage
samples
isolated from lavage fluids of patients with cervical cancer and patients with
ovarian cancer.
These tests are performed with the patient's own tumor cells as target cells
in the assay. As
before, an optimal stimulating concentration of the composition and an optimal
stimulating
concentration of IFN-y plus LPS are compared. Also, the effector/target cell
ratio can be
reduced to 15/1 to conserve patient tumor cells.
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These test results highlight the fact that the local tumor environment may be
a determinant of
the response of immune cells to immunological activators. In this case of
cervical cancer,
there is reduced pathological evidence of malignant disease within the
peritoneal cavity and
the development of tumoricidal function against the autologous tumor is better
with IFN-y
and LPS combined than with the composition. The response against the patient's
own tumor
to IFN-'y and LPS combined is minimal at best, whereas the response to the
composition was
greater.
Tumoricidal function in alveolar macrophages from lung cancer patients and
control subjects
~is tested. These tests can be performed on alveolar macrophage samples
isolated from
bronchoalveolar lavage fluids of a patient with non-small cell lung cancer and
patients with
non-malignant diseases of the lung. These tests utilize an optimal,
stimulating concentration
of the composition and an optimal stimulating concentration of IFN-y and LPS
combined.
The target cells in these studies were the Chang Hepatoma cells and the
effectorltarget cell
ratio is 2011.
The results are consistent with the observation that alveolar macrophages from
lung cancer
patients are impaired in their development of tumoricidal function in response
to conventional
macrophage activators such as IFN-'y + LPS. The results show that the
tumoricidal function
of alveolar macrophages from lung cancer patients is greatly reduced compared
to control
subjects.
Accordingly, the composition can activate tumoricidal activity in alveolar
macrophages.
The preliminary i~ vitro tests with the composition demonstrate that it is a
macrophage
activator. The material provided is able to elicit tumoricidal activity in a
standard
cytotoxicity assay against both an NK insensitive cell line and against
freshly dissociated
human tumor cells. The activity elicited is also found to be concentration-
dependent in these
tests. The capacity of the composition to active macrophage tumoricidal
function ivy vitro is
comparable to that of the best macrophage activating combination presently
available,
namely, IFN-y and endotoxin (i.e., LPS) combined. As stated above, the
capacity of the
composition to elicit this level of tumoricidal function in the absence of
endotoxin would be
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considered important biologically if the material is free of endotoxin
contamination. The
composition is free of endotoxin contamination when tested for pyrogens by the
United States
Pharmacoepeia (LTSP) rabbit pyrogen test.
As has been found fox other macrophage activators, the activity of the
composition in
stimulating macrophage tumoricidal function varies with the source of the
macrophages. It
appears that the composition is an excellent activator of peripheral blood
monocytes being
equivalent to IFN-y + LPS with normal donors and possibly superior to IFN-y +
LPS with
cancer patient donors. Malignant disease has a significant impact on the
development of
monocyte tumoricidal function depending on the activator used (Braun et al.,
(1991)). One
determinant of the biological activity of different macrophage activators in
cancer patients
monocytes is the sensitivity of the activator to arachidonic acid metabolism
and the secretion
by the cell of prostaglandins. From these initial studies with the
composition, it appears that
activity elicited with the compound is not sensitive to the inhibitory effects
of prostaglandins.
If prostaglandin insensitivity can be proven definitively for cancer patient
monocytes
stimulated with the composition, this would be considered important
therapeutically because
the effectiveness of many other biological activators is limited by
prostaglandins. Preliminary
studies with 2 specimens indicate that the composition may have good activity
in peritoneal
macrophages, particularly when malignant disease is present in the peritoneal
cavity.
E~MPLE 14: EVALUATION OF NATURAL KILLER (NK) CELL
INFILTRATION IN MICE HARBORING HUMAN MELANOMA XENOGRAFTS
The mouse xenograft model of human melanoma was used in these studies to
demonstrate the
effect of treatment with a syn-BRM composition (Syn-BRM#4) on NK cell
infiltration into
tumors isolated from mice harboring tumors. The cell line used in this
experiment to
inoculate mice were human melanoma (C8161) cells, although any carcinoma cell
line
capable of tumor formation upon inoculation could be used. The tumor cell
lines were grown
as outlined above and SCID mice ware inoculated with these cell lines as
described in the
previous examples.
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A human melanoma cell line (C8161) was grown as monolayer culture~in Minimum
essential
medium,(-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 p.g/ml
streptomycin and 0.25
p.g/ml amphotericin B and 2mM L-alanyl-1-glutamine at 37°C in an
atmosphere of 5% C02 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.
Tumor Inoculation: An acclimation period of at least 7 days was allowed
between receipt of
the immunocompromised 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.) or
Nat-BRM (0.2
ml/mouse/day, i.p.).
The major endpoint was to determine the extent to which NIA cells infiltrated
tumors in the
presence and absence of various syn-BltM compositions. Human tumor xenografts
from
mice treated with syn-BRM or saline were isolated after perfusion of mice with
saline,
homogenized in PBS and filtered with a Cell Strainer to produce a cell
suspension. Dead cells
and red blood cells were removed by gradient centrifugation with Histopaque-
1077. Cells
were washed twice with PBS and resuspended in culture medium supplemented with
2%
FBS. Cells/sample (1x106) were stained with anti-DXS, a NK cell specific
antibody
conjugated with FITC and/or anti-CDllb, a macrophage specific antibody
conjugated with
PE, and placed on ice for 30 minutes. The cells were subsequently washed once
with
medium and once with PBS, fixed with 1% paraformaldehyde .in PBS and analysed
by FACS
(fluorescence activated cell sorting).
The results shown in Figure 8 are from cells isolated from mice harboring
C8161 human
melanoma xenografts. This two-2 dimensional plot shows the .cell numbers that
stain with
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the macrophage specific antibodies in the y-axis and the NK specific
antibodies in~the x-axis.
Animals treated with syn-BRM showed significantly increased NK infiltration
into tumors
compared to those treated with saline, suggesting a role for syn-BRM in NK
cell activation
and an association between the efficacy of syn-BRM and NK cell activity.
Accordingly, as shown in the aforestated in vitro studies, the composition of
the present
invention is demonstrated to be able to activate monocytes and macrophages to
increase their
immune system function.
EXAMPLE 15: PREPARATION OF Nat-BRM
Bovine bile was collected from the gall bladders removed from healthy cows
(both males and
females) that were at least one and one-half years old. These cows were
slaughtered for food
use at a licensed and inspected abattoir. The slaughtered animals had been
inspected and
evaluated as healthy prior to slaughter and the gall bladders were separated
from the livers
and examined by a veterinarian to confirm that the gall bladders were free of
parasites and
evidence of infection, and thus suitable for use as a source of bile.
Gall bladders that passed this inspection were subjected to the following
procedure: Gall
bladders were wiped with a solution of 70% ethanol to sanitize the exterior of
the bladders
and bile was removed from the bladders with a syringe. The bile removed was
visually
examined in the syringe by the veterinarian to assure that it contained no
blood or pus and
was otherwise satisfactory. Bile from a healthy bovine is a greenish fluid
substantially free of
blood and pus. Fragments of livers, spleen, and lymph nodes were also
collected from the
animals whose bile was collected and the fragments were examined for the
presence of
parasites and other indications of disease.
For species that do not have a defined gall bladder (such as shark), bile is
obtained directly
from the hepatic organ.
Bile found to be satisfactory was transferred into a graduated amber bottle
containing ethanol
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to give a 50% bile/50% ethanol solution by volume. The bile/ethanol solution
was a greenish
fluid substantially free of foreign material and tested positive for ethanol
in accordance with
methods recited at United States Pharmacopeia XXII, Part B (1994). These
bottles were
labeled with a lot number. Bile collected from a minimum of fifty animals was
collected for
each lot.
The bile/ethanol solution was then centrifuged at 4200 rpm for at least 2-1/2
hours at 20 ~
2°C. The supernatant liquid was decanted, filtered through a filter
having, for example, a 2.5
~m retention, and checked for pH and ethanol content. The decanted liquid was
then
subjected to an activated charcoal treatment. The treated liquid was then
monitored for
Optical Density (OD) at 280 nm and conductivity. OD levels andlor conductivity
levels
outside specified ranges necessitated additional treatment of the liquid with
activated carbon
to achieve an OD and conductivity within specified ranges.
Following activated carbon treatment, the treated liquid filtered through a
filter having, for
example, a 2.5 p,m retention, the ethanol was evaporated off (for example, by
heating up to
about 85°C), and the treated liquid was concentrated to approximately
one-eighth of the
original bile/ethanol solution volume. The concentrated liquid was then cooled
to 20-25°C,
filtered through a filter having, for example, a 2.5 pm retention, and mixed
with ethyl ether
and the ether phase was discarded. This step can be repeated once. The aqueous
phase was
heated to remove residual ether (for example, by heating up to about
55°C for about 10 hrs)
and further reduced in volume to one-tenth of the original bile/ethanol volume
by heating to
around 80-85°C. The resultant composition was then tested for
appearance, biological
activity, and ethanol and ether content. The composition was a clear,
yellowish solution,
essentially free of foreign matter, and contained less than 10 ppm ethanol and
less than 5 ppm
ether.
Identity and purity were determined using reverse-phase high pressure liquid
chromatography
(reverse-phase HPLC). Potency is assayed using the monocyte/macrophage
activation test
referred to herein as the peripheral blood mononuclear cell-tumor necrosis
factor assay
(PBMN-TNF assay or, simply, TNF assay), as described in Example 2.
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Initial batches of the composition were manufactured as a non-buffered liquid.
Subsequent
batches were manufactured as a buffered liquid, prepared by adjusting the pH
of the
composition to about 7.4 ~ 0.2, using hydrochloric acid (1%) solution and
sodium hydroxide
(1% solution), as well as using dibasic and monobasic sodium phosphate salts
as buffers.
Bioburden reduction was conducted in a steam autoclave at 104 ~2°C for
60 mins. The bulk
solution was filled into 5 ml or 10 mI sterile bottles and capped. The filled
and capped bottles
were subjected to three sterilization cycles by autoclaving them at
104°C ~ 2°C for 60 mins
followed by incubation at 35°C for 23 ~ 1 hrs. Between each cycle of
sterilization (autoclave
plus incubation), samples were taken and tested for bioburden. Following the
last cycle of
sterilization, the bottles were visually inspected against a black and a white
background to
detect the presence of particulates. ~ .
Following inspection, the lot was. sampled and tested for conformance to
specifications.
Tests included identity, sterility, pyrogenicity, endotoxin, bioassay, HPLC
and general safety.
The table below summarizes the data obtained for the various tests performed
on the bile
extract including normal ranges of data, where appropriate.
Characteristics of Batch Compositions
Obtained In Accordance with Method of Example 15
FINAL PRODUCT TEST BATCH # BATCH # BATCH #
BC0248 BC0249 BC0250
Potency (pg/ml)* 210 183 304
Identity/Purity Pass Pass Pass
Agrees with reference
Safety (passes test according to U.S. 81 CFR ~ Pass Pass Pass
610.11)
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FINAL PRODUCT TEST BATCH # BATCH BATCH
# #
BC0248 BC0249 BC0250
Pyrogenicity (temp. increase Pass Pass Pass
shall not exceed
0.4C)
Endotoxin 0.4 EU/ml 0.25 0.25 0.25
Sterility (no growth) Pass Pass Pass
pH (7.40 ~ 0.2) 7.20 7.27 7.22
Appearance - Visual (clear,
light yellowish
Pass Pass Pass
liquid with little or no precipitate)
Appearance - OD (passes test) 1.34 . 1.38 1.85
Osmolarity (< 1000) 877 854 832
Solids (23 +/- 7mg/ml) 18 ~ 15 - 20
Ethanol (not more than 10 ppm) Pass Pass Pass
Ethyl Ether (not more than 5 Pass Pass Pass
ppm)
Conductivity (35 +/- 5 mMho) 33 35 38
* Potency was measured with activation
respect to monocyte/macrophage as described
in
Example 8; normal TNF-a release
is at least 100 pg/ml.
Accordingly, Nat-BRM can be prepared from readily available sources of bile,
using standard
laboratory methods, resulting in a standardized final product.
EXAMPLE 16: ANALYSIS OF Nat-BRM
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Preparations of the Nat-BRM composition of Example 15 have been analyzed using
methods
known in the art to identify organic, inorganic and amino acid components of
the
composition.
EXAMPLE 16.1: ANALYSIS OF ORGANIC COMPONENTS OF Nat-BRM
LC/MS
Liquid chromatography coupled with mass spectrometry (LC/MS) was used to
detect the
presence of organic components in Nat-BRM, including 3-hydroxybutyric acid,
lactic acid,
acetic acid, creative, creatinine, carnitine, taurineltaurocholic acid,
choline, and urea (Table
3A). The presence of formic acid was also established using this methodology
(Table 3B).
NMR Studies
1D and 2D NMR techniques were also used to identify of organic components in
Nat-BRM.
In deciding which compounds to analyse, biological relevance and profiles of
already
identified compounds and the theoretical structure and chemical shift range of
the candidate
compounds were considered. Biologically relevant compounds that fell within
the molecular
weight range found in Nat-BRM were considered. A large number of these
compounds were
metabolites found in the liver and biological fluids. Finally, compounds were
screened based
on their chemical shift profiles found in NMR databases (Aldrich, Sadder, at
the Chemistry
library of University of Toronto and Internet-based databases). A summary of
the proton
chemical shift assignments for identified compounds in Nat-BRM is given in
Table 5.
Pre-treatment and preparation of NMR samples: Unless otherwise indicated,
untreated Nat-
BRM was used to acquire NMR data. For 2-dimensional NMR spectroscopy, Nat-BRM
was
completely dried by low-heat evaporation and re-dissolved in deuterated water.
Pure
authentic compounds were added into Nat-BRM, and resonance peaks compared to
unknown
peaks in order to determine if the spiked compound is present in Nat-BRM. To
compare the
chemical shifts of peaks from Nat-BRM with those of candidate compounds,
authentic
standard solutions (mixed or single) were prepared (10 mM in deuterated dd-
water). The pH
and osmolarity of the standard were adjusted to that of Nat-BRM prior to NMR
measurements. To minimize data acquisition time and cost, some standards were
mixed.
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Samples for NMR analysis were prepared by mixing 120 p1 of each standard
solution with
500 ~l of Nat-BRM and 180 p1 D20 (containing 0.75% TSP, 3-(trimethylsilyl)-
tetradeutero
sodium propionate).
NMR data acquisition: 1-diriiensional and 2-dimensional proton spectra (COSY
(Correlation
Spectroscopy), TOCSY (Total Correlation Spectroscopy), and NOESY (Nuclear
Overhauser
Enhancement Spectroscopy) for correlation spectroscopy) were obtained using
state-of the-art
high field-strength 400, 500, 600 MHz NMR spectrometers at the University of
Guelph or the
University of Toronto, Ontario, Canada. NMR spectra of one-dimensional 1H, 13C
and 31P,
and two-dimensional COSY, TOCSY, HSQC, and HMBC were recorded and analyzed.
~ectral analysis and interpretations: Spectral data processing and.analyses
were carried out
using PC-based NMR computer software. Raw data, acquired from service
laboratories, were
subjected to transformation, phasing, baseline correction and chemical shift
referencing.
Spectra were aligned with each other, expanded for better viewing, and
plotted. When
required, peak integration was carried out to quantify peak areas. These
analyses allowed
evaluation of chemical shift comparison between resonances in Nat-BRM and
those in
samples of standards. Splitting patterns, ratios of integrals of peaks, lot-to-
lot consistency
and pH dependence on chemical shift of individual peaks were also used to aid
in assignment.
Results of the NMR anal,
1 3-hydroxybutyric acid peaks were identified at 1.20 ppm for CH3, 2.31 and
2.41 ppm
for CH2-CO, and 4.14 ppm for CH-O. The cross peaks of 3-hydroxybutyric acid in
its proton
group connections were obvious in 2D COSY (Figure 9) and TOCSY spectra.
Therefore 3-
hydroxybutyric acid was identified as a component of Nat-BRM.
2 Lactic acid peaks were identified at 1.33 ppm for CH3, and 4.12 ppm for CH-
O, which
were confirmed by COSY experiment. Therefore lactic acid was identified as a
component of
Nat-BRM.
52
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
3 A peak at 1.93 ppm was assigned to a methyl group of acetic acid, which was
surrounded by its two satellite peaks (at +/- 0.1 ppm) identified in the COSY
(Figure 9)
experiment. Therefore acetic acid has was identified as a component of Nat-
BRM.
4 Peaks were assigned at 3.22 ppm to (CH3)3-N, one at 3.54 ppm to CHZ-N, one
at 4.10
ppm to CHz-O, identifying choline as a component of Nat-BRM.
Creatine peaks were identified at 3.04 ppm for CH3-N, and 3.94 ppm for CH2-N.
The
spatial connection between these two groups was observed from their N(JE cross
peak in the
NOESY. spectrum from NMR spectra (Figure 10) thus identifying creatine as a
component of
Nat-BRM.
6 Creatinine peaks were assigned to a peak at 3.05 ppm for CH3-N, and one at
4.07 ppm
for CH2-N. Thus creatinine has been identified as a component of Nat-BRM.
7 The CHZ-N peak of creative was initially assigned to a strong peak at 3.97
ppm (3.96
ppm in the new spectra), however this did not superimpose with the peak in the
spectra of the
standard used to add creative to Nat-BRM. Hence the relatively strong peak at
3.96 ppm was
re-examined and identified as glycolate. The CHZ-N peak of creative was re-
assigned to an
adjacent minor peak at 3.94 ppm.
Carnitine resonance peaks were relatively weak. The (CH3)3-N peak was detected
at
3.23 ppm, and the CHZ-N peak was at 2.43 ppm which overlapped with GHZ-CO peak
of 3-
hydroxybutyric acid. Initially, the peaks from CHZ-CO and CH-O of carnitine
could not be
identified as they overlapped, overwhelmingly, with the peaks from taurine CH2-
S at 3.43
ppm and the residual solvent (water) peak at 4.74 ppm, respectively. TW o NOE
cross peaks
between (CH3)3-N and CHZ-CO, and CH2-N and CHZ-CO were found in the NOESY
spectrum (Figure 10) and the assignment of carnitine was confirmed.
9 Peaks from glycerol are expected at 3.56 and 3.65 ppm for CH2-O, and 3.78
ppm for
CH-O based on the spectra from the spiked authentic sample. Only a single CH2-
O peak at
53
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
3.65 ppm was observed in the spectra of Nat-BRM, in a region overlapped with
ethanol CH2
peaks. Since LC/MS studies demonstrated very low to undetectable levels of
glycerol in Nat-
BRM during method development studies, it is possible that Nat-BRM contains
only trace .
amounts of glycerol.
Peaks at 3.28 and 3.44 ppm have been assigned to CH2-N and CHZ-S of taurine,
respectively. Peaks at 3.11 and 3.51 ppm were originally assigned to the CH2-N
and CHZ-S
moeties of taurine. Using Nat-BRM with taurocholic acid added to it, it was
ascertained that
these peaks belonged to the taurine group in the taurocholic acid molecule.
11 A unique peak at 5.78 ppm was assigned to the NHS- of urea. This was
confirmed by
an increase in the peak from a Nat-BRM sample after standard urea solution was
added to it.
12 Previously identified peaks of phosphorylcholine and methylhydantoin did
not exactly
coincide with those of the phosphorylcholine and methylhydantoin standards.
Additional experiments revealed that a 4.08 ppm phosphorylcholine peak (4.07
in the current
study) was more properly assigned to creatinine. Other peaks previously
assigned to
phospholylcholine became unidentified peaks consistent with LC/MS results in
which these
compounds were 'absent' or at background levels in the Nat-BRM samples tested
.
In summary, analysis of the data from NMR studies confirmed the presence of 9
major
components of Nat-BRM.
Further NMR Studies
It was previously determined that most of the components iri Nat-BRM are of
low
molecular weight (less than 1000 Da). In addition, all the identified organic
components can
be viewed as metabolites from a variety of pathways. To identify further
unknown
components of the Nat-BRM composition small molecular weight compounds can be
considered that are part of the metabolic pathway of some of the already
identified
components, or are related to known components by structure or degradative
pathway. In
54
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
addition, published NMR studies on biological fluid samples and literature
information on
metabolic pathways (in particular liver metabolites), have been used to
determine which
candidate components to investigate (summarized in publications and books such
as: E.
Lynch, et al. J. of Inorganic Biochemistry, (1999), 73, 65-84; Lehninger's
Principles of
Biochemistry, Worth Publishers, Inc., New York, 1982). For example, formic
acid is a
compound whose NMR profiles, from NMR databases, coincided with unknown peaks
in
Nat-BRM. Furthermore, a literature search revealed this compound is found
downstream of
phosphoryl choline and choline in the biosynthetic pathway of glycine (Scheme
1 in
Descampiaux et al, (1997) Che~z. Res: Toxicol. 10: 34-40). As a result, formic
acid was
added to Nat-BRM and 1D proton spectra were recorded in order to observe
whether any
resonance peaks matched an unidentified peaks. The addition of formic acid
increased the
height of an unidentified peak, and accordingly, formic acid was assigned to a
peak at 8.46
ppm, (Figure 11).
In addition, the testing for taurocholic acid (TC) matched peaks at 0.89,
0.93, 1.55, 2.04, and
2.21 ppm, which were typical resonance peaks for C 18, C 19, C20, C22, and C23
from cholic
salts. Because those peaks were connected with two other typical taurocholic
acid peaks (C25
and C26) at 3.09 and 3.52 ppm observed from the COSY (Figure 9) and TOCSY
spectra, the
main cholic salt is likely taurocholic acid.
A typical proton NMR spectrum of Nat-BRM with the assignments is shown in
Figure 14.
The NMR proton chemical shifts of identified compounds of Nat-BRM are
summarized in
Table 4 , and the major peak assignments are summarized in Table 5.
EXAMPLE 16.2: ANALYSIS OF INORGANIC COMPONENTS OF Nat-BRM
Two lots each of Nat-BRM manufactured in two different facilities (the Dalton
and Imutec
facilities) were tested using standard methods known in the art for the
presence of inorganic
components. The detection limit for metals was 0.05 ppm~ and for ions was 1.0
ppm. See
Table 6 for the results of this analysis.
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
Tungsten, barium, nickel, strontium, copper and manganese, if present, were
below'the
detection limit.
EXAMPLE 17: IN VIVO EVALUATION OF Syn-BRM EFFICACY IN MICE
HARBOURING A HUMAN TUMOUR XENOGRAFT
As described in Examples 2-7, and Example 18 the animal models such as the
tumour
xenograft model may be used to assess the antitumour activity of BRM
compositions. Figure
16 summarizes the results of several mouse xenograft experiments in which
various lots of
Nat-BRM and a Syn-BRM composition were shown to have comparable activity in
vivo.
EXAMPLE 18: EVALUATION OF NATURAL HILLER (NIA CELL
INFILTRATION IN Nat-BRM -TREATED MICE HARBOURING HUMAN
TUMOUR XENOGRAFTS
In addition to a mechanism of direct contact (e.g. phagocytosis) for killing
tumor cells,
macrophages may exert their anti-tumor activity by a paracrine mechanism in
which secretion
of inflammatory cytokines, including TNF-alpha and IL-12, recruit and activate
cytotoxic
lymphocytes, such as NK cells, at the tumor site. To assess whether a
paracrine mechanism of
action is functional during Nat-BRM treatment, tumors were isolated from
saline- and Nat-
BRM- treated mice and the number of infiltrated NK cells was assessed first by
immunohistochemistry and then by flow cytometry.
In collaboration with Dr. Hermon Yeger, of the Research Institute at the
Hospital for Sick
Children, Toronto, we have been using an immunohistochemistry approach to
search for
changes in several potential biomarkers, including markers for
differentiation, proliferation,
NK and macrophage cell infiltration and apoptosis in xenografted tumors. Tumor
tissue
sections from excised human tumor xenografts in mice were analyzed and no
measurable
difference between tumors from saline and Nat-BRM treated mice was observed
for a number
of markers. These observations may indicate that changes in numbers of
infiltrating
r
macrophage and NK cells may be relatively small and below the detection
sensitivity of the
method. As an alternative to immunohistochemistry, we are currently using flow
cytometric
56
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
techniques to identify biological markers) in response to Nat-BRM treatment.
In undertaking
this approach we reasoned that flow cytometric methods would be more sensitive
in detecting
changes in the number of infiltrated immune cells than are histological
methods.
Analyses of infiltrated macrophage/monocyte (CD1 1b positive cells and NK)
cells and
assessment of cell cycle condition of tumor cells from Nat-BRM-treated tumors
are ongoing.
Preliminary results are described below.
2.2.2 FACE (fluorescence activated cell sorting) Methodology
Human tumor xenografts from mice treated with saline or Nat-BRM were isolated
after
perfusion of mice with saline. Isolated tissues were homogenized in PBS and
filtered with a
Cell Strainer to produce a cell suspension. Dead cells and red blood cells
were removed by
gradient centrifugation with Histopaque-1077. Cells were washed twice with PBS
and re-
suspended in culture medium supplemented with 2% FBS. 1x106 cells/sample were
stained
with anti-DX5 (NK specific) antibody conjugated with FITC and/or anti-CDl 1b
(macrophage
specific) antibody conjugated with PE,. on ice for 30 minutes. The cells were
subsequently
washed once with medium and once with.PBS, fixed with 1% paraformaldehyde in
PBS and
then analyzed by FACS. The results are presented as 2 dimensional plots of the
cell numbers
that stain with the macrophage specific antibodies in the y axis and the NK
specific antibodies
in the x axis.
Preliminary experiments indicate that the population of infiltrated NK cells
in human
melanoma (C8161) xenograft tumors increased from 6.45%, in the saline-treated
group, to
10.55% in the Nat-BRM treatment.group (Figure 17a). The C8161 experiments have
been
repeated and the averages of three experiments are in close agreement with the
data in
Figurel7a. Hence, the average number of NK cells increased from 6.85% in
saline-treated
tumors to 10.92% in Nat-BRM-treated tumors, which is an increase of 59.4%. In
a separate
experiment, using human pancreatic (Capan-1) xenograft tumors, the population
of infiltrated
NK cells increased from 4.49%, in the saline-treated group, to 9.7% in the Nat-
BRM
treatment group (Figure 17a). These results are consistent with the hypothesis
that Nat-BRM
immune modulation is mediated, at least partially, by NK cells, in vivo.
However, the
population of macrophages in the tumor tissues was too low to be analyzed
accurately by flow
cytometry. These data indicate that Nat-BRM-activated macrophages recruit
and/or activate
57
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
NK cells in tumox tissues.
In addition to the above approach, we are exploring the possibility of using
an RT-
PCR method to examine the expression of TNF-alpha, IL-12, IFN-gamma and iNOS
in cells
from Nat-BRM-treated tumors.
EXAMPLE 19: IN T~ITdO EVALUATION OF MACROPHAGE INVOLVEMENT IN
Nat-BRM ANTITUMOUR EFFECTS
A study is currently under way that will differentiate between macrophage-
mediated and
NK-mediated immune responses after Nat-BRM treatment. Human tumor xenografts
in mice
coupled with a macrophage depletion technique and NK-deficient mice
(SCID/beige) will be
used to assess the respective contributions of macrophage and NK cells in Nat-
BRM-
mediated tumor suppression. We have recently developed the macrophage
depletion
technique and preliminary results are presented below.
2.2.4 Macrophage Depletion Methodology
Selective depletion of macrophages ifs vivo was performed using the
liposomelCl2MBP technique essentially as described by Van Rooijen and Sanders,
1994.
Briefly, 2001 of liposomes prepared with C12MBP were injected (i.v.) the day
before
transplantation of tumor cells. Additional injections, every three days, at
the same dosage,
followed tumor implantation. Mice bearing tumors were randomly separated into
different
groups for treatment with the indicated drugs. Treatment schedules were
similar to the
standard xenograft experiments with Nat-BRM treatment employed at our
laboratory.
Results from an experiment examining the anticancer effect of Nat-BRM on the
development of human melanoma tumors in mice treated with C 12MDP, a chemical
that
selectively destroys macrophages, are presented in Figure 18. The results
indicate that the
anti-tumor effect of Nat-BRM was significantly compromised after macrophage
depletion,
suggesting an important role for macrophages in Nat-BRM-mediated antitumor
efficacy. To
assess the relationship between macrophages and NK cell infiltration into
tumor xenogxafts,
mice were depleted of macrophages, injected with C8161 human melanoma cells
and treated
with saline or Nat-BRM and NK cell infiltration into tumors was assessed as in
Figure 17.
58
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
Depletion of macrophages results in loss of Nat-BRM-induced increase in NK
cell infiltrates
into tumors suggesting that macrophages are involved in NK migration to tumors
(Figure 19).
In addition to investigating the role of macrophages amd NK cells in the anti-
tumor
activity of Nat-BRM, we are investigating the role of IL-12 and/or TNF-a, by
neutralizing
these factors using mono-specific antibodies followed by testing in xenograft
models. We
anticipate that the results of these experiments would be available by July,
2002. In addition
to antibody neutralization we are examining the possibility of testing Nat-BRM
efficacy in
TNF-oc or IL-12 knockout mice.
Moreover, we are attempting to develop cytotoxicity assays to examine the role
of
macrophage/monocyte interactioys with a number of effector cells (NK cells or
Thl sub-type
T lymphocytes). The effects of these interactions on Nat-BRM-mediated anti-
cancer activity
will be assessed by examining phagocytosis and release of inflammatory
cytokine/mediators
such as TNF-oc, IFN-y, IL-12 and perform.
EXAMPLE 20: IN hII~O EVALUATION OF EFFICACY OF Nat-BRM IN MICE
HARBOURING HUMAN TUMOUR XENOGRAFTS
The mouse xenograft model of neoplasia was used in these studies to
demonstrate the
effect of treatment with a Nat-BRM composition on tumor growth in mice. For
comparison,
separate groups of mice were treated with saline (control), a conventional
chemotherapeutic
drug or concurrently with a combination of a Nat-BRM composition and a
chemotherapeutic
drug.
A human carcinoma cell line was grown as monolayer culture in Minimum
essential
medium,(-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 p.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
59
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
inoculation could be used:
pancreatic adenocarcinoma (BxPG-3) (a gemcitabine-resistant cell line)
melanoma (A2058)
melanoma(C 8161 )
breast adenocarcinoma(MDA-MB-231)
prostate carcinoma (PC-3)
ovary adenocarcinoma (SK-OV-3)
large cell lung adenocarcinoma (H460)
small cell lung carcinoma (H209).
Tumor Inoculation: An acclimation period of at least 7 days was allowed
between receipt of
the immunocompromised 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 mI/mouse/day, i.p.),
Nat-BRM (0.2
ml/mouse/day, i.p.), a chemotherapeutic drug, or concurrently with Nat-BRM
(0.2
mllmouse/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.)
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
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
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 tumoux
growth curve,
and a bar graph showing mean tumor weights. -
TABLE: Mouse xenograft experiments with Nat-BRM compositions
and Nat-BRM combinations
Figure Human Mouse combination # mice with total
# carcinoma cell line strain dfU4 eXpt tumor regiression
20, pancreatic CD-1 gemcitabine- BRM: 4 (of 9)
21
22, pancreaticSU.86.86CD-1 gemcitabinegemcitabirie
23
24, melanomaA2058 CD-1 dacarbazinedacarbazine
25
26, melanomaC8161 CD-1 - dacarbazinecomb: 5 (of
27 10)
28, breast MDA-MB-~CD-1 Taxol Taxol
29
30, breast MDA-MB- CD-1 Taxol Taxol BRM: 2; comb:
31 . 5 (of 10)
32, prostatePC-3 ,SLID mitoxantrone-
33
34 pancreaticBxPC-3 CD-1 5-fluorouracil5-fluorouracilcomb: (5 of
10)
35 pancreaticSU.86.86CD-1 5-fluorouracil5-fluorouracil
36, 37 prostate DU145 SCID mitoxantrone
38 ovarian SK-OV-3 CD-1 cisplatin cisplatin
39 ovarian SK-OV-3 CD-1 taxol taxol
41 lung, large H460 CD-1 taxotere taxotere
cell
43 lung, small H209 SCID - -
cel I
The results of the mouse xenograft experiments outlined in the table above are
shown in
Figures 20-43. Nat-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 Nat-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
61
~~ t t ~ . pn
..>;J~:rti .~ ~. / ty .~r 7l ~7 f4;
~'~~(J~~~!A~(]Qg3'~' CA 02451674 2003-12-17
a t t~ ~
~;'a ~3 .,.tM,...1.F~~. x4.3 s d,a.,~,,.,3,~ ' ' s t~ ~.
R~~~i , _~
.~._~~~.
.,.~.....,
treated with a BRM composition alone or with a combination of the Nat-BRM
composition
and_ a chemotherapeutie 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 to 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 animalvweight 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
occurs before hte
tumor reaches 2 g. The animals are autopsied when sacrificed. To study
leukemia, cancerous
cells cari 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 survival
time of the animal. Alternative methods of assessing efficacy, and therapeutic
synergy, can
also be used.
These animal models are recognized in the art to be predictive tests for
anticancer
effects in humans.
EXAMPLE 21: IN TrI~O EVALUATION OF EFFICACY OF Syn-BRM
In addition~to the in vivo xenograft models described in Examples 2-7 and
Example 18, other
animals models of disease can be used assess the effect of Syn-BRM. For
example tumour
growth may be assayed in mice carrying human solid tumour isografts introduced
by fat pad
injection. Other animal models of cancer include an experimental model of
lymphoma and
leukemia in mice (survival assay) which may be applied to, fox
example,'Burkitts lymphoma
(lVan-Hodgkin's) (raji) or marine erythroleukemia (CB7 Friend retrovirus-
induced), and also
an experimental model of lung metastasis in mice as applied to human melanoma
(C8161) or
marine fibrosarcoma (R3).
62
Prated .06 10 2003; ~ epcil~ne File Irispe~~mn ~ .1
... ~.. : ..,~ ...,.,.. ~ ......
~WO~O'02C~OO~S~ ~ .P'wl~~~. '~:~ .~ "~ ~:. 4,
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Any of these or other in vitro or in vivo .models may also be used to assess
the effect of
treatement with Syn-BRM in combination with various anticancer agents, such as
chemotherapeutic drugs, radiation, a gene therapy and an antisense
oligonucleotide.
EXAMPLE 22: EVALUATION OF Syn-BRM ACTIVITIES
A worker skilled in the art can produce Syn-BRM compositions, ~ and assay Syn-
BRIM
compositions for activities such as ih vitro and/oi in vivo monocyte andlor
macrophage
stimulation, modulation of tumor necrosis factor production andlor release,
content of IL-10, IL-
1 CJ, 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. PCTlCA94/00494, published February 16, 1995 as WO
95/07089.
From the foregoing, it will be appreciated that, although specific embodiments
of the inventiow
have been described herein, for purposes of illustration, various
modifications rnay be made
without deviating from the spirit and scope of the invention. Accordingly, the
invention is not
limited except as by the appended claims.
63
Pryhtedy~6.10-X003; epolane ~ F~le~sflnspection 2..=
. ... _ . ...: rr,. ..
'c
f'W~~(~~'~"~r~~~9'r~'~-~CA 02451674 2003-12-17 _ . v, Js- n'L . ~ ~',~;,~~'
~., Af..,..M;~-,~t2~,r~~"~:
. . Table 1. Biological Response Modifier Composition (Syn-BRM #I).
' Compound Concentration
' (mg/L) ~ _ ~
.
3-Hydroxybutyric 560
Acid
Acetic Acid 4gp
Lactic Acid ~ 105 r
Urea 270 '
Creatinine I .5
Creatine I
Choline I . .' .. '
Phosphoryl Choline 1
Taurine . I
Carnitine I
Glycerol I
Methyl Hydantoin I
.
Optionally containing:
Phosphate ~ 15386 mg/L
Sodium 9765 mg/L
Chloride 9137 mg/L .
64
F'~ryted Og 1"0 2003' . epo,fne ~F~le Inspection.
. ,.. ,.r.. . .. , ~ t
~ S
f
W~~O~~F~JAO".~9~~' CA 02451674 2003-12-17 ~~~~~~ °"~
a...;~."ar...,_..z..=..,.;,ri!~;?,.:,.:,~,d.~.M:."~,..?'.:";.:.~:~."..
Table 2. Biological Response Modifier .Composition (Syn-BRM #2) ~ ,
Compound . Concentration
'
~rrtg/L)
3-Hydroxybutyric 560
Acid
Acetic lucid 480
Lactic Acid 105
Urea ' 270
Creatinine L 5
Creatine 1
Choline . 1
Phosphoryl Choline 1
Taurine 1
Carnitine I
Glycerol
Methyl Hydantoin 1
I
65 . .
r
Prni~ted 06.1,0.003 epa,(~ne F~le.lrlspectio~~,
CA 02451674 2003-12-17
~,~0~002CA~0932:° . .., ,;.., ~,.' _.:~ t ~. ~,~ ~ ~,~r
~......: , ~..~L..,:
".. .., ,R9=1, E~~
k.._ f ~. ,~w. ,
Table 3a: Biological Response Modifier Composition (Syn-BRM#3}
Compound ~ ' - m~~L
v v - Urea ' - , 302-930
Acetic acid 140-260
Lactic acid . 33-260
3-hydroxybutyric acid 43-80
Choline 17-29
.. Creatinine 13-23
Taurine. 2.6-12 .
Carriitine~ ' ' 1.5-3
Creatine ~ 1.1-1.6
66
3 , ,
Pr~rited 06 10 2003; epolne..~AFtIe~Inspection
>.
;LWfO2aQ2CA0093~ ~ CA 02451674 2003-12-17 ~~1~~~~ ~'~ s [ ' ,'Y'YY 'f~ ~,'~, t
,M.;
Table 3b: Biological Response Modifier Composition (Syn-BRM #4)
Compound ~ .mglL
. NaCI . 14900
NazHPO4 : - 1390
NaHZP04 780
Urea . . $15
Acetic acid ~ 155
Lactic acid 125
3-l~ydroxybutyric acid ~ 57
Formic acid 40
Creatinine 22
Choline . 20
Taurine 6_g
Carnitine 2.5
Creatine 1,4
Adjusted to = 7.0
Osmolarity ~ Adjusted to - 650 mOsnn
67
t'Pryted 0;6 10 2003; 'epoloe '~Ftle Inspection ~ 0'
~.....w~ ....,~ . ,.~ . ..~,.....
r
,~Q~O~~~r~,~~~~~;,CA 02451674 2003-12-17 . ~ , ?~ ~~;~r~~~~ '~r'. l~ F Pt~'~
;9' "~ r~~'
,;
"" ~,~a~.
' ' . ~ ~x~,:
Table 4: Proton Chemical, Shift Assignments
Compounds ' Chemical Proton groups
shifts(ppmj
1 . 2 CHI
hydroxybutyric 2:31, 2.41 b CHZ-CO
acid 4.14 b CH-O.
1 _ 3 3 ' CH3
lactic acid
4.12. b
CH-O
acetic acid 1.93 CH3
3.04 CH3-N
Creative
3.94 CHZ-N
3 . 0 5 CH3-N
Creatin
ine
. 4 , 07 CHZ-N
2.43 b ~ CHI-N
Carnitine 3.23 ' CH3-N
n CHz-CO; CH-O
3 . 28 CHZ-N
Taurine
3.44 b
CHI-S
3 . 2 2 CH3-N
Choline 3.54 b CHZ-N
4.10 b CHZ-O ,
TJre a 5 . 7 8 NHZ-N
formic acid 8.46 CH-CO
.
Gly 3.58 aCH~
Ala 1.49 ~CH3
n aCH
2.06 b (3CHz
_
Glu 2 . 3 3 b 'yCHa
aCH
2. 1 4, b aCH2
~Gln 2 . 4 5 b yCHz - .
n txCH
0.89, 0.93, C18, C19, C20, C22,
taurocholic 1.55, 2.04 b respectively
acid 2.21 .ta, 3.09,C23, C25, C26;
3.52 b respectively
betaine 3 . 27 . ~ (CH3) 3-N
3 . 91 CHZ-CO
glycolate 3.96 CH3
succinate 2.41 ' Cg~
6$
Printe~~0610..200~~ epo.itne' Ftle',f'nspeetiari:
.... .. ~.".: .. .~ . . ~ . ".
r.
r1/1102002CA(?~932t. CA 02451674 2003-12-17 :,.r"~~~stt~,~.. ' ~ ~ R~~ ~'r y
~.
~r : ~:
...h.."
., . '.. 14t f11
.,x. r:'. .,.,.... ..,.
Table 6: Inorganic analysis
- . , Dalton Imutec
(408) ~ (407)
- . Parameters' ~ ~Od~002~ 08202 . BC031$
~ 'BiC03'17.
m ~ m m
' Sodium ' 5,990 ~E,515 5,960 5,855
Phosphorus 297 292 ' 216 236
Potassium 244 811 277 276
Magnesium 1.20 1.00 0.95 1.05
CaiCium 4.65 2.50 3.65 ' 1.15
i
.
Tungsten N.D. N:D. ~ N.D. ~N.D, ~ .
.
' Barium N. D. N. D. N. D, . . N. t~
D. . ;
~ Tunc . 1.00 ' 3.Ot~ B.00 ~ 3.75
Nickel N. D. . N. D: ~ N. D. N. D, '
.
Chromium 0.05 0.20 0.20 0.20 j
Strontium N.D. N.D. N.D. N.D. .
Copper ' N.D. N.D. N.D. N.D. I
.
Manganese' ~ iJ.D. '...N.D. . ''N.D.. .D,
~ ~ ~ N
'
Molybdenum ' ~N.D. 'N.D, ~ 4.35 1.95
' i
Chlaride 9,90 11,880 9,715. 10,460
Sulphate 88.7 90.2 . 109 111
Phasphafe 909 894 fi62 724
Fluoride 72.0 58.0 a2.2 63.5 ;
. .
Bromide 8.50 10.9 11,1 '.11.8
~ .
Nitrate ' 1:3~'I N. D. 3.40 ~ ' 1.20 1
~ ~ ' -
N.D.
=
Not
Detected
69
Printed 0~.'IO 2003'' ~epolme File Ipspection
' . ~ ... .... . ~..::... ., ,.1.,.. ., ..e..: ; n ... F ~'.
' ~, i !',.~, , ;, F~~ ~ '!~ ~IL9'n2 r
t,~~~2~,d~ .~'r~l~~~~2y CA 02451674 2003-12-17 ' i ' ~< '"~' c2
~~. » E~, ~~ ;E
AM..d.~ ~. , Y k :7 ... ~m ,..~ s, ~,.7 f :y'
~ ri,.3."..wr..:.
'liable 7: Nat-BRM anti-tumor activity: human cancer xenopIants in mice
Cancer type Mouse . Nat-BRM Combination
~
tumor cell line Strain Mono- Therapy .
. therapy (clinical-use
drug)
Skin Cancer
Melanoma A2058 CD- 1 Nude + + (DTIC).
Melanoma C8969 CD- 1 Nude + + (DTIC)
Pancreatic
Adenocarcinoma BxPc-3. CD- 1 Nude + .~- (Gemcitabine)
-h ( 5-FU)
CD- 1 Nude +
Carcinoma SU 86_86 -~- (Gemcitabine)
-t-- ( 5-FU)
CD- 1 Nude + + (Gemcitabine)
MIA-PaCa-2
Breast
Adenocarcinoma MDA- CD- 1 Nude + -E- (Taxol).
MB-237
- (Doxorubicin)
CD- 1 Nude +
N/A
Adenocarcinoma MVB-9
Ovarian
Adenocarcinoma SK-OV- CD- 1 Nude + + (Taxol)
3
* (Cisplatin)
Prostatic
Carcinoma DU945 SCID +
(Novantrone )
Carcinoma PC-3 SCID + -
(Novantrone}
Lung Cancer .
Smal1 cell lung SCID + +(Carboplatin)
carcinoma NCI-H209
CD- 1 Nude +/- .~.~_ (Taxotere)
,Large cell lung
carcinoma NCI-H460
Colon
Adenocarcinoma HT29 CD- 1 Nude - + (5-FU)
Lymphoma
Non-.Hodgkin's SCID . - N/A
lymphoma Raji
Printed 06 1~0 ~003T epolirt~ ~ Frle l}nspection ° ' . ~ 9
. , r ~ .. ...,: . ~:. . ..~.. .....,.,.- .-.:n., _. v .ri . , .s ... : -..,
.. ,_........ G. ,....A. ',.. ~:..
CA 02451674 2003-12-17
WO 02/102363 PCT/CA02/00932
Table 7: Nat-BRM anti-tumor activity: human cancer xenoplants in mice
Cancer type Mouse Nat-BRM Combination
tumor cell line Strain Mono- Therapy
therapy (clinical-use
drug)
Skin Cancer
Melanoma A2056 CD- 1 Nude + + (DTIC)
Melanoma C8161 CD- 1 Nude . + + (DTIC)
Pancreatic
Adenocarcinoma BxPc-3 CD- 1 Nude + -I-~(Gemcitabine)
+ (5-FU)
Carcinoma SU 86.86 CD- 1 Nude + + (Gemcitabine)
+ (5-FU)
CD- 1 Nude + + (Gemcitabine)
MIA-PaCa-2
Breast
Adenooarcinoma MDA- CD- 1 Nude + + (Taxol)
MB-23l
- (Doxorubicin)
CD- 1 Nude +
N/A
Adenocarcinoma MVB-9
Ovarian -
Adenocarcinoma SK-OV- CD- 1 Nude + ~- (Taxol)
3
* (Cisplatin)
Prostatic
Carcinoma DU145 SCID + -.(Novantrone)
Carcinoma PC-3 SCID +
(Novantrone)
Lung Cancer , -.
Small cell lung SCID + +(Carboplatin)
carcinoma NCI-H209
CD- 1 Nude +/-
'E~- (Taxotere)
.Large cell lung .
carcinoma NCI-H460
Colon
Adenocarcinoma HT29 CD- 1 Nude - + (5-FU)
Lymphoma
Non-Hodgkin's SLID - N/A
lymphoma .Raji
71
