U.S. patent application number 10/806621 was filed with the patent office on 2004-12-02 for chemotherapeutic composition and method.
Invention is credited to Filion, Mario C., Phillips, Nigel C..
Application Number | 20040242524 10/806621 |
Document ID | / |
Family ID | 33162851 |
Filed Date | 2004-12-02 |
United States Patent
Application |
20040242524 |
Kind Code |
A1 |
Phillips, Nigel C. ; et
al. |
December 2, 2004 |
Chemotherapeutic composition and method
Abstract
The present invention relates to a composition and method
comprising Mycobacterium phlei (M. phlei)-DNA (M-DNA), M-DNA
preserved and complexed on M. phlei cell wall (MCC), a
chemotherapeutic agent and a pharmaceutically acceptable carrier,
wherein the M-DNA and the MCC induce cell cycle arrest in
proliferating cancer cells, inhibit proliferation of cancer cells,
induce apoptosis in cancer cells and potentiate the antineoplastic
effect of the chemotherapeutic agent on cancer cells.
Inventors: |
Phillips, Nigel C.;
(Pointe-Claire, CA) ; Filion, Mario C.; (Laval,
CA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Family ID: |
33162851 |
Appl. No.: |
10/806621 |
Filed: |
March 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10806621 |
Mar 23, 2004 |
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09857332 |
Sep 17, 2001 |
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09857332 |
Sep 17, 2001 |
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PCT/CA99/01157 |
Dec 3, 1999 |
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60111019 |
Dec 4, 1998 |
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60127320 |
Apr 1, 1999 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 45/06 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 048/00 |
Claims
1-16. (Cancelled)
17. A composition comprising M-DNA and a chemotherapeutic agent,
wherein the M-DNA potentiates the anti-cancer activity of the
chemotherapeutic agent in treating cancer in an animal having
cancer.
18. A composition comprising MCC and a chemotherapeutic agent,
wherein the MCC potentiates the anti-cancer activity of the
chemotherapeutic agent in treating cancer in an animal having
cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/111,109 filed Dec. 4, 1998 and to U.S.
Provisional Application Serial No. 60/127,320 filed Apr. 1,
1999.
FIELD OF INVENTION
[0002] The present invention relates to a composition and method
comprising Mycobacterium phlei (M. phlei)-DNA (M-DNA), M-DNA
preserved and complexed on M. phlei cell wall (MCC) and a
chemotherapeutic agent, wherein the M-DNA and the MCC are effective
for treating cancer and for potentiating the antineoplastic effect
of the chemotherapeutic agent on the cancer.
BACKGROUND OF THE INVENTION
[0003] Cancer is an aberrant net accumulation of atypical cells,
which results from an excess of proliferation, an insufficiency of
cell death, or a combination of the two.
[0004] Proliferation is the culmination of a cell's progression
through the cell cycle and is characterized by replication of total
cellular DNA and the division of one cell into two cells. For cell
division, mammalian cells pass through an organized series of
controlled events, referred to as the cell cycle. The initiation of
an event during cell cycle progression is dependent on the
successful completion of an earlier event. The cell cycle can be
divided into 5 major phases. These are Go, G.sub.1, S, G.sub.2 and
M. During the Go phase, cells are quiescent. Most cells in the
body, at any one time, are in this stage. During the G.sub.1 phase,
cells, responding to signals to divide, produce the RNA and the
proteins necessary for DNA synthesis. During the S-phase (SE, early
S-phase; SM, middle S-phase; and SL, late S-phase) the cells
replicate their DNA. At the end of the S phase, each cell contains
twice its original DNA content but is still bound by one external
cell membrane. During the G.sub.2 phase, proteins are elaborated in
preparation for cell division. During the mitotic (M) phase, the
cell divides into two daughter cells.
[0005] Alterations in cell cycle progression occur in all cancers
and may result from over-expression of genes, mutation of
regulatory genes or abrogation of DNA damage checkpoints, and may
modulate the cellular response to treatment with chemotherapeutic
agents (Hochhauser D. Anti-Cancer Chemotherapeutic Agents 8:903,
1997).
[0006] Cell death is effected by immune-mediators that initiate
cytolytic processes and that promote apoptosis, and from apoptosis
inducers that directly initiate pathways leading to cell death.
Apoptosis is an active cellular death process characterized by
distinctive morphological changes that include condensation of
nuclear chromatin, cell shrinkage, nuclear disintegration, plasma
membrane blebbing, and the formation of membrane-bound apoptotic
bodies (Wyllie et al. Int. Rev. Cytol. 68:251, 1980). A molecular
hallmark of apoptosis is degradation of the cell's nuclear DNA into
oligonucleosomal-length fragments as the result of activation of
endogenous endonucleases (Wyllie A. Nature 284:555, 1981).
[0007] Caspases have been implicated as key enzymes in the
execution phase of apoptosis. The caspase family consists of at
least fourteen related cysteine aspartane proteases. All the
caspases contain a conserved QACXG (where X is R, Q or G) (SEQ ID
NO: 1) pentapeptide active-site motif (Cohen G. Biochim. Biophys.
Acta 1366:139, 1997). A number of caspases are synthesized as
inactive proenzymes, which are activated following cleavage at
specific aspartate cleavage sites (Cohen G. Biochim. Biophys. Acta
1366:139, 1997) or as inactive enzymes that require association
with regulatory molecules for activation (Stennicke et al. J. Biol.
Chem. 274:8359, 1999). Activation of the initiator procaspase
activates downstream effector caspases triggering the cell death
cascade (Pan et al. J. Biol. Chem. 273:5841, 1998; Earnshaw W.
Nature 397:387, 1999).
[0008] Most currently used chemotherapeutic agents are
nonspecifically cytotoxic. Many of these chemotherapeutic agents
have toxic side effects, are debilitating and often compromise the
quality of life of the patient. Moreover, alterations in the
transport and metabolism of chemotherapeutic agents by the cancer
cells result in the development of resistance to the
chemotherapeutic agents by the cancer cells.
[0009] Therefore, there is a continuing need for novel compositions
and methods that induce cell cycle arrest in cancer cells, that
inhibit proliferation of cancer cells, that induce apoptosis in
cancer cells and that potentiate the antineoplastic effect of
chemotherapeutic agents on cancer cells. Moreover, such
compositions should be simple and relatively inexpensive to
prepare, their activity should remain therapeutically stable over
time and they should be effective at dose regimens that are
associated with minimal toxicity even upon repeated
administration.
SUMMARY OF THE INVENTION
[0010] The present invention satisfies these needs by providing a
composition and method comprising Mycobacterium phlei (M.
phlei)-DNA (M-DNA), M-DNA preserved and complexed on M. phlei cell
wall (MCC), a chemotherapeutic agent and a pharmaceutically
acceptable carrier, wherein the M-DNA and the MCC induce cell cycle
arrest in cancer cells, inhibit proliferation of cancer cells,
induce apoptosis in cancer cells and potentiate the antineoplastic
effect of the chemotherapeutic agent on cancer cells. Moreover,
M-DNA and MCC are simple and relatively inexpensive to prepare,
their activity is reproducible among preparations, remains
therapeutically table over time, and is effective at dose regimens
that are associated with minimal toxicity even upon repeated
administration.
[0011] To prepare MCC, M. phlei are grown in liquid medium and
harvested. The M. phlei are disrupted, and the solid components of
the disrupted M. phlei are collected by centrifugal sedimentation.
The solid components are deproteinized, delipidated, and washed.
All reagents are selected to enhance conservation of DNA during MCC
preparation. M-DNA is prepared from MCC or directly from M. phlei.
Again, all reagents are selected to enhance conservation of DNA
during M-DNA preparation.
[0012] A composition comprising M-DNA, MCC, M-DNA+chemotherapeutic
agent or MCC+chemotherapeutic agent is administered to an animal,
including a human, having cancer in an amount effective to treat
the cancer in the animal. The unexpected and surprising ability of
M-DNA and of MCC to induce cell cycle arrest in cancer cells, to
inhibit proliferation of cancer cells, to induce apoptosis in
cancer cells and to potentiate the antineoplastic effect of
chemotherapeutic agents on cancer cells addresses a long felt
unfulfilled need in the medical arts and provides an important
benefit for animals, including humans.
[0013] Another object of the present invention is to provide a
composition and method that induces cell cycle arrest in cancer
cells.
[0014] Another object of the present invention is to provide a
composition and method that inhibits proliferation of cancer
cells.
[0015] Another object of the present invention to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent on cancer cells.
[0016] Another object of the present invention to provide a
composition and method that potentiates the effect of a
chemotherapeutic agent on cancer cells by synchronizing the cell
cycle of the cancer cells.
[0017] Another object of the present invention is to provide a
composition and method that potentiates the effect of a
chemotherapeutic agent on proliferation of cancer cells.
[0018] Another object of the present invention is to provide a
composition and method effective to treat cancer in an animal,
including a human.
[0019] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent in treating cancer in an animal,
including a human.
[0020] Another object of the present invention is to provide a
composition and method effective to eliminate cancer in an animal,
including a human.
[0021] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent in eliminating cancer in an animal,
including a human.
[0022] Another object of the present invention is to provide a
composition and method that induces cell cycle arrest in malignant
melanoma cells.
[0023] Another object of the present invention is to provide a
composition and method that inhibits proliferation of malignant
melanoma cells.
[0024] Another object of the present invention to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent on malignant melanoma cells.
[0025] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent on cell cycle arrest in malignant
melanoma cells.
[0026] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent on proliferation of malignant melanoma
cells.
[0027] Another object of the present invention is to provide a
composition and method that induces apopotosis in malignant
melanoma cells.
[0028] Another object of the present invention is to provide a
composition and method that activates caspases in malignant
melanoma cells.
[0029] Another object of the present invention to provide a
composition and method effective to treat malignant melanoma in an
animal, including a human.
[0030] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent in treating malignant melanoma in an
animal, including a human.
[0031] Another object of the present invention to provide a
composition and method effective to eliminate malignant melanoma in
an animal, including a human.
[0032] Another object of the present invention is to provide a
composition and method that potentiates the antineoplastic effect
of a chemotherapeutic agent in eliminating malignant melanoma in an
animal, including a human.
[0033] Another object of the present invention is to provide a
composition and method that potentiates the effect of radiation in
treating cancer in an animal, including a human.
[0034] Another object of the present invention is to provide a
composition and method that potentiates the effect of radiation in
eliminating cancer in an animal, including a human.
[0035] Another object of the present invention to provide a
composition and method that potentiates the effect of radiotherapy
on cancer cells by synchronizing the cell cycle of the cancer
cells.
[0036] Another object of the present invention is to provide a
composition that can be prepared in large amounts.
[0037] Another object of the present invention is to provide a
composition that is relatively inexpensive to prepare.
[0038] Another object of the present invention is to provide a
composition that has reproducible activity among preparations.
[0039] Another object of the present invention is to provide a
composition that remains stable over time.
[0040] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiment and the
appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1. Inhibition of B-16 melanoma cell division by MCC,
M-DNA, sonicated M-DNA and herring sperm DNA.
[0042] FIG. 2. Inhibition of B-16 melanoma cell division by
mitomycin-C, MCC and mitomycin-C+MCC.
[0043] FIG. 3. Inhibition of B-16 melanoma cell division by
5-fluorouracil, MCC and 5-fluorouracil+MCC.
[0044] FIG. 4. Inhibition of B-16 melanoma cell division by
cisplatin, MCC and cisplatin+MCC.
[0045] FIG. 5. S-phase assessment of B-16 melanoma cells (A) and
S-phase assessment of B-16 melanoma cells after treatment with MCC
(B).
[0046] FIG. 6. Induction of apoptosis in B-16 melanoma cells by MCC
and M-DNA.
[0047] FIG. 7. Induction of caspase-1 activity in B-16 melanoma
cells by MCC.
[0048] FIG. 8. Cytotoxicity of MCC.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention is a composition and method comprising
Mycobacterium phlei (M. phlei)-DNA (M-DNA), M-DNA preserved and
complexed on M. phlei cell wall (MCC), a chemotherapeutic agent and
a pharmaceutically acceptable carrier, wherein the M-DNA and the
MCC induce cell cycle arrest in cancer cells, inhibit proliferation
of cancer cells, induce apoptosis in cancer cells and potentiate
the antineoplastic effect of the chemotherapeutic agent on cancer
cells. M-DNA and MCC are simple and relatively inexpensive to
prepare, their activity is reproducible among preparations, remains
therapeutically stable over time, and is effective at dose regimens
that are associated with minimal toxicity even upon repeated
administration.
[0050] As used herein, "M-DNA" includes DNA isolated from M. phlei
directly and DNA isolated from MCC prepared from M. phlei.
[0051] As used herein, "MCC" is M-DNA preserved and complexed on
deproteinized, delipidated M. phlei cell wall.
[0052] As used herein, "chemotherapeutic agent" is any agent
approved by a regulatory agency of a country or a state government
or listed in the U.S. Pharmacopoeia or other generally recognized
pharmacopoeia for use in treating cancer in an animal, including a
human.
[0053] As used herein, "antineoplastic" relates to preventing the
development, maturation, proliferation and spread of cancer
cells.
[0054] As used herein, "potentiates" relates to a degree of
synergism that is greater than additive.
[0055] As used herein, "synergism" relates to the coordinated
action of two or more chemotherapeutic agents.
[0056] As used herein, "enhances" relates to the additive action of
two or more chemotherapeutic agents.
[0057] Methods to increase the therapeutic effectiveness of M-DNA
and MCC include, but are not limited to, chemically supplementing
or biotechnologically amplifying stimulatory sequences or
confirmations of M-DNA and complexing the M-DNA, MCC
M-DNA+chemotherapeutic agent and MCC+chemotherapeutic agent to
natural or synthetic carriers. Optionally, agents including, but
not limited to, immunological agents and receptor-binding agents
can be included in the M-DNA and the MCC.
[0058] M-DNA, MCC, M-DNA+chemotherapeutic agent and
MCC+chemotherapeutic agent are administered in a pharmaceutically
acceptable carrier including, but not limited to, a liquid carrier
and a solid carrier. Liquid carriers are aqueous carriers,
non-aqueous carriers or both and include, but are not limited to,
aqueous suspensions, oil emulsions, water in oil emulsions,
water-in-oil-in-water emulsions, site-specific emulsions,
long-residence emulsions, sticky-emulsions, microemulsions,
nanoemulsions and liposomes. Solid carriers are biological
carriers, chemical carriers or both and include, but are not
limited to, microparticles, nanoparticles, microspheres,
nanospheres, minipumps, bacterial cell wall extracts and
biodegradable or non-biodegradable natural or synthetic polymers
that allow for sustained release of the M-DNA, MCC,
M-DNA+chemotherapeutic agent or MCC+chemotherapeutic agent. Such
polymers can be implanted in the vicinity of where delivery is
required. Polymers and their use are described in, for example,
Brem et al., J. Neurosurg. 74:441-446 (1991).
[0059] Preferred aqueous carriers include, but are not limited to,
DNase-free water, DNase-free saline and DNase-free physiologically
acceptable buffers. Preferred non-aqueous carriers include, but are
not limited to, mineral oil or neutral oil including, but not
limited to, a diglyceride, a triglyceride, a phospholipid, a lipid,
an oil and mixtures thereof, wherein the oil contains an
appropriate mix of polyunsaturated and saturated fatty acids.
Examples include, but are not limited to, soybean oil, canola oil,
palm oil, olive oil and myglyol, wherein the number of fatty acid
carbons is between 12 and 22 and wherein the fatty acids can be
saturated or unsaturated. Optionally, charged lipid or phospholipid
can be suspended in the neutral oil.
[0060] In an example, M-DNA is suspended in DNase-free sterile
water and is sonicated at 20% output for 5 minutes (Model W-385
Sonicator, Heat Systems-Ultrasonics Inc). Optionally, the sonicated
M-DNA is homogenized by microfluidization at 15,000-30,000 psi for
one flow-through (Model M-110Y; Microfluidics, Newton, Mass.) and
is transferred to an autoclaved, capped bottle for storage at
4.degree. C.
[0061] In an example, DNase free phosphatidylcholine is added to
DNase free triglyceride soybean oil at a ratio of 1 gram of
phospholipid to 20 ml of triglyceride and is dissolved by gentle
heating at 50.degree.-60.degree. C. Several grams of MCC are added
to a dry autoclaved container and the phospholipid-triglyceride
solution is added at a concentration of 20 ml per 1 gram of MCC.
The suspension is incubated at 20.degree. C. for 60 min. and is
then mixed with DNase-free PBS in the ratio of 20 ml MCC suspension
per liter of DNase-free PBS. The mixture is sonicated at 20% output
for 5 minutes (Model W-385 Sonicator, Heat Systems-Ultrasonics
Inc.). Optionally, the sonicated MCC mixture is homogenized by
microfluidization at 15,000-30,000 psi for one flow-through (Model
M-110Y; Microfluidics) and is transferred to an autoclaved, capped
bottle for storage at 4.degree. C.
[0062] A chemotherapeutic agent can be added to M-DNA or to MCC
before, during or after sonication or microfluidization or before
or after storage. Moreover, other methods known to those skilled in
the art for preparing deoxyribonucleic acids, bacterial cell wall
extracts and chemotherapeutic agents for administration to an
animal, including a human can be used.
[0063] Further, M-DNA, MCC, M-DNA+chemotherapeutic agent and
MCC+chemotherapeutic agent can be used with any one, all, or any
combination of excipients regardless of the carrier used to present
the composition to the responding cells. These include, but are not
limited to, anti-oxidants, buffers and bacteriostats, and may
include suspending agents, thickening agents and stabilizing
agents. Stabilizing agents include, but are not limited to,
non-ionic and ionic polymers such as, for example,
polyoxyethylenesorbitan monooleate (Tween) or hyaluronic acid.
[0064] M-DNA, MCC, M-DNA+chemotherapeutic agent and
MCC+chemotherapeutic agent are administered to an animal having
cancer in an amount effective to induce cell cycle arrest in cancer
cells, to inhibit proliferation of cancer cells, to induce
apoptosis in cancer cells and to potentiate the antineoplastic
effect of the chemotherapeutic agent on cancer cells. The
chemotherapeutic agent can be administered before, at the same time
as, or after administration of the M-DNA or the MCC. The
chemotherapeutic agent used and the amount of M-DNA, MCC and
chemotherapeutic agent administered per dose, the number of doses
and the dose schedule will depend on the type of cancer, the
severity of the cancer, the location of the cancer and other
clinical factors such as the size, weight and physical condition of
the recipient and the route of administration and can be determined
by the medical practitioner using standard clinical techniques and
without undue experimentation. In addition, in vitro assays may
optionally be employed to help identify optimal ranges for M-DNA,
MCC, M-DNA+chemotherapeutic agent and MCC+chemotherapeutic agent
administration.
[0065] Preferably, the amount of M-DNA administered is from about
0.00001 to 500 mg/kg per dose, more preferably from about 0.0001 to
100 mg/kg per dose, and most preferably from about 0.001 to 40
mg/kg per dose. Preferably, the amount of MCC administered is from
about 0.00001 to 500 mg/kg per dose, more preferably from about
0.0001 to 100 mg/kg per dose, and most preferably from about 0.001
to 40 mg/kg per dose. Preferably, the M-DNA content of the MCC is
between about 0.001 and 90 mg/100 mg dry MCC, more preferably
between about 0.01 and 40 mg/100 mg dry MCC, and most preferably
between about 0.1 and 30 mg/100 mg dry MCC. The protein content of
the MCC should be less than about 20 mg/100 mg dry MCC and the
extractable M-DNA should be at least about 4.5% of the dry weight
of MCC.
[0066] Chemotherapeutic agents include, but are not limited to,
DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic
agents, tubulin stabilizing agents, tubulin destabilizing agents,
hormone antagonist agents, topoisomerase inhibitors, protein kinase
inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors,
caspase inhibitors, metaloproteinase inhibitors, antisense nucleic
acids, triple-helix DNAs, nucleic acids aptamers, and molecular
biologically modified viral, bacterial and extotoxic agents.
[0067] DNA-alkylating agents include, but are not limited to,
nitrosureas, heavy metal agents and cross-linking agents; antitumor
antibiotic agents include, but are not limited to, mitomycin-C;
antimetabolic agents include, but are not limited to,
5-fluorouracil and methotrexate; topoisomerase inhibiting agents
include, but are not limited to, CPT-11; tubulin stabilizing agents
include, but are not limited to, taxol; tubulin destabilizing
agents include, but are not limited to, vincristine and
vinblasitne; and, hormone antagonist agents include, but are not
limited to, tamoxifen.
[0068] Preferably, the amount of chemotherapeutic agent
administered per dose is from about 0.0001 to 1000 mg/m.sup.2 or
from about 0.0001 to 1000 mg/kg, more preferably from about 0.5 to
70 mg/m.sup.2 or about 0.5 to 70 mg/kg and most preferably from
about 1 to 50 mg/m.sup.2 or about 1 to 50 mg/kg.
[0069] Routes for administration of the composition of the present
invention include, but are not limited to, oral, topical,
subcutaneous, transdermal, subdermal, intra-muscular,
intra-peritoneal, intra-articular, intra-vesical, intra-arterial,
intra-venous, intra-dermal, intra-cranial, intra-lesional,
intra-tumoral, intra-ocular, intra-pulmonary, intra-spinal,
placement within cavities of the body, nasal inhalation, pulmonary
inhalation, impression into skin and electrocorporation. Depending
on the route of administration, the volume per dose is preferably
about 0.001 to 500 ml per dose, more preferably about 0.01 to 100
ml per dose and most preferably about 0.1 to 50 ml per dose.
[0070] The following examples will serve to further illustrate the
present invention without, at the same time, however, constituting
any limitation thereof. On the contrary, it is to be clearly
understood that resort may be had to various other embodiments,
modifications, and equivalents thereof which, after reading the
description herein, may suggest themselves to those skilled in the
art without departing from the spirit of the present invention
and/or the scope of the appended claims.
EXAMPLE 1
[0071] Preparation of M-DNA and MCC from M. phlei
[0072] M-DNA and MCC were prepared from M. phlei (strain 110) as
described in International Patent Application No. PCT/CA98/00744,
which is included by reference herein. All reagents were selected
to enhance conservation of the DNA. Unless stated otherwise, M-DNA
and MCC were resuspended in DNase-free water or in a
pharmaceutically acceptable DNase-free buffer and sonicated at 20%
output for 5 minutes (Model W-385 Sonicator, heat
systems-Ultrasonics, Inc.). M-DNA and MCC did not contain
endotoxins as determined using a Limulus amebocyte lysate QCL-1000
kit (BioWhittaker, Walkersville, Md.).
[0073] For DNase treatment, M-DNA and MCC were digested with 1
International Unit of RNase-free DNase I (Life Technologies) for 1
hour at 25.degree. C. in 20 mM Tris HCl, pH 8.4, 2 mM MgCl.sub.2
and 50 mM KCl. DNase I was activated by the addition EDTA to a
final concentration of 2.5 mM and heating for 10 min at 65.degree.
C. DNase I digests both double stranded and single stranded DNA and
provides almost total degradation of the DNA.
EXAMPLE 2
[0074] Preparation of Mycobacterial DNA (B-DNA) and of
Mycobacterial DNA Preserved and Complexed on Mycobacterial Cell
Wall (BCC) from Species Other than M. phlei
[0075] BCC and B-DNA were prepared from mycobacterial species
including, but not limited to, M. vaccae, M. chelonei, M.
smegmatis, M. terrae, M. duvalii, M. tubeculosis, M. bovis BCG, M.
avium, M. Szulgai, M. scrofulaceum, M. xenopi, M. kansaii, M.
gastr, M. fortuitous and M. asiaticum as in Example 1.
EXAMPLE 3
[0076] Cells and Reagents
[0077] Human Jurkat T cell leukemia cells (Jurkat), human
promyelocytic HL60 leukemia cells (HL-60), mitoxantrone resistant
human promyelocytic HL-60MX1 leukemia cells (HL-60MX1), murine EL-4
lymphoma cells (EL-4) and murine B-16 melanoma cells were obtained
from the American Type Tissue Culture Collection (ATCC Rockville,
Md., USA) and were grown in medium recommended by the ATCC. Unless
stated otherwise, cells were seeded in 6 well flat-bottom tissue
culture plates at concentrations of 5.times.10.sup.3 to
1.times.10.sup.6 cells/ml and were maintained at 37.degree. C. for
24 to 72 hours.
[0078] Mitomycin-C, 5-fluorouracil, cisplatin, methotrexate and
herring sperm-DNA were obtained from Sigma Aldrich Canada
(Oakville, Ontario, Canada)
EXAMPLE 4
[0079] Cell Cycle Analysis
[0080] Cell cycle stage was determined using a CYCLETEST.TM. PLUS
DNA commercial kit (Becton Dickinson, San Jose, Calif., USA).
Briefly, nuclei from treated cells were obtained by dissolving the
cell membrane in a nonionic detergent, eliminating the cell
cytoskeleton and nuclear proteins with trypsin, digesting the
cellular RNA with RNase, and stabilizing the nuclear chromatin with
spermine. Propidium iodide was added to the cell nuclei and their
fluorescence was analyzed in a flow cytometer equipped with
electronic doublet discrimination capability (FACSCalibur, Becton
Dickinson). Accumulation of cells in GO/G.sub.1, S (SE, SM, SL) or
G.sub.2/M phases of the cell cycle was analyzed using MODFIT LT
software (Verity Software House Inc., Topsham, Mass., USA). Results
are expressed as percentage of cells in each phase of the cell
cycle.
EXAMPLE 5
[0081] Synchronization of Cell Populations with Methotrexate
[0082] To synchronize cell populations, exponentially growing cells
were incubated in tissue culture medium containing 0.04 to 0.16
.mu.M methotrexate (MTX) for 20 hours. The MTX medium was removed,
cells were washed extensively with phosphate buffered saline (PBS),
fresh medium was added, incubation was continued for 8 hours and
cell cycle analysis was performed as in Example 4.
EXAMPLE 6
[0083] Induction of Cell Cycle Arrest in Synchronously Dividing
Cancer Cells by MCC
[0084] Exponentially growing Jurkat, HL-60, HL-60MX1 and EL-4
cells, at 1.times.10.sup.6 cells/ml, and B-16 cells, at
3.times.10.sup.5 cells/ml, were prepared for analysis as in Example
5 with 0, 10 and 100 .mu.g/ml of MCC in the MTX medium.
[0085] Table 1 shows that MCC, at both 10 and 100 .mu.g/ml, induced
arrest at the SL+G.sub.2M phase of the cell cycle in synchronously
dividing Jurkat, HL-60, HL-60MX1, EL-4 and B-16 cancer cells.
Accumulation of cells in the SL+G.sub.2M phase was accompanied by a
reduction of cells in the GO/G.sub.1+SE phase or in the
GO/G.sub.1+SE and SM phases of the cell cycle.
1TABLE 1 Induction of cell cycle arrest in synchronously dividing
cancer cells by MCC PERCENTAGE OF CELLS IN EACH PHASE MTX alone MTX
+ MCC 10 .mu.g/ml MTX + MCC 100 .mu.g/ml Cells G.sub.0G.sub.1 + SE
SM SL + G.sub.2M G.sub.0G.sub.1 + SE SM SL + G.sub.2M
G.sub.0G.sub.1 + SE SM SL + G.sub.2M Jukat.sup.a 44.6 25.3 30.1
35.6 17.6 43.8 29.4 21.0 49.6 EL-4.sup.b 18.3 18.4 63.3 7.5 7.3
85.2 6.6 7.4 86.0 B-16 48.3 5.4 46.3 38.3 5.9 55.8 31.2 8.1 60.7
HL-60.sup.c 50.3 24.2 25.5 48.1 24.2 27.7 35.3 24.7 40.0 HL-60 46.1
35.5 18.4 41.4 30.7 27.9 38.0 25.1 36.9 MX1.sup.c .sup.a0.08 .mu.M,
.sup.b0.16 .mu.M or .sup.c0.04 .mu.M
[0086] These data demonstrate that MCC induced arrest in
synchronously dividing cancer cells, including HL-60MX1 cancer
cells, which display atypical multi-chemotherapeutic agent
resistance, altered topoisomerase II catalytic activity and reduced
levels of topoisomerase II alpha and beta proteins (Harker et al.
Cancer Res 49:4542-4549, 1989).
EXAMPLE 7
[0087] Induction of Cell Cycle Arrest in Synchronously Dividing
Jurkat Leukemia Cells by MCC and DNase I-treated MCC
[0088] Exponentially growing Jurkat leukemia cells, at
1.times.10.sup.6 cells/ml, were prepared for analysis as in Example
5 with 0 and 10 .mu.g/ml of MCC and 10 .mu.g/ml DNase I-treated MCC
in the MTX medium.
[0089] Table 2 shows that MCC induced arrest at the SL+G.sub.2M
phase of the cell cycle in synchronized Jurkat cells. Accumulation
of cells in the SL+G.sub.2M phase was accompanied by a reduction of
cells in the GO/G.sub.1+SE phase or in the GO/G.sub.1+SE and SM
phases of the cell cycle. Table 2 also shows that DNase I-treated
MCC did not induce cell cycle arrest in synchronously dividing
Jurkat cells.
2TABLE 2 Induction of cell cycle arrest in synchronously dividing
Jurkat Cells by MCC and by DNase I treated MCC PERCENTAGE OF CELLS
IN EACH PHASE MTX + MCC + MTX.sup.a + MCC 0 .mu.g/ml MTX + MCC 10
.mu.g/ml Dnase I 10 .mu.g/ml Cells G.sub.0G.sub.1 + SE SM SL +
G.sub.2M G.sub.0G.sub.1 + SE SM SL + G.sub.2M G.sub.0G.sub.1 + SE
SM SL + G.sub.2M Jurkat 43.0 25.6 31.4 34.9 11.4 53.7 36.7 26.0
37.3 .sup.aMTX = 0.08 .mu.M
[0090] MCC is M-DNA preserved and complexed on M. phlei cell wall
and DNase I digests the M-DNA of MCC. Therefore, that DNase I
treated MCC did not induce arrest in synchronously dividing Jurkat
leukemia cells demonstrates the importance of M-DNA for MCC
induction of cell cycle arrest in dividing cancer cells.
EXAMPLE 8
[0091] Induction of Cell Cycle Arrest in Cancer Cells by MCC
[0092] Exponentially growing Jurkat, HL-60, HL-60MX1 and EL-4
cells, at 1.times.106 cells/ml, and B-16 cells, at 3.times.105
cells/ml, were prepared for analysis as in Example 5 in the absence
of MTX and in the presence of 0, 10 and 100 .mu.g/ml of MCC
[0093] Table 3 shows that MCC, at both 10 and 100 .mu.g/ml, induced
arrest at the SL+G.sub.2M phase of the cell cycle in asynchronously
dividing Jurkat, HL-60, HL-60MX1, EL-4 and B-16 cancer cells.
Accumulation of cells in the SL+G.sub.2M phase was accompanied by a
reduction of cells in the GO/G.sub.1+SE phase or in the
GO/G.sub.1+SE and SM phases of the cell cycle.
3TABLE 3 Induction of cell cycle arrest in asynchronously dividing
cancer cells by MCC PERCENTAGE OF CELLS IN EACH PHASE MCC 0
.mu.g/ml MCC 10 .mu.g/ml MCC 100 .mu.g/ml Cells G.sub.0G.sub.1 + SE
SM SL + G.sub.2M G.sub.0G.sub.1 + SE SM SL + G.sub.2M
G.sub.0G.sub.1 + SE SM SL + G.sub.2M Jukat 47.2 10.7 42.1 31.8 11.5
56.7 34.1 11.9 54.0 EL-4 55.6 15.8 28.6 47.2 14.8 38.0 44.3 12.8
42.9 B-16 64.2 9.4 26.4 65.8 6.6 27.6 61.9 7.3 30.8 HL-60 64.0 8.6
27.4 54.8 9.8 35.4 49.5 7.8 42.7 HL 60 56.4 11.3 32.3 45.3 8.0 46.7
43.3 9.3 47.4 MX1
[0094] MCC induced arrest in asynchronously dividing cancer cells,
including in HL-60MX1 cells, which display an a typical
multi-chemotherapeutic agent resistance, altered topoisomerase II
catalytic activity and reduced levels of topoisomerase II alpha and
beta proteins (Harker et al. Cancer Res 49:4542-4549, 1989).
EXAMPLE 9
[0095] Induction of Cell Cycle Arrest in Jurkat Leukemia Cells by
M-DNA, MCC and DNase I-treated MCC
[0096] Exponentially growing Jurkat leukemia cells, at
1.times.10.sup.6 cells/ml, were prepared for analysis as in Example
5 in the absence of MTX and in the presence of 200 .mu.g/ml M-DNA,
10 .mu.g/ml MCC and 10 .mu.g/ml of DNase I-treated MCC.
[0097] Table 4 shows that M-DNA and MCC both induced arrest at the
SL+G.sub.2M phase of the cell cycle in asynchronously dividing
Jurkat cells. Accumulation of cells in SL+G.sub.2M phase was
accompanied by a reduction of cells in GO/G.sub.1+SE and SM phases
of the cell cycle. Table 3 also shows that, after DNase I
treatment, MCC induced less cell cycle arrest in asynchronously
dividing Jurkat cells.
4TABLE 4 Induction of cell cycle arrest in asynchronously dividing
Jurkat leukemia cells by M-DNA, MCC and DNase I treated MCC
PERCENTAGE OF CELLS IN EACH PHASE G.sub.0G.sub.1 + SL +
G.sub.0G.sub.1 + SL + Cells SE SM G.sub.2M SE SM G.sub.2M No
Treatment M-DNA 200 .mu.g/ml Jurkat 35.2 9.1 55.7 20.2 4.0 75.8 MCC
10 .mu.g/ml MCC + Dnase I 10 .mu.g/ml Jurkat 22.9 4.9 72.2 32.1 7.3
60.0
[0098] M-DNA and MCC induced cell cycle arrest in asynchronously
dividing Jurkat cells. MCC is M-DNA preserved and complexed on M.
phlei cell wall and DNase I digests the M-DNA of MCC. Therefore,
that DNase I treated MCC induced less arrest in asynchronously
dividing Jurkat cells again demonstrates the importance of M-DNA
for MCC induction of cell cycle arrest in dividing cancer
cells.
EXAMPLE 10
[0099] Inhibition of B-16 Melanoma Cell Proliferation by MCC,
M-DNA, Sonicated M-DNA and Herring Sperm-DNA
[0100] Exponentially growing B-16 melanoma cells, at
3.times.10.sup.5 cells/ml, were prepared for analysis as in Example
5 in the absence of MTX and in the presence 1 to 100 .mu.g/ml MCC,
M-DNA or M-DNA sonicated for 20 min on ice in a Model W-38
ultrasonic processor (HeatSystems-Ultrasonics, Inc.) to reduce
oligonucleotide length, and herring sperm-DNA.
[0101] As shown in FIG. 6, at 1 .mu.g/ml, MCC inhibited
proliferation about 25%, M-DNA about 5%, sonicated M-DNA about 10%
and herring sperm-DNA 0%. At 10 .mu.g/ml, MCC inhibited
proliferation about 50%, M-DNA about 30%, sonicated M-DNA about 25%
and herring sperm-DNA 0%. At 100 .mu.g/ml, MMC inhibited
proliferation about 80%, M-DNA and sonicated M-DNA about 50% and
herring sperm-DNA about 5%.
[0102] MCC, M-DNA and sonicated M-DNA each inhibited proliferation
of asynchronously dividing B-16 melanoma cells, whereas herring
sperm-DNA did not inhibit proliferation of asynchronously dividing
B-16 melanoma cells.
EXAMPLE 11
[0103] Inhibition of B-16 Melanoma Cell Proliferation by
Chemotherapeutic Agents.+-.MCC
[0104] Malignant melanoma is among the most chemotherapy-refractory
cancers and many chemotherapeutic agents do not appear to modify
the prognosis of this disease. Melanoma derived cells lines also
demonstrate significant resistance to most chemotherapeutic agents,
suggesting the presence of intrinsic cellular resistance. This
resistance may be mediated by mechanisms including, but not limited
to, P-glycoprotein, the glutathione/glutathione S-transferase
system multi-chemotherapeutic agent resistance-associated protein,
mutated N-Ras, Bcl-2 and p53 oncogenes and topoisomerase II enzyme
(Serrone et al. Melanoma Res. 9:51, 1999).
[0105] B-16 melanoma cells, at 3.times.10.sup.5 cells/ml, were
incubated for 72 h with the chemotherapeutic agents mitomycin-C,
5-fluorouracil and cisplatin in the absence and in the presence of
MCC. Cell proliferation was determined using
dimethylthiazol-diphenyltetrazolium bromide (MTT) reduction (Mosman
et al. Journal of Immunological Methods 65:55-63, 1983). After 72
h, 20 .mu.l of MTT in culture medium was added to each well and
incubated for 3 h. Medium was then aspirated from each well, 100
.mu.l of acidified isopropyl alcohol was added to each sample and
reduced MTT was solubilized by mixing. The absorbency of the
reaction product was determined using an ELISA microplate reader at
a wavelength of 570 nm.
[0106] B-16 melanoma cells were incubated with 0.01 to 100 .mu.g/ml
of mitomycin-C, with 1 to 100 .mu.g/ml of MCC and with 0.01 to 10
.mu.g/ml of mitomycin-C+1 .mu.g/ml MCC. Mitomycin-C is an
anti-tumor antibiotic produced by Streptomyces caespitosus, which
cross-links DNA, depolymerizes DNA and forms free radicals.
[0107] FIG. 2 shows that, with B-16 cells, 0.1 .mu.g/ml mitomycin-C
inhibited proliferation about 5%, 1 .mu.g/ml about 10%, and 10 and
100 .mu.g/ml 100%, whereas 1 .mu.g/ml MCC inhibited proliferation
about 25%, 10 .mu.g/ml about 50% and 100 .mu.g/ml about 80%. FIG. 2
also shows that, in the presence of 1 .mu.g/ml MCC, 0.1 .mu.g/ml
mitomycin-C inhibited proliferation about 40%, 1 .mu.g/ml about 65%
and 100 .mu.g/ml 100%. These data show that MCC potentiates the
antineoplastic effect of mitomycin-C on proliferating cancer
cells.
[0108] B-16 melanoma cells were incubated with 0.01 to 100 .mu.g/ml
of 5-fluorouracil, with 1 to 100 .mu.g/ml of MCC and with 0.01 to
10 .mu.g/ml of 5-fluorouracil+1 .mu.g/ml MCC. 5-fluorouracil is an
antimetabolite, which interferes with DNA and RNA synthesis.
[0109] FIG. 3 shows that, with B-16 cells, 0.01 .mu.g/ml
5-fluorouracil inhibited proliferation about 8%, 0.1 .mu.g/ml about
50%, 1 .mu.g/ml about 90%, and 10 and 100 .mu.g/ml 100%, whereas 1
.mu.g/ml MCC inhibited proliferation about 25%, 10 .mu.g/ml about
50% and 100 .mu.g/ml about 80%. FIG. 3 also shows that, in the
presence of 1 .mu.g/ml MCC, 0.01 .mu.g/ml 5-fluorouracil inhibited
proliferation about 75%, 0.1 .mu.g/ml about 85%, 1 .mu.g/ml about
90% and 10 .mu.g/ml 100%. These data show MCC potentiates the
antineoplastic effect of 5-fluorouracil on proliferating cancer
cells.
[0110] B-16 melanoma cells were incubated with 0.01 to 100 .mu.g/ml
of cisplatin, with 1 to 100 .mu.g/ml of MCC and with 0.01 to 10
.mu.g/ml of cisplatin+1 .mu.g/ml MCC. Cisplatin is an alkylating
agent that cross-links DNA and inhibits DNA precursors.
[0111] FIG. 4 shows that, with B-16 cells, 0.01 .mu.g/ml cisplatin
inhibited proliferation 0%, 0.1 .mu.g/ml about 8%, 1 .mu.g/ml about
62%, 10 .mu.g/ml about 90% and 100 .mu.g/ml 100%, whereas 1
.mu.g/ml MCC inhibited proliferation about 25%, 10 .mu.g/ml about
50% and 100 .mu.g/ml about 80%. FIG. 4 also shows that, in the
presence of 1 .mu.g/ml MCC, 0.01 .mu.g/ml cisplatin inhibited
proliferation about 40%, 0.1 .mu.g/ml about 50%, 1 .mu.g/ml about
70% and 10 .mu.g/ml about 90%. These data show that MCC enhances
the antineoplastic effect of cisplatin on proliferating cancer
cells.
[0112] Table 5 shows the concentrations of mitomycin-C,
5-fluorouracil, and cisplatin required for 50% inhibition of B-16
melanoma cell division in the absence and in the presence of 1
.mu.g/ml MCC.
5TABLE 5 Concentration of mitomycin-C, 5-fluorouracil and cisplatin
required for 50% inhibition of B-16 melanoma cell proliferation in
the absence and in the presence of 1 .mu.g/ml MCC IC.sub.50*,
.mu.g/ml Treatment Drug Alone Drug + MCC at 1 .mu.g/ml MCC 10 Not
applicable Cisplatin 0.6 0.16 5-Fluorouracil 0.12 0.005 Mitomycin-C
2.2 0.12 *concentration for 50% inhibition
[0113] Table 5 shows the dose dependent inhibition of B-16 cell
melanoma proliferation by MCC at 10 to 100 .mu.g/ml (IC.sub.50=10
.mu.g/ml) and by mitomycin-C, 5-fluorouracil and cisplatin at 0.1
to 10 .mu.g/ml (IC.sub.50=2.2, 0.12 and 0.6 .mu.g/ml respectively).
Table 5 also shows that 1 .mu.g/ml MCC potentiated mitomycin-C
(IC.sub.50=0.12 .mu.g/ml) and 5-fluorouracil (IC.sub.50=0.005
.mu.g/ml) inhibition of B-16 melanoma cell proliferation and that 1
.mu.g/ml MCC enhanced cisplatin (IC.sub.50=0.16 .mu.g g/ml)
inhibition of B-16 melanoma proliferation.
[0114] These data show that MCC not only inhibits cancer cell
proliferation, but also potentiates the antineoplastic effects of
mitomycin-C and 5-fluorouracil on cancer cell proliferation and
enhances the antineoplastic effect of cisplatin on cancer cell
proliferation.
EXAMPLE 12
[0115] Induction of Apoptosis in B-16 Melanoma Cells by MCC and
M-DNA
[0116] Fragmentation of cellular DNA into nucleosome-sized
fragments is characteristic of cells undergoing apoptosis (Newell
et al. Nature 357:286-289, 1990). To assess DNA fragmentation, B-16
cells were lysed with 0.5 ml of hypotonic lysing buffer (10 mM Tris
buffer, 1 mM EDTA, 0.2% t-octylphenoxypolyethoxyethanol (Triton
X-100), pH 7.5). The lysates were centrifuged at 13,000 g for 10
min and the supernatants, containing fragmented DNA, were
precipitated overnight at -20.degree. C. in 50% isopropanol and 0.5
M NaCl. The precipitates were collected by centrifugation and were
analyzed by electrophoresis in 0.7% agarose gels for 3 h at
100V.
[0117] B-16 melanoma cells, at 3.times.10.sup.5 cells/ml, were
incubated for 72 h with 1 .mu.g/ml M-DNA (FIG. 6, lane 1) and with
100 (lane 2), 10 (lane 3) and 1 .mu.g/ml MCC (lane 4). M-DNA and
MCC treated B-16 melanoma cells showed significant DNA
fragmentation, whereas untreated B-16 melanoma cells (FIG. 6, lane
5) showed no DNA fragmentation. A 123-bp DNA ladder (Gibco Life
Science) was used to determine the molecular weight of the
nucleosome-sized DNA fragments (FIG. 6, lane L). These data show
that M-DNA and MCC induce apoptosis in B-16 melanoma cells.
EXAMPLE 13
[0118] Activation of Caspase-1 in B-16 melanoma Cells by MCC
[0119] Interleukin-1-converting enzyme (ICE/caspase-1) is a
cysteine protease involved in the sequential activation of the
caspase cascade required for apoptosis. ICE/caspase-1 is rapidly
and transiently activated by various pro-apoptotic stimuli. To
determine if MCC can directly activate the ICE/caspase-1, the
effect of MCC on ICE/caspase-1 activity was assayed in B-16
melanoma cells.
[0120] B-16 melanoma cells, at 3.times.10.sup.5 cells/ml, were
plated in 6 well tissue culture plates in a volume of 1 ml and were
incubated for 3 h with 1 to 100 .mu.g/ml MCC. The cells were
washed, lysed in 50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1%
3-[3-cholamidopropyl)dimethylammonio]-1-propane- -sulfonate
(CHAPS), 10 mM DTT, 1 mM EDTA and 10% glycerol and centrifuged at
11,000 g for 10 minutes. ICE/caspase-1 activity in the supernatant
was determined using the fluorogenic synthetic substrate
Z-tyr-val-ala-asp[OMe]-7-amino-4-methylcoumarin [ Calbiochem # 6
88225]. Fluorescence was determined at an excitation wavelength of
400 nm and an emission wavelength of 505 nm.
[0121] As shown in FIG. 7, incubation of B-16 melanoma cells with
MCC resulted in a significant dose-dependent increase in
ICE/caspase-1 activity, whereas incubation of B-16 melanoma cells
without MCC resulted in no change in ICE/caspase-1 activity.
EXAMPLE 14
[0122] Cytotoxic Effects of MCC on Malignant Melanoma Cells
[0123] Cell cytotoxicity is characterized by the loss of plasma
membrane integrity and release of cytoplasmic enzymes such as, but
not limited to, LDH (Phillips et al. Vaccine 14:898-904).
[0124] To assess the cytotoxicity of MCC, B-16 melanoma cells were
incubated for 48 h with 100 .mu.g/ml MCC or with lysing buffer (10
mM Tris, 1 mM EDTA, 0.2% Triton X-100, pH 7.5) as a control for
total LDH release (Filion et al. Biochim Biophys Acta 1329:345-356,
1997). LDH was determined by commercial assay (Sigma-Aldrich).
[0125] As shown in FIG. 8, MCC was not cytotoxic to the B-16
melanoma cells. These data demonstrate that MCC does not act by
disrupting the membrane of the cells, but that MCC acts directly on
the B-16 melanoma cells to induce apoptosis.
EXAMPLE 15
[0126] Effects of M-DNA, MCC and DNase I Treated MCC on B-16
Melanoma Tumors in Mice
[0127] B-16 melanoma cells are implanted subcutaneously into 20
male nude BALB/c mice and allowed to grow for 10 days. The mice are
divided into 4 groups and tumor mass is measured in each mouse. On
day 0, Group 1 mice receive saline, Group 2 mice receive MCC, Group
3 mice receive M-DNA and Group 4 mice receive DNase I treated MCC.
After 4 weeks of treatment, the mice are sacrificed and tumor mass
is measured. Group 2 and Group 3 mice have less tumor mass than
Group 1 and Group 4 mice.
EXAMPLE 16
[0128] Effects of M-DNA and MCC in Combination with Mitomycin-C on
B-16 Melanoma Tumors in Mice
[0129] B-16 melanoma cells are implanted subcutaneously into 30
male nude BALB/c mice and allowed to grow for 10 days. The mice are
divided into 6 groups and tumor mass is measured in each mouse. On
day 0, Group 1 mice receive saline, Group 2 mice receive M-DNA,
Group 3 mice receive MCC, Group 4 mice receive mitomycin-C, Group 5
mice receive M-DNA and mitomycin-C and Group 6 mice receive MCC and
mitomycin-C. After 4 weeks of treatment, the mice are sacrificed
and the tumor mass and number of metastases are determined. Group 1
mice have the most tumor mass. Group 4 mice have less tumor mass
than Group 1 mice. Group 2 and Group 3 mice have less tumor mass
than Group 4 mice. Group 5 and Group 6 mice have the least tumor
mass.
[0130] It should be understood, of course, that the foregoing
relates only to a preferred embodiment of the present invention and
that numerous modifications or alterations may be made therein
without departing from the spirit and the scope of the invention as
set forth in the appended claims.
* * * * *