MXPA00001272A - Composition and method for regulating cell proliferation and cell death - Google Patents

Abstract

The present invention relates to a composition and method useful for regulating cell proliferation and cell death in a multicellular organism. The present invention particularly relates to a composition comprising a bacterial DNA (B-DNA) and a first pharmaceutically acceptable carrier, wherein the B-DNA induces a response in responsive cells of an animal. The present invention more particularly relates to a composition comprising a mycobacterial DNA (M-DNA) and a first pharmaceutically acceptable carrier, wherein the M-DNA inhibits proliferation of responsive cells of an animal, induces apoptosis in responsive cells of an animal, and stimulattes responsive cells of the immune system of an animal to produce bioactive molecules. Methods of making the M-DNA composition and methods of using the M-DNA composition also are disclosed.

Description

COMPOSITION AND METHOD TO REGULATE CELLULAR PROLIFERATION AND CELLULAR DEATH FIELD OF THE INVENTION The present invention relates to a composition comprising a mycobacterial DNA (B-DNA) and a first pharmaceutically acceptable carrier, wherein B-DNA is effective to induce a response in responder cells from an animal. Particularly, the present invention relates to a composition comprising Mycobacterium phl ei DNA (M-DNA) and a first pharmaceutically acceptable carrier, wherein M-DNA inhibits the proliferation of responder cells and induces apoptosis therein as well. that stimulates the same in the system in the immune system to produce bioactive molecules. Methods for making M-DNA and methods for using M-DNA are also discussed.

BACKGROUND OF THE INVENTION In multicellular organisms, the number of tissue cells is determined by the rate of cell proliferation minus the rate of cell elimination. Apoptosis is a genetically programmed, non-inflammatory, energy-dependent form of cell death in P1068 / 00MX tissues, including, of course, adult tissue. Apoptosis consists of four sequential steps: (1) committed death by extracellular or intracellular activators; (2) Cell death by activation of intracellular proteases and nucleases, (3) the dead cell is submerged under other cells, and (4) degradation of the dead cell within the lysosomes of phagocytic cells (Steller H. Science 267: 1445-1449 , nineteen ninety five). Aberrations in the regulation of cell proliferation, cellular apoptosis or a combination of the two, are associated with the pathogenesis of several diseases including, but not limited to, cancer, neurodegeneration, autoimmunity and cardiac disease. Apoptosis can be initiated by ligands that bind to cell surface receptors, including, but not limited to, Fas (CD95) (French et al, Journal of Cell Biology 133: 355-364, 1996) and the receptor. 1 of tumor necrosis factor (TNFRl). The binding of FasL to Fas and TNF to TNFR1 initiates the production of intracellular signals that result in the activation of cysteine aspartyl proteases (caspases) that initiate the lethal proteolytic cascade of apoptosis execution that is associated with DNA fragmentation. nuclear (NuMA) and loss of contact with the cell substrate (Muzio et al., Cell 85: 817-827, P1068 / 00MX 1996). Apoptosis can also be induced by intercellular proteins including, but not limited to, p53 / p21 regulators (Levine, A. Cell 88: 323-331, 1997). p53 / p21 acts as transcription factors to activate the expression of genes mediated by apoptosis, including, enunciatively, genes that encode proteins that generate free radicals that, in turn, damage the cellular mitochondria, whose content leaks into the cytoplasm and activates apoptotic caspases (Polyak et al., Nature 389: 300-305, 1997). Cancer is an aberrant net accumulation of atypical cells that causes an excess of proliferation, an insufficiency of apoptosis or a combination of the two. Mutations in genes related to apoptosis, namely, Fas, TNFR1 and p53 / p21 each are involved in the pathogenesis of various types of cancer (Levine A. Cell 88: 323-331, 1997; Fisher D. Cell 78: 529-542, 1994). Apoptosis is important not only for the pathogenesis of cancer but also for the likelihood of resistance to anti-cancer therapies. The resistance of the induction of apoptosis has emerged as an important category of multidrug resistance (MDR-multiple drug resistance), which most likely explains a significant portion of the P1068 / 00MX treatment. NMR, the simultaneous resistance to structurally and functionally unrelated chemotherapeutic agents, they can be both inherent and acquired. That is, some cancers never respond to therapy, while other types of cancer are initially sensitive to therapy, but develop resistance to the drug. Since chemotherapeutic agents are mainly based on the induction of apoptosis in cancer cells to have their therapeutic effect, drug resistance, which decreases the effectiveness of chemotherapeutic agents, leads directly or indirectly to reduced apoptosis and is generally associated with an uninspiring diagnosis in several types of cancer. Cytolysis is the partial or complete destruction of a cell and is mediated by the immune system. In the sense used here, monocytes, macrophages and leukocytes are included in the immune system. Monocytes and activated macrophages produce bioactive molecules that accelerate, amplify and modulate responses in responder cells from an animal. By producing is meant to synthesize and secrete. These bioactive molecules, include, but are not limited to, cytokines and reactive oxygen species. Cytokines include, inter alia, interleukin-1 (IL-1), interleukin-6 P1068 / 00MX (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12) and GM-CSF. IL-12, alone or in combination with other cytokines, promotes the maturation of leukocytes including, but not limited to, B-lymphocytes, CD4 + T cells, CD8 + T cells, and NK cells and induces the secretion of interferon-gamma. IL-12 is reported to have anti-cancer activity in some cancer cells (Stine et al Annals NY Academy of Science 795: 420-421, 1996; Chen et al., Journal of Immunology 159: 351-359, 1997). This activity includes, but is not limited to, activation of specific T-cytolytic lymphocytes, activation of natural killer cells (NK-natural killer) and induction of anti-angiogenic proteins IP-10 and MiG. IP-10 inhibits cancer growth and metastasis, inhibits cancer-induced neovascularization and also activates cells (NK) (Angillo et al Annals NY Academy of Sciences 795: 158-165, 1996. GMCSF reports with a pro-cancer activity in some cancer cells (Hawkyard et al., Journal of Urology 150: 514-518, 1993.) Active oxygen species include, but are not limited to, nitric oxide, superoxide radicals, and hydroxyl radicals. , superoxide radicals and hydroxyl radicals, among other activities, induce apoptosis and cytolysis in target cells.

P1068 / 00MX biological and chemical origin include, but are not limited to, bacterial preparations, and have been used to stimulate or inhibit responder cells in an animal. The cell wall preparation of Mycobacterium species has been used to treat diseases including, but not limited to, various types of cancer (U.S. Patent No. 4,503,048). However, the therapeutic benefit obtained using these preparations is variable and inconsistent and apparently depends on the method by which the preparation is prepared, purified and administered, and on the stability of the preparation. The anti-cancer agents of the prior art have proven inadequate for clinical applications. Many of these agents are ineffective (Bischoff et al., Science 274: 373-376, 1996) or toxic, have significant side effects (Lamm et al., Journal of Urology 153: 1444-1450, 1995), and cause the development of immunosensitization or resistance to drugs and are also debilitating to the recipient. In addition, many of these agents depend on Fas, TNFRl or p53 / p21 for their effectiveness. Therefore, there is a need for a novel therapeutic agent that inhibits the proliferation of cancer cells and induces apoptosis thereof, and that stimulates cells P1068 / 00MX immune system responders to produce cytokines and reactive oxygen species. This therapeutic agent should be useful as an anticancer agent and as an adjuvant for other anticancer agents. By adjuvant is meant to be useful with other anti-cancer agents to increase the effectiveness of the treatment. Furthermore, this therapeutic agent must be simple and relatively inexpensive in its preparation, its activity must be reproducible between the different preparations, its activity must remain stable over time, and its effects on cancer cells must be achievable with dose regimens that they are associated with minimal toxicity.

SUMMARY OF THE INVENTION The present invention satisfies the above needs by providing a therapeutic composition comprising a mycobacterial DNA (B-DNA) and a first pharmaceutically acceptable carrier, wherein the composition is effective to induce a response in responder cells that come from a animal. Particularly, the present invention provides a therapeutic composition comprising a DNA of Mycobacterium phl ei (M-DNA) and a first pharmaceutically acceptable carrier, wherein the responses, include, but are not limited to, inhibition of the P1068 / 00MX proliferation of responder cells and induction of apoptosis in them, including, but not limited to, cancer cells and the stimulation of immune system responsive cells to produce bioactive molecules. The composition of M-DNA is simple and relatively inexpensive in its preparation, its activity is reproducible among the various preparations, it remains stable over time and is effective at dosing regimes that are associated with minimal toxicity. M-DNA is prepared from Mycobacterium um phl ei (M. phl ei) by lysosome digestion, proteinase K and sodium dodecyl sulfate, and phenolic extraction and ethanolic precipitation (M. phl ei-DNA). Alternatively, M-DNA is prepared with the breakdown of M. phl ei, collecting the solid components and preparing an M-DNA and cell wall complex of M. phl ei (MCC). The DNase-free reagents are used to decrease the degradation of M-DNA during the preparation. M-DNA is prepared from MCC by phenolic extraction and ethanolic precipitation (MCC-DNA). The composition of M-DNA, comprising M-DNA and a first pharmaceutically acceptable carrier, is administered to an animal in sufficient dose to inhibit the proliferation of responder cells and induce apoptosis therein and to stimulate P1068 / 00 X the responsive cells of the immune system so that they produce bioactive molecules. These pharmaceutically acceptable first carriers include, but are not limited to, liquid carriers, solid carriers and both. The liquid carrier includes, but is not limited to, aqueous carriers, non-aqueous carriers or both. Solid carriers include, but are not limited to, chemically synthesized carriers and natural carriers. Aqueous carriers include, but are not limited to, water, saline and physiological regulators. Non-aqueous carriers include, but are not limited to, oils or other hydrophobic liquid formulations and liposomes. Natural carriers include, but are not limited to, cell wall of M. phl ei washed, delipidated and deproteinized, where M-DNA is complexed in the cell wall of M. phlei (MCC). In addition, the M-DNA composition can be administered in a second pharmaceutically acceptable carrier. This second pharmaceutically acceptable carrier includes, but is not limited to, liquid carriers and solid carriers. Once again, liquid carriers include aqueous carriers, non-aqueous carriers, or both. Also solid carriers include chemically synthesized carriers and natural carriers.

P1068 / 00MX The M-DNA composition of the present invention is useful for preventing, treating and eliminating a disease. It is particularly useful for treating a disease mediated by unwanted and uncontrolled cell proliferation, for example, cancer. The M-DNA composition is also effective as an adjuvant to enhance the effectiveness of other anti-cancer agents. These agents include, but are not limited to, drugs, immunostimulants, antigens, antibodies, vaccines, radiation and chemotherapeutic agents, genetic agents, biologically manipulated and chemically synthesized, and agents that target the molecules of the dead cell for activation or inactivation. and that inhibit the proliferation of responding cells and induce their apoptosis. Accordingly, an object of the invention is to provide a composition and method that induces a therapeutic response in responder cells that come from an animal. Another objective of the present invention is to provide a composition and method that inhibits the proliferation of responder cells. Another objective of the present invention is to provide a composition and a method that induces apoptosis in responder cells. Another object of the present invention is to provide a composition and method that induces P1068 / 00MX apoptosis independent of Fas. Another objective of the present invention is to provide a composition and method that induces apoptosis independent of TNFR. Another objective of the present invention is to provide a composition and method that induces apoptosis independent of p53 / p21. Another objective of the present invention is to provide a composition and method that induces apoptosis independent of drug resistance. Another objective of the present invention is to provide a composition and method that induces caspase activity in responder cells. Another objective of the present invention is to provide a composition and a method that induces responses in immune system responsive cells to produce bioactive molecules. Another objective of the present invention is to provide a composition and method that induces caspase activity in responder cells. Another objective of the present invention is to provide a composition and a method that induces responses in cells in cells responsive to the immune system to produce bioactive molecules. Another objective of the present invention is to provide a composition and method that induces responsive cells of the immune system to P1068 / 00MX that produce bioactive molecules. Another objective of the present invention is to provide a composition and method that induces responsive cells of the immune system to produce cytokines. Another objective of the present invention is to provide a composition and method that induces responsive cells of the immune system to produce IL-6. Another objective of the present invention is to provide a composition and method that induces responsive cells of the immune system to produce IL-10. Another objective of the present invention is to provide a composition and method that induces immune system responsive cells to produce IL-12. Another objective of the present invention is to provide a composition and method that induces responsive cells of the immune system to produce species and reactive oxygen. Another objective of the present invention is to provide a composition and method that is effective in preventing cancer in an animal. Another objective of the present invention is to provide a composition and method that is effective in treating a cancer in an animal. Another object of the present invention is P1068 / 00 X provide a composition and method that is effective in eliminating a cancer in an animal. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for other anti-cancer therapies. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for anti-cancer chemical agents. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for biological anti-cancer agents. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for biologically manipulated anti-cancer agents. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for anti-cancer vaccines. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for anti-cancer vaccines based on nucleic acids. Another objective of the present invention is to provide a composition and method that is effective as an adjuvant for radiation therapy. Another objective of the present invention is to provide a composition and method that induces terminal cell differentiation incompletely P1068 / 00MX differentiated. Another objective of the present invention is to provide a composition that can be prepared in large quantities. Another object of the present invention is to provide a composition which can be prepared relatively cheaply. Another objective of the present invention is to provide a composition having reproducible activity between the various preparations. Another objective of the present invention is to provide a composition that remains stable over time. Another objective of the present invention is to provide a composition that maintains its effectiveness over time. Another object of the present invention is to provide a composition that is minimally toxic to the recipient. Another objective of the present invention is to provide a composition that is will not cause anaphylaxis in the recipient. Another objective of the present invention is to provide a composition that does not sensitize the recipient to tuberculin skin tests. These and other objectives, features and advantages of the invention will become apparent after a review of the following detailed description P1068 / 00MX of the methods set forth and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS OR FIGURES Figure 1 is an oligonucleotide distribution of M. phl ei-DNA and MCC-DNA. Figure 2 is the inhibition of the proliferation of cancer cells of HT-1376, HT-1197, B-16 Fl, THP-1, RAW 264.7, Jurkat, HL-60, HL-60 MX-1 by DNA of M. phl ei (2A) and MCC DNA (2B), calf thymus DNA (2A and 2B) and herring sperm DNA (2A and 2B). The results are the mean ± SD (standard deviation) of 3 independent experiments. Figure 3 is the inhibition of the proliferation of cancer cells HT-1376, HT-1197, B-16F1, THP-1, RAW 264.7, Jurkat, HL-60, HL-60 MX-1 by MCC. The results are the mean ± SD of 3 independent experiments. Figure 4 depicts the inhibition of the proliferation of human leukemic THP-1 monocytes by M. phl ei DNA, MCC DNA, MCC and hIL-12. The results are the mean ± SD of 3 independent experiments. Figure 5 depicts the inhibition of the proliferation of human bladder cancer cells HT-1197 (5A) and HT-1376 (5B) by MCC and LPS. The results are the mean ± SD of 3 experiments P1068 / 00MX independent. Figure 6 depicts the induction of DNA fragmentation in human leukemic THP-1 monocytes by PBS, herring sperm DNA and untreated M. phl ei DNA treated with DNase I and MCC. The results shown are from 1 to 3 experiments, each of which gave similar results. Figure 7 shows the induction of DNA fragmentation in monocytes of human leukemic THP-1 by PBS, herring sperm DNA treated with DNase I and untreated, and DNA of M. phl ei and by hIL-12. The results shown are for 1 of 3 experiments, each of which gave similar results. Figure 8 shows the induction of DNA fragmentation in human bladder cancer cells HT-1197 (8A) and HT-1376 (8B) by MCC and hIL-12. The results shown are for 1 of 3 experiments, each of which gave similar results. Figure 9 shows the release of NuMA from human leukemic THP-1 monocytes by MCC, M. phl ei DNA, MCC DNA and herring sperm DNA. The results are the mean ± SD of 3 independent experiments. Figure 10 illustrates the release of NuMA from human leukemic THP-1 monocytes by untreated M. phl ei DNA and treated with DNase I, DNA P1068 / 00MX of MCC and MCC. The results are the mean ± SD of 3 independent experiments. Figure 11 illustrates the release of NuMA from human bladder cancer cells HT-1376 and HT-1197 with increasing concentrations of MCC. The results are the mean ± SD of 3 independent experiments. Figure 12 illustrates the release of NUMA from human bladder cancer cells HT-1197 (12A) and HT-1376 (12B) with 1 μg / ml MCC or 100 μg / ml MCC within 48 hours . The results are the mean ± SD of 3 independent experiments. Figure 13 illustrates the release of NuMA from Jurkat cells incubated with PBS, CH-11 antibodies, ZB4 antibodies, M. phl ei DNA, CH-11 antibodies + M. phl ei DNA and ZB4 antibodies DNA of M. phl ei. Figure 14 illustrates the release of NuMA from human leukemic THP-1 monocytes by PBS, M. phlei DNA, sonicated M. phl ei DNA, digested with BstU 1, autoclaved and methylated. Figure 15 illustrates the anti-cancer activity of MCC and MCC treated with DNase I in line 10 of hepatoma in guinea pigs. The results are the mean ± SD of 7 animals in each experimental group.

P1068 / 00MX Figure 16 illustrates the percentage of LDH release from human bladder cancer cells HT-1197 and HT-1376 as an indicator of cytotoxicity of MCC. The results are the mean ± SD of 3 independent experiments. Figure 17 shows the stimulation of MCC in the production of IL-6, IL-12 and GM-CSF by human bladder cancer cells HT-1197 and HT-1376, THP-1 monocytes, human, murine macrophages, murine RAW 264.7 monocytes and murine spleen cells. The results are the mean ± SD of 3 independent experiments. Figure 18 illustrates the effect of antibodies to CD14 receptors on MCC DNA and on the production of IL-12 induced by MCC by human THP-1 monocytes. Figure 19 illustrates the effect of cytochalasin D on M. phl ei DNA, MCC DNA and IL-12 stimulated by MCC by human THF-1 monocytes. Figure 20 illustrates the effect of untreated and treated DNase DNA, MCC and Regressin® on the production of IL-12 by murine macrophages. Figure 21 illustrates the MCC stimulation of NO production by murine monocytes RAW 264.7. Figure 22 illustrates the stimulation of IL-6, IL-10, IL-12 and GM-CSF in CD-1 mice by P1068 / 00MX intraperitoneal injection of 50 mg / kg of MCC. Figure 23 illustrates the stimulation of IL-12 production in CD-1 mice by intravenous injection of 6.6 mg / kg MCC. Figure 24 illustrates the stability of MCC for 6 months of storage.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a therapeutic composition comprising a bacterial deoxyribonucleic acid (DNA) and a first pharmaceutically accble carrier, wherein the composition is effective to induce a response in responder cells from an animal, including human. More particularly, the present invention relates to a composition comprising a mycobacterial DNA (B-DNA) and a first pharmaceutically accble carrier, wherein the composition is effective to induce a response in responder cells of an animal. More particularly, the present invention relates to a composition comprising M. phlei DNA (M-DNA) and a first pharmaceutically accble carrier, wherein the composition is effective to inhibit the proliferation of responder cells and induce apoptosis therein. , and to stimulate the responsive cells of the immune system so that they produce bioactive molecules.

P1068 / 00MX The M-DNA composition of this invention is prepared in a relatively simple and inexpensive manner, its activity is reproducible between different preparations and it remains stable over time. In addition, it is minimally toxic, if it does, for the recipient, does not cause a positive tuberculin reaction in the recipient and rarely causes an anaphylactic response in the recipient even with repeated administration. Many bacterial species can be used to practice the present invention, including, but not limited to, Coryneform bacteria, Corynebacterium species, Rhodococcus species, Eubacterium species, Borde tel la species, Escheri chia species, Lis teria species, species Nocardia and Mycobacterium species. Preferably, the bacterial DNA is prepared from a Mycobacterium spice which includes, but is not limited to, M. phlei, M. smegma tis, M. fortui tum, M. kansaasi i, M. tubercul osis, M. bovi s, M. vacciae and M. avium. More preferably, mycobacterial DNA is prepared from the species Mycobacterium M. phl ei. Methods for increasing the therapeutic activity of M-DNA include, but are not limited to, chemically supplementing or biotechnically amplifying stimulatory sequences or DNA confirmations derived therefrom or P1068 / 00MX different bacterial species, or using bacterial plasmids containing an appropriate stimulatory sequence or DNA confirmations derived from the same or different bacterial species. Other methods for increasing the therapeutic activity of M-DNA include, but are not limited to, complexing the M-DNA with synthetic or biological carriers or coupling the M-DNA with ligands or antibodies directed to the type of tissue or to the cell type. M-DNA is provided in a first pharmaceutically accble carrier that can be prepared by putting into association the M-DNA and its carrier. Preferably, the M-DNA composition is prepared by uniformly and intimately linking the M-DNA with a liquid carrier, with a solid carrier, or both. Liquid carriers include, but are not limited to, aqueous carriers, non-aqueous carriers or both. Solid carriers include, but are not limited to, natural carriers and chemically synthesized carriers. For administration in an aqueous carrier the M-DNA is suspended in water, in saline or in a pharmaceutically accble regulator by techniques including, but not limited to, mixing, sonication and microfluidization. For administration in a carrier not Aqueous P1068 / 00MX, the M-DNA is emulsified with a neutral oil as for example, enunciatively, a diglyceride, a triglyceride, a phospholipid, a lipid, an oil and mixtures thereof, wherein the oil contains an adequate mixture of saturated and polyunsaturated fatty acids. Examples include, but are not limited to, soybean oil, canola oil, palm oil, olive oil and miglyol, where the fatty acid carbon number is between 12 and 22 and where the fatty acids may be saturated or unsaturated. Optionally, the phospholipid or charged lipid can be suspended in the neutral oil. To be administered in a natural carrier, such as, for example, MCC, the M-DNA is conserved and complexed on the cell wall of M. phi ei during the preparation of MCC. In MCC, the amount of M-DNA is enriched in relation to the amount of M-DNA in an intact M. phl ei cell and the M-DNA is more accessible to the responding cells than the M-DNA within a M cell. phlei intact. It should be understood that regulatory methods for using M-DNA can also be used for MCC. The composition of M-DNA, which comprises M-DNA and a pharmaceutically acceptable first carrier, can, in addition, be administered in a second pharmaceutically acceptable carrier. The composition of M-DNA and the formulations of the second carrier Pharmaceutically acceptable P1068 / 00MX can be prepared by uniformly and intimately bringing into association the M-DNA composition with a liquid carrier, with a solid carrier or both. Liquid carriers include, but are not limited to, aqueous carriers, non-aqueous carriers or both. Solid carriers include, but are not limited to, natural carriers and chemically synthesized carriers. The compositions of M-DNA and M-DNA can be administered as an oil emulsion, a water-in-oil emulsion, or a water-in-oil-in-water emulsion. In addition, they can be administered in carriers, including, but not limited to, liposomes, site-specific emulsions, long-residence emulsions, sticky emulsions, microemulsions, nanoemulsions, microparticles, microspheres, nanospheres, nanoparticles and minipumps, and with various natural polymers or synthetics that allow the sustained release of M-DNA, the minipumps or polymers are implanted in the vicinity where the administration of the drug is required. Polymers and their use are described in, for example, Brem et al. (Journal of Neurosurgery 74: 441-446, 1991). In addition, the M-DNA and the M-DNA composition can be used with any of the excipients, with all or with a combination of them, regardless of which is the first pharmaceutically acceptable carrier or the second P1068 / 00MX pharmaceutically acceptable carrier that is used to present the M-DNA to the responder cells. These excipients include, but are not limited to, antioxidants, regulators and antistats, and may include suspending agents and thickening agents. While not wishing to be limited by the following hypothesis, it is believed that the therapeutic effects of M-DNA include, but are not limited to, initiation of intracellular signaling which results in apoptosis in responder cells and cytokine stimulation and production of oxygen species reagent resulting in cytolysis and in the apoptosis of responding cells. Apoptosis and cytolysis, both individually and in combination, have anti-cancer activities and adjuvant activities. That is, the M-DNA composition of the present invention can be used alone as an anticancer agent or can be used before, at the same time or after administering another anti-cancer agent in order to increase the effectiveness of the treatment. The M-DNA composition is administered in an amount effective to induce a therapeutic response. The dose of M-DNA administered will depend on the donation to be treated, the particular formulation and other clinical factors such as weight and condition of the recipient and route of administration. Preferably, the amount of M-DNA P1068 / 00MX administered is between about 0.0001 mg / kg to about 100 mg / kg per dose, more preferably between about 0.0001 mg / kg to about 50 mg / kg per dose and more preferably from about 0.001 mg / kg to about 10 mg / kg per dose. When the M-DNA is administered as MCC, preferably the MCC DNA content is between about 0.001 mg of DNA / 100 mg of dry MCC and about 90 mg of DNA / 100 mg of dry MCC, more preferably between about 0.01 DNA / 100 mg of dry MCC and about 40 mg of DNA / 100 mg of dry MCC, more preferably between about 0.1 mg of DNA / 100 mg of dry MCC and about 30 mg of DNA / 100 mg of dry MCC. It is also preferred that the protein content of the M. phlei cell wall be less than about 2 mg / 100 mg of dry MCC and that the fatty acid content be less than about 2 mg / 100 mg of dry MCC. In addition, when the M-DNA is administered as MCC, the amount of MCC preferably ranges from about 0.00001 mg / kg to about 100 mg / kg per dose, more preferably from about 0.0001 mg / kg to about 50 mg / kg MCC per dose and still more preferably between about 0.001 mg / kg to about 10 mg / kg MCC per dose.

P1068 / 00MX Administration routes include, but are not limited to, oral, topical, subcutaneous, intra-muscular, intra-peritoneal, intra-venous, intra-dermal, intra-thecal, intra-lesional, intra-oral, intra -vejag, intra-vaginal, intra-ocular, intra-rectal, intra-pulmonary, intra-spinal, transdermal, subdermal, placement within body cavities, nasal inhalation, pulmonary inhalation, skin impression and electrocorporation. Depending on the route of administration, the volume per dose of preference is from about 0.001 ml to about 100 ml per dose, more preferably about 0.01 ml to about 50 ml per dose and still more preferably 0.1 ml to about 30 ml per dose . The composition of M-DNA can be administered in a single-dose treatment or in a multiple-dose treatment in a treatment scheme and in a suitable period according to the cancer being treated, the condition of the recipient and the route of treatment. administration . The administration of M-DNA is not an immunization process but is a therapeutic treatment that prevents, treats or eliminates a disease, including, but not limited to, various types of cancer. Cancer includes, but is not limited to, squamous cell carcinoma, P1068 / 00MX fibrosarcoma, sarcoid carcinoma, melanoma, breast cancer, lung cancer, colorectal cancer, renal cancer, osteosarcoma, cutaneous melanoma, basal cell carcinoma, pancreatic cancer, bladder cancer, ovarian cancer, leukemia, lymphoma and metastasis derived from the same. MCC retains its effectiveness after sonication and autoclaving, which reduces the base-paired length of M-DNA and after methylation with GpC, which eliminates the activity of the palindromic, purine-purine-oligonucleotide sequence CG-pyrimidine-pyrimidine. In addition, the unexpected and surprising ability of M-DNA to induce apoptosis in various cancer cell lines including, but not limited to, abnormal Fas, abnormal p52 / 21 and drug-resistant cancer cell lines, resolves a need not Satisfied for a long time in the medical field and provides an important benefit for animals, including man. The following examples serve to further illustrate this invention without constituting any limitation thereto. On the contrary, it is clearly understood that different modalities, modifications and equivalents of the same could have been used, that after reading this description, will occur to those experts of this P1O68 / 00MX without departing from the spirit of the present invention and the scope of the appended claims.

EXAMPLE 1 Preparation of MCC from Mycobacterium phl i MCC was prepared from Mycobacterium phl ei (strain 110). M. phl ei was obtained from the Institut fur Experimental Biologie and Medizin, Borstel, Germany and stored as a suspension in sterile milk at -60 ° C. M. phl ei was grown in Petragnani medium (Difeo Labs, Detroit, MI) and grown in a Bacto AC (Difco Labs) broth for 10 to 20 days. The cells were harvested by centrifugal sedimentation. All reagents used in the following procedures were selected to enhance the conservation of M. phl ei DNA. Approximately 400 grams of the wet cell mass was placed in an autoclave blender with a capacity of 1200 ml. The cell mass was mixed at high speed between 30 to 60 seconds. After mixing, 6 ml of DNase-free Tween 80 (Sigma Chemical Co., St. Louis, MO) and between 200 and 400 ml of water autoclaved were added to the cell mixture. The entire cell suspension was mixed again in the mixer at low speed for approximately 10 seconds. The disruption of the cells was achieved by P1068 / OOMX sonication. Five hundred ml of cell suspension, where the cells comprised approximately 50% to 70% of the volume, they were placed in a liter beaker, autoclaved, and sonicated. The sonicate was stored in a flask passed through an autoclave, placed on ice, during the fractionation process. The non-disintegrated cells were removed by low speed centrifugation. The supernatant from the low speed centrifugation was centrifuged for 1 hour at 27,500 g at 15 ° C and the supernatant from this centrifugation was discarded. The sediment from a centrifugation of 27,000 g was transferred to an autoclaved mixer and suspended in deionized water by autoclaving, with low speed mixing. This suspension was again centrifuged at 27,000 g at 15 ° C for 1 hour, and the supernatant was discarded again. The sediment was suspended in deionized water by autoclaving and rotated for 5 minutes at 350 g to pellet any remaining non-disintegrated cells. The supernatant was decanted and centrifuged at 27,000 g for 1 hour at 15 ° C to pellet the crude fraction of the cell wall. The crude fraction of the cell wall was deproteinized by digestion with proteolytic enzymes, taking care to use DNase-free fragments, where possible, to optimize the amount of DNA in the preparation and to P1068 / 00MX preserve the DNA structure in the preparation. The crude fraction of the cell wall derived from approximately 400 g of whole cells was suspended in 1 liter of Tris-HCl free of 0.05 M DNase, pH 7.5, mixing at low speed. After the crude fraction of the cell wall was completely suspended, 50 mg of DNase-free trypsin (Sigma Chemical Co.) was added and the suspension was stirred using a magnetic stir bar at 35 ° C for 6 hours. After treatment with trypsin, 50 mg of DNase-free pronase (Amersham Canada Limited, Oakville, Ontario) was added to each liter of crude cell wall suspension treated with trypsin. The suspension was stirred using a magnetic stir bar for 12 to 18 hours at 35 ° C. After the proteolytic digestion, the crude fraction of the cell wall was delipidated with detergent and phenol. To each liter of suspension, 60 g of DNase-free urea (Sigma Chemical Co.), 2.0 ml of DNase-free 100% phenol or 150 ml of 90% w / v phenol (Sigma Chemical Co.) were added. The flask containing the suspension was loosely covered with aluminum foil, heated to 60 ° -80 ° C and stirred for 1 hour. The suspension was rotated for 10 minutes at 16,000 g. The supernatant fraction and the fluid below the granule were discarded. The granule was washed 3 times by resuspension in P1068 / 00 X approximately 1 liter of deionized water passed through an autoclave and centrifuged for 10 minutes at 16,000 g. The delipidated, de-protected and washed MCC was lyophilized by transferring the suspension to a autoclaved lyophilization flask with a small amount of water passed by autoclave. A 300 ml flask of lyophilization was used for every 30 grams of the starting material of the wet cell complex. The MCC suspension was frozen by forming a shell or shell by rotating the matras in ethanol cooled with solid carbon dioxide. After the contents of the flask was frozen, the flask was connected to a lyophilization apparatus (Virtis Co., Inc., Gardiner, NY) and the contents were lyophilized. After lyophilization, the sample was transferred to a screw cap container, autoclaved and stored at -20 ° C in a desiccator containing anhydrous calcium sulfate. Unless otherwise mentioned, the lyophilized MCC was resuspended in deionized water passed by autoclave or in a pharmaceutically acceptable DNase-free regulator, for example, saline and PBS, and emulsified by sonication. Optionally, the homogenized MCC mixture was homogenized by microfluidization at 15,000-30,000 psi through a through flow. The MCC suspension was either processed under conditions P1068 / 00HX aseptic or sterilized by autoclave by autoclave.

EXAMPLE 2 Purification of M-DNA from MCC and from M. phl ei MCC was prepared as in Example 1. M-DNA was purified from MCC (MCC DNA) by extraction with phenol / chloroform / alcohol isoamylic acid and ethanol precipitation (Short Protocols in Molecular Biology, 3rd Edition, Ausubel et al., Eds., John Wiley &Sons Inc., New York, USA). Unexpectedly, it was found that at least about 3.6% of the dry weight of MCC can be extracted from M-DNA. M-DNA was purified from M. phl ei (M. phl ei DNA) by suspending M. phl ei (strain 110) in 5 ml of 50 mM Tris-HCl free of DNase, 5 mM EDTA, pH 8.0, adding DNase-free lysosome (Sigma Chemical Co.) at a concentration of 1 mg / ml and incubating for 90 minutes at 37 ° C. Dnasa-free proteinase K (Life Technologies, Burlington, Ontario, Canada) was added at a concentration of 0.1 mg / ml, Dnasa-free sodium dodecylsulfate (BioRad, Richmond, CA) was added at a 1% yield and the incubation was continued for 10 minutes at 65 ° C. The M-DNA was extracted with phenol / chloroform / isoamyl alcohol and precipitated with ethanol.

P1068 / 00MX Unless otherwise stated, the M-DNA is sonicated in autoclaved deionized water or in a pharmaceutically acceptable DNase-free regulator, such as, for example, saline and PBS. MCC, MCC DNA and M. phl ei DNA do not contain endotoxins as determined using the Limulus amebocyte lisate kit QCL-1000 (BioWhittaker, Walkersville, MD).

EXAMPLE 3 Preparation of the bacterial DNA-Bacerian cell wall and bacterial DNA complex from other bacterial species The bacterial DNA-bacterial cell wall complex is prepared from M. smegma tis, M. fortui tous, Nocardia rubra, Nocardia asteroides, Cornybacterium parvum, M. kansaasii, M. tuberculosis and M. bovis as in Example 1. The bacterial DNA is purified from the bacterial DNA-bacterial cell wall complex and from intact bacteria as in Example 2.

EXAMPLE 4 Treatment with DNase. MCC DNA, M-phlei DNA and MCC each contain 1 M-DNA and Regressin® (U.S. Patent No. 4, 744, 9840 [sic]) and were digested with an international unit (IU) of RNase I-free DNase (Life Technologies) for 1 hour at P1068 / 00MX 25 ° C in 20 mM Tris-HCl, pH 8.4, 2 M MgCl 2 and 50 mM KCl. DNase I is inactivated by the addition of EDTA to a final concentration of 2.5 mM and heating for 10 minutes at 65 ° C. DNase I digests both single-stranded and double-stranded DNA. Digestion with DNase I results in almost total degradation of the DNA. Regressin® (Bioniche, Inc. London, Ontario, Canada) is a formulation containing 1 mg of mycobacterial cell wall extract, 10 ml of NF mineral oil in 1 ml of PBS and 0.5% v / v of Tween 80.

EXAMPLE 5 Comparison of MCC DNA and M. phl DNA and MCC DNA and M. phl ei DNA were electrophoresed on a 5% agarose gel (3h, 100V) using procedures known to those skilled in the art. this field. The molecular weight distribution of the DNA was analyzed by gel photoexploration using the ID Main Program software (Advance American Biotechnology, Fullerton, CA). As shown in Figure 1, M. phlei DNA contains a wide range of oligonucleotides between about 5 and about 10,000 base pairs (bp) as well as genomic DNA (> 12,000 pd). The MCC DNA contains a range of oligonucleotides between about 5 and about 250 base pairs, 1 main peak P1068 / 00MX at approximately 4 base pairs and little genomic DNA.

EXAMPLE 6 Cells and reagents All cell lines, except 0C2 and SW260, were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in the medium recommended by the ATCC. OC2 and SW260 were obtained from Dr. J.K. Collins (University College Cork, Cork, Ireland) and were cultured in DMEM medium supplemented with 10% FCS. Table 1 shows the cell lines, their origins and their properties.

TABLE 1 Cell lines P1068 / 00MX Murine macrophages were obtained from CDI female mice injected intraperitoneally with 1.5 ml sterile Brewer's thioglycolate broth (Difco, Detroit, MI). Peritoneal exudate (> 85% macrophages) was collected at day 4, washed with centrifugation in HBSS and cultured in RPMI-1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 20 mM HEPES (Life Technologies). The cells were allowed to adhere for 18 hours after which the non-adhered cells were removed by gentle washing with warm medium. The murine spleen cells were prepared by gently tossing them through a sterile stainless steel mesh. Cell suspensions were layered on a Lympholyte-M cell separation medium (Cedar Lane, Hornby, Ontario, Canada) and centrifuged at 220 rpm for 30 minutes to remove the cells from erythrocytes and dead cells. These cells were cultured in RPMI- P1068 / 00MX 1640 supplemented with 10% FCS, 2mM L-glutamine and 20mM HEPES (Life Techologies). Unless otherwise stated, the cells were seeded in 6-well flat bottom tissue culture plates at concentrations between 3 x 10 5 and 10 6 cells / ml and maintained at 37 ° C in a 5% C0 atmosphere. . The calf thymus DNA, the herring sperm DNA and the lipopolysaccharide of Escherichia coli (LPS) were obtained from Sigma Chemical Co. Recombinant human IL-12 (hIL-12) was obtained from R &D Systems (Minneapolis, MN).

EXAMPLE 7 Inhibition of cell proliferation Cell proliferation was determined using dimethylthiazole-diphenyltetrazolium bromide (MTT) reduction (Mosman et al., Journal of Immunological Methods 65: 55-63, 1983). Briefly, 100 ml of MTT (Sigma-Aldrich) was dissolved in PBS and 5 mg / ml was added to the wells of the plates. After 4 hours, isopropanol-acid, 1 ml of 0.04 N HCl in isopropanol and reduced MTT were added and measured at a wavelength of 570 nm. Cells HT-1376, HT-1197, B-16 Fl, THP-1, RAW 264.7, Jurkat, HL-60 and HL-60 MX-1 were incubated for 24 hours with from 0 μg / ml to 10 μg / ml of M. phlei DNA, MCC DNA, herring sperm DNA and P1068 / 00MX DNA of calf thymus. The DNA of M. phl ei (Figure 2A) and the MCC DNA (Figure 2B) inhibited proliferation in each of the cancer cell lines tested in a dose-dependent manner, whereas the DNA from herring sperm (FIGS. 2A and 2B) and the calf thymus DNA (Figures 2A and 2B) did not inhibit proliferation in any of the cell lines tested. Cells HT-1376, HT-1197, B-16 Fl, THP-1, RAW 264-7, Jurkat, HL-60 and HL-60 MX-1 were incubated for 24 hours with from 0 μg / ml to 10 μg / ml of MCC. MCC inhibited proliferation in each of the cancer cell lines tested in a dose-dependent manner (Figure 3). Human leukemic THP-1 monocytes were incubated for 24 hours with from 9 μg / ml to 10 μg / ml of M. phl ei DNA, MCC DNA, MCC and hIL-12. M. phlei DNA, MCC DNA and MCC inhibited proliferation in a dose-dependent manner. The hIL-12 did not inhibit proliferation (Figure 4). The human bladder cancer cells HT-1197 (Figure 5A) and HT-1376 (Figure 5B) were incubated for 20 hours with 0 to 100 μg / ml of MCC and LPS. MCC inhibited proliferation. LPS did not inhibit proliferation. The M. phl ei DNA, the MCC DNA and the MCC wherein the first pharmaceutically acceptable carriers are the M.phlei cell wall; the P1068 / 00MX inhibition of proliferation of each of the cancer cell lines was tested. In contrast, LPS, which is a non-specific immunostimulant, reported inducing apoptosis in some cancer cell lines (Izquierdo et al., Anticancer Drugs 7: 275-2801996); hIL-12, which is a cytokine reported to induce apoptosis in some of the cancer lines (Stine et al., Annals NY Academy of Science 795: 420-421, 1996) and herring and herbal sperm thymus DNA they did not inhibit the proliferation of any of the cell lines tested. These data show that .M-DNA is responsible for the inhibition of proliferation in the cancer cell lines tested and that other DNAs (herring sperm DNA and calf thymus DNA) can not replace M-DNA. These data also show that the inhibition of M-DNA in cell proliferation does not originate from nonspecific immunostimulation (LPS) or cytokine activity (hIL-12). In addition, abnormal p53 / p21 and human bladder cancer cells HT-1376 resistant to drugs and human promyelocytic leukemia cells HL-60 MX-1 atypically drug resistant are found among the inhibited cancer cells.

P1068 / 00MX EXAMPLE 8 Induction of apoptosis as indicated by DNA fragmentation Fragmentation of cellular DNA into fragments of the size of the nucleosome is characterized by cells undergoing apoptosis. Fragments of the size of the nucleosome are fragments of DNA having a difference of about 20 base pairs in length as determined by agarose gel electrophoresis (Newell et al., Nature 357: 286-289, 1990). To assess DNA fragmentation, non-adherent cells were harvested by centrifugation at 200 g for 10 minutes. The granules of the non-adherent cells and the remaining adherent cells were lysed with 0.5 ml of hypotonic lysate regulator (10 mM Tris buffer, 1 mM EDTA, 0.2% Triton X-100, pH 7.5). The lysates were centrifuged at 13,000 g for 10 minutes and the supernatants, which contain fragmented DNA, were precipitated overnight at -20 ° C in 50% isopropanol and 0.5 M NaCl. The precipitates were collected by centrifugation and analyzed by electrophoresis in 0.7% agarose gels for 3 hours at 100V. A suspension culture of human leukemic THP-1 monocytes was incubated for 48 hours with PBS and with 1 μg / ml of M. phlei DNA treated with DNase I and MCC and with untreated herring sperm DNA.

P1068 / 00MX (Figure 6). DNA from M. phlei (band 2) and MCC (band 4) induced significant DNA fragmentation, whereas PBS (band 1), DNA from M. phl ei treated with DNase I (band 3), MCC treated with DNase I (band 5) and herring sperm DNA (band 6) did not induce DNA fragmentation. The DNA ladder of 123 base pairs (Life Technologies) was used to determine the molecular weight of DNA fragments of the nucleosome size (L band). A THP-1 monocyte suspension culture was incubated for 48 hours with PBS and with 1 μg / ml DNA from M. phl ei treated with DNase I or untreated and herring sperm DNA and with hIL-12 (Figure 7 ). DNA from M. phl ei induced significant fragmentation of DNA (band 5), whereas DNA from M. phl ei treated with DNase I (band 4), herring sperm DNA (band 3), DNA from herring sperm treated with DNase I (band 2), hIL-12 (band 1) and PBS (band 6) did not induce DNA fragmentation. The DNA ladder of 123 base pairs (Life Technologies) was used to determine the molecular weight of DNA fragments of the nucleosome size (L band). Human bladder cancer cells HT-1197 and HT-1376 were incubated for 48 hours with 1 μg / ml of MCC or with hIL 12 (Figure 8). MCC induced significant fragmentation of DNA in cells not P1068 / 00MX adherent HT-1197 (Figure 8A, band 2) and HT-1376 (Figure 8B, band 2), but not in adherent cells HT-1197 (Figure 8A, band 3) and HT-1376 (Figure 8B, band 3). Cells in PBS (Figure 8A and 8B, band 5), hIL-12 (Figure 8A and 8B, band 4), MCC treated with DNase I (Figure 8A and 8B, band 7) and untreated cells (Figure 8A and 8B , band 1) did not induce DNA fragmentation in HT-1197 cells or HT-1376 not adherents. The DNA ladder of 123 base pairs (Life Technologies) was used to determine the molecular weight of DNA fragments of the size of the nucleosome (Figure 8A and 8B, L band). The DNA of M. phlei and MCC, where M-DNA was conserved and complexed with the cell wall of M. phl ei, induced apoptosis in human leukemic THP-1 monocytes and in human vein cancer cells HT-1197 and HT -1376, while herring sperm DNA, hIL-12, and M. phl ei DNA treated with DNase I and MCC did not induce apoptosis in these cells. These data demonstrate that M-DNA is responsible for the induction of apoptosis in the cancer cell lines tested, that M-DNA oligonucleotides must be intact (treatment with DNase I) and other DNAs (herring sperm DNA) can not replace M-DNA. These data also show that the induction of apoptosis by M-DNA is not the result of non-specific immunostimulation (LPS).

P1068 / 00 X EXAMPLE 9 Induction of apoptosis as indicated by solubilization of myotonic nuclear protein apparatus (NuMA-nucl ear my totic protein appara tus) The strong morphological changes in the cell nucleus caused by the solubilization and release of NuMA are characteristic of the apoptose To determine the solubilization and release of NuMA, the media from the cultured cells were removed and centrifuged at 200 g for 10 minutes. Supernatants were collected and 100 μl of each supernatant was used to quantitate the NuMA released in units / ml (U / ml) using a commercial ELISA assay (Calbiochem, Cambridge, MA) (Miller et al. Biotechniques 15: 1042-1047, 1993). The human leukaemic TBP-1 monocytes were incubated for 48 hours with 0 μg / ml to 10 μg / ml of M. phlei DNA, MCC DNA, MCC and herring sperm DNA. DNA, from M. phl ei, MCC DNA and MCC induced the release of NuMA in a dose-dependent manner, whereas herring sperm DNA did not induce the release of NuMA (Figure 9). Treatment with DNase I of M. phlei DNA, MCC DNA and MCC significantly inhibited their induction of NuMA release from these cells (Figure 10).

P1068 / 00MX The human bladder cancer cells HT-1197 and HT-1376 were incubated with 0 μg / ml at 100 μg / ml MCC. MCC induced the release of NuMA in. a dose-dependent manner (Figure 11) and in a time-dependent manner (Figure 12A and 12B). The enhanced release of NuMA was detected within 24 hours after the induction of HT-1197 cells (Figure 12A) and HT-1376 (Figure 12B) with 100 μg / ml MCC. The DNA of M. phl ei, the MCC DNA and the MCC, each of which contains M-DNA, induce the apoptosis of the cancer cells. MCC, wherein the first pharmaceutically acceptable carrier is the cell wall of M. phl ei, induces more apoptosis than M. phl ei DNA or MCC DNA. This suggests that the carrier that presents the M-DNA to the responding cells effects the induction of apoptosis by the M-DNA.

EXAMPLE 10 Independent Induction of Fas in Apoptosis in Jurka Human T lymphoblast Cells by M. phl ei DNA Jurkat human T lymphoblast cells were incubated for 1 hour with PBS, with 1 μg / ml of CH-11 antibody, an antibody that binds to Fas and induces apoptosis (+ control) (Coulter-Immunotech, Hialeah, FL) or with 1 μg / ml of the ZB4 antibody, a P1068 / 00MX antibody that binds Fas and that inhibits apoptosis (- control) (Coulter-Immunotech). The M. phl ei DNA, 1 μg / ml, was added and the NuMA released was determined after 48 hours. As shown in Figure 13, DNA from M. phl ei induced apoptosis both in the absence of ZB4 and in the presence of ZB4. These data demonstrate that the induction of apoptosis by M. phl ei DNA is independent of Fas.

EXAMPLE 11 Summary of the effects of M. phlei DNA, MCC DNA and MCC on cell proliferation and on apoptosis. Table 2 summarizes the effects of M. phlei DNA, MCC DNA and MCC on the inhibition of cancer cell proliferation and on the induction of apoptosis in them, as determined by DNA fragmentation, NuMA release and flow cytometric analysis in human and murine cancer cell lines.

P1068 / 00 X TABLE 2 Inhibition of proliferation of human and murine cancer cell lines and induction of apoptosis thereof.

ND = Not done.

The DNA of M. phl ei, the MCC DNA and the MCC, wherein the first pharmaceutically acceptable carrier is the cell wall of M. phl ei and where the M-DNA complexes on the cell wall, inhibit the proliferation of each of these cancer cell lines and induce their apoptosis. These cancer cell lines include P1068 / 00MX human promyelocytic leukemia cells HL-60 MX-1 resistant to atypical drugs, human bladder HT-1376 cells resistant to drugs and abnormal p53 / p21, human colon cells SW260 Abnormal cells and human cecum carcinoma cells LS1034 resistant to conventional drugs.

EXAMPLE 12 Activation of caspase-3 by MCC in human leukemic THP-1 monocytes caspase 3 is a key enzyme in the apoptotic pathway underneath Fas-FasL signaling. To determine whether MCC can deviate from Fas and directly activate the caspase cascade in cancer cells, the effect of MCC on caspase-3 activity was analyzed in human leukemic THP-1 monocytes. The THP-1 monocytes (2 x 10 7 cells) were incubated for 48 hours with MCC (100 μg / ml). THP-1 cells were used in 50 M HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA and 10% glycerol. The activity of caspase-3 was determined with a commercial ELISA assay (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), using the included substrate, the inhibitor and the purified caspase-3 enzyme. The results are expressed as reading the optical density at 405 nm.

P1068 / 00 X TABLE 3 Activation of caspase 3 activity by MCC (100 μg / ml) in human leukemic THP-1 monocytes.

As shown in Table 3, incubation with MCC resulted in an increase of 232% (3 hours) and 333% (6 hours) in caspase-3 activity and activity similar to caspase-3 in human leukemic THP-1 monocytes. The induction of caspase-3 activity and caspase-3 activity by MCC was stopped with the treatment of DNase I. The specificity of the induction of caspase-3 activity and activity similar to caspase-3 by MCC was demonstrated using the caspase-3 inhibitor. The P1068 / 00MX addition of the caspase-3 inhibitor to the THP-1 cell extract treated with MCC completely eliminated the measurable activity. The ability of MCC to specifically and directly induce caspase-3 activity and caspase-3-like activity in human leukemic THP-1 monocytes is totally unexpected. To specifically stimulate caspase-3 activity and caspase-3-like activity, MCC must enter cells by one or more mechanisms and initiate the lethal proteolytic cascade of the execution of apoptosis.

EXAMPLE 13 The effect of tamoxi fen on MCC induced apoptosis in human THP-1 monocytes. Human leukemic THP-1 monocytes were incubated for 90 minutes in control medium or medium containing 10 μg / ml tamoxifen (Sigma-Aldrich), an anti-estrogen used in the palliative treatment of advanced breast cancer. Cells were washed extensively with ice-cooled medium (2X), resuspended to approximately 10 6 cells / ml in the medium and incubated for 48 hours with 0, 1, 10 and 100 μg / ml MCC. Apoptosis was quantified by measuring NuMA.

P1068 / 00MX TABLE 4 The effect of tamoxifen on MCC induced apoptosis in human leukemic THP-1 monocytes determined by the release of NuMA in U / ml As shown in Table 4, preincubation on tamoxifen significantly increased the apoptosis induced by MCC at each of the used concentrations of MCC. These data demonstrate that MCC, where M-DNA is complexed on the cell wall of M. phl ei, can be used as an adjuvant as other anti-cancer agents in order to increase the efficacy of the treatment.

EXAMPLE 14 Modification of DNA from M. phlei by methylation, sonication and au toclave Nucleic acid preparations from bacillus Calmette-Guerin (BCG) inhibit the growth of cancer (US Pat.

No. 4,579,941; Tokunaga et al. Microbiology and Immunology 36: 55-666, 1992). The active constituent P1068 / 00MX in the BCG nucleic acid has been identified as the palindromic purine-purine-C-G-pyrimidine-pyrimidine oligonucleotide sequence (CG motif). Methylation of cytosine with CpG methylase eliminates the activity of this DNA (Krieg et al., Nature 374: 546-549, 1995). Therefore, the effect of CpG methylation on the DNA ability of M. phlei to induce apoptosis was determined. M. phlei DNA, 1 μg was methyl using 2.5 U of CpG Sss I methylase (New England Biolabs, Mississauga, Ontario, Canada) in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl 2, 1 mM DTT and 160 μM of S-adenosylmethionine for 1 hour at 37 ° C. Native and methylated M. phl ei DNAs were excised by restriction endonuclease BstU I (New England Biolabs) for 1 hour at 60 ° C in 50 mM NaCl, 10 mM Tris HCl, 10 mM MgCl 2, and DTT 1 mM, pH 7.9. Electrophoretic analysis in 0.5% agarose gel for 3 hours at 100 V showed that native M. phl ei DNA was digested by restriction endonuclease BstU I, whereas methylated M. phl ei DNA was not digested by this endonuclease. This confirmed that DNA methylation of M. phl ei was complete. As shown in Figure 14, methylation did not modify the NuMA release induced by M. phl ei from human leukemic THP-1 monocytes. These data demonstrate that, unlike BCG, CG motifs are not the induction of apoptosis P1068 / 00MX required by the DNA of M. phl ei. The length in DNA base pairs of M. phl ei (1 μg) was reduced by sonication for 15 seconds or 20 minutes on ice in an ultrasonic processor Model W-38 (HeatSystems-Ultrasonics, Inc.), by digestion with restriction endonuclease BstU I or by autoclaving at 121 ° C for 30 minutes (Castle Sybron MDT, Dubuque, Iowa). As shown in Figure 14, sonication, digestion with BstU I and passage through the autoclave, each reduced the length of base pairs to a range of approximately 5 base pairs to approximately 250 base pairs, and did not affect the induction caused by M. phl ei DNA in the NuMA release from THP-1 monocytes. These results demonstrate that M. phl ei DNA induces apoptosis in cancer cells, and even at short oligonucleotide lengths (about 5 base pairs up to about 250 base pairs). Human THP-1 monocytes were incubated for 48 hours with untreated M. phlei DNA and MCC and with M. phl ei DNA and MCC autoclaved for 30 minutes at 121 ° C. The autoclave process, which reduces the length of the DNA base pairs, did not affect the ability of M. phl ei or MCC DNA to inhibit the proliferation (Table 5A) of these cells or to induce apoptosis P1068 / 00MX Table 5B) thereof.

TABLE 5A Effect of the autoclave process on the inhibition of DNA proliferation of M. phl ei and MCC TABLE 5B Effect of autoclaving process on the induction of apoptosis by MCC and M. phl ei DNA as determined by the release of NuMA in U / ml P1068 / 00MX EXAMPLE 15 MCC inhibits cancer growth in vivo MCC and MCC treated with DNase I were emulsified to a final concentration of 1 μg / ml in PBS containing 2% w / v mineral oil and 0.02% p / v Tween 80 (Fisher Chemical Co.) by sonication at 4 ° C for 5 minutes (Heat Systems-Ultrasonics, Inc.). The hepatoma cells of line 10, syngeneic for guinea pig strain 2, were thawed rapidly, washed by centrifugation and resuspended in M 199 medium to a concentration of 10 6 cells / ml. One tenth of ml containing 1.5 x 106 was injected intradermally into the back muscles of guinea pig strain 2 of 3 months of age. The treatment started 6 to 7 days after injection when the cancer was between about 0.5 and about 0.8 cm in diameter. 7 animals were treated with emulsification regulator alone (control) 7 with emulsification regulator containing MCC and 7 with emulsification regulator containing MCC treated with DNase I. Emulsions were administered directly by instillation into the cancer and surrounding normal tissue . Half ml of emulsion was administered at 0 hours and 6 hours to achieve a total volume of 1 ml containing 1 mg of MCC or MCC treated with DNase I. Diameters of cancer (major diameter + P1068 / 00MX smaller diameter) were recorded weekly for 3 weeks. The cancer volumes were calculated as mm3 as 0.5 x a (smaller diameter) x b2 (smaller diameter) and the increase in cancer volume in relation to day 0 of the treatment was calculated in each guinea pig. Statistical analysis was performed using bidirectional ANOVA with replicas (PHARM / PCS version 4.2, MCS, Philadelphia, PA). The differences in the treatment were considered significant at p < 0.05. As shown in Figure 15, with the control emulsion, the volume of cancer increased by approximately 22 times by week 3, whereas with MCC, cancer growth was significantly inhibited compared to the control emulsion (Figure 15, Table 6). With the MCC treated with DNase I, the growth of the cancer was not significantly different from that of the control (Figure 15, Table 6).

TABLE 6 These data show that the administration by instillation of MCC in the tumor site gives P1068 / 00MX resulted in tumor regression. In addition, the significant difference (p <0.01) in the inhibition of cancer growth between MCC and MCC treated with DNase I shows that the non-degraded M-DNA is necessary for the anti-cancer activity of MCC in vivo. EXAMPLE 16 Cytotoxicity of MCC Cellular cytotoxicity is characterized by loss of integrity of the plasma membrane and release of cytoplasmic enzymes, for example, LDH (Wyllie et al., International Review of Cytology 68: 251-306, 1980; Phillips et al., Vaccine, 14: 898-904, 1996). Human bladder cancer cells release LDH when treated with cytotoxic agents (Rahman M. Urology International 53.12-17, 1994). To assess the cytotoxicity of MCC, human bladder cancer cells HT-1197 and HT-1376 were incubated for 48 hours with 0 μg / ml to 100 mg / ml MCC or lysate buffer (10 mM Tris, 1 mM EDTA , Triton X-100 0.2%, pH 7.5) as a control for the total release of LDH (Filion et al., Biochim Biophys Acta 1329: 345-356, 1997). The release of LDH towards the culture supernatant was determined using a commercial assay (Sigma-Aldrich). As determined by the release of LDH, MCC was not cytotoxic to HT-1197 or HT- cells P1068 / OOMX 1376 (Figure 17). The absence of cytotoxicity demonstrates that MCC acts directly to inhibit the proliferation of cancer cells and to induce apoptosis thereof.

EXAMPLE 17 Stimulation of cytokine synthesis in vi tro It was reported that IL-12 had anticancer activity in some cancer cells (Voest et al., Journal National Cancer Institute 87: 581-586, 1995; Stine et al., Annals NY Academy. of Science 795: 420-421, 1996), whereas GM-CSF is reported to have pro-cancer activity in some cancer cells (Hawkyard et al, Journal of Urology 150: 514-518, 1993). In addition, it is reported that some cancer cells secrete cytokines (De Reijke et al., Urology Research 21: 349-352, 1993; Bevers et al., British Journal of Urology 80: 35-39, 1997). And therefore, the effect of MCC on the synthesis of IL-6, IL-12 and GM-CSF by human bladder cancer cells HT-1197 and HT-1376 and by human THP-1 monocytes, murine macrophages, monocytes RAW 264.7 murine and murine spleen cells, was determined. The cytokine determination is in pg / ml in 100 μl of culture supernatant using the appropriate commercial ELISA assay (BioSource, Camarillo CA). The IL-12 ELISA assay measures both the p70 IL-12 complex and the p40 free subunit.

P1068 / 00MX The HT-1197 and HT-1376 cells, the THP-1 macrophage, the RAW 264.7 cells and the murine spleen cells were incubated for 48 hours with 1 g / ml MCC. As shown in Figure 1, MCC stimulated the production of IL-6 and IL-12 by human monocytes and murine macrophages, but not by human bladder cancer cells, murine monocytes or spleen cells, MCC did not stimulate the production of GM-CSF in none of the cancer cells tested. These data demonstrate that MCC, where the M. phlei cell wall is the first pharmaceutically acceptable carrier, stimulates the production of anti-cancer cytokines IL-6 and IL-12 by human monocytes and murine macrophages. MCC does not stimulate cytokine production by human bladder cancer cells. MCC does not stimulate the production of pro-cancer cytokine GM-CSF.

EXAMPLE 18 Effect of DNA from untreated M. phl ei and treated with DNase I, MCC DNA, MCC and Regressin® on the production of IL-12 by human THP-1 monocytes THP- monocytes 1 human were incubated for 48 hours with M. phl ei DNA, MCC DNA, MCC and Regressin®, before treatment with DNase I, after treatment with DNase I and after the addition of M-DNA to DNA from M. phl ei treated with DNase I, P1068 / 00MX DNA from MCC and to MCC.

TABLE 7 Production of IL-12 in pg / ml by human THP-1 monocytes As shown in Table 7, M. phl ei DNA, MCC DNA and MCC each stimulate the THP-1 monocytes to produce IL-12. MCC stimulated more IL-12 production than either M. phlei DNA or MCC DNA. Regressin® minimally stimulated the production of IL-12. Treatment with DNase I reduced the production of IL-12 stimulated by M. phlei DNA, MCC DNA and MCC by approximately 50%. Regressin® was not affected by treatment with DNase I. The addition of DNA from M. phl ei to M. phlei DNA treated with DNase I or MCC DNA to MCC DNA treated with DNase I established its production stimulation of IL-12. The addition of the MCC DNA to the MCC treated with DNase I did not re-establish its stimulation of IL-12 production.

P1O68 / 00 X MCC wherein the cell wall of M. phl ei is the first pharmaceutically acceptable carrier stimulates more the production of IL-12 than the M. phl ei DNA or the MCC DNA suggesting that the carrier used to present M-DNA against the responding cells effects the production of IL-12 stimulated by M-DNA. Treatment with DNase I, which degrades M-DNA, significantly reduces the production of IL-12 stimulated by M. phl ei DNA, MCC DNA and MCC suggesting that the M-DNA oligonucleotide form must be retained for stimulation optimal production of IL-12. This addition of M-DNA to MCC treated with DNase I did not re-establish its stimulation of IL-12 production, suggesting that the way in which M-DNA complexes with the cell wall of M. phlei in. MCC is important for the optimal stimulation of IL-12 production by human monocytes.

EXAMPLE 19 Effect of treatment with au toclaves of IL-12 production by M. phlei DNA and MCC, by human THP-1 monocytes. Human THP-1 monocytes were incubated for 48 hours with MCC and M. phl ei DNA and with MCC and M. phl ei DNA autoclaved for 30 minutes in sterile water.

P1068 / 00MX TABLE 8 Effect of autoclaving on the production of IL-12 stimulated by MCC and M. phl ei DNA, in pg / ml by monocytes THP-1 As shown in Table 8, autoclaving, which reduces the size of DNA base pairs, did not affect the stimulation of IL-12 production by MCC or M. phlei DNA in monocytes.

EXAMPLE 20 Effect of the treatment of CD14 antibody on the production of IL-12 is stimulated by MCC DNA and by MCC by human THP-1 monocytes Human THP-1 monocytes were incubated with PBS or with 10 mg / ml anti-CDl4 antibody (clone MY4, Coulter-Immunotech) for 1 hour. Then, 5μg / ml of the MCC or MCC DNA was added, and the incubation continued for 48 hours. The antibodies of P1068 / 00MX CD14, which bind to CD14 receptors on the cell surface, caused approximately an 85% decrease in the production of IL-12 stimulated by MCC DNA and approximately a 20% decrease in IL-12 production stimulated by MCC (Figure 18).

EXAMPLE 21 Effect of cytosine D on IL-12 production is stimulated by M. phlei DNA, MCC DNA and MCC, by human THP-1 monocytes. Human THP-1 monocytes were incubated for 48 hours with PBS or with 1 μg / ml of M. phlei DNA, MCC DNA or MCC in the absence and in the presence of 10 μg / ml of cytochalasin D (Sigma Chemical Co.) . Cytochalasin D, which inhibits phagocytosis, caused a 64% decrease in DNA from M. phl ei, or a 50% decrease in MCC DNA and a 55% decrease in IL-12 production stimulated by MCC (Figure 19). Although it is not desired to be limited by the following hypothesis, but based on the data shown in Figures 18 and 19, it is considered that M. phl ei DNA, MCC DNA and MCC interact as monocytes by multiple mechanism. Figure 18 suggests that they interact with the CD14 membrane receptor bound to GPI and internealize. These mechanisms are more specific for the DNA of M.

P1068 / 00MX phl ei and MCC DNA, soluble, than for insoluble MCC. Figure 19 suggests that they interact with phagocytic receptors, such as the purifying receptor, and internealize. This mechanism is more specific for insoluble MCC than for M. phlei DNA and MCC DNA.

EXAMPLE 22 Effect of CG and MCC sequences on the production of IL-12 by human THP-1 monocytes. BCG nucleic acid preparations are reported to stimulate lymphocyte proliferation, secretion of IL-6 and IL-12 by B-lymphocytes, secretion of IL-12 by monocytes, secretion of IL-6 and interferon-gamma by T-lymphocytes and the secretion of interferon-gamma by NK cells (Klinman et al., Proceeding of the National Academy of Science USA 93: 2879-2883, 1996). As the active constituent of the BCG nucleic acid has already been identified as the CG motif, the THP-1 monocytes were incubated for 48 hours with 0.5, 1 and 5 μg / ml MCC or the DNA sequence 5'-GCTAGACGTTAGCGT- 3 'prepared by solid phase synthesis using an automated DNA synthesizer.

P1068 / 00MX TABLE 9 Effect of the oligonucleotide containing GC and MCC in the production of IL-12 in Pg / ml by monocytes THP-1 As shown in Table 9, the oligonucleotide sequence containing GC did not stimulate the production of IL-12 at any of the three concentrations tested, while MCC at 1 μg / ml had a significant stimulatory effect on IL-1 production. 12 EXAMPLE 23 Effect of the heat treatment and treatment of DNase I on M. phlei DNA, MCC DNA, MCC and stimulation with Regressin®, of the production of IL-12 by murine macrophages. Murine perifoneal macrophages were incubated for 48 hours with M. phlei DNA, MCC DNA, MCC and Regressin® and with M. phl ei DNA, DNA from MCC and Regressin®, which had been heated to 100 ° C by minutes and then they had cooled on ice by 2 minutes .

P1068 / 00MX TABLE 10 Production of IL-12 in pg / ml by murine macrophages As shown in Table 10, at a concentration of 5 μg / ml, the production of IL-12 was further stimulated by MCC, less by M. phl ei DNA and MCC DNA and still less Regressin®. The heat treatment of M. phlei DNA, MCC DNA and Regressin® had no significant effect on its stimulation of IL-12 production. Although not shown, the heat treatment of MCC caused a slight but significant increase in IL-12 production. Murine perifoneal macrophages were incubated for 48 hours with MCC DNA, MCC and Regressin®, treated or not treated with DNase I (Figure P1068 / 00MX 20). The production of IL-12 was stimulated more with MCC, less with MCC DNA and much less with Regressin®. DNase I treatment of MCC DNA and MCC significantly reduced its stimulation of IL-12 production by murine macrophages. Regressin® DNase I treatment had no effect on its activity. These data suggest once that, as with monocytes (Example 18), the oligonucleotide structure of M-DNA must be conserved for optimal stimulation of IL-12 production by murine macrophages.

EXAMPLE 24 Effect of DNA from M. phl ei, MCC DNA, MCC and Regressin®, on production of nitric oxide (NO) by peri-toneal macrophages is murine. The activation of the macrophages stimulated the production of the reactive oxygen species including, without limitation, nitric oxide (NO), superoxide radicals and hydroxyl radicals. These reactive oxygen species induce cytolysis and apoptosis in responder cells and, therefore, have anti-cancer activity. The murine peritoneal macrophages were incubated for 48 hours with 0.1, 5.0 or 12.5 μg / ml of DNA of M. phl ei, DNA of MCC, MCC and Regressin®. The production of NO was measured in nmol / L by reaction of N02- with gray reagent using 100 μl of P1068 / 00MX culture supernatant.

TABLE 11 Effect of M. phlei DNA, MCC DNA, MCC and Regressin® on the production of NO by murine macrophages As shown in Table 11, at 5 μg / ml, MCC stimulated significantly more NO production than M. phlei DNA, MCC DNA or Regressin®. The murine macrophages were incubated for 48 hours with 1 μg / ml of MCC treated or not treated with DNase I and with untreated M. phl ei DNA and untreated MCC DNA.

P1068 / 00MX TABLE 12 Stimulation of NO production in murine macrophages by MCC, MCC treated with DNase I, MCC DNA and M. phl ei DNA As shown in Table 12, M-DNA, complexed on the cell wall of M. phl ei as well as MCC, stimulated significant NO production. Treatment with DNase I of MCC, which degrades M-DNA, eliminated stimulation of NO production caused by MCC. MCC DNA and M. phl ei DNA stimulated minimal NO production. These data demonstrate that both the intact structure of the M-DNA oligonucleotide and the carrier that presents these nucleotides to macrophages are important for optimal stimulation in the production of NO.

EXAMPLE 25 Effect of MCC on the production of nitric oxide (NO) by murine monocytes RAW 264. 7. RAW 264.7 murine monocytes were incubated P1068 / 00MX for 24 hours with 0.5 to 95 μg / ml of MCC. Higher concentrations of MCC stimulated higher amounts of NO production (Figure 21). This was unexpected since the receptors for the induction of NO are optimally expressed in monocytes and, therefore, NO production is not normally associated with monocytes. Under the same conditions, Regressin® did not stimulate NO production.

EXAMPLE 26 Stimulation of in vivo cytokine synthesis Four groups of CD-1 mice, each containing 5 mice, were injected intraperitoneally with 50 mg / kg of MCC. The blood was collected at 0, 3, 6 and 24 hours after the injection and the concentrations (pg / ml) of IL-6, IL-10, IL-12 and GMSF in the sera were determined at 0, 3, 6 and 24 hours after the injection (Figure 22). With intraperitoneal MCC serum concentrations of IL-6, IL-10 and IL-12 were significantly increased at 3 and 6 hours after injection and declined to approximately control values (O hours) at 24 hours after the injection. injection. Serum concentrations of GM-CSF remained at approximately control values (0 hours), at 3, 6 and 24 hours after injection. Five groups of CD-1 mice containing P1068 / 00MX each 5 mice, 6.6 mg / kg of MCC were injected intravenously. Blood was collected at 0, 3, 6 and 24 hours after the injection and the concentrations (pg / ml) of IL-10 and IL-12 in the sera were determined at 0, 3, 6 and 24 hours after the injection (Figure 23). With intravenous MCC, serum IL-12 concentrations increased significantly at 3 and 6 hours after injection and declined to approximately control values (0 hours) at 24 hours after injection. The serum concentrations of IL-10 remained approximately in the control values (0 hours) at 3, 6 and 24 hours after the injection. These data demonstrate that the in vivo administration of M-DNA, where the M-DNA was complexed on the cell wall of M. phlei, like MCC, stimulates the production of anti-cancer cytokines IL-6, IL-10 and IL-12, but not the pro-cancer cytokine GM-CSF. In addition, these data demonstrate that the amount of MCC administered and the route by which it is administered, both effect the ability of MCC to stimulate cytokine production in vi vo. Four groups of CD-1 mice, each containing 4 mice, were injected intraperitoneally with MCC and M. phl ei DNA treated and not treated with DNase I. After 3 hours, the mice were sacrificed blood P1068 / 00 X was collected by cardiac microarray and the concentration (pg / ml) of IL-12 in the sera was measured (Table 13).

TABLE 13 Effect of MCC and DNA from M. phl ei ± tra tami on DNase I on the production of IL-12 in pg / ml by CD-1 ra tones As shown in Table 13, in vivo administration of MCC and M. phl ei DNA stimulates the production of anti-cancer cytokine IL-12. After treatment with DNase I, the production of P1068 / 00MX IL-12 stimulated by MCC decreased to 40.5% and the production of IL-12 stimulated by M. phlei DNA decreased 46.5%. This demonstrates that the oligonucleotide structure of M-DNA must be conserved for optimal stimulation of IL-12 production in vivo.

EXAMPLE 27 The MCC MCC stability at 1 mg / ml was stored as a sterile suspension at 0.85% w / v NaCl in the dark at 4 ° C for 6 months. The average particle diameter was calculated using photonic correlation spectroscopy (N4 Plus, Coulter Electronics Inc.). The MCC suspension was diluted with 0.85% w / v NaCl at a particle count rate between 5 x 104 and 106 counts / second. The average particle diameter was calculated in a size distribution processor (SDP) mode using the following conditions: fluid replacement ratio 1.33, temperature 20 ° C, viscosity 0.93 centipoise, measuring angle 90.0 °, sampling time 10.5 μs and run time of the sample 100 seconds. The potential, the electrical charge at the hydrodynamic interface between the particles and the bulk solvent, was measured on a Delsa 440SX (Coulter Electronics Inc.) using the following conditions: current 0.7 mA, range of P1068 / 00MX frequency 500 Hz, temperature 20 ° C, fluid refractive index 1.33, viscosity 0.93 centipoise, dielectric constant 78.3, conductivity 16.7 ms / cm, ignition time 2.5 seconds, off time 0.5 seconds and run time of the shows 60 seconds As shown in Figure 4, the MCC load and the MCC diameter remained relatively unchanged for 6 months of storage. In addition, the stimulation of MCC for the production of IL-12 and the induction of MCC for apoptosis in THP-1 monocytes remained unchanged during 6 months of storage.

EXAMPLE 28 Trase with MCC DNA and with MCC in human colon cancer. Human colon cancer cells (ICM12C) have been established as an ectopic solid tumor in the subcutaneous tissues of athymic and immunodeficient nude mice (nu / nu mice) and the mice were divided into 5 groups. Group 1 received only the vehicle. Group 2 received MCC DNA. Group 3 received MCC DNA treated with DNase I. Group 4 received MCC. Group 5 received MCC treated with DNase I. The cancer mass was measured before treatment and weekly for 4 weeks of treatment. The mice of group 2 and P1068 / 00MX the mice of group 4 show regren of the cancer mass.

EXAMPLE 29 Treatment with M. phlei DNA and with MCC in human ovarian cancer Human ovarian cancer cells (36M2) are established as ascites in the peritoneal cavity of nude immunodeficient nude mice (ranotes nu / nu) and the mice are divided into 5 groups. Group 1 received only vehicle. Group 2 received DNA from M. phlei, group 3 received DNA from M. phl ei treated with DNase I, group 4 received MCC, group 5 received MCC treated with DNase I. The cancer mass was measured before treatment and weekly for 4 weeks. Mice from group 2 and mice from group 4 showed inhibition of ascites cancer cell proliferation.

EXAMPLE 30 Treatment with MCC of canine transmile canine cancer dogs Dogs with venereal cancer (VT) were divided into 4 groups. Group 1 received vincritin. Group 2 received vincritine combined with methotrexate and cyclophosphamide. Group 3 received MCC DNA complexed with a carrier and presented P1068 / 00MX on a carrier, where the carrier is the mycobacterial cell wall (MCC). Group 4 received vincristine and MCC. The cancerous mass was measured before treatment and weekly for 12 weeks of treatment. Group 4 dogs showed regren of VT.

EXAMPLE 31 Aqueous carrier suspension. The M-DNA was suspended in a first pharmaceutically acceptable carrier and sonicated at 20% output for 5 minutes (Model W-385 Sonicator, Heat Systems-Ultrasonics Inc.). Optimally, the sonicated composition was homogenized by microfluidization at 15,000-30,000 psi through a through flow (Model M-110Y, Microfluidics, Newton, MA).

P1068 / 00MX

Claims (47)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A composition comprising: a. Mycobacterium phl ei DNA (M-DNA), and b. a first pharmaceutically acceptable carrier wherein the M-DNA has anti-cancer activity.
2. 2. The composition according to the claim 2, wherein the anti-cancer activity of M-DNA is the inhibition of the proliferation of cancer cells.
3. 3. The composition according to the claim 1, wherein the anti-cancer activity of M-DNA is the induction of apoptosis in cancer cells.
4. 4. The composition according to the claim 3, wherein the induction of cancer cell apoptosis is independent of a factor selected from the group consisting of Fas, p53 / p21, and drug resistance. The composition according to claim 3, wherein the induction of apoptosis in cancer cells is the induction of caspase-3 activity in cancer cells. 6. The composition according to claim 1, wherein the anti-cancer activity of M-DNA is the P1068 / 00MX stimulation of cytokine production by cells of the immune system. The composition according to claim 6, wherein the cells of the immune system are selected from the group consisting of macrophages and monocytes. The composition according to claim 6, wherein the cytokine is selected from the group consisting of IL-6, IL-10 and IL-12. 9. The composition according to the claim 1, wherein the anti-cancer activity of M-DNA is the stimulation of the production of reactive oxygen species by cells of the immune system. 10. The composition according to claim 9, wherein the cells of the immune system are macrophages. 11. The composition according to the claim I, wherein the first pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier, a non-aqueous carrier and a cell wall of Mycobacterium Phl ei (M. phl ei). 12. The composition according to the claim II, wherein the first pharmaceutically acceptable carrier is the cell wall of M. phl ei. 13. The composition according to the claim 1, which further comprises a second pharmaceutically acceptable carrier, wherein the second pharmaceutically acceptable carrier is selected P1068 / 00MX of the group consisting of an aqueous carrier and a non-aqueous carrier. 14. A composition that comprises: a. M-DNA, b. cell wall of M. phl ei, where the M-DNA is conserved and complexed on the cell wall of M. phl ei (MCC) and c. a pharmaceutically acceptable carrier, wherein the MCC has anti-cancer activity. 15. The composition according to the claim 14, wherein the anti-cancer activity of MCC is the inhibition of the proliferation of cancer cells. 16. The composition according to claim 4, wherein the anti-cancer activity of MCC is the induction of apoptosis in cancer cells. The composition according to claim 16, wherein the induction of apoptosis in cancer cells is independent of a factor selected from the group consisting of Fas, p53 / p21, and drug resistance. 18. The composition according to claim 14, wherein the induction of apoptosis in cancer cells is the induction of caspase-3 activity in cancer cells. 19. The composition according to the claim 1, wherein the anti-cancer activity of MCC is the stimulation of cytokine production by cells of the immune system. P1068 / 00MX 20. The composition according to claim 19, wherein the cells of the immune system are selected from the group consisting of macrophages and monocytes. The composition according to claim 19, wherein the cytokine is selected from the group consisting of IL-6, IL-10 and IL-12. 22. The composition according to claim 14, wherein the anti-cancer activity of MCC is the stimulation of the production of reactive oxygen species by cells of the immune system. 23. The composition according to claim 22, where the cells of the immune system are macrophages. 24. A method for treating cancer in an animal, comprising administering to the animal in need of treatment, an amount of M-DNA and a first pharmaceutically acceptable carrier effective to treat cancer in the animal. 25. The method according to claim 24, wherein the M-DNA inhibits the proliferation of cancer cells in the animal, to treat cancer in the animal. 26. The method according to claim 24, wherein the M-DNA induces apoptosis in cancer cells in an animal, to treat cancer in the animal. The method according to claim 26, wherein the induction of apoptosis in cancer cells P1068 / 00MX in an animal is dependent on a factor selected from the group consisting of Fas, p53 / p21 and drug resistance. The method according to claim 26, wherein the induction of apoptosis in cancer cells in an animal is the induction of caspase 3 activity in cancer cells in the animal, to treat cancer in the animal. 29. The method according to claim 24, wherein M-DNA stimulates the production of cytokine by the cells of the immune system in an animal, to treat the cancer of the animal. 30. The method according to claim 29, wherein the cells of the immune system are selected from the group consisting of macrophages and monocytes. The method according to claim 30, wherein the cytokine is selected from the group consisting of IL-6, IL-10 and IL-12. 32. The method according to claim 24, wherein M-DNA stimulates the production of reactive oxygen species by cells of the immune system in an animal, to treat cancer in the animal. 33. The method according to claim 32, wherein the cells of the immune system are macrophages. 34. The method according to claim 24, wherein the first pharmaceutically acceptable carrier P1068 / 00MX is selected from the group consisting of an aqueous carrier, a non-aqueous carrier and the cell wall of M. phl ei. 35. The method according to claim 34, wherein the first pharmaceutically acceptable carrier is the cell wall of M. phl ei. 36. The method according to claim 24, further comprising a second pharmaceutically acceptable carrier, wherein the second pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier and a non-aqueous carrier. 37. A method for treating cancer in an animal, comprising administering to the animal in need of such treatment, an amount of a composition comprising M-DNA and M. phlei cell wall, wherein the M-DNA is conserved and complexed on the cell wall of M. phl ei (MCC) and a pharmaceutically acceptable carrier effective to treat cancer in the animal. 38. The method according to claim 37, wherein the MCC inhibits the proliferation of cancer cells in an animal, to treat cancer in an animal. 39. The method according to claim 37, wherein the MCC induces apoptosis of cancer cells in an animal, to treat cancer in the animal. 40. The method according to claim 39, in P1068 / 00MX where the induction of apoptosis in cancer cells in an animal is independent of a factor selected from the group consisting of Fas, p53 / p21, and drug resistance. 41. The method according to claim 39, wherein the induction of apoptosis in cancer cells in an animal is the induction of caspase 3 activity in cancer cells in the animal to treat cancer in the animal. 42. The method according to claim 37, wherein MCC stimulates cytokine production by cells of the immune system in an animal to treat cancer in the animal. 43. The method according to claim 42, wherein the cells of the immune system are selected from the group consisting of macrophages and monocytes. 44. The method according to claim 43, wherein the cytokine is selected from the group consisting of IL-6, IL-10 and IL-12. 45. The method according to claim 38, wherein MCC stimulates the production of reactive oxygen species by cells of the immune system in an animal to treat cancer in the animal. 46. The method according to claim 45, wherein the cells of the immune system are macrophages. P1068 / 00MX 47. The method according to claim 37, wherein the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier and a non-aqueous carrier. P1068 / 00MX