MXPA00008142A - Composition and method for the treatment of bladder cancer - Google Patents

Abstract

The present invention relates to a composition and method for treating cancer in the urinary bladder. More particularly, the present invention relates to a composition comprising a Mycobacterium phlei (M. phlei) deoxyribonucleic acid (M-DNA)-M. phlei cell wall complex (MCC), wherein the M-DNA is preserved and complexed on the M. phlei cell wall, and a pharmaceutically acceptable carrier. The MCC composition inhibits proliferation of and induces apoptosis in the cancer cells in the urinary bladder of the animal.

Description

COMPOSITION AND METHOD FOR THE TREATMENT OF BLADDER CANCER.

Field of the Invention This application is incorporated by reference and claims the total benefit of the North American Provisional Application 60/075, 11, issued on February 18, 1998. The present invention comprises a microbacterial acid-deoxyribonucleic acid cell (BCC) complex (B). -ADN), where the B-DNA is conserved and made composed in the bacterial cell wall so that the BCC is effective in the treatment of bladder cancer. More particularly, the present invention comprises a cell wall complex (MCC) Microbacterium Phlei (M. Phlei) -ADN (M-DNA) -M. Phlei, where the M-DNA is conserved and made composed in the M cell wall. Phlei, for MCC to be effective in inhibiting proliferation, and to induce apoptosis in bladder cancer cells.

Antecedents of the Invention. Cancer is an accumulation of aberrant networks of abnormal cells, which results from an excess of proliferation, or an insufficiency of apoptosis, or a combination of the two. Apoptosis can be initiated by ligands that bind to receptors on the surface of the cell (Muzio et al. Cell 85: 817-827, 1996). Mutations in these receptors can cause a failure of apoptosis. Apoptosis can also be induced by intracellular proteins including, but not limited to, p53 / p21 regulators (Levine A. Cell 88: 323-331, 1997). The loss of functional p53 / p21 correlates with the aggressiveness of a variety of cancers (Fisher D. Cell 78: 529-542, 1994). The chemotherapeutic agents depend mainly, due to their therapeutic effect, on the induction of apoptosis in cancer cells. Drug resistance, which decreases the effectiveness of chemotherapeutic agents, directly or indirectly leads to reduction to reduced apoptosis and is generally associated with poor prognosis in a variety of cancers. Bladder cancer is particularly difficult to treat successfully (Lamm and associates Journal of Urology 153: 1444-1450, 1995). Preparations of bacterial origin used to treat bladder cancer, include but are not limited to Basillus Calmette-Guerin (BCG) (Pryor and associated British Journal of Cancer 71: 801-807, 1995) and Regressin® (Bioniche, Inc. London, Ontario, Canada), a microbacterial cell wall extract (MCWE) formulated as a mineral oil emulsion (U.S. Patent No. 4,744,984; Chin and associates Journal of Urology 156: 1 189-1 193, 1996).

BCG does not induce apoptosis directly (Sasaki and associates Urology International 59: 142-148, 1997). Instead, BCG stimulates the cells of the immune system to produce bioactive molecules, such as, but not limited to, cytokines and reactive oxygen species (Kudoh and associated British Journal of Urology 80S2: 40, 1997), which subsequently induce apoptosis and cytolysis. MCWE, which is composed mainly of peptidoglycan and glycolipid, which contains N-acetylmuramyl-L-alanyl-D-isoglutamine and mycolic acid derivatives (Chin and associated Journal of Urology 156: 1 189-1 193, 1996), is considered to stimulate the immune system by activating macrophage and monocyte-mediated reactions (Teware et al., Veterinary Parasitology 62: 223-230, 1996). The therapeutic benefits obtained using BCG and MCWE are variable and inconsistent and appear to depend on the method of preparation and method of administration and the variability in immunogenicity and stability of the resulting preparation. In addition, live BCG can cause serious side effects including, but not limited to, fever, syndrome similar to serum sickness, granulomatous infection, sepsis and even death (Lamm et al. Journal of Urology 147: 596-600, 1992). Other previous bladder cancer agents have also proven to be less than adequate for clinical applications. Many of these agents are inefficient or toxic, have significant side effects, result in the development of drug resistance or immunosensitization, and weaken the recipient. In addition, many of these agents depend on the surface receptors of ligand or p53 / p21 for their effectiveness.

Therefore, there is a need for a novel therapeutic agent that inhibits proliferation and induces apoptosis in cancer cells of the bladder. This therapeutic agent should be useful as an anti-bladder cancer cell agent and as an adjunct agent to other anti-bladder cancer cell agents. By "adjunct agent" is meant a useful agent with other bladder cancer cell agents to increase the effectiveness of the treatment. Furthermore, said therapeutic agent must be simple and relatively inexpensive in its preparation, its activity must be reproducible between preparations, its activity must remain stable over time, and its effects on the bladder cancer cells must be successful with dose regimens that they are associated with minimal toxicity.

Summary of the Invention. The present invention satisfies the above needs by providing a composition and method comprising a complex of microbacterial cell wall (BCC) - microbacterial deoxyribonucleic acid (B-DNA), wherein the B-DNA is conserved and becomes complex in the cell wall bacterial, so that BCC is effective in the treatment of cancer cells of the bladder. More particularly, the present invention satisfies the above needs by providing a cell wall complex M. Phlei (MCC) -ADN-Microbacterium Phlei (M. Phlei) (M-DNA) where the M-DNA is conserved and becomes a complex in the M. Phlei cell wall, so that the MCC is effective in the inhibition of proliferation and in inducing apoptosis in cancer cells of the bladder. MCC is simple and relatively inexpensive in its preparation, its activity is to reproduce within preparations, remains therapeutically stable over time, and is effective in dose regimens that are associated with minimal toxicity even in repeated administration. To prepare MCC, M. Phlei are developed in a liquid medium and are harvested. M. Phlei are interrupted, and the interrupted solid components of M. Phlei are collected by centrifugal sedimentation. The solid components are deproteinized, delipidated, and washed. DNase-free reagents are used to minimize the degradation of M-DNA during preparation. MCC, in combination with a pharmaceutically acceptable carrier, is administered in an animal, including a human, in a dose sufficient to prevent, treat and eliminate cancer cells within the bladder. The unexpected and surprising ability of MCC to inhibit proliferation and induce apoptosis in cancer cells of the bladder including, but not limited to abnormal p52 / 21 and drug-resistant cells directed to a need not met in the medical arts and provides a benefit important.

MCC is also effective as an adjunct agent to improve the effectiveness of other anti-cancer agents. These include, but are not limited to, medications; immunostimulants; antigens; antibodies; vaccines; radiation; chemotherapeutic agents; nucleic acid agents; biologically constructed agents, chemically synthesized agents; agents that focus on molecules that kill cells by activation or inactivation; agents that inhibit proliferation and induce apoptosis in response cells. Therefore, it is an object of the present invention to provide an effective composition and method for preventing bladder cancer. Another object of the present invention is to provide an effective composition and method for treating bladder cancer. Another object of the present invention is to provide an effective composition and method for eliminating bladder cancer. Another object of the present invention is to provide an effective composition and method for eliminating bladder cancer. Another object of the present invention is to provide a composition and method that inhibits the proliferation of cancer cells of the bladder. Another object of the present invention is to provide a composition and method that induces apoptosis in cancer cells of the bladder.

Another object of the present invention is to provide a composition and method that stimulates the response cells of the immune system, within the bladder to produce bioactive molecules. Another object of the present invention is to provide a composition and method that is effective in inhibiting angiogenesis in bladder cancer. Another object of the present invention is to provide a composition and method that is effective as an adjunct agent for other anti-bladder cancer therapies. Another object of the present invention is to provide a composition of particle size and optimal formulation for recognition by the response cells. Another object of the present invention is to provide a composition of optimum particle size and formulation to be taken up by the response cells. Another object 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 that remains stable over time. Another object 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.

These and other objects, features and advantages of the present invention will be appreciated after a review of the following detailed description of the described embodiment and the appended claims.

Brief Description of the Figures. Figure 1 . Inhibition of proliferation of HT-1376, HT-1 197, B-16 F1, THP-1, NATURAL 264.7, Jurkat, cancer cells HL-60 and HL-60 MX-1 by MCC. The results are the meaning of ± SD of 3 independent experiments. Figure 2. Inhibition of proliferation of HT-1376, HT-1 197, B-16F1, THP-1, NATURAL 264.7, Jurkat, bladder cancer cells HL-60 and HL-60MX-1 by M. Phlei-DNA (2A), MCC-DNA (2B), calf thymus-DNA (2 A &2B) and herring-DNA sperm (2A &2B). The results are the ± SD meaning of 3 independent experiments. Figure 3. Inhibition of proliferation of human bladder cancer cells HT-1 197 (3A) and HT-1376 (3B) by MCC and LPS. The results are the ± SD meaning of 3 independent experiments. Figure 4. Induction of DNA fragmentation in human bladder cancer cells HT-1 197 (4A) and HT-1376 (4B) by MCC treated by DNase and untreated and by hIL-12.

The results shown are for 1 of 3 experiments, each of which provided similar results.

Figure 5. NuMA release of human bladder cancer cells HT-1 197 and HT-1376 with increased concentrations of MCC. The results are the meaning of ± SD of 3 independent experiments. Figure 6. NuMA release of human bladder cancer cells HT-1 197 (6A) and HT-1376 (6B) with 1 μg / ml MCC or with 100 μg / ml MCC for 48 hours. The results are the meaning of ± SD of 3 independent experiments. Figure 7. NuMA release of human bladder HT-1376 cancer cells with 1 μg / ml MCC-DNA treated by DNase I and untreated and MCC. The results are the meaning of ± SD of 3 independent experiments. Figure 8. Percentage of LDH release through human bladder cancer cells HT-1 197 and HT-1376 as an indicator of cytotoxicity of MCC. The results are the meaning of ± SD of 3 independent experiments. Figure 9. Stability of MCC during 6 months of storage.

Detailed Description of the Invention. The present invention comprises a complex bacterial cell wall (BCC) - bacterial deoxyribonucleic acid (B-DNA), where the B-DNA is conserved and complexed in the bacterial cell wall, so that the complex is effective to prevent, treat and eliminate cancer cells in the urinary bladder of an animal, including a human. Many bacterial species can be used to practice the present invention including, but not limited to Coryneform species, Corynebacterium species, Rhodococcus species, Eubacterium species, Bordetella species, Escherichia species, Listeria species, Nocardia species and Mycobacterium species. Preferably a Mycobacterium species is used including, but not limited to M. smegmatis, M. fortuitum, M. kansaasii, M. tuberculosis, M. bovis, M. vacciae, M. avium and M.Phlei. more preferably M. Phlei of Mycobacterium species is used. Therefore, the present invention more particularly comprises a cell wall complex M. Phlei (MCC) - deoxyribonucleic acid, M. Phlei (M-DNA), wherein M-DNA is conserved and becomes complex in the cell wall M . Phlei, for MCC to be effective in preventing, treating and eliminating cancer cells in the urinary bladder. In MCC, the amount of M-DNA is enriched relative to the amount of M-DNA found in an intact M .Phlei cell. In addition, M-DNA is conserved and becomes complex in the M .Phlei cell wall to make it more accessible to the response cells, than it is M-DNA within an M cell. Phlei intact. Although not wishing to be linked by the following hypothesis, it is believed that M-DNA, in the form of short oligonucleotides, and its physical association with the M. Phlei cell wall, both will contribute to the optimal expression of the therapeutic activity of MCC. . Methods for enhancing the therapeutic activity of MCC include, but are not limited to, DNA sequences or confirmations of chemical supplementation or biotechnological amplification stimulation, derived from the same or different bacterial species and from making M-DNA or MCC complexes for transporters. natural or synthetic In addition, MCC can be administered before, at the same time or after another anti-bladder cancer agent to increase the therapeutic effectiveness. The compositions comprising MCC and its pharmaceutically acceptable carrier are prepared by uniform and intimate conduction in the association of MCC with liquid carriers, with solid carriers, or with both. Liquid carriers include, but are not limited to, aqueous carriers, non-aqueous carriers, or both. Solid carriers include, but are not limited to, biological transporters, chemical transporters and biotechnologically constructed transporters. Among its pharmaceutically acceptable carriers, MCC can be supplied in aqueous suspension, oil emulsion, water in oil emulsion, water in oil in water emulsion, liposomes, microparticles, site specific emulsions, long-stay emulsions, sticky emulsions, microemulsions nanoemulsions, microspheres, nanospheres, nanoparticles, microbombs, and with several natural or synthetic polymers that allow the sustained release of MCC. In addition, MCC can be used with any with all or with any combination of excipients, regardless of the carrier used to present MCC to the response cells. These include, but are not limited to anti-oxidants, regulators, and bacteriostats, and may include suspending agents and thick agents. Preferably, MCC is supplied in an aqueous suspension. MCC is suspended in a pharmaceutically acceptable carrier such as but not limited to water free of DNase, saline or phosphate regulated salt (PBS) and is emulsified by sonication. Optionally, the amulsified mixture is homogenized by microfluidization. For example, MCC used is suspended in DNase-free water and sonicated in a 20% yield for 5 minutes (Model W-385 Sonicator, Heat Systems-Ultrasonics Inc). The sonicated composition is homogenized by microfluidization at 15,000-30,000 psi for a flow path (Model M-1 10Y; Microfluidics, Newton, MA). The mixture is either aseptically processed or finely sterilized. For administration in an aqueous carrier, MCC is emulsified with a mineral oil or with neutral oil such as but not limited to a diglyceride, tri-glyceride, a phospholipid, or lipid, an oil and mixture thereof, wherein the oil contains a mixture of unsaturated polyunsaturated and saturated polyunsaturated fatty acids. Examples include but are not limited to soybean oil, canola oil, palm oil, olive oil and miglyol, wherein the number of fatty acid carbons is between 12 and 22 and where the fatty acids can be saturated or unsaturated. Optionally, the charged lipid or phospholipid can be suspended in the neutral oil. For example, DNase-free phosphatidylcholine is added to DNase-free soybean triglyceride oil in a ratio of 1 gram of phospholipid to 20 ml of triglyceride and is dissolved by light heating at a temperature of 50 ° -60 ° C. add a few grams of MCC to a dry autoclave container and the phospholipid-triglyceride solution is added at a concentration of 20 ml per 1 gram of MCC. The suspension is incubated for 60 minutes at a temperature of 20 ° C and then mixed with PBS free of DNase in the proportion of 20 ml of MCC suspension per liter of PBS. The mixture is emulsified by sonication at a 20% yield for 5 minutes (Model W-385 Sonicator, Heat Systems-Ultrasonics Inc.). Optionally, the emulsified MCC mixture is homogenized by microfluidization at 15,000-30,000 psi for a flow step (Model M-1 10Y; Microfluidics). The MCC emulsion is transferred to a covered autoclave bottle for storage at a temperature of 4 ° C. The size of the MCC particles should be optimal for recognition and understanding by the response cells. Preferably the average diameter of the MCC particles is between about 10 and about 10,000 nm, more preferably between about 100 and about 1000 nm and most preferably between about 250 and about 600 nm. The M-DNA content of MCC is preferably about 0.001 and about 90 mg / 100 mg of MCC, more preferably between about 0.01 and about 40 mg / 100 mg of MCC, most preferably between about 0.1 and about 30 mg / 100. mg of MCC. It is also preferred that the protein content be less than about 2 mg / 100 mg of MCC, and that the fatty acid content be less than about 2 mg / 100 mg of MCC. Unexpectedly, it was found that at least about 3.6% of the dry weight of MCC is extractable M-DNA. MCC and / or M-DNA are administered in an amount effective to induce a therapeutic response in bladder response cells. The dose of MCC and / or M-DNA administered will depend on the condition being treated, the particular formulation and other clinical factors such as weight and condition of the recipient and route of administration. Preferably, the amount of MCC and / or M-DNA administered, is from about 0.00001 to about 100 mg / kg per dose, more preferably from about 0.0001 to about 50 mg / kg per dose, and most preferably from about 0.001 to about 10 mg / kg per dose.

Routes of administration include but are not limited to oral, intra-venous, intra-lesion, intra-tumor, and intra-bladder.

Preferably, MCC and / or M-DNA are administered by instillation in the urinary bladder by, but not limited to, a urinary tract catheter. Other methods of instilling MCC and / or M-DNA in the urinary bladder are known to those skilled in the art. Depending on the route of administration, the volume per dose is preferably from about 0.001 to about 100 ml, more preferably from about 0.01 to about 70 ml, and most preferably from about 0.1 to about 40 ml. MCC and / or M-DNA can be administered in a single dose treatment or in multiple dose treatment or in a program or during an apriate period of time for the treatment of the disease, the condition of the recipient and the route of administration. When administered to a urinary bladder, depending on the volume administered, the dose should remain in the bladder preferably from about 1 minute to about 8 hours, more preferably from about 15 minutes to about 4 hours, and most preferably from about 30 minutes to aximately 2 hours. The following examples will serve to further illustrate the present invention and at the same time however constitute any limitation thereof. But on the contrary, it will be clearly understood that the resource may have several or other modalities, modifications and equivalents thereof which, after reading the description of the present invention, may be suggested by themselves to those skilled in the art without departing of the spirit of the present invention and / or of the scope of the appended claims.

EXAMPLE 1 Preparation of MCC from Microbacterium Phlei and purification of M-DNA from MCC and from M. Phlei. MCC was prepared from Microbacterium Phlei (M: Phlei) (strain 10), M-DNA was purified from MCC (M-DNA) and M-DNA was purified from M. Phlei (M. Phlei-DNA) as described in application number PCT / CA981 / 00744. All reagents were selected to improve DNA conservation. Unless otherwise stated, MCC, MCC-DNA and M. Phlei-DNA were resuspended in DNase-free water or in a pharmaceutically acceptable DNase-free regulator and emulsified by sonication. MCC, MCC-DNA and M, Phlei-DNA did not contain endotoxins as determined using a QCL-1000 Limisa amebocyte lysate kit (BioWhittaker, Walkersville, MD).

ELEMENT 2 Preparation of bacterial-DNA-bacterial cell wall complex and bacterial DNA from another bacterial species. The bacterial cell-DNA cell complex was prepared (BCC) and bacterial-DNA (B-DNA), from M. smegmatis, M. fortuitous, Nocardia rubra, Nocardia asteroides, Cornybacterium parvum, M. kansaasii, M tuberculosis and M. bovis, such as the example 1 .

EXAMPLE 3 Treatment of DNase MCC-DNA and MCC were digested, each containing one μg of M-DNA, and Regressin® (US Patent No. 4,744,984), with an international unit (IU) of DNase I free from RNase (Life Technologies ) for one hour at a temperature of 25 ° C in 20mM Tris HCl, pH 8.4, 2mM MgCl2 and 50mM KCL. DNase I was deactivated by the addition of EDTA for a final concentration of 2.5 mM and heating it for 10 minutes at a temperature of 65 ° C.

EXAMPLE 4 Cells and reagents Human bladder cancer cells HT-1 197 and HT-1376 ????? were obtained from the Type Culture Collection American (ATCC, Rockville, MD) and were cultured in MEM supplemented with amino acids and non-essential vitamins containing 10% FCS (MEM-FCS) (Gibco Life Science). HT-1 Cells 197 are 4 cells grade anaplastic transition bladder carcinoma, developed from a human male. HT-1 197 cells are sensitive to chemotherapeutic agents such as but not limited to doxorubicin. HT-1376 cells are 3 anaplastic transition carcinoma grade cells, developed from a human female. HT-1376 cells are abnormal p53 / p21 and are resistant to chemotherapeutic agents such as, but not limited to, cisplatin and mitomycin. Cells B-16F1, THP-1, NATURAL 264.7, Jurkat, # L-60 and HL-60MX-1, were obtained from ATCC and were cultured in the medium recommended by the ATCC. Unless otherwise stated, cells were seeded in 6 flat-bottom well tissue culture plates at cell concentrations between 3 X 10 5 and 10 6 / ml and were maintained at a temperature of 37 ° C in an atmosphere from C02 to 5%. Calf-DNA thymus, herring-DNA sperm and Escherichia coli lipopolysaccharides (LPS) were obtained from Sigma Chemical Co. (St Louis, MO). I L-12 of recombinant human (hIL-12) was obtained, coming from R & amp; amp;; D Systems (Minneapolis, MN).

EXAMPLE 5 Inhibition of cell proliferation.

Cell proliferation was determined using diphenyltetrazoline-dimethylthiasol bromide (MTT) reduction (Mosman et al., Journal of Immunological Methods 65: 55-63, 1983). HT-1376, HT-1 197, B-16F1, THP-1, NATU RAL 264.7, Jurkat, HL-60 and HL-60MX-1 were incubated for 24 hours, with from 0 to 10 μg / ml of MCC, M. Phlei-DNA, MCC-DNA, herring-DNA sperm and calf thymus-DNA. MCC (Figure 1), M. Phlei-DNA (Figure 2A) and MCC-DNA (Figure 2B), inhibited proliferation at a dose that depends on the form of administration. The herring-DNA sperm (Figures 2A &2B) and the calf thymus DNA (Figures 2A &2B), did not inhibit proliferation. The HT-1 197 cells (Figure 3A) and HT-1376 (Figure 3B) were also incubated for 24 hours from 0 to 100 μg / ml of MCC and LPS. MCC inhibited proliferation in a dose that depends on the form of administration, where LPS does not inhibit proliferation (Figures 3A &3B). MCC and M-DNA inhibit the proliferation of HT-1 197 and of abnormal p53 / p21, bladder cancer cells HT-1376 resistant to the drug. In addition, inhibition of cell proliferation is not common for all DNAs and (herring sperm-DNA and calf thymus-DNA), and does not result from non-specific immunostimulation (LPS). EXAMPLE 6 Induction of apoptosis as indicated by DNA fragmentation.

Fragmentation of cellular DNA into size fragments of nucleosomes is characteristic of cells undergoing apoptosis (Newell et al., Nature 357: 286-289, 1990). To evaluate DNA fragmentation, non-adherent cells were collected by centrifugation at 200 grams for 10 minutes. The beads of nonadherent cells and the remaining adherent cells were lysed with 0.5 ml of hypotonic lysis buffer (10 mM Tris buffer, 1 mM EDTA, 0.2% Triton X-100, pH 7.5). The lysates were centrifuged at 13,000 grams for 10 minutes and the floating ones, containing fragmented DNA, were precipitated overnight at a temperature of -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. Bladder cancer cells HT-1 197 and HT-1376 were incubated for 48 hours with μg / ml MCC or with ng / ml hl L-12 (Figure 4). MCC induced significant DNA fragmentation in HT-1 cells 197 (Figure 4A, band 2) and HT-1376 (Figure 4B, band 2), but not in adherent cells HT-1 197 (Figure 4A, band 3) and HT -1376 (Figure 4B, band 3). PBS (Figures 4A &4B, band 5), hIL-12 (Figures 4A &4B, band 4) and MCC treated by DNase I (Figure 4A &4B, band 7), do not induce DNA fragmentation in cells HT-1 197 or HT-1376 non-adherent. The untreated cells (Figures 4A &4B, band 1) showed no DNA fragmentation. A 123-bp DNA step (Gibco Life Science) was used to determine the molecular weight of the nucleosome size DNA fragments (Figures 4A &4B, L band). MCC induces apoptosis in bladder cancer cells HT-1 197 and HT-1376, where MCC treated by DNase I does not induce apoptosis in these cells. This suggests that the structure of the intact oligonucleotides of M-DNA is necessary for the induction of apoptosis. hl L-12 does not induce apoptosis in cells in HT-1 197 or in HT-1376 cells.

EXAMPLE 7 Induction of apoptosis as indicated by solubilization of the mitotic protein nuclear apparatus (NuMA). The surprising morphological changes in the nuclei of the cell caused by the solubilization and release of NuMA are characteristic of apoptosis. The NuMA release of cultured cells was determined in units / ml (U / ml), using a commercial ELISA (Calbiochem, Cambridge, MA) (Miller et al. Biotechniques 15: 1042-1047, 1993). Bladder cancer cells HT-1 197 and HT-1376, incubated for 48 hours from 0 to 100 μg / ml of MCC, released NuMA in a dose-related manner (Figure 5). HT-1 cells 197 (Figure 6A) and HT-1376 (Figure 6B), incubated with 1 μg / ml or with 100 μg / ml MCC for 48 hours, showed an improved release of NuMA for 24 hours. The HT-1 197 cells incubated for 48 hours with MCC and with MCC-DNA, showed a significantly higher NuMA release with MCC, than with MCC-DNA (Figure 7). The DNase I treatment of MCC and MCC-DNA significantly reduced NuMA release (Figure 7). The MCC induction of apoptosis in bladder cancer cells HT-1 197 and HT-1376 are both dose and time dependent. Although not wishing to be bound by the following hypotheses, it is believed that both the oligonucleotide structure of M-DNA and the presentation of M-DNA in the M. Phlei cell wall is important for the induction of optimal apoptosis.

EXAMPLE 8 MCC cytotoxicity Cytotoxicity of the cell is characterized by loss of plasma membrane integrity and release of cytoplasmic enzymes such as but not limited to LDH (Phillips et al., Vaccine 14: 898-904). To evaluate the cytotoxicity of MCC, human bladder cancer cells HT-1 197 and HT-1376 were incubated for 48 hours from 0 to 100 μg / ml MCC or with lysis buffer (10mM Tris, 1mM EDTA, 0.2% Triton X-100, pH 7.5) as a control for total LDH release (Filion and associates Biochim Biophys Acta 1329: 345-356, 1997). LDH was determined by a commercial assay (Sigma-Aldrich). The fact that MCC was not cytotoxic (Figure 8) demonstrates that MCC acts directly on bladder cancer cells HT-1 197 and HT-1376 to inhibit proliferation and induce apoptosis.

EXAMPLE 9 MCC and MCC-DNA treatment of human bladder cancer in nu / nu mice Human bladder cancer (HT-1 197) is established as an ectopic solid tumor in the subcutaneous tissues of immune-deficient shaved nude mice (mice nu / nu) and the mice are divided into 5 groups. Group 1 receives a single vehicle. Group 2 receives MCC. Group 3 receives MCC treated by DNase I. Group 4 receives MCC-DNA. Group 5 receives MCC-DNA treated by DNase I. The cancer mass is measured before treatment and on a weekly basis for 4 weeks of treatment. Mice from group 2 and mice from group 4 show regression of the cancer mass. The mice of group 1, 3 and 5 do not show regression of the cancer mass.

EXAMPLE 10 MCC treatment of human bladder cancer. Ten patients with stage 3 orthotic bladder cancer are divided into 2 groups. Group 1 receives intravesicular instillation of MCC on a weekly basis, for 8 weeks. Group 2 receives standard chemotherapy treatment. Patients in group 1 show a significant regression of bladder cancer and do not report side effects of weakening. Patients in group 2 show minimal regression of bladder cancer and report side effects of significant weakening.

EXAMPLE 1 1 MCC stability MCC was stored at 1 mg / ml, in the form of a sterile suspension at 0.85% w / v NaCl in the dark at a temperature of 4 ° C or for 6 months. The average particle diameter was calculated using photon correlation spectroscopy (N4 Plus, Coulter Electronics Inc.). The MCC suspension was diluted with 0.85% w / v of NaCl for a range of counting counts between 5 x 10 (4) and 10 (6) / sec. The diameter of the average particle was calculated in a size distribution processor (SDP) using the following conditions: fluid refractive index 1.33, temperature 208 C, viscosity 0.93 centipoise, measurement angle 90. O8, time of the shows 10.5 μs, and run time of the sample 100 seconds. The power, 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, frequency range 500 HZ, temperature 208 C, fluid refractive index 1 .33, viscosity 0.93 centipoise, dielectric constant 78.3, conductivity 16.7 ms / cm, in time of 2.5 seconds, out of time 0.5 seconds, and run time of the sample 60 seconds.

As shown in Figure 9, the MCC load and the MCC diameter remained relatively unchanged for 6 months of storage. In addition, MCC stimulation of IL-12 production and induction of apoptosis in THP-1 monocytes remained unchanged for 6 months of storage. It should be clear, of course, that the foregoing relates only to a preferred embodiment of the present invention and that numerous modifications or alterations thereof may be made, without departing from the spirit and scope of the present invention, as set forth in Claims attached.

Claims (16)

R E I V I N D I C A C I O N S 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 that includes: a. A microbacterial deoxyribonucleic acid (B-DNA); b. A deproteinized and delipidated microbacterial cell wall, wherein B-DNA is conserved and complexed in the microbacterial cell wall (BCC); and, c. A pharmaceutically acceptable carrier, wherein the BCC is effective in inducing a response in the cancer cells of the urinary bladder of an animal.

2. - The composition as described in the Claim 1, further characterized because the microbacterial DNA is M.Phlei DNA (M-DNA).

3. - The composition as described in the Claim 1, characterized further because the microbacterial cell wall is M. Phlei cell wall.

4. - The composition as described in the Claim 1, characterized in that the response is selected from the group consisting of inhibition of cancer cell proliferation and induction of apoptosis in cancer cells.

5. - The composition as described in the Claim 4, characterized further because the response is inhibition of proliferation of cancer cells.

6. - The composition as described in the Claim 4, characterized further because the response is induction of apoptosis in cancer cells.

The composition as described in claim 1, further characterized in that the pharmaceutically acceptable carrier is selected from the group consisting of an aqueous carrier and a non-aqueous carrier.

8. A method for inducing a response in cancer cells in the urinary bladder of an animal, comprising administering to the animal a composition comprising: a. A mycobacterial deoxyribonucleic acid (B-DNA); b. A deproteinized and delipidated mycobacterial cell wall, wherein B-DNA is conserved and becomes complex in the mycobacterial cell wall C BCC; and, c. A pharmaceutically acceptable carrier, in an amount effective to induce a response in cancer cells in the urinary bladder of the animal, which has cancer cells in the urinary bladder.

9. - The method as described in Claim 8, further characterized in that B-DNA is M. Phlei-DNA (M-DNA).

10. The method as described in claim 8, further characterized in that the mycobacterial cell wall is M .

Phlel cell wall. 1 - The method as described in claim 8, further characterized in that the response is selected from the group consisting of inhibition of cancer cell proliferation and induction of apoptosis in cancer cells.

12. - The method as described in Claim 1 1, further characterized in that the response is inhibition of proliferation of cancer cells.

13. The method as described in Claim 1 1, further characterized in that the response is Induction of apoptosis in cancer cells.

14. The method as described in Claim 8, further characterized in that the * vehicle is pharmaceutically acceptable selected from the group consisting of an aqueous carrier and a non-aqueous carrier.

15. - A composition, comprising: a. MCC, where M Phlei-DNA is conserved and becomes complex in the M .Phlei cell wall; and, b. A pharmaceutically acceptable carrier.

16. A method for treating cancer cells in the urinary bladder of an animal, which has cancer cells in the urinary bladder, comprising administering to an animal in need of said treatment, a composition comprising: to. MCC, where M. Phlei-DNA is conserved and becomes complex in the M cell wall. Phlei and b. a pharmaceutically acceptable carrier in an amount effective to treat cancer cells in the urinary bladder of the animal, which has cancer cells in the urinary urinary bladder. SUMMARY The present invention relates to the composition and method for the treatment of cancer in the urinary bladder. More particularly, the present invention relates to a composition comprising a cell wall complex M. Phlei (MCC) -deoxyribonucleic acid (M-DNA) Micobacterium phlei (M .phlei), wherein the M-DNA is conserved and makes complex in the M. phlei cell wall, and a pharmaceutically acceptable carrier. The MCC composition inhibits proliferation and induces apoptosis in the cancer cells found in the animal's urinary bladder.