FIELD OF THE INVENTION The present invention relates to the pharmaceutical and oncology field and it is mainly focused on the treatment of bladder cancer. Specifically, the invention relates to an oil-in-water microemulsion (O/W) preferably, olive oil-in-water microemulsion (OO/W) and compositions comprising the same, which comprises a bacterium of Mycobacterium genus having an immunopotentiating activity as an active ingredient, as well as olive oil with application in bladder cancer treatment.

STATE OF THE ART

Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the usual treatment for the superficial carcinoma of the bladder after transurethral resection (TUR). It is known that BCG induces two different antitumor effects. On the one hand, BCG shows cytotoxic activity against bladder cancer cells. On the other hand, BCG triggers both the production of cytokines and the expression of cell surface activation markers on immune cells, leading to the activation of immune cells, which in turn kills bladder cancer cells.

Although the benefits of the BCG immunotherapy are widely known: prevents recurrence, tumor progression and prolong survival (van Rhijn et al., 2009), the drawbacks are not underestimable. Toxicity problems are associated with its use. Most of the patients experience some irritable bladder symptoms (cystitis) during BCG therapy, roughly 40% develop hematuria and 30% experience flu-like symptoms including fever, malaise and nausea or vomiting. Moreover, around 5% of the patients suffer considered serious side effects, including sepsis due to BCG infection (Lamm., 2000). Because of these disadvantages, it is necessary to find new alternatives to the current therapy.

Recently, it has been known that a non-pathogenic mycobacterium, Mycobacterium brumae, which compared with BCG has increased ability on inhibiting the proliferation of grade 1 and grade 2 bladder cancer cells in vitro, and similar effect to BCG on inhibiting grade 3 bladder cells growth. Moreover, M. brumae triggers immune response activation, crucial for an antitumor action. In this sense, M. brt/mae-activated peripheral cells show antitumor activity against bladder cancer cells. In addition because of the rapid growth of M. brumae in culture medium it is easier to prepare preparations produced by using M. brumae.

Besides these findings, it is known that mycobacteria have a highly hydrophobic cell wall which makes them form clumps. The high content of lipids in the mycobacteria cell wall (over 60% of its weight) provides an elevated hydrophobic character. It is then understandable that aggregation could interfere in the interaction between host cells and mycobacteria, and consequently could influence in the efficacy of mycobacteria in inhibiting tumor growth. Moreover, this tendency for clumping makes difficult to get stable and homogeneous solutions of mycobacteria.

Different oil-in-water (C7W) or water-in-oil (W/O) formulations have been assayed in order to avoid aggregation, and to enhance induced immune response. In fact, the well-known Complete Freund Adjuvant (CFA) is a heat-killed preparation of Mycobacterium tuberculosis H37Ra made using mineral oil (Wright., 1989). CFA is usually mixed with water soluble antigens to make water-in-oil emulsions, which enhance and prolonged the immune response against the antigen.

Specifically, researchers who proposed cell wall extracts (CWS) o heat-killed mycobacteria for treating bladder cancer developed formulations using oils to create microemulsions of mycobacteria. Water-in-oil strategy has been mainly taken by researchers that studied hydrophilic antitumor chemical compounds (Wu, 2001 ; Hwang, 2009). Oil-in-water strategies have been mainly followed for hydrophobic compounds. Moreover, the development of O/W preparations to incorporate antitumor agents such as mitomycin has recently carried out in order to enhance its antitumor effect. Microemulsion of mitomycin provides longer stability to the drug (longer biological half-life), and reduce its severe side effects after systemic administration (Kotmakchiev, 2012).

As mentioned previously, the O/W emulsions comprising bacteria or CWS or combinations thereof, although have gained a good reputation in utility, they are extremely unstable to generate insoluble aggregates. Such a tendency is especially pronounced in hydrophilic environments. In general, in all the O/W emulsions or formulations, the oils used were mineral oil, squalene and soybean oil (Table 1 ). Only Regiardo Z., studied the administration of extracts from killed mycobacteria using olive oil, but in that case without making any formulation, the mycobacteria extract was directly resuspended on olive oil (Reggiardo, Z., 1980).

Table 1. Review of W/O and O/W formulations for treating cancer.

Mycobacteria/drug Microemulsion Oil Treatment Reference

BCG cell wall skeleton O W mineral oil hepatoma (Meyer et al.,1974) M. phlei cell wall bladder

(Morales et al.,

O/W mineral oil cancer

extract plus DNA 2001 )

M. phlei cell wall O/W (Gray et al., 1975) Killed BCG, M. phlei fibrosarcoma

(Yarkoni & Rapp., O/W squalene

and M. smegmatis 1980)

BCG cell wall hepatoma. (Akazawa et al.,

O/W mineral oil

2004)

Melanoma (Murata., 2008,

BCG cell wall O/W squalene Uenishi et al.,

2009)

Cysplatin W/O soybean oil lung (Hwang et al., carcinoma 2009)

Plasmid W/O olive oil bladder

(Wu et al., 2001 ) cancer

Mitomycin-C (Kotmakchiev et

O/W soybean oil topical

delivery al., 2012)

Cord factor antigen lung cancer

peanut oil (Leclerc et al.,

O/W

(M. smegmatis) 1976)

BCG cell wall extract non emulsion olive oil L1210

(Reggiardo., 1978) leukemia

Therefore, there is a need in the art to provide a stable and homogeneous oil-in- water microemulsion, which does not generate insoluble aggregates, comprising Mycobacterium species, preferably non-pathogenic Mycobacterium species, such as, M. brumae. In this sense, the present invention gives a new step forward, toward the solution of the above problems, developing an olive oil-in-water (OO/W) microemulsion comprising Mycobacterium species, preferably non-pathogenic bacteria, M. brumae, exhibiting high stability and homogeneity and which have also a therapeutic effect in the treatment of bladder cancer. The olive oil-in-water microemulsion discloses in the present invention remain stable and ensures also the mycobacteria viability in the microemulsion. DESCRIPTION OF THE INVENTION

Brief description of the invention The present invention mainly refers to a stable and homogeneous oil-in-water microemulsion comprising Mycobacterium species, preferably, BCG or M. brumae, more preferably M. brumae which is environmental and non-pathogenic mycobacteria. The olive oil-in-water microemulsion discloses in the present invention inhibits proliferation of bladder cancer cells, and stimulates responsive cells of the immune system to produce bioactive molecules, such as cytokines. This microemulsion should be useful as an anti-bladder cancer agent and as an adjunct to other anti- bladder cancer agents, such as mitomycin C.

The present invention therefore provides, according to a first aspect, a microemulsion of a Mycobacterium species comprising:

a) An aqueous phase,

b) An oily phase comprising at least a hydrophilic surfactant and olive oil.

The surfactant is generally present in amounts sufficient to increase the kinetic stability of the microemulsion by stabilizing the interface between the hydrophobic and hydrophilic components of the microemulsion.

In another preferred embodiment, the olive oil-in-water microemulsion disclosed in the present invention is used as medicament, preferably in the treatment of bladder cancer and more preferably in the treatment of non-invasive superficial bladder cancer, more preferably in Grade 1 and Grade 2.

In another preferred embodiment, the olive oil-in-water microemulsion disclosed in the present invention stimulates the responsive cells of the immune system to produce bioactive molecules, preferably cytokines, in a statistically significant increase compared to the emulsion disclosed in the state of the art, such as, mineral oil emulsion comprising mycobacterium species. The microemulsion discloses in the present invention triggers a statistically significant increase in cytokines, IL-6, IL-8 and KC, compared to basal levels produced by non-infected cells or cells infected with the microemulsion without bacteria. Furthermore, the presence of olive oil- formulated bacteria favors the immune response against both, BCG and M brumae. In all cases, olive oil mycobacteria formulations trigger the highest levels of cytokine production.

The microemulsion of the present invention is also effective as an adjunct to enhance the effectiveness of other anti-cancer agents including, but not limited to, anti-bladder cancer agents. Such agents include, but are not limited to, drugs, immunostimulants, antigens, antibodies, vaccines, radiation and chemotherapeutic, genetic, biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation and that inhibit proliferation of and induce apoptosis in responsive cells.

The second aspect of the present invention refers to a composition, preferably a pharmaceutical composition, comprising Mycobacterium species, preferably, BCG or M. brumae, more preferably M. brumae in an olive oil-in water microemulsion according to the present invention and pharmaceutically-acceptable carrier or excipient.

In a preferred embodiment of the invention, the pharmaceutical composition disclosed herein further comprises at least one therapeutic agent to enhance the effectiveness of the composition. In a more preferred embodiment, the therapeutic agent is a cytostatic compound, preferably, mitomycin C.

The third aspect of the present invention discloses a composition, preferably a pharmaceutical composition comprising Mycobacterium species, preferably, BCG or M. brumae, more preferably M. brumae, in an olive oil-in water microemulsion according to the present invention, for use as medicament. Moreover, the present invention discloses said pharmaceutical composition for treatment of bladder cancer. In a preferred embodiment the bladder cancer is non-invasive superficial bladder cancer. Another aspect of the present invention refers to a method for the treatment of a patient or subject suffering a bladder cancer comprising the administration of a pharmaceutically effective amount of the pharmaceutical composition comprising Mycobacterium species, preferably, BCG or M. brumae, more preferably M. brumae in an olive oil-in water microemulsion according to the present invention, to the subject or patient. In a preferred embodiment the bladder cancer is non-invasive superficial bladder cancer.

The term "subject", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a male or female human of any age or race. In the context of the present invention, the subject is a subject suffering from cancer or previously diagnosed with cancer, preferably, bladder cancer and most preferably non-invasive superficial bladder cancer. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation.

The treatment methods of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects.

Another aspect of the present invention refers to a process for the preparation of a microemulsion of a Mycobacterium according to the present invention comprising the step of:

i. mixing the Mycobacterium species with hydrophilic surfactant and olive oil ii. emulsifying the oily phase by adding aqueous solution, mixing and sonicating iii. adding aqueous solution until obtain the final volume of the microemulsion. Typically, the ingredients are mixed together to form a mixture which is then subjected to a sonication process to form the microemulsion. The emulsification step is continued for the time necessary to produce an oil-in-water microemulsion. Moreover, in a preferred embodiment, the process is carried out at RT temperature, preferably at a temperature of 22 °C or less. In a preferred embodiment, the olive oil-in-water microemulsion is stored at a temperature of 4-30 °C after preparation, more preferably at a temperature of 22 °C.

The olive oil-in water microemulsions and pharmaceutical formulation thereof, according to the present invention, allow maintaining mycobacteria viable, disaggregate mycobacteria clumps, inhibit tumor cell growth similarly to mycobacteria diluted in culture media, and more efficiently than mycobacteria on mineral oil microemulsion and also trigger higher production of cytokines in bladder tumor cell cultures than mycobacteria diluted in the rest of formulations.

M. brumae and BCG microemulsionated in olive oil is a suitable alternative to the current BCG treatment for non-invasive bladder cancer.

The terms "comprise" or "comprising" are meant not to be limiting to any subsequently stated elements but rather to encompass non-specified elements of major or minor functional importance. In other words the listed steps, elements or options need not be exhaustive. Whenever the words "including" or "having" are used, these terms are meant to be equivalent to "comprise" or "comprising" as defined above. Moreover, the terms "comprise" or "comprising" are intended to include embodiments encompassed by the terms "consisting essentially of" and "consisting of". Similarly, the term "consisting essentially of" is intended to include embodiments encompassed by the term "consisting of".

Description of the figures

Figure 1. M. brumae cfus from microemulsions containing different oils.

Figure 1 shows cfu/ml obtained after platting on Middlebrook 7H10 medium a 4mg of microemulsionated M. brumae, using the same formulation but made by using different oils. OO: olive oil; SO: soybean oil; SE: squalene; MO: mineral oil; C: control solution (complete culture medium). ^* p<0.05, ^** p<0.001

Figure 2. Percentage of live and death M. brumae measured by confocal microscopy after staining with fluorocroms.

Figure 2 shows the percentage of live (gray scale part) and death (white part) M. brumae in each formulation made with different oils. OO: olive oil; SO: soybean oil; SE: squalene; MO: mineral oil; C: control solution (complete culture medium); 00 hk: heat-killed M. brumae microemulsionated in olive oil as negative control.

Figure 3. Confocal capture images and ImageJ binary mask from M. brumae microemulsionated using different oils.

Figure 3A shows images from live (green) and death (red) M. brumae in each formulation made with different oils. 00: olive oil; SO: soybean oil; SE: squalene; MO: mineral oil; C: control solution (complete culture medium) as positive control; 00 hk: heat-killed M. brumae microemulsionated in olive oil as negative control. (A color Figure 3 accompanies to the present Figure 3).

Figure 3B shows ImageJ binary mask from confocal images of the clumps.

Figure 4. Tumor growth inhibition by microemulsionated or non-microemulsionated mycobacteria.

Human T24 (A) and murine MB49 (B) bladder cancer cells were cultured in medium alone as negative control (which was considered 100% growth), formulations without mycobacteria (00 or MO), or infected with BCG or M brumae diluted in culture medium (C) or microemulsionated (00 + Mycobact; MO + Mycobact). Results from MTT assay are shown as percent of cell survival related to negative control cells, and are expressed as mean ± SD of triplicate preparations. Data represent mean of 3 independent experiments. ^* p <0.05, significant differences on growth inhibition.

Figure 5. IL-6 (A) and IL-8 (B) production by infected human T24 cell line and, the murine homologues: IL-6 (C) and KC (D) produced by mouse MB49 cancer cells. Results are shown as mean ± SD of triplicate preparations. Data represent mean of 3 independent experiments. ^* p <0.05; ^** p<0.01 .

Figure 6. Schematic schedule of the in vivo experiments. Protocol time points of the tumor induction and the subsequent recurrent intravesical treatments of the animals. After tumor induction, tumor-bearing mice were subjected weekly to intravesical instillation of 100 μΙ PBS/emulsion, 10 ⁷ CFU irradiated-M. brumae, or 10 ⁷ CFU live- M. brumae. Figure 7A and 7B. M. brumae emulsionated enhanced survival of tumor-bearing mice. Kaplan-Meier analysis of tumor-bearing mice survival after different treatments. C57BL/6 mice (n=6-7) bearing orthotopic MB49 tumors were treated with emulsionated M. brumae on days 1 , 8, 15, and 22 post-tumor implantation. ^* , p<0.05; ^** , p<0.01 (log-rank test) versus control group. The survival of treated mice is shown in Figure 7A and the control group is shown in Figure 7B. Mouse survival data were recorded dairy. The difference in survival rate was analyzed by Log-rank (Mantel-Cox) test.

Detailed description of the invention

In a first aspect, the present invention discloses a microemulsion of a Mycobacterium species comprising:

a) An aqueous phase,

b) An oily phase comprising at least a hydrophilic surfactant and olive oil.

The term "oil-in-water microemulsion" as used herein refers to a microemulsion which is a dispersed oil phase in a continuous water phase. It does not include water-in-oil-in-water microemulsions. The oil phase used in the present invention is olive oil.

In a preferred embodiment of the present invention, the aqueous phase in the microemulsion represents from 95% to 99.7% by volume relative to the total volume of the microemulsion (v/v) (considering a final volume of 4 ml). In a more preferred embodiment, the aqueous phase is an aqueous solution comprising deionized water and NaCI or phosphate buffer saline salts, being more preferred deionized water and NaCI. In another preferred embodiment, the oily phase represents from 0.3-5% by volume relative to the total volume of the microemulsion (v/v) (considering a final volume of 4 ml).

In another preferred embodiment of the present invention, the Mycobacterium species are selected from the group comprising: Mycobacterium bovis bacillus Calmette-Guerin (BCG) or Mycobacterium brumae, being more preferred M. brumae.

In another preferred embodiment of the invention, the Mycobacterium species are present in a range of concentration of 0.0001 -100 mg/ml of dry weight expressed as a weight by volume of microemulsion (w/v), preferably in a range of 0.001 -50, more preferably in a range of 0.01 -10, even more preferably in a range of 0.1 -1 , and even more preferably, the concentration is 1 mg/ml of dry weight expressed as a weight by volume of microemulsion (w/v).

In another preferred embodiment, the olive oil is present at a concentration of 0.3- 5% by volume relative to the total volume of the microemulsion (v/v).

In another preferred embodiment, the olive oil-in-water microemulsion of a Mycobacterium species discloses in the present invention having a hydrophilic surfactant selected from the group comprising: Tween 80, Tween 20, Span 80, Brij 98, Pluronic F68, and Pluronic F127, being the more preferred hydrophilic surfactant Tween 80. The surfactant is generally present in amounts sufficient to increase the kinetic stability of the microemulsion by stabilizing the interface between the hydrophobic and hydrophilic components of the microemulsion. In this sense the surfactant in the olive oil-in-water composition is present in a concentration of 0.001 %-20% (v/v), more preferred in a concentration of 0.2%.

In another preferred embodiment, the olive oil-in-water microemulsion of a Mycobacterium species discloses in the present invention is used as medicament, preferably in the treatment of bladder cancer and more preferably in the treatment of non-invasive superficial bladder cancer, and more preferably, the grading of bladder cancer is Grade 1 and Grade 2. The microemulsion of the present invention is also effective as an adjunct to enhance the effectiveness of other anti-cancer agents including, but not limited to, anti-bladder cancer agents. Such agents include, but are not limited to, drugs, immunostimulants, antigens, antibodies, vaccines, radiation and chemotherapeutic, genetic, biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation and that inhibit proliferation of and induce apoptosis in responsive cells.

The terms "adjunct" refers to a useful compound with or in combination with other therapeutic agents, preferably, anti-bladder cancer agents, and more preferably, the grading of bladder cancer is Grade 1 and Grade 2 to increase treatment effectiveness.

The second aspect of the present invention refers to a composition, preferably a pharmaceutical composition, comprising an olive oil-in water microemulsion of a Mycobacterium species according to the present invention and pharmaceutically- acceptable carrier or excipient.

Examples of pharmaceutically-acceptable carriers or excipients are well known in the art and include those conventionally used in pharmaceutical compositions, such as, but not limited to, antioxidants, buffers, chelating agents, flavorants, colorants, preservatives, absorption promoters to enhance bioavailability, antimicrobial agents, and combinations thereof. The amount of such carriers or excipients depends on the properties desired, which can readily be determined by one skilled in the art. The pharmaceutical compositions of the present invention may routinely contain salts, buffering agents, preservatives, and compatible carriers, optionally in combination with other therapeutic ingredients. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically-acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically- and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, palicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Standard pharmaceutical texts, such as "Remington's Pharmaceutical Sciences, "1990 may be consulted to prepare suitable preparations, without undue experimentation. In a preferred embodiment of the invention, the composition disclosed herein further comprises at least one therapeutic agent to enhance the effectiveness of the composition. The therapeutic agents including, but not limited to, anti-bladder cancer agents. Such agents include, but are not limited to, drugs, immunostimulants, antigens, antibodies, vaccines, radiation and chemotherapeutic, genetic, biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation and that inhibit proliferation of and induce apoptosis in responsive cells. In a more preferred embodiment, the therapeutic agent is a cytostatic compound, preferably, mitomycin C.

The third aspect of the present invention disclosed the pharmaceutical composition mentioned above for use as medicament and more preferably for treatment of bladder cancer. In a preferred embodiment the bladder cancer is non-invasive superficial bladder cancer, and more preferably, the grading of bladder cancer is Grade 1 and Grade 2. In another preferred embodiment, the pharmaceutical composition disclosed herein trigger higher production of cytokines in bladder tumor cells than mycobacteria diluted in the rest of compositions, such as pharmaceutical compostion comprising mycobacteria emulsionated in mineral oil.

For therapeutic applications, the pharmaceutical composition can be administered directly to a patient when combined with a pharmaceutically-acceptable carrier or excipient. This method may be practiced by administering the therapeutic composition alone or in combination with an effective amount of another therapeutic agent, which may be, but is not limited to, anti-bladder cancer agents. Such agents include, but are not limited to, drugs, immunostimulants, antigens, antibodies, vaccines, radiation and chemotherapeutic, genetic, biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation and that inhibit proliferation of and induce apoptosis in responsive cells, and combinations thereof. Many such therapeutic agents are known in the art. In a more preferred embodiment, the therapeutic agent is a cytostatic compound, preferably, mitomycin C.

The present invention additionally provides methods for treating a patient or subject suffering a bladder cancer by delivering a pharmaceutically effective amount of the olive-in-oil microemulsion or the composition disclosed in the present invention. Improved treatments of non-invasive superficial bladder cancer are specially contemplated. The methods of the present invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Although formulations specifically suited for intravesical are preferred, the compositions of the present invention can also be formulated for parenteral, intravenous, muscular, inhalational, oral, topical, subcutaneous, intraperitoneal, intratumoral administration, and the like.

Those skilled in the art will recognize that the particular mode of administering the therapeutic or diagnostic agent depends on the particular agent selected; whether the administration is for treatment, diagnosis, or prevention of a disease, condition, syndrome, or symptoms thereof; the severity of the medical disorder being treated or diagnosed; and the dosage required for therapeutic efficacy. For example, a preferred mode of administering an anticancer agent for treatment of leukemia would involve intravenous administration, whereas preferred methods for treating skin cancer could involve topical or intradermal administration.

As used herein, "pharmaceutically effective amount" refers to the dosage or multiple dosages of the microemulsion or composition discloses in the present invention at which the desired therapeutic effect is achieved. Generally, an effective amount of the therapeutic or diagnostic agent may vary with the activity of the specific agent employed; the metabolic stability and length of action of that agent; the species, age, body weight, general health, dietary status, sex and diet of the subject; the mode and time of administration; rate of excretion; drug combination, if any; and extent of presentation and/or severity of the particular condition being treated. The precise dosage can be determined by an artisan of ordinary skill in the art without undue experimentation, in one or several administrations per day, to yield the desired results, and the dosage may be adjusted by the individual practitioner to achieve a desired therapeutic effect or in the event of any complication. Importantly, when used to treat cancer, the dosage amount of the therapeutic agent used should be sufficient to inhibit or kill tumor cells while leaving normal cells substantially unharmed. Another aspect of the present invention refers to a process for the preparation of an olive oil-in-water microemulsion of a Mycobacterium species, according to the present invention, comprising the step of:

i. mixing the Mycobacterium species with hydrophilic surfactant and olive oil

ii. emulsifying the oily phase by adding aqueous solution, mixing and sonicating

iii. adding aqueous solution until obtain the final volume of the microemulsion.

Typically, the ingredients are mixed together to form a mixture which is then subjected to a sonication process to form the microemulsion. The emulsification step may typically take between 0.5 and 15 minutes, more preferably between 1 and 7 minutes, most preferably between 2 and 6 minutes, depending upon the level of shear applied and the ingredients. The emulsification step is continued for the time necessary to produce an oil-in-water microemulsion. Moreover, in a preferred embodiment, the process is carried out at RT temperature, preferably at a temperature of 22 °C or less. In a preferred embodiment of the present invention, the aqueous phase represents from 95% to 99.7% by volume relative to the total volume of the microemulsion (v/v). In a more preferred embodiment, the aqueous phase is an aqueous solution comprising deionized water and NaCI or phosphate buffer saline salts, being more preferred deionized water and NaCI.

In another preferred embodiment, the oily phase represents from 0.3-5% by volume relative to the total volume of the microemulsion (v/v).

In another preferred embodiment of the present invention, the Mycobacterium species are selected from the group comprising: Mycobacterium bovis bacillus Calmette-Guerin (BCG) or Mycobacterium brumae, being more preferred M. brumae.

In another preferred embodiment of the invention, the Mycobacterium species are present in a concentration of 0.0001 -100 mg/ml of dry weight expressed as a weight by volume of microemulsion (w/v), preferably in a range of 0.001 -50, more preferably in a range of 0.01 -10, even more preferably in a range of 0.1 -1 , and even more preferably, the concentration is 1 mg/ml of dry weight expressed as a weight by volume of microemulsion (w/v).

In another preferred embodiment, the olive oil is present at a concentration of 0.3- 5% by volume relative to the total volume of the microemulsion

In another preferred embodiment, the NaCI or phosphate buffer saline salts being more preferred deionized water and NaCI, is present at a concentration of 0.001 - 10% expressed as a weight by volume of the microemulsion (w/v).

In another preferred embodiment, the olive oil-in-water microemulsion discloses in the present invention having a hydrophilic surfactant selected from the group comprising: Tween 80, Tween 20, Span 80, Brij 98, Pluronic F68, and Pluronic F127, being the more preferred hydrophilic surfactant Tween 80. The surfactant is generally present in amounts sufficient to increase the kinetic stability of the microemulsion by stabilizing the interface between the hydrophobic and hydrophilic components of the microemulsion. In this sense the surfactant in the olive oil-in- water composition is present in a concentration of 0.001 -20% (v/v), more preferred in a concentration of 0.2%.

EXAMPLES The purpose of the examples listed below is to illustrate the invention without limiting the scope thereof.

Example 1. Bacterial strains and cell lines M. bovis BCG Connaught (ATCC 35745) was purchased from Aventis Pasteur Laboratories (ImmuCyst). M. brumae (ATCC 51384) was obtained from our laboratory strain collection (Luquin et al., 1993). BCG and M. brumae were grown on Middlebrook 7H10 agar (Difco Laboratories, Surrey, UK) supplemented with 10% oleic-albumin-dextrose-catalase enrichment medium at 37C for four and one weeks, respectively. The human transitional carcinoma cell line T24 which represent histopathological tumor grade 3, was kindly provided by the Cancer Cell Line Repository of the Pare de Recerca Biomedica de Barcelona (as part of a Cooperative Cancer Research Network [RTICC] project funded by the Spanish Health Ministry, C03/010). Mouse bladder cancer cell line MB49 was kindly given to us by Dra. Sara Mangsbo and by Dr. Thomas Totterman (Rudbeck Laboratory, Department of Immunology, Genetics and Pathology of the University of Uppsala, Sweden). T24 and MB49 cell monolayers were maintained in Dulbecco's Modified Eagle's medium (DMEM)/Ham's F-12 nutrient mixture (Gibco BRL, Grand Island, NY) and DMEM high glucose (4.5 g/l) with stable glutamine (PAA Laboratories GmbH, Austria), respectively, both supplemented with 10% fetal bovine serum (FBS, Lonza, Switzerland), containing 100 U/ml penicillin G (Lab ERN, Barcelona, Spain) and 100 μg ml streptomycin (Lab Reig Jofre, Barcelona, Spain) (complete medium), at 37C in a humidified atmosphere with 5% C0 ₂ .

Example 2. Microemulsion preparations

In order to improve M. brumae antitumor activity, we designed different formulations and tested their efficacy on disaggregating mycobacteria clumps and on inhibiting tumor growth, ensuring also mycobacteria viability.

Different formulations were prepared following previous described protocols with some modifications (Table 2). Four mg dry-weight of M. brumae was emulsified using oil-in-water (o/w) and water-in-oil (w/o) formulations to a final volume of 4 mL of microemulsion (Table 2). The microemulsions were prepared following two different sonication protocols based on previous studies. On the one hand, a simple sonication protocol which consists in placing the mycobacteria, the oily phase and then the aqueous phase in a tube, and proceeding to sonicate for 5 minutes at 4°C On the other hand, the rod sonication protocol explained later (next paragraph). Furthermore, four different oils: olive oil (OO), soybean oil (SO), squalene (SE) and mineral oil (MO), were used for making the different mycobacteria formulations. Thus, in total 32 different formulations were assayed.

The olive oil-in-water microemulsion of the invention was obtained from 4 mg of dry- weight of mycobacteria which were placed in a sterile conical glass tube (Duran Group, Wertheim/Main, Germany). Then, a sterile mixture of 16.52% v/v Tween 80 and 83.47% v/v oil (47.92 μΙ_) was added and a sterile glass rod was introduced to mix them. 500 μΙ_ of 0.85% w/v NaCI were added to the mixture and sonicated (BANDERLIN electronic, Berlin, Germany) for 3 minutes at room temperature (RT). The final volume of the microemulsion was adjusted with the aqueous phase depending on the volume necessary for each experiment.

Table 2. Percentage of hydrophilic and hydrophobic phases and percentage of components in different water-in-oil and oil-in-water microemulsions showed in the present invention.

Phase Component Water-in-oil Oil-in-water

Wu Hwang Yarkoni Morales Invention

Hydrophilic 6 8 99 98 99

Deionized

6 8 99 98 99 water

Tween 80 - - 0.2 0.50 -

"PBS salts" - 9.36

NaCI - - 0.84 - 0.84

Hydrophobic 94 92 1 2 1

Oil 46.0 50.0 1 2 1

Tween 80 18.0 - - 0.2

Span 80 30.0 30.0 - - Phase Component Water-in-oil Oil-in-water

Wu Hwang Yarkoni Morales Invention

Brij 98 12.0

Example 3. Viability of microemulsioned mycobacteria M. brumae viability was determined by using two methods: counting colony-forming units (cfu) and observing stained live/dead mycobacteria using confocal microscopy. Serial dilutions of each formulation were platted on Middlebrook 7H10 agar. After one week at 37°C, M. brumae colonies were counted and cfu calculated. LIVE/DEAD® BacLight™ Bacterial viability kit (Invitrogen™, Molecular Probes®, Oregon, USA) was used to stain M brumae in each formulation. Stained mycobacteria were observed by using a TCS-SP5 confocal laser scanning microscope (Leica, Germany). HCX PL APO lambda blue 63.0x1 .40 oil UV objective operating at a zoom of 1 .8 was used. To determine both M. brumae viability and clump size (see Example 4) five horizontal (x-z) optical sections (stepsize 1 .51 μηη) of twenty fields for each condition were captured. Digital images were processed with Metamorf software (Molecular Devices, LLC, US) to calculate the percentage of life and death M. brumae.

Example 4. Clumping of mycobacteria

M. brumae clumps in each microemulsion were observed by confocal microscopy as described above. Clumps sizes were analyzed from the images taken in the previous viability study. Size ranges were initially chosen based on the number of bacteria present in each clump, but finally automatically classified from confocal microscopy data as clump area size with ImageJ (National Institutes of Health, US) (Figure 3 and Table 3).

Table 3. Aggregate areas considered.

Aggregate size Clump area (A) size Mm ²

Considered single bacteria A < 2

Small aggregate 2 < A < 10 Aggregate size Clump area (A) size _M m ²

Medium aggregate 10 < A < 60

Large aggregate 60 < A

Example 5. In vitro antitumor activity of the selected microemulsions Tumor cells were infected with live BCG and M. brumae. Briefly, MB49 or T24 cells (3x10 ⁴ per well) were seeded onto 96-well tissue culture plates in complete medium without antibiotics. Three hours later cells were infected with the mineral oil-in water (MO) microemulsion or with the olive oil-in-water (00) microemulsion at a multiply of infection (MOI) of 10:1 for 3 h. After three successive washes with complete medium without antibiotics to remove extracellular bacteria, cells were incubated with fresh complete medium at 37°C. At 72 h after infection, cell culture supernatants were harvested, centrifuged at 100 x g for 10 minutes, and stored at -40°C until used. After removing cell culture supernatant, cell colorimetric assay 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) was carried out in order to measure cell growth. Briefly, after removing culture supernatants, 10% MTT in complete medium was added and incubated for 3 hours at 37°C. After removing the medium water insoluble dark blue formazan was dissolved by adding acidic isopropanol. Absorbance was measured at 550 nm (Tecan, Mannedorf, Switzerland). Non-infected control cultures were always included, and all experiments were repeated at least three times.

Example 6. Analysis of cytokines from cell culture supernatants

Cytokine production was measured by enzyme-linked immunosorbent assays (ELISA) using commercially available kits in accordance with the manufacturer's instructions (human IL-8 and IL-6, and mouse IL-6 (BD Pharmingen, San Diego, CA, USA) and CXCL1/KC (R&D systems, Minneapolis, MN, USA)).

Example 7. Survival of emulsionated BCG and M. brumae inside T24 bladder cells To evaluate if being formulated on microemulsion affects mycobacteria pathogenicity, the capacity of survival of each mycobacteria inside bladder cancer cells were studied. Briefly, T24 cells were seeded onto 48-well tissue culture plates (6 x 10 ⁴ per well), and infected with mycobacteria (MOI 10:1 ) three hours later, as described before. At 3, 24, 48 and 72 hours after infection, cell culture supernatants were removed and T24 cells were lysed by adding a solution of 0.1 % Triton X-100 (Sigma-Aldrich Germany). Serial dilutions of cell lysates were platted on Middelbrock 7H10 plates, and after incubation for 1 and 4 weeks, M. brumae and BCG bacteria counts, respectively, were determined.

RESULTS

Example 8. Microemulsion preparations/selection of the most suitable oil Criteria initially chosen to optimize the mycobacteria formulation were based on homogeneity of the suspension and simplicity on making the microemulsion. In this sense, we found more homogeneous and stable by visual inspection formulations based in oil-in-water mixtures. A first mixture of Tween 80 and oil with the mycobacteria and a later mixture with the water phase provide a more homogeneous solution after visual and microscopic observation.

After choosing the protocol for making the formulation, we tried the efficacy of the different oils on maintaining mycobacteria viable (Figure 1 and Figure 2) and disaggregating mycobacteria clumps (Figure 3 and Table 4).

From the results obtained, we decided to analyze the antitumor ability of both olive oil (OO) and mineral oil (MO) microemulsions. The formulations do not affect mycobacteria viability and are able to disaggregated mycobacteria clumps. As Figure 1 shows, OO and MO formulations not only maintain viability of mycobacteria but also significantly increase the M. brumae cfu/ml with respect to the control solution.

Table 4. Percentage of M. brumae small, medium and large aggregates in different oil formulations. Size clumps 00 SO SE MO C

Small 85.81 86.07 65.04 93.14 93.38

Medium 1 1.49 13.26 28.62 6.86 6.62

Large 2.70 0.67 6.35 0.00 0.00

Example 9. In vitro antitumor activity of mineral oil or olive oil microemulsions

After selecting the two most efficacious formulations, mineral oil and olive oil microemulsions, T24 human and MB49 murine bladder cancer cell lines were infected with emulsionated mycobacteria and tumor growth inhibition and IL-6 and IL-8 cytokine production were measured.

Firstly, we observed microemulsions (using mineral oil or olive oil) without mycobacteria do not have cytotoxic activity on bladder tumor cells (Figure 4A and 4B). Our results also indicate that both BCG and M. brumae emulsified in oil-in-water solution reduced efficiently tumor cell proliferation (Figure 4A and 4B). But the efficacy depends on the oil used in the formulation. As Figure 4A and 4B show, both BCG and M brumae olive oil formulated have a not significantly different antiproliferative activity compared to the control. But the antitumor effect of mineral oil formulations was lower than both olive oil formulation or mycobacteria diluted in culture medium ( ^* , p<0.05).

Thus, olive oil microemulsion is more effective than mineral oil emulsion in inhibiting both human and mouse bladder cancer cells proliferation (Figure 4A and 4B).

While emulsions without mycobacteria did not affect tumor growth, emulsionated mycobacteria inhibits tumor proliferation. Olive oil emulsionated mycobacteria inhibit cell growth similarly to control mycobacteria. However, mineral oil emulsionated mycobacteria is less efficient than olive oil preparation in inhibiting cells growth ( ^* p<0.05). Example 10. Analysis of cytokines from cell culture supernatants

Cytokine production was evaluated in supernatants from infected and non-infected cells. As Figure 5 shows, the presence of emulsionated bacteria on the cultures triggers a statistically significant increase in all cytokines compared to basal levels produced by non-infected cells or cells infected with the microemulsion without bacteria. Furthermore, the presence of oil-formulated bacteria favors the immune response against both, BCG and M brumae. In all cases, olive oil mycobacteria formulations trigger the highest levels of cytokine production.

Example 11. Pathogenicity of microemulsionated mycobacteria

After platting lysates from mycobacteria-infected T24 cells, the results show that M. brumae is killed by bladder cells 72 hours after infection, whereas BCG persist inside bladder cancer cells. Thus, the behavior is the same between emulsionated and non-emulsionated mycobacteria. This result confirms the non-pathogenicity of M. brumae even formulated in microemulsion.

The data disclosed in the present invention show that the olive oil-in water microemulsions and a pharmaceutical formulation thereof, according to the present invention, allow maintaining mycobacteria viable, disaggregate mycobacteria clumps, inhibit tumor cell growth similarly to mycobacteria diluted in culture media, and more efficiently than mycobacteria on mineral oil microemulsion and also trigger higher production of cytokines in bladder tumor cell cultures than mycobacteria diluted in the rest of formulations.

M. brumae and BCG microemulsionated in olive oil could be a suitable alternative to the current BCG treatment for bladder cancer, preferably for non-invasive bladder cancer.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Example 12. Orthotopic model of bladder cancer and intravesical treatment

Animal experiments were performed according to procedures approved by the Animal Care Committee at the Universitat Autonoma de Barcelona. The orthotopic murine model of bladder cancer was done as previously described (Zaharoff, 2009). In brief, C57BI/6 female mice at 7 to 9 weeks of age (Charles River Laboratories, France) were anaesthetized with isoflurane, and chemical lesions to the bladder urothelium by installating intravesically 50 μΙ of L-poly-lysine (Sigma) through a 24- gauge catheter were done. Subsequently tumor was induced by instillating 10 ⁵ MB49 tumor bladder cells suspended in 100 μΙ of culture medium. Cells were retained in the bladder for 60 min. One day following tumor implantation, mice were randomly divided into 3 treatment groups (non-treated, irradiated-M. brumae or live- M. brumae) each containing 6-7 animals. Mice received 10 ⁷ mycobacteria cells intravesically in 100 μΙ of PBS or olive-oil in water microemulsion. Previously, part of bacteria cells were subjected to irradiation treatment in order to compare irradiated and live M. brumae treatments (Secanella-Fandos, 2014). Control mice received in 100 μΙ of PBS or emulsion. Each treatment remained the bladder lumens for one hour while animals were kept under anesthesia. Animals were treated weekly for four weeks (figure 6). A death was recorded if animals met approved guidelines for euthanasia as a result of tumor burden. After 60 days, surviving animals were sacrificed. Log rank tests determined statistical significance of Kaplan-Meier survival curves (KaleidaGraph software) Significance was defined as p<0.05.

M. brumae intravesical treatment enhances mice-bearing tumor survival

In vivo antitumor activity of olive-oil-in-water emulsionated mycobacteria in the orthotopic model of bladder cancer was evaluated. After inducing bladder cancer by intravesical instillation of MB49 BC cells, mice were treated weekly with emulsion, irradiated or live M. brumae as indicated in Figure 6. All animals presented hematuria some days after tumor induction, which is a hallmark of the presence of tumor (Chade, 2008). As shown in Figure 7A, survival rates are significantly higher in mice treated with mycobacteria than in non-treated mice (p<0.05) (Figure 7B). While 100% of non-treated animals succumbed within 35 days post-tumor implantation, 100% of animals treated with live M. brumae in olive oil-in-water emulsionated survived. No significant differences were observed between irradiated and live bo/mae-treated groups (p>0.05). REFERENCES

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