BRPI1004208A2 - hybrid tuberculostatic agent, pharmaceutical composition, preparation process of hybrid tuberculostatic agent, tuberculosis treatment method and use - Google Patents

Abstract

HYBRID TUBERCULOSTATIC AGENT, PHARMACEUTICAL COMPOSITION, HYBRID TUBERCULOSTATIC AGENT PREPARATION PROCESS, TUBERCULOSIS TREATMENT METHOD AND USE The present invention relates to tuberculostatic and easy-to-syntactic drug-containing agents for tuberculostatic and fast-acting syncope-containing compounds. pharmaceutical, biotechnological, therapeutic, biomedical or preventive applications.

Description

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HYBRID TUBERCULOSTATIC AGENT, PHARMACEUTICAL COMPOSITION, HYBRID TUBERCULOSTATIC AGENT PREPARATION PROCESS, TUBERCULOSIS TREATMENT METHOD AND USE

The present invention relates to quaternary ammonium compound hybrid tuberculostatic agents and readily obtainable synergistically acting tuberculostatic drugs for pharmaceutical, biotechnological, therapeutic, biomedical or preventive applications. State of the art Tuberculosis

Tuberculosis is one of the longest documented diseases

in the history of mankind and until the middle of the twentieth century was the leading cause of death from infectious diseases and annually responsible for seven million deaths worldwide (DUCATI, RG; BASSO, LA; SANTOS, DS (2008) In: Trabulsi LR Alterthum, F. Mycobacteria, 5th ed. Sao Paulo: Atheneu). According to the World Health Organization (WHO) in 2008 there were 9.4 (8.9 - 9.9) million cases (equivalent to 139 cases per 100,000 inhabitants) of tuberculosis in the world. There was an increase in the number of cases compared to 2007, with an incidence of 9.3 million new cases. Of the estimated cases in 2008, 55% occurred in Asia and 31% in Africa, and to a lesser extent in the eastern Mediterranean (6%), Europe (5%) and the Americas (3%). Most cases of tuberculosis occurred in developing countries, which are considered priorities for disease control. Asia and Africa are the countries with the highest incidence coefficients, reaching all age groups, with a predominance of economically active individuals (WORLD HEALTH ORGANIZATION, 2009. Global Tuberculosis Control. A short update to the report). Brazil is the 18th country in the world in number of new cases of tuberculosis. The WHO estimated that in 2007 there were 92,000 new cases and 49,000 new BAAR + (acid-fast bacillus resistant) cases in the country, with an incidence rate of 48 and 26 cases per 100,000 population, respectively. In addition, 14% of all tuberculosis cases (all forms) are expected to be in HIV - positive patients. Regarding mortality, it is estimated that about 8,400 deaths are attributed to tuberculosis, ie 4.4 deaths per 100,000 population (WORLD HEALTH ORGANIZATION, 2009. Global Tuberculosis Control. A short update to the report. MINISTRY OF HEALTH (2009) ) HEALTH SURVEILLANCE SECRETARIAT (Epidemiological Electronic Bulletin). The risk of developing active tuberculosis varies according to the time the patient came into contact with the bacillus, age, host immune response, and the risk of developing active tuberculosis was estimated at 10% (COMSTOCK, GW; LIVESAY, VT; WOOLPERT, SF (1974) The prognosis of a positive tuberculin reaction in childhood and adolescence Am J. Epidemiol., 99, 131-138. DUCATI, RG; BASSO, LA; SANTOS, DS (2008) ) In: Trabulsi LR, Alterthum, F. Mycobacteria, 5th ed São Paulo: Atheneu).

Causative agent of tuberculosis: Mvcobacterium tuberculosis

Tuberculosis is an infectious disease of chronic evolution that mainly affects the lungs, but can affect other organs and tissues. Five species of bacteria belonging to the genus Mycobacterium are the causative agents of tuberculosis: M. tuberculosis, M. bovis, M. africanum, M. mieroti and M. eanetti. Pulmonary tuberculosis has as its main causative agent Myeobacterium tuberculosis. Over 60 species of mycobacteria are currently known, the vast majority being part of the environment classified as atypical mycobacteria or non-tuberculous mycobacteria (DUNLAP, NE; BASS, J .; FUJIWARA, P.; HORSBURG, CR; SALFINGER, M .; SIMONE, PM Diagnostic standards and classification of tuberculosis in adults and children (2000) Am J. Respiratory Crit Care Care Med 161, 1376-95 DUCATI, RG; BASSO LA; SANTOS, DS (2008) In: Trabulsi LR (Alterthum, F. Mycobacteria, 5th ed. Sao Paulo: Atheneu). M. tuberculosis is a slightly curved small bacillus of 1.0 to 4.0 μιη in length and 0.3 to 0.6 μιτι in diameter, is an intracellular pathogen remaining mainly in alveolar macrophages, is a strict aerobic non-spore, It produces no toxins and no capsules or flagella. It grows slowly, about 3 to 4 weeks to form visible colonies even in special culture media (ANDERSEN, P. (1997). Host responses and antigens involved in protective immunity to Mycobacterium tuberculosis. Seconde J. Immunol., 115-31. DUCATI, RG; BASSO, LA; SANTOS, DS (2008) In: Trabulsi LR; Alterthum, F. Mycobacteria. 5th ed. Sao Paulo: Atheneu). The bacillus cell wall has an extremely unique structure composed of peptide glycan- (N - glycolylmuramic acid) and mycolic acids (long chain fatty acids), which are covalently linked to polysaccharide (arabinogalactane) which in turn binds to the peptide glycan via phosphodiester bridges. The cell wall contains some types of free lipids, not covalently associated with this basal skeleton (the complex i).

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arabinogalactario-peptideoglycan), in addition to some proteins DUCATI, R.G .; BASSO, L.A .; SANTOS, D. S. (2008) In: Trabulsi L. R .; Alterthum, F. Mycobacteria. 5th ed. Sao Paulo: Atheneu).

Among these is Mycobacterium smegmatis, which can be considered a good Mycobacterium model because it is naturally resistant to traditional antibiotics such as rifampicin and isoniazid. It is found in the environment and is rarely pathogenic (BROWN-ELLIOTT, B.; WALLACE, R. (2002). Clinical and Taxonomic Status of Pathogenic Nonpigmented or Late-Pigmented Rapidly Growing Mycobacteria. Clin. Mierobiol. Rev., 15, 716-746) despite having the typical and unique wall of the genre to which it belongs. Different species of Myeobaeterium are sensitive to different antimicrobial agents. Pathogenic species subjected to antibiotics often develop antibiotic resistance mechanisms. Hence the need for multiple and conjugated drugs for tuberculosis therapy. Recent literature shows that antibiotics to which microorganisms have already developed resistance may have their activity restored by the addition of cationic compounds such as polyamines or polyguanidines (BERA, S.; ZHANEL, GG; SCHWEIZER, F. (2008). Design, synthesis and antibacterial activities of neomycin-lipid conjugates: polycationic lipids with potent gram-positive activity (J. Med. Chem., 51: 6160-64). Tuberculosis drugs

The standard chemotherapy treatment recommended by the World Health Organization for tuberculosis control is based on the combination of isoniazid, rifampicin, pyrazinamide and streptomycin (or ethambutol), as well as other drugs in cases of multidrug resistance (DUCATI, RG; BASSO, LA; SANTOS, DS (2008) In: Trabulsi LR; Alterthum, F. Mycobacteria 5th ed. Sao Paulo: Atheneu). Rifampicin and isoniazid are among the most potent drugs used to treat the disease, killing more than 99% of bacilli within two months of initiation of treatment (ISEMAN, MD; MADSEN, LA (1989). Drug-resistant tuberculosis. Clinical Chest Medical, 10, 341-353 (MITCHISON, DA (1995), Rifabutin in the treatment of newly diagnosed pulmonary tuberculosis (Tuber. Lung Dis., 76). Rifampicin acts by inhibiting the β subunit of RNA polymerase (BURMAN, W.J .; GALLICANO.K .; PELOQUIN, C. (2001). Comparative pharmacokinetics and pharmacodynamics of the rifamycin antibacterials. Clin. Pharmacokinet., 40, 327-341). It irreversibly combines with RNA polymerases, blocking DNA transcription. The chemical structure of rifampicin or 3 - [[(4-methyl-1-piperazinyl) imino] methyl] rifamycin is shown in Figure 1A. Its chemical formula is C43H58N4Oi2; has a molecular weight of 822.95 g / mol; It is poorly soluble in H 2 O and acetone, soluble in ethyl acetate, methanol, tetrahydrofuran (THF), chloroform and dimethyl sulfoxide (DMSO). The limit of solubility of the drug in water is 1.3 g / l or 1.6 mM. It has maximum light absorption at 237, 255, 334 and 475 nm (0.002 mg / mL in pH 7.4 phosphate buffer). Its 1% suspensions in water have a pH between 4.5 and 6.5. It has two pKka values, the first being 1.7 for hydroxyl at position four and the second at 7.9 for piperazine nitrogen three. Its DMSO solutions are very stable and H2O are poorly stable (ΤΉΕ MERCK INDEX (2000) Merck & Co Inc. 12th ed. NJ: Whitehouse station. UNITED STATES PHARMACOPEIA (2006) 29th ed. Rockville: United States Pharmacopeia Convention ). Isoniazid rapidly eradicates most bacilli that are replicating in the first two weeks of tuberculosis treatment. Its biochemical effect occurs by inhibiting the early stages of mycolic acid synthesis. Isoniazid is isonicotinic acid hydrazide: 4-pyridinecarboxylic acid hydrazide. Its chemical structure is in Figure 1B. Its molecular weight is 137.14 g / mol (THE MERCK INDEX (2000) Merck & Co Inc. 12th ed. N.J: Whitehouse station. UNITED STATES PHARMACOPEEA (2006) 29th ed. Rockville: United States Pharmacopeia Convention). It is presented as a white, water soluble crystalline powder (water solubility limit is 140 g / l or 1 M), slightly soluble in ethanol and practically insoluble in ether and benzene. Its solutions in 1% water have a pH of 5.5 to 6.5. It has two protonable clusters with pKa values of 2.0 and 3.5. It is poorly stable in H2O and very stable in DMSO. Maximum absorption at 266, 265 nm (0.01 mg / mL in 0.01 N HCl) (THE MERCK INDEX (2000) Merck & Co Inc. 12th ed. NJ: Whitehouse station UNITED STATES PHARMACOPEEA (2006) 29a Ed Rockville: United States Pharmacopeia Convention).

Quaternary ammonium compounds as bactericidal agents and carriers of antimicrobial agents

The antimicrobial effect of quaternary ammonium grouping is widely known (MERIANOS, JJ (1991) Quaternary ammonium antimicrobial compounds, Disinfection, sterilization and preservation, SS Block, ed. Lea & Febiger, Philadelphia. RUSSELL, AD; HUGO, WB; AYLIFFE, GAJ (1999) Principles and Practice of Disinfection, Preservation and Sterilization Blackwell Science: Oxford DAVIES, A. FIELD, BS (1969) Action of Biguanides, Phenols and Detergents on Escherichia coli and its Spheroplasts J. Appl. 32, 233-238. KANAZAWA, A., IKEDA, T., ENDO, T. (1995) A novel approach to the mode of action of cationic biocides - morphological effect on antibacterial activity J. Appl Bacteriol., 78 , 55-60). Other examples of antimicrobial compounds with the quaternary ammonium group in their structure are:

1) Amphiphiles such as cetyltrimethylammonium bromide (SALT, W.G .; WISEMAN, D. (1970) Relationship between uptake by Escherichia coli and its effects on cell growth and viability. J. Pharm. Pharmacology, 22, 261-264); 2) Cationic lipids such as DODAB (TAPIAS, G.N .; SICCHIEROLLI, S.M .;

MAMIZUKA1 E.M .; CARMONA-RIBEIRO, A.M. (1994) Interactions between Cationic Vesicles and Escherichia coli. Langmuir, 10, 3461-3465. SICCHIEROLLI, S.M .; MAMIZUKA, E.M .; CARMONA-RIBEIRO, A.M. (1995) Bacterium Flocculation and Death by Cationic Vesicles. Langmuir, 11, 2991-2995. MARTINS, L.M.S .; MAMIZUKA, E.M .; CARMONA-RIBEIRO / A.M. (1997) Cationic Vesicles as Bactericides Langmuir, 13, 5583-5587. CARMONA-RIBEIRO, A.M .; ORTIS, F .; SCHUMACHER, R.I .; ARMELIN, M.C.S. (1997) Interactions between Cationic Vesicles and Cultured Mammalian Cells. Langmuir, 13, 2215-2218. CAMPAIGN, M.T.N .; MAMIZUKA, E.M .; CARMONA-RIBEIRO, A.M. (1999) Interaetions Between Cationic Liposomes And Bacteria: The Physical-Chemistry Of The Bactericidal Action. J. Lipid Res., 40, 1495-1500. CAMPAIGN, M.T.N .; MAMIZUKA, E.M .; CARMONA-RIBEIRO, A.M. (2001) Interactions between cationic vesicles and Candida albicans. J. Phys. Chem. B, 105, 8230-8236. VIEIRA, D. B .; LINCOPAN, N .; MAMIZUKA, E. M .; PETRI, D.F.S .; CARMONA-RIBEIRO, A.M. (2003) Competitive Adsorption of Chitosan and Cationic Bilayers on Polymeric Microspheres: Optimization of the Biocidal Action. Langmuir, 19, 924-932. CARMONA-RIBEIRO, A.M. (2003) Bilayer-Forming Synthetic Lipids: Drugs or Carriers? Curr. Med. Chem., 10, 2425-2446. CARMONA-RIBEIRO, A.M .; VIEIRA, D.B .; LINCOPAN, N. (2006) Cationic Lipids and Surfactants as Anti-infective Agents. Anti-infective Agents in Medicinal Chemistry, 5, 33-54. LINCOPAN, N .; CARMONA-RIBEIRO, A.M. (2006) Lipid-covered drug particles: combined action of dioctadecyldimethylammonium bromide and amphotericin B or miconazole. J. Antimicrob. Chemoth., 55, 727-734. VIEIRA, D.B .; CARMONA-RIBEIRO, A.M. (2006) Cationic lipids and surfactants as antifungal agents: mode of action. J. Antimicrob. Chemoth., 58, 760-767. PACHECO, LF, VIEIRA, DB, BELT, FM, CARMONA-RIBEIRO, AM (2004) Interactions between cationic bilayers and Candida albicans cells, 175-177, D0l: 10.1007 / b97059, In Surface and Colloid Science, Vol. 128, edited by Fernando Gajembeck, Springer-Verlag, Heidelberg, Germany. KANAZAWA, A .; IKEDA, T .; ENDO, T (1995) A novel approach to the mode of action of cationic biocides - morphological effect on antibacterial activity. J. Appi Bacteriol., 78, 55-60. RUSSELL, A.D .; CHOPRA, I. (1996) Understanding antibacterial action and resistance, Ellis Horwood: Chichester, 2-5);

3) Cationic polymers (polyelectrolytes) (THOME, J .; HOLLANDER, A.; JAEGER, W .; TRICK, I .; OEHR1 C. (2003) Ultrathin antibacterial polyammonium

coatings on polymer surfaces. Surf Coat. Tech., 174-175, 584-587. TILLER, J. C .; LIAO, C.J .; LEWIS, K .; KLIBANOV, A.M. (2001) Designing surfaces that kill bacteria on contact. Proceedings of the National Academy of Sciences of the United States of America., 98, 5981-5985. VIEIRA, D.B .; CARMONA-RIBEIRO, A.M. (2006) Cationic Iipids and surfactants as antifungal agents: mode of action. J. Antimicrob.

Chemoth., 58, 760-767. CEN, L .; NEOH, K.G .; KANG, E.T. Surface functionalization technique for conferring antibacterial properties to polymeric and ceilulosic surfaces (2003). Langmuir, 19, 10295-10303) or dendrimers with varying degrees of branching (CHEN, C.Z.S. & COOPER, S.L. (2000) Recent Advances in Antimicrobial Dendrimers. Adv. Mater., 12, 843-846. CHEN, C.Z.S .; COOPER, S.L.

(2002) Interactions between dendrimer biocides and bacterial membranes. Biomaterials, 23, 3359-3368). Cationic polymers are proving to be potent antimicrobial agents (Codling, CE; Maillard, J.; Russell, AD (2003). Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine. J. Antimicrob. Chemother., 51, 1153-1158. KUEGLER, R .;

BOULOUSSA, O .; RONDELEZ, F. (2005) Evidence of a charge density threshold for optimum efficiency of biocidal cationic surfaces. Microbiology, 151, 1341-1348);

4) Organic-inorganic hybrid materials containing quaternary ammonium salts (MARINI, M .; BONDI, M .; ISEPPI, R .; TOSELLI, M .; PILATI, F. (2007) Preparation and antibacterial activity of hybrid materials containing quaternary.

ammonium salts via sol-gel process. Eur: Polym. J., 43, 3621-3628. PEREIRA, E.M.A .; KOSAKA, P.M .; ROSA, H .; VIEIRA, D.B .; KAWANO, Y .; PETRI, D.F.S .; CARMONA-RIBEIRO, A.M. (2008). Hybrid materials from intermolecular associations between cationic Iipids and polymers. J. Phys. Chem. B, 112, 9301-9310). The mechanism of biocidal action for cationic compounds has been related to the reversal of cell charge (from negative to positive) (CAMPAIGN, MTN; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1999). Interactions between cationic Iiposomes and bacteria: the physical- chemistry of the bactericidal action J.

Lipid Res., 40, 1495-1500). Adsorption of cationic bilayers composed of dioctadecyldimethylammonium bromide (DODAB) on bacterial or fungal cells changed the cell charge signal from negative to positive, with a close relationship between the positive charge of bacteria or fungi and the bactericidal or fungicidal effect (CAMPAIGN, MTN; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1999) Interactions between cationic Iiposomes and bacteria: the physical chemistry of the bactericidal action J. Lipid Res., 40, 1495-1500 VIEIRA, DB; CARMONA-RIBEIRO, AM (2006) Cationic Lipids and surfactants as antifungal agents: mode of action (J. Antimicrob. Chemother., 58, 760-767). Regarding the mechanism of action of DODAB, no cell lysis or cationic vesicle rupture were observed (MARTINS, LMS; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1997). Cationic vesicles as bactericides. Langmuir, 13, 5583-5587; VIEIRA , DB; CARMONA-RIBEIRO, AM (2006) Cationic Lipids and surfactants as antifungal agents: mode of action (J. Antimicrob. Chemother., 58, 760-767). This investigation was extended to the effect of DODAB on the viability of normal and cultured fibroblasts (CARMONA-RIBEIRO, AM; ORTIS, F.; SCHUMACHER, RI; ARMELIN, MCS (1997) Interactions between cationic vesicles and cultured mammalian cells. , 13, 2215-2218). After half an hour of interaction with eukaryotic cells in subconfluent monolayer, cell death was observed from 0.1 millimolar DODAB for both normal and transformed cells. Table 1 illustrates the cytotoxicity of DODAB against mammalian cells and

some bacteria or fungi (CARMONA-RIBEIRO, A.M. (2003) Bilayer-Forming Synthetic Lipids: Drugs or Carriers - Curr. Med. Chem., 10, 2425-2446). Mammalian cells are more resistant to DODAB than bacteria or fungi, leaving 50% of viable fibrolasts in 1 mM DODAB while for microorganisms 50% viability occurs in the micromolar range of DODAB concentrations. About 100% of mammalian cells stay alive at DODAB concentrations where bacteria and fungi do not survive.

Dioctadecyldimethylammonium bromide (DODAB) organization in water: bactericidal bilayers Cationic synthetic bilayer-forming lipids such as dioctadecyldimethylammonium (DODA) salts 1 when dispersed in water form positively charged bromide (DODAB) or chloride (DODAC) bilayers counter ion. Linked to a quaternary ammonium group are two long carbon chains with 18 carbons each, thus giving it a high hydrophobic character (Figure 1C). Due to this character, DODAB (PM 631) is poorly soluble in water, but, like other lipids, has the property of self-associating in aqueous dispersion as open or closed bilayers depending on the dispersion method (CARMONA-RIBEIRO, AM (1992) Synthetic amphiphile vesicles (Chem. Soc. Rev., 21, 209-214). By sonication with probe, bilayer fragments can be obtained while by evaporation of chloroform solution, or by other methods, closed bilayers or vesicles can be obtained. To obtain fragments the method employed is tip sonication (CARMONA-RIBEIRO, AM; CASTUMA, CE; SESSO, A.; SCHREIER, S. (1991). Bilayer strucutre and stability in dihexadecylphosphate dispersions. J. Phys. Chem 95, 5361-5366) and to obtain vesicles, the chloroform vaporization method is employed (CARMONA-RIBEIRO, AM; CHAIMOVICH, H. (1983). Preparation and characterization of Iarge dioctadecyldimethylammonium chloride Iiposomes and comparison with small sonicated vesicles. Biochem. Biophys. Res. Commun., 733, 172-179) or heating - SNIPPE method (KATZ, D .; KRAAIJEVELD, CA; SNIPPE, H. (1995). Synthetic Iipid compound as antigen-specific immunostimulators for improving the efficacy of vaccine-killed viruses. In: Theory and Practical Application of Adjuvants. Chichester: Stewart-Tull, DES, Ed .; John Willey & Sons).

Synthetic cationic bilayers composed of DODAB can have multiple

uses (CARMONA-RIBEIRO, A.M. (2000) Interactions between cationic Iiposomes and drugs or biomolecules. An. Acad. Bras. Cienc., 72, 39-43). Silica particles or polystyrene microspheres can be functionalized by coating with DODAB bilayers (CARMONA-RIBEIRO, AM; MIDMORE, BR (1992). Synthetic bilayer adsorption onto polystyrene microspheres. Langmuir, 8, 801-806. VIEIRA, DB; LINCOPAN, N. MAMIZUKA, EM; PETR1, DFS; CARMONARIBEIRO, AM (2003) Competitive adsorption of chitosan and cationic bilayers on polymeric microspheres: optimization of the biocidal action Langmuir, 19, 924-932 RAPUANO, R .; CARMONA-RIBEIRO, AM (1997) Physical adsorption of bilayer membranes on silica J. Colloid Interface Sci., 193, 104-111 RAPUANO, R .;

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CARMONA-RIBEIRO, Α.Μ. (2000). Supported bilayers οη silica. J. Colloid Interface Sci., 226, 299-307. MOURA, S.P .; CARMONA-RIBEIRO, Α. Μ (2003). Cationic bilayer fragments οη silica at Iow ionic strength: competitive adsorption and colloid stability. Langmuir, 19, 6664-6667. LINCOPAN, N .; ROSA, H .; CARMONA-RIBEIRO, A.M. (2006) Biomimetic Particles. Macromol. Symp., 245-6, 485-490). Adhesion of vesicles or DODAB or phospholipid bilayer deposition on silicon oxide surfaces or on polymeric particles or films in the absence or presence of oppositely charged polymers can be an important tool for research on the development of biosensors and immunological kits. (SALAY, LC; CARMONA-RIBEIRO AM (1998). Synthetic bilayer wetting on SiO2 surfaces. J. Phys. Chem. B, 102, 4011-4015. SALAY, LC .; CARMONA-RIBEIRO, AM (1999). SiO2 surfaces by phospholipid dispersions J. Adhesion Sci. Technol., 13, 1165-1179. PEREIRA, EMA; PETR1 DFS; CARMONA-RIBEIRO.

A.M. (2002) Synthetic Vesicles at Hydrophobic Surfaces. J. Phys. Chem. B, 106, 8762-8767. PEREIRA, E.M.A .; VIEIRA, D.B .; CARMONA-RIBEIRO, A.M. (2004)

Cationic Bilayers on Polymeric Particles: Effect of Low NaCI Concentration on Surface Coverage. J. Phys. Chem. B, 108, 11490-11495. PEREIRA, Ε. BAD.; PETRI, D.F.S .; CARMONA-RIBEIRO, A.M. (2002) Assemblies of cationic Iipids on polymeric films. Molecular Crystals and Liquid Crystals, 374, 617-622). Also, a remarkable solubilization ability of hydrophobic drugs such as amphotericin B and myconazole in DODAB bilayer fragments has been described (VIEIRA, DB; CARMONA-RIBEIRO, AM (2001). Synthetic bilayer fragments for amphotericin BJ Colloid lnterf. Sci., 244, 427-431. PACHECO, LF; CARMONARIBEIRO, AM (2003) Effects of synthetic lipids on solubilization and colloidal stability of hydrophobic drugs (J. Colloid Interf. Sc / "., 258, 146-154). amphotericin

B, an evident colloidal stabilization of the drug particle in aqueous dispersion was also demonstrated by deposition of cationic fragments on the antifungal anionic granules (LINCOPAN, N .; CARMONA-RIBEIRO, AM (2006). dioctadecyldimethylammonium bromide

and amphotericín B or miconazole. J. Antimicrob. Chemother., 58, 66-75). Synergism between cationic and antibiotic compounds

The various resistance mechanisms developed for antibiotics (DZIDIC, S .; SUSKOVIC, J .; KOS, B. (2008) Antibiotic resistance mechanisms in bacteria: biochemical and genetic aspects. Food Technoi Biotech., 46, 11-21) have awakened interest for the development of new formulations with modes of action capable of preventing or reducing the emergence of resistance. A currently studied class of antibacterial agents are cationic amphiphiles including:

1) Polycationic lipids (BERA. S .; ZHANEL, G.G .; SCHWEIZER, F. (2008). Design, synthesis and antibacterial activities of neomycin-lipid conjugates:

polycationic Iipids with potent gram-positive activity. J. Med. Chem. 2008; 51, 6160-64. VIEIRA D.B .; CARMONA-RIBEIRO, A.M. (2006) Cationic Iipids and surfactants as antifungal agents: mode of action. J Antimicrob. Chemother., 58, 760-67);

2) Cationic peptide antibiotics (HANCOCK, R.E.W .; SAHL1 H. (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies.

Nat Biotechnol., 24, 1551-57), cationic lipopeptides (MALINA, A.; SHAI, Y. (2005) Conjugation of fatty acids with different modulated antimicrobial and antifungal activity of a biologically inactive peptide cationic., Biochem., 390 , 695-702);

3) Synthetic compounds that mimic cationic peptide antibiotics

(SCOTT, R.W .; DEGRADO, W.F .; TEW, G: N. (2008) Newly designed synthetic mimics of antimicrobial peptides. Current Opinin. Biotechnol., 19, 620-27);

4) Cationic polymers (KÜGLER, R.; BOULASSA, O .; RONDELEZ, F. (2005) Evidence of a charge density threshold for optimum efficiency of biocidal cationic surfaces. Microbiol., 151, 1341-48).

Several modes of action for these cationic compounds have been proposed (GILBERT. P .; MOORE L.E. (2005). Cationic antiseptics: diversity of action under a common epithet. J. Appl. Mierobiol., 99, 703-15). The antibacterial effect of cationic amphiphilics involves a disorganization of the bacterial wall induced by removal or replacement of counterions of alkylammonium ion-charged groups (HANCOCK, REW; SAHL1 H. (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat. Bioteehnol., 24, 1551-57 Kügler, R., Boulassa, R. Ronddez, F. (2005) Evidence of a charge density threshold for optimum efficiency of biocidal cationic surfaces.Mierobiology, 151, 1341-48. GILBERT, P. MOORE, LE (2005) Cationic antiseptics: diversity of action under a common epithet (J. Appl. Mierobiol., 99, 703-15). This mode of action has been shown to limit the risk of cross resistance (ZASLOFF., M. (2002). Antimicrobial peptides of multicellular organisms. Nature, 415, 389-95. CHOPRA, I.; HODGSON, J .; METCALF, B .; POSTE, G. (1997) The search for antimicrobial agents effective against bacteria resistant to multiple antibiotics (Antimicrob. Agents Chemother., 41, 497-503). Over the years, cationic lipids formulated as cationic liposomes have been used for various applications, including drug delivery system against infectious diseases and gene transfection (SAINLOS, M .; BELMONT, P .; VIGNERON, J.P .; LEHN, P .;

LEHN, J.M. (2003) Aminoglycoside-derived cationic Iipids for gene transfection: transfection: synthesis of kanamycin A derivatives. Eur. J. Org. Chem., 2764-74).

Among the cationics best known for exhibiting a broad spectrum of antibacterial activity are:

1) Benzalkonium chloride derivatives (JONO, K .; ΤΑΚΑΥΑΜΑ, Τ .; KUNO,

M. et al. (2004) Effect of alkyl chain Iength of benzalkonium chloride on the

bactericidal activity and binding to organic materials. Chem. Pharm. Bull., 34, 4215-24);

2) Sphingosine (DRAKE, R.D .; BROGDEN, K.A.; DAWSON, D.V. et al. (2008) Antimicrobial Iipids at the skin surface. J. Lipid Res., 49, 4-11);

3) Fatty acid amines (KITAHARA, T .; KOYAMA, N .; MATSUDA, J.

et al. (2004) Antimicrobial activity of saturated fatty acids and fatty amines against methicillin-resistant Staphylococcus aureus. Biol. Pharm. Bull., 27, 1321-26);

4) Chlorhexidine (COOKSON, B.D .; BOLTON, M.C .; PLATT, J.H. (1991) Chlorhexidine resistance in methicillin-resistant Staphylococcus aureus or just an

elevated MIC? An in vitro and vivo assessment. Antimicrob. Agents Chemother., 35, 1997-2002);

5) Cationic polymers (KÜGLER, R .; BOULASSA, O .; RONDELEZ, F. (2005) Evidence of a charge density threshold for optimum efficiency of biocidal cationic surfaces. Mierobiol., 151, 1341-48., 1818. PALERMO , EF; KURODA, K.

(2009) Chemical structure of cationic 194 groups in amphiphilic polymethacrylates modulates the antimicrobial and hemolytic activities. Biomacromolecules, 10, 1416-1428).

Many of these cationic agents have been used as antiseptics and disinfectants for several decades with little or no resistance.

(GILBERT, P .; MOORE, L.E. (2005) Cationic antiseptics: diversity of action under a common epithet. J. Appl. Mierobiol., 99, 703-15). Recently, cationic lipid-conjugated neomycin B restored the antibacterial activity of this antibiotic against multiresistant S. aureus (MIC = 8 pg / mL) when compared to neomycin B (MIC = 256 pg / mL) (BERA. S .; ZHANEL, GG; SCHWEIZER, F.

(2008). Design, synthesis and antibacterial activities of neomycin-lipid conjugates: polycationic Iipids with potent gram-positive activity. J. Med. Chem., 51, 6160-64). In addition, the cationic lipid DODAB was combined with the antifungal miconazole leading to a synergistic formulation against Candida albicans (VIEIRA, DB; PACHECO LF; CARMONA-RIBEIRO AM (2006). Colloid Interface Sci., 293, 240-247. LINCOPAN, N.; CARMONA-RIBEIRO AM (2006) Lipid-covered drug particles: combined action of dioctadecyldemethylammonium bromide and amphotericin J. Antimicrob. Chemother., 58, 66-67). Goals

The objectives of the present invention are:

- production of synergistically acting tuberculostatic agents involving low-dose components in the combination;

- production of low cost and easily obtainable agents for incorporation of tuberculostatic drugs;

- perfect control of the proportion of microbicidal agents in the composition;

- dose reduction and consequent decrease of toxicity of antimicobacterial agents in the composition due to the synergistic action between the components of the formulation;

- incorporation of liposoluble and water-soluble drugs in the same formulation.

Short Description

The present invention relates to a hybrid tuberculostatic agent comprising (a) one or more dialkyl dimethyl ammonium salts and (b) rifampicin adsorbed in (a). In addition to the process of preparing the hybrid tuberculostatic agent, the invention further contemplates pharmaceutical composition containing such agent, method of treating tuberculosis and its use. Detailed Description

The strategy of restoring activity of tuberculostatic agents through the use of amphiphilic compounds containing the quaternary ammonium group is the object of the present invention. Thus, the invention deals with formulations with antimicobacterial properties, featuring a cationic compound of amphiphilic character and a liposoluble tuberculostatic drug acting synergistically.

The present invention relates to a hybrid tuberculostatic agent comprising (a) one or more dialkyl dimethyl ammonium salts and (b) rifampicin adsorbed in (a). Hybrid tuberculostatic agent means the composition of two or more agents of proven activity against Mycobacterium but having chemical structures belonging to different groups of substances such as compounds containing the quaternary ammonium group, rifampicin and rifampicin-like compounds. liposoluble tuberculostatic drugs, cationic polymers or surfactants, etc.

The dialkyl dimethyl ammonium salt, which is an amphiphilic compound containing the quaternary ammonium group, is a dioctadecyl or dihexadecyl dimethyl ammonium salt, especially halide salts, preferably chlorides, bromides and iodides. As a preferred embodiment of the invention, the hybrid tuberculostatic agent comprises dioctadecyl dimethyl ammonium bromide (DODAB).

The dialkyl dimethyl ammonium salts are in the form of bilayer fragments or large vesicles (liposomes) presenting rifampicin (the liposoluble drug) adsorbed on the amphiphilic compound.

The hybrid tuberculostatic agent comprises dialkyl dimethyl ammonium (a) salt in a concentration ranging from 0.1 to 100.0 pg / mL. Preferably, the dialkyl dimethyl ammonium salt (a) is present in a concentration ranging from 1.0 to 10.0 μg / mL; more preferably 2.0 or 4.0 pg / ml.

Regarding the liposoluble drug concentration, the hybrid tuberculostatic agent comprises rifampicin in the concentration ranging from 0.01 to 50.0 pg / mL. Preferably, rifampicin is present at a concentration ranging from 0.1 to 5.0 pg / mL; more preferably 1.0 or 2.0 pg / mL

According to the invention, the ratio of dialkyl dimethyl ammonium salt (a): rifampicin (b) present in the tuberculostatic agent ranges from 2: 1 to 2: 2 by mass; preferably 2: 1.

Tuberculostatic agents, according to the invention, have a mean diameter and zeta-potential varying according to the form of the dialkyl dimethyl ammonium salt. The rifampicin-containing bilayer fragments have a mean diameter ranging from 60 to 90 nm, preferably 80 ± 1 nm; and zeta potential ranging from 20 to 60 mV, preferably 30 ± 1 mV. Rifampicin-containing vesicles have a mean diameter ranging from 400 to 500 nm, preferably 460 ± 2 nm; and zeta potential between 30 and 80 mV, preferably 43 ± 1 mV. The present invention further relates to a pharmaceutical composition comprising the hybrid tuberculostatic agent described herein and pharmaceutically acceptable excipients. Such a composition may be in the following pharmaceutical forms: aerosol, sterile parenteral injection dispersion, topical ointments or gels.

Specifically, the composition may comprise inhalation aerosols for pulmonary tuberculosis or multiple drug formulations for parenteral injection in the case of tuberculosis affecting other organs.

Excipients are selected from stabilizing agents such as polyethylene glycol, polysorbates, chitosan, carboxymethylcellulose and other biopolymers or thickening agents or gelling agents such as gums, gelatins or polysaccharides such as heparansulfate.

Optionally, the pharmaceutical composition further comprises a second tuberculostatic drug selected from isoniazid, pyrazinamide, ethambutol, streptomycin, etionamide, terizidone, ofloxacin, clofazimine or mixtures thereof.

The process of preparing hybrid tuberculostatic agent comprises the following steps:

(i) obtaining dialkyl dimethyl ammonium salt bilayer fragments (a) by tip sonication or dialkyl dimethyl ammonium salt vesicles (a)

by vortexing, both in low ionic strength solution containing

rifampicin (b);

(ii) dialysis of the dispersions obtained in (i) for 12 to 24 hours.

The temperature employed in step (i) ranges from 55 ° to 70 ° C; preferably 60 ° C is employed. The solution employed in (i) has an ionic strength of 0.01 to 1 mM NaCl as the monovalent salt, such that the aqueous solution contains the liposoluble drug at a concentration below its water solubility limit.

Dialysis step (ii) aims to eliminate unincorporated (i.e. adsorbed) drug to bilayer fragments or vesicles, as well as to exclude the toxic solvent.

According to the invention, dialkyl dimethyl ammonium salt (a) is employed in a concentration ranging from 0.1 to 2.0 mM; preferably, the concentration of the dialkyl ammonium salt (a) ranges from 0.2 to 1.0 mM; more preferably from 0.3 to 0.5 mM. As a preferred form, 0.5 mM dialkyl dimethyl ammonium salt (a) is employed. Rifampicin is employed in a concentration ranging from 0.1 to 2.0 mM; preferably between 0.2 and 1.0 mM; more preferably from 0.3 to 0.5 mM. As a preferred form, 0.5 mM rifampicin (b) is employed.

The process described herein employs isotonic solution which may be chosen from sucrose, fructose or glucose, preferably 0.264 M D-glucose solution being employed.

The present invention further relates to a method of treating tuberculosis comprising administering the hybrid tuberculostatic agent and pharmaceutical composition described herein to a patient in need of said treatment.

The tuberculosis described herein refers to infections caused by bacteria of the genus Mycobacterium, especially M. tuberculosis, M. bovis, M. africanum, M. microti and M. canetti.

The invention also relates to the use of the hybrid tuberculostatic agent in the preparation of medicament for treating tuberculosis. The antimicobacterial agent described herein has very low toxicity due to the low concentrations of synergistically acting components and is suitable for pharmaceutical, medical, veterinary, biotechnological, biomedical or preventive applications.

Following are examples to further illustrate the invention, however, these are not

are intended to restrict the invention described herein. Examples

1. Materials

99.9% pure dioctadecyldimethylammonium bromide (DODAB) was purchased from Sigma (USA). Rifampicin (RIF) and isoniazid (ISO) were obtained from Fluka (Sigma-AIdrich (USA)). Anhydrous D-glucose (MW 180.16) was purchased from Merck (Darmstadt, Germany).

2. Preparation of DODAB bilayer fragments

Tip sonication was the method employed to disperse amphiphilic powder (DODAB) into 0.264M D-glucose, producing bilayer fragments with a final concentration of 2mM. The sonication process was carried out at 50 ° to 70 ° C for 20 minutes. The dispersion was filter sterilized prior to interaction with bacterial cells.

3. Preparation of the large DODAB vesicles To prepare the large DODAB vesicles, the amphiphilic powder was analytically weighed and transferred to a vial, adding the required amount of 0.264M D-glucose solution to obtain large vesicles with concentration of 2 mM and left in a water bath (30 minutes; 60 ° C), ie above the phase transition temperature of DODAB vesicles which was determined to be 44.3 ° C in water (VIEIRA, DB; PACHECO LF; CARMONA-RIBEIRO AM (2006) Assembly of a Model Hydrophobic Drug into Cationic Bilayer Fragments (J. Colloid Interface Sci.: 293, 240-247). The sample was taken from the water bath three times and vortexed for vesicle formation. 4. Determination of size and zeta potential of dispersions

Size and zeta potential of the dispersions were determined by the ZetaPIus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY1 USA) equipped with a 570 nm laser and 90 ° dynamic light scattering (GRABOWSKI, E .; MORRISON, I (1983), Particle size distribution from analysis of quasi-elastic scattering data (Measurements of Suspended Particles by Quasi-Elastic Light Scattering, 199-236). The average diameter measurements referred to in this work should be understood as hydrodynamic mean diameter (Dz). The zeta potentials (ζ) were determined by the electrophoretic mobility μ and the Smoluchowski equation ζ = μη / ε, where η and ε correspond to the medium viscosity and the dielectric constant of the medium, respectively.

5. Determination of RIF and ISO optical spectra in different solvents

A stock solution of rifampicin 1 mg / mL in dimethyl sulfoxide: methanol (DMSO: methanol) was prepared, which is the best organic solvent for rifampicin. This solution was diluted and the UV-visible spectrum was determined on different media (water, DMSO, DODAB fragments and large DODAB vesicles) against a white lipid dispersion and solvent (no drug), scanned at wavelength. 260 to 600 nm using a 1 cm quartz cuvette on a Hitachi U-2000 spectrophotometer.

Isoniazid was prepared at a concentration of 1 mg / mL in water (the best solvent for this drug). This solution was diluted and the UV-visible spectrum in water and large DODAB vesicles determined against a white lipid dispersion and water (drug free) at a wavelength of 240 to 400 nm.

6. Preparation of bilayer (BF) fragments in the presence of rifampicin

Rifampicin stock solution (20 mM) was diluted at various concentrations for interaction with DODAB bilayer fragments. Measurements of drug size and zeta potential alone or incorporated into the DODAB bilayer fragments were determined using the ZetaPIus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holtsville, NY1 USA) equipped with a 570 nm laser and dynamic light scattering. 90 ° (GRABOWSKI, E.; MORRISON, I. (1983). Particle size distribution from analysis of quasi-elastic scattering data. Measurements of Suspended Particles by Quasi-Elastic Light Scattering, 199-236).

7. Preparation of DODAB Large Vesicles (LV) with Rifampicin and Isoniazid

Rifampicin and isoniazid were solubilized in 0.264M D-glucose solution at 0.1mM concentration, and added to the heavy DODAB powder to a final concentration of 2mM. After mixing DODAB with rifampicin or isoniazid (prepared alone), the vesicles were prepared as described above. In the process of vesicle formation, drugs are incorporated into the inner aqueous compartment of the liposome or lipid bilayer, depending on the solubility characteristic of each drug.

8. Dialysis method to determine drug incorporation percentage in DODAB vesicles

Cellulose dialysis membranes were obtained from Sigma-AIdrich, cut to a length of approximately 10 cm and boiled in a 0.1 M solution of ethylenediamine tetraacetic acid (EDTA) for 30 minutes to open the pores. They were then thoroughly washed with Milli-Q water and stored in a bottle of Milli-Q water in the refrigerator. For dialysis, 3 2 mL aliquots of DODAB-rifampicin solution were transferred to the cellulose sachets. The sachets were tied to a glass pipeline bag to form a 90 ° angle and left in a beaker with 250 mL 0.264M D-glucose under gentle stirring for 24 hours, exchanging the D solution. - glucose after 1, 2, 3 and 8 hours. Prior to dialysis the absorbance readings of the 3 samples were taken at a wavelength of 334 mm and after dialysis the absorbance readings of the samples were also taken to determine the concentration of rifampicin incorporated into the DODAB. The liposomes were treated with 50% ethanol for gallbladder disruption and determination of the incorporated rifampicin concentration. Cellulose sachets containing only 0.1 mM RIF solution and only 2 mM DODAB solution were dialyzed under the same conditions to control the experiment for these Λ 5

10

15

20

25

18/35

Samples were also taken absorbance readings before and after dialysis in 50% ethanol.

Samples of 2 ml of 0.1 mM isoniazid were also placed in cellulose sachets and tied as above. For this drug the dialysis time required for incorporation was shorter, the samples were dialysed for a period of 12 hours, with two D-glucose solution changes. Absorbance readings were taken before and after dialysis to determine the incorporated isoniazid concentration. Prior to dialysis the absorbance readings of the 3 samples were taken at a wavelength of 260 mm and after dialysis the absorbance readings of the samples were also taken to determine the concentration of isoniazid incorporated into the DODAB. The samples were treated with 50% ethanol for gallbladder disruption and determination of isoniazid concentration. Cellulose sachets containing only 0.1 mM ISO solution and only 2 mM DODAB solution were dialyzed under the same conditions for control of the experiment, for these samples absorbance readings were taken before and after dialysis in 50% ethanol. .

9. Incorporation of rifampicin into DODAB liposome by the ultracentrifusion method

The liposomal preparation (DODAB / RIF) was centrifuged in an ultracentrifuge (BECKMAN optima TLX ultracentrifuge) for 5 hours at 80,000 rpm to separate drug incorporated into the drug-free vesicles (at the bottom of the tube) to determine drug uptake. The 2 mM DODAB lipid dispersion and the drug solution alone were centrifuged for control of the experiment, the DODAB dispersion supernatant alone was blank for reading the free drug supernatant (which was not retained on the liposome). Absorbance readings were taken at 334 nm on Hitachi 2000 spectrophotometer. Incorporated drug concentration (Cmc) was calculated using the formula below:

Cinc - Ctotal "Csupernatant

The embed percentage (% lnc) was calculated as follows:

% lnC = (Ctotal "C5Obout) / Ctotal x 100

10. Cultivation of Mvcobacterium smegmatis and Mvcobacterium tuberculosis

M. smegmatis (ATCC mc2155) was reactivated in Middlebrook 7H9 Broth (Difco - Detroit, USA) for 48 hours at 30 ° C while shaking, plated with Mueller-Hinton Agar (Hi-Media Laboratories Pvt, India) at incubated for 72 hours in an oven at 30 ° C. An elevation of this culture was inoculated in 10 ml Middlebrook 7H9 broth and incubated at 30 ° C with shaking for 48 hours during which the strain is in the exponential phase of growth.

M. tuberculosis H37Rv bacterial suspension was prepared

dispensing an elevation of the growing sample for approximately 28 days in Lowestwein-Jensen Medium (BBL ™ - Becton Dickinson Microbiology System, Sparks, MD, USA) in screw-capped glass tubes containing sterile glass beads in 2 mL of sterile distilled water. After homogenization on a mechanical shaker, 200 μί was transferred to a tube containing 2 mL Middlebrook 7H9 culture medium supplemented with 10% OADC (BBL ™ - Becton Dickinson Microbiology System, Sparks, MD, USA) and 0.2% glycerin . These cultures were incubated in shaker under shaking at 37 ° C for 7 to 10 days to reach exponential growth phase. Bacterial suspensions were centrifuged for 10 minutes and washed

in 0.264 M D-glucose solution, this centrifugation and washing process being repeated twice more. The D-glucose suspension was standardized with McFarIand scale tube 1 to be used in interaction with DODAB vesicles or bilayer fragments to determine cell viability. 11. Determination of cell viability of Mycobacterium smegmatis as a function of DODAB concentration.

One hour interaction between 0.5 mL of the bacterial suspension and 0.5 mL of DODAB LV (large vesicle) or BF (bilayer fragment) (0.0005 - 1.0 mM DODAB) was performed. After the interaction time, 100 μί of each concentration, diluted in 0.264M glucose, in triplicate was plated in triplicate to inoculate about 100 cells per plate. The plates were incubated at 30 ° C for 72 hours and colony forming units were counted. Positive control was performed by interaction of 0.5 mL of the bacterial suspension and 0.5 mL of 0.264M D-glucose solution, and plated 100 μί of the diluted suspension.

12. Determination of cell viability of Mvcobacterium tuberculosis as a function of time of interaction with DODAB.

Assays were performed with interaction time variation between 0.5 mL of standard bacterial suspension and 0.5 mL of DODAB LV or BF (0.1 mM DODAB). After interaction time, triplicate plating was performed on A 20/35

.. plates of Middlebrook 7H10 Agar supplemented with OADC and then incubated in an oven with 5% CO2, 10% humidity and 37 ° C for a period of 3 to 4 weeks. After incubation time the plates were removed from the greenhouse for counting colony forming units and determination of cell viability.

13. Determination of minimum bactericidal concentration (CBIW DODAB or RIF against M. smeqmatis

The macrodilution method was used to determine the minimum bactericidal concentration (CBM) 1 following the recommendations of the Cinical Laboratory Standards Institute M07-A8 and M24-A. An adaptation to the macrodilution test was required. Culture media components (eg amino acids) could inactivate the action of DODAB (LINCOPAN, N .; CARMONA-RIBEIRO AM (2006). Lipid-covered drug particles: combined action of dioctadecyldemethylammonium bromide and amphotericin B or miconazole J. Antimicrob Chemother., 58, 66-67). The 0.264M D-glucose solution was used to replace the culture medium for drug dilution and bacterial inoculum standardization. RIF and DODAB concentrations ranged from 512 to 1 pg / mL to a volume of 1 mL, each tube was inoculated with 1 mL of the standard bacterial suspension (105 CFU / mL). The tubes were incubated at 30 ° C for 72 hours. A 100 μί aliquot was taken from each tube and plated onto Mueller-Hinton agar (in duplicate), the plates were incubated at 30 ° C for 72 hours and CBM determined as the lowest concentration of the drug that killed> 99.9%. of bacterial inoculum.

14. Determination of synergistic effect between rifampicin and DODAB for M. smeqmatis

The macrodilution method was used to perform the

synergism between DODAB and RlF. Serial ratio two dilution to RIF at final concentrations ranging from 512 to 1 μg / mL was made to a volume of 500 μί per tube. In the tubes containing the serial dilution of RIF, 500 μί DODAB 2 Mg / mL was added. On the other hand, serial DODAB dilutions were made at final concentrations ranging from 512 µg / ml to a volume of 500 µg per tube. In tubes containing DODAB RIF at a concentration of 2 pg / ml was added. RIF and DODAB BF were mixed and allowed to interact for 1 hour before addition of bacterial cells. In each tube was added 1 mL of the standard bacterial suspension to a final concentration of 105 CFU / mL. The tubes were incubated in an oven at 30 ° C for 72 hours. A 100 μί aliquot of each tube was plated on Muller Hinton agar plates (in duplicate) and incubated at 30 ° C for a further 72 hours. CBM was determined as the lowest concentration of the drug that killed> 99.9% of the bacterial inoculum. CBM of the drugs alone and in combination was used to determine the synergistic effect. According to Odds, 2003 (ODDS, FC Synergy1 antagonism, and what the checkerboard puts between them (2003). J. Antimicrob. Chemother., 52.1), synergism can be determined by the bactericidal fractional concentration sum (ICFB). ) of the two drugs in combination. CFB is obtained by the CBM ratio of the drug used in combination with the CBM of the drug alone. When ICFB <0.5 occurs drug synergism, while an index> 4 indicates antagonism, a value between 0.5 and 4 indicates drug independent behavior. Results and discussion

Cell viability curves after 1 hour of interaction of M. smegmatis 3.5 χ 106 CFU / mL as a function of dispersed DODAB concentration as DODAB BF (·) or DODAB LV (■), for a range of 0.0005 to 1 mM DODAB1 are shown in Figure 2. As already described, DODAB has a microbicidal effect against bacteria and fungi due to its positive charges on the quaternary ammonium group present in its chemical structure. (CAMPAIGN, MTN; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1999) Interactions Between Cationic Liposomes And Bacteria: The Physical-Chemistry Of The Bactericidal Action. J. Lipid Res., 40, 1495-1500. CAMPAIGN, TN. TN ; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (2001) Interactions between cationic vesicles and Candida albicans (J. Phys. Chem. B, 105, 8230-8236).

Micromolar DODAB concentrations were effective against M. smegmatis. For the two different dispersions (DODAB BF and DODAB LV), in 0.002 mM DODAB there was 50% of cell viability for a final viable cell concentration of 3.5 χ 106 CFU / mL.

Cell viability curves for M. tuberculosis 1.8 χ 1Ό6 CFU / mL as a function of dispersed DODAB concentration as DODAB BF (·) or DODAB LV (■) at 0.1 mM, varying the time For M. tuberculosis it was necessary to vary the interaction time because this microorganism is slow growing, so the interaction time with DODAB and other antibiotics should be longer than for other fast growing microorganisms. . In this case, 50% of cell viability occurred with approximately 45 hours of interaction at »1. *

22/35

concentration of 0.1 mM. DODAB has activity for M. tuberculosis requiring higher concentrations and interaction time.

Quaternary ammonium compounds (also known as cationic detergents) are widely used as antiseptics or disinfectants as membrane active agents. A sequence of events has been proposed for microorganisms exposed to cationic agents: a) adsorption and penetration of the agent within the cell wall; b) reaction with the cytoplasmic membrane (lipid or protein) followed by membrane disorganization; c) extravasation of low molecular weight material; d) degradation of proteins and nucleic acids; e) wall lysis caused by autolytic enzymes. There is thus a loss of the structural organization and integrity of the cytoplasmic membrane in the bacterium, along with other detrimental effects on the bacterial cell (Maçdonnell, G.; Russell, AD (1999). Antiseptics and disinfectants: activity, action, and resistance. Clin. Microbiol Rev. 12). The mechanism of cationic lipid biocidal action (DODAB) has been related to the reversal of charge from negative to positive cell (CAMPAIGN, MTN; MAMIZUKA, EM; CARMONA-RiBEIRO1 AM (1999) Interactions Between Cationic Liposomes And Bacteria: The Physical Chemistry Of The Bactericidal Action (J. Lipid Res., 40, 1495-1500). Adsorption of cationic bilayers composed of dioctadecyldimethylammonium bromide (DODAB) on bacterial or fungal cells changes the cell charge signal from negative to positive, with a close relationship between the positive charge of bacteria or fungi and the bactericidal or fungicidal effect (CAMPANHA MTN ; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1999) Interactions Between Cationic Liposomes And Bacteria: The Physical Chemistry Of The Bactericidal Action J. Lipid Res., 40, 1495-1500 VIEIRA, DB; PACHECO LF; CARMONA- Ribeiro AM (2006) Assembly of a Model Hydrophobic Drug into Cationic Bilayer Fragments (J. Colloid Interface Sci., 293, 240-247). Regarding the mechanism of action of DODAB, there was no cell lysis or cationic gallbladder rupture (MARTINS, LMS; MAMIZUKA, EM; CARMONA-RIBEIRO, AM (1997). Cationic Vesicles as Bactericides. Langmuir, 13, 5583-5587. VIEIRA DB; CARMONA-RIBEIRO, AM (2006) Cationic Lipids and Surfactants as Antifungal Agents (Rifode of Action J. Antimicrob. Chemother., 58, 760-767).

The interaction of cationic vesicles composed of phosphatidylcholine, cholesterol and dioleoyloxytrimethylammonium propane (PC: Col: DOTAP) was studied in P. aeruginosa cells with evaluation of the influence of bacterial cell surface charge on the fusion process between vesicles and cells. Bacteria are negatively charged, which favors their electrostatic interaction with cationic lipid vesicles, but other factors may also be responsible for fusion. For example, an 18-kDA P. aeruginosa outer membrane protein favored the cell fusion process with rhodamine-labeled cationic vesicles and thus facilitated fluorescence microscopy fusion visualization (DRULLIS-KAWA, Z .; DOROTKIEWICZ -JACH, A.; GUBERNATOR, J .; GULA, G .; BOCER, T .; DOROSZKIEWICZ, W. (2009) The interaction between Pseudomonas aeruginosa and cationic PC: Chol: DOTAP Iiposomal vesicles versus outer-membrane structure and envelope properties of bacteria (Cell. Int. J. Pharm. 367, 211-219). Notably, the zeta potential of cationic vesicles has been related to cell viability. Cell membranes have negatively charged domains in their structure, which can attract cationic vesicles. A high affinity of cationic vesicles for cell membranes is expected, primarily by electrostatic interaction. Then cationic vesicles can be captured by the membranes by various mechanisms, including endocytosis, pinocytosis and phagocytosis (WEINSTEIN, JN (1981). Liposomes as a targeted drug carrier: a physical chemical perspective. Pure Appl. Chem., 53, 2241 -2254 LORENZ, MR; HOLZAPFEL, V.; MUSYANOVYCH, A.; NOTHELFER, K.; WALTHER, P.; FRANK H. (2006). Uptake of functionalized, fluorescent polymerized particles in different cell Iines and stem cells, Biomaterials, 27, 2820-2828). Other interaction studies between cationic vesicles with cells have shown that the behavior of liposomes in cells depends primarily on surface charge (Miller, Cr; Bondurant, B.; Mclean, SD; McGOVERN, KA; O'BRIEN, DF (1998)). In vitro liposome-cell interactions: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes Biochemistry, 37, 12875-12883 JUBEH, TT; BARENHOLZ, Y .; RUBISNSTEIN, A. (2004) Differential adhesion of normal and inflamed rat colonic mucosa by charged liposomes Pharmaceutical Research, 21, 447-453 LIANG, CH .; CHOU, TH (2009) Effect of chain Iength on physicochemical properties and cytotoxicity of cationic vesicles composed of phosphatidylcholines and dialkyldimethylammonium bromides. Chem. Phys. Lipids 158 (2): 81-90).

Figure 4 shows the absorption spectrum of solubilized rifampicin in its best organic solvent (Figure 4A), in water (Figure 4B), DODAB bilayer fragments (Figure 4C) and large DODAB vesicles (2 D), to 16 (a); (B); (C); and 64 (d) μο / ml rifampicin. The absorption spectrum of RIF in the presence of dispersions obtained by sonication, vortexing or in water are similar to the spectrum of RIF in its best organic solvent (Figure 4A-D). In the presence of these solvents the RIF remains in its monomeric form, so DODAB dispersions are good solubilizers for this drug. The RIF solubility in water is 1.3 mg / mL, so at the concentrations that were used to read the absorption spectrum, this drug is water soluble, so the water spectra are similar to the spectrum at their best. organic solvent. Above this limit of solubility the drug would be aggregated. The hydrophobic area of the bilayer fragments is believed to provide additional sites for interaction between the drug and the bilayer. The molar absorptivity (ε) of the drug in dimethyl sulfoxide (DMSO) was determined to be 15,130 M "1cm" 1. Similar values were obtained for the drug in DODAB dispersions: for DODAB BF, 14,840 M'1 cm1 and for DODAB LV (15,000 M1Cm1) (attached Table 2).

The solubilization of two hydrophobic antifungal drugs, amphotericin B (AnB) and miconazole in DODAB bilayer fragments, showed the possibility of using DODAB as a carrier of these drugs (PACHECO, LF; CARMONA-RIBEIRO, AM (2003) Effects of synthetic Iipids J. Colloid Interface Sci., 258, 146-54 VIEIRA, DB; CARMONA-RIBEIRO AM (2001) Synthetic bilayer for solubilization of amphotericin BJ Colloid Interface Sci., 244, 427-431 ). Physicochemical studies characterized the solubilization of AnB in DODAB and DHP nanofragments, evaluated by spectrophotometry and size distribution in the mixtures by dynamic light scattering. After interaction, the visible UV absorption spectrum of AnB in DODAB fragments was similar to that obtained by solubilization of the drug in its best organic solvent. Size distribution revealed the disappearance of drug aggregates, postulating that bilayer fragments are excellent solubilizers for AnB (VIEIRA, DB; CARMONA-RIBEIRO AM (2001) Synthetic bilayer for amphotericin BJ Colloid Interface Sci., 244, 427-431). Later, these experiments were extrapolated to the miconazole antifungal, giving identical results (PACHECO, LF; CARMONA-RIBEIRO, AM (2003). Effects of synthetic Iipids on solubilization and colloid stability of hydrophobic drugs. J. Interface Colloid Sci., 258,146-54). From the results of our experiments we can also say that rifampicin, a drug used in the treatment of tuberculosis, is also solubilized in DODAB bilayer fragments when added at concentrations greater than their limit of water solubility (1.6 mM).

Figure 5 shows the size distribution of 1 mM DODAB BF in water (Figure 5A), 2 mM RIF in water (1.6) (Figure 5B), and 1 mM dispersed DODAB as DODAB BF in the presence of 2 mM of RIF (Figure 5C). Therefore, the drug was added in aqueous solution at a concentration value above its water solubility limit. Therefore, in 2 mM RIF in water, an aggregate size distribution of the drug is observed (Figure 5B). However, in the presence of DODAB fragments, there was a typical fragment size distribution only, with total solubilization of the drug aggregates. There was a large change in the size distribution of the drug alone in water relative to that of the drug in the presence of DODAB bilayer fragments. Rifampicin at a concentration of 2 mM has an average size of 760 ± 20 nm and zeta potential 0 ± 10 mV (aggregate drug). When solubilized in the bilayer fragment, the average size decreases to 80 ± 1 nm and zeta potential becomes 31 ± 1 mV (Figure 5C). The size is the same as for the drug-free DODAB fragments: mean diameter 84 ± 1 nm. However, the zeta-potential was lower than that obtained for drug-free fragments (65 ± 1 mV). Large aggregates of the drug were solubilized by DODAB fragments decreasing zeta potential. The size (Dz) and zeta potential (ζ) values of Figure 5A-C are presented in the attached Table 3. This same 2 mM drug solution was also solubilized at lower DODAB concentrations. In Figure 6 the complete solubilization of drug aggregates is shown, even at 0.5 mM DODAB. Table 4 (attached) shows the size (Dz) and potential zeta (ζ) values of Figure 6A-C. In addition to hydrophobic interactions between DODAB BF and rifampicin, there was also a change in the zeta potential of the preparations, the zeta potential of the fragments changed from 65 ± 1 mV to 31 ± 1 mV in the presence of rifampicin, suggesting possible insertion of the drug into the bilayer, promoting separation cationic lipid heads and decreased charge density and zeta potential. Other authors have also observed a decrease in zeta-potential modulus by interaction of rifampicin and ofloxacin-loaded bilayers (BERMUDEZ, M.; MARTINEZ, E.; MORA, M .; SARIGRAS, ML; MADARIAGA, MA (1999). Molecular and physicochemical aspects of the interactions (the tuberculostatics of oxoxacin and rifampicin with Iiposomal bílayers: a P-NMR and DSC study. Colloid Surf. A 158, 59-66). Rifampicin is partially ionized (-40%) to anionic form at pH (7.4). This point is important to know if electrostatic interactions with charged bilayers occur. Rifampicin interacts strongly with DMPC (dimyristoyl-La-phosphatidylcholine) liposomes by inserting into the bilayer (RODRIGUES, C.; GAMEIRO, P.; PIETRO, M .; CASTRO, B. (2003) Interaction of rifampicin and isoniazid with Iarge unilamellar liposomes: spectroscopic Iocation studies Biochim.

Biophys. Acta, 1620,151-159).

In Figure 7 are the optical spectra of light absorption by isoniazid in water (Figure 7A) or in large DODAB vesicles (Figure 7B), for drug concentrations of 10 (a), 20 (b), 30 (c), 40 (d) μg / mL As isoniazid is a water-soluble drug, incorporation occurs in the internal aqueous compartment of the DODAB liposome, unlike rifampicin, which incorporates both in the internal aqueous compartment of the vesicles and in the bilayer because it is of a drug with hydrophobic characteristics. There is great similarity between the water isoniazid spectra and the large DODAB vesicles. Table 5 (attached) shows the maximum peak absorbance and molar absorbance values of isoniazid (20 μg / mL) in water and large DODAB vesicles. Isoniazid has only one peak maximum absorbance. The ε values of isoniazid in water and large vesicles are similar. As isoniazid is quite water soluble and is a small molecule, it could eventually be incorporated into the inner aqueous compartment of the vesicles as long as it was not permeable through the bilayer. To evaluate its permeability, dialysis and ultracentrifugation experiments were performed for the separation between drug incorporated into vesicles and free drug. The percentage of drug incorporation into the 2 mM DODAB liposome (% lnc) was determined by the amount of drug retained by DODAB dispersion after extensive dialysis. The same liposomal preparation (DODAB / RIF) was ultracentrifuged to determine the percentage of incorporation by this alternative method.

Table 6 (attached) shows the absorbance values before and after dialysis for free rifampicin and liposomal rifampicin. The samples were treated with 50% ethanol for gallbladder rupture before absorbance reading. Readings were performed in triplicate and the mean and standard deviation were calculated. Percent drug uptake (% lnc) in DODAB large vesicles was calculated from absorbance readings before and after dialysis from the equation:

% lnc = 100 X [(Abs dd sample / AbS ad sample) ~ (AbS dd control / AbS ad control)]

Since% lnc depends on lipid concentration, another characteristic parameter of a given vesicle population that would depend only on lipid size and not on its concentration would be the incorporation efficiency (Ef Inc) defined as the% lnc normalized to molar concentration of vesicles. lipid (C) in M whose unit will be in liters per mol of lipid:

Ef Inc =% Inc / (100 C)

The drug alone freely crosses the dialysis membrane. Prior to dialysis, the absorbance value of the 0.1 mM RIF solution was 1.984. After dialysis this value decreased to 0.120. When this same drug solution is used to prepare DODAB liposomes, virtually all of the drug is incorporated as inferred by the absorbance value of 1.621, totaling 81% incorporation of this drug into the 2 mM DODAB vesicles (Table 6 attached). Considering the mean DODAB gallbladder radius as 230 nm, the area per DODAB molecule is 0.6 nm2 (CLAESSON, P.; CARMONA-RIBEIRO, AM; KURIHARA, K. (1989). Dihexadecyl phosphate monolayers: intralayer and interlayer J. Phys. Chem. 93, 917-22) and its self-association as closed bilayers (vesicles), one can assume a spherical shape for the vesicle and calculate the area and volume per vesicle to obtain a volume. encompassed by one mol of DODAB molecules equal to 5.5 LVmoI. This theoretical incorporation efficiency (Ef Inc) would reflect the ability of this vesicle population to encompass a fraction of the total aqueous volume of the solution per mole of DODAB. The experimentally obtained Ef Inc for rifampicin is 405 L / mol. This value is 100 times higher than the previously calculated theoretical value (5.5 LVmoI). Therefore, it can be directly inferred that the incorporation is not only in the internal aqueous compartment occurring mainly in the DODAB bilayer itself. To confirm this drug incorporation preferentially into the bilayer, the dialysis experiment and determination of incorporation efficiency was also done with a DODAB dispersion that has no internal aqueous compartment: the sonicated DODAB bilayer fragments (DODAB BF). As shown in Table 6, also in this case, Ef Inc was very high, 375 L / mol indicating adsorption and / or insertion of IFR in the bilayer.

DODAB liposome samples were also dialyzed under the same conditions to control the experiments. DODAB vesicles or bilayer fragments do not pass through the dialysis membrane because the concentration of DODAB within the dialysis bag was the same before and after dialysis.

The percentage of IFR incorporation into the DODAB liposome by ultracentrifugation was obtained by concentration of drug present in the supernatant after centrifugation. Total drug concentration prior to centrifugation (0.075 mM) minus supernatant concentration (0.017 mM) resulted in the incorporated drug concentration (0.058 mM). With this concentration of incorporated drug it was possible to calculate the percentage of incorporation: 77.3% of incorporated RIF in DODAB liposomes, in excellent agreement with the results obtained by dialysis. Due to the hydrophobicity of rifampicin, this drug has a high affinity for DODAB lipid bilayers, resulting in a good percentage of incorporation. Figure 8 illustrates the size of the 2 mM DODAB liposome alone (Figure 8A), and with the incorporated RIF (Figure 8B). In this case, 0.1 mM RIF did not cause significant change in the physical properties of DODAB vesicles due to the low drug concentration. Table 7 (attached) shows the size and zeta potential measurements of liposomes in rpesença and in the absence of RIF.

Different liposomal formulations were prepared with isoniazid to verify the efficiency of incorporation into different lipids, these formulations were made because this drug was permeable to DODAB liposomes, unlike rifampicin in which there was 75% incorporation by the dialysis method. The attempt was to use a neutral lipid DPPC, together with DODAB and a natural lipid (asolecithin) that forms multilamellar liposomes. In Table 8 (attached) are the incorporation efficiency results of the preparations of 2 mM DODAB with 0.1 mM isoniazid solution, 1: 1 DODAB / DPPC liposome with 0.1 mM isoniazid solution and asolecithin 2 liposomes. mM with a higher concentration of 10 mM isoniazid. Isoniazid was permeable to different liposomal formulations because it is a small molecule. Incorporation percentages were close to 0% in all liposomal preparations tested. The percentage of rifampicin incorporation was evaluated in different liposomal formulations (PC - phosphatidylcholine; DPPC-dipalmitoyloglycero-PC; DSPC-disteroyloglycero-PC) containing or not cholesterol. The incorporation percentage is influenced by liposome composition, DPPC and DSPC have higher incorporation percentages than PC1 with the addition of cholesterol in the three formulations the incorporation percentage decreases. The lipid composition of the liposome is very important in determining the percentage of drug incorporation. The amount of rifampicin incorporated increases with the degree of lipid saturation (DPPC <DSPC) compared with PC (ZARU1 M .; MOURTAS, S .; KLEPETSANIS, P .; FADDA1 A.M .;

ANTIMISIARIS, S.G. (2007) Liposomes for drug delivery to the Iungs by nebulization. Eur. J. of Pharm. and Biopharm., 67, 655-666). Similar results were observed in DPPC / Chol compound liposomes compared to PC / Chol, the incorporation percentage is higher in DPPC / Chol liposomes than PC / Chol, lipid chain length is an important factor in drug incorporation ( GURSOY, A.; KUT, E .; OZKIRIMLI, S. (2004) Co-encapsulation of isoniazid and rifampin in Iiposomes and characterization of Iiposomes by derivative spectroscopy. Int. J. Pharm., 271, 115-123). A significant difference in the percentage of incorporation was observed for both tuberculostatic drugs (rifampicin and isoniazid), rifampicin presented a higher percentage of incorporation in relation to isoniazid (GURSOY, A .; KUT, E .; OZKIRIMLI, S. (2004) Co- encapsulation of isoniazid and rifampin in Iiposomes and characterization of Iiposomes by derivative spectroscopy (Int. J. Pharm., 271, 115-123). The percentage of isoniazid incorporation also depends on the phospholipid composition, phosphatidylcholine and phosphatidylcholine liposomes showed higher incorporation percentages (approximately 3.2 and 3.4% respectively) than in phosphatidylcholine compounds with phosphatidylethanolamine and lysophosphatidylcholine (approximately 1 %), the low percentages of isoniazid incorporation is reported by the high membrane permeability of the drug (SOROKOUMOVA, GM; SELISHCHEVA, AA; MALIKOVA, NM; MININA, AS; SHVETS, Vl (2004). Incorporation of isoniazid into Iiposomes with different Iipid composition (Bull. Exp. Bioi Med., 137, 24-26). Some studies have shown that the% incorporation of hydrophobic drugs is higher than for hydrophilic drugs (BETAGARI, GV; PARSONS, PL (1992). Drug encapsulation and release from multilamellar and unilamellar liposomes. Int. J. Pharm., 81, 235- 241. DI GIULIO, Α .; MAURIZI, G .; ODOARDI, P .; SALETTI, Μ.Α .; AMICOSANTI, G .; ORATORE1 Α. (1993) Encapsulation of ampicillin in reverse-plane evaporation liposomes: a direct evaluation by derivative spectrophotometry (Int. J. Pharm., 74, 159-164). These results are in agreement with those obtained in this work, rifampicin a hydrophobic drug is well incorporated in the DODAB liposome whereas isoniazid a hydrophilic drug was permeable to

different liposomal preparations.

The minimum bactericidal concentration (CBM) of rifampicin and DODAB and the combination of DODAB / RIF against M. smegmatis are presented in Table 9 (attached). The RIF CBM for M. smegmatis is 32 μg / mL and when combined with DODAB this value decreased to 2 μg / mL in the presence of 2 Mg / m L DODAB. The DODAB CBM in the two preparations: large vesicles (DODAB LV) or bilayer fragments (DODAB BF) was determined at 4 Mg / mL1. This value was reduced to 2 Mg / mL when in combination with 2 Mg / mL. rifampicin in the DODAB BF / RIF formulation and the DODAB LV / RIF formulation with the combination of 2 Mg / mL DODAB LV and 1 Mg / mL RIF. With the data presented in the table it was possible to verify that CBM values are lower when DODAB is present than those when the drug is alone, so DODAB enhances the action of the drug. The DODAB / RIF formulation has synergistic effect, ICBF resulted in 0.5 which was calculated by summing the CBF of the two drugs in combination. According to Odds1 2003 (ODDS1 FC Synergy, antagonism, and what the checkerboard puts between them (2003) J. Antimicr. Chemother., 52, 1) an ICBF <0.5 corresponds to drug synergism, while an index> 4 indicates antagonism and between 0.5 and 4 indicates independent behavior. DODAB alone is a potent bactericidal agent, because DODAB is a cationic lipid and has in its structure quaternary ammonium group, in fact, quaternary ammoniums have bactericidal properties (CARMONA-RIBEIRO, AM; VIEIRA, DB; LINCOPAN, N. (2006). ) Cationic Lipids and Surfactants as Anti-infective Agents, Anti-Infective Agents in Medicinal Chemistry, 5, 33-54). Other studies have shown that conjugation of drugs with lipid increases its activity. Neomycin B shows low activity against strains of MRSA (methicillin resistant S. aureus) and P. aeruginosa, whereas kanamycin A has low activity against MRSA1 MRSE (methicillin resistant S. epidermidis), and P. aeruginosa. Lipid conjugation with cationic group restored MRSA activity of both aminoglycosides and kanamycin A activity for MRSE. Lipid conjugation in drugs increased the activity of the two aminoglycosides against resistant strains: the minimal inhibitory concentration (MIC) of neomycin B to MRSA was 256 Mg / mL, whereas neomycin B associated with lipid had an MIC of 8 Mg / mL ( BERA, S., ZHANEL1 GG; SCHWEIZER, F. (2008) Design, synthesis and antibacterial activities of neomycin-lipid conjugates: polycationic lipids with potent gram-positive activity (J. Med. Chem., 51, 6160-64) . The results show that conjugation of rifampicin with cationic lipid DODAB improved bacterial activity, reducing CBM from 32 Mg / mL to 2 Mg / mL. Similar results were obtained by Lincopan et al., 2006 (LINCOPAN, N .; CARMONA-RIBEIRO AM (2006) Lipid-covered drug particles: combined action of dioctadecyldemethylammonium bromide and amphotericin B or miconazole. J. Antimicrob. Chemother., 58, 66-67), the combination of DODAB BF with miconazole (MCZ) and amphotericin B (AMB) against strains of Candida albicans and Cryptococcus neoformans showed that the minimum fungicidal concentration (CFM) is lower in the liposomal formulation than when the drugs were administered. tested alone (AMB CFM against C. albicans was 4 mg / L and in the DODAB / AMB combination this concentration was reduced to 1 mg / L, MCZ CFM against the same strain was 8 mg / L, whereas in the formulation DODAB / MCZ CFM reduced to 1 mg / L), and for C. neoformans (AMB CFM was 2 mg / L, and in DODAB / AMB formulation reduced CFM to 1 mg / L on the other hand MCZ was 1 mg / L, this concentration was reduced to 0.5 mg / L). From the synergism calculation for C. albicans, which was 0.65 and 0.325 for DODAB / AMB and DODAB / MCZ, respectively, the combined action of cationic lipid and drugs could be considered synergistic only for miconazole. In the case of C. neoformans the combined action resulted in a value of 0.1 for DODAB / AMB and DODAB / MCZ, suggesting lipid and drug independent action, according to the interpretation of Odds, 2003. For the formulation developed in this work ( DODAB / RIF), the possible synergistic effect of the RIF and the cationic lipid was calculated, the synergism index was 0.5, this value represents a synergistic effect among the drugs tested for M. smegmatis. These results are similar to those obtained for the DODAB / MCZ formulation for C. albicans fungus. In DODAB and drug combinations, a small concentration of DODAB is required, just enough to produce a lipid layer surrounding the hydrophobic granules of the drugs. For future preclinical tests such as toxicity, it is believed that toxicity will be minimized given the very low doses in the combination. It is important to highlight the differential cytotoxicity of DODAB as it is much less cytotoxic to mammalian cells than to bacteria and fungi. Cell Type Initial Concentration of Viable Cell Concentration / DODAB for 50% (cells / mL) surviving / (mM) Normal Balb-c 3T3 mouse fibroblasts (clone A31) 1 χ 104 1,000 SVT2 mouse fibroblasts (transformed SV40) 1 χ 104 1,000 Candida albicans 2 χ 106 0,010 Escherichia coli 2 x 107 0,028 Salmonella typhimurium 2 χ 107 0,010 Pseudomonas aeroginosa 3 χ 107 0.005 Staphylococcus aureus 3 χ IO7 0.006 TABLE 1 Wavelengths (λ) Means of corresponding maximum absorbance solubilization (nm) (max) nm ε (W1Cnrr1) λΐ A2 DMSO 336 477 336 15.130 WATER 331 468 331 13,890 DODAB BF 336 476 336 14,840 DODAB LV 334 476 334 15,000 TABLE 2 Dispersion D ± δ (nm) ζ ± δ (mV)% RIF solvent (2 mM) 760 ± 20 0 ± 10 10 DODAB BF (1 mM DODAB) 84 ± 1 65 ± 1 - RIF (2 mM) / DODAB BF (1 mM DODAB) 80 ± 1 31 ± 1 10

TABLE 3 Dispersion D ± δ (nm) ζ ± δ (mV)% RIF solvent (2 mM) 760 ± 20 0 ± 10 10 DODAB BF (0.5 mM DODAB) 86 ± 2 61 ± 1 - RIF (2 mM) / DODAB BF (0.5 mM DODAB) 89 ± 1 26 ± 2 10

TABLE 4

Wavelengths (λ)

Means of corresponding to λ __ o / M-vm-iv

..... _,. ,. . -. , »A (max) nm ε (Μ cm)

maximum absorbance solubility (nm) v '

λ

WATER 260 260 2,995

DODABLV 260 260 2,717

TABLE 5

ABS334 ABS334 o / Eflnc

K before after / 0, nc (L / mol)

O1ImMRIF 1.984 ± 0.050 0.120 ± 0.050

DODAB LV 2 mM / RIF 0.1 mM 1.992 ± 0.050 1.621 ± 0.050 81 405

0.1mMRIF 2.300 ± 0.050 2.300 ± 0.050

DODAB BF 2 mM / RIF 0.1 mM 2,300 ± 0.050 1,750 ± 0.050 75 375

TABLE 6

Dz ± δ ζ ± δ

Dispersed (nm) (mV)

DODAB (2mM) 459 ± 2 45 ± 1

DODAB 2mM / RIF 0.1mM 473 ± 1 45 ± 1

TABLE 7 I 35/35

Absorbance 260 nm Before Dialysis After Dialysis

ISO 0.1 mM (Control) 0.542 0.061 - 2 mM DODAB / ISO 0.1 mM 0.540 0.052 0 DODAB / DPPC 1: 1 / ISO 0.1 mM 0.504 0.058 0 ISO 10 mM (Control) 20.0 0.017 - ASO 2 mM / ISO 10 mM 19.5 0.400 0.1

TABLE 8

CBM Microorganism (pg / mL) CFB ZCFB Drug Combination M. smegmatis 32 (RIF) 2 (2 DODAB BF) 0.0625 4 (DODAB BF) 2 (2 RlF) 0.5 0.5 4 (DODAB LV) 2 ( 1 RIF) 0.5

TABLE 9

Claims (32)

Hybrid tuberculostatic agent comprising (a) one or more dialkyl dimethyl ammonium salts and (b) rifampicin adsorbed in (a). Hybrid tuberculostatic agent according to claim 1 characterized in that the dialkyl dimethyl ammonium salt (a) is a dioctadecyl dimethyl ammonium salt. Hybrid tuberculostatic agent according to claims 1 and 2, characterized in that the dialkyl dimethyl ammonium salt (a) is chosen from the group of halides, preferably chloride, bromide and iodide. Hybrid tuberculostatic agent according to claims 1 to 3 characterized in that the dialkyl dimethyl ammonium salt (a) is dioctadecyl dimethyl ammonium bromide (DODAB). Hybrid tuberculostatic agent according to claim 1, characterized in that the dialkyl dimethyl ammonium salt (a) is in the form of bilayer or vesicle fragments. Hybrid tuberculostatic agent according to claim 1 characterized in that it comprises dialkyl dimethyl ammonium (a) salt in a concentration ranging from 0.1 to 100.0 pg / mL. Hybrid tuberculostatic agent according to claim 6 characterized in that it comprises dialkyl dimethyl ammonium salt (a) in a concentration ranging from 1.0 to 10.0 pg / mL; preferably 2.0 or 4.0 pg / ml. Hybrid tuberculostatic agent according to claim 1 characterized in that it comprises rifampicin (b) in a concentration ranging from 0.01 to 50.0 pg / mL. Hybrid tuberculostatic agent according to claim 8 characterized in that it comprises rifampicin (b) in a concentration ranging from 0.1 to 5.0 pg / mL; preferably 1.0 or 2.0 pg / mL. Hybrid tuberculostatic agent according to claim 1 characterized in that the ratio of dialkyl dimethyl ammonium salt (a): rifampicin (b) ranges from 2: 1 to 2: 2 by mass; preferably 2: 1. Hybrid tuberculostatic agent according to claims 1 and 5 characterized in that the bilayer fragments containing rifampicin have a mean diameter ranging from 60 to 90 nm. Hybrid tuberculostatic agent according to claim 11, characterized in that the rifampicin-containing bilayer fragments have an average diameter of 80 ± 1 nm. Hybrid tuberculostatic agent according to claims 1 and 5 characterized in that the rifampicin-containing vesicles have a mean diameter ranging from 400 to 500 nm. Hybrid tuberculostatic agent according to claim 13 characterized in that the rifampicin-containing vesicles have a mean diameter of 460 ± 2 nm. Hybrid tuberculostatic agent according to claims 1 and 5 characterized in that the rifampicin-containing bilayer fragments have zeta potential ranging from 20 to 60 mV. Hybrid tuberculostatic agent according to claim 15, characterized in that the rifampicin-containing bilayer fragments have a potential of 31 ± 1 mV. Hybrid tuberculostatic agent according to claims 1 and 5 characterized in that rifampicin-containing vesicles have a zeta potential ranging from 30 to 80 mV. Hybrid tuberculostatic agent according to claim 17 characterized in that rifampicin-containing vesicles have a zeta potential of 43 ± 1 mV. Pharmaceutical composition comprising hybrid tuberculostatic agent according to claims 1 to 18 and pharmaceutically acceptable excipients. Pharmaceutical composition according to Claim 19, characterized in that it comprises excipients selected from stabilizing agents such as polyethylene glycol, polysorbates, chitosan, carboxymethylcellulose and other biopolymers or gelling agents such as gums, gelatins or polysaccharides such as heparansulfate. Pharmaceutical composition according to claim 19, optionally comprising a second tuberculostatic drug selected from isoniazid, pyrazinamide, ethambutol, streptomycin, etionamide, terizidone, ofloxacin, clofazimine or mixtures thereof. Process for preparing a hybrid tuberculostatic agent according to claims 1 to 18, comprising the following steps: (i) obtaining dialkyl dimethyl ammonium salt bilayer fragments (a) by tip sonication or dialkyl dimethyl ammonium salt vesicles (a) by vortexing, both in low ionic strength solution containing rifampicin (b); (ii) dialysis of the dispersions obtained in (i) for 12 to 24 hours. Process for preparing a hybrid tuberculostatic agent according to claim 22, characterized in that it employs dialkyl dimethyl ammonium salt (a) in a concentration ranging from 0.1 to 2.0 mM. Process for the preparation of a hybrid tuberculostatic agent according to claim 23, characterized in that the dialkyl dimethyl ammonium salt (a) is employed in the concentration ranging from 0.2 to 1.0 mM; preferably 0.3 and 0.5 mM. Process for preparing a hybrid tuberculostatic agent according to claim 22, characterized in that it employs rifampicin (b) in a concentration ranging from 0.1 to 2.0 mM. Process for preparing a hybrid tuberculostatic agent according to claim 25, characterized in that it employs rifampicin (b) in a concentration ranging from 0.2 to 1.0 mM; preferably from 0.3 to 0.5 mM. Process for preparing a hybrid tuberculostatic agent according to claim 22, characterized in that the temperature in step (i) ranges from 55 ° to 70 ° C; preferably 60 ° C. Process for preparing a hybrid tuberculostatic agent according to claim 22, characterized in that it employs isotonic solution. Process for preparing a hybrid tuberculostatic agent according to claim 28, characterized in that the isotonic solution is chosen from sucrose, glucose or fructose; preferably 0.264M D-glucose solution is employed. A method of treating tuberculosis comprising administering the hybrid tuberculostatic agent according to claims 1 to 18. Use of hybrid tuberculostatic agent according to claims 1 to 18, characterized in that it is used in the preparation of medicament for treating tuberculosis. Use of a hybrid tuberculostatic agent according to claims 1 to 18, characterized in that it is employed in the pharmaceutical, medical, veterinary, biotechnological, biomedical or preventive areas.