MXPA97004679A - Liposomal antibacterial composition of bajarigi - Google Patents

Abstract

The present invention relates to a liposomal formulation containing at least one therapeutic agent such as an antibiotic and to a method for the treatment of bacterial infections through the administration of said formulation. A low cholesterol, cholesterol free, multilamellar, liposomal formulation comprising a neutral lipid, an anionic lipid and at least one therapeutic agent is provided, wherein the liposomal formulation improves the penetration of the therapeutic agent into the bacterial cell. A preferred lipid composition is dipalmitoylphosphatidylcholine (DPPC): dimyristoylphosphatidylglycerol (DMPG) at a ratio of 10: 1 to 15: 1, with a total lipid concentration ranging from 5 to 85

Description

LIPOSOMAL ANTIBACTERIAL COMPOSITION OF LOW RIGIDITY BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates to a formulation containing a therapeutic agent. In addition, it relates to an original liposomal formulation that allows a modulated release of the therapeutic agent over time, as well as an increased penetration of a therapeutic agent such as an antibiotic to the bacterial cells. The invention further relates to a method for the treatment of bacterial infections in an animal through the administration of the formulation of the present invention. 2. Description of the Prior Art The encapsulation of bioactive compounds in natural or synthetic matrices has been extensively studied during the last decades. The advantages of said administration strategy are numerous. First, it provides protection from inactivation or degradation of the bioactive compound. Secondly, it controls the kinetics of the release of the compound, allowing the optimization of the blood concentration profile. This decreases the damaging effects of bioactive compounds with short half-lives. In addition, it allows a reduction in the risk of toxicity. Liposomes are microscopic vesicles that form spontaneously from phospholipids above their transition temperature, in the presence of an excess of water. Vesicles with a diameter ranging from 20 nanometers to several micrometers can be prepared. Multilamellar liposomes are made of double layers of concentric phospholipids separated by aqueous layers. Unilamellar liposomes consist of a single layer of phospholipid surrounding an aqueous core. Liposomes can adapt hydrophilic molecules in aqueous spaces and lipophilic molecules in double layers of lipid. The potential of liposomes as vehicles for therapeutic agents, or therapeutic liposomal formulations has been studied by several investigators. Successful treatment with liposomes against intracellular bacteria has been demonstrated (Lopez-Berestein et al., 1987, J. Clin. Oncology, 5: 310-317; and Popescu et al., 1991, US 4,981,692). A number of studies have also shown that antibacterial agents trapped in liposomes increase the therapeutic indices of these agents, as a result of reduced toxicity, modification of pharmacokinetics and tissue distribution parameters (Lagacé et al., 1991, J. Microencapsulation 8: 53-61 and references there, Omri et al., 1994, Antimicrob Agents Chemorther, 38: 1090-1095). The most widely used type of antibacterial agent is very certainly antibiotics. Antibiotics can be subdivided into different groups, which include ß-lactams, aminoglycosides, macrolides, lincomycin, clindamycin, tetracyclines, chloramphenicol, vacomycin, rifampin, quinolones, and sulfonamides. Aminoglycosides are all potent bactericidal agents that share the same general scale of antibacterial activity and pharmacokinetic behavior. The members of the group are represented by the presence of amino sugars glycosidically bound to the aminocyclitols. The main agents fall into two groups: the small group consisting of streptomycin, and its closest derivatives; and the large group, which is subdivided into the neomycin group, the kanamycin group, which is again subdivided into kanamycins, tobramycin and its semi-synthetic derivatives amikacin and dibecacin and the important subgroup of gentamicins and their Relative, netilmicin and sisomicin. Aminoglycosides inhibit protein synthesis in a variety of microorganisms and are used primarily to treat infections caused by organisms that are resistant to other antibiotics, particularly gram-negative bacteria, such as, but not limited to, Escherichia species, Enterobacter, Klebsiella , Pseudomonas, Salmonella. At different degrees, the aminoglycosides are also active against Staphylococcus aureus, Staphilococcus epidermidis, Listeria and bacteria of the genus Mycobacteria.

Since aminoglycosides are highly polar cationic compounds, the diffusion through the bacterial cell membrane is very limited and the intracellular accumulation of antibacterial agents is caused by the transport of the active ingredient. Many organisms exhibit resistance to the older aminoglycosides. In addition, an increase in the resistance of microorganisms to recently introduced aminoglycosides is passively growing. Growing evidence suggests that acquired antibiotic resistance is usually due to a balance between the rate of penetration of the outer membrane and the rate of inactivation of the subsequent enzyme. Thus, the outer membrane barrier and the antibiotic degradation enzymes are strongly synergistic. In addition, since a newer aminoglycoside, by virtue of its non-susceptibility to bacterial degradation enzymes, is active against strains resistant to older members of the group, and can not be used to predict its activity in general, in view of impermeability relative of a significant number of strains. Although aminoglycosides are useful for the treatment of infections, their use may be accompanied by toxicity and side effects. The most important toxic effects are ototoxicity and nephrotoxicity. Since aminoglycosides can produce oto- and nephrotoxicity relative to the concentration, it is important to ensure that their concentrations in the plasma do not exceed the toxic levels. This is equally important to ensure that the danger of toxicity does not result in a therapeutically inappropriate dose. The encapsulation of aminoglycosides and β-lactam antibiotics in liposomal formulations has been described by the dehydration-rehydration vesicle (DRV) method, (Lagacé et al., 1991, J. Microencapsulation 8: 53-61). Diesteroil phosphatidyl choline (DSPC) and dimyristoyl phosphatidyl-glycerol (DMPG), two synthetic phospholipids, were used at a molar ratio of 10: 1 and at a lipid concentration of 16.5 umoles / ml. The same liposomal formulation was tested "in situ" in an animal model with chronic pulmonary infection with Pseudomonas aeruginosa and allowed a marked increase in the residence time of the antibiotic in the lungs and a reduced absorption of the systemic antibacterial agent. However, this liposomal aminoglycoside formulation did not show an improvement in bactericidal activity compared to free antibiotics and other controls (Omri et al., 1994, Antimicrob Agents Chemother, 3_8: 1090-1095). Other groups have described liposomal aminoglycoside formulations (Da Cruz et al., 1993, WO 93/23015 and Proffitt et al., 1994, WO 94/12155). However, the described formulations fail to exhibit a very drastic improvement in the therapeutic activity of the antibiotic, compared to its activity in the free form. In fact, the liposomal formulation of aminoglycoside (netilmicin) from Da Cruz et al, which comprises phosphatidylcholine (PC), cholesterol and phosphatidyl-inositol (Pl), only shows a modest increase activity, in vivo, with the aminoglycoside as a part of the liposomal formulation, compared to the free aminoglycoside (at best by a factor of 3). Proffitt et al. Describe a liposomal formulation (amikacin) of a different aminoglycoside comprising PC, cholesterol and distearoyl phosphatidyl glycerol (DSPG). Although the formulation of Proffitt et al seems to be superior in increasing the therapeutic activity, in vivo, of the aminoglycoside compared with that of Da Cruz, this increase is still relatively low and tissue-dependent (10-fold increase in spleen, 5-fold increase in liver and only 2 times in lung). Importantly, liposomal formulations available for use in the treatment of bacterial infections do not appear to significantly increase the passage of the therapeutic agent through the bacterial membrane. Cystic fibrosis (CF) is one of the most common lethal genetic diseases in humans. While the course of the CFIt varies enormously from patient to patient, it is determined enormously by the degree of pulmonary involvement. In CF, deterioration seems inevitable, and eventually leads to death. Although a prognosis of a patient with CF has improved dramatically in the second half of this century, the average survival rate is only 30 years of age. Of importance, a correlation has been observed between the early colonization of Pseudomonas and a worse prognosis for patients with CF. In addition, chronic lung infection, due to Pseudomonas aeruginosa, is the main cause of morbidity and mortality in patients with cystic fibrosis (Omri et al., 1994, Antimicrob Agents Chemother 38: 1090-1095; and Merck Manual, 1992, 16th. edition, Merck Res. Lab). In patients with CF, Staphylococcus aureus, and Haemophilus influenza, other gram-negative strains are usually early pathogens. These bacterial infections in patients with CF are, in most cases, efficiently treated with antibiotics. A number of antibiotics are used for antibacterial therapy, either alone or in combination. The choice of a particular antibiotic regimen depends on a number of factors, which include the site and severity of the infection as well as the resistance / sensitivity profile of the organism. It is important that high doses of antibiotics, especially aminoglycosides, as well as a long-term antibiotic treatment, are generally indicated in patients with CF. Pseudomonas aeruginosa colonize more than 90% of adolescents with CF. Efficient therapy directed against Pseudomonas aeruginosa remains difficult and controversial (Omri et al., Antimicrob, Agents Chemother, 38: 1090-1095). The usual normal therapy for CF patients, colonized with this microorganism, involves the use of an aminoglycoside or ß-lactam, only in combination. These antibacterial agents require frequent parenteral administration of high doses, in order to obtain therapeutically effective concentrations in the serum, particularly against biofilm cells formed by the mucoid phenotype of P. aeruginosa. It should be noted that the permeability of the outer membrane (OM) of P. aeruginosa is only about 1-8% of that of E. coli, as analyzed by the rates of antibiotic penetration (Yoshimura et al., 1992, J. Bacteriol 152: 636-642; Nicas et al. 1983; J. Bacteriol., 153: 281-285; and Angus et al., 1984, Antimicrob. Agents Chemother., 1_4: 349-357). It has also been reported that prolonged or repeated treatment with antibiotics has been associated with the gradually decreasing susceptibility of this organism and with the accelerated tolerance of antibiotics in these patients (Omri et al., 1994, Antimicrob Agents Chemother., 38: 1090-1095 and references in it). Thus, although the use of liposomes as a vehicle for antibiotics has been shown in "in vitro" experiments that are a promising avenue for the treatment of P. aeruginosa (Lagacé et al., 1991, J. Microencapsulation 8_: 53- 61; and Nacucchio et al., 1988, Microencapsulation, 5: 303-309), the design of a liposomal formulation that allows a significant improvement in the activity of the antibiotic, as well as a significantly improved penetration within the bacterial cell, continues to emerge. The design of said liposomal formulation could be of enormous importance in the treatment, and / or prophylaxis of bacterial infections in patients with CF, and perhaps in the prognosis of these patients. Although microorganisms resistant to antibiotics have long been recognized, this continues to be a very important global health problem. In addition, based on the relative impermeability of numerous strains to antibiotics, the design of newer, more efficient versions of them, which can overcome strain-based enzymatic degradation, does not yet solve the significant obstacle of obtaining the antibiotic to through the impermeable membrane or through an exopolysaccharide layer of the bacteria and into its site of action. In addition, the problem of increased resistance to antibiotics is mixed with the misuse of these agents (Merck Manual, 1992, 16th edition, Merck Res. Lab. For example, since antibiotic resistance of microorganisms, which is more acute With older types of antibiotics, practitioners have usually accelerated the use of a newer generation antibiotic, thus contributing to the increased resistance of microorganisms to newer generation antibiotics.The use of large scale antibiotics in animals, including, but not limited to, dairy cows, and the presence of these antibiotics in milk or in the environment, is yet another contributor to the increased resistance of the microorganism to antibiotics. Chris W.M. Grant et al. (1989, Biochimica et Biophysica Acta 984 (1): 11-20) describes the results of thermodynamic studies (at 35 ° C and 22 ° C) and electronic freeze attack microscopy, to characterize amphotericin liposomes. B made of DMPC / DMPG comparatively with DPPC and DEPC, containing 0-25 mol% of amphotericin B. It was demonstrated in this document that the fluid liposomes made of DMPC / DMPG, which have a Tc of 23 ° C, showed considerable dissolution, particularly at high drug concentrations. Second, Grant and others tried to solve the problem caused by the relative immiscibility of amphotericin B with the rigid phospholipid. Finally, his study suggests that fluidity alone can not explain the protective nature of double phospholipid layers against the toxicity of amphotericin B. This may be of enormous importance for the physician to be able to increase the activity of antibiotics, allowing thus potentially a reduction of the doses required to achieve the objective antibacterial action. Furthermore, this increase in antibiotic activity could allow a more efficient use of the older generation antibiotics, thus moderating the increase in the resistance of the microorganism to the new generation antibiotics. This could be a very significant advantage for the physician, veterinarian, or the like, to be able to use a liposome formulation containing an antibacterial agent, such as an antibiotic, wherein the liposomal formulation significantly improves the antibacterial activity of the agent, not only due to the increased circulation time, and lower toxicity, but also because this formulation comprises phospholipids that markedly improve the penetration of the agent into the bacterial cell. In addition it could be of enormous advantage, if the formulation also allows a remarkable increase in the penetration of the antibacterial agent through the outer membrane (OM) and mucoid exopolysaccharides, such as those secreted by mucoid variants of bacteria such as those of Pseudomonas aeuroginosa. In addition, it would be advantageous to provide an antibacterial liposomal formulation that is effective against a wide variety of bacterial strains that exhibit significant variations in their outer membrane composition. Finally, it could be a huge advantage to have access to a therapeutic liposomal formulation, wherein the composition of the formulation allows the modulated release of the therapeutic agent, over time, thereby reducing side effects and prolonging the action of the agent.

COMPENDIUM OF THE INVENTION Based on the physico-chemical properties of the phospholipids, many new liposomal formulations were designed, in order to promote the "in vivo" bactericidal efficacy of the liposomal aminoglycosides, while maintaining an encapsulation efficiency, prolonged antibiotic residence time in the target organ and low toxicity. These new liposomal formulations were subjected to different tests, "in vitro" and "in vivo". The present invention relates to the successful design of liposomal formulations, which contain an aminoglycoside in one embodiment, exhibit a bactericidal activity, "in vivo", very effective, compared to free antibiotics and satisfy other of the following several needs: modulated release of the therapeutic agent over time, maintenance of the encapsulation efficiency of the therapeutic agent, prolonged residence time of the antibiotic in the target organ, and low toxicity. The present invention also relates to liposomes containing a therapeutic agent, and is characterized by an original formulation that allows for the increased penetration of the therapeutic agent into bacterial cells and through bacterial mucoid exopolysaccharides. An example of a therapeutic agent is an antibiotic, but it is not limited thereto. Through its increased penetration of bacterial cells, the liposomal formulation of the present invention showed a marked improvement in bactericidal efficacy "in vivo", while the free antibiotic showed nothing or showed very little bactericidal activity. In addition, the present invention relates to the pharmaceutical or veterinary use of the liposomal formulations of the present invention, in the treatment or prophylaxis of bacterial infections. It is an object of the present invention to provide a low-rigidity liposomal formulation comprising a therapeutic agent, wherein the interaction between the components of the formulation allows a slow but constant release of the therapeutic agent over time, as well as improved penetration of the agent inside a bacterial cell.

It is another object of the invention to provide a liposomal formulation for the treatment of bacterial infections, wherein the liposomal formulation comprises a combination of lipids together with a therapeutic agent. Furthermore, it is a further object of the present invention to provide an antibacterial liposomal formulation effective against bacterial strains that have significant variations in their outer membrane and / or bacterial wall composition. The liposomal formulations of the present invention have not been specifically described in the prior art. Although such formulations appear to fall widely within the claims of WO 93/23015, WO 94/12155, US 4,235,871 and US 4,981,692, they are not specifically identified there and there is no suggestion of any special activity inherent therein. In addition, before obtaining the appropriate formulations of the invention, a large number of formulations were prepared, also generally described in WO 93/23015, WO 94/12155, US 4,235,871 and US 4,981,692. These include DSPC: DMPG, DSPC: DPPC, DPPC: DMPC, in a molar ratio of 15: 1 and 10: 1, with or without cholesterol (at a molar ratio of 1, ie, 10: 1: 1). None of these formulations, also comprising tobramycin, showed a marked improvement in antibacterial activity when compared to free tobramycin. Additionally, these experiments could suggest that the presence of cholesterol in the therapeutic liposomal formulation improves liposomal stability in a way that goes against the desired therapeutic activity of the formulation. Thus, it is an object of the invention to provide a liposomal formulation, which is free of stabilizing agents that could affect the desired therapeutic activity of the formulation and the desired kinetics of the release of the therapeutic agent from the liposomes. . In accordance with an aspect of the present invention, a cholesterol-free, low-rigidity, multilamellar liposomal formulation comprising a neutral lipid, an anionic lipid and at least one therapeutic agent is provided, wherein the liposomal formulation improves the penetration of the therapeutic agent into a bacterial cell. According to another aspect of the present invention, there is provided a method for the treatment of a bacterial infection in an animal, comprising the administration of a pharmaceutically or veterinarily adequate dose of the liposomal formulation. According to a further aspect of the present invention, a liposomal formulation is provided which allows penetration of the trapped therapeutic agent, through the exopolysaccharide layer of a bacterium. Therefore, the liposomal formulation of the present invention provides increased efficacy in the treatment of mucoid bacteria. According to yet another aspect of the present invention, the use of a liposomal formulation is provided for the treatment, prophylaxis and diagnosis of a bacterial infection in an animal, comprising the administration of a pharmaceutically or veterinarily suitable form of the formulation . Since a multitude of therapeutic agents can be trapped within the liposomes of the invention, in the specification and appended claims, it is to be understood that the term, therapeutic agent, is designed to include, but is not limited to, antibiotics, bioactive molecules. , such as proteins or parts thereof, nucleic acids or part thereof, amino acid analogs or nucleoside analogs, as well as other medically or veterinarily useful agents, such as contrast materials (eg, dyes) and materials of diagnosis, as well as growth factors, hormones such as corticosteroids, or the like. Furthermore, it should be understood that the term, therapeutic agent, should be taken in a broad sense to also include a combination of at least two therapeutic agents.

In the specification and appended claims, the term lipid is designed to include, but is not limited to, saturated or unsaturated lipids, or synthetic or derived from natural sources, provided that the lipid-therapeutic agent composition exhibits fluidity / stability, which is compatible with the penetration of the therapeutic agent into a bacterial cell and / or its modulated release. Similarly, the term bacterial infections should be constructed to include, but is not limited to, Gram-negative bacteria, such as Salmonella, or Pseudomonas, to Gram-positive bacteria such as of the Mycobacteria genus. Other aspects and advantages of the invention will be apparent from the following description of the preferred embodiments. However, it should be understood that the detailed description, while indicating the preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications, within the spirit and scope of the invention, will be apparent to those skilled in the art. in the technique.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a graphic representation of the bacterial counts of Pseudomonas aeruginosa (429) in proteose peptone (MIC> 60 μg / ml) under different conditions; Figure 2 shows a graphic representation of the bacterial counts of B? Rkholderia cepacia (LSPQ I D 28369) in proteose peptone (MIC> 26 μg / ml) under different conditions; Figure 3 shows a graphic representation of the bacterial counts of Escherichia coli (nm 88 1061) in protease peptone (MIC> 5 μg / ml) under different conditions; Figure 4 shows a graphic representation of the bacterial counts of Staphylococcus aureus (LS PQ 2499) in proteose peptone (MIC> 9 μg / ml) under different conditions.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a therapeutic liposomal formulation that allows increased penetration of a therapeutic agent into bacterial cells and through bacterial mucoid exopolysaccharides. The liposomal formulation is prepared by lyophilization, rehydration and extrusion under pressure. The liposomes have, in a preferred embodiment, an average size of 0.6 μm to 0.2 μm, and are composed of a neutral lipid and a negatively charged lipid. The molar amount of the negatively charged lipid is from 6.5% to 11% of the total lipid, and the encapsulation efficiency is typically greater than 20%. When administered "in situ" to animals, the formulation of liposomal therapeutic agent not only prolongs the residence time of the therapeutic agent and reduces its toxicity, but also increases its therapeutic activity. One embodiment of said formulation contains an antibiotic as the therapeutic agent. In another embodiment, the liposomal formulation serves for the treatment of bacterial infections, and comprises a combination of phosphatidylcholine, a neutral phospholipid, and phosphatidylglycerol, an anionic phospholipid, at a ratio of 10: 1 to 15: 1, together with a therapeutic agent . In another preferred embodiment, the formulation contains an aminoglycoside as the antibiotic. An example of the aminoglycoside is tobramycin. Said formulation of liposomal aminoglycoside shows: 1) a high bactericidal activity against microorganisms, which are resistant during antibiotic therapy in mammals; 2) a high efficiency of encapsulation of the therapeutic agent; 3) a prolonged residence time of the antibiotic in the target organ; 4) low toxicity; and 5) a modulated, gradual release of the encapsulated therapeutic agent, over time. The present invention also provides a therapeutic liposomal formulation, which allows a modulated release of the therapeutic agent over time, and thus allows for a well controlled release of the therapeutic agent. The present invention also provides a liposomal release that can serve as a diagnostic tool. Numerous types of bioactive agents can be coupled to the liposomes of the invention, for example, antibodies, in order to target a specific tissue or cell type. The detection of the target can be determined according to known methods, including, for example, the use of a label, radioactive or not, or a dye entrapped in the liposomes. One of the numerous examples of the diagnostic use of the liposomal formulations of the invention is to target a tumor antigen, through an antibody specific to this antigen, in order to detect, quantify or analyze the presence of metastasis. The selected therapeutic agent will depend on the organism that causes the infection. Suitable antibiotics include, but are not limited to: penicillin, ampicillin, netacycline, carbencillin, tetracycline, tetracycline hydrochloride, oxtetracycline hydrochloride, chlortetracycline hydrochloride, 7-chloro-6-dimethyltetracycline, doxycycline, doxycycline monohydrate, metacycline hydrochloride Minocycline hydrochloride, rolitetracycline, dihydrestreptomycin, streptomycin, gentamicin, kanamycin, neomycin, erythromycin, carbomycin, oleandomycin, troleandomycin, Polymycin B, colistin, cefalotin sodium, cephaloridin, cephaloglycine dehydrate, and cephalexin monohydrate. If the site of infection or affliction is external or accessible, the therapeutic agent trapped in the liposome can be applied topically. Bacterial agents contemplated herein include, but are not limited to: Moraxella spp., Costridium spp., Corynebacterium spp., Diplococcus spp., Flavobacterium spp., Hemophilus spp., Klebsiella spp., Leptospira spp., Mycobacterium spp. , Neisseria spp., Propionibacterium spp., Proteus spp., Pseudomonas spp., Serratia spp., Escherichia spp., Staphylococcus spp., Streptococcus spp., And organism of the bacterium type, including Mycoplasma spp., And Rickettsia spp. It will be understood that the aminoglycoside represents aminoglycosides and analogs and derivatives thereof, including streptomycin, dehydroesteptomycin, tobramycin, neomycin B, paromycin, ribostramycin, lividomycin, kanamycin A and B, viomycin, gentamicin, (including Ci, C? A, and C2) , sisomicina, netilimicina and amicacin. It will be understood that β-lactams refer to synthetic, semi-synthetic and natural penicillins, cephalosporins, monobactams and tinamycins such as oxacillin, cephapirin, aztreonam, and imipenem. Depending on the purpose of delivery, the liposomal formulation can be administered through a number of routes: in humans and animals these include, but are not limited to, injection (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, intra-auricular, intramammary, intraurethral, etc.), topical application (eg, on affected areas), and by absorption through epithelial or mucocutaneous coatings (eg, ocular epithelium, oral mucosa, epithelial coatings of the rectum and vagina, coatings of the respiratory tract, nasopharyngeal mucosa, intestinal mucosa, etc.). The mode of administration of the preparation can determine the sites and cells in the organisms to which the compound will be administered. The liposomes can be administered alone, but will generally be administered in admixture with a selected pharmaceutical carrier with respect to the intended route of administration and normal pharmaceutical practice. The preparations can be injected parenterally, for example, intraperitoneally, intra-arterially or intravenously. The preparations can also be administered via oral, subcutaneous, intramuscular and, of course, intramammary routes. For parenteral administration, they may be used, for example, in the form of a sterile aqueous solution, which may contain other solutes, for example, enough salts or glucose for the solution to be isotonic. Those skilled in the art can design other uses, depending on the particular properties of the preparation. The supply of the liposomal formulation through an aerosol, it is also contemplated as a preferred method of administration. For example, but not limited to, the formulations of the present invention can be used in the treatment of respiratory diseases. Asthma is one of the many diseases for which these formulations can be used. For administration to animals, including humans, in the curative treatment of disease states, the prescribing medical professional will finally determine the appropriate dose for a given subject, and it is expected that this will vary according to the agent, weight, and response of the animal, as well as the nature and severity of the disease. The dose of therapeutic agent in liposomal form may be, according to the present invention, lower than that used for the free therapeutic agent. In some cases, however, it may be necessary to administer equal or greater doses. It is also contemplated that periodic treatments or different cycles of treatment may be beneficial. The liposome delivery path can also affect its distribution in the body. A passive delivery of liposomes involves the use of several routes of administration, v. gr., intravenous, subcutaneous and topical. Each route produces differences in the location of the liposomes. Two common methods used to actively direct the liposomes to selected target areas are to bind either antibodies or specific receptor ligands to the surface of the liposomes. It is known that antibodies have a high specific character for their corresponding antigen and have been shown to be capable of binding to the surface of liposomes, thus increasing the specific target character of the liposome encapsulated in the drug. The present invention also provides liposomal formulations of aminoglycoside or β-lactam preferably containing tobramycin and the following synthetic lipids: di-palmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG). Other suitable phosphatidylcholines and phosphatidylglycerols include those obtained from soybean, egg or plant sources, or those that are partially synthetic. Depending on the desired application, the purpose of delivery, the route of delivery, the objective, and other parameters related to the use of the formulation, the size of the liposomes can be adapted according to well-known methods. For example, it is well known that large liposomes are better for topical application, while smaller liposomes are preferred for intravenous administration. In addition, the size of the liposomes affects their ability to be phagocytosed by macrophages. In this way, the size of the liposomes can be adapted, in order to favor a route of administration, promote retention in the reticuloendothelial organs or favor phagocytosis (to treat bacteria within the macrophage, for example). The sizes of the contemplated liposomes vary from nanometer to miera, preferably from 100 nm to 1 μm. In a preferred embodiment, the size of the liposomes varies from about 200 nm to 600 nm. Said liposomal formulation is compatible with an aerosol administration of the formulation to be delivered to the lungs of an animal. One formulation includes liposomes comprising an encapsulated aminoglycoside, wherein the liposomes are multilamellar vesicles having an average size ranging from 0.2 μm to 0.6 μm. A preferred ratio of DPPC: DMPG is from about 5: 1 to 20: 2, and a preferred ratio of therapeutic agent to total lipid is from about 1: 1 to 1:10. Other preferred formulations include suitable lipids such as phosphatidylcholine and / or phosphatidylglycerols present individually or as a mixture, in a molar ratio ranging from about 0.01 to 20. Other preferred formulations include formulations wherein the ratio of therapeutic agent to total lipid is 1:10 to 1: 1. In accordance with the present invention, the method of preparation of multilamellar liposomes can be divided into 5 main steps. The lipids are dissolved in chloroform (approximately 1 mg of lipid / ml of chloroform or more) and the solution is evaporated to form a lipid film between room temperature and 60 ° C. The lipid mixture is preferably negatively charged and the resulting lipid concentration varies from about 5 mM to 130 mM. Liposomal preparations are typically mixtures of two or more components: a phosphatidylcholine and a negatively charged molecule, such as a phosphatidylglycerol, with each component of the liposomal preparation in molar ratios of 40-90% and 5-60%, respectively. A preferred combination is dipalmitoylphosphatidyl choline (DPPC): dimyristoylphosphatidylglycerol (DMPG) at a ratio of 10: 1 to 15: 1, with a total lipid concentration ranging from 5 to 85 mM. The resulting negatively charged lipid induces high efficiencies of antibiotic encapsulation, while the lipid formulation promotes the increased penetration of antibiotics into bacterial cells. The lipid film is hydrated with an aqueous solution of antibiotic or saline regulated at its phosphate pH (PBS) diluted 1:20. The concentration of the antibiotic can vary from 0.01 mg / ml to 150 mg / ml. The preferred concentration is 10 mg / ml to 40 mg / ml. The antibiotic is preferably an aminoglycoside, as cited herein, or a β-lactam, but other antibiotic or non-antibiotic therapeutic agents may also be beneficial for the methods of the present invention. After hydration of the lipid film and the formation of multilamellar liposomes, the preparation is subjected to freezing either in liquid nitrogen (-170 ° C) or two hours in a deep freezer (-70 ° C) followed by lyophilization in a freezer dryer at 5 mtorr for 24 hours. The lyophilized samples were stored at -70 ° C or -20 ° C, until used. For use, a powder is rehydrated with an antibiotic solution (10 mg / ml to 40 mg / ml) to a 1/8 portion of the initial volume, with vigorous stirring, followed by incubation at 65 ° C for 60 minutes , stirring every 10 minutes. The suspension was then brought to the initial volume of 50% with a saline solution regulated in its pH and stirred vigorously again. Preferably, the multilamellar vesicles are extruded, successively, through smaller pore polycarbonate membranes, from 1 μm to below 0.2 μm, or as desired to obtain a gradual reduction in liposome size. Finally, the sized mixture was centrifuged twice, for 20 minutes, at 5,000 g and the pellet was resuspended in the saline solution. The determination of tobramycin in liposomes was carried out through high performance liquid chromatography (HPLC). A particularly important embodiment of the invention produces a liposome / aminoglycoside formulation, allowing a remarkable increased penetration of the antibiotic to the bacterial cells. In this embodiment, the lipid mixture is dipalmitoyl-phosphatidylcholine (DPPC): dimyristoylphosphatidylglycerol (DMPG) at a ratio of 1:10 and 1:15, with the total lipid concentration varying from 5 to 85 mM. The final liposomal / aminoglycoside formulation had a diameter of approximately 0.4 uM and possessed an encapsulation efficiency of 20% and a therapeutic agent to lipid ratio of 1: 1. The improved bactericidal efficacy that resulted is related to the fact that the therapeutic agent is not only incorporated into the liposomes, but is incorporated into an original combination of phospholipids that remarkably improves the penetration of the therapeutic agent into bacterial cells and through exopolysaccharides mucoids secreted by Pseudomonas aeruginosa. The liposomal / antibiotic formulations of the invention may target monoclonal antibodies or other molecules for a particular tissue or cell, such as a bacterial cell. The present method for the encapsulation of the aminoglycoside is a very significant improvement over the previous protocols using encapsulated aminoglycoside, since a low concentration of the encapsulated aminoglycoside kills the bacteria, whereas with a free antibiotic, 107 c.f.u. (See later).

EXAMPLE 1 Tobramycin Liposomal Formulation The following examples describe the analysis of liposome / aminoglycoside formulations, prepared as described above, wherein the aminoglycoside was tobramycin, the lipid mixture was dipalmitoylphosphatidylcholine (DPPC): dimyristoylphosphatidylglycerol (DMPG) at a ratio of 10: 1 or 15: 1, with the total lipid concentration varying from 5 to 85 mM. The hydration was presented with saline regulated in its pH of phosphate, diluted to 1:20, followed by freezing at -70 ° C and lyophilization. Rehydration was done by adding the antibiotic solution (10 mg / ml) to a 1/8 portion of the initial volume, followed by filling at 50% of the initial volume with saline regulated at its phosphate pH. The liposomes were first extruded through a 1 u filter, followed by extrusion through 0.6 and 0.4 μm polycarbonate membranes and centrifugation, twice at 5,000 x g for 20 minutes and resuspended in PBS.

EXAMPLE 2 Physical and Biological Characteristics of Different Liposomal Formulations of Tobramycin Different liposomal formulations were prepared according to Example 1, and analyzed by differential scanning calorimetry. Using differential scanning calorimetry, the phase transition temperatures (Tc) were calculated for the liposomal formulations of tobramycin listed in Table 1. All these formulations were then tested in vitro to determine the antibiotic kinetics of release from the liposomes. In addition, these formulations were tested in an uninfected mouse model as previously described (Omri et al, 1994, Antimicrob Agents Chemother, 38: 1090-1095) to determine the persistence of liposomes in the lung. Only the liposomal formulations of DPPC / DMPG 10: 1, 15: 1 and DSPC (distearylphosphatidylcholine) / DMPC (dimyristoylphosphatidyl col ina) 15: 1 (shown in Table 1) exhibited the following characteristics: release of gradual and convenient amounts of the antibiotic by virtue of its fluidity / stability characteristics. These liposomal formulations were further tested in an animal model with chronic lung infection to examine their antibacterial efficacy. Contrary to the two formulations of DPPC / DMPG, the formulation of DSPC / DMPG was shown to be inactive in this animal model. In addition, some formulations exhibiting a phase transition temperature comparable to that of the two formulations of DPPC / DMPG, while showing the desired fluidity / stability characteristics, proved to be inefficient in the uninfected animal model. Note that the addition of cholesterol to the formulation described in Table 1 brought the Tc to a minimum value of 60 ° C. These formulations were incompatible with the modulation of gradual antibiotic release and appropriate iterations with the bacteria. Thus, in order to maintain the desired characteristic of the liposome formulation, a low liposome rigidity appears to be required. This low stiffness can be obtained by maintaining a low phase transition temperature (below the body temperature of the animal to which the formulation is being administered) and avoiding the use of cholesterol in the formulation.

TABLE 1 Tc Phase Transition Temperature of Different Tobramycin Liposomal Formulation Phospholipids Ratio Tc DSPC / DMPG 15: 1 44 DSPC / DMPC 15: 1 42 DSPC / DPPC 15: 1 46 DSPC / DMPG 10: 1 40 DSPC / DMPC 10: 1 42 DSPC / DPPG 10: 1 45 DPPC / DMPG 10: 1 29.5 DPPC / DMPG 15: 1 35 EXAMPLE 3 Pulmonary Retention of The Therapeutic Agent As briefly alluded to in Example 2, lung retention studies were performed with liposomes prepared with a 10: 1 molar ratio of DPPC: DMPG, as prepared in the Example, in BALB / c mice (Charles River), and using tobramycin as control. The animals were injected intratracheally as described previously (Omri et al., 1994, Antimicrob. Agents Chemother. 38: 1090-1095) with a dose of 50 ul (200 ug) of the liposomal preparations of tobramycin and the free ones and the lungs, kidneys and blood were collected in fixed times (Table 2). The lungs and kidneys were removed aseptically, weighed and then homogenized in cold sterile PBS (40% w / v) for 30 s with a Plytron homogenizer. Through to HPLC, tobramycin levels were measured, both in homogenized tissues and in sera. Groups of three mice were used for each time value.

TABLE 2 Comparative Antibiotic Concentrations After the Administration of Free Tobramycin and Encapsulated in Liposome, in Mice Conc. (Ug / pair of Conc. (Ug / pair of Serum lungs) kidneys) ug / ml Time Tobra. Tobra liposomes. Tobra liposomes. Liposomes (h) free free free 0.25 43 58 ND * ND ND ND 1 11 27 25 19 UDf 5 8 UD 46 ND ND ND ND 24 UD 73 ND ND ND ND 32 UD 17 ND ND ND ND 48 UD 15 UD 13 UD UD * ND: not done; t UD: not detected Administration of the aminoglycoside liposomal formulation prepared according to this invention resulted in a prolonged pulmonary retention time of the encapsulated form of tobramycin in the lungs, compared to that of the free therapeutic agent. However, it should be noted that the concentration of tobramycin is reduced over time, with the formulation of DPPC: DMPG, shown in Table 2. This result is in contrast to that of a DSPC formulation: DMPG (10: 1) , which showed a constant concentration of tobramycin over time, and, therefore, a high stability of the liposomes (Omri et al., 1994, Antimicrob Agents Chemother. 38_: 1090-1095, see also below).

EXAMPLE 4 In Vivo Analysis of the Bactericidal Activity of Tobramycin Encapsulated in Liposome To evaluate the bactericidal efficacy of a liposomal aminoglycoside formulation, produced in accordance with the present invention, male, pathogen-free Spragu-Dawley rats weighing 175 to 225 g (Charles River) were used. Chronic infection was established in the lungs through the intra-tracheal administration of 5x105 CFU of Pseudomonas aeruginosa PA 508 (mucoid phenotype) prepared on agar beads. It should be noted that this rat model for chronic lung infection is widely recognized as the most appropriate animal model for chronic pulmonary infections in human patients with CF. After 3 days, three doses (600 ug) of free tobramycin or encapsulated in liposome were given, intra-tracheally at 16-hour intervals. The lipid mixture was DPPC: DMPG at a molar ratio of 10: 1 (formula No. 1) and DPPC: DMPC at a ratio of 15: 1 (formula No. 2). Sixteen hours after the last treatment, the animals were sacrificed and the entire lungs aseptically removed, weighed and homogenized as previously described for the mice. Serial 10-fold dilutions of the homogenates were made in cold PBS and extended in triplicate onto protease peptone agar plates. The identification of P. aeruginosa was confirmed through specific cultures. CFU were counted after incubations of 24 hours at 37 ° C under 5% CO2. The counts were expressed in log CFU by pairs of lungs. As controls, PBS and PBS-liposomes were used. The results are shown in Table 3.

TABLE 3 Bactericidal Effect of Liposomal Tobramycin in P. aeruginosa in Rat Lung Infected Tissues Regimen # Rats CFU / pair of CFU of log / pair lungs of lungs Only PBS 2 1.40 x 106 6.15 liposome-PBS 2 2.32 x 107 7.36 (formula No. 1) without tobramycin liposome-tobra. 5 < account < count (formula No. ^ t) significant * significant liposome-PBS 3 2.11 x 107 7.32 (formula No. 2) f tobra. liposomal 6 1.83 x 106 6.26 (formula No. 2) * free tobramycin 5 1.25 x 107 7.10 t formula No. 1: formula according to the present invention used here at a molar ratio of 10: 1, DPPC: DMPG. * none or only a rare CFU (0 to 4) were visible on plates sprayed in triplicate with undiluted samples of lung. According to the American Society for Microbiology, Manual of Methods for General Bacteriology. Washington, D.C., 1981, p. 185, CFU counts < 30 are not statistically significant, t Formula No. 2 was prepared with DSPC: synthetic DMPC at a molar ratio of 15: 1. This formulation, as a previously described formulation (Omri et al., 1994, Antimicrob. Agents Chemother. 38: 090-1095) at a molar ratio of 10: 1 of DSPC: DMPG represents other liposomal formulations without improved bactericidal efficacy, when compared to the activity of the free antibiotic against P. aeruginosa. A second experiment was carried out to study the bactericidal effect of the liposomal tobramycin preparation, produced according to the present invention, as set forth in Table 3 with the following modifications: 1) the liposomes were prepared with a molar ratio of DPPC 15: 1: DMPG (Formula No. 3); and 2) only two doses of 240 ug of tobramycin encapsulated in liposome were administered to the rats.

TABLE 4 Bactericidal effect of Tobramycin Liposomal in P. aeruginosa in Infected Tissues of Rat Lung Regimen # Rats CFU / pair of CFU of log / pair lungs of lungs Only PBS 3 1.05 x 108 8.02 liposome-PBS 3 1.24 x 10e 8.93 (formula No. 3 without tobramycin) tobra. liposomal < account < account (formula No. 3) significant * significant free tobramycin 1.07 x 106 6.03 No CFU or only scarce CFU (0 to 6) was visible on triplicate plates sprayed with undiluted lung samples.

The results of the experiments show that the "in situ" administration of low doses of tobramycin in lungs drastically increases the bactericidal efficacy of the encapsulated aminoglycoside comparatively with the free therapeutic agent. The very strong increase in the bactericidal efficacy of the encapsulated tobramycin indicates that the liposomal formulation allows increased diffusion through the bacterial cell membrane and intracellular accumulation of the therapeutic agent. The drastic increase in the antibacterial activity of relatively low doses of tobramycin as part of the liposomal formulation compared to the free one, further suggests that the lipids of the formulation promote a fusion between the liposome and the bacterial cells. The specific liposomal formulation according to this invention has original properties not shared by other prior liposomal formulations. One case in question is the significant bactericidal activity of the liposomal formulation of tobramycin in the P. aeruginosa mucoid strain used. In this way, the formulations of the invention appear to not only improve the passage of the antibiotic through OM of the bacterium, but also through its exopolysaccharide. Thus, the liposomal formulations herein can be used successfully to treat non-mucoid and mucoid forms of bacteria. The fact that low doses of aminoglycosides are sufficient to present a source of bactericidal efficacy reduces the toxicity of the antibacterial agent. In fact, the results in Tables 3 and 4 showed a drastic bactericidal activity of the antibiotic-liposomal formulation with as little as 1.37 mg of tobramycin per kg of the animal. The previously described formulations used 35-120 mg / kg of antibiotic with substantially lower bacterial activity (WO 94/12155 and US 4,981,692). In addition, the therapeutic liposomal formulations of the present invention are not strictly dependent on phagocytosis by macrophages, such as those of Popescu et al. (US 4,981,692), specifically designated for the treatment of intracellular infections. In addition, the fact that the concentrations of tobramycin observed in the kidneys were reduced when they were used when encapsulated antibiotics were used, comparatively to free antibiotics, indicates a reduced toxicity. In patients with CF, Burkholderia cepacia is recognized as the most resistant bacterium. It has been reported that B.cepacia (formerly Pseudomonas) in the early 1980s caused an accelerated and fatal deterioration of pulmonary function, fever, pneumonia of necrosis and, in some cases, septicemia in patients with cystic fibrosis (Govan et al., 1993, Royal Soc. Med. Suppl. No. 20, 86: 11-18). One of the clinically important features of ß. cepacia is its intrinsic resistance to structurally unrelated antimicrobial agents (Gotoh et al., 1994, Microbiol.140: 3285-3291). Important differences between P. aeruginosa and ß were observed. cepacia with respect to its outer membrane (Gotoh et al., 1994).

Xanthomonas maltophilia is another type of bacteria, which is refractory to conventional treatments. A parallel can be drawn between X. maltophilia and B. cepacia with respect to their intrinsic resistance to antimicrobial agents. Being relatively impermeable, X. maltophilia infections usually lead to death. The bacterial walls of E. coli and S. aureus have very different characteristics compared to those of the Pseudomonas. The outer membrane of Enterobacteriacae as E. coli, have distinct porins and the lipopolysaccharide side chains are interlaced, thus conferring an unusually low permeability for the lipid double layer region of the outer membrane for hydrophobic solutes (Nikaido, 1988, Rev. Infecí. Dis. 1_0, Sup. 2 : S279-S281). The cell wall of Gram-positive bacteria such as S. aureus, consists of peptidoglycan, polysaccharides and polymers such as teichoic acids. In contradistinction to the cell walls of Gram-negative bacteria, which contain lipid material, that of Gram-positive bacteria such as that of S. aureus, lack lipid material. The porosity of Gram-positive cell wall preparations has apparently not been analyzed with modern technology, but it is reasonable to assume that they are absolutely porous (Nikaido, 1994, J.-M. Ghuysen and R. Hakenbeck (Eds.) Bacterial Cell Wall). It has been shown that the outer membrane of all Gram-negative bacteria species contain porin channels. Hydrophilic molecules of sizes below a given exclusion limit can pass through channels filled with water of protein called porins. In the case of aminoglycosides, a polycationic antibiotic, the mechanism of consumption through the outer membrane, has been proposed, being different for P. aeruginosa and E. coli. For P. aeruginosa, the aminoglycosides are taken through the autopromovida consumption route (Hancock et al 1981, Antimicrob Agents Chemother. 1_9_: 777-785; and Nicas et al., 1980, J. Bacteriol. 143: 872-878). In this trajectory, the polycations act to competitively displace divalent cations that cross-bridge adjacent lipopolysaccharide molecules (LPS), thus breaking these important outer membrane stabilization sites. Although this invention is not restricted to a particular theory, it is believed that, in turn, it permeabilizes the outer membrane and promotes the consumption of other molecules of the permeabilization polycation. This is consistent with the polycationic nature of the aminoglycosides, which carry three to five positive charges. The porins of E. coli appear to be particularly complex, since the trimeric arrangements form three small pores, which converge in an individual channel filled with water (Engel et al., 1985, Nature (London) 317: 643-645). For £. coli, two modes of penetration of aminoglycoside have been suggested; 1) the aminoglycosides are taken by the path of the porin; and 2) the penetration of the aminoglycosides may be due to the aggregation-disintegration of OmpF (porin F), mediated by the interaction to a divalent cation binding site on OmpF (Hancock et al., 1991, Antimicrob. Agents Chemother. 1309-1314). In order to demonstrate that the liposomal formulations produced according to the present invention are effective against a broad array of bacterial strains, bactericidal tests were performed using P. aeruginosa, B. cepacia, E. coli, S. aureus and X maltophilia.

EXAMPLE 5 Bactericidal Activity, in vitro, against Different Bacterial Families To evaluate the bactericidal efficacy of the liposomal tobramycin formulation, produced according to the present invention (DPPC / DMPG), in vitro tests were performed against different clinical strains: Pseudomonas aeruginosa (strain 429) MIC > _60 μg / ml, Burkholderia cepacia (strain ID-28369) MIC > _27 μg / ml, Escherichia coli (strain 1061 mn 88) MIC > 5 μg / ml, Staphylococcus aureus (strain LSPQ 2499) MIC > 9 μg / ml and Xanthomonas maltophilia MIC > 5 μg / ml. To culture tubes containing protease penton (29 ml), a minimum number of 108 CFU of logarithmic phase bacteria (1 ml) and one of the following preparations (100 μl) were added at zero time: free tobramycin, tobramycin encapsulated in liposome, control liposomes or PBS. Experiments were done in triplicate. At time, 1, 3, 6 and 16 hours after the addition of the antibiotic or controls, 2 ml of samples were collected and 10-fold serial dilutions were made and extended in triplicate onto proteose peptone agar plates for strains. Gram-negative and on MacConkey agar plates for S. aureus. CFUs were counted after incubations of 24 hours and 48 hours at 37 ° C under 5% CO2. The counts were expressed in CFU log per ml of culture medium. The results are presented in Figures 1-4 and Table 5. As can be seen from these, for the five (5) bacterial families, the amount of tobramycin encapsulated in each experiment was lower than the MIC of the bacteria used.

TABLE 5 Viable Bacterial Counts (CFU) of Xanthomonas maltophilia in Protein Peptone (MIC> 5 μg / ml) The results presented in Figures 1-4 and Table 5 showing significant bactericidal efficacy of the liposomal formulation of tobramycin comparatively to tobramycin with the five different bacteria used, show that the antibacterial property of this formulation can not be restricted to one type Particular of bacterial cell wall and suggest that the liposomal formulations of the present invention can be effectively used for the treatment of bacterial infections in general. In summary, the liposomal formulations of the present provide a very significant improvement in the delivery of therapeutic agents, compared to those of the prior art. These formulations can be used in numerous animal systems with bacterial infections. The bactericidal efficacy of the tobramycin encapsulated in the liposome against different families of bacteria, as demonstrated in Figures 1-4 and Table 5, shows that the liposomal formulation of the present invention can be effective against a large number of bacteria presenting important variations in its outer membrane. In addition, the liposomal formulation of the present provides a promising alternative for the treatment of chronic lung infections in patients with cystic fibrosis. Since the invention has been described with particular reference to the illustrated embodiment, it will be understood that those skilled in the art can make numerous modifications thereto. Accordingly, the foregoing description and the accompanying drawings should be taken as illustrative of the invention and not in a limiting sense.

Claims (21)

1. - A multilaminar, low-stiff liposomal formulation, which is characterized by being free of cholesterol and phospholipids with a high phase transition temperature (Tc), and said formulation comprises neutral and anionic phospholipids at a molar ratio of 5: 1 to 20: 1, whose mean value of Tc is below 37 ° C or below the body temperature of an animal to be treated and at least one antimicrobial agent.

2 - The liposomal formulation according to the claim 1, wherein the formulation is in a powdered state.

3. The liposomal formulation according to claim 1, wherein the phospholipid and the anionic phospholipid are present at a ratio of 5: 1 to 20: 1.

4. The liposomal formulation according to the claim 3, wherein the neutral phospholipid and the anionic phospholipid are present at a ratio of 7.5: 1 to 17.5: 1.

5. The liposomal formulation according to claim 3, wherein the neutral phospholipid and the anionic phospholipid are present at a ratio of 10: 1 to 15: 1.

6. The liposomal formulation according to claim 5, wherein the neutral phospholipid and the anionic phospholipid form a negatively charged lipid mixture.

7. The liposomal formulation according to claim 5, wherein the neutral phospholipid is dipalmitoylphosphatidylcholine (DPPC) and the anionic phospholipid is dimyristoylphosphatidylglycerol (DMPG).

8. The liposomal formulation according to the claim 1, wherein the antimicrobial agent is tobramycin at a concentration of 10 mg / ml to 40 mg / ml.

9. The liposomal formulation according to the claim 5, wherein the antimicrobial agent is tobramycin at a concentration of 10 mg / ml to 40 mg / ml.

10. The liposomal formulation according to the claim 7, wherein the antimicrobial agent is tobramycin at a concentration of 10 mg / ml to 40 mg / ml.

11. The liposomal formulation according to the claim 1, wherein the formulation improves passage through direct interaction with bacteria of at least one antimicrobial agent through at least the bacterial outer membrane and the exopolysaccharide layer.

12. A method for the treatment or prevention of a bacterial infection in mammals, comprising administering a suitable antimicrobial dose of the liposomal formulation of claim 1, to said mammals.

13. A method for the treatment or prevention of a mucoid variant of a bacterial infection in mammals, comprising the administration of a suitably acceptable form of the liposomal formulation of claim 10 to said mammals.

14. A method for the treatment or prevention according to claim 13, wherein the bacterium is Pseudomonas aeruginosa and the mammal is a human being with cystic fibrosis or chronic infection.

15. A method for the treatment or prevention according to claim 12, wherein the infection is caused by at least one type of bacteria, wherein said type of bacteria is selected from Pseudomonas, Burkholderia, Escherichia, Staphylococcus and Xanthomonas.

16. A method for the treatment or prevention according to claim 15, wherein the infection is caused by at least one type of bacteria, wherein said type of bacteria is selected from Pseudomonas aeruginosa, Burkholderia cepacia, Escherichia coli, Staphylococcus aureus and Xanthomonas maltophilia.

17. - The use of the liposomal formulation of claim 1 for the treatment or prevention of a bacterial infection in mammals, comprising administering a suitable antimicrobial dose of the formulation to said mammals.

18. The use of the liposomal formulation of claim 1 for the manufacture of a medicament for the treatment or prevention of a bacterial infection in mammals.

19. An antibacterial formulation comprising the liposomal formulation of claim 1 in an antimicrobial dose for the treatment or prevention of bacterial infections.

20. The antibacterial formulation according to claim 19, wherein said bacterium is selected from the group consisting of Pseudomonas, Burkholderia, Escherichia, Staphylococcus and Xanthomonas.

21. The antibacterial formulation according to claim 20, wherein said bacterium is selected from the group consisting of Pseudomonas aeruginosa, Burkholderia cepacia, Escherichia coli, Staphylococcus aureus and Xanthomonas maltophilia.