ES2574615B1 - Tobramycin lipid nanoparticle - Google Patents

Abstract

The present invention relates to a lipid nanoparticle comprising at least one tobramycin antibiotic, a lipid fraction and one or more surfactants, and the use of the nanoparticle in the prevention and / or treatment of respiratory tree infections.

Description

"Tobramycin lipid nanoparticle ~

SECTOR OF THE TECHNIQUE

The present invention relates to a lipid nanoparticle comprising at least one tobramycin antibiotic, a pharmaceutical composition comprising said nanoparticle and the use of the nanoparticle in the prevention and / or treatment of infections by tobramycin-sensitive bacteria, preferably in the tree. respiratory.

PREVIOUS STATE OF THE TECHNIQUE

Tobramycin is a water-soluble antibiotic of the aminoglycoside family that is used in severe cases of infection due to its bactericidal activity against numerous gram-negative and gram-positive bacteria. However, tobramycin should be used strictly and occasionally limited, due to its narrow therapeutic margin, as it may result in a nephrotoxic, ototoxic and / or neurotoxic effect.

Tobramycin is rarely used as monotherapy. However, different studies have shown that its stability is affected by the presence of other components. Temperature dependence of the stability of tobramycin mixed with penicillins in human serum. Am J Hosp Pharm. 1982 Jun; 39 (6): 1005-8.

It is also known that tobramycin is chemically unstable. EP238800BA 1 describes different formulas that favor the stability of tobramycin.

One of the diseases in which chronic and recurrent infections by gram-negative multi-resistant bacteria (BGN-MR) have the greatest impact, for example, by Pseudomonas aeruginosa, is cystic fibrosis. The persistence of this bacterium is associated, among other causes, with its growth in a biofilm, which consists of a collective structure of bacteria that adheres to surfaces, covered by a protective layer that secrete the bacteria themselves, and that provides the capacity to resist biocides and antibiotics more effectively, withstanding significantly higher doses of antibacterial products, and causing a deterioration of lung function.

Some antibiotics for the treatment of these infections have adverse effects, so the use of microsystems or nanosystems to administer such antibiotics presents a particular interest. Different uses of these systems comprising some of these antibiotics are described in the literature, as is the case of US2009169635, in which nanoparticles of biodegradable polymers of polyester type for systemic administration are described.

The direct administration of antibiotics to the lower airways through the administration of aerosols and dry powder has potential advantages such as the higher local concentration that can be achieved by deposition in the alveolar location where the infection is, and, therefore, inhaled drugs can reduce the appearance of systemic adverse effects by reducing the dose administered. Previous studies have shown that the concentration of aerosolized tobramycin in sputum for suppression of Pseudomonas aeruginosa, in patients with cystic fibrosis requires levels of 10 times the MIC to suppress growth and 25 times the MIC to have a bactericidal effect. However, a small proportion of tobramycin can be absorbed generating unwanted toxic effects.

Another case study of patients treated with tobramycin in an inhaled solution is that if they are taking other inhaled medications, they should take special care not to mix the tobramycin with the rest of the medications, the administration of tobramycin should be ultimately place.

In view of these data, there is therefore a need to develop tobramycin drugs for the treatment of infections, preferably in the respiratory tree, that overcome the drawbacks of the prior art.

EXHIBITION OF THE INVENTION

The inventors have developed lipid nanoparticles comprising at least one tobramycin antibiotic that are able to adhere to or interact with the mucous layer of the respiratory tract or the biofilm generated by the bacteria themselves, favoring that at a lower therapeutic dose of antibiotic results are obtained of minimum optimal inhibitory concentration.

The nanoparticles of the invention are protected against premature degradation and also have a sustained release of the antibiotic in the epithelia, more specifically in the alveolar and / or bronchial epithelium.

Thus, one aspect of the invention relates to a lipid nanoparticle comprising at least one tobramycin antibiotic, a lipid fraction comprising a mixture of one or more solid lipids whose melting point is equal to greater than 25 ° C and one or more liquid or semi-solid lipids whose melting point is less than 25 ° C, and one or more surfactants.

Another aspect of the invention relates to a pharmaceutical composition comprising the lipid nanoparticles defined above together with one or more pharmaceutically acceptable carriers or carriers.

Another aspect of the invention relates to a method of preparing the lipid nanoparticles defined above comprising the following steps:

a) Prepare a mixture of lipids and at least one antibiotic by heating at a temperature slightly higher than the solid lipid melting point.

b)

Prepare an aqueous solution with one or more surfactants.

C)

Heat the aqueous solution b) at the same temperature as the oil phase a).

d)

Add the aqueous phase b) over the oil phase a) and mix to obtain a

emulsion.

e) Maintain at a temperature 5 ° C ± 3 ° C until the lipids recrystallize giving rise to nanoparticles. f) Wash the nanoparticles obtained by centrifugation and / or ultrafiltration.

The lipid nanoparticles of the invention may be useful in the treatment of infections, in particular respiratory tree infections. Therefore, another aspect of the invention is directed to the lipid nanoparticle defined above, for its Use as medicine.

Another aspect of the invention relates to the lipid nanoparticle defined above, for use in the treatment and / or prevention of infections in the respiratory tree. Thus, this aspect refers to the use of the lipid nanoparticle defined above to prepare a medicament for the treatment and / or

prevention

from a infection, preferably in he tree respiratory,

preferably

caused by P. aeruginosa I species related I

Tobramycin sensitive microorganisms.

Another aspect of the invention is directed to a method of treatment and / or prevention of an infection, preferably in the respiratory tree, preferably caused by P. aeruginosa and / or related species and / or microorganisms sensitive to tobramycin, which comprises administering a therapeutically effective amount of the lipid nanoparticle defined above, together with pharmaceutically acceptable excipients or carriers, in a subject in need of such treatment and / or prevention, including a human.

In this sense, the studies carried out by the inventors have demonstrated the ability of these lipid nanoparticles, as well as pharmaceutical compositions and / or drugs comprising these nanoparticles, to:

obtain a stable lipid nanoparticle with a sustained and / or regulated antibiotic release effect, protect the antibiotic from premature degradation, have the ability to penetrate the biofilm generated by the bacteria themselves, and obtain better values of minimum inhibitory concentration that The free antibiotic.

lead to less toxic effects due to tobramycin.

These and other advantages and features of the invention will become apparent in view of the figures and the detailed description of the invention.

DESCRIPTION OF THE DRAWINGS

Figure 1. Microscopic photograph of an embodiment of the lipid nanoparticles of the present invention.

Figure 2. Antibiotic release curve over time of lipid nanoparticles.

DETAILED EXHIBITION OF THE INVENTION

The lipid nanoparticle that the inventors have developed comprises at least one tobramycin antibiotic, a lipid fraction comprising a mixture of one or more solid lipids whose melting point is equal to greater than 25 ° e and one or more liquid or semi-solid lipids whose melting point it is less than 25 ° C, and one or more surfactants.

In the context of the present invention, the term "lipid nanoparticle" refers to a matrix comprising a core of lipid and / or lipophilic nature based on mixtures of solid and liquid lipids.

The scope of the invention includes nanoparticles comprising nanostructured lipid transporters.

In one embodiment, the nanoparticles of the invention are characterized by having an average size, approximately, between 10 nm and approximately 1000 nm, preferably between approximately 100 nm and approximately 500 nm.

By "average size" is meant the average diameter of the population of lipid nanoparticles of the present invention. The average size can be measured by standard procedures known to the person skilled in the art, and which are described, for example, in the part of the examples below.

The particle size is one of the factors that determines the sustained release of the antibiotic. In general, the antibiotic located on the surface of the nanoparticle is the first to be released. The smaller the nanoparticle size, the greater the specific surface area of interaction, so there will be a greater initial release of antibiotic.

In one embodiment, the nanoparticles have a surface charge (measured by the zeta potential), the magnitude of which can vary from about 30 mV to about -5 mV and preferably between -30 mV and -16 mV. Generally, the zeta potential is one of the parameters that affect the stability of the lipid nanoparticles. The fact that they are negatively or positively charged will favor that the repulsion forces between the nanoparticles avoid agglomerations presenting better dispersion properties.

Considering the positive charge of the lipopolysaccharides of the bacterial membranes present in the biofilm generated by the bacteria themselves and / or present in the lung mucosa, the negative surface charge of the nanoparticles of the present invention favors the nanoparticle-bacterium junction, optimizing retention and adhesion of the nanoparticle in the place of action, favoring a sustained and therapeutic effect of the antibiotic better.

In one embodiment, the nanoparticles of the invention have polydispersion index (PDI) values equal to or less than 0.5. This index gives us an idea of the diversity of nanoparticle sizes in a mixture. The closer it is to zero, the more homogeneous the nanoparticles will be, indicative of a homogeneous size distribution of the manufacturing batches.

Both the average size, as well as the zeta potential, and the PDI value of the nanoparticles are mainly influenced by the amount of the lipid component, by the amount of surfactants and by the parameters of the preparation procedure, such as the power and type of agitation , the temperature of both phases or the duration of the mixing phase.

Lidic Fraction

The lipid nanoparticle of the present invention comprises as a lipid fraction a mixture of one or more solid lipids whose melting point is equal to greater than 25 ° C and one or more liquid or semi-solid lipids whose melting point is less than 25 ° C.

In the context of the present invention, "solid lipid at room temperature" is understood as a lipid having a melting point equal to or greater than 25 ° C.

In one embodiment, the solid lipid or solid lipids of the lipid fraction have a melting point that is between 25 ° C and 80 ° C, preferably between 40 ° C and 75 ° C, most preferably between 53 ° C and 72 ° C

In the context of the present invention, "liquid lipid at room temperature" is understood as a lipid having a melting point below 25 ° C.

In one embodiment, the liquid or semi-solid lipid of the lipid fraction has a melting point of less than 17 ° C, preferably less than 4 ° C, most preferably less than O ° C

In a preferred embodiment, the lipid fraction comprises lipids selected from the group consisting of oils and / or monoglycerides and / or diglycerides and / or triglycerides and / or fatty acids and / or esters of fatty acids and / or their derivatives and / or mixtures thereof.

As a solid lipid, being able to be saturated or unsaturated, triglycerides (for example triestearyl), and / or mono or diglycerides (for example derivatives and mixtures of mono and diglycerides) and / or fatty acids (for example stearic acid, for example) ) and / or waxes (for example cetyl palmitate). The definition of fatty acid derivatives includes those fatty acids, their esters or their amides that have hydroxyl groups as substituents of the hydrocarbon chain.

In one embodiment, the solid lipid fraction comprises a mixture of glycerol dibehenal (for example, Compritol® 888 and a mixture of glyceryl palmitoestearate monoglycerides, diglycerides and / or triglycerides (for example, Precirol® ATO 5). of the invention, the weight ratio (weight / weight) of glycerol dibehenate and the mixture of glyceryl palmosteterate glycerides is comprised approximately between 0.5: 10 and approximately 5:10, preferably 1:10.

In another embodiment, the solid lipid fraction comprises a mixture of monoglycerides, diglycerides and triglycerides of glyceryl palmostearate (for example, Precirol® ATO 5).

As a liquid lipid, oils, and / or triglycerides, and / or monoglycerides and / or diglycerics and / or fatty acids and / or esters of fatty acids and / or mixtures thereof may be included, without limitation.

In one embodiment, the liquid lipid fraction comprises triglycerides.

In one embodiment, an oleic acid and / or an oleic acid derivative is used as a liquid lipid.

In a preferred embodiment, a triglyceride of caprylic acid and capric acid (for example MyglioI812) is used as a liquid lipid.

The liquid lipid provides a less ordered structure increasing the carrying capacity of the antibiotic in the nanoparticle matrix.

In one embodiment of the invention, the weight (weight / weight) ratio of liquid lipid to solid lipid is between 0.1: 10 and 10:10.

In a preferred embodiment, the weight (weight / weight) ratio of liquid lipid to solid lipid is 1:10.

The use of solid and liquid lipids provide the following advantages to the nanoparticle: improved organism and tissue tolerance due to the use of

5 physiologically accepted lipids or generally recognized as safe, possibility of encapsulating drugs, using different preparation methods,

do not show biological toxicity, and

10 It is possible to modulate antibiotic release as needed. The nanoparticles with an antibiotic-rich shell have an important initial release while the nanoparticles with a drug-rich nucleus allow a sustained release thereof.

15 Surfactant

As stated above, the nanoparticles of the invention comprise one or more surfactants. In a particular embodiment, the hydrophilic phase of the nanoparticles surrounding the lipophilic core comprises a surfactant. At

In the context of this invention, a surfactant is an emulsifier or emulsifier that reduces the surface tension of the different phases that are required for the manufacture of the nanoparticles, achieving a better interposition of them and thus, the formation of the nanoparticles.

25 The surfactants can be cationic, ionic or non-ionic and their classification will depend on the charge of the surface part. Examples of cationic surfactants include, without limitation, cetrimide and / or cetylpyridinium chloride; Examples of anionic surfactants include, without limitation, sodium docusate and / or sodium lauryl sulfate.

30 The term "non-ionic surfactant" means a compound without any net charge, which has a hydrophobic part and a hydrophilic part.

In a preferred embodiment, the nanoparticle comprises at least one nonionic surfactant 35 whose main functions are to control the particle size and confer stability avoiding the formation of aggregates. Examples of non-ionic surfactants include, without limitation, polysorbates, polyethylene glycol copolymers and / or polypropylene glycol copolymers. In a preferred embodiment, the non-ionic surfactants are polysorbate 80 and / or poloxamer.

In one embodiment, the proportion of non-ionic surfactant is between 0.5% and 10% by weight with respect to the total weight of the nanoparticle, preferably between 0.5% and 2%, most preferably between 0.6% and 1.5%.

Antibiotic

An antibiotic or antimicrobial is an agent that acts against bacterial infections either by inhibiting the growth of the bacteria or giving rise to a chain of biochemical events that will lead to the lysis of the bacteria.

In the present invention, the lipid nanoparticle comprises at least one tobramycin or a prodrug of tobramycin or a derivative of tobramycin, preferably tobramycin.

Antibiotic release as well as antibacterial action can be regulated by the weight ratio of the antibiotic to the lipid fraction. In one embodiment, the weight ratio of the antibiotic to the lipid fraction is from 0.25: 10 to 4:10, preferably 1:10.

The antibacterial action of an antibiotic can be measured by the minimum inhibitory concentration, which consists in the concentration of the antibiotic required to prevent bacterial growth from the incubation of 10 4-5 bacteria in rapid growth phase, in a free medium of proteins with pH 7.2, aerobic, during an overnight incubation period. This term is used to determine bacterial sensitivity to a specific antibiotic agent.

The term sensitive in the context of infection means an inhibition of the growth of the microorganism and / or death of the microorganism, in the case of a therapeutic dose treatment.

The weight ratios of antibiotic-lipid fraction of the different embodiments of the present invention have demonstrated a lower inhibitory concentration lower than the free antibiotic. This fact, in addition to being a cost advantage since a smaller amount of antibiotic is required for the same therapeutic effect, favors a lower probability of acquired bacterial resistance.

By the term bacterial resistance acquired in the context of the invention is understood that resistance acquired by the bacterium through the acquisition of resistance genes from other bacteria and / or by mutation processes. Bacterial resistance is directly related, among other causes, to the use of inadequate dose or duration of antibacterial therapy.

The fact of requiring a smaller amount of tobramycin for the same therapeutic effect favors a lower toxic effect of tobramycin.

Preparation Procedure

The lipid nanoparticles of the present invention can be prepared by heat fusion homogenization technique.

This technique includes the following stages:

a) Prepare a mixture of lipids and at least one antibiotic tobramycin

heating at a temperature slightly higher than the melting point of

solid lipid

b) Prepare an aqueous solution with one or more surfactants.

c) Heat the aqueous solution b) at the same temperature as the oil phase a).

d) Add the aqueous phase b) over the oil phase a) and mix to obtain a

emulsion.

e) Maintain at a temperature 5 ° C ± 3 ° C until the lipids recrystallize.

f) Wash the nanoparticles obtained by centrifugation / ultrafiltration.

In one embodiment, on the one hand the solid and / or liquid lipids and at least the tobramycin type antibiotic are mixed, and heated at a temperature slightly higher than the melting point of the solid lipid. On the other hand, an aqueous solution of at least one surfactant is prepared. The oily solution and the solution

The aqueous solution is heated to the same temperature, and once both phases reach the same temperature, the aqueous solution is added over the oil. The mixture is emulsified by sonication. The size, polydispersion index and encapsulation efficiency of the nanoparticles depend on the sonication power and the sonication time. Preferably it is sonic between 10W and 40W and most preferably between 20W and 30W for between 10 seconds and 45 seconds, preferably between 15 seconds and 31 seconds and most preferably between 29 seconds and 30 seconds. The emulsion obtained is stored at least between 5 hours and 30 hours, preferably between 10 hours and 20 hours and most preferably between 11 hours and 13 hours, 12 hours between 1 ° C and 10 ° C, preferably between 2 ° C and 6 ° C and most preferably between 3 ° C and 5 ° C. In this period, lipids are recrystallized forming the nanoparticles. After time, they are washed by centrifuging and filtering between 1 and 10 times, preferably between 2 and 5 times and most preferably 3 times, between 1000 rpm and 3500 rpm, preferably at 2000 rpm and 3000 rpm and most preferably above 2500 rpm for between 10 minutes and 30 minutes, preferably between 12 minutes and 16 minutes and most preferably between 14 minutes and 15 minutes. One of the advantages of this method is that organic solvents are not used, thus avoiding the need to carry out trace tests for organic solvents prior to the commercialization of nanoparticles for human consumption.

One aspect of this invention is directed to the product obtainable by the technique described above.

lyophilization

In one embodiment, the lipid nanoparticle is a lyophilized nanoparticle. Lyophilization allows a dry powder to be obtained from the lipid nanoparticles, which gives it greater stability than the lipid nanoparticles in suspension, since it prevents the degradation of the nanoparticle and the early release of the antibiotic to the solution in which the nanoparticles are suspended .

Lyophilization can be performed by standard procedures known to the person skilled in the art, and which are described, for example, in the part of the examples below.

In one embodiment, the lipid nanoparticle of the present invention comprises a cryoprotectant. The cryoprotectant favors the stabilization of the nanoparticles during the freeze-drying process. This cryoprotectant can be selected, without limitation, from Si02 colloidal, glycine, lactose, mannitol, trehalose, raffinose, nulin, sodium bicarbonate and sodium borate.

In a preferred embodiment, the nanoparticle comprises trehalose as a cryoprotectant.

In one embodiment, the lipid nanoparticle comprises between about 5% and about 20% of cryoprotectant by weight with respect to the weight of the lipid nanoparticle, preferably between 5% and 15%.

Infection

One aspect of the invention is directed to the lipid nanoparticle of the invention, for use in the treatment and / or prevention of infection, preferably of an infection in the respiratory tree.

Another aspect of the invention is directed to the use of the lipid nanoparticle of the invention, to prepare a medicament for the treatment and / or prevention of infection, preferably of an infection in the respiratory tree.

Another aspect of the invention is directed to a method of treatment or prevention of an infection, preferably of an infection in the respiratory tree, which comprises administering a therapeutically effective amount of the lipid nanoparticle defined above, together with pharmaceutically acceptable excipients or carriers, in a subject in need of such treatment and / or prevention, including a human.

The term infection includes any infection by gram negative and / or gram positive bacteria and / or tobramycin-sensitive bacteria or microorganisms, preferably by Pseudomona aeruginosa. This infection can be located in the respiratory tree, in the skin, in the urinary tract, in the eye, in the bone system, in the joints and / or at the systemic level.

The term respiratory tree includes the nasal cavity, pharynx, larynx, trachea, primary bronchus and lungs.

The term "prevention or treatment" in the context of the specification means the administration of the nanoparticles according to the invention to preserve health in a patient who suffers or is at risk of suffering a bacterial infection described above. Such terms also include the administration of the nanoparticles according to the invention to prevent, improve, alleviate or eliminate one or more symptoms associated with the bacterial infection. The term "improve" in the context of this invention is understood to mean any improvement in the situation of the treated patient, either subjective (sensation of or in the patient) or objectively (measured parameters).

In one embodiment, the infection in the respiratory tree is due to Pseudomonas aeruginosa.

The nanoparticle of the invention has demonstrated its ability to adhere to the biofilm generated by the bacteria or the mucus of the respiratory tree tissue itself. Thus, in a particular embodiment, the invention is directed to the use of the lipid nanoparticle of the invention to lung infection associated with cystic fibrosis and / or bronchiectasis.

The lipid nanoparticles of the present invention may be part of a pharmaceutical composition. Such pharmaceutical compositions include any solid, semi-solid or liquid composition.

The pharmaceutical composition comprises the lipid nanoparticle together with pharmaceutically acceptable excipients or carriers, in a subject in need of such treatment and / or prevention, including a human. The person skilled in the art can determine which additional components can be used and if necessary, many of them being commonly used in pharmaceutical compositions.

The term "therapeutically effective amount" in the context of this invention refers to the amount of composition that once administered, is sufficient to prevent or treat one or more symptoms derived from bacterial infection. The particular dose administered according to the present invention will be determined according to the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated and similar considerations.

The term "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, composition or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications according to a benefit ratio! reasonable risk

The pharmaceutical composition may comprise other ingredients such as viscosity modulators, preservatives, solubilizers, which include, without limitation, cyclodextrins, lecithins and / or glycerol monostearate, antifloculants, which includes, without limitation, leucine, and / or stabilizers, which include, without limitation, alginates, alginic acid and / or trehalose. These components will be added to the lipophilic or hydrophilic phase depending on their nature.

In one embodiment, the pharmaceutical composition comprises the tobramycin lipid nanoparticles, a cryoprotectant, an anti-caking agent and other excipients. The pharmaceutical presentation can be a powder to be nebulized in solution or in dry powder for direct administration.

The lipid nanoparticle of the invention and / or the pharmaceutical composition comprising said nanoparticle and / or the medicament of the invention are administered by the digestive route (enteral, buccal, sublingual or rectal), by topical route (transdermal or ophthalmic), by route parenteral (intradermal, subcutaneous, intramuscular, intravenous,

or intraperitoneal) or direct administration in the respiratory tree.

In a preferred embodiment, it is administered in the respiratory tract, by inhalation.

These administrations by inhalation are liquid or solid preparations containing the nanoparticle and / or pharmaceutical composition and / or medicament of the invention alone or together with more drugs. The size of the particles intended to be inhaled should be adjusted to locate their distribution in the lower part of the respiratory tree and controlled by appropriate methods for determining the size of the particles. The person skilled in the art can determine which processes and / or devices can be applied for optimal administration by means of vapors

or sprays or powders.

In another aspect of the invention, the inhaled particle size is between 1¡1m and 10¡1m, preferably between 2¡1m and 8¡1m and more preferably between 3 ~ m and 5 ~ m . These sizes allow perfect alveolar deposition and pulmonary retention of the therapeutically effective amount. In a particular embodiment these sizes are achieved by adding the nanoparticles, in the case of their application as dry powder, or by generating an aerosol with the appropriate carrier in the case of being administered by nebulization.

In the following, some illustrative examples are described that show the characteristics and advantages of the invention, however, they should not be construed as limiting the object of the invention as defined in the claims.

Examples

Example 1: Preparation of lipid nanoparlicles with antibiotic by heat fusion homogenization technique.

Example 1a:

A mixture of 1000 mg of Precirol® ATO 5 and Miglyof ~ 812 was prepared in a 10: 1 ratio together with 100 mg of tobramycin, at a temperature slightly higher than the solid lipid melting point.

On the other hand, an aqueous solution of the surfactant was prepared (Poloxamer 188 at 0.6% and Polysorbate 80 at 1.3%).

The lipid and aqueous solution were heated at the same temperature. (approximately, at a temperature between 5 ° C and 10 ° C higher than the temperature of lipid fusion). The aqueous phase was added over the oil phase, and the mixture was emulsified. by sonication at 20W for 30 seconds. The emulsion obtained was stored for 18 hours at 4 ° C so that lipids could be recrislalized and form the nanoparticles.

The nanoparticles obtained were washed by centrifuging 3 times at 2500 rpm for 15 minutes using Amicon® Ultra (Millipore) filters.

Example 1b: Lipid nanoparticles were prepared according to the method described in example 2a). Unlike 2a), two types of solid lipids were used: 500mg of Precirol® ATO 5 AND 500mg of Compritol®888 ATO.

Example 1c: A part of the nanoparticles prepared according to 1a) and 1b) were lyophilized subjecting them to the following stages:

a) Addition of 15% trehalose with respect to the total weight of the lipid fraction. b) Freezing at -20 ° C and then at -80 ° C. e) Freezing at -50 ° C, at 10,000 mbar pressure for 3 hours. d) Vacuum at -50 ° C until a pressure of 0.20 mbar is obtained. e) Drying at -50 ° C at 0.20 mbar pressure for 5 hours. f) Drying at 20 ° C at 0.20 mbar pressure for 7 hours. g) Drying at 20 ° C at ambient pressure for 24 hours.

Hereinafter, the nanoparticles prepared according to 1 a and lyophilized according to 1 e, are called 1 to lyophilized, and the nanoparticles prepared according to 1 b and lyophilized according to 1e, are called 1 b lyophilized.

5 Example 2: Preparation of lipid nanoparticles in different weight ratios of liquid lipid with respect to solid lipid.

Several batches of lipid nanoparticles were prepared, with different weight ratios of liquid lipid versus solid lipid according to the method described in Example 18. The ratios were as follows: 0.1: 10; 0.5: 10; 1:10; 2.5: 10; 5:10 and

10:10 The ratios of 0.5, 1, 2.5 and 5 did not carry any antibiotic.

Example 3 Characterization of nanoparticles

15 The particle size and zeta potential were characterized by a Zetasizer Nano ZS. The following table describes the average results obtained with the batches manufactured according to examples 1 and 2:

Lots Example 1a Example 1a lyophilized Example 1b lyophilized Example 2 Example 2 Example 2 Example 2 Example 2 Example 2

Liquid lipid-solid lipid ratio 1:10 1:10 1:10 0.1: 10 0.5: 10 1:10 2.5: 10 5: 10 10:10 Size (nm) 254 ± 15 236 ± 31 279 ± 20 399 ± 75 397 ± 44 248 ± 46 401 ± 56 396 ± 56 254 ± 36 Potential Z index (mV) polydispersion (PDI) -24 ± 3 0.33 -25.6 ± 1 0.22 -22.2 ± 1 0.37 -8.8 ± 1 0.46 -16 ± 1 0.35 -25 ± 2 0.38 -31í2 0.46 -31í1 0.46 -16 ± 4 0.40

20 From this table it can be seen that nanoparticles with optimum size, Z potential and POI have been obtained for good stability and homogeneity of the nanoparticles.

Example 4: Efficiency of encapsulation.

The antibiotic encapsulation efficiency was determined by determining the amount of antibiotic present in the supernatant after the washing process described in example 1a and 1b.

The amount of tobramycin present in the supernatant was determined by fluorescamine derivatization spectrophotometry (0.5% solution in ethanol). Different standard solutions of tobramycin in ultrapure water of type 1 (for example, MiIliQ®) were prepared with the following concentrations of tobramycin: 5 ~ g / ml, 15 ~ g / ml, 25 ~ g / ml, 50 ~ g / ml , 75 ~ g / ml and 100 ~ g / ml.

Each solution, as well as the supernatant were mixed with fluorescamine in equal parts. After an incubation period of 1 hour at room temperature (25 ° C ± 2) the absorbance value at a wavelength of 390 nm of each solution was analyzed. The amount of tobramycin in the supernatant was determined from the absorbances obtained from the standard solutions.

The encapsulation efficiency was determined by the following formula:

Efficiency of Encapsulation (%) = 100 * (initial amount of antibiotic-amount of non-encapsulated antibiotic)! initial amount of antibiotic).

Batches manufactured according to the manufacturing method described in example 1 gave average values of 97% ± 1. These values are indicative that the effectiveness of the preparation procedure is around 100%, ensuring maximum use of the antibiotic added to the manufacturing process.

Example 5: In vitro tests for the determination of the minimum inhibitory concentration (Mle).

100.JI of a concentration of 104 Colony Forming Units (CFU)! Ml of 31 strains of P. aeruginosa obtained from 31 patients with cystic fibrosis, of which 13 strains were producing mucosa, were incubated for 24 hours at 37 ° C in a Mueller Hinton Broth medium with adjusted concentration of cations (MH8CA), corresponding between 20 mg and 25 mg per liter of Calcium and between 10 mg and 12.5 mg per liter of magnesium, which ensured the reproducibility of the results for P. aeruginosa, in the presence of Tobramycin, and nanoparticles obtained according to example 1 to lyophilized, in different concentrations (less than

0.125 g / ml; 0.125 g / ml; 0.25 ~ g / ml; 0.5 ~ g / ml; 1 g / ml; 2 g / ml; 4 ~ g / ml; 8 ~ g / ml; 16 ~ g / ml; 32 ~ g / ml; 64 g / ml; 128 g / ml; greater than 128 ~ g / ml).

For free Tobramycin, for approximately 18% of the strains a MIC of less than 0.25 IJg / ml was obtained, for approximately 67% of the strains a MIC of between 0.26 I1g / ml and 0.50! -I9 / ml was obtained, for approximately 85% a MIC of between 0.51 1J9 / ml and 1 IJg / ml, for approximately 5% a MIC of between 1.11Jg / ml and 2 IJg / ml, for approximately 2.5% a MIC of between 2.1 IJg / ml and 4 IJg / ml. The remainder obtained a MIC greater than 32 1J9 / ml.

In the case of the tobramycin nano particles obtained according to example 1a, for approximately 5% of the strains a MIC of less than 0.125 IJg / ml was obtained, for approximately 30% of the strains a MIC of between 0.126 IJg was obtained / ml and 0.25 IJg / ml, for approximately 40% a MIC of between 0.26 IJg / ml and 0.5 ~ g / ml and for approximately 7%, a MIC of between 0.56 ~ g / ml and 1 ~ g / ml. The remainder obtained a MIC greater than 2 IJg / ml.

These results demonstrate that the nanoparticles of the present invention have a better MIC value than the free antibiotic.

Example 6: Release studies

On the one hand, a sample of 50 mg of nanoparticles 1 a-lyophilized was incubated, and on the other, a sample of 50 mg of nanoparticles 1 b-lyophilized, in 50 ml of PBS each. These samples were previously resuspended in 1 ml of PBS and placed in microdialysis cells that were subsequently sealed with a membrane with a pore size molecular cut size of 6000-8000 Da. At pre-established times, samples were taken from the medium and replaced with fresh PBS (in the same amount as the sample taken). The antibiotic present in the removed medium was analyzed by spectrophotometry according to example 4. Figure 2 shows the percentage of antibiotic released with respect to the total amount

of antibiotic encapsulated in the nanoparticle for each type of nanoparticle in the

time (hours)

Lipid nanoparticles present

a large specific surface given to its

5

size. When be put in Contact with he PBS, first be release the

drug associated to the surface or very close to the nanoparticle superstructure. TO

This first phase of rapid release is called "burst." In a second phase,

The active substance is released by degradation / erosion or core swelling of the

particle, giving rise to the sustained release phase. In the case of therapy

1 o

antimicrobial, antibiotic levels have proven optimal for inhibiting

In vitro growth of P. aeruginosa bacteria.

Claims (16)

1. Lipid nanoparticle comprising: tobramycin, a lipid fraction comprising a mixture of one or more lipids solids whose melting point is equal to greater than 25 ° e and one or more liquid or semi-solid lipids whose melting point is less than 25 ° C, and one or more surfactants.

2.

Lipid nanoparticle according to claim 1, wherein the lipid fraction comprises lipids selected from the group consisting of oils and / or monoglycerides and / or diglycerides and / or triglycerides and / or fatty acids and / or esters of fatty acids and / or their derivatives and / or mixtures thereof.

3.

Lipid nanoparticle according to any of the preceding claims, wherein the weight ratio of tobramycin with respect to the lipid fraction is between 0.25: 10 and 4:10, preferably 1:10.

Four.

Lipid nanoparticle according to any of the claims, wherein the weight ratio of liquid lipid to solid lipid is between 0.1: 10 and 10:10, preferably 1:10.

5.

Lipid nanoparticle according to any of the preceding claims, wherein the proportion by weight of surfactant with respect to the total weight of the nanoparticle is between 0.5% and 10%, preferably between 0.5% and 2%.

6.

Lipid nanoparticle according to any of the preceding claims, wherein the surfactants are nonionic.

7.

Lipid nanoparticle according to any of the preceding claims, wherein the solid lipid has a melting point that is between 25 ° C and 80 ° C, preferably being between 40 ° C and 75 ° C.

8.

Lipid nanoparticle according to any of the preceding claims, in

where the semi-solid or liquid lipid has a melting point of less than 17 ° C, preferably less than 4 ° C.

9.

Lipid nanoparticle according to any of the preceding claims, which comprises a cryoprotectant.

10.

Lipid nanoparticle according to any of the preceding claims, which is lyophilized.

eleven.

Pharmaceutical composition comprising the lipid nanoparticle according to any of the preceding claims together with one or more pharmaceutically acceptable excipients or carriers.

12.

Lipid nanoparticle according to any of the preceding claims, for use as a medicament.

13.

Lipid nanoparticle according to any of the preceding claims, for use in the treatment and / or prevention of infections caused by gram negative and / or gram positive bacteria and / or tobramycin sensitive microorganisms.

14.

Lipid nanoparticle for use according to claim 13, wherein the infections are caused by Pseudomona aeruginosa.

fifteen.

Lipid nanoparticle according to any of the preceding claims, which is administered digestively (enteral, sublingual, buccal or rectal), topically (transdermal or ophthalmic), parenterally (intradermally, subcutaneously, intramuscularly, intravenously, or intraperitoneally) or direct administration in the respiratory tree.

16.

Lipid nanoparticle according to claim 15, which is administered by inhalation in the respiratory tract, in the form of vapors or aerosols or powders, wherein the inhaled particle has a size between 1¡1m and 10¡1m, preferably between 1.5¡ .1m and 5¡1m, and more preferably between 2¡1m and 4¡1m.