RU2428986C2 - Aerosol fluorochinolones and their applications - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to pharmaceutical compositions for inhalation introduction, containing water solution of fluorochinolone antimicrobial medication, preferably levofloxacin or ofloxacin, and bivalent or trivalent cation. Cation is selected from calcium, aluminium, zinc and iron. Compositions also can contain other antimicrobial medication and sweetener. Invention also relates to methods of treating lung infection, chronic obstructive lung disease or cystic fibrosis by introduction of said compositions to patient, who needs it. Invention also describes sets and systems for introduction, including container with composition by invention and sprayer.

EFFECT: medication in high concentration is delivered directly to affected tissue for treatment of bacterial infection in animals and people.

122 cl, 53 dwg, 37 tbl, 13 ex

Description

RELATED APPLICATIONS

This application claims the priority of provisional patent application US 60/682530, registered 05/18/2005, provisional patent application US 60/696160, registered 07/01/2005 and provisional patent application US 60/773300, registered 02/13 / 2006, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Description of the Related Art

Over the past half century, antibiotics have been effective in treating infectious diseases. From the creation of antimicrobial therapy until the late 1980s, most bacterial infections in patients in developing countries could be controlled, unless the infection occurred in an organ or in an environment where antibiotics were difficult to deliver or were ineffective, such as bacterial infections circulatory system in patients with sepsis or bacterial infections in the lungs with cystic fibrosis. However, even in the case of common infections, in response to the increased use of antimicrobials, multiple resistance mechanisms are widespread, which are dangerous in the clinical use of even the most aggressive antibacterial therapy. An increase in the number of antimicrobial resistant strains has become very common in most hospitals and health care centers. The consequences of an increase in the number of resistant strains include increased morbidity and mortality, a longer hospital stay for patients, and an increase in the cost of treatment.

Bacteria have developed various mechanisms to overcome the effects of antimicrobials. These resistance mechanisms may be specific to the molecule or family of antimicrobials or may be non-specific and underlie resistance to unrelated antimicrobials. A single bacterial strain may have several resistance mechanisms, and these mechanisms may act independently or they may act synergistically to overcome the effect of an antimicrobial agent or combination of antimicrobial agents. Specific mechanisms include drug degradation, drug inactivation by enzymatic modification, and drug target modification. However, there are more general mechanisms of drug resistance in which the antimicrobial agent’s access to the target is prevented or reduced by reducing the transfer of the antimicrobial agent to the cell or by increasing the excretion of the drug from the cell into the environment. Both mechanisms can reduce the concentration of the drug in the target area and allow bacteria to survive in the presence of one or more antimicrobial agents that would otherwise inhibit or lyse bacterial cells. Some bacteria use both mechanisms, combining low cell wall permeability (including membranes) with active antimicrobial excretion.

SUMMARY OF THE INVENTION

Various embodiments of the invention relate to compositions and methods for optimal antimicrobial activity for the treatment of infections of the respiratory tract and lungs in humans and veterinary care using short-term rapid aerosol administration and by delivering a high concentration of the drug directly to the affected tissue. In particular, in some embodiments, concentrated doses of the fluoroquinolone class of antibiotic substances are delivered to maximize concentrations of the active drug to the respiratory, pulmonary, and other non-oral local areas, including, but not limited to: skin, rectum, vagina, urethra, bladder , eye and ear. Since it is known that different drugs exhibit different antimicrobial effects depending on the dose, form, concentration and delivery schedule, some embodiments relate to specific drugs and delivery parameters that provide antimicrobial effects that are therapeutically significant. The present invention includes, but is not limited to, specific fluoroquinolone antibiotics, such as levofloxacin, formulated to be aerosolized according to the specific concentrations and criteria of an antimicrobial agent needed to treat patients with various bacterial infections. These preparations and methods are applicable with commercially available inhalers for one or more commodity-market opportunities for aerosol therapeutic agents.

Aerosol administration directly into the nasal cavity, respiratory tract and pulmonary by intranasal or oral inhalation allows drug delivery in high concentration to the site of respiratory infection with a lower risk of non-respiratory toxicity associated with the non-respiratory method of drug delivery. Moreover, direct introduction into the foci of infection allows for very high local levels of the drug, a property that makes possible the damaging effect on the principle of "rapid administration, high concentration, local exposure", specific for this class of antibiotics. Accordingly, since the antimicrobial effect of a particular antibiotic compound and therapeutic composition varies depending on the preparation and delivery parameters, new compositions and delivery methods can be developed for existing drug compounds for which the formulation is changed and administered using new delivery methods. Thanks to this finding, methods of treating other local infections can also benefit from high concentrations, direct exposure of fluoroquinolone to infected skin, rectum, vagina, urethra, bladder, eye and ear.

Members of the fluoroquinolone class of drugs possess unique pharmacological properties, including bioavailability (F), average time of absorption (MAT) from the lungs, maximum drug concentrations in the epithelial lining fluid, bronchial lavage fluid, sputum and / or lung tissue (Cmax) after aerosol administration , retention time in the lungs, area under the curve (AUC), minimum inhibitory concentrations (MIC) of the antibiotic necessary for the manifestation of antibacterial activity, AUC / MIC ratio, as well as Full and system security. A feature of the present invention is the use of short-term rapid aerosol administration delivering the drug in high concentration directly to the affected tissue (ELF, sputum, BAL, tissue) by the aerosol delivery method for treating bacterial infection in animals and humans.

In addition to the clinical and pharmacological requirements for any composition intended for therapeutic administration, many physicochemical features unique to the drug compound should also be considered. These include, but are not limited to: water solubility, viscosity, partition coefficient (LogP), estimated stability in various formulations, osmolality, surface tension, pH, pKa, pKb, degradation rate, spill permeability, phlegm binding / inactivation , taste, irritating effect on the throat and acute tolerance.

Other factors to consider when formulating a product include the physicochemical properties and antibacterial activity of fluoroquinolone, disease symptoms, clinical acceptability, and patient compliance. By way of non-limiting example, if desired, the aerosol fluoroquinolone product may exist in the form of a simple liquid (e.g., soluble fluoroquinolone with non-encapsulating soluble excipients / salts), a complex liquid (e.g., fluoroquinolone, encapsulated or complexing with soluble excipients, such as lipids, liposomes cyclodextrins, microencapsulations and emulsions), complex suspensions (for example, fluoroquinolone per se as a sparingly soluble stable nanosuspension, complexes of co-crystals / s β-precipitates and mixtures with poorly soluble lipids, such as solid lipid nanoparticles), or a dry powder (dry fluoroquinolone powder alone or in a spray-dried complex of co-crystals / co-precipitates or mixtures with poorly soluble fillers / salts or readily soluble mixtures, such as lactose )

Along with the shape of the product, packaging should also be considered. By way of non-limiting example, packaging principles include product-specific stability, the need for freeze-drying to ensure stability, the choice of device (e.g., a liquid nebulizer, a powder inhaler, a metered-dose inhaler), and the packaging form (e.g., a simple liquid or complex liquid preparation in a vial in the form of a liquid or lyophilisate, which should be dissolved previously or during application to the device; complex suspension preparations in a vial in the form of a liquid or lyophilisate with soluble with or without salt / filler, which must be dissolved previously or when added to the device or separate packaging of liquid and solid components; powder preparations in a vial, capsule or blister pack; and other preparations packaged as readily soluble or sparingly soluble solids in separate containers alone or together with readily soluble or sparingly soluble solids, each individually packaged substance will be made to mix beforehand or while entering the delivery device.

In some aspects, the present invention relates to aerosol or topical delivery of fluoroquinolone antimicrobials, such as levofloxacin. Levofloxacin has suitable solubility characteristics that allow the clinically necessary amounts of fluoroquinolone to be administered as an aerosol (e.g. by liquid spray, powder spray or metered dose) or topically (e.g., an aqueous suspension, an oil preparation and the like, or as a drip, spray, suppository, ointment or rubbing or the like) and can be used in methods for emergency or preventive medical care of infected vertebrates For example, a bacterial infection or a subject at risk of infection.

Others include: ofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloksatsin, rufloksatsin, fleroxacin, balofloksatsin, sparfloxacin, trovafloxacin, enoxacin, norfloxacin, klinafloksatsin, grepafloksatsin, sitafloxacin, Marbofloxacin, Orbifloxacin, sarafloksatsin, danofloxacin, difloxacin , enrofloxacin, garenoxacin, prulifloxacin, olamufloxacin, DX-619, TG-873870 and DW-276.

In a preferred embodiment, the method treats a bacterial infection in a subject using concentrated aerosol levofloxacin administered to a subject infected with pathogenic bacteria in the lungs.

The therapeutic method may also include a diagnostic step, such as identifying a patient infected with certain pathogenic bacteria or resistant bacteria. In some embodiments, the method further comprises identifying a patient infected with bacteria that is capable of developing resistance to fluoroquinolone antimicrobial agents. In some embodiments, the delivered amount of aerosol levofloxacin is sufficient to overcome resistance or prevent the development of levofloxacin resistance. In one embodiment, the MIC of the fluoroquinolone antibacterial compound for microbes is greater than about 2 μg / ml.

In another embodiment, the delivered amount of aerosol levofloxacin is sufficient to overcome resistance or prevent further resistance of the body for which the MIC of the fluoroquinolone antibacterial compound is greater than about 4 μg / ml.

In another embodiment, the delivered amount of aerosol fluoroquinolone is sufficient to overcome resistance or prevent further resistance of the body for which the MIC of the fluoroquinolone antibacterial compound is greater than about 8 μg / ml.

In another embodiment, the delivered amount of aerosol fluoroquinolone is sufficient to overcome resistance or prevent further resistance of the body for which the MIC of the fluoroquinolone antibacterial compound is greater than about 16 μg / ml.

In another embodiment, the delivered amount of aerosol fluoroquinolone is sufficient to overcome resistance or prevent further resistance of the body for which the MIC of the fluoroquinolone antibacterial compound is greater than about 32 μg / ml.

In another embodiment, a prophylactic medical care method is provided to a subject, comprising administering to a subject susceptible to a microbial infection or a chronic carrier of an asymptomatic or mild microbial infection of a fluoroquinolone antimicrobial agent to achieve a minimum inhibitory concentration of the antimicrobial agent in the focus of the potential or current infection. In one embodiment, the method further comprises identifying the subject as a subject for which there is a risk of a bacterial infection or there is a risk of an exacerbation of the infection.

In another embodiment, a method is provided for emergency or preventive medical care to a patient by aerosol administration of fluoroquinolone to achieve and maintain a threshold concentration of drug in the lungs, which can be measured as drug levels in the epithelial lining fluid (ELF), sputum, lung tissue, or bronchial fluid lavage (BAL). One embodiment includes the use of short-term rapid aerosol administration delivering the drug in high concentration directly to the affected tissue for treating a bacterial infection in animals and humans.

In another embodiment, a method of treating a microbial infection in a subject is provided, comprising administering to a subject infected with microbes a fluoroquinolone antimicrobial agent to achieve a minimum inhibitory concentration of the antimicrobial agent in the foci of infection. In one embodiment, the method further comprises detecting a subject infected with microbes that are resistant to an antimicrobial substance.

In another embodiment, a method is provided for emergency or preventive medical care to a patient by non-oral or non-nasal topical administration of fluoroquinolone in order to achieve and maintain a threshold concentration of the drug at the site of infection or at risk of infection. One embodiment includes the use of short-term rapid aerosol administration delivering the drug in high concentration directly to the affected tissue for treating or preventing a bacterial infection in the skin, tissues of the rectum, vagina, urethra, eye and auricle.

In another embodiment, a method for administering a fluoroquinolone antimicrobial by inhalation is provided, wherein the inhaled liquid or powder aerosol has an average particle size of from about 1 micron to 10 microns in a mass average aerodynamic diameter and a geometric standard deviation of particle size of less than or equal to about 3 microns. In another embodiment, the particle size is from 2 microns to about 5 microns in the mass average aerodynamic diameter and the geometric standard deviation of the particle size is less than or equal to about 2 microns. In one embodiment, the geometric standard deviation of the particle size is less than or equal to about 1.8 microns.

In some embodiments of the methods described above, the minimum inhibitory concentration of the fluoroquinolone antimicrobial agent is maintained at the site of infection for at least about 5 minutes, at least about 10 minutes, at least about 20 minutes a period of at least about a 30-minute period, at least about a 1-hour period, a 2-hour period, at least about a 4-hour period, or other time values with a quarter hour interval. An effective minimum inhibitory concentration (MIC) of a fluoroquinolone antimicrobial agent is sufficient to induce a therapeutic effect and this effect can be localized at the site of infection. In some embodiments, one or more administrations of levofloxacin can achieve a fluoroquinolone concentration in ELF, BAL and / or sputum of at least 1 to 5,000 times the MIC for infectious or potentially infectious organisms, including all integral values within the range, such as 2x, 4x, 8x, 16x, 32x, 64x, 128x, 256x, 512x, 1028x, 2056x and 4,112x the MIC microbes.

In some embodiments, such as for the pulmonary zone, a fluoroquinolone antimicrobial agent is administered in one or more administrations so as to achieve a respiratory delivered daily dose of at least about 5 mg to about 50 mg, including all integral values in the range such as 10, 15, 20, 25, 30, 35, 40 and 45 mg. Similarly, a fluoroquinolone antimicrobial agent is administered in one or more administrations so as to achieve a respiratory delivered daily dose of at least about 50 to about 100 mg, including all integral values in the range such as 55, 60, 65, 70, 75, 80, 85, 90, and 95 mg. In some embodiments of the methods described above, the fluoroquinolone antimicrobial agent is administered in one or more administrations so as to reach a respiratory delivered daily dose of up to 150 mg, including all integral values in the range such as 105, 110, 115, 120, 125 130, 135, 140 and 145 mg. The fluoroquinolone antimicrobial agent is administered in the desired respiratory delivered dose for less than 20 minutes, less than 10 minutes, less than 7 minutes, less than 5 minutes, less than 3 minutes and less than 2 minutes. In some embodiments of the methods described above, the antimicrobial agent is selected from the group consisting of ofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloksatsina, rufloksatsina, fleroxacin, balofloksatsina, sparfloxacin, trovafloxacin, enoxacin, norfloxacin, klinafloksatsina , grefafloxacin, sitafloxacin, marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, difloxacin, enrofloxacin, garenoxacin, pruliflo satsina, olamufloksatsina, DX-619, TG-873 870 and DW-276, although it is preferred levofloxacin.

In some embodiments of the methods described above, the bacteria are Gram-negative bacteria, such as Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas citeprobia salma pherobiacephaloma, colopheria colepria trophia salmonidae typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella prilellaensi malisensis, Morrisis malisensis, Francisella prularegis malisensisis, Moris frangis malisisis Providencia stuartii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Y ersinia intermedia, Bordetella pertussis, Bordetella parapertussis , Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi , Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides Bacteroidesides, Bacteroideseroides, Bacteroidesides, Bacteroides, bacteroides, bacteroides, bacteroides, bacteroides, bacteroides, bacteroides, bacteroides, bacterides, bacterides, bacterides, eggerthii and Bacteroides splanchnicus. In some embodiments of the methods described above, the bacteria are gram-negative anaerobic bacteria, in a non-limiting example these include Bacteroides fragilis, Bacteroides distasonis, homologous group Bacteroides 3452A, Bacteroides vulgatus, Bacteroides ovalus , Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii and Bacteroides splanchnicus. In some embodiments of the methods described above, the bacteria are gram-positive bacteria, by way of non-limiting example, they include: Corynebacterium diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus milleri; Streptococcus (Group G); Streptococcus (group C / F); Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus saccharolyticus. In some embodiments of the methods described above, the bacteria are gram-positive anaerobic bacteria, but include , but are not limited to, Clostridium difficile, Clostridium perfringens, Clostridium tetini, and Clostridium botulinum. In some embodiments of the methods described above, the bacteria are acid resistant bacteria, but not limited to Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium leprae. In some embodiments of the methods described above, the bacteria are atypical bacteria, but not limited to Chlamydia pneumoniae and Mycoplasma pneumoniae .

In some embodiments of the methods described above, the subject is a human. In some embodiments of the methods described above, the subject is a person suffering from cystic fibrosis. In some embodiments of the methods described above, the subject is a person suffering from pneumonia, chronic obstructive pulmonary disease or sinusitis, or a person undergoing mechanical ventilation.

In another embodiment, a pharmaceutical composition is provided that includes a simple liquid fluoroquinolone antimicrobial preparation (e.g., soluble fluoroquinolone with non-encapsulating water-soluble excipients), as described above, having an osmolality of from about 200 mosmol / kg to about 1250 mosmol / kg. In one such embodiment, the solution has a penetrating ion concentration of from about 30 mM to about 300 mM. In one embodiment, the osmolality ranges from about 250 mosmol / kg to about 1050 mosmol / kg. In one embodiment, the osmolality is preferably from about 350 mosmol / kg to about 750 mosmol / kg and, most preferably, about 300 mosmol / kg.

In another embodiment, a pharmaceutical composition is provided that includes a simple liquid fluoroquinolone antimicrobial agent having a penetrating ion concentration of from about 30 mM to about 300 mM, and preferably from about 50 mM to 200 mM. In one such embodiment, one or more penetrating ions in the composition is selected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided that includes a complex liquid fluoroquinolone antimicrobial preparation (e.g., fluoroquinolone encapsulated or complexed with water-soluble excipients, such as lipids, liposomes, cyclodextrins, microencapsulations and emulsions), as described above, having an osmol solution 200 mosmol / kg to about 1250 mosmol / kg. In one such embodiment, the solution has a penetrating ion concentration of from about 30 mM to about 300 mM. In one embodiment, the osmolality corresponds to from about 250 mosmol / kg to about 1050 mosmol / kg. In one embodiment, the osmolality is preferably from about 350 mosmol / kg to about 750 mosmol / kg and, most preferably, about 300 mosmol / kg.

In another embodiment, a pharmaceutical composition is provided that includes a complex liquid fluoroquinolone antimicrobial agent having a penetrating ion concentration of from about 30 mM to about 300 mM. In one such embodiment, one or more penetrating ions in the composition is selected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided that includes a complex liquid fluoroquinolone antimicrobial agent having a penetrating ion concentration of from about 50 mM to about 200 mM. In one such embodiment, one or more penetrating ions in the composition is selected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided that includes a complex liquid fluoroquinolone antimicrobial preparation (for example, fluoroquinolone alone as a poorly soluble in water stable nanosuspension or co-crystal / co-precipitate complexes, or mixtures with sparingly soluble lipids such as lipid nanosuspensions) as described above, having a solution osmolality of from about 200 mosmol / kg to about 1250 mosmol / kg. In one such embodiment, the solution has a penetrating ion concentration of from about 30 mM to about 300 mM. In one embodiment, the osmolality ranges from about 250 mosmol / kg to about 1050 mosmol / kg. In one embodiment, the osmolality is preferably from about 350 mosmol / kg to about 750 mosmol / kg and, most preferably, about 300 mosmol / kg.

In another embodiment, a pharmaceutical composition is provided that includes a complex suspension fluoroquinolone antimicrobial preparation having a penetrating ion concentration of from about 30 mM to about 300 mM. In one such embodiment, one or more penetrating ions in the composition is selected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided that includes a complex suspension fluoroquinolone antimicrobial preparation having a penetrating ion concentration of from about 50 mM to about 200 mM. In one such embodiment, one or more penetrating ions in the composition is selected from the group consisting of chloride and bromide.

In another embodiment, a pharmaceutical composition is provided that includes a taste masking agent for a drug. As a non-limiting example, a taste-masking agent may include sugar, a divalent or trivalent cation, which complexes with fluoroquinolone, optimizes osmolality, and optimizes the concentration of penetrating ions.

In another embodiment, a pharmaceutical composition is provided that includes a simple powder fluoroquinolone antimicrobial compound (for example, fluoroquinolone alone in the form of a dry powder with or without a mixture component, such as lactose).

In another embodiment, a pharmaceutical composition is provided that includes a complex powder fluoroquinolone antimicrobial preparation (e.g., fluoroquinolone in a spray-dried co-crystal / co-precipitate complex or a mixture with sparingly soluble excipients / salts in the form of a dry powder with a mixture component such as lactose , or without it).

In another embodiment, a system for administering a fluoroquinolone antimicrobial agent is provided that includes a container containing a fluoroquinolone antimicrobial solution and a nebulizer physically connected or packaged with the container and adapted to convert the solution into an aerosol having a particle size of from about 2 microns to about 5 microns in the mass average aerodynamic diameter and geometric standard deviation of the particle size less than or equal to about 2.5 microns on a mass median aerodynamic diameter. In one embodiment, the geometric standard deviation of the particle size is less than or equal to about 2.0 microns. In one embodiment, the geometric standard deviation of the particle size is less than or equal to about 1.8 microns.

In another embodiment, a system for administering a fluoroquinolone antimicrobial agent is provided that includes a container containing a dry powder of a fluoroquinolone antimicrobial agent and a powder inhaler connected to the container and adapted to produce a dispersed powder aerosol having a particle size of from about 2 microns to about 5 microns in mass average aerodynamic diameter and a standard deviation of particle size less than or equal to about 3 microns. In one embodiment, the standard deviation of particle size is less than or equal to about 2.5 microns. In one embodiment, the standard deviation of particle size is less than or equal to about 2.0 microns.

In another embodiment, a kit is provided that includes a container containing a pharmaceutical preparation containing a quinolone antimicrobial agent and an aerosol dispenser adapted to aerosolize the pharmaceutical preparation and deliver it to the lower respiratory tract and pulmonary region after administration to the oral cavity. The drug can also be delivered in the form of a dry powder or by means of a metered-dose inhaler.

In another embodiment, a kit is provided that includes a container containing a pharmaceutical preparation containing a quinolone antimicrobial agent and an aerosol dispenser adapted to aerosolize a pharmaceutical preparation and deliver it to the nasal cavity after intranasal administration. The drug can also be delivered in the form of a dry powder or by means of a metered-dose inhaler.

It should be understood that both the foregoing general description and the following detailed description are by way of example and explanation only and are not limiting of the invention as claimed.

DESCRIPTION OF DRAWINGS

Figure 1 is a graph illustrating the dose: MIC ratio for fluoroquinolones and other antibiotics for bacterial lysis.

Figure 2 is a graph illustrating serum ciprofloxacin concentrations after oral administration in CF patients with relatively healthy controls.

Figure 3 is a graph illustrating ciprofloxacin concentrations in sputum and serum after oral administration.

Fig. 4A is a graph illustrating the lysing effect of levofloxacin over time on PAM 1020 log cells.

4B is a graph illustrating the lysing effect of levofloxacin over time on PAM 1032 logarithmic cells.

5A is a graph illustrating the lysing effect of levofloxacin over time on PAM l020 cells in the stationary phase.

5B is a graph illustrating the lysing effect of levofloxacin over time on PAM l032 cells in the stationary phase.

6A is a graph illustrating the resumption of growth of PAM 1020 after 10 minutes of exposure to levofloxacin.

6B is a graph illustrating the resumption of growth of PAM 1020 after 160 minutes exposure to levofloxacin.

6C is a graph illustrating the resumption of growth of PAM 1032 after 10 minutes exposure to levofloxacin.

Fig.6D is a graph illustrating the resumption of growth of PAM 1032 after 160 minutes of exposure to levofloxacin.

Fig. 7A is a graph illustrating the lysing effect of levofloxacin in time on late log PAM l020 cells under conditions of oxygen deficiency.

Fig. 7B is a graph illustrating the lysing effect of levofloxacin over time on late log PAM l032 cells under conditions of oxygen deficiency.

Fig. 8A is a graph illustrating the kinetics of lysis of levofloxacin PAM 1032 in Meuller-Hinton broth (MHB).

Figv is a graph illustrating the kinetics of lysis of levofloxacin PAM 1032 in sputum with cystic fibrosis.

Fig. 9 is a graph illustrating the lysing effect of levofloxacin on Pseudomonas biofilms.

10 is a graph illustrating the bactericidal effect of levofloxacin with a Cmax of 1000 μg / ml and a half-life of 10 minutes in a hollow fiber model.

11 is a graph illustrating the bactericidal effect of levofloxacin with a Cmax of 600 μg / ml and a half-life of 10 minutes in a hollow fiber model.

12 is a graph illustrating the relationship between the micronization pressure used to micronize the dry levofloxacin powder and the average particle size of the dry levofloxacin powder.

13 is a graph illustrating a DSC profile of a pre-micronized and micronized dry levofloxacin powder.

Fig. 14A is an image illustrating SEM micrographs of premicronized dry levofloxacin powder.

14B is a SEM image showing microphotographs of micronized dry levofloxacin powder.

FIG. 15 is an image illustrating X-ray diffraction of premicronized and micronized dry levofloxacin powder.

16 is a graph illustrating the pH solubility profile of levofloxacin in acid titration.

17 is a graph of pH measurement for titration of levofloxacin HCl.

Fig. 18 is a graph illustrating the dependence of Vt [OH] on Vt of levofloxacin.

Fig. 19 is a graph of pH measurement for titration of levofloxacin NaOH.

FIG. 20 is a graph of dpH / dV measurements versus volume of a titration solution of NaOH (Vt) for titration of levofloxacin.

Fig is a graph of measuring the absorption of a solution of levofloxacin at 257 nm depending on pH.

Fig. 22 is an image illustrating DSC scans of pamic acid, levofloxacin, co-crystallized precipitate of levofloxacin-pamic acid and a physical mixture of levofloxacin and pamic acid.

23 is a view illustrating FTIR spectra of pamic acid, levofloxacin, co-crystallized precipitate of levofloxacin-pamic acid, and a physical mixture of levofloxacin and pamic acid.

Fig. 24 is an image illustrating DSC scans of xinafoic acid and co-crystallized levofloxacin-xinafoic acid precipitate.

Fig. 25 is an image illustrating FTIR spectra of xinafoic acid and co-crystals of levofloxacin xinafoate.

Fig. 26 is an image illustrating DSC scans of stearic acid, co-crystallized precipitate of levofloxacin-stearic acid and a physical mixture of levofloxacin and stearic acid.

27 is an image illustrating FTIR spectra of stearic acid, co-crystallized precipitate of levofloxacin-stearic acid and a physical mixture of levofloxacin and stearic acid.

28 is an image illustrating DSC scans of oleic acid, co-crystallized precipitate of levofloxacin-oleic acid, a physical mixture of levofloxacin and oleic acid (50:50), a physical mixture of levofloxacin and oleic acid (10:90), and a physical mixture of levofloxacin and oleic acid (90:10).

Fig. 29 is an image illustrating FTIR spectra of oleic acid, co-crystallized precipitate of levofloxacin-oleic acid, oleic acid, co-crystallized precipitate of levofloxacin-oleic acid compared to an equimolar physical mixture of levofloxacin and oleic acid.

FIG. 30 is a graph illustrating the kinetic solubility of the co-crystallized levofloxacin precipitate with oleic acid at room temperature, 40 ° C. and an equimolar physical mixture at 40 ° C.

31 is a graph illustrating the solubility profile of levofloxacin xinafoate.

32 is a graph illustrating the solubility profile of levofloxacin xinafoate, with a focus on the period between two and ten minutes.

Fig. 33 is a graph illustrating the solubility profile of levofloxacin xinafoate, with a focus on the period between ten and thirty minutes.

Fig. 34 is a graph illustrating a solubility profile of a levofloxacin base.

Fig. 35 is a graph illustrating the solubility profile of levofloxacin pamoate.

36 is a graph illustrating the solubility profile of levofloxacin pamoate, with a focus on the period between two and ten minutes.

37 is a graph illustrating the solubility profile of levofloxacin pamoate, with a focus on the period between ten and sixty minutes.

Fig. 38 is a graph illustrating the solubility profile of levofloxacin stearate.

Fig. 39 is a graph illustrating the solubility profile of levofloxacin stearate, with a focus on the period between two and ten minutes.

40 is a graph illustrating the solubility profile of levofloxacin stearate, with a focus on the period between ten and thirty minutes.

Fig. 41 is a graph illustrating the complexation of levofloxacin with divalent and trivalent cations.

Fig. 42 is a graph illustrating double titration of the complexation of levofloxacin with Mg2 +.

43 is a graph illustrating double titration of the complexation of levofloxacin with Fe2 +.

Fig. 44 is a graph illustrating double titration of the complexation of levofloxacin with Ca2 +.

Fig. 45 is a graph illustrating double titration of the complexation of levofloxacin with Zn2 +.

Fig. 46 is a graph illustrating levofloxacin in combination with Ca2 + against free levofloxacin.

47 is a graph illustrating levofloxacin in complex with Mg2 + against free levofloxacin.

Fig. 48 is a graph illustrating levofloxacin in combination with Fe2 + against free levofloxacin.

49 is a graph illustrating levofloxacin in combination with Zn2 + against free levofloxacin.

Fig. 50 is a graph illustrating the solubility of levofloxacin in the presence of with Mg2 +.

Fig. 51 is a graph illustrating the solubility of levofloxacin in the presence of Mg2 + at constant ionic strength.

52 is a graph illustrating the complexation of levofloxacin with Fe2 +, measured spectrofluorimetrically.

Fig. 53 is a graph illustrating the complexation of levofloxacin with Zn2 +, measured spectrofluorimetrically.

DETAILED DESCRIPTION

Many problems associated with antimicrobial resistant pathogens can be reduced if it is possible to safely increase the concentration of antimicrobial agents in the foci of infection. For example, pulmonary infections can be treated by direct administration of an antimicrobial agent in high concentrations directly to the site of infection without causing high systemic concentrations of the antimicrobial agent. Accordingly, some of the embodiments described herein are improved methods for delivering drug compositions for treating bacterial infections in the lungs. More specifically, as described herein, the discovery has been made that aerosolized levofloxacin and other fluoroquinolones can be safely delivered by inhalation in quantities sufficient to kill susceptible bacterial infections, reduce the incidence of antimicrobial resistance, and increase efficacy against persistent pulmonary infections.

Definitions

The term “administration” or “administration” refers to a method for delivering a dose of an antimicrobial pharmaceutical composition to a vertebral. The preferred route of administration may vary depending on various factors, for example, the components of the pharmaceutical composition, the site of the potential or current bacterial infection, the microorganism involved, and the severity of the current microbial infection.

A “carrier” or “vehicle” is a compound or material used to facilitate administration of a compound, for example, to increase the solubility of a compound. Solid carriers include, for example, starch, lactose, dicalcium phosphate, sucrose and kaolin. Liquid carriers include, for example, sterile water, saline, buffers, nonionic surfactants, and edible oils such as vegetable oil, peanut and sesame oils. In addition, various adjuvants may be included, such as those commonly used in the art. These and other similar compounds are described in the literature, for example, in the Merck Index, Merck & Company, Rahway, NJ. The principles for incorporating various components in pharmaceutical compositions are described, for example, in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.

A "diagnosticum" as used herein is a compound, method, system or device that facilitates recognition and characterization of a health condition or disease. Diagnosticum can be used in standard assays known in the art.

The term "mammal" is used in its usual biological sense. So, in particular, it includes people, cattle, horses, dogs and cats, but also includes many other species.

The term "microbial infection" refers to the unwanted spread or presence of invasion of pathogenic microbes in the host. It includes the overgrowth of microbes that are usually present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a population (s) of microbes is harmful to the host mammal. Thus, microbial infection occurs when excess amounts of the microbial population are present in or on the body of the mammal, or when the effects of the presence of the microbial population (s) are harmful to the cells or other tissue of the mammal.

The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any or all of solvents, dispersants, coatings, antibacterial and antifungal agents, isotonic or delayed absorption of the substance, and the like. The use of such media and substances for pharmaceutically active substances is well known in the art. The use of any conventional medium or substance in therapeutic compositions is contemplated, unless it is incompatible with the active component. Additional active ingredients may also be included in the composition.

The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acidic and / or basic salts due to the presence of amino and / or carboxyl groups or groups like them. Pharmaceutically acceptable acid addition salts can be prepared using inorganic acids and organic acids. Inorganic acids from which salts can be obtained include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Organic acids from which salts can be prepared include, for example, acetic acid, propionic acid, naphthoic acid, oleic acid, palmitic acid, pamic acid (emboic acid), stearic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid acid, succinic acid, fumaric acid, tartaric acid, citric acid, ascorbic acid, glucoheptonic acid, glucuronic acid, lactic acid, lactobioic acid, tartaric acid, benzoic acid, ary acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be obtained include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like; especially preferred are salts of ammonium, potassium, sodium, calcium and magnesium. Organic bases from which salts can be obtained include, for example, primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins and the like, especially such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, histidine, arginine, lysine, benethamine, N-methylglucamine and ethanolamine. Other acids include dodecylsulfuric acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, and saccharin.

"Solvate" refers to a compound formed by the interaction of a solvent and a fluoroquinolone antimicrobial agent, metabolite, or salt thereof. Suitable solvates are pharmaceutically acceptable solvates, including hydrates.

In the context of the response of a microbe, such as a bacterium, to an antimicrobial substance, the term "susceptibility" refers to the sensitivity of the microbe to the presence of an antimicrobial substance. Thus, increased susceptibility means that lower antimicrobial concentrations will inhibit microbes in the environment surrounding microbial cells. This is equivalent to saying that microbes are more sensitive to antimicrobial agents. In most cases, the minimum inhibitory concentration (MIC) of such an antimicrobial substance will be reduced.

By "therapeutically effective amount" or "pharmaceutically effective amount" is meant a fluoroquinolone antimicrobial substance, as described in this invention, which has a therapeutic effect. Doses of fluoroquinolone antimicrobial agents that are suitable for treatment are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount means such amounts of a fluoroquinolone antimicrobial substance that provide the desired therapeutic effect based on the results of clinical trials and / or studies in animal models of infections. In specific embodiments, the fluoroquinolone antimicrobial is administered at a predetermined dose, and thus a therapeutically effective amount would be the amount administered. This amount and amount of fluoroquinolone antimicrobial can be easily determined by those skilled in the art and it will vary depending on various factors, such as the particular microbial strain involved. This amount may additionally depend on the patient’s height, weight, gender, age and medical history. For prophylactic procedures, a therapeutically effective amount is one that is effective in preventing microbial infection.

A “therapeutic effect” is to alleviate, to a certain extent, one or more symptoms of an infection and includes a cure for the infection. “Cure” means that the symptoms of an active infection have been eradicated, including the complete or substantial elimination of an excess of live microbes from those involved in the infection to or below a threshold for detection by conventional methods. However, certain long-term or permanent effects of infection may exist even after a cure is achieved (such as extensive tissue damage). As used herein, a “therapeutic effect” is defined as a statistically significant reduction in the excess of bacteria in a host, the occurrence of resistance, or an improvement in symptoms of infection, determined based on clinical findings in humans or animal studies.

“Treat,” “treatment,” or “procedure,” as used herein, refers to the administration of a pharmaceutical composition for prophylactic and / or therapeutic purposes. The term “prophylactic procedure” refers to a procedure for a patient who is not yet infected, but who is exposed or otherwise at risk of getting a specific infection. The term "therapeutic procedure" refers to the treatment of a patient who is already suffering from an infection. Thus, in preferred embodiments, the procedure is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of a fluoroquinolone antimicrobial substance.

Pharmacokinetics (PK) studies the changes over time in the concentration of an antimicrobial agent in the body. Pharmacodynamics (PD) studies the relationship between pharmacokinetics and in vivo antimicrobial efficacy. PK / PD parameters establish the relationship between antimicrobial exposure and antimicrobial activity. The degree of lysis by an antimicrobial agent depends on the mode of action of the antimicrobial agent and is determined either by the length of time required for lysis (time-dependent) or by the effect of increasing concentrations (concentration-dependent). Accordingly, various PK / PD parameters can be used to predict the therapeutic efficacy of antimicrobial agents with different mechanisms of action.

The “AUC / MIC ratio” is one example of a PK / PD parameter. AUC is defined as the area under the curve of the concentration of an antimicrobial agent in plasma or in the focus of infection versus time in vivo (in an animal or human). The AUC / MIC ratio is determined by dividing the AUC in 24 hours for a single antimicrobial agent by MIC for the same antimicrobial agent as determined in vitro . Antimicrobial activity with dose-dependent lysis (such as fluoroquinolones) is well predicted based on the magnitude of the AUC / MIC ratio.

The ratio “Cmax: MIC” is another PK: PD parameter. It characterizes the maximum concentration of drug in plasma or tissue relative to MIC. Fluoroquinolones and aminoglycosides are examples of when Cmax: MIC can help predict bacterial lysis in vivo, where resistance can be suppressed.

“Time exceeding MIC” (T> MIC) is another PK / PD parameter. It is expressed as a percentage of the dosing interval, during which the plasma level or the site of infection exceeds MIC. Antimicrobial activity with time-dependent lysis (such as β-lactams or oxazolidinones) is well predicted based on the ratio of T> MIC.

The term "dosage interval" refers to the time between the introduction of two consecutive doses of pharmaceutical preparations in multiple dose regimens. For example, in the case of ciprofloxacin, which is administered twice a day (traditional regimen 400 mg twice a day) and levofloxacin, which is administered once a day (500 mg or 750 mg once a day.), The dosage intervals are 12 hours and 24 hours, respectively.

As used here, the “peak period” of the concentration of pharmaceutical preparations in vivo is defined as the time of the dosage interval of the pharmaceutical preparation when the concentration of the pharmaceutical preparation is not less than 50% of its maximum concentration in the plasma or the focus of infection. In some embodiments, a “peak period” is used to characterize the dosage interval of the antimicrobial agent.

A “respiratory delivered dose” is the amount of medicament inhaled during the inhalation phase of a breath simulator, which is equal to or less than 5 microns, when using a simulator programmed in accordance with European standard at 15 inspirations per minute with a 1: 1 ratio of inspirations .

Benefits of inhaled aerosol and local (non-oral) delivery of fluoroquinolone

The degree of antibiotic lysis depends on the mode of action of the antibiotic and is determined either by the length of time required by the antibiotic for lysis (time-dependent) or by the effect of increasing the concentration of the antibiotic (concentration-dependent). Fluoroquinolones are characterized by concentration-dependent lysis activity over time, when a high local peak concentration above the MIC of the infecting pathogen is required for the therapeutic effect.

The efficacy of fluoroquinolone in humans, animals, and in vitro model infections is related to the AUC: MIC ratio and the Cmax: MIC ratio. Given the a priori uncertainty in the pharmacokinetics of fluoroquinolones in lung tissue, many in vitro studies have been carried out to determine whether high doses of levofloxacin with extremely short half-lives (as predicted from the PK model in rats and humans) lead to more efficient bacterial lysis compared to with what takes place under conditions of longer retention times. In these studies, levofloxacin concentrations that were 0.018-1024 times higher than MIC were evaluated using a standard lysis curve and in vitro analysis using hollow fibers. In both of these analyzes, high concentrations of levofloxacin quickly showed a bactericidal effect and the maximum degree of lysis was reached after 10-20 minutes. This level of lysis was maintained, both when maintaining levofloxacin at the same level, and with a half-life of 10 minutes. Accordingly, high doses and rapid delivery of levofloxacin in a specific form, such as a rapidly delivered dose of 20-50 mg of respirable precipitated aerosol of levofloxacin (which will provide initial ELF concentrations of 800-1600 μg / ml), quickly exhibit a bactericidal effect on susceptible organisms and resistant organisms with MIC up to 32 μg / ml. These unique antimicrobial properties of fluoroquinolones are expected to occur in topical applications, including but not limited to the fight against infection or prophylaxis in the skin, eye, ear, rectum, vagina or urinary tract.

To measure the effectiveness of various delivery methods, levofloxacin preparations improving the AUC configuration were prepared and in vivo measurements were made in comparison with levofloxacin preparations not improving the AUC configuration and other antibiotics using both PK for rats and mouse efficacy after intratracheal administration. As shown previously for the rat system, there are differences between the pharmacokinetic drugs in the lungs, with some substances having lower AUCs (for example, levofloxacin), while others, such as gemifloxacin or tobramycin, have higher concentrations, resulting in slow pulmonary clearance. Studies on a mouse model of a single dose infection with aerosol dosing have shown that the compounds have different efficiencies. As shown in FIG. 1, data analysis by aerosol dose separation according to MIC indicates a strong correlation between dose: MIC and bactericidal activity (R2 = 0.89). These data indicate that the initial bactericidal activity in this model does not depend on the pulmonary clearance of the drug. Although pulmonary clearance was not evaluated in mice, translating the dose into AUC using rat scale values supposedly weakens this relationship. Therefore, these data indicate that optimization of the AUC configuration for levofloxacin may not be necessary for the effectiveness of aerosol levofloxacin in the treatment of respiratory or lung infections.

Recent studies of fluoroquinolones have led to the development of the “mutant selection window” (MSW) concept for bacterial resistance resulting from treatment. This concept helps determine the range of concentrations at which the most frequent selection of mutants in vitro and in vivo . The lower border of the window is the lowest concentration at which most infectious cells are lysed (roughly equivalent to MIC), while the upper border of the window is the concentration of the drug that blocks the growth of the least susceptible first stage mutants. Above the upper concentration limit, the growth of infectious bacteria requires the presence of at least two mutations of resistance. This upper bound is called the concentration of mutation prophylaxis (MPC). MPC values vary with bacteria and fluoroquinolone and can be 10 to 20 times the MIC. Various modeling studies have shown that the greater the concentration of the drug exceeds MPC in the focus of infection, the more effectively the treatment will prevent the development of resistance. Conversely, the longer the antibiotic concentration remains within MSW, the higher the likelihood of selection of resistant mutants. It is important that the current dosing regimen for oral and intravenous administration of levofloxacin contributes to finding this antibiotic within the MSW for more than 20% of the dosing interval for pathogens such as P. aeruginosa (Pa) and S. pneumonia. Accordingly, a high level of resistance to levofloxacin is reported for both of these pathogens.

Consequently, in one embodiment, the concentration of levofloxacin in the foci of infection is increased by delivering it directly to the lungs using inhalation therapy, thereby shortening the length of time that levofloxacin is in MSW. This therapeutic approach allows you to expand the coverage of pathogens (including strains resistant to levofloxacin), prevents the further development of resistance and leads to a decrease in the course of levofloxacin therapy.

Pharmacokinetics of orally administered fluoroquinolones in groups without CF and with CF

Concentrations in sputum of patients with CF

The pharmacokinetics of ciprofloxacin was carefully studied in patients with CF after oral administration. Indeed, it was shown that PK serum ciprofloxacin profiles are very similar in patients with CF and healthy volunteers (figure 2).

Moreover, the profile of ciprofloxacin, depending on time in sputum, was very similar to its profile in serum after oral administration (Fig. 3). After an oral dose of 750 mg, peak concentrations were ~ 4.2 μg / ml and ~ 3.5 μg / ml in serum and sputum, respectively. Serum and sputum drug concentrations reached peak values after 1.5 and 4 hours, respectively. While the total amount of ciprofloxacin in sputum was high relative to serum concentrations, absolute concentrations were low relative to MIC values of target organisms such as Pa. These data are consistent with a low clinical result due to the development of resistance to these low drug concentrations.

Although data on the intrapulmonary pharmacokinetics of levofloxacin for cystic fibrosis are not available, data on closely related ofloxacin were published in the 1980s and 1990s. Ofloxacin consists of a racemic mixture of right- (microbiologically inactive) and levorotatory isomers (levofloxacin - microbiologically active). Studies have shown that the pharmacokinetic properties of the 2 components are similar. In comparative studies with ciprofloxacin, ofloxacin had a longer half-life and greater distribution in sputum (79% versus 21%) than ciprofloxacin.

Lung epithelial lining fluid

Recently, interest in the use and improvement of fluoroquinolones in a society suffering from gram-positive infections has been focused on the study of intrapulmonary phakmakokinetics in the fluid of the epithelial lining of the lungs (ELF). Although the significance of the distribution of the drug in this fluid is not obvious for the situation with cystic fibrosis, these studies may contribute to a better understanding of the pharmacology of drugs. Levofloxanthin easily penetrates into the lung tissue. Typically, concentrations in lung tissue are 2 to 5 times higher than plasma concentrations. The results of some recent studies (presented in Table 1) indicate that the concentrations of levofloxacin in ELF in healthy subjects after an oral dose of 750 mg reach a maximum concentration of about 20 μg / ml. Similar peak concentrations are expected in the sputum of patients with CF after oral or intravenous administration of 750 mg of levofloxacin. In contrast, ciprofloxacin penetrates into the lung tissue much less effectively than levofloxacin. Based on a study of the mutant selection window (MSW), these drug levels of the fluoroquinolone in ELF are not essential to achieve the concentration required for the prevention of mutants, 10-20 times the MIC for infectious organisms.

Table 1 The concentration of levofloxacin in the fluid of the epithelial lining in humans Medicine Dose Way The concentration of the drug in ELF (μg / ml) 0.5 h 1 hour 2 h 4 h 6 h 12 h 24 h Levofloxacin 500 mg in / in eleven 2.5 1.24 Levofloxacin 500 mg Orally 9.9 6.5 0.7 Levofloxacin 500 mg Orally 9.94 6.46 0.7 Levofloxacin 500 mg Orally 4.74 10.8 9 10.9 9.6 Levofloxacin 750 mg in / in 12.94 6.04 1.73 Levofloxacin 750 mg Orally 22.1 9.2 1,5 Levofloxacin 750 mg Orally 22.13 9.19 1.55 Ciprofloxacin 500 mg Orally 1.9 0.4

Quinolones

Non-limiting examples of quinolones for use as described herein include amifloksatsin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, Flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rozoksatsin, temafloxacin, tosufloxacin, sparfloxacin, klinafloksatsin , gatifloxacin, moxifloxacin; hemifloxacin; garenoxacin; olamufloxacin, clinofloxacin, trovafloxacin, halofloxacin, prulifloxacin, moxifloxacin, hemifloxacin, rufloxacin, sitafloxacin (Sato, K, et al., 1992, Antimicrob Agents Chemother. 37: 1491-98, the entire contents of which are hereby incorporated by reference) orbifloxacin, sarafloxacin, danofloxacin, difloxacin, enrofloxacin, TG-873870, DX-619, DW-276, ABT-492, DV-7751a (Tanaka, M, et al., 1992, Antimicrob. Agents Chemother. 37: 2212-18 ), and F-1061 (Kurosaka et al., Interscience Conference on Antimicrobial Agents and Chemotherapy, 2003, 43rd: Chicago, the entire contents of which are incorporated herein by reference).

Methods of treatment or prevention

In some embodiments, a method is provided for treating a microbial infection in an animal, particularly a mammal, inclusive, by treating an animal suffering from such an infection with a fluoroquinolone antimicrobial agent. In some embodiments, fluoroquinolone antimicrobials may be administered after aerosol formation and inhalation. Thus, this method of treatment is particularly suitable for the treatment of pulmonary infections caused by microbial strains that are difficult to treat using an antimicrobial agent delivered parenterally because of the need for high parenteral dose levels (which may cause unwanted side effects), or because there are no either clinically effective antimicrobial agents. In one such embodiment, the method can be used to administer a fluoroquinolone antimicrobial agent directly to the site of infection. A similar method can reduce systemic exposure and maximizes the amount of antimicrobial substance in the focus of microbial infection. This method is also suitable for the treatment of infections caused by microbes that are susceptible to fluoroquinolone antimicrobials, as a way to reduce the frequency of selection of resistant microbes. This method is also suitable for the treatment of infections caused by microbes that are otherwise resistant to fluoroquinolone antimicrobial agents, as a way to increase the amount of antimicrobial agent in the focus of microbial infection. A subject can be defined as being infected with bacteria that are capable of developing resistance, by diagnosing the subject as having symptoms that are characteristic of a bacterial infection with strains of bacteria that are known to have resistant strains, or bacteria that are members of a group that is known to have resistant strains. Alternatively, bacteria can be cultured and defined as species that are known to have resistant strains, or bacteria that are members of a group that is known to have resistant strains.

In some embodiments, the aerosol fluoroquinolone antimicrobial is administered in an amount sufficient to overcome the occurrence of resistance in bacteria or increase the efficiency of lysis so that there is no possibility for the development of resistance.

In some embodiments, aerosol fluoroquinolone therapy can be used as a treatment or prophylaxis in combination or in alternating treatment sequences with other aerosol, oral, or parenteral antibiotics. By way of non-limiting example, they may include aerosol tobramycin and / or another aminoglycoside, aerosol aztreonam and / or other β or monobactam, aerosol ciprofloxacin and / or other fluoroquinolones, aerosol azithromycin and / or other macrolides or ketolides, tetracycline and / or other tetracyclines , kinupristine and / or other streptogramines, linezolid and / or other oxazolidinones, vancomycin and / or other glycopeptides, as well as chloramphenicol and / or other phenols and colisitin and / or other polymyxins.

Pharmaceutical Compositions

For the purposes of the method described herein, a fluoroquinolone antimicrobial agent can be administered using an inhaler. In some embodiments, the fluoroquinolone antimicrobial agent described herein is prepared as a pharmaceutical composition suitable for aerosol formation, having good taste, storage stability, and is safe and tolerated by the patient.

In some embodiments, the implementation of the isoforms of the fluoroquinolone produced can be optimized for tolerance, antimicrobial activity, and stability.

Introduction

The fluoroquinolone antimicrobial agents described herein can be administered in therapeutically effective doses, for example, in a dose sufficient to provide treatment for the disease conditions described previously. Although optimal dose levels for humans in aerosol delivery have yet to be determined, the usual daily aerosol dose of levofloxacin (and for most fluoroquinolone antimicrobials described here) is from about 0.1 to 10 mg / kg body weight, preferably from about 0.20 to 5.0 mg / kg body weight, and most preferably from about 0.4 to 4.0 mg / kg body weight. Thus, for administration to a person weighing 70 kg, the dose range should be from about 7.0 to 700.0 mg per day, preferably from about 14.0 to 350.0 mg per day, and most preferably from about 28.0 to 280 , 0 mg per day. The amount of active compound administered will, of course, depend on the subject and the condition of the disease being treated, the severity of the disease, the method and mode of administration and decision of the attending physician; for example, the likely dose range for aerosol administration of levofloxacin would be from about 20 to 400 mg per day.

The administration of the fluoroquinolone antimicrobial agents described herein or their pharmaceutically acceptable salts may occur by any conventional route of administration for substances that serve similar purposes, including but not limited to aerosol inhalation.

Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as, for example, powders, liquids, suspensions, complexones, liposomes, microparticles and the like. Preferably, the compositions are provided in unit dosage forms suitable for a single, precise dose administration. Dosage units can also be combined and packaged to provide the patient with a weekly or monthly supply, and other compounds, such as saline, flavoring agents, pharmaceutical excipients, and other active ingredients or carriers, may also be included.

The fluoroquinolone antimicrobial substance can be administered either by itself or more commonly in combination with a conventional pharmaceutical carrier, excipient or the like (e.g. mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, croscarmellose sodium, glucose, gelatin, sucrose, magnesium carbonate, magnesium chloride, magnesium sulfate, calcium chloride, lactose, sucrose, glucose and the like). If desired, the pharmaceutical composition may also contain minor amounts of non-toxic adjuvants, such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate and the like like that). Typically, depending on the intended route of administration, the pharmaceutical preparation will contain from about 0.005% to 95%, preferably from about 0.5% to 50%, by weight of the compound of this invention. Current methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

In one preferred embodiment, the compositions will take the form of a unit dosage form, such as a vial containing a liquid, a solid to be suspended, a dry powder, lyophilisate or other composition, and thus the composition may contain, along with the active component, a diluent such as lactose, sucrose, dicalcium phosphate or the like; a lubricant such as magnesium stearate and the like; and a binder, such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives or the like.

Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing and so on of an active substance, as defined above, and pharmaceutical adjuvants of choice in a carrier (e.g., water, saline, liquid dextrose, glycerin, glycol, ethanol and the like) for obtaining a solution or suspension. The solutions to be converted to aerosol can be prepared in conventional forms, either as liquid solutions or suspensions, such as emulsions, or in solid forms suitable for dissolution or suspension in a liquid prior to aerosol preparation and inhalation. The percentage of active compound in such aerosol compositions is highly dependent on its specific nature, as well as on the activity of the compound and the needs of the subject. However, the percentage of active component in the solution can be from 0.01% to 90% and will be higher if the composition is a solid, which will subsequently be dissolved to the above percentage. In some embodiments, the composition will comprise 1.0% -50.0% of the active substance in solution.

Fluoroquinolone preparations can be divided into two groups; those that have a simple composition, and complex preparations with corrected taste, better tolerance and / or composition that improves the configuration of AUC. Simple drugs can be further divided into three groups. 1. Simple preparations may include water-based liquid preparations for spraying. By way of non-limiting example, water-based liquid preparations may consist of fluoroquinolone alone or with non-encapsulating, water-soluble excipients. 2. Simple preparations may also include organic liquid preparations for nebulization or a metered-dose inhaler. By way of non-limiting example, organic-based liquid preparations may consist of fluoroquinolone alone or with non-encapsulating excipients soluble in organic solvents. 3. Simple preparations may also include dry powder preparations for administration with a powder inhaler. By way of non-limiting example, dry powder preparations may consist of fluoroquinolone alone or with either water-soluble or soluble in organic solvents non-encapsulating excipients with or without a component of the mixture, such as lactose. Complex drugs can be further divided into five groups. 1. Complex formulations may include water-based liquid formulations for nebulization. By way of non-limiting example, water-based liquid complex preparations may consist of fluoroquinolone encapsulated or complexed with water-soluble excipients such as lipids, liposomes, cyclodextrins, microencapsulations, and emulsions. 2. Complex formulations may also include organic liquid formulations for nebulization or a metered dose inhaler. By way of non-limiting example, organic-based liquid complex preparations may consist of fluoroquinolone encapsulated or complexed with excipients soluble in organic solvents, such as lipids, microencapsulations, and water-based reversed-phase emulsions. 3. Complex formulations may also include sparingly soluble liquid aqueous preparations for spraying. By way of non-limiting example, water-poorly soluble liquid complex preparations may consist of fluoroquinolone alone as a poorly soluble, stable nanosuspension in water, or in complexes of co-crystals / co-precipitates with a filler, or mixtures with poorly soluble lipids, such as lipid nanosuspensions. 4. Complex formulations may also include sparingly soluble, liquid, organic based formulations for nebulization or a metered dose inhaler. As a non-limiting example, organic-poorly soluble liquid complex preparations can consist of fluoroquinolone per se as poorly soluble in organic solvents of stable nanosuspension, or in complexes of co-crystals / co-precipitates with a filler, or mixtures with poorly soluble lipids, such as lipid nanosuspensions. 5. Complex formulations may also include dry powder formulations for administration with a powder inhaler. By way of non-limiting example, complex dry powder preparations may consist of fluoroquinolone in a spray-dried co-crystal / co-precipitate complex or a mixture with sparingly soluble fillers / salts in the form of a dry powder with or without a mixture component such as lactose. Specific methods for preparing simple and complex preparations are described herein.

Aerosol delivery

The fluoroquinolone antimicrobial agents, as described herein, are preferably administered directly as an aerosol to the site of infection in the respiratory tract. In some embodiments, aerosol delivery is used to treat an infection in the lungs, such as a pulmonary infection of Pseudomonas.

There are various instrumental technologies for delivering either dry powder or liquid aerosol products. Dry powder formulations usually require less time to administer the drug, although a longer and more expensive development effort. In contrast, liquid formulations have historically been deficient in the form of longer administration times, although they have the advantage of shorter and more expensive development efforts. The fluoroquinolone antimicrobial agents described herein have a solubility range, are usually stable, and have a range of tastes. In one such embodiment, the fluoroquinolone antimicrobial agent levofloxacin is soluble in water at a neutral pH, stable in aqueous solution, and has a mild or no taste.

Accordingly, in one embodiment, the particular fluoroquinolone antimicrobial preparation described herein is provided in combination with a specific aerosolizing device for producing an aerosol for inhalation that is optimized for maximum drug deposition at the site of infection and maximum tolerance. Factors that can be optimized include a solution or particulate preparation, delivery rate, and particle size and distribution obtained by an aerosolizing device.

Particle Size and Distribution

Typically, deposition of respirable particles occurs through one of two mechanisms: sedimentation, which usually predominates in the case of larger particles, and sedimentation, which usually prevails in the case of smaller particles. Precipitation occurs when the inertia of the inhaled particle is so large that the particle does not follow in the air stream and encounters a physiological surface. On the contrary, sedimentation occurs mainly deep in the lungs, when very small particles that have made their way in the stream of inhaled air come across physiological surfaces as a result of random diffusion in the air stream.

For pulmonary administration, the upper respiratory tract is bypassed for the middle and lower respiratory tract. Pulmonary drug delivery can be achieved by inhalation of an aerosol through the mouth and throat. Particles with a mass average aerodynamic diameter (MMAD) of greater than about 5 microns usually do not reach the lungs; instead, they tend to settle on the back of the throat and are swallowed and possibly absorbed orally. Particles having a diameter of about 2 to about 5 microns are small enough to reach the upper to middle pulmonary region (conducting airways), but are too large to reach the alveoli. Smaller particles, that is, from about 0.5 to about 2 microns, are able to reach the alveolar zone. Particles with diameters smaller than about 0.5 microns can also be deposited in the alveolar zone by sedimentation, although very small particles can be exhaled. The size of the particles can be defined as volume average diameter (VMD), mass average diameter (MMD), or MMAD. These measurements can be carried out by deposition (MMD and MMAD) or using a laser (VMD). For liquid particles, VMD, MMD, and MMAD can be the same if environmental conditions such as standard humidity are maintained. However, if humidity is not maintained, then the definitions of MMD and MMAD will be less than VMD due to dehydration during impact measurements. For the purposes of this description, it is believed that the VMD, MMD and MMAD measurements are carried out under standard conditions, so that the values of VMD, MMD and MMAD are comparable. Similarly, the sizing of the MMD and MMAD particles of the dry powder is also considered comparable.

In some embodiments, the implementation of the particle size of the aerosol is optimized in order to maximize the deposition of fluoroquinolone antimicrobial substance in the focus of infection and maximize tolerance. Aerosol particle size can be expressed as mass average aerodynamic diameter (MMAD). Large particles (e.g. MMAD> 5 μm) can be deposited in the upper respiratory tract, as they are too large to move following the bends of the upper respiratory tract. Small particles (for example, MMAD <2 μm) can be poorly deposited in the lower respiratory tract and, thus, excreted on the exhale, increasing the likelihood of deposition in the upper respiratory tract. Thus, intolerance (for example, cough and bronchospasm) can occur as a result of deposition in the upper respiratory tract of both precipitated large respirable particles and sedimentary small particles in the process of repeated inspiration and exhalation. Thus, in one embodiment, particles of an optimal size are used (e.g., MMAD = 2-5 μm) in order to maximize deposition in the mid-pulmonary site of infection and minimize intolerance associated with deposition in the upper respiratory tract. Moreover, obtaining particles of a certain size with a limited geometric standard deviation (GSD) will optimize deposition and tolerance. Narrow GSD boundaries limit the number of particles outside the desired MMAD size range. In one embodiment, an aerosol is provided comprising one or more of the compounds described herein having an MMAD of from about 2 microns to about 5 microns with a GSD of less than or equal to about 2.5 microns. In another embodiment, an aerosol having an MMAD of from about 2.8 microns to about 4.3 microns with a GSD of less than or equal to 2 microns is provided. In another embodiment, an aerosol having an MMAD of from about 2.5 microns to about 4.5 microns with a GSD of less than or equal to 1.8 microns is provided.

The fluoroquinolone antimicrobial agents described herein for respiratory delivery (either for systemic or local distribution) can be administered as aqueous preparations, as suspensions or solutions in halogenated hydrocarbon propellants, or as dry powders. Aqueous preparations can be converted into aerosol using liquid nebulizers, where either hydraulic or ultrasonic atomization is used. In propellant-based systems, appropriate pressurized metered dose inhalers (pMDIs) can be used. For dry powders, powder inhalation devices (DPIs) that can efficiently disperse a drug can be used. The desired particle size and distribution can be obtained by selecting the appropriate device.

Liquid atomizer

In one embodiment, the nebulizer is selected based on its ability to aerosol the fluoroquinolone antimicrobial agent described herein, which has an MMAD value of preferably from about 2 to about 5 microns. In one embodiment, the delivered amount of a fluoroquinolone antimicrobial agent provides a therapeutic effect in respiratory infections.

It was previously shown that two types of atomizers, jet and ultrasonic, are able to form and deliver aerosol particles having a particle size of 2 to 4 microns. Particles of this size have been shown to be optimal for the treatment of bacterial pulmonary infections caused by gram-negative bacteria, such as Pseudomonas aeruginosa, Escherichia coli, Enterobacter species, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marophisceria, Haratus marophus hemida, cepacia, Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and multidrug-resistant Pseudomonas aeruginosa. However, unless a specially formulated solution is used, large volumes are usually required for these nebulizers to administer a sufficient amount of the drug to produce a therapeutic effect. In a spray gun, air pressure is used to break up an aqueous solution into aerosol drops. An ultrasonic atomizer uses dissection of an aqueous solution with a piezoelectric crystal. Typically, however, inkjet nebulizers have a clinical efficiency of only about 10%, while an ultrasonic nebulizer has an efficiency of only about 5%. The amount of pharmaceutical product deposited and absorbed in the lungs is thus part of 10%, despite its large amount placed in the nebulizer.

Accordingly, in one embodiment, a vibrating cell nebulizer is used to deliver the aerosol of the fluoroquinolone antimicrobial agent described herein. The vibrating cellular atomizer consists of a container for storing fluid when the fluid contacts the diaphragm, as well as inspiratory and expiratory valves. In one embodiment, from about 1 to about 5 ml of a fluoroquinolone antimicrobial agent are placed in a container and an aerosol generator is launched to create a spray aerosol with a particle size selectively from about 1 to about 5 microns.

By way of non-limiting example, the fluoroquinolone antimicrobial agent described herein is placed in a liquid nebulizer inhaler and formulated in doses for delivery of about 7 to about 700 mg from a metering solution of about 1 to about 5 ml, preferably about 14 to about 350 mg, in from about 1 to about 5 ml, and most preferably from about 28 to about 280 mg, from about 1 to about 5 ml with an MMAD particle size of from about 2 to about 5 microns.

By way of non-limiting example, the nebulized fluoroquinolone antimicrobial agent can be administered at the described respiratory delivered dose for less than about 20 minutes, preferably less than about 10 minutes, more preferably less than about 7 minutes, more preferably less than about 5 minutes, more preferably less than about 3 minutes, and in some cases, more preferably less than about 2 minutes.

By way of non-limiting example, under other circumstances, a nebulized fluoroquinolone antimicrobial agent may have improved tolerance and / or exhibit an advanced AUC configuration property when administered over long periods of time. Under these conditions, the described respiratory delivered dose for more than about 2 minutes, preferably more than about 3 minutes, preferably more than about 5 minutes, more preferably more than about 7 minutes, more preferably more than about 10 minutes and in some cases, more preferably from about 10 to about 20 minutes.

For aqueous and other non-pressurized fluid systems, there are many nebulizers (including small volume nebulizers) for converting the preparations into aerosol. Compressor sprays use jet technology and apply compressed air to produce liquid aerosol. Such devices can be obtained, for example, from Healthdyne Technologies, Inc .; Invacare, Inc .; Mountain Medical Equipment, Inc .; Pari Respiratory, Inc .; Mada Medical, Inc .; Puritan-Bennet; Schuco, Inc., DeVilbiss Health Care, Inc .; and Hospitak, Inc. Ultrasonic nebulizers are based on the use of mechanical energy in the form of a vibration of a piezoelectric crystal to produce respirable liquid droplets and can be obtained, for example, from Omron Heathcare, Inc. and DeVilbiss Health Care, Inc. Vibrating cell nebulizers are based on the use of either piezoelectric or mechanical pulses to produce respirable liquid droplets. Other examples of nebulizers for use with the fluoroquinolone antimicrobial agents described herein are described in US Pat. Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740; 5,586,550; 5758637; 6644304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6192876; 6,230,706; 6349719; 6,367,470; 6,534,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6192876; 6644304; 5,549,102; 6083922; 6161536; 6,264,922; 6,557,549; and 6612303, the full contents of all of which are incorporated herein by reference. Examples of commercial nebulizers that can be used with the fluoroquinolone antimicrobial agents described herein include Respirgard II®, Aeroneb®, Aeroneb® Pro, and Aeroneb® Go manufactured by Aerogen; AERx® and AERx Essence ^TM, manufactured Aradigm; ^{Porta-Neb®, Freeway Freedom TM,} Sidestream, Ventstream and I-neb, manufactured by Respironics, Inc .; and PARI LC-Plus®, PARI LC-Star®, and e-Flow ^7m , manufactured by PARI, GmbH. As a further non-limiting example, US Pat. No. 6,196,219, the entire contents of which are incorporated herein by reference.

In some embodiments, a drug solution is prepared before the patient uses a nebulizer. In other embodiments, the implementation of the drug is stored in a nebulizer in solid form. In this case, the solution is mixed upon activation of the nebulizer, as described in US Pat. No. 6,427,682 and PCT Publication Number WO 03/035030, the entire contents of which are incorporated herein by reference. In these nebulizers, the solid drug, optionally combined with excipients to form a solid composition, is stored in a separate compartment from the liquid solvent.

The liquid solvent is capable of dissolving the solid composition to form a liquid composition that can be aerosolized and inhaled. This ability, among other factors, depends on the amount selected and, potentially, on the composition of the liquid. For ease of handling and dosing reproducibility, a sterile aqueous liquid may be able to dissolve the solid composition in a short period of time, possibly with gentle shaking. In some embodiments, the final liquid is ready for use after no more than about 30 seconds. In some cases, the solid composition dissolves within about 20 seconds, and preferably within about 10 seconds. As used here, the terms "dissolve (s)", "dissolution" and "dilution" refer to the dissociation of the solid composition and the release, that is, the dissolution of the active compound. By dissolving the solid composition with a liquid solvent, a liquid composition is formed which contains the active compound in a dissolved state. As used here, the active compound is in a dissolved state when at least about 90% by weight. are dissolved, and more preferably when at least about 95% by weight. are dissolved.

As for the basic design of the atomizer with separate compartments, it mainly depends on the application, whether it will be more convenient to place the liquid solution and solid composition in separate chambers of the same container or inner packaging, or whether they should be delivered in separate containers. In the case of using separate containers, they are delivered as a set inside the same outer packaging. The use of separate containers is particularly preferred in the case of nebulizers containing two or more doses of the active compound. There are no restrictions on the total number of containers delivered in a multi-dose kit. In one embodiment, the solid composition is delivered as unit doses within multiple containers or within multiple chambers of a container, while a liquid solvent is delivered within a single chamber or container. In this case, in a preferred design, the liquid is delivered in a spray dosimeter, which may consist of a glass or plastic bottle closed with a spray device, such as a mechanical atomizer for measuring liquid. For example, a single trigger of a spray mechanism can spray an exact amount of liquid to dissolve one unit dose of a solid composition.

In another embodiment, for multi-dose nebulizers with separate compartments, both the solid composition and the liquid solvent deliver both conjugate unit doses within multiple containers or within multiple container chambers. For example, you can use two-chamber containers to store one unit of solid composition in one of the chambers and one unit of liquid in another. As used here, one unit denotes the amount of drug present in the solid composition, which is one unit dose. Such two-chamber containers can, however, also be used predominantly for nebulizers containing only one single dose of the drug.

In one embodiment, for dual-compartment nebulizers, a blister pack with two blisters is used, where the blisters are comers for containing the solid composition and the liquid solvent in conjugate amounts to produce a unit dose of the final liquid composition. As used herein, a blister pack is an internal unit pack formed by temperature or pressure, most likely consisting of a polymer packaging material, which optionally includes a metal foil, such as aluminum. The blister pack may be shaped to facilitate easy atomization of the contents. For example, one side of the package may be narrowed or have a narrowed part or portion through which the contents are sprayed into another vessel when the blister pack is opened from the narrowed end. The tapered end may be a tip.

In some embodiments, two blister pack chambers are connected by a channel that is adapted to direct fluid from a blister containing a liquid solvent to a blister containing a solid composition. During storage, the channel is hermetically sealed by a partition. In this sense, a septum is any structure that protects a liquid solvent from contact with a solid composition. The partition is preferably destructible or removable; destruction or removal of the septum when using a spray will allow the liquid solvent to penetrate into another chamber and dissolve the solid composition. The dissolution process can be improved by shaking the blister pack. Thus, the final liquid composition for inhalation is obtained, with the liquid present in one or both of the packaging chambers connected by a channel, depending on how the package is held.

According to a further embodiment, one of the chambers, preferably one that is closer to the narrowed part of the blister pack, is in communication with the second channel, the channel stretches from the camera to a distant point of the narrowed part. During storage, the second channel does not communicate with the outside of the package, but is closed in an airtight manner. Optionally, the distal end of the second channel is closed with a destructible or removable cap or plug, which may, for example, be a screw cap, a breakable cap or a cut off cap.

In one embodiment, a vial or container having two compartments is used, wherein the compartment is chambers for containing the solid composition and the liquid solvent in conjugate amounts to produce a unit dose of the final liquid composition. The liquid composition and the second liquid solvent can be contained in conjugate amounts to produce a unit dose of the final liquid composition (as a non-limiting example, in cases where two soluble excipients or fluoroquinolone and excipient are unstable during storage, although they are intended for a single administration mixture.

In some embodiments, the two compartments are physically separated, but with a fluid contact such that in this case the vial or container is connected by a channel or a destructible partition, the channel or destructible partition is adapted to direct fluid between the two compartments, which allows mixing prior to administration. During storage, the canal is closed by a partition or intact destructible barrier. In this sense, a partition is any structure that prevents the contents of the two compartments from mixing. The partition is preferably destructible or removable; destruction or removal of the septum when using a spray will allow the liquid solvent to penetrate into the other chamber and dissolve the solid composition or, in the case of two liquids, allow mixing. The dissolution or mixing process can be improved by shaking the container. In this way, a final liquid composition for inhalation is obtained, wherein the liquid is present in one or both of the packaging chambers connected by a channel or a breakable barrier, depending on how the package is held.

The solid composition itself can be supplied in various types of dosage forms, depending on the physicochemical properties of the drug, the desired dissolution rate, price considerations, and other criteria. In one embodiment, the solid composition is a single unit. This implies that one standard dose of the drug is made in the form of a single physically formed solid form or product. In other words, the solid composition is one, as opposed to the plural form of a unit dose in which the doses are separated.

Examples of individual units that can be used as dosage forms for a solid composition include tablets such as compressed tablets, film units, foil units, capsules, lyophilized matrix units, and the like. In a preferred embodiment, the solid composition is a highly porous lyophilized form. Such lyophilisates, sometimes also called capsules or lyophilized tablets, are particularly suitable for their rapid disintegration, which also makes possible the rapid dissolution of the active compound.

On the other hand, for some applications, the solid composition can also be obtained in the form of a multiple form of unit doses, as defined above. Examples of multiple forms are powders, granules, microparticles, dragees, granules, lyophilized powders and the like. In one embodiment, the solid composition is a lyophilized powder. Such a dispersed lyophilized system contains many powder particles, and due to the lyophilization process used to obtain the powder, each particle has an irregular porous microstructure, due to which the powder is able to absorb water very quickly, which leads to rapid dissolution.

Another type of multicuspid system, which is also capable of providing rapid dissolution of the drug, are such powders, granules or dragees of water-soluble excipients that are coated with the drug, so that the drug is located on the surface of individual particles. In this type of system, a water-soluble low molecular weight filler is useful for producing kernels of similar coated particles that can subsequently be coated with a coating composition containing a drug and, preferably, one or more additional excipients, such as a binder, blowing agent, saccharide, sugar alcohol, a film-forming polymer, emollient or other excipients used in pharmaceutical coating compositions.

In another embodiment, the solid composition is a coating layer that covers multiple units derived from insoluble material. Examples of insoluble units include granules derived from glass, polymers, metals and mineral salts. Again, the desired effect is mainly the rapid disintegration of the coating layer and the rapid dissolution of the drug, which is achieved by obtaining a solid composition in physical form, which has a particularly high surface to volume ratio. Typically, the coating composition will, in addition to the drug and water-soluble low molecular weight excipient, contain one or more excipients, such as those mentioned above for the coating of soluble particles, or any other excipient known for use in pharmaceutical coating compositions.

To achieve the desired effects, it may be useful to include more than one water soluble low molecular weight filler in the solid composition. For example, one excipient can be selected because of its ability to be a carrier and solvent of a drug, while another excipient can be selected to adjust pH. If the final liquid composition is to be buffered, two excipients can be selected that together form a buffer system.

In one embodiment, the liquid to be used in a split-chamber atomizer is an aqueous liquid, which is defined herein as a liquid whose main component is water. A fluid does not necessarily consist only of water; however, in one embodiment, it is purified water. In another embodiment, the liquid contains other components or substances, preferably other liquid components, but possibly also dissolved solids. Liquid components other than water that can be used include propylene glycol, glycerin and polyethylene glycol. One reason to include a dissolved solid compound is that the presence of such a compound is desirable in the final liquid composition, but is incompatible with the solid composition or its component, such as the active ingredient.

Another desirable characteristic for a liquid solvent is its sterility. Aqueous liquid may be at risk of significant microbiological contamination and seedlings if measures are not taken to ensure sterility. To ensure substantial sterility of the liquid, an effective amount of an acceptable antimicrobial substance or preservative can be added to the composition, or the liquid can be sterilized previously and hermetically sealed with an airtight partition. In one embodiment, the liquid is a sterilized liquid without preservatives and is delivered in an appropriate airtight container. However, in another embodiment, wherein the nebulizer contains multiple doses of the active compound, the liquid may be delivered in a multi-dose container, such as a nebulizer, and a preservative may be required to prevent microbial contamination after first use.

Inhaler Dispenser (MDI)

Propellant-activated inhaler (pMDI) releases a metered dose of the drug with each actuation. The drug is obtained in the form of a suspension or solution of the drug in a suitable propellant, such as a halogenated hydrocarbon. pMDIs are described, for example, in Newman, S. P., Aerosols and the Lung, Clarke et al., eds., pp. 197-224 (Butterworths, London, England, 1984).

In some embodiments, the particle size of the drug in MDI can be optimally selected. In some embodiments, the implementation of the particles of the active ingredient have a diameter of less than about 50 microns. In some embodiments, the particles have a diameter of less than about 10 microns. In some embodiments, the particles have a diameter of from about 1 micron to about 5 microns. In some embodiments, the particles have a diameter of less than about 1 micron. In one preferred embodiment, the particles have a diameter of from about 2 microns to about 5 microns.

The propellants for use with MDI may be any propellants known in the art. Examples of propellants include chlorofluorocarbons (CFC) such as dichlorodifluoromethane, trichlorofluoromethane and dichlorotetrafluoroethane; hydrofluoroalkanes (HFA); and carbon dioxide. It may be preferable to use HFA instead of CFC due to environmental concerns regarding the use of CFC. Examples of medical aerosol formulations containing HFA are presented in US Pat. Nos. 6,585,958; 2868691 and 3014844, the full contents of which are incorporated herein by reference. In some embodiments, the co-solvent is mixed with a propellant to facilitate dissolution or suspension of the drug substance.

In some embodiments, the propellant and the active ingredient are in separate containers as described in US Pat. No. 4,534,345, the entire contents of which are incorporated herein by reference.

In some embodiments, the MDI used herein, the patient activates by pressing a lever, button, or other activator. In other embodiments, aerosol release is activated by respiration, so that after the initial cocking of the device, the aerosol of the active compound is released as soon as the patient begins to inhale, as described in US Pat. Nos. 6672304; 5,404,871; 5,347,998; 5,284,133; 5,217,004; 5,119,806; 5,060,643; 4,664,107; 4,648,393; 3,789,843; 3,732,864; 3,636,949; 3,598,294; 3,565,070; 3,456,646; 3,456,645; and 3,456,644, the entire contents of which are incorporated herein by reference. Such a system helps to get more active compound into the lungs of the patient. Another mechanism that helps the patient to get the appropriate dose with the active ingredient may include a valve mechanism that allows the patient to inhale the drug in more than one breath, as described in US patents Nos. 4,470,412 and 5,385,140, the entire contents of which are incorporated herein by reference .

Additional examples of MDIs known in the art and suitable for use herein include US Pat. Nos. 6,435,177; 6,585,958; 5,642,730; 6,223,746; 4,955,371; 5,404,871; 5,364,838; and 6,523,536, the entire contents of which are incorporated herein by reference.

Powder Inhaler (DPI)

There are two main designs of powder inhalers. One design is a metering device in which a drug reservoir is placed inside the device and the patient adds a dose of the drug to the inhaler chamber. The second is a factory metering device in which each individual dose is made in a separate container. For both systems, it is important that the preparation is obtained in the form of small particles with mass average diameters from about 1 to about 5 microns, and usually the preparation is made up with larger filler particles (usually lactose particles with a diameter of 100 microns). The powder of the drug is placed in the chamber of the inhaler (either by measuring into the device or by opening the factory packaging of the dose) and the air stream inhaled by the patient accelerates the exit of the powder from the device into the oral cavity. Features of the non-laminar flow along the powder pathway cause the disintegration of the filler-drug aggregates, and the mass of larger filler particles contributes to their sedimentation on the back of the throat, while smaller particles of the drug are deposited deep in the lungs.

As with liquid spray and MDI, the particle size of the fluoroquinolone antimicrobial aerosol preparation can be optimized. If the particle size is larger than about 5 μm MMAD, then the particles are deposited in the upper respiratory tract. If the particle size of the aerosol is less than about 1 μm, then it reaches the alveoli and can enter the circulatory system.

By way of non-limiting example, in powder inhalers, the fluoroquinolone antimicrobial agents described herein are prepared in dosages for delivery of from about 7 to about 700 mg in a dosing solution of from about 1 to about 5 ml, preferably from about 14 to about 350 mg, from about 1 up to about 5 ml, and most preferably from about 28 to about 280 mg, from about 1 to about 5 ml, with a resulting MMAD particle size of from about 2 to about 5 microns.

In some embodiments, a powder inhaler (DPI) is used to spray the fluoroquinolone antimicrobial agents described herein. DPIs contain a drug substance in the form of fine dry particles. Typically, when inhaled by a patient, dry particles form an aerosol cloud that enters the patient's lungs. Fine dry particles of the drug can be obtained by any method known in this field. Some well-known methods include the use of a jet mill or other grinding equipment, precipitation from saturated or oversaturated solutions, spray drying, micronization at the time of formation (Hovione), or supercritical fluid methods. Conventional powder preparations include the preparation of spherical granules or adhesive mixtures. In adhesive mixtures, drug particles are attached to larger carrier particles, such as lactose monohydrate, having a size of from about 50 to about 100 microns in diameter. Larger particle sizes increase aerodynamic force on carrier / drug agglomerates to improve aerosol production. Vortex and / or mechanical devices destroy agglomerates into their constituent parts. Smaller particles of the drug then enter the lungs, while larger particles of the carrier are deposited in the mouth or throat. Some examples of adhesive mixtures are described in US Pat. No. 5,478,578 and PCT Publication Nos. WO 95/11666, WO 87/05213, WO 96/23485, and WO 97/03649, the entire contents of which are incorporated herein by reference. Along with the drug substance, additional excipients may also be included.

There are three common types of DPIs, all of which can be used with the fluoroquinolone antimicrobial agents described here. In a single dose DPI, a capsule containing one dose of dry drug substance / excipients is placed in an inhaler. When activated, the capsule is broken, which allows you to spray dry powder and inhale it using a powder inhaler. To spray the following doses, the old capsule must be removed and an additional capsule inserted. Examples of single dose DPIs are described in US Pat. Nos. 3807400; 3,906,950; 3991761; and 4013075, the entire contents of which are incorporated herein by reference. DPIs for multiple unit doses provide a package containing compartments for multiple unit doses. For example, the package may be in the form of a blister pack, where each blister compartment contains one dose. Each dose can be sprayed when the blister compartment ruptures. Any of a variety of ways to arrange compartments in a package can be used. For example, rotating and stripe arrangement methods are generally accepted. Examples of DPI for multiple unit doses are described in EPO patent applications, publication numbers 0211595A2, 0455463A1, and 0467172Al, the entire contents of which are incorporated herein by reference. Multi-dose DPIs use one dry powder tank. There are mechanisms that measure individual doses from a reservoir for aerosolization and inhalation, such as those described in US Pat. Nos. 5,829,434; 5,437,270; 2587215; 5,113,855; 5,840,279; 4,688,218; 4,667,668; 5,033,463; 4805811 and PCT Publication Number WO 92/09322, the entire contents of which are incorporated herein by reference.

In some embodiments, the implementation can be applied additional auxiliary or other than by inhalation of the patient, energy to facilitate DPI. For example, compressed air can be used to facilitate powder deagglomeration, as described in US Pat. Nos. 3,906,950; 5,113,855; 5,388,572; 6029662 and PCT publications numbered WO 93/12831, WO 90/07351 and WO 99/62495, the entire contents of which are incorporated herein by reference. Electric impellers may also be used, as described in US Pat. Nos. 3,948,264; 3,971,377; 4,147,166; 6006747 and PCT Publication Number WO 98/03217, the entire contents of which are incorporated herein by reference. Another mechanism is a piercing piston with an electric drive, as described in PCT publication number WO 90/13327, the full contents of which are incorporated herein by reference. Other DPIs use a vibrator, as described in US Pat. Nos. 5,694,920 and 6,026,809, the entire contents of which are incorporated herein by reference. Finally, a scraper system can be used, as described in PCT publication number WO 93/24165, the entire contents of which are incorporated herein by reference.

Additional examples of DPI for use herein are described in US Pat. Nos. 4,811,731; 5,113,855; 5,840,279; 3507277; 3,669,113; 3,635,219; 3991761; 4,353,365; 4,889,144; 4,907,538; 5,829,434; 6681768; 6,561,186; 5,918,594; 6003512; 5,775,320; 5,740,794 and 6,626,173, the entire contents of which are incorporated herein by reference.

In some embodiments, an intermediate pad or chamber with any of the inhalers described herein can be used to increase the amount of drug that the patient absorbs, as described in US Pat. Nos. 4,470,412; 4,790,305; 4,926,852; 5012803; 5,040,527; 5,024,467; 5,816,240; 5027806 and 6026807, the full contents of which are incorporated herein by reference. For example, an intermediate pad may increase the time from aerosol formation to the moment the aerosol enters the patient’s mouth. Such a delay can improve synchronization between the patient's inspiration and aerosol formation. You can include a mask for children or other patients who have difficulty using traditional mouthpieces, as described in US Pat. Nos. 4,809,692; 4,832,015; 5012804; 5,427,089; 5645049 and 5988160, the full contents of which are incorporated herein by reference.

Powder inhalers (DPIs), in which dry powders are disaggregated and aerosolized, usually depend on the release of inhaled air that passes through the device, delivering a dose of the drug. Such devices are described, for example, in US Pat. No. 4,807,814, which discloses a pneumatic powder ejector that has a suction step and an injection step; SU 628930 (Summary), describing a portable powder atomizer having a longitudinal air tube; Fox et al., Powder and Bulk Engineering, pages 33-36 (March 1988), describing a Venturi ejector having a longitudinal air inlet pipe upstream of a Venturi restriction; EP 347 779, which describes a portable powder nebulizer having a folding expansion chamber, and US Pat. No. 5,785,049, which deals with powder drug delivery devices.

Solution / Dispersed System Preparations

One embodiment relates to aqueous preparations containing soluble or nanoparticulate drug particles. For aqueous aerosol formulations, the drug may be present in a concentration of from about 1 mg / ml up to about 700 mg / ml. Such formulations provide efficient delivery to the corresponding areas of the lungs, with more concentrated aerosol formulations having the added benefit of making it possible to deliver large quantities of the drug to the lungs in a very short period of time. In one embodiment, the preparation is optimized to produce a well-tolerated preparation. Accordingly, in one embodiment, the fluoroquinolone antimicrobial agents described herein are formulated to have a palatable taste, pH from about 5.5 to about 7, osmolality from about 200 to about 1250 mosmol / kg, penetrating ion concentration from about 30 to about 300 mm.

In one embodiment, the solution or solvent used to prepare the aerosol formulations has a pH range of from about 4.5 to about 7.5, preferably from about 5.5 to about 7.0. This pH range improves tolerance. If the aerosol is either acidic or alkaline, it can cause bronchospasm and cough. Although the safe pH range is relative, and some patients can tolerate a slightly acidic aerosol, others will experience bronchospasm. Any aerosol with a pH of less than about 4.5 usually causes bronchospasm. Aerosols with a pH of from about 4.5 to about 5.5 will occasionally cause bronchospasm. Any aerosol with a pH higher than about 7.5 may have low tolerance, as body tissues are usually not capable of buffering alkaline aerosols. Aerosols with a controlled pH lower than about 4.5, and higher than about 7.5, usually lead to lung irritation, accompanied by severe bronchospastic cough and inflammatory reactions. For these reasons, and also in order to avoid bronchospasm, coughing or inflammation in patients, the optimum pH for the aerosol preparation is determined to be from about pH 5.5 to about pH 7.0. Consequently, in one embodiment, the aerosol formulations for use as described herein are adjusted to a pH of from about 4.5 to about 7.5 with a preferred pH range of from about 5.5 to about 7.5. The most preferred pH range is from about 5.5 to about 7.5.

By way of non-limiting example, the compositions may also include a buffer or pH-adjusting substance, usually a salt derived from an organic acid or base. Examples of buffers include salts of organic acids of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, tromethamine, hydrochloride or phosphate buffers.

Many patients are hypersensitive to various chemical tastes, including sensations of bitterness, salinity, sweets, metallic taste. To obtain well-tolerated drug products, as a non-limiting example, the taste can be corrected by adding taste-enhancing fillers, by adjusting osmolality, or by using sweeteners.

Many patients are hypersensitive to various chemicals and often have bouts of bronchospastic, asthmatic and other types of cough. Their airways are particularly sensitive to hypotonic or hypertonic and acidic or alkaline conditions, as well as to the presence of any penetrating ion, such as chloride. Any imbalance of these conditions or the presence of chloride above a certain value leads to bronchospastic or inflammatory phenomena and / or cough, which greatly damages the treatment with inhaled drugs. Both of these conditions interfere with the effective delivery of aerosol drugs to the endobronchial space.

In some embodiments, the osmolality of aqueous solutions of the fluoroquinolone antimicrobial agent described herein is adjusted by adding excipients. In some cases, a certain amount of chloride or other anion is necessary for the successful and effective delivery of an aerosol antibiotic. However, it has been found that such amounts are less than those proposed and commonly used for aerosols of other compounds.

Bronchospasm or cough reflexes do not respond to the same osmolality of the solvent for aerosolization. However, they can be sufficiently controlled and / or suppressed when the osmolality of the solvent is in a certain range. A preferred solution for aerosolization of therapeutic compounds that is safe and tolerable has a total osmolality of from about 200 to about 1250 mosmol / kg with a range of chloride concentrations from about 30 mM to about 300 mM, and preferably from about 50 mM to about 150 mM. This osmolality controls bronchospasm, the concentration of chloride, as a penetrating anion, controls the cough. Being both penetrating ions, anions of both bromine and iodine can be used instead of chloride. In addition, bicarbonate can be used instead of chloride ion.

By way of non-limiting example, a preparation for an aerosol fluoroquinolone antimicrobial substance may contain from about 7 to about 700 mg, preferably from about 14 to about 300 mg, or most preferably from about 28 to about 280 mg of a fluoroquinolone antimicrobial in from about 1 to about 5 ml diluted saline (from 1/10 to 1/1 of normal saline). Accordingly, the concentration of the levofloxacin solution may be higher than about 25 mg / ml, higher than about 35 mg / ml and, preferably, higher than about 40 mg / ml, and as high as 50 mg / ml or higher .

In one embodiment, the osmolality of the solution is from about 100 mosmol / kg to about 600 mosmol / kg. In various other embodiments, the osmolality of the solution is from about 2000 mosmol / kg to about 1250 mosmol / kg; from about 250 mosmol / kg to about 1050 mosmol / kg; and from about 350 mosmol / kg to about 750 mosmol / kg.

In one embodiment, the concentration of the penetrating ion is from about 25 mM to about 400 mM. In various other embodiments, the penetrating ion concentration is from about 30 mM to about 300 mM; from about 40 mm to about 200 mm and from about 50 mm to about 150 mm.

Particulate Matter

Some embodiments relate to the use of solid drug nanoparticles for preparing dry aerosols or for preparing nanoparticles in a liquid suspension. Powders containing the drug in the form of nanoparticles can be obtained by spray drying of aqueous dispersed systems from the drug in the form of nanoparticles and surface modifiers to obtain a dry powder, which consists of aggregated nanoparticles of the drug. In one embodiment, the aggregates may have a size of from about 1 to about 2 microns, which is suitable for delivery deep into the lungs. The particle sizes of the aggregates can be increased to send them to other delivery sites, such as the upper bronchial zone or the mucous membrane of the nasal cavity, increasing the concentration of the drug in the spray-dried dispersed system or increasing the size of the droplets obtained by spray drying.

Alternatively, the aqueous dispersed drug system and surface modifier may contain a diluted diluent, such as lactose or mannitol, which, when spray dried, forms respirable diluent particles, each of which contains at least one interspersed drug nanoparticle and a surface modifier. The interspersed drug diluent particles may have a particle size of from about 1 to about 2 microns, suitable for delivery deep into the lungs. In addition, the particle size of the diluent can be increased to direct them to other delivery sites, such as the upper bronchial zone or mucous membrane of the nasal cavity, increasing the concentration of dissolved diluent in the aqueous dispersion system before spray drying or increasing the size of the droplets obtained by spray drying.

Spray dried powders can be used in DPI or pMDI, either on their own or in combination with a lyophilized powder in the form of nanoparticles. In addition, spray dried powders containing drug nanoparticles can be reconstituted and used either in jet or ultrasonic nebulizers to produce aqueous dispersed systems having respirable particle sizes, where each drop contains at least one drug nanoparticle facilities. Concentrated dispersed nanoparticle systems can also be used in these aspects of the invention.

Dispersed drug systems in the form of nanoparticles can also be lyophilized to obtain powders suitable for nasal or pulmonary delivery. Such powders may contain aggregated particles of the drug in the form of nanoparticles with a surface modifier. Such aggregates may have a size in the range suitable for inhalation, for example from about 2 to about 5 microns MMAD.

Lyophilized powders with an appropriate particle size can also be obtained by lyophilization of aqueous dispersed drug systems and a surface modifier that further comprises a diluted diluent such as lactose or mannitol. In these cases, lyophilized powders consist of respirable diluent particles, each of which contains at least one interspersed drug nanoparticle.

Lyophilized powders can be used in DPI or pMDI, either by themselves or in combination with spray dried powder in the form of nanoparticles. In addition, lyophilized powders containing drug nanoparticles can be reconstituted and used either in inkjet or ultrasonic nebulizers to produce aqueous dispersed systems having respirable particle sizes, where each drop contains at least one drug nanoparticle.

One embodiment of the invention relates to a process and composition for propellant-based systems containing nanoparticle drug particles and a surface modifier. Such preparations can be obtained by wet grinding a coarse drug and a surface modifier in a liquid propellant, either at atmospheric pressure or under high pressure. Alternatively, dry powders containing drug nanoparticles can be prepared by spray drying or lyophilization of aqueous dispersed drug nanoparticle systems and the resulting powders dispersed into suitable propellants for use in conventional pMDI. Similar nanoparticle pMDI preparations can be used for either nasal or pulmonary delivery. For pulmonary administration, such preparations can increase delivery to the deep zones of the lungs due to the small (for example, from about 1 to about 2 microns MMAD) particle size obtained using these methods. Concentrated aerosol preparations can also be used in pMDI.

Another embodiment relates to dry powders that contain nanoparticle compositions for pulmonary or nasal delivery. Powders may consist of nanoparticles suitable for inhalation of drug particle aggregates, or diluent particles suitable for inhalation, which contains at least one interspersed drug nanoparticle. Powders containing drug particles in the form of nanoparticles can be obtained from aqueous dispersed nanoparticle systems by removing water by spray drying or lyophilization (freeze-drying). Spray drying takes less time and is cheaper than lyophilization, and therefore more cost-effective. However, certain drugs, such as biologicals, benefit from freeze-drying compared to spray drying for powder formulations.

Conventional micronized drug particles used in aerosol delivery of dry powder having particle diameters of from about 2 to about 5 microns MMAD are often difficult to dose and distribute in small quantities due to the electrostatic cohesive forces inherent in such powders. Such difficulties can lead to drug losses in the delivery device, as well as incomplete powder distribution and insufficient delivery to the lungs. Many drug compounds, especially proteins and peptides, are intended for deep lung delivery and systemic absorption. Since the average particle sizes of dry powders obtained in the conventional manner are usually in the range of about 2 to about 5 microns MMAD, the portion of the material that actually reaches the alveolar zone can be extremely small. Thus, the delivery of micronized dry powders to the lungs, especially to the alveolar zone, is usually very ineffective due to the properties of the powders themselves.

Powder aerosols that contain drugs in the form of nanoparticles can be made smaller than a comparable micronized drug and, therefore, are suitable for efficient delivery deep into the lungs. Moreover, the nanoparticle-like drug aggregates are spherical in shape and have good flow characteristics, thereby facilitating the dosage and deposition of the administered composition in the lungs or nasal cavity.

Dry nanoparticle compositions can be used in both DPI and pMDI. As used herein, “dry” refers to a composition containing less than about 5% water.

One embodiment relates to compositions containing nanoparticles that have an effective average particle size of less than about 1000 nm, more preferably less than about 400 nm, less than about 300 nm, less than about 250 nm, or less than approximately 200 nm, as determined by light scattering methods. By "effective average particle size less than about 1000 nm" is meant that at least 50% of the particles of the drug have a weight average particle size of less than about 1000 nm when measured by light scattering methods. Preferably, at least 70% of the particles of the drug have an average particle size of less than about 1000 nm, more preferably at least 90% of the particles of the drug have an average particle size of less than about 1000 nm, and even more preferably at least 95% of the particles have a weight average particle size of less than about 1000 nm.

For aqueous aerosol formulations, a nanoparticle substance may be present in a concentration of from about 5.0 mg / ml up to about 700 mg / ml. For powder aerosol preparations, a nanoparticle substance may be present in a concentration of about 5.0 mg / g up to about 1000 mg / g, depending on the desired dose of the drug. Concentrated nanoparticle aerosols, defined as containing a nanoparticle drug at a concentration of about 5.0 mg / ml up to about 700 mg / ml for aqueous aerosol formulations and about 5.0 mg / g up to about 1000 mg / g for powder aerosol preparations are offered especially. Such preparations provide efficient delivery to the corresponding areas of the lungs or nasal cavity in a short administration time, i.e., less than about 3-15 seconds per dose, compared with the administration time up to 4 to 20 minutes, as established for conventional pulmonary nebulization types therapy.

The nanoparticle drug compositions for aerosol administration can be prepared, for example, (1) by spraying a nanoparticle dispersed drug system obtained either by grinding or precipitation; (2) converting the dry powder of drug aggregates in the form of nanoparticles and a surface modifier into an aerosol (the aerosolized composition may further comprise a diluent); or (3) converting into a spray a suspension of a drug in the form of nanoparticles or drug aggregates in a non-aqueous propellant. Aggregates of the drug in the form of nanoparticles and a surface modifier, which may additionally contain a diluent, can be obtained in a non-aqueous system that is not or under pressure. Concentrated aerosol preparations can also be obtained in similar ways.

The grinding of the aqueous drug to obtain the drug in the form of nanoparticles can be accomplished by dispersing the drug particles in a liquid dispersion medium and using mechanical means in the presence of abrasive materials to reduce the particle size of the drug to the desired effective average particle size. Particles can be reduced in size in the presence of one or more surface modifiers. Alternatively, the particles may come into contact with one or more surface modifiers after grinding. Other compounds, such as a diluent, may be added to the drug / surface modifier composition during size reduction. Dispersed systems can be manufactured continuously or in batches.

Another way to obtain a dispersed system of nanoparticles is microprecipitation. It is a method of producing stable dispersed systems of drugs in the presence of one or more surface modifiers and one or more surface active substances that increase the stability of colloidal systems, free of any traces of toxic solvents or solubilized impurities of heavy metals. A similar method includes, for example, (1) dissolving the drug in a suitable solvent with stirring; (2) adding the preparation from step (1) with stirring to a solution containing at least one surface modifier to obtain a clear solution; and (3) precipitating the preparation from step (2) with stirring using a suitable precipitant. After applying this method, you can further remove any salt formed, if any, by dialysis or diafiltration and concentrate the dispersed system by conventional methods.

The resulting nanoparticle dispersed drug system can be used in liquid nebulizers or processed to produce a dry powder for use in DPI or pMDI.

In a non-aqueous, non-pressurized grinding system, a non-aqueous liquid having a vapor pressure of about 1 atm or less at room temperature, and in which the drug substance is substantially insoluble, as a wet grinding medium to obtain a nanoparticle drug composition, can be used. In this process, a thick suspension of the drug and surface modifier can be crushed in a non-aqueous medium to obtain particles of the drug in the form of nanoparticles. Examples of suitable non-aqueous media include ethanol, trichlorormonofluoromethane, (CFC-11) and dichlorotetrafluoroethane (CFC-114). The advantage of using CFC-11 is that it can be used at more or less cool room temperatures, while CFC-114 requires more controlled conditions to avoid evaporation. Upon completion of the grinding, the liquid medium can be removed and restored under vacuum or by heating, resulting in a dry composition in the form of nanoparticles. The dry composition can then be placed in a suitable container and the final propellant added. Examples of final product propellants that ideally do not contain chlorinated hydrocarbons include HFA-134a (tetrafluoroethane) and HFA-227 (heptafluoropropane). Although non-chlorinated propellants can be preferred due to environmental considerations, chlorinated propellants can also be used in this aspect of the invention.

In a non-aqueous, pressurized grinding system, a non-aqueous liquid having a vapor pressure substantially higher than 1 atm at room temperature in the grinding process can be used to obtain drug compositions in the form of nanoparticles. If the grinding medium is a suitable halogenated hydrocarbon propellant, the resulting dispersed system can be immediately placed in a suitable pMDI container. Alternatively, the grinding medium can be removed and restored under vacuum or by heating to obtain a dry composition in the form of nanoparticles. This composition can then be placed in a suitable container and the final propellant added for use in pMDI.

Spray drying is a process used to obtain a powder containing nanoparticle particles of a drug after reducing the particle size of the drug in a liquid medium. Generally, spray drying can be used when the liquid medium has a vapor pressure of less than about 1 atm at room temperature. Spray drying is a device that allows the evaporation of a liquid and the collection of drug powder. A liquid sample, either a solution or a suspension, is placed in a spray nozzle. The nozzle creates sample droplets in the range of about 20 to about 100 microns in diameter, which are then transported by the carrier gas to the drying chamber. The temperature of the carrier gas is usually from about 80 to about 200 ° C. Drops of liquid undergo rapid evaporation, leaving behind dry particles that are collected in a special tank under the cyclone apparatus.

If the liquid sample consists of an aqueous dispersed system of nanoparticles and a surface modifier, the collected product will consist of spherical aggregates of drug particles in the form of nanoparticles. If the liquid sample consists of an aqueous dispersed system of nanoparticles in which an inert diluent material (such as lactose or mannitol) is dissolved, the collected product will consist of diluent particles (e.g. lactose or mannitol) that contain interspersed drug particles in the form of nanoparticles. The final size of the collected product can be controlled, and it depends on the concentration of the drug in the form of nanoparticles and / or diluent in the liquid sample, as well as on the size of the droplets created by the spray nozzle spray drying. The collected products can be used in conventional DPIs for pulmonary or nasal delivery, dispersed in propellants for use in pMDI, or particles can be reconstituted in water for use in nebulizers.

In some cases, it may be desirable to add an inert carrier to the spray dried material to improve the dosage properties of the final product. This may especially be the case when the spray dried powder is very fine (less than about 5 microns) or when the intended dose is extremely small, making dosing difficult. Basically, particles of such a carrier (also known as bulking agents) are too large to be delivered to the lungs and simply stick to the mouth and throat and are swallowed. Such carriers usually consist of sugars such as lactose, mannitol or trehalose. Other inert materials, including polysaccharides and cellulosic polymers, can also be used as carriers.

Spray-dried powders containing drug particles in the form of nanoparticles can be used in conventional DPIs, dispersed in propellants for use in pMDI, or reconstituted in a liquid medium for use in nebulizers.

For compounds that are denatured or destabilized by heating, such as compounds having a low melting point (i.e., from about 70 to about 150 ° C), or, for example, biological preparations, sublimation is more preferable to obtain a powder drug composition in the form of nanoparticles, than evaporation. The reason is that sublimation does not use high process temperatures associated with spray drying. In addition, sublimation, also known as freeze-drying or lyophilization, can increase storage stability, especially for biological products. Freeze-dried particles can also be reconditioned and used in nebulizers. Aggregates of freeze-dried particles of the drug in the form of nanoparticles can be mixed either with a powdered intermediate, or used without additives in DPI and pMDI for nasal or pulmonary delivery.

Sublimation involves freezing the product and placing the sample in a deep vacuum. This makes it possible to directly convert the formed ice from a solid state to a vapor state. A similar process is highly efficient and therefore provides a higher yield than spray drying. The resulting freeze-dried product contains the drug and modifier (s). The drug is usually present in an aggregated state and can be used for inhalation (pulmonary or nasal) either by itself or in combination with diluent materials (lactose, mannitol, etc.), in DPI or pMDI, or reconstituted for use in nebulizers .

Liposome Compositions

In some embodiments, the fluoroquinolone antimicrobial agents described herein can be prepared as liposome particles, which can then be converted to aerosol for inhalation delivery. The lipids used in the present invention may be any of a variety of lipids, including both neutral and charged lipids. Carrier systems having the desired properties can be prepared using appropriate lipid combinations, targeting groups, and circulation enhancers. In addition, the compositions provided herein may be in the form of liposomes or lipid particles, preferably lipid particles. As used here, the term "lipid particle" refers to a lipid bilayer carrier that "covers" the nucleic acid and contains little or no water inside. More specifically, the term is used to describe a self-assembled lipid bilayer carrier, in which the inner layer is cationic lipids that form ionic bonds or ion pairs with negative nucleic acid charges (e.g., phosphodiester skeleton of a plasmid). The inner layer may also be neutral or fusogenic lipids and, in some embodiments, negatively charged lipids. The outer layer of the particle will usually contain lipid mixtures oriented tail-to-tail (as in liposomes) with hydrophobic tails of the inner layer. The polar head groups present on the lipids of the outer layer will form the outer surface of the particle.

Liposomal bioactive substances can be designed so that they have a long therapeutic effect or reduced toxicity, making possible less frequent administration and increased therapeutic index. Liposomes are composed of two layers that hold the desired pharmaceutical preparation. They can be shaped into multilayer bubbles of concentric double layers, with a pharmaceutical preparation held either in lipids of different layers or in the water space between the layers.

By way of non-limiting example, the lipids used in the compositions may be synthetic, semi-synthetic or natural lipids, including phospholipids, tocopherols, steroids, fatty acids, glycoproteins such as albumin, negatively charged lipids and cationic lipids. Phospholipids include egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE) and egg phosphatidic acid (EPA); soybean equivalents, soybean phosphatidylcholine (SPC); SPG, SPS, SPI, SPE and SPA; hydrogenated egg and soy equivalents (e.g., HEPC, HSPC), other phospholipids derived from ester bonds of fatty acids at positions 2 and 3 of glycerol containing chains of 12 to 26 carbon atoms, and various end groups at position 1 of glycerol, which include choline, glycerin, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated and the phospholipid can be formed from fatty acids with different chain lengths and different degrees of unsaturation. In particular, formulation compositions may include dipalmitoylphosphatidylcholine (DPPC), a major constituent of the natural pulmonary surfactant, as well as dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). Other examples include dimyristoylphosphatidylcholine (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG) dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylethanolamine (DOPE) and mixed phospholipids like palmitoilstearoilfosfatidilholin (PSPC) and palmitoilstearoilfosfatidilglitserin (PSPG) , and single acylated phospholipids, such as monooleoylphosphatidylethanolamine (MOPE).

In a preferred embodiment, the modified PEG lipids are included in the compositions of the present invention as an anti-aggregation agent. The use of a modified PEG lipid promotes the positioning of bulk PEG groups on the surface of a liposome or lipid carrier and prevents DNA from binding to the outer surface of the carrier (thereby inhibiting crosslinking and aggregation of the lipid carrier). The use of PEG-ceramide is often preferred and has the additional benefits of stabilizing membrane bilayer structures and prolonging circulation life. In addition, PEG ceramides can be prepared with lipid tails of various lengths to control the lifetime of the PEG ceramide in the lipid double layer. In this way, it is possible to carry out a “programmed” release and, as a result, control the fusion of lipid carriers. For example, PEG ceramides having C ₂₀ acyl groups bound to the ceramide moiety will diffuse from the lipid bilayer carrier with a half-life of 22 hours. PEG ceramides having C ₁₄ and C ₈ acyl groups will diffuse from the same carrier with a half-life of 10 minutes and less than 1 minute, respectively. As a result, the choice of the length of the lipid tail creates a composition in which the double layer becomes destabilized (and therefore fusogenic) at a known rate. Although less preferred, other PEG lipids or lipid-polyoxyethylene conjugates are useful in the present compositions. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-modified diacylglycerols and dialkylglycerols, PEG-modified dialkylamines and PEG-modified l, 2-diacyloxypropan-3-amines. Particularly preferred are conjugates of PEG ceramides (e.g., PEG-Cer-C ₈ , PEG-Cer-C ₁₄ or PEG-Cer-C ₂₀ ) which are described in US Pat. No. 5,820,873, incorporated herein by reference.

The compositions of the present invention can be prepared to provide liposome compositions that have a diameter of from about 50 nm to about 400 nm. One skilled in the art will recognize that the size of the compositions may be larger or smaller depending on the volume that is encapsulated. So, for large volumes, the size distribution will usually be from about 80 nm to about 300 nm.

Surface modifiers

The fluoroquinolone antimicrobial agents described herein can be prepared in a pharmaceutical composition with appropriate surface modifiers that can be selected from known organic and inorganic pharmaceutical excipients. Such excipients include low molecular weight oligomers, polymers, surfactants and natural products. Preferred surface modifiers include nonionic and ionic surfactants. Two or more surface modifiers can be used in combination.

Representative examples of surface modifiers include cetylpyridinium chloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol, gum arabic, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl ether sulphate ether, cetericetherogesterol ether, cetericeterol esteribeterol ether ceterogyloterol ether ceterogyloteriol ether ceterogyloteriol ether ceterogyloterol ether ceterogyloteriol esteribol polyoxyethylene alkyl esters (for example, macrogol esters such as cetomacrogol 1000), castor oil polyoxyethylene derivatives, sorbitan fatty acid polyoxyethylene esters (for example, commercially available Tweens.RTM, such as, for example, Tween 20.RTM and Tween 80.RTM, (ICI Specialty Chemicals)); polyethylene glycols (e.g. Carbowaxs 3350.RTM and 1450.RTM. and Carbopol 934.RTM, (Union Carbide)), dodecyl trimethylammonium bromide, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, HP carboxymethyl cellulose HPC, calcium -L), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA) polyvinyl alcohol (PVA) don (PVP), 4- (l, l, 3,3-tetramethylbutyl) phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol, superion and triton), poloxamers (e.g. Pluronics F68.RTM and F108. RTM., which are block copolymers of ethylene oxide and propylene oxide); poloxamines (for example, Tetronic 908. RTM., also known as Poloxamine 908.RTM., which is a tetrafunctional block copolymer obtained from the sequential addition of propylene oxide and ethylene oxide to ethylene diamine (BASF Wyandotte Corporation, Parsippany, NJ.)); a charged phospholipid such as dimyristoylphosphatidylglycerol, dioctyl sulfosuccinate (DOSS); Tetronic 1508.RTM; (T-1508) (BASF Wyandotte Corporation), sodium sulfosuccinic acid dialkyl esters (e.g. Aerosol OT.RTM., Which is sodium sulfosuccinic acid dioctyl ester (American Cyanamid)); Duponol P.RTM., Which is sodium lauryl sulfate (DuPont); Tritons X-200.RTM, which is an alkylaryl polyethersulfonate (Rohm and Haas); Crodestas F-110.RTM., Which is a mixture of sucrose stearate and sucrose distearate (Croda Inc.); p-isononylphenoxypoly- (glycidol), also known as Olin-log.RTM, or Surfactant 10-G.RTM, (Olin Chemicals, Stamford, Conn.); Crodestas SL-40.RTM, (Croda, Inc.); and SA9OHCO, which is C.sub. 18 H.sub. 37 CH.sub.2 (CON (CH.sub.3) -CH.sub.2 (CHOH) .sub.4 (CH.sub.2 OH ) .sub.2 (Eastman Kodak Co.); decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecide β-D-glucopyranoside; n-dodecide β- D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexyl β-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noyl β-D β-glucopyranoside; octanoyl-N-methylglucarmide; n-octyl-β-D-glucopyranoside; octyl β-D-thioglucopyranoside; and the like. Tiloxapol is a particularly preferred modificato surface for pulmonary or intranasal delivery of steroids, and still more for spray treatment.

Examples of surfactants for use in the solutions described herein include, but are not limited to: ammonium laureth sulfate, cetamine oxide, cetrimonium chloride, cetyl alcohol, cetyl myristate, cetyl palmitate, cocamide DEA, cocamidopropyl betaine, cocamidopropylamine sulfamide amide malamide, amamide laurethamide amide malamide acids, dicetyl dimethyl ammonium chloride, dipalmitoyl ethyl hydroxyethyl ammonium, disodium laureth sulfosuccinate, di (hydrogenated) hard fatty phthalic acid, glyceryl dilaurate, glyceryl distearate, gly Eryl oleate, glyceryl stearate, nf isopropyl myristate, nf isopropyl palmitate, DEA lauramide, MEA lauramide, lauramide oxide, myristamine oxide, octylisononanoate, octyl palmitate, octyldodecyl steptoglide-2-tetra-glomero-2-tetra-glomero-2-nitro-2-tetra-glomero and PEG-150 stearate and distearate, PEG-4 to PEG-150 laurate and dilaurate, PEG-4 to PEG-150 oleate and dioleate, PEG-7 glyceryl cocoate, PEG-8 beeswax, propylene glycol stearate, C 14-16 sodium olefin sulfonate, sodium lauryl sulfoacetate, sodium lauryl sulfate, sodium tridecyl sulfate, stearalconium chlorine id, stearamide oxide, TEA-dodecylbenzenesulfonate, TEA lauryl sulfate.

Most of these surface modifiers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 1986), specifically incorporated by reference. Surface modifiers are commercially available and / or can be obtained by methods known in the art. The relative amount of drug and surface modifier can vary widely and the optimal amount of surface modifier may depend on, for example, the particular drug and surface modifier selected, the critical concentration of micelle surface modifier, if it forms micelles, hydrophilic-lipophilic balance (HLB) modifier surface, the melting temperature of the surface modifier, the solubility in water of the surface modifier and / or drug Twa, the surface tension of water solutions of the surface modifier and so on.

According to the present invention, the optimum ratio of drug to surface modifier is from ~ 0.1% to ~ 99.9% of the fluoroquinolone antimicrobial substance, more preferably from about 10% to about 90%.

Microspheres

Microspheres can be used for pulmonary delivery of fluoroquinolones by first adding the appropriate amount of the drug compound to be solubilized in water. For example, an aqueous solution of levofloxacin can be dispersed in methylene chloride containing a predetermined amount (0.1-1% w / v) of poly (DL-lactide-glycolide) (PLGA) by treating the sample with ultrasound for 1-3 minutes in an ice bath. Separately, levofloxacin is solubilized in methylene chloride containing PLGA (0.1-1% w / v). The obtained primary water-in-oil emulsion or a polymer / drug solution is dispersed in a homogeneous aqueous phase consisting of 1-2% polyvinyl alcohol (pre-cooled to 4 ° C), treating the sample with ultrasound for 3-5 minutes in an ice bath. The resulting emulsion is continuously stirred for 2-4 hours at room temperature to evaporate methylene chloride. Microparticles thus obtained are separated from the homogeneous phase by centrifugation at 8000-10000 rpm for 5-10 minutes. The precipitated particles are washed twice with distilled water and lyophilized. Lyophilized microparticles of levofloxacin are stored at -20 ° C.

By way of non-limiting example, a spray drying approach is used to obtain levofloxacin microspheres. An appropriate amount of levofloxacin is solubilized in methylene chloride containing PLGA (0.1-1%). This solution is spray dried to obtain microspheres.

As a non-limiting example, levofloxacin microparticles are characterized by particle size distribution (requirement: 90% <5 μm, 95% <10 μm), shape, degree of drug loading and drug release using appropriate methods and methods.

As a non-limiting example, this approach can also be used to separate and improve the solubility in water of solid preparations that improve the configuration of AUC, such as poorly soluble salt forms of levofloxacin for nanoparticle-based preparations.

A certain amount of fluoroquinolone can first be dissolved in a minimum amount of 96% ethanol, which is necessary to maintain fluoroquinolone in solution when diluted with water from 96 to 75%. This solution can then be diluted with water to a 75% ethanol solution, and then a certain amount of paracetamol can be added to obtain the following drug / polymer ratios: 1: 2, 1: 1, 2: 1, 3: 1, 4: 1 , 6: 1, 9: 1 and 19: 1. These final solutions are spray dried under the following conditions: feed rate, 15 ml / min; input temperature 110 ° C; discharge temperature 85 ° C; 4 bar pressure and drying air throughput 35 m ³ / h. The powder is then collected and stored under vacuum in a desiccator.

Solid lipid particles

The preparation of solid lipid particles of fluoroquinolone may include dissolving the drug in a lipid melt (phospholipids such as phosphatidylcholine and phosphatidylserine) maintained at at least the melting point of the lipid, followed by dispersion of the drug-containing melt in a hot aqueous surfactant solution (usually 1- 5% w / v) maintained at at least the melting point of the lipid. The coarse disperse system is homogenized for 1-10 minutes using Microfluidizer® to produce a nanoemulsion. When the nanoemulsion is cooled to a temperature within 4-25 ° C, the lipids solidify, which leads to the formation of solid lipid nanoparticles. The preparation parameters (type of lipid matrix, surfactant concentration and manufacturing parameters) are optimized to achieve sustained drug delivery. As a non-limiting example, this approach can also be used to separate and improve the solubility in water of solid preparations that improve the configuration of AUC, such as poorly soluble salt forms of levofloxacin for nanoparticle-based preparations.

Melt Extruded AUC Configurable Preparations

Melt extrudable AUC configuration-improving fluoroquinolone preparations can be prepared by dissolving drugs in micelles by adding surfactants or by preparing a microemulsion, by incorporating inclusion complexes with other molecules such as cyclodextrins, by preparing drug nanoparticles or by encapsulating amorphous drugs in a polymer matrix. By enclosing the drug uniformly in a polymer matrix, a solid dispersion system is obtained. Solid dispersion systems can be obtained in two ways: by dissolution and by melt. The dissolution method uses an organic solvent in which the drug and the corresponding polymer are dissolved and then dried (spray). The main disadvantages of this method are the use of organic solvents and a batch mode of the production method. In the melt method, heat is used in order to disperse or dissolve the drug in the corresponding polymer. The melt extrusion process is an optimized version of the melt process. An advantage of the melt extrusion approach is the absence of an organic solvent and a continuous production process. Since melt extrusion is a new pharmaceutical method, there is little literature on this subject. The technical scheme includes a mixture and extrusion of fluoroquinolone, hydroxypropyl-b-cyclodextrin (HP-b-CD) and hydroxypropyl methylcellulose (HPMC) in order to, as a non-limiting example, obtain a leucofloxacin or other fluoroquinolone form-improving AUC. Cyclodextrin is a toroidal molecule with hydroxyl groups on the outer surface and a cavity in the center. Cyclodextrin captures the drug, forming an inclusion complex. The formation of a complex of cyclodextrin with the drug was carefully studied. It is known that a water-soluble polymer interacts with cyclodextrin and a drug in the process of complex formation to obtain a stabilized complex of a drug and cyclodextrin in complex with a polymer. Such a complex is more stable than the classic cyclodextrin-drug complex. By way of example, HPMC is water soluble; therefore, the use of this polymer with HP-b-CD in the melt is expected to produce a water-soluble AUC configuration improving formulation. As a non-limiting example, this approach can also be used to separate and improve the solubility in water of solid preparations that improve the configuration of AUC, such as poorly soluble salt forms of levofloxacin for nanoparticle-based preparations.

Co-precipitates

Co-precipitate of fluoroquinolone preparations can be obtained by the formation of co-precipitates with pharmacologically inactive polymeric materials. It has been shown that the formation of molecular solid disperse systems or co-precipitates to obtain AUC configuration-improving preparations with various water-soluble polymers can significantly slow their in vitro dissolution and / or in vivo absorption rates. When producing powder products, grinding is usually used to reduce particle size, since the dissolution rate is highly dependent on particle size. Moreover, strong interaction (such as grinding) can increase surface energy and cause deformation of the crystal lattice, as well as a decrease in particle size. Co-grinding the drug with hydroxypropylmethyl cellulose, b-cyclodextrin, chitin and chitosan, crystalline cellulose and gelatin can increase the dissolution ability so that an improved AUC configuration is obtained for otherwise readily bioavailable fluoroquinolones. By way of non-limiting example, this approach can also be used to separate and improve the solubility in water of solid preparations that improve the configuration of AUC, such as poorly soluble salt forms of levofloxacin for nanoparticle-based preparations.

Dispersion Improving Peptides

Compositions may include one or more di- or tripeptides containing two or more leucine residues. As a further non-limiting example, the entire contents of US Pat. No. 6,835,372 describing dispersion improving peptides are incorporated herein by reference. This patent describes the discovery that dileycyl-containing dipeptides (eg, dileucin) and tripeptides are best in their ability to increase the dispersibility of the powder composition.

In another embodiment, well dispersible particles, including an amino acid, are administered. Hydrophobic amino acids are preferred. Suitable amino acids include natural and non-naturally occurring hydrophobic amino acids. Some natural hydrophobic amino acids, including, but not limited to the following, non-naturally occurring amino acids include, for example, β-amino acids. Hydrophobic amino acids of both D, L and racemic configurations can be used. Suitable hydrophobic amino acids may also include amino acid analogues. As used herein, an amino acid analogue includes an amino acid in a D or L configuration having the following formula: —NH — CHR — CO—, where R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and where R does not correspond to the side chain of a natural amino acid. As used herein, aliphatic groups include straight chain, branched or cyclic C1-C8 hydrocarbons that are fully saturated, which contain one or two heteroatoms, such as nitrogen, oxygen or sulfur and / or which contain one or more units of unsaturation. Aromatic groups include carbocyclic aromatic groups such as phenyl and naphthyl and heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituent groups on aliphatic, aromatic or benzyl groups include --OH, halogen (--Br, - Cl, - I and --F) - O (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), - CN, --NO ₂ , --COOH, --NH ₂ , --NH (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), --N (aliphatic group , substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group) _2, --COO (aliphatic group deputy schennaya aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), --CONH _2, --CONH (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), --SH, - S ( aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group) and --NH - C (= NH) - NH ₂ . A substituted benzyl or aromatic group may also have an aliphatic or substituted aliphatic group as a substituent group. A substituted aliphatic group may also have a benzyl, substituted benzyl, aryl or substituted aryl group as a substituent group. A substituted aliphatic, substituted aromatic or substituted benzyl group may have one or more substituent groups. By modifying the amino acid substitution group, for example, the lipophilicity or hydrophobicity of natural amino acids that are hydrophilic can be increased.

Many suitable amino acids, amino acid analogues and their salts are commercially available. Others can be synthesized by methods known in the art.

Hydrophobicity is usually determined taking into account the distribution of the amino acid between a non-polar solvent and water. Hydrophobic amino acids are those that have an affinity for non-polar solvents. The relative hydrophobicity of amino acids can be expressed on the hydrophobicity scale, on which glycine has a value of 0.5. On this scale, amino acids that have an affinity for water have values below 0.5, and those that have an affinity for non-polar solvents have values above 0.5. As used here, the term hydrophobic amino acid refers to an amino acid that has a hydrophobicity scale greater than or equal to 0.5, in other words, tends to be distributed in non-polar acid, which is at least less than or equal to that of glycine.

Examples of amino acids that can be used include, but are not limited to: glycine, proline, alanine, cysteine, methionine, valine, leucine, tyrosine, isoleucine, phenylalanine, tryptophan. Preferred hydrophobic amino acids include leucine, isoleucine, alanine, valine, phenylalanine and glycine. Combinations of hydrophobic amino acids can also be used. Moreover, combinations of hydrophobic and hydrophilic (mainly distributed in water) amino acids, where the final combination is hydrophobic, can also be used.

Amino acids may be present in the particles of this invention in an amount of at least 10% by weight. Preferably, the amino acid may be present in the particles in an amount of from about 20 to about 80% by weight. The hydrophobic amino acid salt may be present in the particles of this invention in an amount of at least 10% by weight. Preferably, the amino acid salt is present in the particles in an amount of from about 20 to about 80% by weight. In preferred embodiments, the particles have a shake density of less than about 0.4 g / cm ³ .

Methods for preparing and delivering particles that contain an amino acid are described in US Pat. No. 6,586,008, entitled Use of Simple Amino Acids to Form Porous Particles During Spray Drying, the teachings of which are incorporated herein by reference in their entirety.

Proteins / Amino Acids

Protein fillers may include albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. Suitable amino acids (other than the dileycyl peptides of this invention), which may also serve as buffers, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine, tryptophan and the like. Preferred are amino acids and polypeptides that act as dispersants. Amino acids that fall into this category include hydrophobic amino acids such as leucine, valine, isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine, histidine, and proline. Dispersion enhancing peptide excipients include dimers, trimers, tetramers and pentamers containing one or more hydrophobic amino acid components, such as those described above.

Carbohydrates

By way of non-limiting example, carbohydrate fillers may include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melesitose, maltodextrins, dextrans, starches and the like; and polyhydric alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucite), pyranosylsorbitol, myoinositol, isomalt, trehalose and the like.

Polymers

By way of non-limiting example, the compositions may also include polymeric fillers / additives, for example polyvinyl pyrrolidones, cellulose derivatives such as hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl methyl cellulose, ficolls (polymeric sugars), hydroxyethyl starch, dextrates (by way of non-limiting example, cyclodextrins may include β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, randomly methylated β-cyclodextrin, dimethyl-α-cyclodextrin, dimethyl-β-cyclodec trine, maltosyl-α-cyclodextrin, glucosyl-1-α-cyclodextrin, glucosyl-2-α-cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and sulphobutylether-β-cyclodextrin), polyethylene glycols, and pectin may also be used.

The fine particles that are introduced contain a bioactive substance and a biocompatible and preferably biodegradable polymer, copolymer or mixture. The polymers can be crosslinked to optimize various particle characteristics, including: i) interactions between the delivered substance and the polymer to stabilize the substance and maintain activity during the delivery; ii) the rate of degradation of the polymer and, therefore, the rate of release profiles of the drug; iii) surface characteristics and guiding abilities through chemical modification; and iv) particle porosity.

To produce particles, surface disruptive polymers such as polyanhydrides can be used. For example, polyanhydrides such as poly [(p-carboxyphenoxy) hexanoic anhydride] (PCHP) can be used. Biodegradable polyanhydrides are described in US Pat. No. 4,857,311. Base-breaking polymers such as those based on polyesters, including polyoxyacids, can also be used. For example, to produce particles, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof can be used. The polyester may also have a charged or functionalizable group, such as an amino acid. In a preferred embodiment, particles with controlled release properties can be prepared from poly (D, L-lactic acid) and / or poly (DL-lactic-co-glycolic acid) ("PLGA"), which comprises a surface-active compound, such as dipalmitoylphosphatidylcholine (DPPC).

Other polymers include polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), polyvinyl compounds such as polyvinyl alcohols, polyvinyl ethers and polyvinyl esters, polymers of acrylic and methacrylic acids, cellulose and other polysaccharides, as well as peptides or proteins, or their copolymers or mixtures. The polymers can be selected or modified so that they have the appropriate stability and degradation rate in vivo for various applications for controlled drug delivery.

Fine particles can be obtained from functionalized grafted copolymers of polyesters, as described in Hrkach et al., Macromolecules, 28: 4736-4739 (1995); and Hrkach et al., "Poly (L-Lactic acid-co-amino acids) Graft Copolymers: A Class of Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M, Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.

In a preferred embodiment of the invention, fine particles containing a bioactive substance and a phospholipid are administered. Examples of suitable phospholipids, among others, include phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, and combinations thereof. Specific examples of phospholipids include, but are not limited to, phosphatidylcholines: dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylglycerol, or limerinium. Other phospholipids are known to those skilled in the art. In a preferred embodiment, the phospholipids are endogenous to the lungs.

The phospholipid may be present in the particles in amounts limited to from 0 to about 90% by weight. More often, it may be present in the particles in amounts limited to from about 10 to about 60% by weight.

In another embodiment, the phospholipids or combinations thereof are selected so as to impart controlled release properties to the finely divided particles. The phase transition temperature of a particular phospholipid may be lower, near or above the physiological temperature of the patient's body. Preferred phase transition temperatures are in the range of 30 ° C to 50 ° C (for example, within ± 10 of the patient’s normal body temperature). By choosing phospholipids or combinations of phospholipids according to their phase transition temperature, particles can be crosslinked so as to obtain controlled release properties. For example, by introducing particles that contain a phospholipid or combination of phospholipids that have a phase transition temperature higher than the patient’s body temperature, the release of a dopamine precursor, agonist, or any combination of precursors and / or agonists can be slowed down. On the other hand, rapid release can be obtained by incorporating phospholipids in particles having lower transition temperatures.

Correction of taste, flavors, other.

By way of non-limiting example, the compositions may further include flavoring agents, taste masking agents, inorganic salts (e.g. sodium chloride), antimicrobials (e.g. benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as " TWEEN 20 ”and“ TWEEN 80 ”), sorbitan esters, saccharin, cyclodextrins, lipids (eg phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines), fatty acids and fatty esters s, steroids (e.g., cholesterol), and chelating agents (e.g., EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and / or additives suitable for use in the compositions of the present invention are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), as well as in " Physician's Desk Reference ", 52.sup.nd ed., Medical Economics, Montvale, NJ (1998).

By way of non-limiting example, a variety of taste masking agents for fluoroquinolone preparations include the addition of flavors, sweeteners, and various other coating techniques. As non-limiting examples, they can be selected from sugars such as sucrose, dextrose and lactose, carboxylic acids, salts such as magnesium and calcium (non-specific or chelation-based taste correction of fluoroquinolones), menthol, amino acids or amino acid derivatives such as arginine , lysine and monosodium glutamate, as well as synthetic aromatic oils and aromatic aromatic substances and / or natural oils, extracts from plants, leaves, flowers, fruits and so on, and combinations thereof. These may include cinnamon oils, pear oil, peppermint oil, clover oil, bay oil, anise oil, eucalyptus, vanilla, citrus oil such as lemon oil, orange oil, grape and grapefruit oil, fruit essences, including apple, peach , pear, strawberry, raspberry, cherry, plum, pineapple, apricot and so on. Additional sweeteners include sucrose, dextrose, aspartame (Nutrasweet®), acesulfame-K, sucrolose and saccharin, organic acids (by way of non-limiting example, citric acid and aspartic acid). Such flavors may be from about 0.05 to about 4 percent. Another way to improve or mask the taste of unpleasant inhaled drugs is to reduce the solubility of drugs, for example, in order to interact with taste buds, drugs must dissolve. Therefore, by delivering the drug in solid forms, a taste response can be avoided and the desired taste correction effect can be achieved. Non-limiting methods for reducing the solubility of fluoroquinolones are described herein, for example, salt forms of levofloxacin or hemifloxacin with xinafoic acid, oleic acid, stearic acid and pamic acid. Additional co-precipitating agents include dihydropyridines and a polymer such as polyvinylpyrrolidone. In addition, taste corrections can be achieved by receiving lipophilic vesicles. Additional coating or coating agents include dextrates (by way of non-limiting example, cyclodextrins may include 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, randomly methylated β-cyclodextrin, dimethyl-α-cyclodextrin, dimethyl-β-cyclodextrin , maltosyl-α-cyclodextrin, glucosyl-1-α-cyclodextrin, glucosyl-2-α-cyclodextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin and sulfobutyl ether-β-cyclodextrin), modified celluloses such as ethyl cellulose, methyl cellulose vine, hydroxypropylcellulose, gidroksilpropilmetiltsellyuloza, polyalkylene glycols, polyalkylene oxides, sugars and sugar alcohols, waxes, shellacs, acrylics and mixtures thereof. By way of non-limiting example, other methods of delivering undissolved forms of fluoroquinolones are by administering the drug in a pure form or in a simple, solubility-free preparation, such as crystalline micronized dry powder spray dried and a drug in the form of nanosuspension. However, an alternative method is to include taste masking agents. These include a taste-masking substance that is mixed with, top-coated, or otherwise combined with a fluoroquinolone active drug. However, such an additive may also serve to improve the taste of another selected additive to the medicinal product, for example, a mucolytic substance. Non-limiting examples of such substances include acid phospholipids, lysophospholipid, tocopherol polyethylene glycol succinate, and embonic acid (pamoate). Many of these substances can be used alone or in combination with fluoroquinolones for aerosol administration.

Mucolytic substances

Methods for the preparation of preparations that combine substances to reduce the viscosity of sputum during aerosol treatment with fluoroquinolones include the following. Such substances can be obtained in fixed combinations or administered after exposure to aerosol fluoroquinolone.

The most common prescribed substance is N-acetylcysteine (NAC), which depolymerizes mucous secretions in vitro by breaking down disulfide bridges between macromolecules. It is believed that such a decrease in the stickiness of sputum facilitates its removal from the respiratory tract. In addition, NAC can act as an acceptor of oxygen radicals. NAC can be taken orally, either orally or by inhalation. The differences between these two routes of administration have not been formally studied. After oral administration, NAC is restored in the liver and intestines to cysteine, the precursor of the antioxidant glutathione. Antioxidant properties may be useful in preventing a decrease in pulmonary function in cystic fibrosis (CF). The nebulized form of NAC is usually prescribed to patients with CF, especially in continental Europe, to improve expectoration of sputum by reducing its stickiness. The ultimate goal of this is to slow the decline in pulmonary function in CF.

L-lysine-N-acetylcysteinate (ACC) or Nazistelin (NAL) is a new mucoactive substance with mucolytic, antioxidant and anti-inflammatory properties. Chemically, it is an ACC salt. This drug appears to have an activity superior to that of the parent ACC molecule due to the synergistic mucolytic activity of L-lysine and ACC. Moreover, its almost neutral pH (6.2) allows it to be injected into the lungs with a very low risk of bronchospasm, in contrast to acidic ACC (pH 2.2). NAL is difficult to obtain in an inhalable form because the required pulmonary dose is very high (approximately 2 mg) and the micronized drug is viscous and sticky, and thus it is problematic to obtain a nebulized preparation. Initially, NAL was developed in the form of a chlorofluorocarbon (CFC) -containing metered-dose inhaler (MDI), as this form was the easiest and fastest to develop for the start of preclinical and first clinical trials. NAL MDI delivered 2 mg in a single injection, of which approximately 10% could reach the lungs in healthy volunteers. One of the main disadvantages of this drug was adherence to the treatment of the patient, since up to 12 injections were necessary in order to obtain the required dose. In addition, the increasing removal of CFC gases from medical products, associated with coordination problems encountered in a significant proportion of patients (12), led to the development of a new galenic form of NAL. A powder inhaler (DPI) was chosen to solve the problems of adherence to treatment in MDI cases and to combine it with the optimal, reproducible and comfortable way of administering the drug to the widest possible range of patients, including young children.

NAL DPI DPI involves the use of unconventional lactose (commonly used for direct compression of tablets), namely, roller-dried (RD) anhydrous β-lactose. When tested in vitro with a single dose from a DPI device, this powder preparation forms a finely divided fraction (FPF) of at least 30% of the nominal dose, namely three times higher than in MDI cases. This approach can be used in combination with a fluoroquinolone antibiotic both for co-administration and for the introduction of fixed combinations in antibiotic therapy.

In addition to mucolytic activity, excessive neutrophil elastase activity in the airways in patients with cystic fibrosis (CF) leads to progressive lung damage. The destruction of disulfide bridges in elastase by reducing substances can alter its enzymatic activity. Three natural dithiol reduction systems were studied in terms of their effect on elastase activity: 1) the thioredoxin (Trx) system from Escherichia coli, 2) the recombinant human thioredoxin system (rhTrx), and 3) dihydrolipoic acid (DHLA). Trx systems consisted of Trx, Trx reductase and NADPH. As shown by spectrophotometric analysis of elastase activity, two Trx systems and DHLA inhibited both purified human neutrophil elastase and elastolytic activity present in the soluble phase (rast.) Of sputum CF. Removal of any of the three components of the Trx system prevented inhibition. Compared with monothiols N-acetylcysteine and reduced glutathione, dithiols showed greater inhibition of elastase. To improve Trx as a research tool, a stable reduced form of rhTrx was synthesized and used as the only component. Reconstituted rhTrx inhibited purified elastase and rust. sputum CF elastase in the absence of NADPH or Trx reductase. Since Trx and DHLA have a mucolytic effect, the authors investigated changes in elastase activity after mucolytic exposure. Untreated CF sputum was directly treated with reduced rhTrx, Trx system, DHLA, or DNase. The Trx system and DHLA did not increase elastase activity, while treatment with reduced rhTrx increased the activity of rast. 60% elastase. In contrast, elastase activity after treatment with DNase increased by 190%. The ability of Trx and DHLA to limit the activity of elastase in combination with their mucolytic effect makes these compounds potential therapeutic agents for CF.

In addition, clots of F-actin and DNA present in the sputum of patients with cystic fibrosis (CF), but absent in the liquid secretions in the normal airways, contribute to the altered viscoelastic properties of sputum, which slows the elimination of infected liquid secretions of the respiratory tract and exacerbates the pathology of CF. One way to alter such detrimental properties is to remove these filamentary clusters using DNase to enzymatically depolymerize DNA to constituent monomers, and gelsolin to separate F-actin into small fragments. High densities of negative surface charge on DNA and F-actin suggest that clumps of such filaments, which individually exhibit strong electrostatic repulsion, can be stabilized by polyvalent cations, such as histones, antimicrobial peptides and other positively charged molecules that predominate in liquid secretions of the respiratory tract. In addition, it was indeed observed that the DNA and F-actin clots formed after the addition of histone Hl or lysozyme are effectively dissolved by soluble polyvalent anions, such as polymeric aspartate or glutamate. The addition of polyaspartate or polyglutamate also decomposes DNA and actin-containing clots in CF sputum and reduces the elasticity coefficients of these samples to levels comparable to those obtained after treatment with DNase I or gelsolin. The addition of polyaspartic acid also increases DNase activity when added to samples containing DNA clots formed using histone Hl. When sputum is added to CF, polyaspartic acid significantly reduced bacterial growth, which suggests activation of endogenous antibacterial factors. These data suggest that soluble polyvalent anions are potentially capable, by themselves and in combination with other mucolytic substances, of selectively cleaving large clots of charged biopolymers formed in CF sputum.

Therefore, NAC, unfractionated heparin, reduced glutathione, dithiols, Trx, DHLA, other monothiols, DNase, dornase alpha, hypertonic drugs (for example, with an osmolality greater than about 350 mOsmol / kg), polyvalent anions such as polymeric aspartate or glutamate , glycosidases and other of the above samples can be combined with fluoroquinoline antibiotics and other mucolytic substances for aerosol administration in order to improve antibacterial activity by better distribution due to reduced sputum viscosity, as well as to improve the clinical result by improving lung function (due to improved sputum mobility and cleansing of the ciliated epithelium), as well as reducing lung tissue damage due to the immune inflammatory response.

EXAMPLES

The following examples serve to more fully describe the method of application of the above invention, as well as to formulate the best options provided for the implementation of various aspects of the present invention. Obviously, these examples in no way serve to limit the true scope of the present invention, but rather are presented for illustrative purposes. The full contents of all references cited herein are hereby incorporated by reference.

Example 1 - High local concentration with short-term exposure to aerosol fluoroquinolone

Aerosol administration of fluoroquinolones, such as levofloxacin, leads to high concentrations in the fluid of the epithelial lining (ELF) in rats and humans. However, according to observations, this dose is rapidly reduced after administration.

In order to determine whether short-term high concentrations of levofloxacin can be effective in the treatment of P. aeruginosa (PA), studies were conducted to measure their bactericidal activity against various strains of the organism that were grown under different conditions. They were selected based on what is known about the conditions and growth of PA in the lung with cystic fibrosis (CF). For these experiments, four isogenic strains of P. aeruginosa were used (Table 2).

table 2 PA strains used in lysis time experiments Strain Genotype MIC of levofloxacin (μg / ml) PAM 1020 dt 0.25 PAM 1032 nalB one

PAM 1020 - original wild-type strain, PAM l032 contains mutation nalB, which leads to increased resistance to levofloxacin at an elevated expression MexAB-OprM, encoding a discharge pump which is capable of outputting cells from levofloxacin.

Experiment 1. Levofloxacin activity against exponentially growing cells

Methods

Inoculum preparation

The strains were grown aerobically overnight in Mueller-Hinton broth (MHB) at 35 ° C. The cultures were then diluted 1: 1000 in 100 ml of fresh MHB and grown to OD ₆₀₀ ~ 0.3 to achieve CFU / ml ~ 10 ⁸ . 10 ml of this culture was transferred into 50 ml vials, each of which contained 10 ml of preheated MHB broth with levofloxacin in appropriate concentrations (2X compared to exposure concentrations).

Impact

All strains were treated for 10 min, 20 min, 40 min, 80 min and 160 min. The following levofloxacin concentrations (μg / ml) were used to affect PAM1020 and PAM1032: 16, 32, 64, 128, and 256. All strains were treated at each concentration for 10 min, 20 min, 40 min, 80 min, and 160 min.

Viable cell count

At appropriate time points, 1 ml of each exposed culture was centrifuged for 2 minutes, the pellet was washed twice with 1 ml of drug-free MHB and resuspended in 1 ml of MHB. The number of viable cells was counted by plating serially diluted samples (in duplicate) on Petri dishes with MHB by the method of dropwise (10 μl) seeding. The detection limit was 100 CFU / ml. Cell lysis is presented as the logarithm of the reduction calculated in relation to the number of cells at the time the antibiotic exposure begins. Used relative concentrations of the antibiotic (relative to the MIC of the respective strains). The number of cells at the time of exposure to the antibiotic are presented in table 3.

Table 3 The number of bacteria at the beginning of exposure to bacteria Strain CFU / ml PAM 1020 4.03E + 07 PAM 1032 5.60E + 07

results

For the most sensitive strain of PAMl020, maximum lysis (a decrease in the number of viable cells of 5.5 logarithmic units) was achieved after incubation for 10 minutes at a concentration of levofloxacin corresponding to 256-fold MIC (tested 64 μg / ml). Logarithms of lysis 5 were already achieved at the lowest concentrations tested (16 μg / ml or 64-fold MIC) (Fig. 4A). For strain PAM1032, until a concentration of greater than 128-fold MIC (128 μg / ml) was reached, a 10-minute exposure was sufficient to result in maximum lysis (more than 5 logarithmic units). With short-term exposure (10 or 20 minutes), less lysis was observed at concentrations below 128-fold MIC. At longer exposure times, concentrations corresponding to a 16-fold MIC and higher resulted in a similar lysis maximum (Fig. 4B). These results indicate that P. aeruginosa logarithmic cells are effectively lysed after short-term exposure to levofloxacin in high concentrations.

Experiment 2. Activity of levofloxacin against cells in the stationary phase

Methods

Inoculum preparation

The strains were grown aerobically overnight in Mueller-Hinton broth (MHB) at 35 ° C (350 ml total). The depleted medium was obtained after centrifugation of overnight cultures and filtration of the supernatant. The cultures were diluted to OD = 0.3 with depleted medium. The same medium was also used to obtain the antibiotic in various concentrations (as in experiment 1).

Impact

Antibiotic concentrations, exposure times and determination of the number of viable cells were the same as in experiment 1.

results

The number of cells at the time of initiation of antibiotic exposure are presented in table 4.

Table 4 The number of bacteria at the beginning of exposure to bacteria Strain CFU / ml PAM 1020 8,0E + 08 PAM 1032 8.50E + 08

For PAM l020 cells in the stationary phase, maximum lysis was observed at the lowest concentration corresponding to 64-fold MIC (16 μg / ml) and the shortest exposure time of 10 min (Fig. 5A). However, in the case of PAM 1032, a clear, dose-dependent lysis was observed with a maximum of lysis (4 log units) at 64 MIC concentrations for short-term exposure. The increase in exposure time did not lead to a higher degree of lysis. However, lower drug concentrations were required to achieve the same lysis with longer exposures (Fig. 5B).

Further, the authors compared the resumption of growth of PAM 1020 and PAM 1032 after treatment for either 10 or 160 minutes with levofloxacin in various concentrations. After appropriate treatments, the cells were washed twice with medium without antibiotic. 150 μl of cells were placed in a 96-well plate and growth was monitored continuously by measuring A ₆₆₀ with SpectraMax (Molecular Devices). The results are presented in figa-6D.

These results show that the growth resumption of both strains was observed at approximately the same time, whether the cells were treated with levofloxacin in high concentrations for 10 minutes or 160 minutes. These results further support the efficacy of short-term treatment with levofloxacin in high concentrations.

Experiment 3. The activity of levofloxacin against cells grown in conditions of oxygen deficiency

Methods

Inoculum preparation

Overnight cultures were aerobically grown overnight in Mueller-Hinton broth and then diluted 1: 10,000 in MBH, which filled the growth flasks to the top. The cultures were grown without shaking to OD ~ 0.3 at 37 ° C. Under these conditions, an average of ~ 20 hours was required to achieve OD = 0.3 compared to ~ 5 hours under aeration conditions (50 ml of medium in 250 ml flasks, vigorous shaking). The analysis found that OD = 0.3 corresponded to the late logarithmic phase of growth. With the exception of reduced aeration, the antibiotic concentration, exposure time, and quantification of viable cells were the same as in experiments 1 and 2.

results

The number of cells at the time of the beginning of exposure to the antibiotic are presented in table 5.

Table 5 The number of bacteria at the beginning of exposure to bacteria Strain CFU / ml PAM 1020 7.5E + 07 PAM 1032 8.5E + 07

In the case of PAM 1020, lysis close to maximum (4 log units versus 4.5 log units observed with normal aeration) was achieved after exposure to levofloxacin at the lowest concentration for the shortest duration (10 min) (Fig. 7A). In the case of PAM 1032, dose-dependent lysis was observed at 10 minutes or 20 minutes of exposure, with the highest lysis observed at concentrations corresponding to 128 to 256-fold MIC. A slightly stronger (difference of less than 1 logarithmic unit) lysis was observed for longer exposure intervals (FIG. 7B). These results show that in conditions of oxygen deficiency, cells in the late logarithmic growth phase are effectively lysed after short-term exposure to levofloxacin in high concentrations.

Experiment 4. The activity of levofloxacin against PAM 1032 in CF sputum

Methods

Cells of strain PAM 1032 (MIC = 1 μg / ml) were grown to OD = 1 (late exponential / early stationary growth phase) in MHB and then concentrated 10-fold in 10-fold concentrated MHB. 10 μl of cells was then added to 90 μl of sputum or water in 96-well round-bottom plates, restoring MHB to its original concentration. The plates for quantitative analysis were preheated for 5 min at 37 ° C, and levofloxacin was added at various concentrations (512 μg / ml, 128 μg / ml, 32 μg / ml, 8 μg / ml, 2 μg / ml and 0.5 μg / ml). At appropriate time points, 10 μl of each treated culture was diluted 100 times in MHB to minimize levofloxacin impurities. The number of viable cells was counted by plating in Petri dishes with MHB serially diluted samples by the method of drop (10 μl) seeding. The detection limit was 10 ⁴ CFU / ml. Cell lysis was presented as a percentage of the initial inoculum surviving after treatment with levofloxacin. The results are presented in figa and 8B.

results

The results show that although sputum had a slight effect on the level of levofloxacin lysis, nevertheless, fast and extensive (up to five orders of magnitude) lysis of levofloxacin in sputum was observed after short-term treatment at high antibiotic concentrations.

Experiment 4. The activity of levofloxacin against colonial biofilms PAM1032

Methods

Obtaining biofilms

Colonial biofilms were grown on polycarbonate membrane filters (25 mm diameter; Poretics, Livermore, CA), placed on Petri dishes with MBH. The overnight culture of PAM 1032 was diluted to OD = 0.3 and then diluted 1: 100 in fresh MBH. 5 μl of this culture was applied dropwise to a membrane filter. Bacteria were incubated at 37 ° C for 48 hours (mature biofilms).

Impact

After germination, the filters were placed in tubes containing 3 ml of saline or saline with levofloxacin at 128 μg / ml and 1024 μg / ml. Each tube was incubated for 10 minutes and 80 minutes. About 5 minutes before the end of the incubation time, the tubes were vigorously shaken (A) or sonicated and shaken (B) to separate the cells. 1 ml of each exposed culture was centrifuged for 2 minutes, the pellet was washed twice with 1 ml of MHB without drug and resuspended in 1 ml of MHB. The number of viable cells was counted by plating in Petri dishes with MHB serially diluted samples (in duplicate) by the method of drop (10 μl) seeding. The results are presented in Fig.9.

results

The data show that maximum lysis (~ 2 logarithmic units) is achieved after 10 minutes at the lowest concentration of levofloxacin tested (128-fold MIC). No additional lysis was observed at a higher concentration of levofloxacin. These data show that colonial biofilms are more resistant to lysis compared to cells in the logarithmic and stationary phase. However, the maximum observed bactericidal activity against biofilms (99% under these conditions) was reached after 10 min exposure to levofloxacin.

Experiment 5. Model short-term, quick aerosol administration, providing the effect of a highly concentrated drug on an in vitro pharmacodynamic model

Pharmacodynamic models of in vitro infection make it possible to expose a growing bacterial inoculum to the drug in varying concentrations, as it could in vivo. The advantage of this approach is that the profile of the dependence of the concentration of the drug in serum on time in the human body can be simulated in vitro in the laboratory to determine the optimal profile of exposure (i.e. dose and dosage interval) for the drug and the intended pathogen.

The following report describes experiments designed to determine C _max and AUC, which provide maximum bactericidal effects after an aerosol dose of fluoroquinolone.

Materials and methods

Pharmacodynamic model of in vitro infection

The in vitro pharmacodynamic model consists of a central (“serum”) compartment and a peripheral (“extravascular”) compartment. The peripheral compartments consist of artificial capillary units (Unisyn, Hopkinton, MA) connected in series with the central compartment. Each capillary unit has a bundle of small semipermeable fibers with a molecular size retention limit of 10,000 MW in order to ensure the passage of nutrients, but not bacteria. The entire system is housed in a dry incubator set to 37 ° C.

Both central and peripheral compartments were filled with Mueller-Hinton broth. Each peripheral compartment (capillary unit and tubes) contained 23 ml of growth medium.

Bacteria were introduced into the peripheral chamber of the model and left to grow for 2 hours before the first "dose" of the drug. Doses of the drug were introduced into the central compartment and pumped by a peristaltic pump to the peripheral chambers. Concentrations in the model were reduced according to the first-order exclusion law (half-life) by diluting the central compartment with a drug-free medium, which was added with an additional peristaltic pump tuned to the desired clearance rate.

Samples (0.3 ml) were collected from peripheral compartments at various time intervals to determine drug and bacterial concentrations. Samples were collected from peripheral compartments and analyzed for drug concentration by HPLC.

Test bacteria strains

Pseudomonas aeruginosa PAM 1032 and PAM 1582. MICs for levofloxacin in these strains were 1.0 and 32 μg / ml, respectively.

Inoculum preparation

The strains were aerobically grown overnight in Mueller-Hinton broth (MHB) at 35 ° C, reseeded in fresh MHB and re-incubated at 35 ° C for 2 hours. After 2 hours, the inoculum was further diluted 1: 1000 to a final concentration of about 1.0 · 10 ⁶ CFU / ml. From the final dilution, 2.3 ml was introduced into each peripheral chamber of hollow fiber bioreactors (Unisyn, Hopkinton, MA).

Pharmacokinetics

The half-life of levofloxacin was adjusted so that it was 10 min, which is equivalent to that observed after aerosol delivery of levofloxacin to the pulmonary region of a person. The target C _max was 1000 and 600 μg / ml in two experiments.

results

As planned, the model showed a half-life of levofloxacin of 10 minutes and a C _{max of} 1000 μg / ml in experiment 5. For comparison, experiment 6 was modified so as to achieve the same half-life as in experiment 5, but with a target C _{max of} 600 μg / ml

The bactericidal effects of these two regimens correlated with C _max . In experiment 5 with C _max 1000 μg / ml, the maximum bactericidal effect was observed as a decrease in bacterial count by 5 logarithmic units in 10 minutes in the case of PAM 1032 and a decrease in bacterial count by 4 logarithmic units in 20 minutes in the case of PAM 1582; and no growth resumption was observed during the remaining 2 hours of the experiment (Fig. 10). In contrast, while C _max 600 μg / ml used in experiment 6, the bacterial count decreased by 5 logarithmic units in the case of PAM 1032, although it took 30 minutes compared to the 10 minutes observed in experiment 1, only the decrease in bacterial scores for 3 logarithmic units were observed for PAM 1582 after 45 minutes (FIG. 11). Moreover, PAM 1582 showed initial growth resumption before the end of the 2-hour experimental window.

findings

Levofloxacin can lead to a decrease in the number of bacteria up to 99.9999% at C _{max of} both 600 and 1000 μg / ml in the case of a strain with MIC equal to 1 μg / ml. However, the maximum bactericidal activity requires 3 times more time at C _max 600 μg / ml. Levofloxacin can also lead to a decrease in the number of bacteria up to 99.99% at a C _{max of} 600 μg / ml in the case of a strain with an MIC of 32 μg / ml. However, the time to reach the maximum effect is 45 minutes. In contrast, levofloxacin can lead to a decrease in the number of bacteria up to 99.999% in this resistant strain at C _max 1000 μg / ml, and the time of the maximum effect is reduced to 20 minutes. Based on these results, the extremely high, but short-term effects of levofloxacin lead to rapid and continuous bacterial lysis both in the flask and in the hollow fiber model. Together, the above results indicate that reaching an initial concentration of 800 μg / ml of levofloxacin or other fluoroquinolone in human ELF or sputum is sufficient to achieve the above antibiotic effects for the MIC99 population, as shown by PAM 1582 (MIC 32 μg / ml).

Example 2 - Determination of the aerosol properties of antibacterial fluoroquinolones

Introduction

Purpose . The purpose of these studies was to evaluate the possibility of obtaining the drug and delivery by spraying a number of fluoroquinolones for the treatment of pulmonary bacterial infections by aerosol administration. Estimated fluoroquinolones are shown in table 6.

Table 6 Proven Fluoroquinolones Fluoroquinolone Sp MIC ₉₀
(mcg / ml) MSSA MIC ₉₀
(mcg / ml) MRSA MIC ₉₀
(mcg / ml) Pa MIC ₉₀
(mcg / ml) Official status ciprofloxacin 2 one 64 8 allowed hemifloxacin 0.06 0.06 2 8 allowed levofloxacin 2 0.5 16 8 allowed marbofloxacin 2 2 but 8 veterinary gatifloxacin 0.5 0.125 four 16 allowed ofloxacin 2 one > 32 16 allowed tosufloxacin 0.5 0.125 > 16 16 Japan lomefloxacin 16 2 > 32 32 allowed moxifloxacin 0.25 0.125 2 32 allowed sparfloxacin 0.5 0.125 16 32 forbidden orbifloxacin 2 2 but > 32 veterinary pefloxacin 32 2 > 32 > 32 Europe trovafloxacin 0.25 0.06 8 > 32 forbidden

These fluoroquinolones were selected based on their availability, official status, and antimicrobial properties. All tested fluoroquinolones are either currently approved for use in the United States, or were previously authorized, but were later banned due to various harmful reactions. In addition, some fluoroquinolones used in veterinary medicine were also evaluated. Among the pathogenic bacteria responsible for respiratory infections, the most resistant to treatment with fluoroquinolones are Pseudomonas aeruginosa (Pa) and methicillin-resistant Staphylococcus aureus (MRSA). Streptocossus pneumonia (Sp) is probably the most important pathogen responsible for respiratory tract infections, and many reports indicate a high degree of resistance to fluoroquinolones in these bacteria. MIC ₉₀ for Pa ranges from 4 μg / ml to 32 μg / ml and from 2 μg / ml to> 32 μg / ml for Pa and MRSA, respectively. Ciprofloxacin, levofloxacin, hemifloxacin and gatifloxacin are most effective against Pa, while against MRSA, hemifloxacin and moxifloxacin.

Table 7 contains a list of additional fluoroquinolones for potential evaluation. The most microbiologically interesting compounds on the list are clinafloxacin and olamufloxacin, which were discontinued due to harmful reactions, and sitofloxacin, which is in phase III of clinical trials.

Table 7 Fluoroquinolones for potential assessment Fluoroquinolone Sp
MIC ₉₀
(mcg / ml) Sa
MIC ₉₀
(mcg / ml) MRSA
MIC ₉₀
(mcg / ml) Pa
MIC ₉₀
(mcg / ml) Market status clinafloxacin 0.06 0.06 2 four discontinued sitafloxacin 0.06 0.125 four 8 phase III olamufloxacin 0.06 one 2 16 discontinued norfloxacin 16 one > 4 16 allowed prulifloxacin one 0.25 32 16 phase III danofloxacin NA 0.125 NA > 16 veterinary enrofloxacin one 0.125 8 > 16 veterinary sarafloxacin NA 0.25 > 16 > 16 veterinary halofloxacin 0.5 0.25 8 32 Korea fleroxacin 8 one > 4 32 Europe difloxacin 2 0.5 NA 32 veterinary rufloxacin 32 2 64 32 Europe, China enoxacin 16 one > 4 > 32 forbidden garenoxacin 0.06 0.06 8 > 32 phase III grefafloxacin 0.5 0.125 32 > 32 forbidden pazufloxacin four 0.5 > 16 > 32 Japan

The fluoroquinolones in these two tables represent one of the many options for selecting a candidate for aerosol fluoroquinolone. Some potent fluoroquinolones, such as DX-619 and DW-286, which are in the early stages of clinical development, would probably also be of interest for future studies.

Specific physical and chemical considerations for spraying include water solubility, viscosity, and surface tension. The solubility of the drug in water should preferably be sufficient to meet or exceed the minimum dosage requirement. The loading concentration of the drug also affects the delivery time. Longer delivery times may be commercially unacceptable or lead to poor patient adherence. Although longer delivery times may in fact alter the configuration of the AUC, as a non-limiting example, the PARI eFlow device was designed to administer 4 ml of aqueous levofloxacin in less than 5 minutes. Moreover, when using such an effective device, high concentrations of levofloxacin will allow delivering the effective doses described here in a time interval that further makes possible the rapid administration of the drug in the required high concentrations necessary for optimal fluoroquinolone therapy.

In the case of fluoroquinolones, pH directly affects solubility. As a rule, solubility decreases significantly with increasing pH in the range from 1.5 to 6.5. Since pH also affects patient tolerance (see below), the optimal choice of fluoroquinolone for delivery to the lungs by aerosol means certain levels of solubility and pH.

For the purpose of this study of technical applicability, the target solubility was established from 10 mg / ml or higher at a pH of 4.5 or higher, which is based on calculations of the therapeutic dose and delivery parameters of available nebulizers. To exceed the concentration preventing mutations (MPC), the peak concentration of fluoroquinolone after aerosol administration advantageously reaches from about 100 μg / ml to about 1000 μg / ml at the site of infection, approaching the MIC of the infecting organism. Based on these considerations, a minimum dose within this therapeutically appropriate range has been proposed such that it is at least about 30-40 mg of a respiratory delivered dose (RDD). Considering the relative half-life of levofloxacin in human lungs, the practical achievement of such a dose by spraying can be obtained with a loading dose of at least about 100 mg in a volume of about 2 ml (about 50 mg / ml) in a highly efficient vibrating cellular device operating with the most effective regimen and delivering this dose in less than 4 minutes. A standard ultrasonic or jet nebulizer may require a loading dose of at least about 400 mg in a volume of about 5 ml (about 80 mg / ml). However, the rate of administration of such less effective devices may not be sufficient to achieve high local concentrations with short exposure times. Similar effective doses can also be achieved by the administration of levofloxacin in the form of a dry powder, when the property of the fast solubility of levofloxacin can allow rapid dissolution, leading to the desired concentrations of the dissolved drug. However, other concentrations or changes in the AUC configuration of fluoroquinolones may be desirable.

Alternatively, although solubility in water is important, it makes sense to provide a preparation using the dispersion or complexation method to make it possible to spray less soluble fluoroquinolones. Unfortunately, more complex drugs increase both the complexity and the cost of developing a drug, and in the case of inkjet and ultrasonic nebulizers significantly reduce the delivery efficiency and limit the ability to introduce other graphic elements into the final drug product.

In addition to the solubility of the drug, in vibrating cellular devices, spraying is also sensitive to the surface tension of the drug. For this reason, in one embodiment, the surface tension is adjusted during preparation, modifying the concentration of the drug, the concentration of the excipient, and / or adding surfactant.

In addition to factors affecting spray efficiency, other factors affecting patient tolerance and treatment adherence can be considered. By way of non-limiting example, such factors may include osmolality, pH, and taste. Osmolality affects acute tolerance in the respiratory tract and can be optimized for most drugs when formulating drugs. Similarly, the pH of the aerosol also affects tolerance, most likely only negatively, when the pH of the drug is less than 4.5. For this reason, since pH directly contributes to the solubility of fluoroquinolone, fluoroquinolones, which require a pH of less than 4.5 for solubility, are apparently poorly tolerated. Finally, the taste of fluoroquinolones can affect patient adherence. As you know, fluoroquinolones usually have an unpleasant, sometimes very intense taste. Although there are affordable methods that mask the poor taste of the drug, such methods increase development complexity and cost, and in the case of fluoroquinolones may not be fully effective. Thus, similar to pH, taste can also be taken into account when identifying fluoroquinolones suitable for spraying.

Preparation and characterization of test solutions

Antibiotics were obtained from one of several sources presented in table 8.

Table 8 Preparation of fluoroquinolone test solutions No. Fluoroquinolone Source ^a Clean ^b Qty Volume H ₂ O The final concentrate. one gatifloxacin Lkt 99.6 8.7 mg 0.87 ml 10 mg / ml 2 hemifloxacin LG 99.6 9.5 mg 0.95 ml 10 mg / ml 3 levofloxacin Lkt 99,2 10.3 mg 1.03 ml 10 mg / ml four moxifloxacin Lkt 99.5 12.5 mg 1.25 ml 10 mg / ml 5 ciprofloxacin Lkt 99.3 19.5 mg 1.95 ml 10 mg / ml 6 ofloxacin Lkt 99.1 11.7 mg 1.17 ml 10 mg / ml 7 lomefloxacin MPI NA 17.0 mg 1.70 ml 10 mg / ml 8 marbofloxacin Vetoquino NA 4.8 mg 0.48 ml 10 mg / ml 9 orbifloxacin MPI NA 4.2 mg 0.42 ml 10 mg / ml 10 pefloxacin MPI NA 15.0 mg 1.50 ml 10 mg / ml eleven sparfloxacin MPI NA 14.5 mg 1.45 ml 10 mg / ml 12 tosufloxacin MPI NA 15.2 mg 1.52 ml 10 mg / ml 13 trovafloxacin MPI NA 2.0 mg 0.20 ml 10 mg / ml

a. LKT: LKT Laboratories. LG: LG Chem. NA. The source does not meet the requirements.

b. Checked the purity of the material. Characterized by GMP or percentage API.

c. The solution is 25 mg / ml.

From 2 to 20 mg of a sample of each antibiotic was weighed into sterile plastic tubes and volumes of sterile water were added to obtain 10 mg / ml antibiotic solutions or suspensions. Before further operations, the samples were incubated for approximately 10 minutes at room temperature and stirring occasionally.

Following the incubation period, antibiotic solutions were examined for their appearance, and the results are presented in table 9.

Five of the tested fluoroquinolones were soluble in appearance and were either colorless or yellowish in color. Eight were insoluble in appearance, being either turbid (finely dispersed), or opaque (dispersion from fine to medium density), or turbid (thick coarse suspension), and in all cases with a visible precipitate. The pH of these stock solutions was determined and ranged from 3.5 to 7.0. Insoluble solutions were titrated with 1 N. HCl to the point of apparent solubility and the pH of the solubilized solutions was determined. In three cases, marbofloxacin, sparfloxacin and tosufloxacin, even at pH 1.5, solubility was not achieved, and further acid addition was stopped. With the exception of ofloxacin, the pH of these titrated solutions ranged from 1.5 to 3.0.

Table 9 Characteristics of fluoroquinolone solutions Stock solution After adjusting the pH No. Fluoroquinolone Appearance pH 1 n HCl (μl) Appearance ^* pH one gatifloxacin white, cloudy, visible precipitate 7.0 5 yellowish, transparent, without sediment 3.0 2 hemifloxacin colorless, transparent, without precipitate 4.7 NT - 3 levofloxacin yellowish, transparent, without sediment 4.7 NT - four moxifloxacin bright yellow, transparent, no precipitate 4.7 NT - 5 ciprofloxacin white, opaque (very thick), visible sediment 5.5 60 colorless, transparent, without precipitate 2.0 6 ofloxacin cloudy, visible precipitate 6.5 10 yellowish, transparent, without sediment 5.2 7 lomefloxacin cloudy, visible precipitate 4.2 - transparent, without precipitate after 10 minutes at room temperature. temperature - 8 marbofloxacin white, very cloudy, visible precipitate 6.5 40 white, cloudy, visible precipitate 1,5 9 orbifloxacin white
cloudy, visible precipitate twenty colorless,
transparent, without sediment 1.7 10 pefloxacin colorless, transparent, without precipitate 4,5 NT - eleven sparfloxacin bright yellow, cloudy, visible precipitate 5,0 twenty bright yellow, densely cloudy, visible precipitate 1,5 12 tosufloxacin white, cloudy, visible precipitate 3,5 twenty white, cloudy, less cloudy, visible precipitate 1,5 13 trovaflok-satsin colorless, slightly cloudy, without sediment 4.2 NT -

a.NT: No pH adjustment required. Fluoroquinolone was soluble at pH ≥ 4 in the initial solution.

After adjusting the pH and following subsequent incubation for 10 minutes with stirring from time to time, the final appearance of the solutions was determined immediately before the tests for aerosol tolerance and taste. The results are presented in table 10.

Table 10 Appearance of final fluoroquinolone solutions No. Fluoroquinolone pH Solubility Color Sediment Turbidity one gatifloxacin 3.0 + Bts no no or very light 2 hemifloxacin + Bts no no or very light 3 levofloxacin 4.7 + Coolant no no four moxifloxacin 4.7 + F +/- no 5 ciprofloxacin 2.0 + Bts no no 6 ofloxacin 5.2 + SJ +/- no 7 lomefloxacin 4.2 + Bts +/- no or very light 8 marbofloxacin 1,5 - B ++ ++ 9 orbifloxacin 1.7 + Bts no light 10 pefloxacin 4,5 + Bts no light eleven sparfloxacin 1,5 --- TJ +++ ++++ 12 tosufloxacin 1,5 - B ++ +++ 13 trovafloxacin 4.2 + Bts + light

W = yellow; SJ = light yellow; Coolant = very light yellow; TJ = dark yellow; BC = colorless; B = white.

Compounds exhibiting preferred solubility for solutions suitable for inhalation delivery (10 mg / ml at pH 4.5 or higher) were levofloxacin, hemifloxacin, moxifloxacin, ofloxacin and pefloxacin. Levofloxacin, ofloxacin and moxifloxacin showed the best solubility / pH characteristics.

Assessment of taste and tolerance

In order to determine how fluoroquinolone solutions are suitable in terms of taste and tolerance, two evaluations were performed.

First, when testing for taste, the taste of a portion of 20 μl of the test sample was determined by one healthy volunteer, to whom the material was placed directly in the center of the front of the tongue. Taste was determined after 1 minute. This test was carried out both on the prepared stock solutions and on the final solutions, after adjusting the pH. The data are presented in table 11.

Table 11 Fluoroquinolone Oral Flavor Test No. Fluoroquinolone Stock solution Final solution one gatifloxacin mildly bitter unpleasant taste, slightly odorous strong unpleasant taste of bitter almonds, strong aftertaste 2 hemifloxacin very bitter unpleasant taste with a strong aftertaste, all the way to the throat did not 3 levofloxacin slightly chemical taste, slightly bitter, light almond flavor did not four moxifloxacin moderately bitter-sweet unpleasant taste, slightly odorous did not 5 ciprofloxacin sweet almond flavor very strong bitter taste all the way to the throat 6 ofloxacin unpleasant taste of bitter almonds moderately bitter unpleasant almond taste 7 lomefloxacin moderate or strong almond taste, not too unpleasant did not 8 marbofloxacin unpleasant taste of bitter almonds moderate or strong bitter unpleasant almond taste 9 orbifloxacin bad taste very strong, very unpleasant bitter taste 10 pefloxacin strong unpleasant taste of bitter almonds did not eleven sparfloxacin light taste strong almond flavor 12 tosufloxacin mild or moderate almond flavor strong almond flavor 13 trovafloxacin very strong unpleasant taste of bitter almonds, strong aftertaste Did not

Lowering the pH usually had the effect of enhancing the taste of the solution. Gatifloxacin, gemifloxacin, ciprofloxacin, orbifloxacin, and trovafloxacin were the least pleasant during taste testing. Of the fluoroquinolones tested, levofloxacin was the only fluoroquinolone that was tolerated in terms of taste at the tested concentrations. Lomefloxacin had a moderately strong almond flavor, and the taste was slightly unpleasant.

In the second test, the tolerance and taste of small aerosol samples from an aliquot of 0.5 ml of the test drug was determined by one healthy volunteer after spraying with a PARI eFlow device (table 12).

Table 12 Test for tolerance and taste of aerosol fluoroquinolones No. Fluoroquinolone Tolerance and taste of aerosol one gatifloxacin moderately bitter unpleasant taste, slight urge to cough 2 hemifloxacin strong unpleasant bitter taste, slight urge to cough 3 levofloxacin chemical taste, a little bitter, slight urge to cough four moxifloxacin moderately bitter unpleasant taste, slight cough, strong bitter aftertaste 5 ciprofloxacin very strong bitter unpleasant taste, instant cough 6 ofloxacin bitter chemical taste, slight urge to cough 7 lomefloxacin chemical taste, a little bitter, slight urge to cough 8 marbofloxacin too soluble for testing 9 orbifloxacin very acidic, strong bitter unpleasant taste, strong cough 10 pefloxacin chemical taste, slight cough eleven sparfloxacin too soluble for testing 12 tosufloxacin too soluble for testing 13 trovafloxacin bitter unpleasant taste, without coughing or the urge to cough, without aftertaste

In the case of orbifloxacin, marbofloxacin and trovafloxacin, smaller portions were tested due to the limited solubility. In a calibration experiment, the nebulizer produced aerosol with a vmd of 4.1 microns and a geometric standard deviation (GSD) of 1.64 microns vmd. In addition to these measurements, the inhaler produced a fraction of fine particles (FPD) equal to 54.9% (the percentage of the emitted dose in the form of particles is less than 5 microns). The tolerance and taste of the drug were tested over a very short period of administration and for a period of 10 minutes after administration. The tolerance parameters were of the following types: (i) coughing, coughing or sneezing; (ii) irritation, burning, or numbness in the throat; (ii) irritation or fluid discharge in the nasal ducts or eyes; (iii) irritation, burning or numbness in the lungs or shortness of breath; and (iv) dizziness, headache, nausea, or other systemic effects.

Marbofloxacin, sparfloxacin and tosufloxacin were too poorly soluble to be evaluated in this test. For the remaining fluoroquinolones tested, no intolerance effects were observed either during or after aerosol exposure in categories ii, iii or iv (see above). Gatifloxacin, moxifloxacin, ciprofloxacin, orbifloxacin and pefloxacin all caused a cough. In cases of ciprofloxacin and orbifloxacin, this could be due to the low pH of the solution. Of the fluoroquinolones tested, levofloxacin at 10 mg / ml had the best palatability. Ofloxacin, lomefloxacin and pefloxacin had a more distinct taste than levofloxacin, which was also acceptable during the short term administration.

Results and conclusions from the taste test of fluoroquinolones

Of the thirteen fluoroquinolones tested in this study, levofloxacin had the preferred physicochemical properties for aerosol administration and showed the best acute tolerance from the fluoroquinolones tested (Table 13). It is also recognized that levofloxacin has one of the best antimicrobial profiles for respiratory pathogens and has the highest in vivo efficacy comparable to ciprofloxacin for the treatment of Pseudomonas aeruginosa infections.

Table 13 General sprayability No. Fluoroquinolone Analysis Overall rating Limitation one gatifloxacin poor solubility and pH, moderately strong bitter taste of aerosol - solubility, taste 2 hemifloxacin sufficient solubility and pH, strong bitter taste of the aerosol, strong aftertaste --- taste 3 levofloxacin excellent solubility and pH, chemical taste of the aerosol, slightly bitter + taste four moxifloxacin sufficient solubility and pH, moderately strong bitter taste of the aerosol, strong aftertaste - taste, activity to RA 5 ciprofloxacin poor solubility and pH, very strong bitter taste of aerosol, cough --- solubility, taste 6 ofloxacin minimum acceptable solubility and pH, bitter chemical taste of aerosol - / + taste 7 lomefloxacin minimum acceptable solubility and pH, chemical taste of the aerosol, strong taste of the liquid - / + activity to RA 8 marbofloxacin very poor solubility even at low pH, inability to test --- solubility 9 orbifloxacin very poor solubility even at low pH, strong bitter unpleasant taste of the aerosol, severe cough --- solubility, taste, activity to RA 10 pefloxacin sufficient solubility and pH, chemical taste of the aerosol, strong unpleasant taste of the liquid - / + activity to RA eleven sparfloxacin very poor solubility even at low pH, inability to test --- solubility, activity to RA 12 tosufloxacin very poor solubility even at low pH, inability to test --- solubility 13 trovafloxacin moderate solubility and pH, bitter taste of aerosol - taste, activity to RA

Ofloxacin, lomefloxacin and pefloxacin showed lower solubility and stronger taste at 10 mg / ml than levofloxacin. Ofloxacin is 2 times less effective than levofloxacin, and lomefloxacin and pefloxacin are 4 times less effective. Higher concentrations of these antibiotics are preferred and have an administration time of less than 15 minutes.

In a separate study conducted in a similar way, norfloxacin was tested and found to have solubility, taste, and efficacy profile very similar to those of gatifloxacin, with the exception of significantly less activity against gram-positive pathogens.

Taste tests of aerosol salt preparations of levofloxacin and hemifloxacin.

Based on the results of the above studies, levofloxacin and its racemate, ofloxacin, as well as gemifloxacin and, to a lesser extent, gatifloxacin and norfloxacin are suitable for aerosol administration during antimicrobial treatment of the lungs. For further testing of the taste and acute tolerance properties (urge to cough and cough) of levofloxacin and hemifloxacin, several preparations with various organic and inorganic acids were obtained and checked by the method described previously. Solutions were prepared by first adding 500 mg of levofloxacin to 10 ml of water, or adding 500 mg of hemifloxacin to 20 ml of physiological saline (due to limited solubility), titrating with HCl or organic acid to a pH of ~ 6.5, then adjusting the osmolality of solutions containing levofloxacin up to ~ 300 mosmol / kg sodium chloride. The tested preparations are presented in table 14.

These drugs were tested on three healthy volunteers in the same manner as described above, with a concentration of levofloxacin of 50 mg / ml and a concentration of hemifloxacin of 25 mg / ml in a carefully controlled, full-time, completely blind test. The results are presented in tables 15 and 16.

These results demonstrate that levofloxacin preparations based on hydrochloric acid, citric acid and ascorbic acid have excellent taste and tolerance compared to levofloxacin preparations based on acetic acid, lactic acid and tartaric acid. Moreover, these levofloxacin preparations have superior taste and tolerance compared to similar hemifloxacin preparations. Regarding hemifloxacin, a citric acid preparation had an excellent taste and tolerance compared to hemofloxacin preparations based on HCl and ascorbic acid and could be used for aerosol administration with further improvement of the preparation.

Table 14 Levofloxacin and hemifloxacin preparations Fluoroquinolone Acid Conc. (mg / ml) pH Osmolality (mosm / kg) levofloxacin Hcl fifty 6.5 181 levofloxacin vinegar fifty 6.41 273 levofloxacin lemon fifty 6.45 286 levofloxacin dairy fifty 6.42 286 levofloxacin ascorbic fifty 6.50 278 levofloxacin wine fifty 6.35 286 hemifloxacin Hcl 25 5,6 330 hemifloxacin lemon 25 5.7 363 hemifloxacin ascorbic 25 5.9 347

Table 15 Taste and tolerance of levofloxacin preparations at 50 mg / ml Tester Acid one 2 3 Hcl moderately bitter taste bitter taste, coughing bitter taste acetic acid very acidic taste strong acid taste, coughing acid taste, aftertaste lemon acid soft finish, slightly sweet soft taste, sweet soft finish lactic acid strong bitter taste mild taste, aftertaste, mild cough bitter, soft finish vitamin C mild flavor, slightly acidic soft taste weak taste or aftertaste wine acid very bitter, strong aftertaste strong bitter taste, bitter aftertaste bitter taste

Table 16 Taste and tolerance to hemifloxacin preparations at 25 mg / ml. Tester Acid one 2 3 hydrochloric acid metallic taste, strong aftertaste slightly bitter taste bitter taste, slightly metallic lemon acid slightly sweet mild cough, slightly bitter very mild taste, without aftertaste vitamin C soft taste cough, soft bitter aftertaste slightly bitter, soft finish

Tests of the taste of additional aerosol preparations of levofloxacin

For further testing in a systematic manner, the taste properties and tolerability of additional combinations of excipients to levofloxacin were obtained and tested a number of drugs. The preparations are shown in table 17. They included sugars, salts, sweeteners and other excipients obtained by mixing levofloxacin with water, adding the excipients shown in table 17, and titration, if necessary, to the desired pH with dilute HCl. Osmolality was not optimized for these studies. However, osmolality was determined with an Advanced Instruments Model 3250 osmometer. This measurement, performed on 250 μl samples, is based on the determination of osmolality by lowering the freezing temperature.

These drugs were tested in total on three healthy volunteers in a series of tests (A-G) in the same manner as described above, in a thoroughly verified, full-time, completely blind way. All tests were performed in a completely blind manner. The test results (tables 19-25) are shown below. The following scoring system was used (table 18).

Test A: Tests of the tastes of sweeteners. Bivalent metal salts and surfactants. This test included sweeteners, calcium and magnesium salts, as well as surfactants (i.e. glycerin and PS-80). As shown in table 17, preparations containing these sweeteners had a mild bitter and metallic taste. Artificial sweeteners seem to give a bitter taste that is different from the bitterness observed in other cases. Most importantly, the preparation containing CaCl ₂ had the most improved taste compared to the control (MgCl _{2 was} not checked in these experiments) (table 19).

Test B: Tests of the tastes of mono- and disaccharides in the presence of calcium chloride. All drugs tested in this experiment were well tolerated and tasted better than the control sample. Preparations containing both calcium salt and sugar were better than containing anything separately, which suggests that these compounds improve the taste by various mechanisms. Of these drugs, the best was 5% CaCl ₂ + 7.5% glucose. It should be noted that lactose is present in concentrations lower than other sugars (table 20).

Table 17 Levofloxacin preparations containing various excipients Fluoroquinolone Conc. (mg / ml) Fillers pH Osmolality (mosmol / kg) Levofloxacin fifty Control A (0.225% NaCl) 6.50 180 Levofloxacin fifty Aspartame (0.1%) 6.49 175 Levofloxacin fifty Sucrulose (0.1%) 6.49 178 Levofloxacin fifty Glucose (5%) 6.5 380 Levofloxacin fifty Sucrose (7.5%); NaCl (0.225%) 6.51 329 Levofloxacin fifty Glycerin (5%) 6.48 880 Levofloxacin fifty PS-80 (0.1%) 6.51 189 Levofloxacin fifty CaCl ₂ (5%) 6.10 784 Levofloxacin fifty MgSO ₄ (5%) 6.41 73 Levofloxacin fifty Control B-E (0.225% NaCl) 6.51 182 Levofloxacin fifty CaCl ₂ (5%) 6.1 735 Levofloxacin fifty CaCl ₂ (5%), Sucrose (7.5%) 6.10 958 Levofloxacin fifty CaCl ₂ (5%), Glucose (7.5%) 6.10 1174 Levofloxacin fifty CaCl ₂ (5%), Glucose (7.5%) 5.25 1246 Levofloxacin fifty CaCl ₂ (5%), Lactose (7.5%) 6.07 864 Levofloxacin fifty MgCl ₂ (5%) 5.90 600 Levofloxacin fifty MgCl ₂ (5%), Sucrose (7.5%) 5.98 815 Levofloxacin fifty MgCl ₂ (5%), Glucose (7.5%) 5.98 999 Levofloxacin fifty MgCl ₂ (5%), Glucose (7.5%) 5.04 1035 Levofloxacin fifty MgCl ₂ (5%), Lactose (7.5%) 5.96 697 Levofloxacin fifty MgSO ₄ (5%), Sucrose (7.5%) 6.20 433 Levofloxacin fifty MgSO ₄ (5%), Glucose (7.5%) 6.21 625 Levofloxacin fifty MgSO ₄ (5%), Glucose (7.5%) 5.40 660 Levofloxacin fifty MgSO ₄ (5%), Lactose (7.5%) 6.18 387 Levofloxacin fifty Control F-G (0.45% NaCl) 6.5 221 Levofloxacin fifty Glucose (5%) 6.5 376 Levofloxacin fifty Sucrose (5%) 6.5 240 Levofloxacin fifty Lactose (5%) 6.62 241 Levofloxacin fifty Lactose (2.5%) 6.55 170 Levofloxacin fifty CaCl ₂ (5%) 6.10 735 Levofloxacin fifty CaCl ₂ (5%), Lactose (5%) 6.21 1037 Levofloxacin fifty CaCl ₂ (2.5%), Lactose (5%) 6.36 565 Levofloxacin fifty CaCl ₂ (2.5%), Lactose (2.5%) 6.41 370 Levofloxacin fifty CaCl ₂ (1.25%), Lactose (2.5%) 6.64 227 Levofloxacin fifty CaCl ₂ (0.625%), Lactose (2.5%) 6.06 163

Table 18 Flavor test score system Points Taste Portability one comparable to saline no coughing, no coughing 1.25 slightly more palpable taste than saline mild coughing, without coughing 1,5 soft bitter / metallic taste urge to cough, mild cough 1.75 between 1.5 and 2 - 2 moderate bitter / metallic taste urge to cough, moderate cough 2.25 between 2 and 2.5 - 2.5 strong bitter / metallic taste - 2.75 between 2.5 and 3 - 3 very strong bitter / metallic taste coughing and coughing four very strong bitter / metallic taste and other unacceptable taste urge to cough, severe cough and other irritation

Table 19 Taste and tolerance of levofloxacin preparations containing sweeteners, divalent metal salts and surfactant Fillers Tester one 2 3 Average Taste Migration Taste Migration Taste Migration Taste Migration Aspartame (0.1%) 2 1.25 2 one 2 one 2 one Sucrulose (0.1%) 2 one 1.75 one 2 one 2 one Sucrose (7.5%); NaCl (0.225%) 2 one 2.25 one 2 one 2 one Glucose (5%) 1,5 2 2.5 one 2 one 2 one Glycerin (5%) 2.25 one 2.25 one 2.5 one 2,3 one PS-80 (0.1%) 1.75 one 2.25 one 2.5 one 2,3 one CaCl ₂ (5%) 1.25 one 1,5 1,5 2 one 1,5 one MgSO ₄ (5%) 1,5 1,5 2.5 2.5 2.5 one 2.5 1,5 Control-A (0.225% NaCl) 3 one 3 one 2.5 one 3 one

Table 20 Taste and tolerance of levofloxacin preparations with CaCl ₂ Fillers Tester one 2 3 Average Taste Migration Taste Migration Taste Migration Taste Migration CaCl ₂ (5%) 1.75 one 2 one 2.75 one 2 one Sucrose (5%) 2 one 2 one 2 one 2 one CaCl ₂ (5%) ₂ , Sucrose (7.5%) 1.75 one 1.75 one 1,5 one 1.8 one CaCl ₂ (5%), Glucose (7.5%) 1,5 one 1,5 one 2 one 1,5 one CaCl ₂ (5%), Lactose (5%) one one 1.75 one 2 one 1.8 one Control B-E (0.225% NaCl) 3 one 2.5 one 3 one 3 one

Test C: Tests of the tastes of mono- and disaccharides in the presence of magnesium chloride. Like the above, all drugs tested in this experiment were well tolerated and tasted better than the control sample. Preparations containing magnesium salt and lactose, apparently, were slightly better than containing anything separately. This experiment confirms that the combination of divalent metal salts and simple sugars are effective in correcting the taste (table 21).

Table 21 Taste and tolerance of levofloxacin preparations with MgCl ₂ Fillers Tester one 2 3 Average Taste Migration Taste Migration Taste Migration Taste Migration MgCl ₂ (5%) 1,5 one - - 1.75 one 1,6 one MgCl ₂ (5%), Sucrose (7.5%) 1,5 one 1.75 one 2 one 1.8 one MgCl ₂ (5%), Glucose (7.5%) 1.25 one 2.25 one 2 one 2 one MgCl ₂ (5%), Lactose (5%) one one 1,5 one 1,5 one 1,5 one Control B-E (0.225% NaCl) 2.25 one - - 2.75 one 2.5 one

Test D: Tests of the tastes of mono- and disaccharides in the presence of magnesium sulfate. As with calcium and magnesium chlorides, formulations containing magnesium sulfate and glucose, sucrose or lactose had a better taste than the control sample. This experiment reaffirms that the combination of divalent metal salts and simple sugars masks the taste (table 22).

Table 22 Taste and tolerance of levofloxacin with MgSO ₄ Fillers Tester one 2 3 Average Taste Migration Taste Migration Taste Migration Taste Migration MgSO ₄ Sucrose 1,5 2 1,5 1.25 1,5 one 1,5 1.3 MgSO ₄ Glucose 1,5 2.75 2 2.25 1,5 1,5 1,5 2.5 MgSO ₄ Lactose 1.25 2.25 1.75 1.25 1.75 one 1.8 1.3 Control B-E (0.225% NaCl) 2.25 one - - 3 one 2.6 one

Test E: Tests of the tastes of divalent metal salts in the presence of glucose at low and high pH. In this experiment, the effect of glucose in combination with each of the three salts of divalent cations was tested for taste and tolerance at low (≤5.5) and high (≥6.0) pH. Small but significant corrections of tastes were noted at higher pH (table 23).

Table 23 Taste and tolerance of levofloxacin preparations with CaCl ₂ at low compared with high pH values Fillers Tester one 2 3 Average Taste Migration Taste Migration Taste Migration Taste Migration CaCl ₂ (5%), Glucose (7.5%), pH 6.1 one one 1,5 one 2 one 1,5 one CaCl ₂ (5%), 1.25 one 1.75 one 2.5 one 1.8 one Glucose (7.5%), pH 5.5 MgCl ₂ (5%), Glucose (7.5%), pH 6.0 1.25 one 2 one 2 one 2 one MgCl ₂ (5%), Glucose (7.5%), pH 5.0 1.75 one 1.75 one 1,5 one 1.8 one MgSO ₄ (5%), Glucose (7.5%), pH 6.2 1.25 2.25 2.25 1.75 1,5 one 1,5 1.8 MgSO ₄ (5%), Glucose (7.5%), pH 5.4 1,5 1.75 1.75 1,5 2 one 1.8 1,5 Control B-E (0.225% NaCl) 2 one - - - - - -

Test F. Tests of the tastes of mono- and disaccharides. All drugs tested in this experiment were well tolerated and had a better taste than the control sample. All three sugars at 5% were better than control, lactose at 2.5% tasted better than control, but not as good as at 5%. This experiment again confirms that simple sugars mask the taste (table 24).

Table 24 Taste and tolerance of levofloxacin with sugars Fillers Tester one 3 Average Taste Migration Taste Migration Taste Migration Glucose (5%) 1,5 1,5 2 one 1.8 1.3 Sucrose (5%) 1,5 1,5 1,5 one 1,5 1.3 Lactose (5%) 1.75 1.25 2 one 1.9 1,1 Lactose (2.5%) 2.25 1,5 2 2.1 1.3 Control F-G (0.45% NaCl) 2.5 one 2.5 one 2.5 one

Test G. Taste and tolerance of levofloxacin preparations with CaCl ₂ in the presence of lactose. In this experiment, levofloxacin was combined with calcium chloride and lactose in various concentrations (table 25). As noted in this series of experiments, all preparations containing divalent metal salts and sugar were more advanced in terms of taste and tolerance compared to the control drug. Most importantly, 5% calcium chloride or 2.5% calcium chloride in the presence of 5% lactose was most effective in reducing the bitterness of levofloxacin. A further decrease in the concentration of these excipients was less effective.

Table 25 Taste and tolerance of levofloxacin preparations with CaCl ₂ in the presence of lactose Fillers Tester one 3 Average Taste Migration Taste Migration Taste Migration CaCl ₂ (5%) 1.25 one 1,5 one 1.4 one CaCl ₂ (5%), Lactose (5%) 1.25 one 2 one 1,6 one CaCl ₂ (2.5%), Lactose (5%) 1.25 one 2 one 1,6 one CaCl ₂ (2.5%), Lactose (2.5%) 1,5 one 2.5 one 2 one CaCl ₂ (1.25%), Lactose (2.5%) 1.75 one 2 one 1.9 one CaCl ₂ (0.625%), Lactose (2.5%) 1.75 1.25 2 one 1.9 1,1 Control F-G (0.45% NaCl) 3 one 2.5 one 2,8 one

Example 3 - Characteristics of aerosol levofloxacin in a spray nozzle PARI LC Plus.

The following studies describe the possibility of aerosol delivery of levofloxacin when administered to a patient using a jet nebulizer. To accomplish this task, a simple preparation of levofloxacin was obtained, and the aerosol was characterized in a jet nebulizer. The results of these studies are summarized below.

Levofloxacin inhalation solution (55 mg / ml) was evaluated using a PARI LC Plus air jet nebulizer with a ProNeb compressor. The emitted dose, particle size distribution, and fine particle fraction were measured in cascade compression using a Marple-Miller impactor. The above parameters were used to evaluate in vitro aerosol drug performance.

Marple-Miller studies.

Goal. Determine the particle size distribution and estimate the amount of drug that the patient is able to inhale (respirable fraction). The secondary goal was to evaluate the emitted dose, which is the amount of levofloxacin exiting the nebulizer.

Methods Preparation: 55 mg / ml levofloxacin, 120 mM chloride, 70 mM sodium, pH = 6.7. The drug is formulated on the basis of maximum solubility, allowing you to get a 300 mg dose in 6 ml at a neutral pH. 5.5 ml of levofloxacin was added to a PARI LC Plus air-jet nebulizer with a ProNeb compressor. The nebulizer beaker contained a total of 302 mg of levofloxacin. The atomizer was connected in series with a Marple Miller Impactor (MMI), which operated at an air flow rate of 60 l / min. Each atomizer (n = 2) was produced to dryness (no aerosol was produced, which was confirmed by visual inspection for 15 minutes). After aerosolization, the MMI was disconnected and levofloxacin was quantitatively extracted with the mobile phase (90/10 ACN: water) from the USP inlet, each of the collector impactor cups (steps), and the fiberglass filter. Any amount of drug remaining in the nebulizer after aerosolization (in a glass and mouthpiece) was also quantified.

results

As shown in table 26, the total average amount recovered after the MMI experiments was 170.2 mg. The expected recovery was 302 mg. This corresponds to a total recovery of ~ 57%, which does not correspond to generally accepted specifications for studies using impactors (85% - 115% of the total recovery). This difference was found to be due to the non-specific adhesion of levofloxacin to the LC Plus spray device. The average percentage of drug released from the atomizer in the form of fine particles was ~ 72%. Thus, the inhaled emitted dose was 89.7 mg. Assuming that ~ 50% is not inhaled during normal intermittent breathing, only ~ 40 mg is deposited in the lungs from a 300 mg dose. However, taking into account the slow administration times for this device, competition with lung clearance will more likely prevent the accumulation of a sufficient amount of levofloxacin, which meets the minimum concentration requirement for dosage according to the principle of “fast administration, high concentration”, which is required for maximum antimicrobial activity of fluoroquinolones and prevent resistance .

Table 26 Marple Miller impactor dataset Sample Name BUT
Issued dose (mg) AT
Amount of drug remaining in a glass of nebulizer (mg) A + B
Amount of drug recovered
(mg) Percentage of the total amount of drug discharged from the nebulizer in the form of a fraction of fine particles (% <5 μm) left run 1 134.70 45.00 179.70 73.5 left run 2 114.40 46.30 160.70 70,4 average 124.6 45.7 170,2 72.0

Example 4 - Animal models and evaluation of fluoroquinolones and fluoroquinolone preparations.

Pharmacokinetic model

Six rats in each study were given a single slow bolus intravenous injection of a dose of 10 mg / kg through the lateral tail vein, or a single micro-spray aerosol dose of 10 mg / kg was administered using a micro-spray device for aerosol generation (PennCentury, Philadelphia, RA). Blood samples were taken at various time intervals for 3 hours to determine the pharmacokinetic parameters of plasma. Two rats were sacrificed 0.5, 1.5, and 3 hours after dosing to determine levels in the lungs, broncho-alveolar lavage (BAL), and epithelial lining fluid (ELF). Plasma and tissue concentrations were determined by HPLC, and then the data were assembled using WinNonlin software. The data are presented in table 27.

Performance model

P. aeruginosa strain PAM 1723 was grown in Mueller-Hinton broth (MHB) at 35 ° C with constant aeration, after 16 hours the inoculum was recultivated into fresh MHB and re-grown at 35 ° C with constant aeration for 4 hours. The inoculum was fed up to approximately 5 · 10 ⁶ CFU / ml, correlating the absorbance at 600 nm with the previously calculated Petri dishes. Male CFW mice (4-6 weeks old, N = 4 / group) were neutropenically injected into the abdominal cavity with 150 mg / kg cyclophosphamide (Cytoxan, Mead Johnson, Princeton, NJ) on days 1 and 4. On the 5th day, the mouse was infected by intratracheal instillation of 0.05 ml of inoculum under isoflurane anesthesia (5% isoflurane in an oxygen flow of 4 l / min). Two hours after infection, mice were injected either intraperitoneally or intratracheally with a dose of each fluoroquinolone at a dosage of 25 mg / kg. Mice were sacrificed 1 and 4 hours after treatment, their lungs were removed, homogenized, and placed on Petri dishes to count the colonies. The data are shown in table 28.

Table 27 Pharmacokinetic modeling Medicine Way Dose (mg / kg) AUC serum (0-inf) Serum and mouth t 1/2 ELF AUC (0.5-3 h) F ,% of pulmonary versus iv levofloxacin in / in 10 3.8 0.5 10.5 NA levofloxacin and / t 10 3.28 0.4 12.07 86% ciprofloxacin in / in 10 2,56 0.53 Nd NA ciprofloxacin and / t 3.3 0.8 0.93 194 82% clinafloxacin and / t 10 3.2 0.74 30.8 gatifloxacin in / in 10 5.31 1.06 5.32 gatifloxacin and / t 10 5.83 1.13 54.7 one hundred% norfloxacin in / in 10 4.65 1.21 3.27 norfloxacin and / t 10 4.46 1.13 41.7 one hundred% hemifloxacin in / in 8 4,54 1,04 3.72 hemifloxacin and / t 10 5.86 1.68 536.5 86% tobramycin in / in 10 15.7 0.5 27.6 NA tobramycin and / t 10 13.82 1,0 5152.0 81%

In pharmacokinetic studies in rats, aerosol administration of fluoroquinolones leads to increased ELF AUC in the range from 0.5 to 3 hours for all tested fluoroquinolones, as well as tobramycin, indicating that the aerosol route of administration is highly effective against pulmonary infections.

In murine models of pulmonary infections, increased efficacy has been confirmed, based on pharmacokinetic studies of rats. For all fluoroquinolones tested, the aerosol route of administration (intratracheal and / and / t) led to a greater decrease in the number of bacteria than the intraperitoneal (ip) route of administration, indicating that the observed increased efficiency is due to the high local concentrations resulting from direct aerosol administration.

Table 28 Performance Modeling Medicine Way Dose (mg / kg) Delta log CFU 1 h ^b Delta log CFU 4 h ^b Levofloxacin in / b 25 -1.00 -0.52 Levofloxacin and / t 25 -1.97 -1.28 Hemifloxacin in / b 25 -0.28 -0.32 Hemifloxacin and / t 25 -2.45 -1.81 Levofloxacin in / b 25 -1.40 -1.14 Levofloxacin and / t 25 -2.48 -1.45 Hemifloxacin in / b 25 -0.74 -0.71 Hemifloxacin and / t 25 -3.20 -2.28 Clinafloxacin in / b 25 -1.32 -1.33 Clinafloxacin and / t 25 -2.86 -2.47 Tobramycin in / b 5 -0.70 0.29 Tobramycin and / t 5 -1.59 -0.94 Ciprofloxacin in / b 25 -1.59 -0.41 Ciprofloxacin and / t 25 -2.32 -1.45 Gatifloxacin in / b 25 -0.34 -0.02 Gatifloxacin and / t 25 -1.48 -2.11 Clinafloxacin in / b 10 -0.96 -1.39 Clinafloxacin and / t 10 -2.71 -2.40 Sparfloxacin in / b 25 -0.85 0.09 Sparfloxacin and / t 25 -1.56 -0.81 Tosufloxacin in / b 25 0.00 1.33 Tosufloxacin and / t 25 -0.48 -0.24 a. Method of drug administration
b. Time after drug administration

Example 5 - Characterization of aerosol levofloxacin in a nebulizer PARI eFIow

Laser measurement of particle size distribution

Productivity of the device was characterized by measuring the size of the emitted particles. By way of non-limiting example, particle size distribution of the emitted levofloxacin solution can be measured using a Malvern Spraytec particle size meter under the following conditions. Environmental conditions were controlled to maintain room temperature between 23 ° C and 24 ° C, and relative humidity from 42% to 45%. Levofloxacin at 25 mg / ml was loaded into two PARI eFlow nebulizers equipped with 40 spray heads. The Malvern Spraytec particle size meter software was used to calculate the following information: A) volume mean diameter (VMD), is the average volume of particles crossing the laser beam; B) geometric standard deviation (GSD), - a diameter of 84 ^th percentile / diameter 50 ^th percentile; C)% of particles ≤5 microns, percentage of particles less than 5 microns or% of particles> 1 microns and <7 microns, percentage of particles between 1 and 7 microns.

The device was loaded with 2 ml of levofloxacin at 25 mg / ml. The mouthpiece of the device was placed so that the mouthpiece tip was 2 cm from the center of the beam along the X axis and as close as possible to the laser optical lens along the Y axis. When conditioning the environment, an oblique flow through the spray gun was provided in an amount sufficient to obtain a total spray flow 20 l / min The spray gun was turned on and allowed to operate continuously for 1 minute before measurement. The measurement sequence began after 1 minute, and the measurements were carried out continuously for 1 minute at intervals of 1 second. At the end of the measurement phase, data from 60 records were averaged over VMD, GSD and% ≤5 microns, as well as%> 1 and <7 microns. At the end, the atomizer was weighed to determine the degree of yield.

Research by simulating breathing

The performance of the device was measured under conditions similar to natural breathing using a PARI Compas Breath Simulator, programmed in accordance with the European standard for a frequency of 15 breaths per minute with an inspiratory / expiratory ratio of 1: 1. Such measurements were carried out under external conditions that can be controlled to maintain room temperature from 23 ° C to 24 ° C and relative humidity from 42% to 45%. For this experiment, a PARI eFlow device was loaded with 4 ml of levofloxacin solution at 25 mg / ml.

We started the simulation of breathing and turned on the nebulizers. The devices were allowed to operate continuously until spraying ceased. Duration was measured from the time the spray started. After spraying, the inhalation and exhalation filters were individually washed with a known amount of solvent (distilled H ₂ O). The spray cup was also washed separately. For quantification, individual washings were analyzed spectrophotometrically at a wavelength of 290 nm, and the final concentration was recounted for the content. Using these quantitative data, the following analysis was performed. A) respirable dose (ID), the total amount of drug detected on the inhalation filter. B) residual dose (RD), the amount of drug detected in the nebulizer after spraying is complete. C) fine particle dose (FPD), ID multiplied by the respirable fraction (e.g.% particles ≤5 microns VMD, depending on the method used to determine the particle sizes emitted by the selected device). D) duration, time from start to finish spraying. E) respiratory delivered dose (RDD),% ID, which is, for example, ≤5 microns VMD.

The results in table 29 show that out of a 100 mg dose of levofloxacin, it appears that ~ 34 mg of fluoroquinolone is deposited in the pulmonary region in ~ 4 min using the PARI eFlow device (table 29) compared to a 300 mg dose from the PARI LC Plus device delivering an equivalent dose in> 15 min. Based on the “fast administration, high concentration” dosage principle and the delivery model described here, since the delivery time of 15 minutes with the LC Plus seems to be unsuitable, a 4-minute delivery time of 35-40 mg of levofloxacin may satisfy the criteria for maximum fluoroquinolone activity. However, increasing the concentration of the drug to enable faster administration (for example, 50 mg / ml at a dosage of 2 ml, delivering 35-40 mg of levofloxacin in ~ 2 min), even more satisfies these minimum requirements. In addition, shorter administration times improve patient adherence to dosing. In addition, it should be noted that hypotonic solutions of levofloxacin at concentrations higher than 10 mg / ml are poorly tolerated by inhalation.

Table 29 Properties of the aerosol levofloxacin (loaded dose - 100 mg) FPD (%) RDD (mg) VMD GSD Osmo

Duration (minutes) Residual dose Inhaled dose ≤5 mk 1-7 microns ≤5 mk 1-7 microns μm μm mos / kg 3.9 ± 0.1 24.8 ± 3.4 61.1 ± 1.6 54.9 73.8 33.5 45.1 4.7 1,6 67 ± 1,0

Example 6 - Tolerance to the aerosol of levofloxacin by a healthy person. Methods

On one person, a healthy volunteer, the applicability of aerosol delivery of levofloxacin was established either using the Aerogen Clinical vibrating mesh device, which created particles with a volume mean diameter (VMD) of 3.4 microns, or with MMAD ~ 2 microns (hereinafter referred to as “ Small Aerogen ”), or using a PARI eFlow atomizer producing particles with a VMD of ~ 4.7 microns (hereinafter referred to as“ Large PARI ”). Levofloxacin was tested at concentrations of 4.25 mg / ml or 18.75 mg / ml in doses of 10 mg, 35 mg and 55 mg in an isotonic solution.

results

In the first test, 6 ml of a 4.25 mg / ml solution was inhaled using a Small Aerogen nebulizer. The expected RDD based on selected in vitro studies on the characteristics of a device using a breathing simulator was estimated to be 10 mg. Delivery time was 22 minutes. No noticeable adverse effects were observed either in the throat, or in the airways or lungs during and after administration, including the urge to cough and cough, and only a mild chemical taste was noted during and after administration. No adverse effects or taste were noted during the 30-minute observation following the administration of the drug. At these low concentrations and doses and slow administration rates, levofloxacin was well tolerated.

In a second test, 4 ml of a solution of 18.75 mg / ml was inhaled using a Small Aerogen nebulizer. The expected RDD based on selected in vitro studies on the characteristics of a device using a breathing simulator was estimated to be 35 mg. Delivery time for drug administration was 14 minutes. Despite the increased dose, acute tolerance was comparable to the first test both during and after administration. A stronger taste was noted, the solution had a more bitter / metallic taste characteristic of levofloxacin. The taste was most distinguishable within a few minutes after the end of the administration, which is again characteristic of levofloxacin.

In a third test, 4 ml of a solution of 18.75 mg / ml was inhaled using a Big PARI device. The expected RDD based on separate in vitro studies on the characteristics of the device using a breathing simulator was ~ 55 mg (using an FPD definition of <5 microns). Delivery time for drug administration was ~ 5 min. Despite the significantly increased particle size and drug delivery rate compared to test 2, no harmful effects were observed either in the throat, or in the airways or lungs, with the exception of the acute taste effect noted earlier, including the urge to cough and cough, during the dosing period and during the 30-minute observation period after dose delivery. Excretion of urine, which is an accurate measure of exposure, confirms that the planned respirable dose of approximately 55 mg was successfully delivered.

These results demonstrate the applicability of aerosol delivery of levofloxacin to humans at the average concentrations tested, and suggest that higher concentrations and doses that are properly formulated for tolerance and taste are achievable.

Example 7 - Micronization of levofloxacin

Micronization of Levofloxacin

Levofloxacin base dry powder can be micronized for therapy with exposure to high local concentrations, taste correction or delivery of levofloxacin with an improved AUC configuration by pulmonary administration of dry powder. Other methods currently under study include spray drying and in situ micronization methods. This method can also be used with other fluoroquinolone antibiotics, including without limitation, ofloxacin, lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloksatsin, rufloksatsin, fleroxacin, balofloksatsin, sparfloxacin, trovafloxacin, enoxacin, norfloxacin, klinafloksatsin, grepafloksatsin, sitafloxacin , Marbofloxacin, Orbifloxacin, Sarafloxacin, Danofloxacin, Difloxacin, Enrofloxacin, Garenoxacin, Prulifloxacin, Olamufloxacin, DX-619, TG-873870 and DW-276.

Description

In order to characterize the applicability of the micronized base of levofloxacin, the following studies were carried out.

Micronization

The drug powder of levofloxacin was micronized using a vortex mill. After micronization, the drug powder was collected in two fractions, one between 5-6 microns and a finer fraction.

Powder characteristic

The drug was characterized by particle size and particle size distribution before and after grinding using laser diffraction. Any changes in the physical form of the drug were evaluated by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Particle morphology was studied using scanning electron microscopy (SEM). The equilibrium moisture content in the drug powder before and after micronization was determined by thermogravimetric analysis (TGA) or Karl Fischer method. Any degradation of the drug during micronization was evaluated by HPLC. Separation conditions were chosen to determine whether new peaks appear after micronization.

Micronization

Experimental methodology

Two batches of levofloxacin were micronized using a vortex mill (Glen Mills). The development of the method was carried out to determine the micronization pressure necessary to achieve fractions of the required size between a) 5-6 microns and b) 2-3 microns. The particle sizes of levofloxacin were determined using a Sympatec HELOS laser diffraction particle size analyzer.

results

12 is a graph of average particle diameter (X50) versus micronization pressure. The first batch of levofloxacin had an average particle diameter of 10.6 microns before micronization. The graph shows that with increasing micronization pressure, the particle size decreased. A pressure of approximately 120 psi was required to achieve a size of 2.5 microns. inch. With a second batch of levofloxacin, which had an average particle size of 12.99 microns before micronization, a pressure of 30 psi was required. inch to achieve particle sizes of 5.2 microns.

Powder characteristic

Differential Scanning Calorimetry

Experimental methodology

Differential scanning calorimetry of premicronized as well as micronized levofloxacin (average particle size 2.5 microns) was performed using TA Instrument DSC Q1000. 1-2 mg of each sample was weighed into a crystallizer, sealed and heated at a rate of 10 ° C / min from 25 ° C to 300 ° C in a nitrogen atmosphere.

results

DSC profiles of premicronized and micronized levofloxacin are shown in FIG. 13. No difference was observed in the DSC profiles of micronized levofloxacin compared with premicronized.

Experimental methodology

Powders (micronized and premicronized) were attached to double-sided carbon plates on aluminum racks, which were then coated with a gold-palladium alloy. Microphotographs were taken from several different zones of powder on a rack using a scanning electron microscope.

results

Representative micrographs of premicronized and micronized levofloxacin taken with a scanning electron microscope are shown in FIGS. 14A and 14B. Levofloxacin crystals are lamellar before micronization. This form remains after micronization.

Experimental methodology

A thin layer of powder sample was placed on a zero background plate in an XRD sample holder. Each sample was analyzed using a Scintag XDS 2000 diffractometer under the following conditions:

Excitation source: Copper K _b X-rays; Scan speed = 1 ° per minute.

Voltage: 40 kV; Current strength: 35 mA.

results

X-ray diffraction graphs of premicronized and micronized levofloxacin are shown in FIG. The diffraction peak intensity at 9 ° is reduced after micronization. These results are consistent with published data for micronization of olanzapine (Stephenson G.A. The Rigaku Journal, 22 (2005): 2-15). A decrease in the relative intensities of the diffraction peaks could occur due to the formation of new surface faces of the crystal. The facet most processed after micronization will be the one for which the intensity is reduced as much as possible.

Experimental methodology

Samples of micronized and premicronized levofloxacin 15-25 mg each were dissolved in methanol (with a predetermined moisture content) and the moisture content of the samples was determined by a Carl Fischer Aquastar 3000 coulometric titrator.

results

The results of the Karl Fischer analysis are presented in table 30.

Table 30 Humidity of premicronized and micronized levofloxacin Humidity (%) premronized 6.16 micronized 5.42

Example 8 - A preliminary study of the base of levofloxacin

The objective of this study was to characterize the base of levofloxacin in order to understand the physicochemical capabilities and limitations of the base of levofloxacin for various methods of preparation of the drug. The ultimate goal of this study was to characterize the physicochemical properties of the base of levofloxacin.

Preliminary research

PH solubility studies

The pH solubility of levofloxacin was determined. First, buffer solutions were prepared in the pH range of 2-10. Small aliquots of each buffer (~ 200-250 μl) were saturated with the drug and shaken until equilibrium solubility was achieved. Then the samples were centrifuged, and the content of the dissolved drug in the supernatant was analyzed by UV spectrophotometry or HPLC. It was shown that the buffer solutions used in this study affect the solubility result (since different buffer counterions can form different salt forms left in the solution). Therefore, the dependence of solubility on pH should be evaluated in the absence of buffer solutions (by titration).

PKa definition

pKa of levofloxacin was determined by titrimetry. The obtained pKa values were confirmed by UV spectrophotometry. This information was used to select the salt form of levofloxacin and to determine the charge of levofloxacin under pH conditions in the lungs.

Preliminary investigation of the fluid system

The applicability of the liquid preparation was studied using: (a) solubility and (b) surface tension as the main parameters for the preparation without any additives in physiological saline.

Preliminary study of levofloxacin

HPLC method transfer

Experimental methodology

The HPLC method was used to assess the linearity, reliability and accuracy of the analysis of levofloxacin. A 50 × 4.6 mm column, Onyx Monolithic C18 (Phenomenex) was used at 30 ° C. The mobile phase consisted of an 85% solution of 0.1% TFA in water and a 15% solution of 0.1% TFA in acetonitrile. The flow rate was adjusted to 3 ml / min. Samples were injected into the chromatographic system and the output stream was controlled at 277 nm.

results

The retention time for levofloxacin was approximately 0.82 minutes. It was found that the analysis was linear in the range of 5 to 15 μg / ml with a correlation coefficient of 1000. The relative standard deviation (RSD) was less than 0.5% and the reliability was in the range of 98 to 102%

PH solubility studies

Titration

Experimental methodology

A saturated solution of levofloxacin in 0.1 N HCl was titrated with NaOH. After each addition of alkali, the solution was shaken on a vortex mixer. An aliquot of the sample was taken, centrifuged, and the supernatant was analyzed by UV spectroscopy at 288 nm. The same solution was titrated in the opposite direction with HCl.

results

The pH solubility profile of levofloxacin is shown in FIG. 16. During titrimetry, levofloxacin had a solubility of 25.4 mg / ml at pH 7.3. However, in contrast to the results of shaking experiments, the solubility during titrimetry decreased at a pH below 6.5, which may be due to the usual ionic effect. Since a solution of levofloxacin was prepared in HCl, a salt of hydrochloric acid and levofloxacin should have formed in the solution. Further addition of chloride ions in the form of hydrochloric acid should suppress the solubility of the hydrochloric acid salt.

PKa definition

Titrimetry

Experimental methodology

A solution of levofloxacin (18 mg / g) was prepared in water (18.45 mg / g). The initial pH of the solution was 7.36. This solution was titrated with 1 n HCl. Measured aliquots of HCl were added and the pH was recorded after each addition. Titration was continued to pH 1.

To determine the acid pKa, a solution of levofloxacin (18.38 mg / g) was prepared in 0.1 N HCl. The initial pH of the solution was 1.32. The solution was titrated with 1 n NaOH. Titration was continued to pH 6.55.

results

17 is a graph of pH versus volume of titrant added during titration of levofloxacin HCl. These data were substituted in the following equation:

V _t [OH ^- ] = K _b · V _ep -K _b · V _t

where V _t = volume of added titrant;

V _ep = volume of titrant added to the equilibrium point;

[OH ^- ] = concentration of hydroxide ions = Kw / [H ⁺ ];

[H ⁺ ] = concentration of hydroxonium ions = 10 ^-pH .

The dependence of V _t [OH ^- ] on V _s V _t is a straight line (Fig. 18). Data from the pre-equilibrium region are presented. Based on the slope, receive:

slope: K _b = 2.09 · 10 ^-8 ,

pK _b = -log K _b = 7.7,

pK _a = 14-pK _b = 6.3.

Fig. 19 is a graph of pH V _s versus titrant volume added during titration of levofloxacin NaOH. Acid pKa was difficult to calculate because it was rather low (<2.0). However, a rough rough estimate of pKa can be defined as pH at the half-equilibrium point. From the graph of dpH / dV versus titrant volume (V _t ) (Fig. 20), the equilibrium point is reached at 250 μl. The pH at the half-equilibrium point (i.e., when V _t = 125 μl) is 1.6. Thus, acid pKa ~ 1.6.

UV spectroscopy

Experimental methodology

Diluted solutions of levofloxacin (0.013 mg / ml) were prepared in several buffer solutions. Buffer solutions with HCl (pH 1.2), acetate (pH 4.5), phosphate (pH 6.7.8) and borate (9.10) were used. Levofloxacin solutions were analyzed by UV spectroscopy at 257 nm.

results

A plot of pH versus absorption of a levofloxacin solution at 257 nm is shown in FIG. These data were substituted into the modified Henderson-Hasselbach equation:

Abs _observed = Abs _HA [H ⁺ ] + Abs _A- [H ⁺ ] Ka + [H ⁺ ] Ka + [H ⁺ ],

where Abs _observed is the absorption of levofloxacin solution;

Abs _HA = absorption of levofloxacin solution at pH 1.2;

Abs _A- = absorption of levofloxacin solution at pH 7.8;

[H ⁺ ] = concentration of hydroxonium ions = 10 ^-pH

Substitution in the equation gives an estimated value pKa = 5.91.

Example 9 - Preparation of Levofloxacin Salts

The aim of this study was to obtain various salt forms of levofloxacin, which can acquire new, improving AUC configuration properties through reduced solubility and / or decomposition. These benefits may alter the pharmacodynamic properties of levofloxacin after pulmonary administration in the form of a suspension of nanoparticles or powder inhalation. These drugs can be optimized to prolong the release of levofloxacin from salt forms with reduced solubility. These properties can be imparted to other fluoroquinolone antibiotics, including but not limited to hemifloxacin, gatifloxacin, norfloxacin, tosufloxacin, sitafloxacin, sarafloxacin, prulifloxacin and pazufloxacin. Currently, studies are underway to characterize various salt forms and co-precipitates of hemifloxacin to correct taste, improve the configuration of AUC, as well as the introduction of nanoparticles in suspension and powder inhalation. Other approaches currently being studied include spray drying and in situ micronization methods.

For suspension and powder formulations, specific salt forms can provide important physical and chemical characteristics that can contribute to the effectiveness of the product. For improving the AUC configuration of the drug, the task of choosing a salt was to decrease the solubility and / or decrease the rate of decomposition of levofloxacin. Acid counterions can be selected by:

changes in the melting point: an increase in the melting point is usually accompanied by a decrease in salt solubility. Salts formed by flat, high melting aromatic acids are typically crystalline salts with a high melting point;

changes in hydrophobicity: salts formed by acids with a hydrophobic conjugate are hydrophobic and poorly wet, and this inevitably leads to a longer decomposition. Examples of acids selected for the preparation of salts are listed below:

a) pamic acid (emboic acid);

b) 2-naphthalenesulfonic acid (napsilic acid);

c) oleic acid;

d) xinafoic acid;

e) stearic acid;

f) lauryl sulfonate (estolate).

Other factors taken into account include surface properties, polymorphic modifications, and chemical stability.

Description

The objective of the study was to obtain salt forms of levofloxacin in order to reduce its solubility and / or decomposition rate. The goal was:

(a) make levofloxacin less soluble by salt formation with suitable excipient (s),

(b) to obtain salt forms of levofloxacin, which will have a lower solubility and / or decomposition rate than the free base.

To accomplish these tasks, efforts were concentrated on obtaining salts from the alkaline portion of the molecule (pKa ~ 6.8).

Pamic acid (t.p. = 280 ° C) and napsilic acid (t.p. = 125 ° C) have planar hydrophobic structures, which, as expected, will give the salt a hydrophobic character. The high melting point of pamic acid can result in a high melting crystalline salt form. Oleic acid was initially chosen because it was approved for pulmonary delivery. It has a low melting point (4 ° C), which may not correspond to the first condition, but there was hope that a long aliphatic chain could give hydrophobicity sufficient to reduce solubility. Xynaphoic acid (mp = 195 ° C) was also chosen to produce the salt, since it also has planar hydrophobic structures, which are expected to make the salt hydrophobic. The main reason for choosing stearic acid and lauryl sulfonate (estolate) was similar to that for oleic acid, only their toxicity to the lungs is unknown. Estolate approved for oral delivery (erythromycin estolate has approximately 1/12 ^th the solubility of free base drug and is in the form of an oral suspension).

Salt production

Typically, the levofloxacin base and acid were dissolved in a suitable volatile organic solvent (1: 1 molar ratio) and stirred at room temperature. Any obtained crystalline product was filtered, dried and characterized. Characterization consisted of DSC, FTIR and elemental analysis.

Obtaining and characterization of co-crystals of levofloxacin with pamic acid

Experimental methodology

Obtaining co-crystals of levofloxacin with pamic acid

0.31 g (0.8 mmol) of pamic acid was dissolved with stirring in 100 ml of tetrahydrofuran (THF). To this was added 0.30 g (0.8 mmol) of levofloxacin, dissolved by stirring, and the final solution was refluxed for 2.5 hours. The resulting suspension was cooled to room temperature, filtered, and the obtained precipitate was dried in vacuo at about 70 ° C for 3 hours.

Characteristic

Thermal analysis. Thermal analysis of (a) pamic acid; (b) levofloxacin; (c) co-crystallized levofloxacin pamoate precipitate; (d) a physical mixture of pamic acid and levofloxacin was performed using a differential scanning calorimeter (TA Instrument DSC Q1000). 2-5 mg of each sample was weighed into a crystallizer, hermetically sealed and heated at 10 ° C / min from 25 ° C to 300 ° C in a nitrogen atmosphere.

Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectroscopy of (a) pamic acid; (b) levofloxacin; (c) co-crystallized levofloxacin pamoate precipitate; (d) a physical mixture of pamic acid and levofloxacin was performed using an FTIR spectrophotometer Model IRPrestige-21, Shimadzu).

Saturated solubility. The saturating solubility of levofloxacin and the co-crystallized precipitate of levofloxacin-pamic acid was determined by balancing the excess amount of solids with water. The pH of the suspensions was adjusted with HCl to 4, 5, 6, and 7, shaken, centrifuged, and the supernatant was analyzed by UV spectroscopy at 288 nm.

results

Thermal analysis. DSC profiles of (a) pamic acid; (b) levofloxacin; (c) co-crystallized levofloxacin pamoate precipitate; (d) a physical mixture of pamic acid and levofloxacin is shown in FIG. Pamic acid and levofloxacin have acute endotherms at 330 ° C and 239 ° C, respectively, which is most likely due to the melting of pamic acid and levofloxacin, respectively. The DSC profile of the co-crystals of levofloxacin pamoate has one main endotherm at 210 ° C, while the 1: 1 molar mixture of levofloxacin and pamic acid had wide endotherms at 129 ° C and 220 ° C.

FTIR. FTIR spectra obtained for (a) pamic acid; (b) levofloxacin; (c) co-crystallized levofloxacin pamoate precipitate; (d) a physical mixture of pamic acid and levofloxacin is shown in FIG. High-intensity absorption bands at 1650 cm ⁻¹ in the FTIR spectra of pamic acid, which are the result of stretching vibrations of the C = O group, are greatly reduced in co-crystals.

Saturated solubility. Table 31 presents saturation solubility data for levofloxacin and levofloxacin pamoate at various pHs. Solubility was determined in water, since buffer acids affected the solubility of levofloxacin. However, after shaking levofloxacin or salt solutions in water, a pH shift occurred, especially in a solution of levofloxacin with a pH of 5, a shift to pH 1.6 occurred. Since the pH of the solution is between two pKa of levofloxacin (~ 1.6 and ~ 6), such a solution will have a reduced buffer capacity and, therefore, a pH shift. Solutions with a pH near the pKa of the drug have a high buffer capacity and resist pH changes. The solubility of levofloxacin pamoate was significantly lower than that of levofloxacin at all pHs.

Table 31 Saturated Solubility Data of Levofloxacin and Levofloxacin Pamoate Levofloxacin pH before shaking 3.97 5.06 5.96 pH after shaking 4.21 1,63 5.70 Solubility (mg / ml) 158.28 225.04 297.20 Levofloxacin Pamoate pH before shaking 4.06 4.98 5.98 7.00 pH after shaking 5.63 6.30 6.66 7.32 Solubility (mg / ml) 0.34 0.29 0.24 0.44

Interpretation

Since the co-crystallized precipitate of levofloxacin pamoate has a melting point and FTIR spectra that are different from those for levofloxacin, pamic acid or their physical mixture, it is possible that the equimolar complex of levofloxacin with pamic acid may be a salt of levofloxacin pamoate having much less solubility compared to levofloxacin .

Obtaining and characterization of co-crystals of levofloxacin with xinafoic acid

Experimental methodology

Obtaining co-crystals of levofloxacin with xinafoic acid

1.004 g (2.7 mmol) of levofloxacin was dissolved by heating under reflux in 80 ml of ethyl acetate. To this was added 0.51 g (2.7 mmol) of xynaphoic acid dissolved in 35 ml of ethyl acetate, and the solution was cooled overnight under stirring to room temperature. The resulting suspension was filtered and the precipitate was dried in vacuo at 75 ° C for about 3.5 hours.

Characteristic

Thermal analysis. Thermal analysis of (a) xinafoic acid; (b) co-crystallized levofloxacin xinafoate precipitate was performed using a differential scanning calorimeter (TA Instrument DSC Q1000). 2-5 mg of each sample was weighed into a crystallizer, hermetically sealed and heated at 10 ° C / min from 25 ° C to 300 ° C in a nitrogen atmosphere.

Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectroscopy of (a) xinafoic acid; (b) co-crystallized levofloxacin xinafoate precipitate was performed using an FTIR spectrometer (Model IRPrestige-21, Shimadzu).

Saturated solubility. The saturating solubility of the co-crystallized precipitate of levofloxacin-xinafoic acid was determined by balancing the excess amount of solids with water. Suspensions were adjusted to pH 4, 5, 6, and 7 with HCl, shaken, centrifuged, and the supernatant was analyzed by UV spectroscopy at 288 nm.

results

Thermal analysis. DSC profiles of (a) xinafoic acid, (b) co-crystallized levofloxacin xinafoate precipitate are shown in FIG. 24. The co-crystallized precipitate of levofloxacin xinafoate has a melting endotherm at 196 ° C, which differs from that for xinafoic acid (216 ° C) and levofloxacin (239 ° C).

FTIR. FTIR spectra obtained for a) xinafoic acid, (b) co-crystallized levofloxacin xinafoate precipitate are shown in FIG. 25. The FTIR spectrum of the co-crystal has minimal transmittance at wavelengths other than for xinafoic acid and levofloxacin.

Saturated solubility. Table 32 presents the saturation solubility data of levofloxacin xinafoate at various pHs. The solubility of the xinafoate salt was intermediate between those for the base of levofloxacin and the co-crystal of levofloxacin pamoate.

Table 32 Saturating solubility data of co-crystal of levofloxacin xinafoate Levofloxacin Xinafoate pH before shaking 4.05 4.94 6.00 6.95 pH after shaking 4.78 5.37 6.30 7.17 Solubility (mg / ml) 21.69 12.95 4,59 5.95

Interpretation

The co-crystallized precipitate of levofloxacin pamoate has a melting point and FTIR spectra that are different from those for levofloxacin and xinafoic acid, which suggests the possible formation of the levofloxacin xinafoate salt. This salt has an intermediate solubility between levofloxacin and levofloxacin pamoate.

Obtaining and characterization of co-crystals of levofloxacin with stearic acid

Experimental methodology

Obtaining co-crystals of levofloxacin with stearic acid

0.77 g (2.07 mmol) of stearic acid was dissolved by heating and sonication in 40 ml of methanol. To this was added 1.00 g (2.07 mmol) of levofloxacin dissolved in 60 ml of methanol. The resulting solution was heated at 55 ° C for about 15 minutes, followed by cooling to room temperature and then to -20 ° C. The resulting suspension was filtered.

Characteristic

Thermal analysis. Thermal analysis of (a) stearic acid, (b) co-crystallized precipitate of levofloxacin stearate, (c) a physical mixture of stearic acid and levofloxacin was performed using a differential scanning calorimeter (TA Instrument DSC Q1000). 2-5 mg of each sample was weighed into a crystallizer, hermetically sealed and heated at 10 ° C / min from 25 ° C to 250 ° C in a nitrogen atmosphere.

Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectroscopy of (a) stearic acid, (b) co-crystallized precipitate of levofloxacin-stearic acid, (d) a physical mixture of stearic acid and levofloxacin was performed using an FTIR spectrometer (Model IRPrestige-21, Shimadzu).

Saturated solubility. The saturating solubility of levofloxacin and the co-crystallized precipitate of levofloxacin-stearic acid was determined by balancing the excess amount of solids with water. Suspensions were adjusted to pH 4, 5, 6, and 7 with HCl, shaken, centrifuged, and the supernatant was analyzed by UV spectroscopy at 288 nm.

results

Thermal analysis. DSC profiles of (a) stearic acid, (b) co-crystallized precipitate of levofloxacin stearate, (d) a physical mixture of stearic acid and levofloxacin are shown in Fig. 26. Stearic acid and levofloxacin have acute endotherms at 76.4 ° C and 239 ° C, respectively, which is most likely a consequence of the melting of stearic acid and levofloxacin, respectively. The DSC profile of the co-crystals of levofloxacin-stearic acid had two sharp endotherms at 88.03 ° C and 138.54 ° C and minor endotherms at 231 ° C and 242.72 ° C. Minor endotherms may result from melting of trace amounts of residual levofloxacin in the original sample. A 1: 1 molar mixture of levofloxacin and stearic acid had endotherms at 68.87 ° C, 134.43 ° C and 240.74 ° C and minor endotherms at 79.73 ° C and 86.74 ° C.

FTIR. FTIR spectra obtained for (a) stearic acid, (b) co-crystallized precipitate of levofloxacin-stearic acid, (c) a physical mixture of stearic acid and levofloxacin are shown in Fig. 27. The stretching band C = O is visible at 1700, 1705 and 1721 cm ^-1 in stearic acid, co-crystallized precipitate and physical mixture, respectively.

Saturated solubility. Table 33 presents the saturation solubility data of the co-crystals of levofloxacin-stearic acid at different pH.

Table 33 Saturated solubility data of co-crystals of levofloxacin-stearic acid Levofloxacin stearate pH before shaking 4.05 5.02 6.01 6.96 pH after shaking 3.36 5.05 6.02 6.98 Solubility (mg / ml) 0.86 1.30 2.20 1,64

Interpretation

The DSC profile of the co-crystals precipitate levofloxacin-stearic acid has two endotherms. One of these endotherms may be due to melting of co-crystals. The nature of the second endotherm remains to be explored. Since the co-crystal precipitate of levofloxacin-stearic acid has solubility values lower than those for levofloxacin, it is possible that the precipitates may be a salt, a salt of levofloxacin stearate.

Obtaining and characterization of co-crystals of levofloxacin with oleic acid

Experimental methodology

Obtaining co-crystals of levofloxacin with oleic acid

0.78 g (2.76 mmol) of oleic acid was dissolved in 10 ml of chloroform. To this was added 1.025 (2.76 mmol) of levofloxacin dissolved in 10 ml of chloroform. The resulting solution was thoroughly mixed and evaporated at 40 ° C.

Characteristic

Thermal analysis. Thermal analysis of (a) oleic acid, (b) precipitate of co-crystals of levofloxacin oleate, (c) a physical mixture of oleic acid and levofloxacin (50:50), (d) a physical mixture of oleic acid and levofloxacin (10:90) and (e ) a physical mixture of oleic acid and levofloxacin (90:10) was performed using a differential scanning calorimeter (TA Instrument DSC Q1000). 2-5 mg of each sample was weighed into a crystallizer, sealed and heated at 1 ° C / min or 10 ° C / min from 25 ° C to 250 ° C in a nitrogen atmosphere.

Fourier Transform Infrared Spectroscopy (FT-IR). FT-IR spectroscopy of (a) oleic acid, (b) co-crystallized levofloxacin-oleic acid precipitate, (d) a physical mixture of oleic acid and levofloxacin was performed using an FTIR spectrometer (Model IRPrestige-21, Shimadzu).

Determination of kinetic solubility. The co-crystallized precipitate of levofloxacin oleate (50 mg) was suspended in 2 ml of water. The suspension was adjusted to pH 7 with HCl and shaken. The solubility of these co-crystals was determined at different time intervals. This study was carried out at room temperature and at 40 ° C. The kinetic solubility of the equimolar physical mixture of levofloxacin and oleic acid was also determined and compared with that for co-crystals at 40 ° C.

results

Thermal analysis. DSC profiles of (a) oleic acid, (b) precipitate of co-crystals of levofloxacin oleate, (c) a physical mixture of oleic acid and levofloxacin (50:50), (d) a physical mixture of oleic acid and levofloxacin (10:90) and (e ) a physical mixture of oleic acid and levofloxacin (90:10) are presented in Fig. 28. A thermogram of oleic acid has endotherms at -6.15 ° C and 13.05 ° C. The endotherm at -6.15 ° C corresponds to the γ-α phase transition of oleic acid (Crowley KJ, 1999). The co-crystallized precipitate of levofloxacin oleate has an endotherm at 127.69 ° C, while the equimolar physical mixture of levofloxacin with oleic acid has endotherms at 123.69 ° C, 179.35 ° C and 224 ° C. The equimolar physical mixture has an endotherm that is close to the melting point of co-crystals, on the basis of which a possible reaction between oleic acid and levofloxacin in the solid state can be suggested. To investigate this phenomenon, DSCs of physical mixtures of levofloxacin and oleic acid (90:10) and (10:90) were performed. The physical mixture of levofloxacin and oleic acid (10:90) has the main isotherm at 10.33 ° C (possible melting of oleic acid) and at 281 ° C. There is no endotherm near the melting point of co-crystals. The physical mixture of levofloxacin-oleic acid (90:10) does not have a melting endotherm at 10 ° C for oleic acid. It possesses endotherms at 79.77 ° C and at 128 ° C (close to the melting point of co-crystals), which suggests a possible reaction of levofloxacin and oleic acid in the presence of large amounts of levofloxacin.

FTIR. The FTIR spectra of oleic acid have an intense peak of C = O stretching vibrations at 1710 cm ^-1 and planar and out-of-plane OH bands at 1462 and 937 cm ^-1 , respectively.

High-intensity absorption bands at 1710 cm ⁻¹ in the FTIR spectra of oleic acid, which are the result of stretching vibrations of the C = O group, are slightly reduced in co-crystals. Planar and out-of-plane OH bands at 1462 and 937 cm ^-1 in oleic acid are absent in co-crystals. Also, the FTIR spectrum of the physical mixture is different from that for the salt (FIG. 29).

Determination of kinetic solubility

On Fig presents data from experiments to determine the kinetic solubility conducted with co-crystallized precipitate at room temperature and at 40 ° C. The solubility of co-crystals at room temperature is approximately 0.9 mg / ml and remains unchanged throughout the study period. At 40 ° C, solubility increases from 1.17 mg / ml after 15 minutes to 1.86 mg / ml after 4 hours and remains almost unchanged until 24 hours. The solubility profile of an equimolar physical mixture at 40 ° C differs from that for co-crystals. The physical mixture has greater solubility (9.16 mg / ml after 24 hours) compared with co-crystals (1.89 mg / ml after 24 hours).

Interpretation

The DSC data of the equimolar physical mixture demonstrate the presence of an endotherm near the melting endotherm of the co-crystallized precipitate. However, the FTIR data and the solubilities of the co-crystals differ from those for the physical mixture, while the co-crystals have less saturating solubility. The saturating solubility of the co-crystals is 0.9 mg / ml, in contrast to 25 mg / ml for the base of levofloxacin.

However, the levofloxacin oleate salt is waxy in nature, which can create difficulties in grinding / micronization and, as a result, in the preparation. It is reported that the stickiness and deformability of the waxy drug salt of the propranolol oleate fatty acid made particle size reduction difficult (Crowley. J., et al, International journal of Pharmaceutics, 2000, 211 (1-2): 9-17.

Dissolution rate study

Levofloxacin Xinafoate

Experimental methodology

50 mg of levofloxacin xinafoate salt was suspended in a dissolution bath containing 500 ml of Tris buffer pH 7.4 at 37 ° C with stirring with a paddle stirrer at a speed of 100 rpm. 5 ml of samples were taken at periodic intervals and replaced with the same volume of simple buffer.

results

The dissolution profile of levofloxacin xinafoate is shown in FIG. It is seen that the rate of dissolution of levofloxacin xinafoate in the early stages of 2-10 minutes is higher than that which takes place after 10-30 minutes. When levofloxacin xinafoate is added to the solvent medium, it disperses as a fine powder, and the dissolution of such small particles occurs faster, about 1.24 mg / min (Fig. 32). Over time, the powder sticks together and moves in a vortex created by the agitator blade, thereby reducing the dissolution rate to 0.28 mg / min (Fig. 33).

Levofloxacin

Experimental methodology

200 mg of levofloxacin was suspended in a dissolution bath containing 500 ml of Tris buffer pH 7.4 at 37 ° C with stirring with a paddle stirrer at a speed of 100 rpm. 5 ml of samples were taken at periodic intervals and replaced with the same volume of simple buffer.

results

The dissolution profile of levofloxacin is presented in Fig. 34. Since levofloxacin has better solubility than its salts, its dissolution rate is very high. In the case of levofloxacin, too, the initial dissolution occurred from finely dispersed particles and, thus, the speed was higher. At later stages, the particles stuck together and reduced its dissolution rate.

Levofloxacin Pamoate

Experimental methodology

10 mg of levofloxacin pamoate salt was suspended in a dissolution bath containing 500 ml of Tris buffer pH 7.4 at 37 ° C with stirring with a paddle stirrer at a speed of 100 rpm. 5 ml of samples were taken at periodic intervals (2, 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 1320 and 1440 minutes) and replaced with the same volume of simple buffer. The study was performed in duplicate.

results

The dissolution profile of levofloxacin pamoate is shown in Fig. 35. It is seen that the rate of dissolution of levofloxacin pamoate in the early stages of 2-10 minutes is higher than that which takes place after 10-60 minutes. When levofloxacin pamoate is added to the solvent medium, it disperses as a fine powder, and the dissolution of such small particles occurs faster, about 0.146 mg / min (Fig. 36). Over time, the powder sticks together and moves in a vortex created by the agitator blade, thereby reducing the dissolution rate to 0.0331 mg / min (Fig. 37).

Levofloxacin stearate

Experimental methodology

25 mg of levofloxacin stearate salt was suspended in a dissolution bath containing 500 ml of Tris buffer pH 7.4 at 37 ° C with stirring with a paddle stirrer at a speed of 100 rpm. 5 ml of samples were taken at periodic intervals (2, 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 1320 and 1440 minutes) and replaced with the same volume of simple buffer.

results

The dissolution profile of levofloxacin stearate is shown in Fig. 38. It is seen that the dissolution rate of levofloxacin stearate in the early stages of 2-10 minutes is 0.499 mg / min (Fig. 39), which is higher than that after 10-30 minutes (0.161 mg / min) (Fig. 40).

The dissolution of levofloxacin and salts was carried out in such concentrations that the concentration of the solution in the dissolution bath never reached more than 10% of its saturating solubility. This was done in an attempt to maintain absorption conditions.

Considering the dissolution rate of these and other salt forms and co-precipitates, these AUC configuration-improving forms of levofloxacin, hemifloxacin, and other fluoroquinolone antibiotics may be best suited for a suspension of nanoparticles (solubility <100 μg / ml, low dissolution rates) or micronized dry powders (solubility > 100 μg / ml, a higher dissolution rate than the data, is most suitable for nanosuspension.Suspensions of nanoparticles can be entered by spraying using jet, ultrasound sow or vibrating cellular technology, while powder preparations can be administered using either active or passive powder inhalers.

Example 11 - solid lipid nanoparticles of levofloxacin

The aim of this study was to obtain solid lipid nanoparticles of levofloxacin in order to achieve the properties of improving the AUC configuration by reducing solubility and degradation. These advantages can improve the pharmacodynamic properties of levofloxacin after pulmonary administration using a suspension of nanoparticles or powder inhalation preparations. These drugs are optimized to prolong the release of levofloxacin from salt forms of reduced solubility. These properties can also be imparted to other fluoroquinolone antibiotics, including without limitation hemifloxacin, gatifloxacin, norfloxacin, tosufloxacin, sitafloxacin, sarafloxacin, prulifloxacin and pazufloxacin. Currently, studies are also being conducted to characterize various forms of hemifloxacin lipid nanoparticles to correct taste, improve AUC configuration, and introduce nanoparticles in suspension and powder inhalation. Other currently explored approaches for solid lipid nanoparticles include spray drying and in situ grinding techniques.

Preliminary research

The distribution of each compound (including salts of levofloxacin and complexes with metal cations) in 1-octanol was determined at various suitable pH values. You can also evaluate the distribution over time to determine whether dissociation of levofloxacin (both from salts and from complexes) takes place, and in the case of salts, also determine whether a selective distribution of the fatty acid component takes place over time. Compound (s) with a significant distribution (log P> 2.0) was evaluated in terms of their solubility in various lipid melts. In addition, the distribution of lipophilic fluoroquinolones (if any) is also being studied, and their solubility in various lipid melts will be evaluated. A lipid in which the drug is relatively soluble will be selected for the preparation of solid lipid nanoparticles. A necessary condition for achieving a sufficient load-bearing capacity of the drug in solid lipid nanoparticles was the good solubility of the drug in the lipid melt.

Obtaining solid lipid nanoparticles

The preparation of solid lipid nanoparticles typically involves dissolving the drug in a lipid melt, followed by dispersion of the drug-containing melt in a hot aqueous surfactant solution. The coarse dispersed system is homogenized using Microfluidizer® to produce a nanoemulsion. Cooling the nanoemulsion to room temperature will cause the solidification of the lipid, which will lead to the formation of solid lipid nanoparticles. The production parameters will be optimized (type of lipid matrix, surfactant concentration and manufacturing parameters) so as to achieve long-term drug delivery.

Characterization of solid lipid nanoparticles

Nanoparticles are characterized on the basis of size and zeta potential, using a device for dynamic light scattering, while laser diffraction is used to detect large microparticles.

After completion of the synthesis, studies are performed using differential scanning calorimetry to study any possible modifications induced in the physical form of the lipid.

In vitro drug release testing is performed using an appropriate methodology.

Example 10 - Complexes of levofloxacin with metal ions

The aim of this study was to obtain levofloxacin in various chelated salt forms in order to achieve the acquisition of taste correction properties, improving the AUC configuration by changing solubility, degradation and / or bioavailability. These benefits can improve the pharmacodynamic properties of levofloxacin after pulmonary administration using a suspension of nanoparticles, powder inhalation preparations, or simple liquid preparations. These drugs can be optimized to create leucofloxacin chelates that improve the AUC configuration of chelates with altered solubility or slow release, or low bioavailability. These properties can also be imparted to other fluoroquinolone antibiotics, including without limitation hemifloxacin, gatifloxacin, norfloxacin, tosufloxacin, sitafloxacin, sarafloxacin, prulifloxacin and pazufloxacin. Research is also ongoing to characterize the various and chelated forms of hemifloxacin to improve taste, improve AUC configuration, administer a suspension of nanoparticles, and powder inhalation.

Obtaining complexes of levofloxacin-metal ion

Preliminary research

A mixture of levofloxacin and salts of this cation was solubilized in deionized water and titrated with sodium hydroxide. The titration curve was compared with that obtained for levofloxacin alone to determine the formation of the levofloxacin-metal complex, as described in Physical Pharmacy (4th Edition) by Alfred Martin (pp 261-263). Then, salts of various metal cations (e.g., Ca2 +, Mg2 +, etc.) were evaluated to identify a suitable candidate (s) for further evaluation. Various molar ratios of cations and levofloxacin were also evaluated.

Getting complexes

Levofloxacin solutions were titrated against aqueous solutions of salts of selected metals. Titrations were performed at constant pH. Complex formation was monitored in various ways, including titrimetry, spectrofluorimetry, solubility, and so on, depending on the circumstances. The endpoint of the complexation reaction depended on the method adopted.

Characterization of levofloxacin complexes

The metal levofloxacin-cation complexes were characterized in terms of stoichiometry, formation constants, and dissociation kinetics using an appropriate methodology.

Goals

Obtain and characterize complexes of levofloxacin with metal cations (divalent and trivalent).

Complexation Assessment

Preliminary studies have indicated that levofloxacin forms soluble complexes with metal cations. As a result, it was not possible to evaluate the complexation process through precipitation. Other approaches that have been tried are described below.

Titrimetry

This approach is based on the assumption that the carboxyl fragment of levofloxacin is involved in the formation of a complex with a cation of this metal and that the formation of the complex leads to the release of a proton from levofloxacin. Thus, the concentration of released protons would be proportional to the degree of complexation (depending on the binding constant) and the stoichiometry of the complex (Physical Pharmacy: 4 ^th Edition by Alfred Martin; pp-261-263).

Experimental methodology

Approximately 0.35 mmol of levofloxacin (in 16 ml of deionized water) was titrated with 6 N NaOH in the presence and absence of a metal cation salt (equimolar). Levofloxacin solutions were acidified to pH values less than 2.0 with 6N HCl before titration with NaOH. Salts of metal cations used included calcium chloride, magnesium chloride, iron chloride, zinc chloride, aluminum sulfate and aluminum chloride.

results

As shown in FIG. 41, titration performed in the presence of metal cations resulted in a positive shift of the titration curves compared to those obtained for levofloxacin alone, indicating that an additional amount of NaOH (titration solution) was needed to obtain a certain pH value solution in the presence of a metal cation. The magnitude of the shift of the titration curve at any point is a moth of protons released as a result of complexation and, therefore, a moth of the formed levofloxacin complex.

The degree of complexation (binding and / or stoichiometry), apparently, increases in the following order: Ca ⁺ <Mg ²⁺ <Zn ²⁺ = Fe ²⁺ <Al ³⁺ , which is quite consistent with existing literature data.

Note: From the literature it follows that aluminum chloride and aluminum sulfate have acid-like properties and can lower the pH of aqueous solutions. Therefore, based on the titration curves obtained with A1C1 ₃ and A1 ₂ (SO ₄ ) _3, it is impossible to obtain conclusive information on complexation with levofloxacin.

Double titration

In this approach, a solution of levofloxacin was titrated with a solution of this metal cation to obtain a drop in pH, presumably due to the release of protons upon complexation. Subsequently, NaOH was added to return the levofloxacin solution to the initial pH (before the cation solution was added). This allows you to determine the fraction of levofloxacin in the form of a complex at a given pH.

Experimental methodology

Approximately 1.55-1.72 mmol of levofloxacin was solubilized in deionized water and the resulting solution was acidified with 6 N HCl to the desired initial pH. This acidified levofloxacin solution was titrated with a known volume of a concentrated solution of this metal cation (Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ ). The pH change was neutralized (to the initial pH) by adding 6 N NaOH and the added volume of NaOH solution was recorded. The addition of the metal cation solution followed by the neutralization of NaOH was continued until the further addition of the metal cation solution ceased to cause a change in the pH of the levofloxacin solution, which would indicate the end point of complexation. A graph was plotted for the cumulative amounts of the added metal cation added to the cumulative amounts of NaOH needed to neutralize the pH changes (Figs. 42-45).

results

Based on FIGS. 42-45, plateau portions were extrapolated to obtain the total amount of NaOH needed to neutralize pH changes due to complexation. These values also represented the amount of levofloxacin in the form of a complex (assuming that complexation of levofloxacin leads to equimolar proton release). The amounts of levofloxacin in the form of a complex with Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ are 0.8; 1.0; 1.3 and 1.1 mmol, respectively. This corresponds to 46.5; 64.5; 77.8 and 64.5% of complexation for Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ , respectively. It should be noted that% of complexation will depend on the total concentration of levofloxacin.

The binding constants, as well as the stoichiometry of complexation for complexes of levofloxacin with metal cations, were determined as follows:

M + nA⇔MA _n K _b

where M, A and MA _n are a metal cation, levofloxacin and a complex, respectively. K _b is the equilibrium binding constant. It is believed that in the above reaction, “n” moles of levofloxacin react with one mole of metal, which leads to the formation of one mole of the complex.

K _b = [MA _n ] / {[M] [A] ⁿ } (units M ^-n ), Lv. 1

where [MA _n ] represents the concentration of the formed complex;

[M] and [A] are the concentrations of unbound metal and unbound levofloxacin, respectively.

Converting Lv. 1,

[MA _n ] / [A] ⁿ = K _b * [M], Lv. 2

where [A] = [A] _total - [A] _bound = [A] _total - [NaOH] _used ;

[M] = [M] _total - [M] _linked = [M] _total - [NaOH] _used / n;

[MA _n ] = [A] _bound / n = [NaOH] _used / n.

Note [NaOH] _used is the concentration of sodium hydroxide used at any given point to neutralize the pH change caused by the addition of a metal cation (presumably due to complexation).

Lv.2 can be converted to receive

[A] _linked / [A] ⁿ = nK _b * [M]. Lv. 3

From Eq. 3, we can conclude that the graph of the dependence of [M] on [A] _connected / [A] ⁿ will be a straight line with a slope of nK _b when

n = 1 for the complex 1: 1,

n = 2 for the complex 2: 1,

n = 3 for the complex 3: 1 and so on.

Figures 46-49 below show such plots for Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ , respectively.

As shown in Figs. 46-49, for each of the cations studied, the graph of [A] _bound / [A] ⁿ versus nK _b * [M] was linear for n = 2 (for Ca ²⁺ n = 2, the correspondence was the best, than for n = 1). Based on these results, it can be assumed that complexes of levofloxacin with Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ are formed with stoichiometry of 2 moles of the drug per 1 mol of cation (2: 1).

For n = 2, the binding constants of the above complexes can be determined based on the slopes of the corresponding linear graphs.

The binding constants for the 2: 1 complexes, represented as log (K _b ), are as follows: Ca ²⁺ = 2.75, Mg ²⁺ = 3.69, Zn ²⁺ = 4.44, Fe ²⁺ = 4.54 .

Solubility

This method allows the stoichiometry of complexation to be determined in a relatively simple way. The approach includes evaluating the solubility of the drug (levofloxacin) in the presence of a complexing agent (a given metal cation) in increasing concentrations. It is expected that the overall solubility of the drug (as part of the complex and not as part of the complex) will increase linearly due to complexation and reach a plateau corresponding to the saturating solubility of both the drug and the complex. The determination of stoichiometry based on such a solubility curve is described in detail elsewhere (Physical Pharmacy: 4 ^th Edition by Alfred Martin; pp 265).

Experimental methodology

Excessive amounts of levofloxacin (amounts recorded) were mixed in the presence of MgCl ₂ at increasing concentrations with 25 mM MES buffer (pH 5.99) using a vortex mixer. Then the samples were filtered, and the filtrate was diluted, respectively, and analyzed spectrophotometrically to determine the concentrations of levofloxacin (Fig. 50).

results

As shown in FIG. 50, the solubility of levofloxacin did indeed increase with increasing concentrations of MgCl ₂ . However, beyond the solubility plateau (~ 650 mM levofloxacin), a further increase in solubility was observed, which did not coincide with the expected profile. This was attributed to the effect of ionic strength on the solubility of levofloxacin. It is important to note that the final pH of all solutions was unchanged, although more than 5.99 (final pH ~ 7.0).

Subsequently, the experiment was repeated at a constant ionic strength of ~ 1.0 M (adjusted with NaCl) and with 0.5 M MES buffer (pH 5.99) to increase the buffer capacity of the solution (Fig. 51).

Spectrofluorimetry

This approach was used to assess the complexation of levofloxacin, based on existing literature data that the complexation process is associated with a change in the fluorescence properties of fluoroquinolone. By controlling the changes in the fluorescence of levofloxacin in the presence of a given metal cation in various concentrations, one can determine the binding constant of complexation, as well as stoichiometry.

Experimental methodology

Fluorescence of levofloxacin was evaluated at excitation and emission wavelengths of 298 nm and 498 nm, respectively. The studies were carried out at two different pH values, namely 5.0 (acetate) and 9.0 (histidine). A series of solutions with a constant concentration of levofloxacin, but increasing concentrations of this cation, were analyzed in relation to the fluorescence of levofloxacin. Studied metal salts included CaC1 ₂ , MgC1 ₂ , FeC1 ₂ , ZnC1 ₂ and A1 ₂ (SO ₄ ) ₃ .

results

As shown in table 34, significant data were obtained only for Fe ²⁺ and Zn ²⁺ . For the remaining cations, the relative concentrations of levofloxacin and the cation must be further optimized in order to observe a specific tendency in the fluorescence of levofloxacin.

The effect of increasing concentrations of Fe ²⁺ and Zn ²⁺ on the fluorescence of levofloxacin is shown in FIGS. 52 and 53, respectively.

As described above, both Fe ²⁺ and Zn ²⁺ obviously form 2: 1 complexes with levofloxacin; however, their effects on the fluorescence of levofloxacin are different (FIGS. 52 and 53). The exact reason for this is currently unclear.

Table 34 Fluorescence characteristics of levofloxacin in the presence of cations Cation Fluorescence of Levofloxacin results Comments pH 5.0 pH 9.0 Ca ²⁺ Minor changes Minor changes N / a - Mg ²⁺ Minor changes Minor changes N / a - Fe ²⁺ Emission decrease with increasing Fe ²⁺ N / a Fig. 3.12 (pH 5.0) FeC1 ₂ insoluble at pH 9.0 Zn ²⁺ Minor changes Emission increase with increasing Zn ²⁺ Figure 3.13 (pH 9.0) - A1 ³⁺ Minor changes N / a N / a A1 ₂ (SO ₄ ) ₃ insoluble at pH 9.0

Samples of levofloxacin complexes

The efficacy and pharmacokinetics of seven samples of levofloxacin complexes were evaluated in vivo. Information on tested samples is given in the following table 35.

Table 35 The molar ratio of the complexes of levofloxacin Individual sample number Cation Used molar ratio The total content of levofloxacin (mg / ml) The final pH of the solution NB-049-001-06-066A Mg ²⁺ 1: 1 40,2 6.24 NB-049-001-06-066B Fe ²⁺ 1: 1 40.1 6.30 NB-049-001-06-066C Mg ²⁺ 1: 1 202 5.98 NB-049-001-06-081A Ca ²⁺ 1: 1 40.1 6.53 NB-049-001-06-081B Ca ²⁺ 1: 1 201 6.04 NB-049-001-06-081C Zn ²⁺ 1: 1 40 6.33 NB-049-001-06-081D Zn ²⁺ 1: 1 200 5.69

Conclusions and next steps

The results obtained in the studies of the authors with double titration indicate that levofloxacin forms 2: 1 complexes with all divalent metal cations. The binding constants (log K _b ) for complexation with Ca ²⁺ , Mg ²⁺ , Fe ²⁺ and Zn ²⁺ are 2.75, 3.69, 4.44 and 4.54, respectively.

Example 11 - Preparations of levofloxacin and hemifloxacin with organic acids

Experimental methodology

A solution of levofloxacin was prepared by dissolving either 50 or 100 mg of levofloxacin base in 15-20 ml of water. The initial pH of the solution of levofloxacin in water was approximately 7.3. The pH of the solution was adjusted with an approximately 10% solution of acid in water. The following acids were used to adjust the pH of the levofloxacin solution: acetic acid, ascorbic acid, citric, lactic, tartaric and propionic acids. After adjusting the volume of the solution to approximately 90% of the final volume, the osmolality of the solution was measured and adjusted to 300 mosm / kg using approximately 20% sodium chloride in water. After adjusting the pH and osmolality, the volume of the solution was adjusted to about 25 ml with water and its surface tension was measured. After adjusting the volume, pH and osmolality were measured, and the measurement results are presented in table 36. (The exact amounts of suspended levofloxacin, the acid needed to adjust the pH, sodium chloride needed to adjust the osmolality, and the final solution volume are shown in table 36). The content of levofloxacin in solutions was determined by HPLC.

results

Detailed information on levofloxacin preparations with organic acids is presented in table 36. The HPLC results are presented in table 37.

When tartaric acid was used to adjust the pH of the levofloxacin solution to 100 mg / ml, a precipitate formed.

Note: Solutions with acetic acid, citric acid and ascorbic acid were prepared anew for HPLC analysis, and therefore the theoretical concentrations for these solutions in table 36 and table 37 are different.

Organic base hemifloxacin preparations

Experimental Methodology and Results

Hemifloxacin with sodium ascorbate

50.30 mg of hemifloxacin mesylate (equivalent to 40.37 mg of hemifloxacin) was added to 1.5 ml of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. After filtration, 1.3 ml of a solution having a pH of 4.28 was obtained. The pH of this solution was adjusted to 5.48 with 400 μl of a 10% solution of sodium ascorbate in water (the amount of base required to adjust pH = 0.04 g). The osmolality of this solution was 308 mosm / kg, and therefore, sodium chloride was not used to increase the osmolality. The final solution volume was 1.7 ml. * The theoretical concentration of hemifloxacin in this formulation would be 20.59 mg / ml.

Table 36

Organic Levofloxacin Preparations

* Theoretical concentration = theoretical amount of hemifloxacin in the filtered solution (in this case 35 mg of hemifloxacin in the filtered 1.3 ml) / final solution volume (in this case 1.7 ml).

Table 37 Theoretical and measured concentrations of levofloxacin in preparations Acid Theoretical conc. (mg / ml) Concentration (mg / ml) measured by HPLC Acetic acid 50.05 51.45 Acetic acid 99.9 102.32 Lemon acid 49.91 50.31 Lemon acid 99.86 102,99 L-ascorbic acid 49.95 50.01 L-ascorbic acid one hundred 102,49 Lactic acid 50.05 50.07 Lactic acid 97.54 95.27 Wine acid 49.74 51.07

Note: Solutions with acetic acid, citric acid and ascorbic acid were prepared anew for HPLC analysis, and therefore the theoretical concentrations for these solutions in table 36 and table 37 are different.

Hemifloxacin with sodium lactate

50.05 mg of hemifloxacin mesylate (equivalent to 40.17 mg of hemifloxacin) was added to 1.8 ml of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. After filtration, 1.52 ml of a solution having a pH of 4.21 was obtained. The pH of this solution was adjusted to 5.42 with 180 μl of a 20% solution of sodium lactate in water (the amount of base required to bring pH = 0.036 g). The osmolality of this solution was 478 mosm / kg. The final solution volume was 1.7 ml. The theoretical concentration of gemifloxacin in this formulation would be 19.95 mg / ml.

Hemifloxacin with sodium acetate

50.47 mg of hemifloxacin mesylate (equivalent to 40.50 mg of hemifloxacin) was added to 2.0 ml of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. After filtration, 1.77 ml of a solution having a pH of 4.40 was obtained. The pH of this solution was adjusted to 5.40 with 50 μl of a 10% solution of sodium acetate in water (the amount of base required to bring pH = 0.005 g). The osmolality of this solution was 192 mosm / kg. The osmolality of this solution was adjusted to 295 mosm / kg with 28 μl of a 20% solution of sodium chloride in water.

Hemifloxacin with sodium propionate

50.00 mg of hemifloxacin mesylate (equivalent to 40.13 mg of hemifloxacin) was added to 1.9 ml of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. After filtering, 1.39 ml of a solution having a pH of 4.32 was obtained. The pH of this solution was adjusted to 5.50 with 30 μl of a 20% solution of sodium propionate in water (the amount of base required to adjust pH = 0.006 g). The osmolality of this solution was 183 mosm / kg. The osmolality of this solution was adjusted to 296 mosm / kg with 25 μl of a 22% solution of sodium chloride in water. This solution was prepared again with an increase in osmolality to 237 mosm / kg.

Hemifloxacin with sodium citrate

49.92 mg of hemifloxacin mesylate (equivalent to 40.06 mg of hemifloxacin) was added to 1.9 ml of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. After filtration, 1.63 ml of a solution having a pH of 4.20 was obtained. The pH of this solution was adjusted to 5.39 with 15 μl of a 20% solution of sodium citrate in water (the amount of base required to adjust pH = 0.003 g).

Example 12 Microspheres of Levofloxacin

The aim of this study was to obtain levofloxacin in various forms of microspheres, which can acquire the properties of improving taste and improving the configuration of AUC by reducing solubility and / or decomposition. These advantages can improve the pharmacodynamic properties of levofloxacin after pulmonary administration using either a suspension of nanoparticles or powder inhalation preparations. These drugs are optimized to increase the duration of levofloxacin release from forms with less solubility or decomposition. These properties can also be imparted to other fluoroquinolone antibiotics, including without limitation hemifloxacin, gatifloxacin, norfloxacin, tosufloxacin, sitafloxacin, sarafloxacin, prulifloxacin and pazufloxacin. Currently, studies are underway to characterize hemifloxacin microspheres to correct taste, improve AUC configuration, as well as introduce nanoparticles in suspension and powder inhalation. Other approaches for powder administration currently under study include spray drying and in situ micronization methods.

Preparation for the preparation of levofloxacin

Preliminary research

Preliminary studies were performed to determine the solubility of levofloxacin and polymers in various solvents, which are suggested for use in the processing process.

Obtaining microspheres

To obtain polymer microparticles loaded with levofloxacin, the spray drying method is used. The preparation of microspheres typically involves dissolving the drug and polymer in a suitable solvent. The solution is spray dried by spray drying to evaporate the solvent, thus capturing the drug in the polymer matrix. Optimization of the production parameters (drug: polymer ratio, polymer solution concentration and manufacturing parameters) is carried out to achieve the desired microparticle size, optimal drug loading and in vitro drug release.

Microsphere characteristics

Microparticles are characterized with respect to their morphology using SEM microscopy or using the appropriate method (laser diffraction) to estimate their size.

The drug load is determined by extracting the drug from the microspheres in an appropriate solvent and analyzing the extract using UV / HPLC.

The release of the drug from the microspheres is carried out using a device for USP dissolution.

Example 13 - Inhalation toxicology in rats

In a 4-day non-GLP study of the effect of increasing doses of aerosolized levofloxacin on male and female Sprague-Dawley rats, a 25 mg / ml levofloxacin solution was administered for one hour on the first day, and a 50 mg / ml levofloxacin solution was administered for two hours in days on days 2 through 4. During the period of application of the procedures, no clinical signs of toxicity were found. Necropsy 24 hours after administration did not reveal any abnormalities.

In a GLP study of the effect of aerosol levofloxacin on male and female Sprague-Dawley rats, aerosol levofloxacin was administered daily at an average dose of 6.92 mg / kg / day to males and 10.04 mg / kg / day to females for 4 days, using a device for aerosol delivery only through the nose. The total exposure was 29 and 42 mg / kg for males and females, respectively, during the study period. Each dose was administered for 2 hours daily. The dose for this study was chosen based on the maximum solubility of levofloxacin, which can be entered into the device for 2 hours. No clinical signs of toxicity were observed, and all animals survived for a 4-day treatment period. Animal necropsy did not reveal any abnormalities after the last dose.

In a 28-day GLP study on Sprague-Dawley rats, randomized groups of animals were compiled for 3 dose levels of aerosol levofloxacin or saline. Additional recovery groups were also injected with the control vehicle medium and the highest dose, and were observed for a 14 day recovery period after the last dose. The average dose of aerosol levofloxacin was 1.49; 3.63 and 7.29 mg / kg / day for males and 2.20; 5.35 and 11.01 mg / kg / day for females. The total effects over the 28-day period of the procedures ranged from 41.7 to 204.1 mg / kg for males and from 61.6 to 308.3 mg / kg for females. Each dose was administered for 2 hours daily. No evidence of toxicity associated with the administered doses was observed, and all animals survived for a 28-day treatment period. Animal necropsy after the last dose was administered revealed respiratory throat flat cell hyperplasia associated with the administered doses, the severity of which decreased over the 14-day recovery period.

Claims (122)

1. A pharmaceutical composition for inhalation administration comprising an aqueous solution of levofloxacin or ofloxacin and a divalent or trivalent cation, the solution being suitable for inhalation into the lung. 2. The composition according to claim 1, where the specified solution is presented in the form of an aerosol. 3. The composition according to claim 1 or 2, where the divalent or trivalent cation is magnesium. 4. The composition according to claim 1 or 2, where the solution contains magnesium chloride. 5. The composition according to claim 4, where the concentration of magnesium chloride is from about 25 mmol to about 400 mmol. 6. The composition according to claim 4, where the concentration of magnesium chloride is from about 100 mmol to about 250 mmol. 7. The composition according to claim 4, where the concentration of magnesium chloride is from about 125 mmol to about 250 mmol. 8. The composition according to claim 1 or 2, where the divalent or trivalent cation is selected from one or more of calcium, aluminum, zinc and iron. 9. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin greater than about 10 mg / ml 10. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin greater than about 25 mg / ml 11. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin greater than about 35 mg / ml 12. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin greater than about 40 mg / ml 13. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin greater than about 50 mg / ml 14. The composition according to claim 1 or 2, where the solution has a concentration of levofloxacin or ofloxacin 100 mg / ml 15. The composition according to claim 1 or 2, where the solution has an osmolality of from about 200 mosmol / kg to about 1250 mosmol / kg. 16. The composition according to claim 1 or 2, where the solution has an osmolality of from about 250 mosmol / kg to about 1050 mosmol / kg 17. The composition according to claim 1 or 2, where the solution has an osmolality of from about 350 mosmol / kg to about 750 mosmol / kg 18. The composition according to claim 1 or 2, where the solution has a pH from about 4.5 to about 7.5. 19. The composition according to claim 1 or 2, where the solution has a pH from about 5 to about 6.5. 20. The composition according to claim 1 or 2, where the solution has a pH from about 5.5 to about 6.5. 21. The composition according to claim 1 or 2, where the solution contains levofloxacin in a concentration of from about 100 mmol to about 625 mmol. 22. The composition according to claim 1 or 2, where the solution contains more than about 50 mg / ml levofloxacin, has a pH from about 5.5 to about 6.5 and an osmolality from about 350 mosmol / kg to about 750 mosmol / kg. 23. The composition according to claim 1 or 2, containing a sweetener. 24. The composition according to item 23, where the sweetener is aspartame or sucralose. 25. The composition according to item 23, where the sweetener is a mono - or disaccharide. 26. The composition according to item 23, where the sweetener is lactose, sucrose, dextrose or glucose. 27. The composition according to claim 1 or 2, containing another antimicrobial agent. 28. The composition according to item 27, where another antimicrobial agent is an aminoglycoside. 29. The composition according to p, where the aminoglycoside is tobramycin. 30. The composition according to item 27, where another antimicrobial agent is a polymyxin. 31. The composition of claim 30, wherein the polymyxin is colistin. 32. The composition according to item 27, where another antimicrobial agent is a monobactam. 33. The composition according to p, where the monobactam is aztreonam. 34. The composition according to item 27, where another antimicrobial agent is a macrolide or ketolide. 35. The composition according to item 27, where another antimicrobial agent is a glycopeptide. 36. The composition of claim 35, wherein the glycopeptide is vancomycin. 37. The composition of claim 27, wherein the other antimicrobial agent is fluoroquinolone. 38. The composition of claim 37, wherein fluoroquinolone is selected from the group consisting of lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloksatsina, rufloksatsina, fleroxacin, balofloksatsina, sparfloxacin, trovafloxacin, enoxacin, norfloxacin, klinafloksatsina, grepafloksatsina, sitafloxacin, temafloxacin, marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, difloxacin, enrofloxacin, garenoxacin, prulifloxacin, olamufloxacin, DX-619, TG-873870 and DW. 39. The composition according to claim 1 or 2, containing one or more of dornase alpha, hypertonic drug, mannitol and sodium chloride. 40. The composition according to claim 1 or 2, where the solution contains levofloxacin and magnesium cations, has a concentration of greater than about 50 mg / ml, has an osmolality of from about 350 mosmol / kg to about 750 mosmol / kg and has a pH of from about 5, 5 to about 6.5. 41. The composition according to claim 2, having a mass average aerodynamic diameter of from about 2 microns to about 5 microns with a geometric standard deviation of less than or equal to about 2.5 microns. 42. The composition according to claim 2, having a mass average aerodynamic diameter of from about 2.8 microns to about 4.3 microns with a geometric standard deviation of less than or equal to about 2 microns. 43. The composition according to claim 2, having a mass average aerodynamic diameter of from about 2.5 microns to about 4.5 microns with a geometric standard deviation of less than or equal to about 1.8 microns. 44. A sterile disposable container containing the composition according to claim 1. 45. The container according to item 44, containing from about 1 ml to about 5 ml of solution. 46. The container according to item 44, containing from about 20 mg to about 400 mg of levofloxacin. 47. The container according to item 44, containing from about 28 mg to about 280 mg of levofloxacin. 48. The container according to item 44, containing at least about 100 mg of levofloxacin. 49. The container according to item 44, containing at least approximately 400 mg of levofloxacin. 50. A kit comprising: a container according to any one of claims 44-49 and a nebulizer adapted to be aerosolized for delivery to the lung by oral inhalation. 51. The kit according to item 50, where the atomizer operates on the principle of ultrasonic atomization. 52. The kit according to item 50, where the spray operates on the principle of hydraulic atomization. 53. The kit according to item 50, where the atomizer operates on the principle of vibrating cells. 54. The kit according to item 50, containing the second container, which contains another antimicrobial agent. 55. The kit according to item 54, where another antimicrobial agent is an aminoglycoside. 56. The kit of claim 55, wherein the aminoglycoside is tobramycin. 57. The kit of claim 54, wherein the other antimicrobial agent is polymyxin. 58. The kit of claim 57, wherein the polymyxin is colistin. 59. The kit of claim 54, wherein the other antimicrobial agent is monobactam. 60. The kit according to § 59, where monobactam is aztreonam. 61. The kit of claim 54, wherein the other antimicrobial agent is macrolide or ketolide. 62. The kit of claim 54, wherein the other antimicrobial agent is glycopeptides. 63. The kit of claim 62, wherein the glycopeptide is vancomycin. 64. The kit of claim 54, wherein the other antimicrobial agent is fluoroquinolone. 65. The kit of claim 64, wherein fluoroquinolone is selected from the group consisting of lomefloxacin, pefloxacin, ciprofloxacin, gatifloxacin, gemifloxacin, moxifloxacin, tosufloxacin, pazufloksatsina, rufloksatsina, fleroxacin, balofloksatsina, sparfloxacin, trovafloxacin, enoxacin, norfloxacin, klinafloksatsina, grepafloksatsina, sitafloxacin, temafloxacin, marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, difloxacin, enrofloxacin, garenoxacin, prulifloxacin, olamufloxacin, DX-619, TG-873870 and DW. 66. The kit of claim 50, comprising one or more of dornase alpha, a hypertonic drug, mannitol, and sodium chloride. 67. A system comprising:
a reservoir containing an aqueous solution of levofloxacin or ofloxacin and a divalent or trivalent cation, the solution being suitable for inhalation into the lung; and
a nebulizer adapted for aerosolization of a solution for delivery to the lung by oral inhalation. 68. The system of claim 67, wherein the atomizer operates on the principle of ultrasonic atomization. 69. The system of claim 67, wherein the atomizer operates on the principle of hydraulic atomization. 70. The system of claim 67, wherein the atomizer operates on the principle of vibrating cells. 71. The system of claim 70, wherein the atomizer operating on the principle of vibrating cells is a RAM E-FLOW® atomizer. 72. The system of claim 67, having a mass average aerodynamic diameter of from about 2 microns to about 5 microns with a geometric standard deviation of less than or equal to about 2.5 microns. 73. The system of claim 67, having a mass average aerodynamic diameter of from about 2.8 microns to about 4.3 microns with a geometric standard deviation of less than or equal to about 2 microns. 74. The system of claim 67, having a mass average aerodynamic diameter of from about 2.5 microns to about 4.5 microns with a geometric standard deviation of less than or equal to about 1.8 microns. 75. The system of claim 67, wherein the solution contains levofloxacin at a concentration of from about 100 mmol to about 625 mmol and a concentration of magnesium chloride is from about 125 mmol to about 250 mmol, has a pH of from about 5.5 to about 6.5, and an osmolality from about 350 mosmol / kg to about 750 mosmol / kg. 76. A method for the treatment of pulmonary infection, chronic obstructive pulmonary disease, or cystic fibrosis, comprising administering the composition of claim 2 to a patient in need thereof. 77. The method according to claim 76, wherein the pulmonary infection is caused by one or more of the following bacteria: Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Aeromonas hydrophilium coli salii, Colomilla salmonidae, Escherichia coli salmonidae, typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Morganella morgani maleniens Proteus Princi Proteus Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, H aemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurella multocida, Pasteurella haemolytica, Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholera, Vibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhoeae , Neisseria meningitidis, Burkholderia cepacia, Francisella tularensis, Kingella and Moraxella. 78. The method of claim 76, wherein the pulmonary infection is caused by one or more of the following bacteria: Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Haemophilus influenzae, Burkholderia cepacia, and Moraxella. 79. The method according to p, where the pulmonary infection is caused by the bacterium Burkholderia. 80. The method according to p, where the pulmonary infection is pneumonia. 81. The method according to p, where the pulmonary infection is caused by gram-negative anaerobic bacteria. 82. The method of claim 76, wherein the pulmonary infection is caused by one or more bacteria selected from the group consisting of Bacteroides fragilis, Bacteroides distasonis, homologous group Bacteroides 3452A, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Biieroides egg and Bacteroides splanchnicus. 83. The method according to p, where the pulmonary infection is caused by gram-positive bacteria. 84. The method of claim 76, wherein the pulmonary infection is caused by one or more bacteria selected from the group consisting of Corynebactermm diphtheriae, Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus milleri; Streptococcus (Group G); Streptococcus (group C / F); Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp.hyicus, Staphylococcus haemolyticus, Staphylococcusphomichis. 85. The method according to p, where the pulmonary infection is caused by gram-positive anaerobic bacteria. 86. The method of claim 76, wherein the pulmonary infection is caused by one or more bacteria selected from the group consisting of Clostridium difficile, Clostridium perfringens, Clostridium tetini, and Clostridium botulinum. 87. The method according to p, where the pulmonary infection is caused by acid-resistant bacteria. 88. The method of claim 76, wherein the pulmonary infection is caused by a Mycobacterium bacterium. 89. The method of claim 76, wherein the pulmonary infection is caused by one or more bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium leprae. 90. The method according to p, where the pulmonary infection is caused by atypical bacteria. 91. The method of claim 76, wherein the pulmonary infection is caused by one or more bacteria selected from the group consisting of Chlamydia pneumoniae and Mycoplasma pneumoniae. 92. The method according to p, where the solution contains levofloxacin and magnesium cations, has a concentration of greater than about 50 mg / ml, has an osmolality of from about 350 mosmol / kg to about 750 mosmol / kg and has a pH of from about 5.5 to approximately 6.5. 93. The method according to any one of claims 76-91, wherein the divalent or trivalent cation is selected from one or more magnesium, calcium, aluminum, zinc and iron. 94. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2 microns to about 5 microns with a geometric standard deviation of less than or equal to about 2.5 microns. 95. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2.5 microns to about 4.5 microns with a geometric standard deviation less than or equal to about 1.8 microns. 96. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2.8 microns to about 4.3 microns with a geometric standard deviation of less than or equal to about 2 microns. 97. The method according to p, where the aerosol is formed due to the atomizer, acting on the principle of vibrating cells. 98. The method according to p. 97, where the atomizer, operating on the principle of vibrating cells, is a PARI E-FLOW® atomizer. 99. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2 microns to about 5 microns with a geometric standard deviation less than or equal to about 2.5 microns. 100. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2.8 microns to about 4.3 microns with a geometric standard deviation of less than or equal to about 2 microns. 101. The method according to p, where the aerosol has a mass average aerodynamic diameter of from about 2.5 microns to about 4.5 microns with a geometric standard deviation of less than or equal to about 1.8 microns. 102. The method of claim 97, wherein the nebulizer is designed to deliver at least about 20 mg of levofloxacin or ofloxacin to the lung. 103. The method of claim 97, wherein the nebulizer is designed to deliver at least about 100 mg of levofloxacin or ofloxacin to the lung. 104. The method of claim 97, wherein the nebulizer is designed to deliver at least about 125 mg of levofloxacin or ofloxacin to the lung. 105. The method of claim 97, wherein the nebulizer is designed to deliver at least about 150 mg of levofloxacin or ofloxacin to the lung. 106. The method of claim 97, wherein the nebulizer is designed to deliver aerosol to the lung for less than about 10 minutes. 107. The method of claim 97, wherein the nebulizer is designed to deliver aerosol to the lung for less than about 5 minutes. 108. The method of claim 97, wherein the nebulizer is designed to deliver aerosol to the lung for less than about 3 minutes. 109. The method of claim 97, wherein the nebulizer is designed to deliver aerosol to the lung for less than about 2 minutes. 110. The method according to p, in which the aerosol is intended for alternate administration with a second inhaled antimicrobial agent. 111. The method according to p, in which the second inhaled antimicrobial agent is an aminoglycoside. 112. The method according to p. 111, in which the aminoglycoside is tobramycin. 113. The method according to p, in which another antimicrobial agent is a polymyxin. 114. The method according to p, in which the polymyxin is a colistin. 115. The method according to p, where another antimicrobial agent is a monobactam. 116. The method according to p. 115, where monobactam is aztreonam. 117. A method of masking the taste of fluoroquinolone, which is a combination of fluoroquinolone with a divalent cation. 118. The method according to p. 117, where the divalent cation is selected from one or more of magnesium, calcium, zinc and iron. 119. The method of claim 117, wherein the fluoroquinolone is levofloxacin or ofloxacin. 120. A method according to p.117, where fluoroquinolone is selected from the group consisting of ciprofloxacin, gatifloxacin, gemifloxacin, norfloxacin, moxifloxacin, Marbofloxacin, Orbifloxacin, sparfloxacin, tosufloxacin, trovafloxacin, pefloxacin, lomefloxacin, klinafloksatsina, prulifloksatsina, sarafloksatsina, and sitafloxacin pazufloksatsina. 121. The method of claim 117, wherein the fluoroquinolone and divalent cation are combined in one solution. 122. The method of claim 117, further comprising aerosolizing the combination.

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