Aerosolized fluoroquinolones and uses thereof

ES 2 599 313 T3

DESCRIPTION

Aerosol fluoroquinolones and their uses

Related requests

Background of the invention

Description of Related Art

Antibiotics have been effective tools in the treatment of infectious diseases for the last half century. Since the development of antimicrobial therapy in the late 1980s, most bacterial infections present in patients in developed countries could be controlled unless the infection occurs in an organ or environment where antibiotics are ineffective or difficult to deliver. , such as bacterial infections of the circulatory system in patients with sepsis or bacterial infections of the lungs in cystic fibrosis. However, even in ordinary infections, in response to the pressure of antimicrobial use, multiple mechanisms of resistance have become widespread and threaten the clinical utility of even the most aggressive antibacterial therapy. The rise of resistant antimicrobial strains has been particularly common in major hospitals and care centers. The consequences of the increase in resistant strains include increased morbidity and mortality, longer patient hospitalization, and increased treatment costs.

Bacteria have developed several different mechanisms to overcome the action of antimicrobials. These resistance mechanisms may be specific to a molecule or family of antimicrobials, or they may be non-specific and involved in resistance to unrelated antimicrobials. Several mechanisms of resistance can exist in a single bacterial strain, and these mechanisms can act independently or they can act synergistically to overcome the action of an antimicrobial or a combination of antimicrobials. Specific mechanisms include degradation of the drug, inactivation of the drug by enzymatic modification, and alteration of the drug's target. There are, however, more general mechanisms of drug resistance, in which antimicrobial access to the target is prevented or reduced by decreasing antimicrobial transport into the cell or by increasing drug efflux from the cell. to the outside environment. Both mechanisms can reduce the drug concentration at the target site and allow bacterial survival in the presence of one or more antimicrobials that could otherwise inhibit or kill bacterial cells. Some bacteria use both mechanisms, combining a low permeability of the cell wall (which includes membranes) with an active efflux of antimicrobials.

Patent document WO0218345 describes certain 7-substituted amino-6-fluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylic acids and pharmaceutically acceptable salts or esters thereof having a solubility enhancing portion at the 1-positions and / or 7 or prodrugs thereof, and the uses of the compounds in the treatment of bacterial infections. Compounds are stated to be suitable for inhalation delivery for the treatment of lung infections.

Brief description of the invention

According to one aspect of the invention, there is provided a pharmaceutical composition comprising a solution of a fluoroquinolone and a divalent or trivalent cation, wherein the fluoroquinolone is levofloxacin or ofloxacin.

Further provided is an aerosol of a solution comprising a fluoroquinolone and a divalent or trivalent cation, wherein the fluoroquinolone is levofloxacin or ofloxacin.

In addition, a sterile single-use container is provided, comprising the pharmaceutical composition.

In addition, a kit is provided that comprises a pharmaceutical composition comprising a solution of levofloxacin or ofloxacin and a divalent cation in a sterile container, wherein the solution of levofloxacin or ofloxacin has an osmolality greater than approximately 150 mOsmol / kg, and a nebulizer adapted to aerosolize the concentrated solution of levofloxacin or ofloxacin for delivery to a lower respiratory tract through oral inhalation.

Further provided is the use of the pharmaceutical composition in the preparation of a medicament, preferably an aerosol, to treat: (i) a pulmonary infection or cystic fibrosis, preferably cystic fibrosis; or (ii) pneumonia, a chronic obstructive pulmonary disease, or sinusitis.

Further provided is the use of the pharmaceutical composition in the preparation of an aerosol for treating or preventing a microbial infection in a patient with a concentration in a patient's lung of at least 32 pg / ml of the levofloxacin or ofloxacin, preferably a concentration of the lung of at least 128 pg / ml of levofloxacin or ofloxacin, more preferred a concentration in the lung of at least 512 pg / ml of levofloxacin or ofloxacin, even more preferred is a lung concentration of 800 pg / mL to 1600 pg / mL of levofloxacin or ofloxacin.

ES 2 599 313 T3

Further provided is a method for masking the taste of a fluoroquinolone selected from levofloxacin or ofloxacin, which comprises complexing the fluoroquinolone with a divalent or trivalent cation.

Various modalities provide compositions and methods for optimal antimicrobial activity for the treatment of lung and respiratory tract infections in human and / or veterinary subjects using rapid, short-term aerosol administration, and through delivery of drug exposure to high concentration directly to the affected tissue. Specifically, in some embodiments, concentrated doses of agents from the fluoroquinolone class of antibiotics are delivered to produce peak concentrations of the active drug in the respiratory, pulmonary, and other non-oral topical compartments, including, but not limited to, skin, rectum, vagina, urethra, urinary bladder, eye and ear. Because different pharmaceuticals are known to produce different antimicrobial effects depending on dose, form, concentration, and delivery profile, some modalities provide specific formulation and delivery parameters that produce antimicrobial results that are therapeutically significant. The invention includes, but is not limited to, specific fluoroquinolone antibiotics, such as levofloxacin, formulated to allow aerosol administration meeting specific concentrations and antimicrobial criteria necessary to treat patients with various bacterial infections. These formulations and methods are useful with commercially available inhalation devices for one or more therapeutic aerosol opportunities.

Aerosol administration directly to the nasal, sinus, respiratory tract and pulmonary compartments through intranasal or oral inhalation allows the delivery of high concentration drugs to a site of respiratory infection with decreased risk of extra-respiratory toxicity associated with the non-respiratory routes of drug delivery. In addition, direct administration to the site of infection allows very high local drug levels, a property that allows rapid administration, high concentration, local exposure eliminating the special effect of this class of antibiotic. Consequently, because the microbial killing effect of a particular antibiotic compound and therapeutic composition varies depending on formulation and delivery parameters, new compositions and delivery methods can be developed for existing drug compounds that are reformulated. and they manage through new existing supply techniques. Other topical infections may also benefit from discovery through direct exposure to high-concentration fluoroquinolone on infected skin, rectum, vagina, urethra, urinary bladder, eye, and ear.

Members of the fluoroquinolone drug class exhibit unique pharmacological properties, including bioavailability (F), mean absorption time (MAT) from lungs, peak drug concentrations in epithelial lining fluid, bronchial lavage fluid, sputum and / or lung tissue (Cmax) after aerosol administration, lung retention time, area under the curve (AUC), minimum inhibitory concentrations (MIC) of the antibiotic required for antibacterial activity, the AUC / MIC ratio, and local and systemic safety. Specific to the invention is the use of short-term, rapid aerosol administration, which delivers high concentration of drug exposure directly to the affected tissue (ELF, sputum, BAL, tissue) through the delivery of the aerosol for treatment. of bacterial infection in animals and humans.

In addition to the clinical and pharmacological requirements present in any composition intended for therapeutic administration, many physicochemical factors unique to a pharmaceutical compound must also be considered. These include, but are not limited to, aqueous solubility, viscosity, partition coefficient (LogP), predicted stability in various formulations, osmolality, surface tension, pH, pKa, pKb, dissolution rate, sputum permeability, binding / inactivation of the sputum, taste, throat irritability and acute tolerance.

Other factors to consider when designing the product form include the physical chemistry of fluoroquinolone and antibacterial activity, indication of disease, clinical acceptance, and patient compliance.

Combined with the shape of the product is the consideration of the packaging. By non-limiting example, considerations for packaging include the intrinsic stability of the product, the need for lyophilization that provides stability, device selection (eg, liquid nebulizer, dry powder inhalers, metered dose inhaler), and packaging form. (eg, simple liquid or complex liquid formulation in a vial in liquid or lyophilized form that dissolves before or after insertion into the device; complex suspension formulations in either a liquid or lyophilized bottle with or without a soluble excipient / salt component that dissolves before or after insertion into the device, or in separate packaging of the liquid and solid components ; dry powder formulations in a bottle, capsule or blister pack, and other formulations packaged as low solubility or readily soluble solid agents in separate containers alone or together with easily soluble or low solubility solid agents. Any agent packaged separately will be manufactured to mix before or after insertion into the delivery device).

In some aspects, the present invention relates to the aerosol and topical delivery of antimicrobials of fluoroquinolone, such as levofloxacin. Levofloxacin has favorable solubility characteristics that allow dosing of clinically desirable levels of fluoroquinolone by aerosol (eg, via liquid nebulization, dry powder dispersion, or metered dose administration) or topically (eg, suspension aqueous, oily preparation or the like or as a drip, spray, suppository, ointment, or an ointment or the like) and can be used in methods for the acute or prophylactic treatment of an infected vertebrate, for

ES 2 599 313 T3 example, a bacterial Infection, or a subject at risk of an Infection. Other fluoroquinolone antimicrobials include ofloxacin.

A method of treating a bacterial infection in a subject is described using concentrated aerosol levofloxacin which is administered to a subject infected with a pathogenic bacteria in the lungs.

The therapeutic method may further include a diagnostic step, such as identifying a patient infected with a particular pathogenic bacterium, or a resistant bacterium. In some embodiments, the method further includes identifying a patient who becomes colonized with a bacterium that is capable of developing resistance to the fluoroquinolone antimicrobials. In some embodiments, the delivered amount of levofloxacin aerosol is sufficient to overcome resistance or prevent the development of resistance to levofloxacin. In one embodiment, the MIC of the fluoroquinolone antibacterial compound for the microbe is greater than about 2 ug / ml.

In another embodiment, the delivered amount of the aerosolized levofloxacin is sufficient to overcome resistance or further prevent resistance of an organism exhibiting an MIC of the antibacterial compound of fluoroquinolone that is greater than about 4 ug / ml.

In another embodiment, the delivered amount of aerosolized fluoroquinolone is sufficient to overcome resistance or further prevent resistance of an organism exhibiting an MIC of the antibacterial compound of fluoroquinolone that is greater than about 8 ug / ml.

In another embodiment, the delivered amount of aerosolized fluoroquinolone is sufficient to overcome resistance or further prevent resistance of an organism exhibiting an MIC of the antibacterial compound of fluoroquinolone that is greater than about 16 ug / ml.

In another embodiment, the delivered amount of aerosolized fluoroquinolone is sufficient to overcome resistance or further prevent resistance of an organism exhibiting an MIC of the antibacterial fluoroquinolone compound that is greater than about 32 ug / ml.

Further described is a method for the prophylactic treatment of a subject, which includes administering to a subject, susceptible to microbial infection or a chronic carrier of an asymptomatic or poorly symptomatic microbial infection, a fluoroquinolone antimicrobial to achieve a minimal inhibitory concentration. of antimicrobials at a potential or current site of infection. In one embodiment, the method further comprises identifying a subject as a subject at risk for a bacterial infection or at risk for an exacerbation of an infection.

Also described is a method for the acute or prophylactic treatment of a patient through the administration of aerosolized fluoroquinolone to produce and maintain threshold drug concentrations in the lung, which can be measured as drug levels in the lining fluid. epithelial (ELF), sputum, lung tissue, or bronchial lavage fluid (BAL). One embodiment includes the use of short-term, rapid aerosol delivery that delivers high concentration of drug exposure directly to affected tissue for the treatment of bacterial infections in animals and humans.

Further described is a method of treating a microbial infection in a subject, which includes administering to a subject infected with a microbe, a fluoroquinolone antimicrobial to achieve a minimal inhibitory concentration of the antimicrobials at a site of infection. In one embodiment, the method further comprises identifying the subject as infected with a microbe that is resistant to an antimicrobial agent.

Further described is a method for the acute or prophylactic treatment of a patient through non-oral or non-nasal topical administration of fluoroquinolone to produce and maintain threshold drug concentrations at the site of infection or at risk of infection. One modality includes the use of short-term, rapid aerosol delivery that delivers high concentration of drug exposure directly to affected tissue for the treatment or prevention of bacterial skin, rectal, vaginal, urethral, ​​ocular infections. , and the ear tissues.

Further described is a method for administering a fluoroquinolone antimicrobial agent by inhalation, wherein the inhaled dry powder or liquid aerosol has a mean particle size of between approximately 1 micron to 10 microns in mass median aerodynamic diameter and a standard deviation geometric 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 mass median aerodynamic diameter and a geometric standard deviation of the particle size of 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 above-described methods, the minimum antimicrobial inhibitory concentration of fluoroquinolone remains at the site of infection for at least a period of about 5 minutes, at least about a period of 10 min, at least about a period of 20 minutes. min, at least about a 30 min period, at least about a 1 hour period, 2 hour period, at

ES 2 599 313 T3 minus approximately a 4 hour period or other time values ​​in the Quarter Hour Interval. The effective antimicrobial minimum inhibitory concentration (MIC) of fluoroquinolone is sufficient to cause a therapeutic effect and the effect can be localized to the site of infection. In some modalities, one or more administrations of levofloxacin achieve a concentration of fluoroquinolone in ELF, BAL, and / or sputum of at least 1 time to 5000 times the MIC of infecting or potentially infecting organisms, including all integer values. in it, such as 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 128 times, 256 times, 512 times, 1028 times, 2056 times, and 4112 times the microbial MICs.

In some embodiments, such as a lung site, the fluoroquinolone antimicrobial is administered in one or more administrations to achieve a daily delivered respirable dose of at least about 5 mg to about 50 mg, including all integer values ​​therein as such as 10, 15, 20, 25, 30, 35, 40, and 45 milligrams. Similarly, the fluoroquinolone antimicrobial agent is administered in one or more administrations to achieve a daily delivered respirable dose of at least about 50 to about 100 mg including all integer values ​​therein, such as 55, 60, 65 , 70, 75, 80, 85, 90, and 95 mg. In some embodiments of the above-described methods, the fluoroquinolone antimicrobial is administered in one or more administrations to achieve a delivered respirable daily dose of up to 150 mg including all integer values ​​therein, such as 105, 110, 115, 120, 125, 130, 135, 140 and 145 mg. The fluoroquinolone antimicrobial is administered in the given delivered respirable dose in less than 20 minutes, less than 10 minutes, less than 7 minutes, less than 5 minutes, in less than 3 minutes, and in less than 2 minutes. In some embodiments of the above-described methods, the antimicrobial agent is preferably levofloxacin.

In some embodiments of the methods described above, the bacterium is a gram-negative bacterium such as Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes, Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas hydrophiliai, Salcherichia collibii typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartinia, Yuberniacinetobacteris colitis, Yuberniacinetobacteriscolus, Providencia stuartiniatobacteriscolus, Acuberiniaytobacteriscolus psebonytobacteria pseudiniatobacteris colitis , Yersinia intermedia, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus, Haemophilus parahaemolyticus, 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 distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii, and Bacteroides splanchnicus. In some embodiments of the above-described methods, the bacterium is a gram-negative anaerobic bacterium, by non-limiting example, these include Bacteroides fragilis, Bacteroides distasonis, homology group Bacteroides 3452A, Bacteroides vulgatus, Bacteroides ovalus, Bacteroides thetaiotaomicron eg Bacteroides uniformis, Bacteroides uniformisgerth , and Bacteroides splanchnicus. In some embodiments of the above-described methods, the bacterium is a gram-positive bacterium, by non-limiting example this includes: 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 above-described methods, the bacterium is an anaerobic Gram-positive bacterium, by non-limiting example, these include Clostridium difficile, Clostridium perfringens, Clostridium tetini, and Clostridium botulinum. In some embodiments of the above-described methods, the bacterium is an acid-fast bacterium, by non-limiting example, these include Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium leprae. In some embodiments of the above-described methods, the bacterium is an atypical bacterium, by non-limiting example, these include Chlamydia pneumoniae and Mycoplasma pneumoniae.

In some embodiments of the above-described methods, the subject is a human. In some embodiments of the above-described methods, the subject is a human with cystic fibrosis. In some embodiments of the above-described methods, the subject is a human with pneumonia, chronic obstructive pulmonary disease, or sinusitis, or a mechanically ventilated human.

In another embodiment, a pharmaceutical composition is provided that includes an antimicrobial formulation of the simple liquid fluoroquinolone (eg, soluble fluoroquinolone with non-encapsulated 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 permeating ion concentration of from about 30 mM to about 300 mM. In one embodiment, the osmolality is from about 250 mOsmol / kg to about 1050 mOsmol / kg. In one embodiment, the osmolality is preferably

ES 2 599 313 T3 about 350 mOsmol / kg and about 750 mOsmol / kg and with superlative preference about 300 mOsmol / kg.

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

In another embodiment, a pharmaceutical composition is provided that includes a taste masking agent. By non-limiting example, a taste masking agent can include a sugar, a divalent or trivalent cation that complexes with a fluoroquinolone, optimized osmolality, and / or an optimized permeating ion concentration.

In another mode, A system is provided for administering a fluoroquinolone antimicrobial including a container comprising a solution of a fluoroquinolone antimicrobial and a nebulizer physically coupled or co-packaged with the container and adapted to produce an aerosol of the solution having a particle size from about 2 microns to about 5 microns in mass median aerodynamic diameter and a geometric standard deviation of particle size of less of or equal to approximately 2.5 microns in 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 kit is provided that includes a container comprising a pharmaceutical formulation comprising a quinolone antimicrobial agent and an aerosolizer adapted to aerosolize the pharmaceutical formulation and deliver it to the lower respiratory tract and lung compartment after intraoral administration.

In another embodiment, a kit is provided that includes a container comprising a pharmaceutical formulation comprising a quinolone antimicrobial agent and an aerosolizer adapted to aerosolize the pharmaceutical formulation and deliver it to the nasal cavity after intranasal administration.

It should be understood that both the above general description and the following detailed description are illustrative and explanatory only and are not restrictive of the claimed invention.

Description of the figures

Figure 1 is a graph showing the dose: MIC ratio of fluoroquinolones and other antibiotics to kill bacteria.

Figure 2 is a graph showing serum concentrations of ciprofloxacin after oral dosing in both CF patients and healthy controls.

Figure 3 is a graph showing the concentrations of ciprofloxacin in sputum and serum after oral dosing.

Figure 4A is a graph showing that the destruction over time of levofloxacin affects the log phase of PAM1020 cells.

Figure 4B is a graph showing that the destruction over time of levofloxacin affects the log phase of PAM1032 cells.

Figure 5A is a graph showing that the destruction over time of Levofloxacin affects the stationary phase of PAM1020 cells.

Figure 5B is a graph showing that the destruction over time of Levofloxacin affects the stationary phase of PAM1032 cells.

Figure 6A is a graph showing re-growth of PAM 1020 after exposure to Levofloxacin for 10 minutes.

Figure 6B is a graph showing re-growth of PAM 1020 after exposure to Levofloxacin for 160 minutes.

Figure 6C is a graph showing re-growth of PAM 1032 after exposure to Levofloxacin for 10 minutes.

Figure 6D is a graph showing re-growth of PAM 1032 after exposure to Levofloxacin for 160 minutes.

Figure 7A is a graph showing that the destruction in time of Levofloxacin affects the late log phase of PAM1020 cells under oxygen limiting conditions.

Figure 7B is a graph showing that the destruction in time of Levofloxacin affects the late log phase of PAM1032 cells under oxygen limiting conditions.

Figure 8A is a graph showing the Levofloxacin kill kinetics of PAM1032 in MeullerHinton broth (MHB).

Figure 8B is a graph showing the Levofloxacin destruction kinetics of PAM1032 in cystic fibrosis sputum.

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Figure 9 is a graph showing that the destruction of Levofloxachine affects the bloofilms in Pseudomonas.

Figure 10 is a graph showing the bactericidal effects of Levofloxacin with a Cmax of 1000 pg / ml and a half-life of 10 minutes in a hollow fiber model.

Figure 11 is a graph showing the bactericidal effects of Levofloxacin with a Cmax of 600 pg / ml and a half-life of 10 minutes in a hollow fiber model.

Figure 12 is a graph showing the pH solubility profile of Levofloxacin by acid titration.

Figure 13 is a graph measuring pH while titrating Levofloxacin with HCl.

Figure 14 is a graph showing Vt [OH] vs. Vt of Levofloxacin.

Figure 15 is a graph measuring pH while titrating Levofloxacin with NaOH.

Figure 16 is a graph that measures dpH / dV vs. NaOH titrant volume (Vt) for Levofloxacin titration.

Figure 17 is a graph that measures the absorbance of a Levofloxacin solution at 257 nm vs. pH.

Figure 18 is a graph showing the complexation of Levofloxacin with divalent and trivalent cations.

Figure 19 is a graph showing the double titer of the complexation of Levofloxacin with Mg2 +.

Figure 20 is a graph showing the double titer of the complexation of Levofloxacin with Fe2 +.

Figure 21 is a graph showing the double titer of the complexation of Levofloxacin with Ca2 +.

Figure 22 is a graph showing the double titer of the complexation of Levofloxacin with Zn2 +.

Figure 23 is a graph showing Ca2 + complexed Levofloxacin vs. Free levofloxacin.

Figure 24 is a graph showing Mg2 + complexed Levofloxacin vs. Free levofloxacin.

Figure 25 is a graph showing Fe2 + complexed Levofloxacin vs. Free levofloxacin.

Figure 26 is a graph showing Zn2 + complexed Levofloxacin vs. Free levofloxacin.

Figure 27 is a graph showing the solubility of Levofloxacin in the presence of Mg2 +.

Figure 28 is a graph showing the solubility of Levofloxacin in the presence of Mg2 + at constant ionic strength.

Figure 29 is a graph showing the complexation of Levofloxacin with Fe2 +, as measured by spectrofluorometry.

Figure 30 is a graph showing the complexation of Levofloxacin with Zn2 + measured by spectrofluorometry.

Detailed description

Many of the problems associated with antimicrobial resistant pathogens can be alleviated if the concentration of the antimicrobial can be safely increased at the site of infection. For example, pulmonary infections can be treated by administering the antimicrobial agent directly, at high concentrations directly to the site of infection, without incurring large systemic concentrations of the antimicrobial. Accordingly, some of the modalities described in the present disclosure are improved methods for delivering drug compositions to treat bacterial lung infections. More specifically, as described herein, it has been discovered that aerosolized levofloxacin and other fluoroquinolones can be safely delivered by inhalation at levels sufficient to destroy susceptible bacterial infections, to decrease the frequency of antimicrobial resistance, and to increase efficacy against resistant lung infections.

Definitions

The term "administration" or "administering" refers to a method of giving a dosage of an antimicrobial pharmaceutical composition to a vertebrate. The preferred method of administration may vary depending on various factors, for example, the components of the pharmaceutical composition, the site of the potential or actual bacterial infection, the microbe involved, and the severity of an actual microbial infection.

A carrier or excipient is a compound or material used to facilitate administration of the compound, for example, to increase the solubility of the 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 oil, peanut and sesame oils. In addition, various adjuvants can be included, such as those commonly used in the art. These and other such compounds are described in the literature, for example, in the Merck Index, Merck & Company, Rahway, NJ. Considerations for the inclusion of 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 diagnosis as used in the present description is a compound, method, system or device that aids in the identification and characterization of a health condition or disease. The diagnosis can be used in standard tests as is known in the art.

The term mammal is used in its usual biological sense. Therefore, humans, cattle, horses, dogs, and cats are specifically included, but many other species are also included.

The term microbial infection refers to unwanted proliferation or the presence of invasion by microbes.

ES 2 599 313 T3 pathogens in a host organism. This includes the overgrowth of microbes that are normally 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 microbial population (s) is detrimental to a host mammal. Therefore, a microbial infection exists when an excessive number of a microbial population is present in or on the body of a mammal, or when the effects of the presence of a microbial population (s) damage cells or other tissues. of a mammal.

The term "pharmaceutically acceptable carrier or pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplemental active ingredients can further be incorporated into the compositions.

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

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

In the context of the response of a microbe, such as a bacterium, to an antimicrobial agent, the term "susceptibility" refers to the sensitivity of the microbe to the presence of the antimicrobial agent. Thus, increasing susceptibility means that the microbe will be inhibited by a lower concentration of the antimicrobial agent in the environment surrounding the microbial cells. This is equivalent to saying that the microbe is more sensitive to the antimicrobial agent. In most cases, the minimum inhibitory concentration (MIC) of this antimicrobial agent will have been reduced.

By therapeutically effective amount or pharmaceutically effective amount is meant a fluoroquinolone antimicrobial agent, as described for this invention, which has a therapeutic effect. Doses of the fluoroquinolone antimicrobial agent that are useful in treatment are therapeutically effective amounts. Therefore, as used herein, a therapeutically effective amount means those amounts of the fluoroquinolone antimicrobial agent that produce the desired therapeutic effect, as judged by the results of clinical trials and / or infection studies in the model. animal. In particular embodiments, the fluoroquinolone antimicrobial agent is administered in a predetermined dose, and therefore, a therapeutically effective amount can be an administered dose amount. This amount and the amount of the fluoroquinolone antimicrobial agent can be routinely determined by one of ordinary skill in the art, and will vary, depending on various factors, such as the particular microbial strain involved. This amount may also depend on the height, weight, sex, age and medical history of the patient. For prophylactic treatments, a therapeutically effective amount is that amount that can be effective in preventing a microbial infection.

A therapeutic effect alleviates, to some extent, one or more of the symptoms of the infection, and includes the cure of an infection. Cure means that the symptoms of active infection are eliminated, including the total or substantial removal of excess members of viable microbes from those involved in the infection to a point at or below the threshold of detection by traditional measurements. However, there may be certain long-term or permanent effects of the infection, even after a cure is obtained (such as extensive tissue damage). As used in the present description, a therapeutic effect is defined as a statistically significant reduction

ES 2 599 313 T3 of bacterial load in a host, emergence of resistance, or improvement in symptoms of infection, as measured by clinical results in humans or animal studies.

Treat, treat or treat, as used herein refers to administering a pharmaceutical composition for prophylactic and / or therapeutic purposes. The term "prophylactic treatment" refers to treating a patient who is not yet infected, but who is susceptible to, or otherwise at risk for, a particular infection. The term "therapeutic treatment" refers to administering treatment to a patient already suffering from an infection. Therefore, in preferred embodiments, treating is the administration to a mammal (either for therapeutic or prophylactic purposes) of therapeutically effective amounts of a fluoroquinolone antimicrobial agent.

Pharmacokinetics (PK) refers to the evolution over time of the antimicrobial concentration in the body. Pharmacodynamics (PD) refers to the relationship between pharmacokinetics and antimicrobial efficacy in vivo. PK / PD parameters correlate antimicrobial exposure with antimicrobial activity. The rate of destruction by the antimicrobial is dependent on the mode of action of the antimicrobial and is determined by either the time taken to destroy (time dependent) or the effect of increasing concentrations (concentration dependent). Consequently, PK / PD parameters can be used to predict the therapeutic efficacy of antimicrobials with several different mechanisms of action.

The AUC / CMI ratio is an example of a PK / PD parameter. AUC is defined as the area under the infection concentration curve at the site of infection or plasma over time of an antimicrobial agent in vivo (in animal or human). The AUC / MIC ratio is determined by dividing the 24-hour AUC for an individual antimicrobial by the MIC for the same antimicrobial determined in vitro. The activity of antimicrobials with dose-dependent killing (such as fluoroquinolones) is well predicted by the magnitude of the AUC / MIC ratio.

The Cmax: MIC ratio is another PK: PD parameter. Describes the maximum drug concentration in plasma or tissue relative to MIC. Fluoroquinolones and aminoglycosides are examples where Cmax: MIC can predict bacterial kill in vivo where resistance can be suppressed.

Time above MIC (T> MIC) is another PK / PD parameter. A percentage of a dosage range is expressed in which the level in the plasma or site of infection exceeds the MIC. The activity of antimicrobials with time-dependent killing (such as beta-lactams or oxazolidinones) is well predictable by the magnitude of the T> MIC relationship.

The term "dosage interval" refers to the time between administrations of the two sequential doses of a pharmaceutical product during multiple dosage regimens. For example, in the case of ciprofloxacin, which is given twice a day (traditional regimen of 400 mg twice a day) and levofloxacin, which is given once a day (500 mg or 750 mg each day), the dosing intervals are 12 hours and 24 hours, respectively.

As used herein, the maximum period of a concentration of the pharmaceutical product in vivo is defined as the time of the pharmaceutical dosage interval when the pharmaceutical concentration is not less than 50% of its maximum concentration in plasma or at the site. of infection. In some embodiments, maximum period is used to describe an antimicrobial dosage range.

The delivered respirable dose is the amount of drug inhaled during the inspiratory phase of the breath simulator that is equal to or less than 5 microns using a simulator programmed to the European Standard pattern of 15 breaths per minute, with an inspiration to expiration ratio of 1: 1.

Advantages of providing inhaled Fluoroquinolone in aerosol and topical use (Not Oral)

The rate of destruction of the antibiotic is dependent on the mode of action of the antibiotic and is determined by either the time required for the antibiotic to destroy (time dependent) or the effect of increasing the antibiotic concentration (concentration dependent). Fluoroquinolones are characterized by concentration-dependent killing activity where a therapeutic effect requires a high local maximum concentration above the MIC of the infecting pathogen.

The efficacy of fluoroquinolone in humans, animals, and in vitro infection models is linked to the AUC: MIC ratio and the Cmax: MIC ratio. Given the above uncertainty of the pharmacokinetics of fluoroquinolones in lung tissue, a number of in vitro studies have been performed to determine whether high doses of levofloxacin with very short half-lives (as predicted from a rat PK model and human) result in bacterial kill greater than that seen in conditions with longer residence times. In these studies, Levofloxacin concentrations that were 0.018 times - 1024 times the MIC were evaluated in a standard kill curve and in the in vitro hollow fiber assay. In both studies, high concentrations of levofloxacin were rapidly bactericidal and reached their maximum level to destroy in 10-20 minutes. This level of destruction was maintained if levofloxacin was maintained at that level or giving a half-life of 10 minutes. Consequently,

ES 2 599 313 T3 high dose and rapid delivery of specially formulated levofloxacin, such as a rapidly delivered dose of 20-50 mg aerosolized respirable Levofloxacin (which will produce initial ELF concentrations of 800 - 1600 ug / ml) is rapidly bactericidal for susceptible organisms and resistant organisms with MICs up to 32 ug / ml. These unique antimicrobial properties of fluoroquinolones are expected to further carry over to topical administrations, including, but not limited to, infections or prophylaxis of the skin, eye, ear, rectum, vagina, or urinary tract.

To measure the efficacy of different delivery models, formulations of levofloxacin that improve AUC conformation were prepared and measured in vivo compared to formulations of Levofloxacin that do not improve AUC conformation and other antibiotics using both the PK of rat as mouse efficacy after intratracheal administration. As previously demonstrated in a rat system, there were differences between drugs in lung pharmacokinetics, with some agents showing lower AUCs (e.g. levofloxacin), while others such as gemifloxacin or tobramycin showing higher concentrations resulting from clearance. slower lung. Studies in a single dose aerosol dose mouse infection model have shown variable efficacy between compounds. Referring to Figure 1, data analysis dividing aerosol dose by MIC indicated a strong correlation between dose: MIC and bactericidal activity (R2 = 0.89). These data suggest that the initial bactericidal activity in this model is not affected by lung clearance of the drug. Although pulmonary clearances have not been estimated in mice, it can be expected that the dose transformation ratio for AUC will be degraded using the rat scaled values. Therefore, these data suggest that optimization of the Levofloxacin AUC profile may not be necessary for aerosolized levofloxacin which is effective in the treatment of respiratory tract and lung infections.

Recent research with fluoroquinolones resulted in the development of the concept of a mutant selection window (MSW) for bacterial resistance that arises during therapy. This concept helps to identify a concentration range where mutants are most frequently selected in vitro and in vivo. The lower limit of the window is the lowest concentration that kills most of the cells that infect (approximately by MIC), while the upper limit of the window is the concentration of drug that blocks the growth of at least the mutant susceptible to the first stage. Above the upper limit concentration, the growth of the infecting bacteria requires the presence of at least two resistance mutations. This upper limit designates the mutant preventive concentration (MPC). MPC values ​​vary depending on bacteria and fluoroquinolones, and can be 10 to 20 times higher than MIC. Several modeling studies have shown that the more the drug concentration exceeds the MPC at the site of infection, the more effective treatment will be in preventing the development of resistance. On the contrary, the more the antibiotic concentration remains within the MSW, the greater the probability of selecting resistant mutants. Importantly, the currently approved dosing regimen for oral or intravenous levofloxacin has placed this antibiotic in the MSW for more than 20% of the dosage range for pathogens such as P. aeruginosa (Pa) and S. pneumonia. Consequently, a high level of resistance to levofloxacin was reported for both pathogens.

Therefore, in one embodiment, the concentration of Levofloxacin at the site of infection is increased by delivering it directly to the lung using inhalation therapy, thereby decreasing the amount of time that Levofloxacin is in the MSW. Such a therapeutic approach achieves broader coverage of pathogens (including strains resistant to levofloxacin), further prevents the development of resistance, and results in shorter courses of levofloxacin therapy.

Pharmacokinetics of Orally Administered Fluoroquinolones in Populations without CF and with CF

Sputum concentrations in CF patients

The pharmacokinetics of ciprofloxacin have been extensively studied in CF patients after oral administration. In fact, it has been shown that the serum PK profile of ciprofloxacin is very similar in CF patients compared to healthy volunteers (Figure 2).

Furthermore, the sputum profile vs. time of ciprofloxacin is very similar to its serum profile after oral administration (Figure 3). Following a 750 mg oral dose, peak concentrations of ~ 4.2 pg / ml and ~ 3.5 pg / ml were achieved for serum and sputum, respectively. Serum and sputum drug concentrations peaked at 1.5 and 4 hours, respectively. While the total amount of ciprofloxacin in sputum is high relative to serum concentrations, the absolute concentrations are low relative to the MIC of target organisms, such as Pa. This data is constant with a poor clinical result due to to the development of resistance to these low drug concentrations.

Although data on the intrapulmonary pharmacokinetics of Levofloxacin in cystic fibrosis are not available, data on the closely related ofloxacin were published in the 1980s and 1990s. Ofloxacin is composed of a racemic mixture of dextro (microbiologically inactive) and levo -rotatory (levofloxacin-microbiologically active). Studies have shown that the pharmacokinetic properties of the 2 components are similar. On

ES 2 599 313 Τ3 comparative studies with clprofloxachine, ofloxachine had a longer half-life and a greater distribution in sputum (79% vs 21%) than clprofloxachine.

Lung epithelial lining fluid

The most recent emphasis on the use and development of fluoroquinolones in community-acquired Gram-positive infections has focused on Intrapulmonary pharmacoclinical studies in lung epithelial lining fluid (ELF). Although the relevance of drug distribution in this fluid is unclear in the context of cystic fibrosis, clarification of drug pharmacology can be obtained from these studies. Levofloxachine penetrates well into lung tissues. Concentrations in lung tissues are generally 2 to 5 times higher than plasma concentrations. Several recent studies (summarized in Table 1) demonstrated that concentrations of Levofloxachine in the ELF of healthy subjects after a 750 mg oral dose reach a maximum concentration of around 20 pg / mL. Similar peak concentrations are expected in the sputum of CF patients after oral or IV administration of 750 mg of levofloxachine. In contrast, clprofloxachine penetrates lung tissues much less efficiently than levofloxachine. Based on mutant selection window (MSW) studies, these fluoroqulnolone drug levels in the ELF are insufficient to achieve the concentration required for mutant prevention which is 10 to 20 times the MIC for the infecting organism. .

Table 1. Concentration of Levofloxachine in the Fluid of the Epithelial Lining in Man.

<td rowspan="2">Drug</td><td rowspan="2">Dose</td><td rowspan="2">Route</td><td colspan="7">ELF drug concentration (pg / ml)</td>

<td>0.5 hr</td><td>1 hr</td><td>2 hr</td><td>4 hr</td><td>6 hr</td><td>12 hr</td><td>24 hr</td>

<td>Levofloxachine</td><td>500 mg</td><td>IV</td><td></td><td></td><td></td><td> 11</td><td></td><td> 2,5</td><td> 1.24</td>

<td>Levofloxachine</td><td>500 mg</td><td>oral</td><td></td><td></td><td></td><td> 9,9</td><td></td><td> 6,5</td><td> 0,7</td>

<td>Levofloxachine</td><td>500 mg</td><td>oral</td><td></td><td></td><td></td><td> 9,94</td><td></td><td> 6,46</td><td> 0,7</td>

<td>Levofloxachine</td><td>500 mg</td><td>oral</td><td> 4,74</td><td> 10,8</td><td> 9</td><td> 10,9</td><td> 9.6</td><td></td><td></td>

<td>Levofloxachine</td><td>750 mg</td><td>IV</td><td></td><td></td><td></td><td> 12,94</td><td></td><td> 6,04</td><td> 1,73</td>

<td>Levofloxachine</td><td>750 mg</td><td>oral</td><td></td><td></td><td></td><td> 22,1</td><td></td><td> 9,2</td><td> 1,5</td>

<td>Levofloxachine</td><td>750 mg</td><td>oral</td><td></td><td></td><td></td><td> 22,13</td><td></td><td> 9,19</td><td> 1,55</td>

<td>clprofloxachine</td><td>500 mg</td><td>oral</td><td></td><td></td><td></td><td> 1,9</td><td></td><td> 0,4</td><td></td>

Qulnolones

Non-limiting examples of qulnolones as described in the present description include amlfloxaclna, clnoxaclna, clprofloxaclna, enoxaclna, fleroxaclna, flumequlna, lomefloxaclna, nalldixlco acid, norfloxachine, ofloxaclna, levofloxxaclnacl, tospaffloxanalcine, pelafloxacin acid, pelafloxacin. , cllnafloxaclna, gatlfloxaclna, moxlfloxaclna; gemlfloxaclna; garenoxaclna; olamufloxaclna, cllnofloxaclna, trovafloxaclna, balofloxaclna, prullfloxaclna, moxlfloxaclna, gemlfloxaclna, rufloxaclna, sltafloxaclna (Sato, K, et al., 1992, Antlmlcrob Agents Chemother. 37: 1491-98 herein incorporated as reference in its entirety) , marbofloxaclna, orblfloxaclna, sarafloxaclna, danoflaxaclna, dlfloxaclna, enrofloxaclna, TG-873870, DX-619, DW-276, ABT-492, DV-7751a, (Tanaka, M, et al., 1992, Antlmlcrob. Agentsother. 37: 2212-18), and F-1061 (Kurosaka et al., Intersclence Conference on Antlmlcroblal Agents and Chemotherapy, 2003, 43^to: Chlcago, which is incorporated herein by reference in its entirety).

Treatment or Prophylaxis Methods

In some embodiments, a method is described for treating a microbial infection in an animal, specifically including a mammal, by treating an animal suffering from such an infection with a fluoroquinolone anti-microbial agent. In some embodiments, fluoroquinolone antimicrobials can be administered after aerosol formation and inhalation. Therefore, this method of treatment is especially suitable for the treatment of lung infections involving microbial strains that are difficult to treat with a parenterally administered anti-microbial agent due to the need for high levels of parenteral doses (which can cause secondary effects). unwanted), or due to a lack of clinically effective antimicrobial agents. In such an embodiment, this method can be used to deliver an anti-microbial fluoroquinolone directly to the site of Infection. Such a method can reduce the chemical exposure and maximize the amount of anti-microbial agent at the site of microbial infection. This method is also suitable for treating Infections Involving microbes that are susceptible to fluoroquinolone antimicrobial agents as a way to reduce the frequency of selection for resistant microbes. This method is also suitable for

ES 2 599 313 T3 treat infections involving microbes that are otherwise resistant to fluoroquinolone antimicrobials as a way to increase the amount of antimicrobial at the site of microbial infection. A subject can be identified as infected with bacteria that are capable of developing resistance by diagnosing the subject as having symptoms that are characteristic of a bacterial infection with a known species of bacteria that has resistant strains or one with a bacterium that is a member of the group. known to have resistant strains. Alternatively, the bacteria can be cultured and identified as a species known to have resistant strains or a bacterium that is a member of the group known to have resistant strains.

In some embodiments, the aerosolized fluoroquinolone antimicrobial agent is administered at a level sufficient to overcome the emergence of resistance in bacteria or increase clearance efficiency so that resistance does not have a chance to develop.

In some embodiments, fluoroquinolone aerosol therapy can be administered as a treatment or prophylaxis in combination or alternating the therapeutic sequence with other aerosol, oral, or parenteral antibiotics. By non-limiting example, this may include aerosolized tobramycin and / or other aminoglycoside, aerosolized aztreonam and / or other beta or mono-bactam, aerosolized ciprofloxacin and / or other fluoroquinolones, aerosolized azithromycin and / or other macrolides or ketolides, tetracycline and / or other tetracyclines, quinupristin and / or other streptogramins, linezolid and / or other oxazolidinones, vancomycin and / or other glycopeptides, and chloramphenicol and / or other phenicols, and colisitin and / or other polymyxins. Pharmaceutical compositions

For purposes of the method described in the present description, a fluoroquinolone antimicrobial agent can be administered using an inhaler. In some embodiments, a fluoroquinolone antimicrobial agent described herein is produced as a pharmaceutical composition suitable for aerosol formation, good taste, storage stability, and patient safety and tolerance.

In some embodiments, the content of the manufactured fluoroquinolone isoform can be optimized for tolerance, antimicrobial activity, and stability.

Administration

The fluoroquinolone antimicrobials described herein can be administered in a therapeutically effective dosage, eg, a dosage sufficient to provide treatment for the disease states described above. While optimal dosage levels in humans have not yet been determined for aerosol administration, generally, a daily aerosol dose of levofloxacin (and for most of the fluoroquinolone antimicrobial agents described herein) is from about 0.1 to 10 mg / kg of body weight, preferably from about 0.20 to 5.0 mg / kg of body weight, and most preferably about 0.4 to 4.0 mg / kg of body weight. Thus, for administration to a 70 kg person, the dosage range would be from about 7.0 to 700.0 mg per day, preferably about 14.0 to 350.0 mg per day, and most preferably about 28 0 to 280.0 mg per day. The amount of active compound administered will, of course, be dependent on the condition of the disease and the subject being treated, the severity of the affliction, the manner and schedule of administration, and the opinion of the prescribing physician; for example, a likely dose range for aerosol administration of levofloxacin would be about 20 to 400 mg per day.

Administration of the fluoroquinolone antimicrobial agents described herein or the pharmaceutically acceptable salts thereof may be via any of the accepted modes of administration for agents that serve similar utilities, including, but not limited to, inhalation. aerosol.

Pharmaceutically acceptable compositions include solid, semi-solid, liquid, and aerosol dosage forms, such as, for example, powders, liquids, suspensions, complexes, liposomes, particles, or the like. Preferably, the compositions are provided in unit dosage forms suitable for single administration of a precise dose. The unit dosage form can further be assembled and packaged in unison to provide a patient with a weekly or monthly supply and furthermore other compounds such as saline, masking agents, pharmaceutical excipients, and other active ingredients or carriers can be incorporated.

The fluoroquinolone antimicrobial agent can be administered either alone or more typically in combination with a conventional pharmaceutical carrier, excipient, or the like (eg, mannitol, lactose, starch, magnesium stearate, saccharin, 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 further contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents, and the like (for example, sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like). Generally, depending on the mode of administration

Intended for ES 2 599 313 T3, the pharmaceutical formulation will contain about 0.005% to 95%, preferably about 0.5% to 50% by weight of a compound of the invention. True methods for preparing those dosage forms are known, or will be apparent, to those of skill in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

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

Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier, (eg, water, saline, aqueous dextrose, glycerol, glycols, ethanol, or the like) to form a solution or suspension. Aerosolized solutions 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 the liquid prior to aerosol production and inhalation. The percentage of active compound contained in such aerosol compositions is highly dependent on the specific nature of the aerosol, as well as on the activity of the compound and the needs of the subject. However, percentages of active ingredient from 0.01% to 90% in solution can be employed, and will be higher if the composition is a solid which will subsequently be diluted to the above percentages. In some embodiments, the composition will comprise 1.0% -50.0% of the active agent in solution.

Fluoroquinolone formulations can be separated into two groups; those of simple formulation and complex formulations that provide properties of masking of the flavor, improvement of the tolerance and / or a formulation that improves the profile of AUC. Simple formulations can be further separated into three groups. 1. Simple formulations can include water-based liquid formulations for nebulization. By non-limiting example, water-based liquid formulations can consist of the fluoroquinolone alone or with non-encapsulated water-soluble excipients. 2. Simple formulations may further include organic-based liquid formulations for nebulization or metered dose inhaler. By non-limiting example, organic-based liquid formulations may consist of fluoroquinolone or non-encapsulating organic soluble excipients. 3. Simple formulations can further include dry powder formulations for administration with a dry powder inhaler. By non-limiting example, dry powder formulations may consist of the fluoroquinolone alone or with either non-encapsulated water soluble or organic soluble excipients with or without a mixing agent such as lactose. Complex formulations can further be separated into five groups. 1. Complex formulations can include water-based liquid formulations for nebulization. By non-limiting example, water-based liquid complex formulations can consist of the fluoroquinolone encapsulated or complexed with water-soluble excipients such as lipids, liposomes, cyclodextrins, microencapsulations, and emulsions. two. Complex formulations may further include organic-based liquid formulations for nebulization or metered dose inhaler. By non-limiting example, organic-based complex liquid formulations may consist of the fluoroquinolone encapsulated or complexed with soluble organic excipients such as lipids, microencapsulations, and reversed phase water-based emulsions. 3. Complex formulations may further include low solubility, water-based liquid formulations for nebulization. By non-limiting example, complex low solubility water-based liquid formulations may consist of fluoroquinolone alone as a stable nanosuspension, poorly soluble in water or in complexes with cocrystal / coprecipitate excipient, or mixtures with low solubility lipids, such as as lipid nanosuspensions. Four. Complex formulations may further include low solubility organic-based liquid formulations for nebulization or metered dose inhaler. By non-limiting example, complex liquid formulations based on low solubility organics may consist of fluoroquinolone alone as a stable nanosuspension, poorly soluble in organic or in co-crystal / coprecipitate excipient complexes, or mixtures with low solubility lipids, such as as lipid nanosuspensions. 5. Complex formulations can further include dry powder formulations for administration using a dry powder inhaler. By non-limiting example, complex dry powder formulations may consist of the fluoroquinolone in complex or cocrystal / coprecipitate / spray dry blend with poorly water soluble excipients / dry powder salts with or without a mixing agent such as lactose. Specific methods for the preparation of the simple and complex formulation are described in the present description.

Aerosol supply

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

Various device technologies exist to deliver either liquid aerosol or dry powder products. Dry powder formulations generally require less time for drug delivery, although

ES 2 599 313 T3 longer and more costly development efforts. In contrast, liquid formulations have historically suffered from longer administration times, although they have the advantage of shorter and less costly development efforts. The fluoroquinolone antimicrobial agents described in the present disclosure in the range of solubility are generally stable and have a variety of flavors. In such embodiments, the antimicrobial fluoroquinolonal levofloxacin is soluble in water at neutral pH, is stable in aqueous solution, and has been limited to no flavor.

Accordingly, in one embodiment, a particular formulation of the fluoroquinolone antimicrobial agent described in the present disclosure is combined with an aerosolization device particularly to provide an aerosol for inhalation that is optimized for maximum deposition of the drug at a site of infection. and maximum tolerance. Factors that can be optimized include solution or solid particle formulation, delivery rate, and particle size and distribution produced by the aerosolization device.

Particle size and distribution

Generally, inhaled particles are subject to deposition by one of two mechanisms: impaction, which generally predominates for larger particles, and sedimentation, which is frequent for smaller particles. Impaction occurs when the momentum of an inhaled particle is great enough that the particle does not follow the air stream and meets a physiological surface. In contrast, sedimentation occurs primarily in the deep lung when very small particles that have traveled with the inhaled air stream meet physiological surfaces as a result of random diffusion within the air stream.

For pulmonary administration, the upper airways are bypassed in favor of the middle and lower airways. Delivery of pulmonary drugs can be done by inhalation of an aerosol through the mouth and throat. Particles having a mass median aerodynamic diameter (MMAD) of more than about 5 microns generally do not reach the lung; instead, they tend to impact the back of the throat and are swallowed and possibly absorbed orally. Particles having diameters of about 2 to about 5 microns are small enough to reach the upper-than-middle lung region (conducting airways), but are too large to reach the alveoli. The smallest particles, ie, about 0.5 to about 2 microns, are capable of reaching the alveolar region. Particles having diameters less than about 0.5 microns can further deposit in the alveolar region by sedimentation, although very small particles can be exhaled. Particle size measurements can be referred to as volume median diameter (VMD), mass median diameter (MMD), or MMAD. These measurements can be made by impaction (MMD and MMAD) or by laser (VMD). For liquid particles, VMD, MMD and MMAD can be the same if ambient conditions are maintained, eg standard humidity. However, if humidity is not maintained, the MMD and MMAD determinations will be smaller than VMD due to dehydration during impactor measurements. For the purposes of this description, the VMD, MMD and MMAD measurements are considered to be under standard conditions, so the VMD, MMD and MMAD descriptions will be comparable. Similarly, dry powder particle size determinations by MMD and MMAD are further considered comparable.

In some embodiments, the aerosol particle size is optimized to maximize fluoroquinolone antimicrobial agent deposition at the site of infection and to maximize tolerance. The particle size of the aerosol can be expressed in terms of the mass median aerodynamic diameter (MMAD). Large particles (eg, MMAD&gt; 5 pm) can be deposited in the upper airways because they are too large to navigate the curvature of the upper airways. Small particles (eg, MMAD &lt;2pm) can be poorly deposited in the lower airways and thus become exhaled, providing the additional opportunity for deposition in the upper airways. Therefore, intolerance (for example, cough and bronchospasm) can result from deposition in the upper respiratory tract both by impaction from inhalation of large particles and by sedimentation of small particles during repeated inhalation and expiration. Thus, in one embodiment, an optimal particle size (eg, MMAD = 2-5 pm) is used to maximize deposition at a mid-lung infection site and to minimize intolerance associated with deposition. in the upper respiratory tract. Furthermore, generating a defined particle size with limited geometric standard deviation (GSD) can optimize deposition and tolerance. Narrow GSD limits the number of particles outside the desired MMAD size range. In one embodiment, an aerosol is provided containing one or more compounds described in the present disclosure having a 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 is provided having a MMAD of from about 2.8 microns to about 4.3 microns with a GSD less than or equal to 2 microns. In another embodiment, an aerosol is provided having a MMAD of from about 2.5 microns to about 4.5 microns with a GSD less than or equal to 1.8 microns.

The fluoroquinolone antimicrobial agents described herein intended for respiratory delivery (either for systemic or local delivery) can be administered as aqueous formulations, as suspensions or solutions in the halogenated hydrocarbon propellants, or as dry powders. Aqueous formulations can be aerosolized using liquid nebulizers that employ either hydraulic atomization or

ES 2 599 313 Τ3 ultrasonic. Propellant-based systems can use suitable pressurized metered dose inhalers (pMDI). Dry powders can use Dry Powder Inhaler (DPI) devices, which are able to disperse the drug substance effectively. A desired particle size and distribution can be obtained by choosing a suitable device.

Liquid nebulizer

In one embodiment, a nebulizer is selected on the basis of allowing the formation of an aerosol of an anti-microblan agent from the fluoroquinolone described in the present disclosure having a predominantly MMAD between about 2 to about 5 microns. In one embodiment, the supplied amount of the fluoroquinolone anti-microbial agent provides a therapeutic effect for respiratory infections.

Previously, two types of jet nebulators, and ultrasonic, have been shown to be capable of producing and delivering aerosol particles having sizes between 2 and 4 um. These particle sizes have been shown to be optimal for the treatment of lung bacterial infection caused by gram-negative bacteria such as Pseudomonas aeruglnosa, Escheñchla coll, Enterobacter species, Klebslella pneumonlae, K. oxytoca, Proteus mlrabllls, Pseudomonas aeruglnosa, Serratla marcescens, Haemophllus nfluenzae, Burkholderla cepacla, Stenotrophomonas maltophllla, Alcallgenes xylosoxldans, and multidrug resistant Pseudomonas aeruglnosa. However, unless a specially formulated solution is used, these nebulizers typically need large volumes to deliver a sufficient amount of drug to obtain a therapeutic effect. A jet nebulizer uses the breaking of the air pressure of an aqueous solution into aerosol droplets. An ultrasonic nebulizer uses the slow cooling of the aqueous solution by means of a plezoelectric crystal. Typically, however, jet nebulizers are only about 10% efficient in clinical conditions, whereas the ultrasonic nebulizer is only about 5% efficient. The amount of pharmaceutical product deposited and absorbed in the lungs is therefore a fraction of 10% despite the large amounts of the drug placed in the nebulizer.

Accordingly, in one embodiment, a vibrating mesh nebulizer is used to deliver an aerosol of the fluoroquinolone anti-microbial agent described in the present disclosure. A vibrating mesh nebulizer consists of a liquid storage container in contact with the fluid, with a diaphragm and Inhalation and Exhalation valves. In one embodiment, about 1 to about 5 ml of the fluoroqulnolone anti-microblane agent is placed in the storage container and the aerosol generator is dedicated to the production of atomized aerosol of selectively particle sizes between about 1 and about 5 um.

By non-limiting example, a fluoroqulnolone anti-microbial agent described in the present disclosure is placed in a liquid mist inhaler and prepared in dosages to deliver from about 7 to about 700 mg from a dosage solution of about 1 to about 5 ml, preferably from about 14 to about 350 mg in about 1 to about 5 ml and most preferably from about 28 to about 280 mg in about 1 to about 5 ml being produced with MMAD particle sizes between about 2 to about 5 µm.

By non-limiting example, a nebulized fluoroquinolone anti-microbial can be administered in the described delivered respirable dose in less than about 20 min, preferably less than about 10 min, more preferably less than about 7 min, most preferably less than about 5 min. , more preferably less than about 3 min, and in some cases most preferably less than about 2 min.

By non-limiting example, in other circumstances, a nebulized fluoroquinolone anti-microbial may achieve better tolerance and / or exhibit a characteristic that improves the AUC profile when administered for longer periods of time. Under these conditions, the delivered respirable dose described in more than about 2 min, preferably more than about 3 min, more preferably more than about 5 min, more preferably more than about 7 min, most preferably more than about 10 min, and in some cases with superlative preference from about 10 to about 20 min.

For aqueous systems and other non-pressurized liquids, a variety of foggers (including small volume foggers) are available to spray formulations. Compressor driven foggers incorporate jet technology and use compressed air to generate the liquid aerosol. Such devices are commercially available from, for example, Healthdyne Technologies, Inc .; Invacare, Inc .; Mountaln Medical Equlpment, Inc .; Parí Resplratory, Inc .; Mada Medical, Inc .; Purltan-Bennet; Schuco, Inc., DeVIlblss Health Care, Inc .; and Hospltak, Inc. Ultrasonic nebulators rely on the mechanical energy in the form of vibration of a plezoelectric crystal to generate breathable droplets of liquid and are commercially available from, for example, Omron Heathcare, Inc. and DeVIlblss Health Care, Inc. either plezoelectric or mechanical pulses to generate breathable liquid droplets. Other examples of nebulizers for use with fluoroquinolone antimicrobial agents described in the present disclosure are described in US Pat. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911; 4,510,929; 4,624,251; 5,164,740;

IS 2 599 313 Τ3

5,586,550; 5,758,637; 6,644,304; 6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575; 6,192,876;

6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971; 6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304; 5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and 6,612,303 which are incorporated herein by reference in their entirety. Commercial examples of nebulizers that can be used with the fluoroquinolone antimicrobial agents described in the present disclosure include Resplrgard II®, Aeroneb®, Aeroneb® Pro, and Aeroneb® Go produced by Aerogen; AERx® and AERx Essence ™ produced by Aradlgm; PortaNeb®, Freeway Freedom ™, Sldestream, Ventstream and l-neb produced by Resplronlcs, Inc .; and PARI LC-Plus®, PARI LC-Star®, and e-Flow^7m produced by PARI, GmbH. For additional non-limitation see US Patent No. 6,196,219.

In some embodiments, the drug solution is formed prior to use of the nebulizer by a patient. In other embodiments, the drug is stored in the nebulizer in solid form. In this case, the solution is mixed after activation of the fogger, as described in US Patent No. 6,427,682 and PCT Publication No. WO 03/035030. In these nebulizers, the solid drug, optionally combined with excipients to form a solid composition, is stored in a separate compartment of a liquid solvent.

The liquid solvent is capable of dissolving the solid composition to form a liquid composition, which can be aerosolized and inhaled. Such capacity is, among other factors, a function of the quantity selected and, potentially, the composition of the liquid. To allow easy handling and reproducible dosing, the sterile aqueous liquid may be able to dissolve the solid composition within a short period of time, possibly under gentle agitation. In some embodiments, the final liquid is ready to use after no more than about 30 seconds. In some cases, the solid composition dissolves within about 20 seconds, and advantageously, within about 10 seconds. As used herein, the terms dissolve (n), dissolve, and dissolve refer to the disintegration of the solid composition and the release, ie, dissolution, of the active compound. As a result of dissolving the solid composition with the liquid solvent, a liquid composition is formed in which the active compound is contained in the dissolved state. As used herein, the active compound is in a dissolved state when at least about 90% by weight is dissolved, and more preferably when at least about 95% by weight is dissolved.

Regarding the basic compartmentalized nebulizer design, this depends mainly on the specific application if it is more useful to accommodate the aqueous liquid and the solid composition within the separate chambers of the same container or primary packaging, or if they should be provided in separate containers. IF separate containers are used, they are provided as a set within the same secondary packaging. The use of separate containers is especially preferred for foggers containing two or more doses of active compound. There is no limit to the total number of containers provided in a multldosls kit. In one embodiment, the solid composition is provided as a unit dose within multiple containers or within multiple chambers of a container, while the liquid solvent is provided within one chamber or container. In this case, a favorable design provides the liquid in a metered dose dispenser, which may consist of a glass or plastic bottle closed with a dispensing device, such as a mechanical pump to measure the liquid. For example, an activation of the pump mechanism can dispense the exact amount of liquid to dissolve a unit dose of the solid composition.

In another embodiment for multiple dose, separate compartment nebulizers, both the solid composition and the liquid solvent are provided as collateral unit doses within multiple containers or within multiple chambers of a container. For example, two-chamber containers can be used to hold a unit of the solid composition in one chamber and a unit of liquid in the other. As used herein, a unit is defined by the amount of drug present in the solid composition, which is a unit dose. Such two-chamber containers can, however, also be used advantageously for nebulizers that only contain a single dose of the drug.

In one embodiment of a separate compartment nebulizer, an blister pack having two blisters is used, the blisters represent the chambers containing the solid composition and the liquid solvent in collateral amounts to prepare a unit dose of the final liquid composition. As used herein, a blister pack represents a thermoformed or pressure formed primary packaging unit, most likely comprising a polymeric packaging material that optionally includes a metal foil, such as aluminum. The blister pack can be shaped to allow easy dispensing of the contents. For example, one side of the container may be tapered or it may have a conical portion or ridge through which the contents are dispensed into another glass after the blister is opened at the conical end. The conical terminal can represent a point.

In some embodiments, the two chambers of the blister pack are connected by a channel, the channel which is adapted to discharge fluid from the ampoule containing the liquid solvent to the ampoule containing the solid composition. During storage, the channel is closed with a seal. In this sense, a seal is any structure that prevents contact of the liquid solvent with the solid composition. The seal is preferably breakable or removable; Breaking or removing the seal when the fogger is to be used will allow the liquid solvent to enter the other chamber and dissolve the solid composition. The dissolution process can be improved by stirring the

ES 2 599 313 T3 blister pack. Therefore, the final liquid composition for Inhalation is obtained, the liquid will be presented in one or both chambers of the container connected by the channel, depending on how the container is gripped.

According to another embodiment, one of the chambers, preferably the one that closes in the conical portion of the blister pack, communicates with a second channel, the channel that extends from the chamber to the distal position of the conical portion. During storage, this second channel does not communicate with the outlet of the container but is closed in a hermetic manner. Optionally, the distal end of the second channel is closed by a breakable or removable cap or closure, which may be, for example, a screw cap, a breakaway cap, or a shear cap.

In one embodiment, a bottle or container having two compartments, the compartment representing the chambers, is used to contain the solid composition and the liquid solvent in matching amounts to prepare a unit dose of the final liquid composition. The liquid composition and a second liquid solvent can be contained in matching amounts to prepare a unit dose of the final liquid composition (by non-limiting example in cases where two soluble excipients or the fluoroquinolone and one excipient are unstable for storage, yet are you want in the same mix for administration.

In some embodiments, the two compartments are physically separated but are in fluid communication such as when the bottle or container is connected by a breakable channel or barrier, the breakable channel or barrier being adapted to direct the fluid between the two compartments allowing for prior mixing. of the administration. During storage, the channel is closed with an intact breakable seal or barrier. In this sense, a seal is any structure that avoids mixing the contents in the two compartments. The seal is preferably breakable or removable; breaking or disassembling the seal when the nebulizer is to be used will allow the liquid solvent to enter the other chamber and dissolve the solid composition or allow mixing in the case of two liquids. The mixing or dissolving process can be improved by shaking the container. Therefore, the final liquid composition for inhalation is obtained, the liquid that is present in one or both chambers of the container is connected by the channel or breakable barrier, depending on how the container is gripped.

The solid composition itself can be provided in various types of different dosage forms, depending on the physicochemical properties of the drug, the desired dissolution rate, cost considerations, and other criteria. In one of the modes, the solid composition is a single unit. This implies that a unit dose of the drug is comprised in a single, solid, physically shaped form or article. In other words, the solid composition is consistent, unlike what is a multiple unit dosage form, where the units are inconsistent.

Examples of single units that can be used for the solid composition include tablets, such as compressed tablets, film-like units, sheet-like units, wafers, lyophilized matrix units, and the like. In a preferred embodiment, the solid composition is a highly porous lyophilized form. Such lyophilizates, sometimes also referred to as lyophilized wafers or tablets, are particularly useful for their rapid disintegration, which also allows for rapid dissolution of the active compound.

On the other hand, for some applications the solid composition may be further formed as a multiple unit dosage form as defined above. Examples of multiple units are powders, granules, microparticles, pellets, beads, lyophilized powders, and the like. In one embodiment, the solid composition is a lyophilized powder. Such a freeze-dried dispersed system comprises a multitude of powder particles, and due to the freeze-drying process used in the formation of the powder, each particle has an irregular porous microstructure, through which the powder is capable of absorbing water very quickly, which results in rapid dissolution.

Another type of multi-particle system that is capable of also achieving rapid dissolution of the drug is that of powders, granules or pellets of water-soluble excipients that are coated with the drug, so the drug is located on the outer surface of the individual particles. In this type of system, the water-soluble low molecular weight excipient that is useful for the preparation of the cores of said coated particles, which can be subsequently coated with a coating composition comprising the drug and, preferably, one or more additional excipients, such as a binder, an ex pore, a saccharide, a sugar alcohol, a film-forming polymer, a plasticizer, or other excipients used in pharmaceutical coating compositions.

In another embodiment, the solid composition resembles a coating layer that is coated in multiple units of insoluble material. Examples of insoluble units include glass beads, polymers, metals, and mineral salts.Once again, the desired effect is primarily rapid disintegration of the coating layer and rapid dissolution of the drug, which is achieved by providing the solid composition in a physical form. which has a particularly high surface-to-volume ratio. Typically, the coating composition, in addition to the drug and the water soluble low molecular weight excipient, will comprise one or more excipients, such as those mentioned above for coating soluble particles, or any other excipient known to be useful in the compositions. coating of pharmaceutical products.

To achieve the desired effects, it may be useful to incorporate more than one low molecular weight excipient soluble in

ES 2 599 313 T3 water in the solid composition. For example, one excipient can be selected for its drug carrier and binding capacity, while another excipient can be selected for pH adjustment. If the final liquid composition needs to be regulated, two excipients can be selected which together form a buffer system.

In one embodiment, the liquid that is used in a separate compartment nebulizer is an aqueous liquid, which in the present description is defined as a liquid whose major component is water. The liquid does not necessarily consist of water only; 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 may be useful include propylene glycol, glycerol, and polyethylene glycol. One of the reasons for incorporating a solid component as a solute is that such a compound is desirable in the final liquid composition, but is incompatible with the solid composition or with a component thereof, such as the active ingredient.

Another desirable feature for the liquid solvent is that it is sterile. An aqueous liquid may be subject to the risk of microbiological contamination and considerable growth if steps are not taken to ensure sterility. To provide a substantially sterile liquid, an effective amount of an acceptable antimicrobial agent or preservative can be incorporated or the liquid can be sterilized prior to delivery and to seal with an airtight seal. In one embodiment, the liquid is a sterile preservative-free liquid and is provided in an appropriate airtight container. However, in accordance with another embodiment in which the nebulizer contains multiple doses of the active compound, the liquid can be delivered in a multi-dose container, such as a metered-dose dispenser, and may require a preservative to avoid microbial contamination afterward. of the first use.

Metered dose inhaler (MDI)

A propellant-powered inhaler (pMDI) delivers a metered dose of medicine after each run. The medicine is formulated as a suspension or solution of a drug substance in a suitable propellant such as a halogenated hydrocarbon. PMDIs are described in, for example, Newman, SP, Aerosols and the Lung, Clarke et al., Eds., Pp. 197-224 (Butterworths, London, England, 1984).

In some embodiments, the particle size of the drug substance in an MDI can be optimally selected. In some embodiments, the active ingredient particles have diameters of less than about 50 microns. In some embodiments, the particles have diameters of less than about 10 microns. In some embodiments, the particles have diameters from about 1 micron to about 5 microns. In some embodiments, the particles have diameters of less than about 1 micron. In an advantageous embodiment, the particles have diameters from about 2 microns to about 5 microns.

The propellants for use with the MDIs can be any of the propellants known in the art. Examples of propellants include the chlorofluorocarbons (CFCs) such as dichlorodifluoromethane, trichlorofluoromethane, and dichlorotetrafluoroethane; hydrofluoroalkanes (HFA); and carbon dioxide. It may be advantageous to use HFAs instead of CFCs due to the environmental concerns associated with the use of CFCs. Examples of medicinal aerosol preparations containing the HFAs are presented in US Patent Nos. 6,585,958; 2,868,691 and 3,014,844. In some embodiments, a cosolvent is mixed with the propellant to facilitate dissolution or suspension of the drug substance.

In some embodiments, the propellant and active ingredient are contained in separate containers, as described in US Patent No. 4,534,345.

In some embodiments, the MDI used in the present disclosure is activated by a patient pushing a lever, button, or other actuator. In other embodiments, the delivery of aerosol activates the breath such that initially, after assembling the unit, the aerosol active compound is released once the patient begins to inhale, as described in US patents. United Nos. 6,672,304; 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. Such a system allows more of the active compound to enter the lungs of the patient. Another mechanism that helps a patient take an adequate dose with the active ingredient may include a valve mechanism that allows a patient to use more than one breath to inhale the drug, as described in U.S. Patent Nos. . 4,470,412 and 5,385,140.

Additional examples of MDIs known in the art and suitable for use in the present disclosure include US Patent 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.

Solution / dispersion formulations

Aqueous formulations containing soluble drug particles or nanoparticles are further described. For aqueous aerosol formulations, the drug can be presented at a concentration of approximately 1

ES 2 599 313 T3 mg / mL to about 700 mg / mL. Such formulations provide effective delivery to the appropriate areas of the lung, with the more concentrated aerosol formulation having the additional advantage of allowing large amounts of the drug substance to be delivered to the lung in a very short period of time. In one embodiment, a formulation is optimized to provide a well-tolerated formulation. Accordingly, in one embodiment, the fluoroquinolone antimicrobial agents described in the present disclosure are formulated to have a good taste, pH from about 5.5 to about 7, osmolarity from about 200 to about 1250 mOsmol / kg, ion concentration permeant from about 30 to about 300 mM.

In one embodiment, the solution or diluent used for the preparation of aerosol formulations has a pH range of from about 4.5 to about 7.5, from about 5.5 to about 7.0. This pH range improves tolerance. When the spray is either acidic or basic, it can cause coughing and bronchospasm. Although the safe pH range is relative, some patients can tolerate a slightly acidic spray, while others will experience bronchospasm. Any aerosol with a pH of less than about 4.5 typically induces bronchospasm. Sprays with a pH of about 4.5 to about 5.5 will occasionally cause bronchospasm. Any aerosol that has a pH greater than about 7.5 may have a low tolerance because body tissues are generally unable to regulate alkaline aerosols. Aerosols with a controlled pH below about 4.5 and above about 7.5 typically result in lung irritation accompanied by severe brochospasm, coughing, and inflammatory reactions. For these reasons, as well as for the prevention of bronchospasm, cough, or inflammation in patients, the optimum pH for the aerosol formulation was determined to be between about pH 5.5 to about pH 7.0. Consequently, in one embodiment, aerosol formulations for use as described herein are adjusted to pH between about 4.5 and about 7.5 with the preferred pH range of about 5.5 to about 7.5. Most preferably the pH range is from about 5.5 to about 7.5.

By non-limiting example, the compositions may also include a buffer or pH adjusting agent, typically a salt prepared from an organic acid or base. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or buffers of phthalic acid, Tris, tromethamine, hydrochloride, or phosphate.

Many patients have increased sensitivity to various chemical tastes, including bitter, salty, sweet, metallic sensations. By the non-limiting example of taste masking it is possible to create well tolerated drug products through the addition of taste masking excipients, adjusted osmolarity, and sweeteners.

Many patients have increased sensitivity to various chemical agents and have a high incidence of bronchospasm, asthma, or other incidents of coughing. Your airways are particularly sensitive to hypotonic or hypertonic and acidic or alkaline conditions and to the presence of any permeable ion, such as chloride. Any imbalance in these conditions or a presence of chloride above a certain value leads to bronchospasmic or inflammatory events and / or cough that greatly impair treatment with inhalable formulations. These two conditions impede the efficient delivery of aerosol drugs to the endobronchial space.

In some embodiments, the osmolality of the aqueous solutions of the fluoroquinolone antimicrobial agent described herein are adjusted by providing excipients. In some cases, a certain amount of chloride or other anion is needed for the successful and effective delivery of the aerosol antibiotic. However, such amounts have been found to be less than the amounts provided and typically used in aerosols of other compounds.

Bronchospasm or cough reflexes do not respond to the same osmolality of the diluent for aerosolization. However, they can be sufficiently controlled and / or suppressed when the osmolality of the diluent is in a certain range. A preferred solution for aerosolization of therapeutic compounds that is safe and tolerated has a total osmolality of about 200 to about 1250 mOsmol / kg with a chloride concentration range of about 30 to about 300 mM, and preferably about 50 to about 50 mM. 150 mM. This osmolality controls bronchospasm, the chloride concentration, such as a permeating anion, controls coughing. Because both ions are permeable, both bromide and iodide ions can be substituted for chloride. Also, the bicarbonate can be substituted for the chloride ion.

By non-limiting example, the formulation for an aerosol fluoroquinolone antimicrobial agent may comprise from about 7 to about 700 mg, preferably from about 14 to about 300 mg, or superlatively preferably from about 28 to about 280 mg of antimicrobial agent from fluoroquinolone for about 1 to about 5ml of diluted saline (between 1/10 to 1/1 of normal saline). Accordingly, the concentration of a levofloxacin solution may be greater than about 25mg / ml, greater than about 35mg / ml, and preferably, it is greater than about 40mg / ml, and is as high or greater than 50 / ml.

In one embodiment, the osmolality of the solution is from about 100 mOsmol / kg to about 600

ES 2 599 313 T3 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 embodiments, the permeating ion concentration is from about 25 mM to about 400 mM. In various other embodiments, the concentration of the permeating ion 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.

Surface modifiers

The fluoroquinolone antimicrobial agents described herein can be prepared into a pharmaceutical composition with suitable surface modifiers which 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 ionic and nonionic surfactants. Two or more surface modifiers can be used in combination.

Representative examples of surface modifiers include cetyl pyridinium chloride, gelatin, casein, lecithin (phosphatides), dextran, glycerol, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, alcohol. ketostearyl, cetomacrogol emulsifying wax, sorbitan esters, alkylpolyoxyethylene ethers, (for example macrogol esters such as acetomacrogol 1000), polyoxyethylene castor oil derivatives, polyoxyethylene fatty acid esters (eg, the commercially available Tweens.RTM, such as, for example, Tween 20.RTM, and Tween 80.RTM, (ICI Specialty Chemicals)); polyethylene glycols (for example, Carbowaxs 3350.RTM, and 1450.RTM., and Carbopol 934.RTM, (Union Carbide)), dodecyl trimethyl ammonium bromide, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, calcium carboxymethylcellulose , hydroxypropyl cellulose (HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polymer 4- (1,1,3,3-tetramethylbutyl) -phenol with ethylene oxide and formaldehyde (also known as tyloxapol, superion, and triton ), poloxamers (eg Pluronic 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 derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Wyandotte Corporation, Parsippany, New Jersey), a charged phospholipid, such as dimyristoyl phosphatidylglycerol, dioctylsulfosuccinate (DOSS), Tetronic 1508.RTM; (T-1508) (BASF Wyandotte Corporation), sodium dialkyl ether of sulfosuccinic acid (for example, Aerosol OT.RTM., Which is a sodium dioctyl ether of sulfosuccinic acid (American Cyanamid)); Duponol P.RTM., Which is a sodium lauryl sulfate (DuPont); Tritons X200.RTM., Which is an alkyl arylsulfonate polyether (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 Olinlog.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 .beta.-D-glucopyranoside; n-decyl .beta.-D-maltopyranoside; ndodecyl .beta.-D-glucopyranoside; n- dodecyl .beta.-D-maltoside; heptanoyl-N-methylglucamide; n-heptyl-.beta.-D-glucopyranoside; n-heptyl .beta.-D-thioglycoside; n-hexyl .beta.-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noyl .beta.D-glucopyranoside; octanoyl-N-methylglucamide; n-octyl-.beta.-D-glucopyranoside; octyl .beta.-D-thiooglycopyranoside; and the like. Tyloxapol is a particularly preferred surface modifier for pulmonary or intranasal steroid delivery, even more so for nebulization therapies.

Examples of surfactants for use in the solutions described herein include, but are not limited to, ammonium lauryl ether sulfate, ketamine oxide, cetrimonium chloride, cetyl alcohol, cetyl myristate, cetyl palmitate, DEA cocamide, cocamidopropyl betaine, cocamidopropylamine oxide, cocamide MEA, DEA lauryl sulfate, di-stearylphthalic acid amide, dicetyl dimethyl ammonium chloride, dipalmitoylethyl hydroxethylmonium, disodium lauryl ether sulfosuccinate, di (hydrogenated) sebophthalic acid, glyceryl dilaurate, glyceryl distearate, glyceryl oleate, glyceryl stearate, isopropyl myristate nf, isopropyl palmitate nf, lauramide oxide, lauramide MEA, lauramide MEA myistamine, octyl isononanoate, octyl palmitate, octyldodecyl neopentanoate, olealkonium chloride, PEG-2 stearate, PEG-32 glyceryl caprylate / caprate, PEG-32 glyceryl stearate, PEG-4 and PEG-150 stearate and distearate, PEG-4 to PEG-150 and dilaurate laurate, PEG-4 oleate &amp; dioleate to PEG-150, PEG-7 glyceryl cocoate, PEG-8 beeswax, stearate Propylene Glycol, C14-16 Sodium Olefin Sulfonate, Sodium Lauryl Sulfoacetate, Sodium Lauryl Sulfate, Sodium Tridecyl Ether Sulfate, Stearalkonium Chloride, Stearamide Oxide, TEAdodecylbenzene Sulfonate, TEA Lauryl Sulfate

Most of these surface modifiers are known pharmaceutical excipients and are described in detail in the Handbook of Pharmaceutical Excipients, jointly published by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain (The Pharmaceutical Press, 1986). Surface modifiers are commercially available and / or can be prepared by techniques known in the art. The relative amount of drug and surface modifier can vary widely and the optimal amount of surface modifier can depend, for example, on the particular drug and surface modifier selected, micellar concentration

ES 2 599 313 T3 critical of the surface modifier if it forms micelles, the hydrophilic-lipophilic balance (HLB) of the surface modifier, the melting point of the surface modifier, the water solubility of the surface modifier and / or drugs, the surface tension of aqueous solutions of the surface modifier, etc.

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

Proteins / Amino Acids

Protein excipients can include albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. Suitable amino acids (outside the dileucylpeptides of the invention), which can also function in regulatory capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine , phenylalanine, aspartame, tyrosine, tryptophan, and the like. Amino acids and polypeptides that function as dispersing agents are preferred. Amino acids included in this category include hydrophobic amino acids such as leucine, valine, isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine, histidine, and proline. Peptide excipients that enhance dispersibility include dimers, trimers, tetramers, and pentamers comprising one or more hydrophobic amino acid components such as those described above.

Carbohydrates

By non-limiting example, carbohydrate excipients can 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, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, sorbitol, xylitol (glucitol), pyranosyl sorbitol, myoinositol, isomalt, trehalose, and the like.

Polymers

By non-limiting example, the compositions may also include polymeric excipients / additives, eg, polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethyl starch, dextrates (by non-limiting example cyclodextrins, may include, 2-hydroxypropyl-beta-cyclodextrin, 2-hydroxypropyl-gamma-cyclodextrin, randomly methylated beta-cyclodextrin, dimethyl-alpha-cyclodextrin, dimethyl-beta-cyclodextrin, maltosyl-alpha-cyclodextrin, glucosyl-1-alpha-cyclodextrin, glucosyl -2-alpha-cyclodextrin, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and sulfobutyl etherbeta-cyclodextrin), polyethylene glycols , and pectin can be used as well.

Masking taste, flavor, other

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

By non-limiting example, the classes of flavor masking agents for fluroquinolone formulation include the addition of flavorings, sweeteners, and various other coating strategies. By non-limiting examples, they can be chosen from sugars such as sucrose, dextrose, and lactose), carboxylic acids, salts, such as magnesium and calcium (chelation-based or nonspecific flavor masking of fluoroquinolone), menthol, amino acids or derivatives of amino acids such as arginine, lysine, monosodium glutamate, and synthetic flavor oils and aromatic flavors and / or natural oils, extracts of plants, leaves, flowers, fruits, etc., and their combinations. These can include cinnamon oils, wintergreen oil, peppermint oils, clove oil, bay oil, anise oil, eucalyptus, vanilla, citrus oil, such as lemon oil, orange oil, grapefruit oil, and grapefruit. , fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, apricot, etc. Additional sweeteners include sucrose, dextrose, aspartame (Nutrasweet®), acesulfame-K, sucralose and saccharin, organic acids (by non-limiting example citric acid and aspartic acid). Such flavors can be present at about 0.05 to about 4 percent. Another approach to improve or mask the unpleasant taste of inhaled drugs is to decrease the solubility of drugs, for example, drugs must dissolve to interact with taste receptors. Therefore, supplying the solid forms of the drug can avoid the affected taste response and acquire the desired improved taste. Non-limiting methods for decreasing the solubility of fluoroquinolones are described herein, for example, the forms

ES 2 599 313 T3 salines of levofloxacin or gemifloxacin with xinafoic acid, oleic acid, stearic acid and pamoic acid. Additional co-precipitating agents include dihydropyridines and a polymer such as polyvinylpyrrolidone. On the other hand, taste masking can be carried out by creating lipophilic vesicles. Additional coating or encapsulating agents include dextrates (by non-limiting example, cyclodextrins may include, 2-hydroxypropyl-beta-cyclodextrin, 2-hydroxypropyl-gamma-cyclodextrin, randomly methylated beta-cyclodextrin, dimethyl-alpha-cyclodextrin, dimethyl- beta-cyclodextrin, maltosyl-alpha-cyclodextrin, glucosyl-1-alpha-cyclodextrin, glucosyl-2-alpha-cyclodextrin, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and sulfobutyl ether-beta-cyclodextrin), modified celluloses such as ethyl cellulose, methyl cellulose alcohols, hydroxypropyl cellulose, hydroxy propyl methyl cellulose, polyalkylene glycols, polyalkylene oxides, sugars and sugar alcohols, acrylics and their lacquers. By non-limiting example, other methods for delivering undissolved forms of the fluoroquinolones deliver the drug alone or in a simple formulation that does not affect solubility, such as a spray-dried, dry powder, crystalline micronized, and nanosuspension formulation. However, an alternative method is to include flavor modifying agents. These include flavor masking substances that are mixed with, coated on, or otherwise combined with the active fluoroquinolone drug. However, this addition can also serve to enhance the taste of another addition of the chosen drug product, for example a mucolytic agent. Non-limiting examples of such substances include the acidic phospholipids, lysophospholipid, polyethylene glycol, tocopherol succinate, and embonic acid (pamoate). Many of these agents can be used alone or in combination with the fluoroquinolones for aerosol administration.

Mucolytic agents

Methods for producing formulations combining agents to reduce sputum viscosity during treatment with an aerosol fluoroquinolone include the following. These agents can be prepared in fixed combination or administered in succession with fluoroquinolone aerosol therapy.

The most commonly prescribed agent is N-acetylcysteine ​​(NAC), which depolymerizes mucus in vitro by breaking the disulfide bonds between macromolecules. Such a reduction in the toughness of sputum is supposed to facilitate its removal from the respiratory tract. Additionally, NAC can act as an oxygen radical scavenger. NAC can be taken either orally or by inhalation. The differences between these two methods of administration have not been formally studied. After oral administration, NAC is reduced to cysteine, a precursor to the antioxidant glutathione, in the liver and intestine. The antioxidant properties may be useful in preventing the deterioration of lung function in cystic fibrosis (CF). Nebulized NAC is commonly prescribed for CF patients, particularly in continental Europe, to improve sputum expectoration by reducing its toughness. The ultimate goal of this is to slow the decline in lung function in CF.

L-lysine-N-acetylcysteinate (ACC) or Nacysteline (NAL) is a new mucoactive agent that has mucolytic, antioxidant, and anti-inflammatory properties. Chemically, it is a salt of ACC. This drug appears to exhibit superior activity than its parental ACC molecule due to synergistic mucolytic activity of L-lysine and ACC. Furthermore, its almost neutral pH (6.2) allows its administration to the lungs with a very low incidence of bronchospasm, which is not the case with acidic ACC (pH 2.2). NAL is difficult to formulate in an inhaled form because the required pulmonary dose is very high (approximately 2 mg) and the micronized drug is adherent and cohesive and is therefore problematic to produce a redispersible formulation. NAL was first developed as a metered dose inhaler (MDI) containing chlorofluorocarbon (CFC) because this form was the easiest and fastest to develop to begin preclinical studies and early clinical studies. NAL's MDI delivers 2 mg per puff, of which approximately 10% was able to reach the lungs in healthy volunteers. One of the main drawbacks of this formulation was patient compliance due to the fact that a maximum of 12 puffs were necessary to obtain the required dose. Additionally, the progressive elimination of CFC gases from medicinal products combined with coordination problems satisfied a large proportion of the patient population (12) leading to the development of a new galenic form of NAL. A dry powder inhaler (DPI) formulation was selected to solve the problems in accordance with the MDIs and combined with an optimal, reproducible, and comfortable way to deliver the drug to the widest possible patient population, including young children.

NAL's DPI formulation involved the use of an unconventional lactose (normally reserved for direct tablet compression), namely a roll-dried anhydrous β-lactose (RD). When tested in vitro with a single-dose DPI device, this powder formulation produces a fine particle fraction (FPF) of at least 30% of the nominal dose, that is, three times higher than with MDIs. This approach can be used in combination with a fluoroquinolone antibiotic, either for co-administration or administration of fixed combination antibiotic therapy.

In addition to mucolytic activity, excessive neutrophil elastase activity within the airways of cystic fibrosis (CF) patients results in progressive lung damage. The breaking of the disulfide bonds of elastase by reducing agents can modify its enzymatic activity. Three naturally occurring dithiol reducing systems were examined for their effects on elastase activity: 1) Escherichia coli thioredoxin system (Trx), 2) recombinant human thioredoxin (rhTrx), and 3) dihydrolipoic acid ( DHLA). Trx systems consist of Trx, Trx reductase, and NADPH. As shown by the spectrophotometric assay of the activity of

ES 2 599 313 Τ3 elastase, the two Trx systems and DHLA inhibited purified human neutrophil elastase, as well as the elastolytic activity present in the soluble (sol) phase of CF sputum. Removal of any of the three constituents of the Trx system prevented inhibition. Compared to the monothiols N-acetylcysteine ​​and reduced glutathione, the dithiols show greater inhibition of elastase. To simplify Trx as a research tool, a stable reduced form of rhTrx was synthesized and used as a single component. Reduced rhTrx inhibits purified elastase and CF sputum sol elastase without NADPH or Trx reductase. Because Trx and DHLA have mucolytic effects, changes in elastase activity after mucolytic treatment were investigated. Raw CF sputum was directly treated with reduced rhTrx, Trx system, DHLA, or DNase. The Trx and DHLA system did not increase elastase activity, while treatment with reduced rhTrx increased elastase sol activity by 60%. In contrast, elastase activity increased by 190% after DNase treatment. The ability of Trx and DHLA to limit elastase activity combined with their mucolytic effects makes these compounds potential therapies for CF.

In addition, the F-actin and DNA assemblages present in the sputum of patients with cystic fibrosis (CF), but absent from the normal airway fluid contribute to the altered viscoelastic properties of sputum that inhibit the clearance of the airway fluid. infected airway and exacerbate CF pathology. One approach to alter these adverse properties is to remove these filamentous aggregates using DNase to enzymatically depolymerize DNA to form monomers and gelsolin that cut F-actin into small fragments. High negative surface charge densities in DNA and F-actin suggest that assemblages of these filaments, which only exhibit strong electrostatic repulsion, can be stabilized with multivalent cations such as histones, antimicrobial peptides, and other prevailing positively charged molecules. in the airway fluid. Furthermore, in fact, it has been observed that the DNA or F-actin pools formed after the addition of histone H1 or lysozyme are efficiently dissolved by soluble multivalent anions, such as polymeric aspartate or glutamate. The addition of the polyaspartate or polyglutamate also disperses the assemblages containing DNA and actin in the CF sputum and decreases the modulus of elasticity of these samples to levels comparable to those obtained after treatment with DNase I or gelsolin. The addition of polyaspartic acid also increased DNase activity when added to samples containing DNA pools formed with histone H1. When added to CF sputum, polyaspartic acid significantly reduced the growth of bacteria, suggesting activation of endogenous antibacterial factors. These results suggest that soluble multivalent anions have the potential alone or in combination with other mucolytic agents to selectively dissociate the large assemblages of charged biopolymers that form in CF sputum.

Thus, NAC, unfractionated heparin, reduced glutathione, dithiols, Trx, DHLA, other monothiols, DNase, dornase alfa, hypertonic formulations (for example, osmolalities greater than approximately 350 mOsmol / kg ), multivalent anions such as polymeric aspartate or glutamate, glycosidases and other examples mentioned above can be combined with fluoroquinolone antibiotics and other mucolytic agents for aerosol administration to enhance antibacterial activity through better distribution of sputum viscosity reduction, and a better clinical outcome by improving lung function (sputum mobility and mucociliary clearance) and decreasing lung tissue damage from the inflammatory immune response.

EXAMPLES

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. Examples in accordance with the invention are those that fall within the scope of the claims in the present description.

Reference Example 1 - high local concentration with short duration of exposure to aerosol fluoroquinolone.

Aerosol administration of fluoroquinolones such as levofloxacin produces high concentrations in the epithelial lining fluid (ELF) of rats and humans. However, this dose has been observed to decrease rapidly after administration.

To determine if high concentrations of short-lived levofloxacin can be effective in the treatment of P. aeruginosa (PA), studies were carried out to measure its bactericidal activity on several strains of this organism that were cultured under different conditions. They were chosen on the basis of what is approximately known about the conditions and growth of AP in cystic fibrosis (CF) of the lung. Four isogenic strains of P. aeruginosa were used for these experiments (Table 2).

Table 2. PA strains used in destruction experiments

<td>Strain</td><td>Genotype</td><td>Levofloxacin MIC (ug / ml)</td>

<td>PAM1020</td><td>Wild type</td><td> 0.25</td>

<td>PAM1032</td><td>nalB</td><td> 1</td>

IS 2 599 313 Τ3

PAM1020 is the parent wild-type strain, PAM1032 contains the na / 6 mutation that results in increased resistance to levofloxacin due to overexpression of the efflux pump MexAB-OprM that can extrude levofloxacin out of cells.

Experiment 1. Activity of levofloxacin against exposed cells.

Methods

Inoculum Preparation

The strains were cultured aerobically overnight in Mueller-HInton broth (MHB) at 35 ° C. They were then diluted 1: 1000 in 100 ml of fresh MHB and cultured at DC&gt; 6oo ~ O, 3 until reaching the CFU / ml ~ 10⁸. 10 ml of this culture was transferred to 50 ml flasks, each containing 10 ml of MHB broth pre-warmed with appropriate concentrations of levofloxacin (2X, compared to exposure concentrations).

Exposition

All strains were treated for 10 mln., 20 mln., 40 mln., 80 mln. and 160 minutes. The following concentrations of levofloxacin (ug / ml) were used for the challenge of PAM1020 and PAM1032: 16, 32, 64, 128 and 256. All strains were treated at each concentration for 10 mln., 20 mln., 40 mln. ., 80 mln. and 160 minutes.

Determination of the number of viable cells

At appropriate time intervals, 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 were enumerated by plating samples diluted in serle (in duplicate) on MHB plates by the droplet plating method (10 ul). The detection limit was 100 CFU / ml. The destruction is presented as the log of the reduction calculated in relation to the cell count at the time of ice from exposure to antibiotics. Relative antibiotic concentrations (in relation to the MIC of the corresponding strains). Cell numbers on ice from antibiotic exposure are shown in Table 3.

Table 3. Number of bacteria at the time of initial bacterial exposure.

<td>Strain</td><td>CFU / ml</td>

<td>PAM1020</td><td>4.03E + 07</td>

<td>PAM1032</td><td>5.60E + 07</td>

Results

For the most susceptible strain, PAM1020, maximum kill (5.5 log decrease in viable cell counts) was achieved after incubation for 10 minutes with the levofloxacin concentration corresponding to the MIC of 256 times (64 ug / ml tested). 5-logs of destruction were already achieved with the lowest concentration tested (16 ug / ml or 64-fold MIC) (Figure 4A). For strain PAM1032, as long as the concentration was reached above 128 times the MIC (128 ug / ml), 10 minutes of exposure was sufficient to give rise to the maximum destruction (more than 5 logs). In short exposures (10 or 20 minutes), less destruction was observed at concentrations below 128 times the MIC. At longer exposure times, the concentration corresponding to MICs of 16 times and above resulted in similar maximum kill (Figure 4B). These results indicate that the log phase cells of P. aeruginosa are efficiently killed after short-term exposures to high concentrations of levofloxacin.

Experiment 2. Activity of levofloxacin against cells in stationary phase

Methods

Inoculum Preparation

The strains were grown aerobically overnight in Mueller-Hnton broth (MHB) at 35 ° C (350 ml total). The spent medium was obtained after centrifugation of the overnight cultures and the supernatant was filtered. The cultures were diluted to OD = 0.3 in spent medium. The same medium was also used to prepare the antibiotic concentrations (the same as in Experiment 1).

Exposition

IS 2 599 313 Τ3

The antibiotic concentrations, the exposure time, as well as the determination of the viable cell counts were the same as in Experiment 1.

Results

The number of cells at the start of antibiotic exposure is shown in Table 4.

Table 4. Numbers of bacteria at the time of initial bacterial exposure.

<td>Strain</td><td>CFU / ml</td>

<td>PAM1020</td><td>8.0E + 08</td>

<td>PAM1032</td><td>8.50E + 08</td>

For PAM1020 cells in stationary phase, the maximum destruction was observed at the lowest concentration corresponding to 64 times the MIC (16 ug / ml) and the shortest duration of exposure, 10 minutes (Figure 5A) . However, PAM1032 demonstrated an evident dose-dependent kill with maximum kill (4 logs) at 64 MIC concentrations in a short exposure time. The extension of the exposure times did not result in a greater degree of destruction. However, lower drug concentrations were required to achieve the same destruction at longer exposure times (Figure 5B).

The re-growth of PAM1020 and PAM1032 was then compared either after 10 minutes or 160 minutes of treatment with various concentrations of levofloxacin. After the corresponding treatments, the cells were washed twice with antibiotic-free medium. 150 µl of cells were placed in the 96-well plate and growth was continuously monitored at Aeeo using SpectraMax (Molecular Devices). The results are shown in Figures 6A-6D.

The results demonstrate that re-growth of both strains was observed at approximately the same time when the cells were treated with high concentrations of levofloxacin for 10 minutes or 160 minutes. These results further support the efficiency of short-term treatment with high concentrations of levofloxacin. Experiment 3. Activity of levofloxacin against cells grown under oxygen-limiting conditions

Methods

Preparation of the inoculum

The overnight cultures were grown aerobically overnight in Mueller-Hinton broth and then diluted 1: 10,000 in MHB by filling the growth flasks to the top. The cultures were grown without shaking at 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 aerated conditions (50 ml of medium in 250 ml flasks, vigorous shaking). After analysis, it was found that an OD = 0.3 corresponds to a late logarithmic phase of growth. In addition to decreased aeration, antibiotic concentration, exposure time, and determination of viable cell counts were the same as in Experiments 1 and 2.

Results

Cell numbers at the start of antibiotic exposure are shown in Table 5.

Table 5. Number of bacteria at the time of initial bacterial exposure.

<td>Strain</td><td>CFU / ml</td>

<td>PAM1020</td><td>7.5E + 07</td>

<td>PAM1032</td><td>8.5E + 07</td>

In the case of PAM1020, close to maximum destruction (4 logs vs 4.5 logs observed under normal aeration) was achieved after exposure with the lowest concentration of levofloxacin for the shortest duration of time (10 minutes) (Figure 7A ). In the case of PAM1032, dose-dependent killing was observed during 10 minutes or 20 minutes of exposure with the highest killing observed at concentrations corresponding to 128 to 256 times the MIC. Slightly stronger destruction (less than 1 log difference) observed during exposure intervals

Longer ES 2 599 313 T3 (Figure 7B). These data indicate that under oxygen-limited conditions, cells in the late logarithmic growth phase are efficiently destroyed after short-term exposure to high concentrations of levofloxacin.

Experiment 4. Activity of levofloxacin against PAM1032 in CF sputum.

Methods

Cells of strain PAM1032 (MIC = 1 ug / ml) were cultured to OD = 1 (late exponential / early stationary phase of growth) in MHB and then 10-fold concentrated in 10-fold concentrated MHB. 10 ul cells were then added to 90 ul sputum or water in 96-well round bottom plates, restoring the MHB to its original concentration. The quantification plates were preheated for 5 minutes at 37 ° C and different concentrations of levofloxacin (512 ug / ml, 128 ug / ml, 32 ug / ml, 8 ug / ml, 2 ug / ml, and 0, 5 ug / ml) were added. At appropriate times, 10 ul of each treatment culture was diluted 100-fold in MHB to minimize carry-over of levofloxacin. Viable cell numbers were enumerated by plating serially diluted samples on MHB plates by the drop plating method (10 ul). The detection limit was 10⁴ CFU / ml. Kill is presented as the percentage of the starting inoculum that survived after treatment with levofloxacin. The results are shown in Figures 8A and 8B.

Results

The results indicate that while sputum slightly affected the degree of destruction by levofloxacin, rapid and extensive destruction (up to five orders of magnitude) by levofloxacin in sputum was still observed after short-term treatment at high concentrations of antibiotic. .

Experiment 4. Activity of levofloxacin against biofilms of PAM1032 colonies.

Methods

Preparation of the biofilm

Colony biofilms are grown on polycarbonate membrane filters (diameter, 25 mm; Poretics, Livermore, California) resting on MHB plates. The overnight culture of PAM1032 was diluted to OD = 0.3, and then diluted 1: 100 in fresh MHB. 5 ul of this culture was spotted on the membrane filter. The bacteria were incubated at 37 ° C for 48 hours (biofilm maturation).

Exposition

After growth filters were placed in the tubes containing 3 ml of saline or saline and levofloxacin at 128 ug / ml and 1024 ug / ml. Each tube was treated for 10 minutes and 80 minutes. Approximately 5 minutes before the end of the incubation time, the tubes were vigorously vortexed (A) or sonicated with ultrasound and vortex (B) to separate the cells. 1 ml of each challenge 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 were enumerated by plating serially diluted samples (in duplicate) on MHB plates by the droplet plating method (10 ul). The results are shown in Figure 9.

Results

The data demonstrate that the maximum kill (~ 2 logs) is obtained after 10 min with the lowest concentration of the levofloxacin tested (128-fold MIC). No additional destruction was observed at the highest levofloxacin concentration. These data indicate that colony biofilms are more resistant to destruction compared to logarithmic or stationary phase cells. However, the maximum bactericidal activity observed against biofilms (99% under these conditions) was achieved after 10 minutes of exposure to levofloxacin. Experiment 5. Rapid aerosol administration, short-term simulated,

Delivering high concentration of drug exposure in the in vitro pharmacodynamic model.

In vitro pharmacodynamic models of infection allow the exposure of a growing bacterial inoculum to changes in drug concentrations as may occur in vivo. The strength of this approach is that serum concentration vs. the time profile of a drug in man can be simulated in the laboratory in vitro to determine the optimal exposure profile (ie, dose and dosing interval) for a target drug and pathogen.

The following report describes experiments designed to determine the Cmax and AUC that will provide maximum bactericidal effects after an aerosol dose of a fluoroquinolone.

ES 2 599 313 T3

Materials and methods

Pharmacodynamic model of in vitro infection

The in vitro pharmacodynamic model consists of a central (serum analog) and peripheral (extravascular) compartment. The peripheral compartments consist of artificial capillary units (Unisyn, Hopkinton, Massachusett) arranged in series with the central compartment. Each capillary unit has a set of small semi-permeable fibers with a molecular size retention of about 10,000 MW to allow the passage of nutrients but not bacteria. The entire system is set up in a dry heat incubator set at 37 ° C.

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

Bacteria were introduced into the peripheral chamber of the model and grown for 2 hours prior to the first drug dose. The doses of the drugs were administered in the central compartment and pumped to the peripheral chambers by a peristaltic pump. The concentrations in the model were reduced according to the first order of elimination (half-life) by dilution of the central compartment with drug-free medium introduced by an additional peristaltic pump adjusted to the desired clearance.

Samples (0.3 ml) were collected from peripheral compartments at various intervals for determination of bacteria and drug concentrations. Samples were collected from peripheral compartments and assayed for drug concentrations by HPLC.

Bacterial test strains

Pseudomonas aeruginosa PAM1032 and PAM1582. The MICs of these strains for levofloxacin were 1.0 and 32 ug / ml, respectively.

Preparation of the inoculum

Strains were grown aerobically overnight in Mueller-Hinton broth (MHB) at 35 ° C and subcultured 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 approx. 1.0 x 10⁶ CFU / ml. From the resulting dilution, 2.3 ml was injected into each peripheral chamber of the hollow fiber bioreactors (Unisyn, Hopkinton, Massachusett).

Pharmacokinetics

The half-life of levofloxacin was adjusted to be 10 minutes to be equivalent to that observed after administration of aerosol levofloxacin to the lung compartment of man. The target Cmax was 1000 and 600 ug / ml in two experiments.

Results

As an objective, the model showed a half-life of levofloxacin of 10 minutes and the Cmax of 1000 ug / ml for Experiment 5. In comparison, Experiment 6 was modified to achieve the same half-life as Experiment 5, but with a Cmax 600 pg / ml target.

The bactericidal effects of these two regimens were correlated with Cmax. In Experiment 5 with a Cmax of 1000 ug / ml, the maximum bactericidal effect was observed as a 5 log reduction in bacterial counts within 10 minutes with PAM1032 and a 4 log reduction in bacterial counts within 20 minutes with PAM1582 and no regrowth observed during the remaining 2 hours of the experiment (Figure 10). In contrast, while the Cmax of 600 ug / ml used in Experiment 6 maintained the 5 log reduction in bacterial counts for PAM1032, although taking 30 minutes instead of the 10 min observed in Experiment 1, only a reduction of 3 log in bacterial counts was observed for PAM1582 after 45 min (Figure 11). Furthermore, PAM1582 exhibited initial regrowth before the final 2 hours of the experimental window.

Conclusions

Levofloxacin can produce a bacterial reduction of up to 99.9999% with a Cmax of both 600 and 1000 ug / ml versus a strain with a MIC of 1 ug / ml. However, maximum bactericidal activity requires 3X longer at a Cmax of 600 ug / ml. Levofloxacin can also produce a bacterial reduction of up to 99.99% with a Cmax of 600 ug / ml compared to a strain with a MIC of 32ug / ml. However, the time to reach the maximum effect is 45 minutes. In contrast, levofloxacin can produce up to 99.999% of the bacterial reduction of this resistant strain with a Cmax of 1000 ug / ml and the time to maximum effect is reduced to 20

ES 2 599 313 Τ3 minutes. From these results, very high, but short-term exposures of levofloxacin produce rapid and sustained bacterial kill, in both hollow fiber and vial models. Taken together, the above results indicate that the achievement of 800 ug / ml of Initial levofloxacin or another concentration of fluoroquinolone in sputum or human ELF is sufficient to achieve that the above antibiotic has an impact on the MIC99 population as represented by PAM 1582 (MIC of 32 ug / ml).

Example 2 - Determination of the aerosol properties of antibacterial fluoroquinolones.

Introduction

Objective. The purpose of these studies was to evaluate the ability to formulate and deliver by nebulization a variety of fluoroquinolones for the treatment of lung bacterial infections by aerosol administration. The fluoroquinolones evaluated are shown in Table 6.

Table 6. Fluoroquinolones Tested.

<td>Fluoroquinolone</td><td>Sp MICgo (ug / mL)</td><td>MSSA MICgo (ug / mL)</td><td>MRSA CMIg₀(ug / mL)</td><td>Pa CMIg₀(ug / mL)</td><td>approval status</td>

<td>Ciprofloxacin *</td><td> 2</td><td> 1</td><td> 64</td><td> 8</td><td>approved</td>

<td>Gemlfloxaclna *</td><td> 0,06</td><td> 0,06</td><td> 2</td><td> 8</td><td>approved</td>

<td>Levofloxacin</td><td> 2</td><td> 0,5</td><td> 16</td><td> 8</td><td>approved</td>

<td>Marbofloxachine *</td><td> 2</td><td> 2</td><td>ND</td><td> 8</td><td>Veterinary</td>

<td>Gatlfloxaclna *</td><td> 0,5</td><td> 0,125</td><td> 4</td><td> 16</td><td>approved</td>

<td>Ofloxaclna</td><td> 2</td><td> 1</td><td> &gt;32</td><td> 16</td><td>approved</td>

<td>Tosufloxachine *</td><td> 0,5</td><td> 0,125</td><td> &gt;16</td><td> 16</td><td>Japan</td>

<td>Lomefloxachine *</td><td> 16</td><td> 2</td><td> &gt;32</td><td> 32</td><td>approved</td>

<td>Moxlfloxaclna *</td><td> 0,25</td><td> 0,125</td><td> 2</td><td> 32</td><td>approved</td>

<td>Sparfloxachine *</td><td> 0,5</td><td> 0,125</td><td> 16</td><td> 32</td><td>retired</td>

<td>Orblfloxaclna *</td><td> 2</td><td> 2</td><td>ND</td><td> &gt;32</td><td>Veterinary</td>

<td>Pefloxachine *</td><td> 32</td><td> 2</td><td> &gt;32</td><td> &gt;32</td><td>Europe</td>

<td>Trovafloxachine *</td><td> 0,25</td><td> 0,06</td><td> 8</td><td> &gt;32</td><td>retired</td>

<td colspan="2">'(described but not claimed)</td><td></td><td></td><td></td><td></td>

These fluoroquinolones were chosen based on their availability, approval status, and antimicrobial properties. All fluoroquinolones tested are either currently approved in the United States or approved but later withdrawn due to various adverse reactions. In addition, fluoroquinolone vapors, which are in use for veterinary applications, have also been evaluated. Among the bacterial pathogens responsible for respiratory tract infections, Pseudomonas aeruginosa (Pa) and metallic-resistant Staphylococcus aureus (MRSA) are the most intractable to fluoroquinolone treatment. Streptococcus pneumonia (Sp) is probably the most important pathogen responsible for respiratory tract infections and numerous reports show high rates of resistance to fluoroquinolones in these bacteria. The MICgo for Pa is in the range of 4 ug / ml to 32 ug / ml and from 2 ug / ml to&gt; 32 ug / ml for Pa and MRSA, respectively. Ciprofloxacin, levofloxacin, gemlfloxacin, and gatlfloxacin vs gemlfloxacin and moxlfloxacin are the most potent against Pa and MRSA, respectively.

Table 7 contains a list of additional fluoroquinolones (disclosed but not claimed) for potential evaluation. The most microblologically interesting compounds on the list are cllnafloxachine and olamufloxachine, which was discontinued due to adverse reactions, and sltofloxachine, which is in phase III clinical trials.

IS 2 599 313 Τ3

Table 7. Fluoroquinolones for potential evaluation.

<td>Fluoroquinolone</td><td>Sp MICgo (ug / mL)</td><td>Sa MICgo (ug / mL)</td><td>MRSA CMIg₀(ug / mL)</td><td>Pa CMIg₀(ug / mL)</td><td>State market</td>

<td>Clinafloxacin</td><td> 0,06</td><td> 0,06</td><td> 2</td><td> 4</td><td>discontinued</td>

<td>Sitafloxacin</td><td> 0,06</td><td> 0,125</td><td> 4</td><td> 8</td><td>phase III</td>

<td>Olamufloxacin</td><td> 0,06</td><td> 1</td><td> 2</td><td> 16</td><td>discontinued</td>

<td>Norfloxacin</td><td> 16</td><td> 1</td><td> &gt;4</td><td> 16</td><td>approved</td>

<td>Prulifloxacin</td><td> 1</td><td> 0,25</td><td> 32</td><td> 16</td><td>Phase III</td>

<td>Danofloxacin</td><td>NA</td><td> 0,125</td><td>NA</td><td> &gt;16</td><td>Veterinary</td>

<td>Enrofloxacin</td><td> 1</td><td> 0,125</td><td> 8</td><td> &gt;16</td><td>Veterinary</td>

<td>Sarafloxacin</td><td>NA</td><td> 0,25</td><td> &gt;16</td><td> &gt;16</td><td>Veterinary</td>

<td>Balofloxacin</td><td> 0,5</td><td> 0,25</td><td> 8</td><td> 32</td><td>Korea</td>

<td>Fleroxacin</td><td> 8</td><td> 1</td><td> &gt;4</td><td> 32</td><td>Europe</td>

<td>Difloxacin</td><td> 2</td><td> 0.5</td><td>NA</td><td> 32</td><td>Veterinary</td>

<td>Rufloxacin</td><td> 32</td><td> 2</td><td> 64</td><td> 32</td><td>Europe, China</td>

<td>Enoxacin</td><td> 16</td><td> 1</td><td> &gt;4</td><td> &gt;32</td><td>retired</td>

<td>Garenoxacin</td><td> 0,06</td><td> 0,06</td><td> 8</td><td> &gt;32</td><td>Phase III</td>

<td>Grepafloxacin</td><td> 0,5</td><td> 0,125</td><td> 32</td><td> &gt;32</td><td>retired</td>

<td>Pazufloxacin</td><td> 4</td><td> 0,5</td><td> &gt;16</td><td> &gt;32</td><td>Japan</td>

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

Specific physicochemical considerations for fogging include aqueous solubility, viscosity, and surface tension. The aqueous solubility of the drug should advantageously be sufficient to meet or exceed the minimum dosage requirements. Drug loading concentration further affects delivery time. Longer delivery times can be commercially unacceptable or lead to poor patient compliance. Although longer delivery times can, in effect, modify the AUC profile, by non-limiting example, the PARI eFlow device has been found to deliver 4 ml of the aqueous levofloxacin in less than 5 min. Furthermore, using such an efficient device, the high concentration of levofloxacin may be able to deliver the effective doses described by the present invention in a time frame that further allows rapid administration, the high drug concentration requirements necessary to optimal fluoroquinolone therapy.

In the case of fluoroquinolones, pH directly affects solubility. In general, the solubility decreases significantly with increasing pH in the range of 1.5 to 6.5. Because pH also affects patient tolerance (see below), the optimal choice of fluoroquinolone for pulmonary aerosol delivery has certain levels of solubility and pH.

For the purpose of this feasibility study, the target solubility was set at 10 mg / ml or at a pH greater than 4.5 or more, based on calculations of therapeutic dose and delivery metrics for available nebulizers. To overcome the preventive concentration of the mutant (MPC), the peak concentration of fluoroquinolone after aerosol administration advantageously reaches from approximately 100 ug / ml to approximately 1000 ug / ml at the site of infection, pending the MIC of the infecting organism. Based on these considerations, the minimum dose to be in this therapeutically relevant range is anticipated to be at least about 30-40 mg Respirable Dose Delivered (RDD). Given the relative half-life of levofloxacin in the human lung, the practical realization of this dose by nebulization can be obtained with a loading dose of at least approximately 100 mg in a volume of approximately 2 mL (approximately 50 mg / mL) in a high-efficiency vibrating mesh device that works at its maximum performance efficiency delivering this dose in less than 4 minutes. A standard jet or ultrasonic 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 speed of administration by these less efficient devices may not be sufficient to achieve a high

ES 2 599 313 Τ3 local concentration with short-term exposure. Similar effective dosages can also be achieved by administering levofloxacin as a dry powder, where the rapid solubility properties of levofloxacin can allow for rapid dissolution resulting in these desired concentrations of the soluble drug. However, alternative concentrations or alteration of the AUC shape profile of fluoroquinolone may be desirable.

Alternatively, although aqueous solubility is important, it is reasonable to predict a formulation that uses complexing or particle technology to allow nebulization of less soluble fluoroquinolones. Unfortunately, more complex formulations increase both the complexity and cost of drug development, and in the case of ultrasonic and jet nebulizers, cause a significant reduction in delivery efficiency, and limit the ability to introduce other design elements. in a final drug product.

In addition to the solubility of the drug, for vibrating mesh nebulizer devices the surface tension of the drug formulation is also sensitive. Thus, in one embodiment, the surface tension is adjusted during formulation by modifying the drug concentration, the excipient concentration, and / or the addition of the surfactant.

In addition to factors that affect efficient nebulization, other factors can be considered for patient tolerance and compliance. By non-limiting example, these factors can include osmolality, pH, and taste. Osmolality affects acute tolerance in the respiratory tract and can be optimized for most drugs during formulation. Similarly, the pH of an aerosol further contributes to tolerance, however, only negatively when the pH of the formulation is less than 4.5. Therefore, because pH contributes directly to the solubility of fluoroquinolone, fluoroquinolones that require a pH of less than 4.5 for solubility are likely to be poorly tolerated. Lastly, the taste of fluoroquinolones can affect better patient compliance. Fluoroquinolones are generally known to be associated with an unpleasant, sometimes very intense taste. While there are technologies available that can mask the bad taste of drugs, these technologies increase the complexity and cost of development, and may not be fully effective in the case of fluoroquinolones. Therefore, similar to pH, taste can be considered to identify a suitable fluoroquinolone for fogging.

Preparation and characterization of test solutions

The antibiotics were purchased from various sources, as shown in Table 8.

Table 8. Preparation of fluoroquinolone test solutions (described, but not claimed).

<td>Not.</td><td>Fluoro-quinolone</td><td>Fountain ^to</td><td>Purity¹⁵</td><td>Quantity</td><td>Volume H2O</td><td>Final Conc.</td>

<td> 1</td><td>Gatifloxacin</td><td>LKT</td><td> 99,6</td><td>8.7 mg</td><td>0.87 mL</td><td>10 mg / mL</td>

<td> 2</td><td>Gemifloxacin</td><td>LG</td><td> 99,6</td><td>9.5 mg</td><td>0.95 mL</td><td>10 mg / mL</td>

<td> 3</td><td>Levofloxacin</td><td>LKT</td><td> 99,2</td><td>10.3 mg</td><td>1.03 mL</td><td>10 mg / L</td>

<td> 4</td><td>Moxifloxacin</td><td>LKT</td><td> 99,5</td><td>12.5 mg</td><td>1.25 mL</td><td>10 mg / mL</td>

<td> 5</td><td>Ciprofloxacin</td><td>LKT</td><td> 99,3</td><td>19.5 mg</td><td>1.95 mL</td><td>10 mg / mL</td>

<td> 6</td><td>Ofloxacin</td><td>LKT</td><td> 99,1</td><td>11.7 mg</td><td>1.17 mL</td><td>10 mg / mL</td>

<td> 7</td><td>Lomefloxacin</td><td>ΜΡΙ</td><td>NA</td><td>17.0 mg</td><td>1.70 mL</td><td>10 mg / mL</td>

<td> 8</td><td>Marbofloxacin</td><td>Vetoquino</td><td>NA</td><td>4.8 mg</td><td>0.48 mL</td><td>10 mg / mL</td>

<td> 9</td><td>Orbifloxacin</td><td>ΜΡΙ</td><td>NA</td><td>4.2 mg</td><td>0.42 mL</td><td>10 mg / mL</td>

<td> 10</td><td>Pefloxacin</td><td>ΜΡΙ</td><td>NA</td><td>15.0 mg</td><td>1.50 mL</td><td>10 mg / mL</td>

<td> 11</td><td>Sparfloxacin</td><td>ΜΡΙ</td><td>NA</td><td>14.5 mg</td><td>1.45 mL</td><td>10 mg / mL</td>

<td> 12</td><td>Tosufloxacin</td><td>ΜΡΙ</td><td>NA</td><td>15.2 mg</td><td>1.52 mL</td><td>10 mg / mL</td>

<td> 13</td><td>Trovafloxacin</td><td>ΜΡΙ</td><td>NA</td><td>2.0 mg</td><td>0.2 mL</td><td>10 mg / mL</td>

<td colspan="3">to. LKT: LKT Laboratories. LG: LG Chem</td><td colspan="3">NA Font not available.</td><td></td>

<td colspan="6">b. Purity of the material tested. Described as GMP or API percentage.</td><td></td>

<td colspan="2">c. 25 mg / ml solution.</td><td></td><td></td><td></td><td></td><td></td>

IS 2 599 313 Τ3

A sample of 2-20 mg of each antibiotic was weighed into sterile plastic tubes and made up with a volume of sterile water to obtain a 10 mg / mL solution or suspension of the antibiotic. The samples were incubated for approximately 10 minutes at room temperature with occasional mixing, before further handling.

After the Incubation period, the antibiotic solutions were observed for their visible appearance, with results as shown in Table 9.

Five of the fluoroquinolones tested were visibly soluble, and either colorless, or with a yellow shade. Eight were visibly insoluble, appearing cloudy (fine particles), opaque (fine to medium dense particle), or cloudy (coarse, large particle suspension), in all cases with visible sediment. The pH of these Initial solutions were determined, and the Range was 3.5 to 7.0. Insoluble solutions were titrated with 1N HCl to the point of visible solubility, and the pH of the solubilized solution was determined. In three cases, marbofloxacin, sparfloxacin and tosufloxacin, the solubility was not reached at pH 1.5, and the addition of acid was also stopped. With the exception of ofloxacin, the pH of these titrated solutions ranged from 1.5 to 3.0.

Table 9. Characteristics of the flouroquinolone solution (described, but not claimed).

<td></td><td></td><td colspan="2">Initial solution</td><td colspan="3">After pH adjustment</td>

<td>Not.</td><td>Fluoroquinolone</td><td>Appearance</td><td>PH</td><td>1N of HCI (uL)</td><td>Appearance ^to</td><td>H</td>

<td> 1</td><td>Gatifloxacin</td><td>white, cloudy, visible sediment</td><td> 7,0</td><td> 5</td><td>slightly yellow color, transparent, without precipitate</td><td> 3.0</td>

<td> 2</td><td>Gemifloxacin</td><td>colorless, transparent, without sediment</td><td> 4,7</td><td></td><td>NR</td><td> -</td>

<td> 3</td><td>Levofloxacin</td><td>slightly yellow color, transparent, without sediment</td><td> 4,7</td><td></td><td>NR</td><td></td>

<td> 4</td><td>Moxifloxacin</td><td>bright yellow color, transparent, without sediment</td><td> 4,7</td><td></td><td>NR</td><td></td>

<td> 5</td><td>Ciprofloxacin</td><td>white, opaque (very dense), visible sediment</td><td> 5,5</td><td> 60</td><td>colorless, transparent, without sediment</td><td> 2.0</td>

<td> 6</td><td>Ofloxacin</td><td>cloudy, visible sediment</td><td> 6,5</td><td> 10</td><td>slightly yellow color, transparent, without sediment</td><td> 5.2</td>

<td> 7</td><td>Lomefloxacin</td><td>cloudy, visible sediment</td><td> 4,2</td><td></td><td>clear, without precipitate, after 10 mln. at room temperature.</td><td></td>

<td> 8</td><td>Marbofloxacin</td><td>white, very cloudy, visible sediment</td><td> 6,5</td><td> 40</td><td>white, cloudy, visible sediment</td><td> 1.5</td>

<td> 9</td><td>Orbifloxacin</td><td>white, cloudy, visible sediment</td><td></td><td> 20</td><td>colorless, transparent, without sediment</td><td> 1.7</td>

<td> 10</td><td>Pefloxacin</td><td>Colorless, transparent, without precipitate</td><td> 4,5</td><td></td><td>NR</td><td> -</td>

<td> 11</td><td>Sparfloxacin</td><td>bright yellow, cloudy, visible sediment</td><td> 5,0</td><td> 20</td><td>bright yellow, thickly cloudy, visible sediment</td><td> 1.5</td>

<td> 12</td><td>Tosufloxacin</td><td>white, cloudy, visible sediment</td><td> 3,5</td><td> 20</td><td>white, cloudy, less cloudy, visible sediment</td><td> 1.5</td>

<td> 13</td><td>Trovafloxacin</td><td>colorless, slightly cloudy, without precipitate</td><td> 4,2</td><td></td><td>NR</td><td> -</td>

<td colspan="2">to. NR: initial pH adjustment.</td><td colspan="5">not required. Fluoroquinolone was soluble at pH&gt; 4 in the solution</td>

IS 2 599 313 Τ3

After pH adjustment, and followed by an additional 10 minute incubation period with occasional mixing, the final appearance of the solutions was determined, just prior to aerosol tolerance and taste testing. The results are shown in Table 10.

Table 10. Final solution appearance of fluoroquinolones (described, but not claimed).

<td>Not.</td><td>Fluoroquinolone</td><td>PH</td><td>Solubility</td><td>Colour</td><td>Sediment</td><td>Opacity</td>

<td> 1</td><td>Gatifloxacin</td><td> 3,0</td><td> +</td><td>C</td><td>None</td><td>none to very light</td>

<td> 2</td><td>Gemifloxacin</td><td></td><td> +</td><td>C</td><td>None</td><td>none to very light</td>

<td> 3</td><td>Levofloxacin</td><td> 4,7</td><td> +</td><td>VLY</td><td>None</td><td>None</td>

<td> 4</td><td>Moxifloxacin</td><td> 4,7</td><td> +</td><td>Y</td><td> +/-</td><td>None</td>

<td> 5</td><td>Ciprofloxacin</td><td> 2,0</td><td> +</td><td>C</td><td>None</td><td>None</td>

<td> 6</td><td>Ofloxacin</td><td> 5,2</td><td> +</td><td>Ly</td><td> +/-</td><td>None</td>

<td> 7</td><td>Lomefloxacin</td><td> 4,2</td><td> +</td><td>C</td><td> +/-</td><td>none to very light</td>

<td> 8</td><td>Marbofloxacin</td><td> 1,5</td><td> -</td><td>W</td><td> ++</td><td> ++</td>

<td> 9</td><td>Orbifloxacin</td><td> 1,7</td><td> +</td><td>C</td><td>None</td><td>light</td>

<td> 10</td><td>Pefloxacin</td><td> 4,5</td><td> +</td><td>C</td><td>None</td><td>light</td>

<td> 11</td><td>Sparfloxacin</td><td> 1,5</td><td> ...</td><td>DY</td><td> +++</td><td> ++++</td>

<td> 12</td><td>Tosufloxacin</td><td> 1,5</td><td> -</td><td>W</td><td> ++</td><td> +++</td>

<td> 13</td><td>Trovafloxacin</td><td> 4,2</td><td> +</td><td>C</td><td> +</td><td>light</td>

Y = yellow; LY = light yellow; VLY = very light yellow; DY = dark yellow; C = colorless; W = white.

Compounds exhibiting the preferred solubility of solutions suitable for inhalation administration (10 mg / mL at pH 4.5 or higher) were levofloxacin, gemifloxacin, moxifloxacin, ofloxacin, and pefloxacin. Levofloxacin, ofloxacin, and moxifloxacin exhibited the best solubility / pH characteristics.

Taste and Tolerance Assessment

Two evaluations were performed to determine the suitability of the fluoroquinolone solutions with respect to taste and tolerance.

First, an oral taste test determined the taste of a 20 ul portion of the test sample in a single, healthy human volunteer by placing the material directly on the front center portion of the tongue. The taste was then monitored for a period of 1 minute. This test was carried out with the initial solutions prepared, as well as the final solutions after pH adjustment. The data is shown in Table 11.

Table 11. Oral fluoroquinolone taste test.

<td>Not.</td><td>Fluoroquinolone</td><td>Initial solution</td><td>Final solution</td>

<td> 1</td><td>Gatifloxacin</td><td>unpleasant moderate bitter taste, slightly aromatic</td><td>strong bitter taste, almond-like, unpleasant, strong bad taste in the mouth</td>

<td> 2</td><td>Gemifloxacin</td><td>Very bitter unpleasant taste with a strong bad taste in the mouth, up to the end of the throat.</td><td>unrealized</td>

<td> 3</td><td>Levofloxacin</td><td>slightly chemical taste, slightly bitter, slightly almond-like taste</td><td>unrealized</td>

<td> 4</td><td>Moxifloxacin</td><td>moderate bitter-sweet taste, unpleasant, slightly aromatic</td><td>unrealized</td>

IS 2 599 313 Τ3

<td> 5</td><td>Ciprofloxacin</td><td>sweet almond-like taste</td><td>bitter taste, very strong in all parts of the throat</td>

<td> 6</td><td>Ofloxaclna</td><td>bitter, unpleasant, almond-like taste</td><td>moderate bitter, unpleasant, almond-like taste</td>

<td> 7</td><td>Lomefloxaclna</td><td>moderate to strong taste, almond-like, not very unpleasant</td><td>unrealized</td>

<td> 8</td><td>Marbofloxaclna</td><td>unpleasant amrago taste, similar to almonds</td><td>moderate to strong, bitter, unpleasant, almond-like taste</td>

<td> 9</td><td>Orbifloxacin</td><td>strong, unpleasant taste</td><td>very strong bitter taste, very unpleasant</td>

<td> 10</td><td>Pefloxaclna</td><td>strong bitter taste, unpleasant almond-like</td><td>unrealized</td>

<td> 11</td><td>Spreading</td><td>light taste</td><td>strong almond-like flavor</td>

<td> 12</td><td>Tosufloxachine</td><td>medium to moderate almond-like flavor</td><td>strong almond-like flavor</td>

<td> 13</td><td>Trovafloxacin</td><td>very strong bitter taste, unpleasant, almond-like, strong bad taste in the mouth</td><td>undone</td>

Lowering the pH generally had the effect of improving the taste properties of the solution. Gatifloxacin, gemifloxacin, ciprofloxacin, orbifloxacin, and trovafloxacin were the least desirable in the taste test. Of the fluoroquinolones tested, levofloxacin was the only fluoroquinolone that was tolerable with respect to taste, at the concentration tested. Lomefloxachine had a moderately strong almond-like flavor, and the taste was a bit off-putting.

In the second test, a small 0.5 ml aerosol sample from an aliquot of the test formulation was determined for tolerance and taste in a single healthy human volunteer, after nebulization in a PARI eFlow nebulizer (Table 12 ).

Table 12. Aerosol Fluoroquinolone Tolerance and Taste Test (described, but not claimed).

<td>Not.</td><td>Fluoroquinolone</td><td>Aerosol tolerance and taste</td>

<td> 1</td><td>Gatifloxacin</td><td>moderate bitter taste, unpleasant, mild coughing sensation</td>

<td> 2</td><td>Gemifloxacin</td><td>strong bitter taste, unpleasant, strong bad taste in the mouth, mild coughing sensation</td>

<td> 3</td><td>Levofloxacin</td><td>chemical taste, sometimes bitter, mild coughing sensation</td>

<td> 4</td><td>Moxlfloxaclna</td><td>moderate bitter taste, unpleasant, some cough, bad taste strong and bitter in the mouth</td>

<td> 5</td><td>Ciprofloxacin</td><td>very strong, unpleasant bitter taste, immediate coughing sensation</td>

<td> 6</td><td>Ofloxaclna</td><td>bitter chemical taste, mild coughing sensation</td>

<td> 7</td><td>Lomefloxaclna</td><td>chemical taste, sometimes bitter, mild coughing sensation</td>

<td> 8</td><td>Marbofloxaclna</td><td>too insoluble to test</td>

<td> 9</td><td>Orbifloxacin</td><td>very acidic, unpleasant strong bitter taste, strong cough</td>

<td> 10</td><td>Pefloxaclna</td><td>chemical taste, some cough</td>

<td> 11</td><td>Spreading</td><td>too insoluble to test</td>

IS 2 599 313 Τ3

<td> 12</td><td>Tosufloxachine</td><td>too insoluble to test</td>

<td> 13</td><td>Trovafloxaclna</td><td>unpleasant bitter taste, no cough or coughing sensation, no bad taste in the mouth</td>

In the case of orblfloxacin, marbofloxaxin, and trovafloxacin, smaller portions were tested, due to solubility limitations. In a calibration experiment, the Inhaler produced an aerosol output of 4.1 microns VMD, with a geometric standard deviation (GSD) of 1.64 microns VMD. In addition to these measurements, the Inhaler produced a fine particle dose (FPD) of 54.9% (percent of the emitted dose in particles less than 5 microns). The tolerance and taste of the drug were monitored for a very short period of administration and for a period of 10 minutes after administration. Tolerance parameters were of the following types: (I) cough, coughing or sneezing sensation (¡I) Irritation, burning or tightness in the throat, (¡I) Irritation or drainage in the nasal passages or eyes, (¡II) Irritation, burning, or a feeling of tightness in the lungs or lack of amplitude, and (¡v) dizziness, headache, nausea, or other systematic effects.

Marbofloxachine, sparfloxachine, and tosufloxachine were too insoluble to be evaluated in this trial. For the rest of the fluoroquinolones tested, no tolerance effects were observed during or after aerosol exposure in categories II, III, or IV (above). Gatlfloxachine, moxlfloxachine, orblfloxachine, and pefloxachine have been associated with cough. In the case of clprofloxachine and orblfloxachine this could be associated with the low pH of the solution. Of the fluoroquinolones tested, levofloxachine at 10 mg / ml had the best flavor characteristics. Ofloxachine, lomefloxachine, and pefloxachine had a more perceptible taste than levofloxachine, which were also acceptable, during the short cycle of administration.

Summary and Conclusions of the Fluoroquinolone Taste Test

Of the thirteen fluoroquinolones tested in this study, levofloxachine had preferred physicochemical properties for aerosol administration and a demonstration of better acute tolerance of the fluoroquinolones tested (Table 13). Levofloxachine is also recognized as having one of the best antimicrobial profiles for respiratory pathogens and has the highest in vivo efficacy comparable to clprofloxachine for the treatment of Pseudomonas aeruginosa Infections.

Table 13. General convenience for nebulization (described, but not claimed).

<td>Not.</td><td>Fluoroqulnolone</td><td>Evaluation</td><td>Punctuation total</td><td>Limitation</td>

<td> 1</td><td>Gatlfloxaclna</td><td>poor solubility and pH, moderately strong bitter spray taste</td><td> —</td><td>solubility, flavor</td>

<td> 2</td><td>Gemlfloxaclna</td><td>sufficient solubility and pH, spray taste strong bitter, strong bad taste in the mouth</td><td></td><td>Taste</td>

<td> 3</td><td>Levofloxachine</td><td>excellent solubility and pH, chemical spray taste, sometimes bitter</td><td> +</td><td>Taste</td>

<td> 4</td><td>Moxlfloxaclna</td><td>sufficient solubility and pH, moderately strong bitter spray taste, strong bad taste in the mouth</td><td></td><td>Taste, Pa's Activity</td>

<td> 5</td><td>Clprofloxachine</td><td>poor solubility and pH, very strong bitter spray taste, presence of cough</td><td></td><td>solubility, flavor</td>

<td> 6</td><td>Ofloxaclna</td><td>minimally acceptable solubility and pH, bitter chemical spray taste</td><td> -/+</td><td>Taste</td>

<td> 7</td><td>Lomefloxaclna</td><td>minimally acceptable solubility and pH, chemical spray flavor, strong liquid flavor</td><td> -/+</td><td>Activity Pa</td>

<td> 8</td><td>Marbofloxaclna</td><td>very little solubility Even at low pH, Unable to test</td><td> -</td><td>Solubility</td>

<td> 9</td><td>Orblfloxaclna</td><td>very poor solubility Even at low pH, unpleasant bitter strong aerosol taste, strong cough</td><td></td><td>solubility, flavor, Activity Pa</td>

IS 2 599 313 Τ3

<td rowspan="2"> 10</td><td rowspan="2">Pefloxacin</td><td colspan="2">sufficient solubility and pH, flavor spray</td><td rowspan="2"> -/+</td><td rowspan="2">Activity Pa</td>

<td>chemical flavor unpleasant</td><td>strong liquid</td>

<td> 11</td><td>Spreading</td><td>very little solubility Unable to test</td><td>Even at low pH,</td><td> —</td><td>solubility, Actlvladad Pa</td>

<td> 12</td><td>Tosufloxachine</td><td>very little solubility Unable to test</td><td>Even at low pH,</td><td> —</td><td>Solubility</td>

<td> 13</td><td>Trovafloxaclna</td><td colspan="2">moderate solubility and pH, bitter spray taste</td><td> —</td><td>Flavor, Activity Pa</td>

Ofloxacin, lomefloxacin, and pefloxacin exhibited lower solubility and stronger flavor at 10 mg / ml of levofloxacin. Ofloxacin is 2 times less potent than levofloxacin, and lomefloxacin and pefloxacin are 4 times less potent. The highest concentrations of these antibiotics have the preferred potency and administration times of less than 15 minutes.

In a separate study, carried out in a similar manner, norfloxachine was tested and found to have a very similar solubility, taste, and potency profile to gatlfloxachine, with the exception of significantly less activity against gram-posthlve pathogens. .

Taste test of saline formulations of levofloxachine and aerosol gemlfloxachine

Based on the results of previous studies, levofloxachine and its racemate ofloxacin, as well as gemlfloxachine, and to a lesser extent gatlfloxachine and norfloxachine are susceptible to aerosol administration for pulmonary antibacterial therapy. To test the taste properties and acute tolerance (cough and cough sensation) of levofloxachine and gemlfloxachine, various formulations were prepared with different organic and inorganic acids and tested as described above. Solutions were prepared by first adding 500 mg of levofloxachine to 10 ml of water or adding 500 mg of gemlfloxachine to 20 ml of saline (due to solubility limitations), titrating the pH to ~ 6.5 with HCl or acid. organic, then adjusting the osmolality of the solutions containing the levofloxachine to -300 mOsmol / kg with sodium chloride. The formulations tested are shown in Table 14.

These formulations were tested in a total of three healthy human volunteers in the same manner as described above, at a levofloxachine concentration of 50 mg / ml, and a gemlfloxachine concentration of 25 mg / ml, in a carefully controlled trial. point to point, totally blind. The results are shown in Table 15 and 16.

These results demonstrate that the hydrochloric acid, citric acid, and ascorbic acid formulations of levofloxachine have superior taste and tolerance compared to the acetic acid, lactic acid, and tartaric acid formulations of levofloxachine. Furthermore, these levofloxachine formulations have superior taste and tolerance over equivalent gemlfloxachine formulations. With respect to gemlfloxachine, the citric acid formulation had superior taste and tolerance compared to the HCl and ascorbic acid formulations of gemlfloxachine, and with further formulation refinement, may be susceptible to aerosol administration.

Table 14. Levofloxachine and gemlfloxachine formulations (described but not claimed).

<td>Fluoroquinolone</td><td>Acid</td><td>Cone (mg / mL)</td><td>PH</td><td>osmolality (mOsm / kg)</td>

<td>Levofloxachine</td><td>HCl</td><td> 50</td><td> 6,5</td><td> 181</td>

<td>Levofloxachine</td><td>Acetic</td><td> 50</td><td> 6,41</td><td> 273</td>

<td>Levofloxachine</td><td>Citric</td><td> 50</td><td> 6,45</td><td> 286</td>

<td>Levofloxachine</td><td>Lactic</td><td> 50</td><td> 6,42</td><td> 286</td>

<td>Levofloxachine</td><td>Ascorblco</td><td> 50</td><td> 6,50</td><td> 278</td>

<td>Levofloxachine</td><td>Tartaric</td><td> 50</td><td> 6,35</td><td> 286</td>

<td>Gemlfloxaclna</td><td>HCl</td><td> 25</td><td> 5,6</td><td> 330</td>

<td>Gemlfloxaclna</td><td>Citric</td><td> 25</td><td> 5,7</td><td> 363</td>

<td>Gemlfloxaclna</td><td>Ascorblco</td><td> 25</td><td> 5,9</td><td> 347</td>

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Table 15. Taste and tolerance of 50 mg / ml aerosol levofloxachine formulations (described, but not claimed).

<td></td><td colspan="3">Taster</td>

<td>Acid</td><td> 1</td><td> 2</td><td> 3</td>

<td>HCI</td><td>Moderate bitter taste</td><td>Bitter taste, coughing sensation</td><td>Bitter taste</td>

<td>Acetic acid</td><td>Very sour taste</td><td>Strong acidic taste, coughing sensation</td><td>sour taste, aftertaste</td>

<td>Citric acid</td><td>mild taste then slightly sweet</td><td>mild, sweet taste</td><td>smooth aftertaste</td>

<td>Lactic acid</td><td>Strong bitter taste, aftertaste</td><td>Mild taste, aftertaste, slight cough</td><td>bitter, mild aftertaste</td>

<td>Acid ascorblco</td><td>mild, slightly sour taste</td><td>Smooth flavor</td><td>little taste or aftertaste</td>

<td>Tartaric acid</td><td>very bitter, strong aftertaste</td><td>strong bitter taste, bitter aftertaste</td><td>bitter taste</td>

Table 16. Taste and tolerance of Gemlfloxaclna formulations at 25 mg / mL (described but not claimed).

<td></td><td colspan="3">Taster</td>

<td>Acid</td><td> 1</td><td> 2</td><td> 3</td>

<td>Acid hydrochloric</td><td>metallic taste, strong aftertaste</td><td>slight bitter taste</td><td>bitter, slightly metallic taste</td>

<td>Citric acid</td><td>slightly sweet</td><td>Mild cough slightly bitter</td><td>very mild flavor, no aftertaste</td>

<td>Ascorbic acid</td><td>Smooth flavor</td><td>Cough, mild bitter aftertaste</td><td>Slightly bitter, smooth aftertaste</td>

Taste Test Additional Formulations of Levofloxachine Aerosol

To test the flavor and tolerance properties of the additional levofloxachine excipient combinations in a systematic way, a number of formulations were prepared and tested. The formulations are listed in Table 17. They included sugars, salts, sweeteners and other excipients prepared by mixing levofloxachine with water, adding the excipients listed in Table 17, and titrating if necessary to the desired pH with dilute HCl, osmolality was not optimized for these studies. However, osmolality was determined using an Advanced Instruments Model 3250 osmometer. This measurement, carried out on 250 µl of sample, is based on lowering the freezing point to determine osmolality.

These formulations were tested on a total of three healthy human volunteers with a test signal (AG) in the same manner as described above, in a carefully controlled, point-to-point, completely blind manner. All tests were carried out in a completely blind manner. Test results (Tables 19-25) are described below. The following scoring system was used (Table 18).

Test A: Taste test of sweeteners, dlvalent metal salts, and surfactants. This test includes sweeteners, calcium and magnesium salts, and surface active agents (ie, glycerin and PS-80). As shown in Table 17, the formulations containing sweeteners were shown to be mildly bitter and have a metallic taste. The artificial sweeteners appeared to produce a bitter taste that is distinct from the bitterness otherwise observed. More significantly, the formulation containing CaCl2 had the best taste relative to the control (MgCl2 was not tested in this experiment) (Table 19).

Test B: Taste test of monkey and sugarcane in the presence of calcium chloride. All the formulations tested in this experiment were well tolerated and tasted better than the control sample. Formulations containing both the calcium salt and sugar performed better than either of them alone, suggesting that these compounds improve flavor through different mechanisms. Of these formulations, 5% of

IS 2 599 313 Τ3

CaCl2 + 7.5% glucose performed better. Note that lactose is present in a lower concentration than the other sugars (Table 20).

Table 17. Levofloxacin formulations containing various excipients.

<td>Fluoroquinolone</td><td>Cone (mg / ml)</td><td>Excipients</td><td>PH</td><td>Osmolality (mOsm / kg)</td>

<td>Levofloxacin</td><td> 50</td><td>Control A (0.225% NaCl)</td><td> 6,50</td><td> 180</td>

<td>Levofloxacin</td><td> 50</td><td>Aspartame (0.1%)</td><td> 6,49</td><td> 175</td>

<td>Levofloxacin</td><td> 50</td><td>Sucralose (0.1%)</td><td> 6,49</td><td> 178</td>

<td>Levofloxacin</td><td> 50</td><td>Glucose (5%)</td><td> 6,5</td><td> 380</td>

<td>Levofloxacin</td><td> 50</td><td>Sucrose (7.5%); NaCI (0.225%)</td><td> 6,51</td><td> 329</td>

<td>Levofloxacin</td><td> 50</td><td>Gllcerlna (5%)</td><td> 6,48</td><td> 880</td>

<td>Levofloxacin</td><td> 50</td><td>PS-80 (0.1%)</td><td> 6,51</td><td> 189</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5 %)</td><td> 6,10</td><td> 784</td>

<td>Levofloxacin</td><td> 50</td><td>MgSO₄ (5 %)</td><td> 6,41</td><td> 73</td>

<td>Levofloxacin</td><td> 50</td><td>Control - BE (0.225% NaCI)</td><td> 6,51</td><td> 182</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5 %)</td><td> 6,1</td><td> 735</td>

<td>Levofloxacin</td><td> 50</td><td>CaCl2 (5%), Sucrose (7.5%)</td><td> 6,10</td><td> 958</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5%), Glucose (7.5%)</td><td> 6,10</td><td> 1174</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5%), Glucose (7.5%)</td><td> 5,25</td><td> 1246</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5%), Lactose (5%)</td><td> 6,07</td><td> 864</td>

<td>Levofloxacin</td><td> 50</td><td>MgCI₂ (5 %)</td><td> 5,90</td><td> 600</td>

<td>Levofloxacin</td><td> 50</td><td>MgCl2 (5%), Sucrose (7.5%)</td><td> 5,98</td><td> 815</td>

<td>Levofloxacin</td><td> 50</td><td>MgCI₂ (5%), Glucose (7.5%)</td><td> 5,98</td><td> 999</td>

<td>Levofloxacin</td><td> 50</td><td>MgCI₂ (5%), Glucose (7.5%)</td><td> 5,04</td><td> 1035</td>

<td>Levofloxacin</td><td> 50</td><td>MgCI₂ (5%), Lactose (5%)</td><td> 5,96</td><td> 697</td>

<td>Levofloxacin</td><td> 50</td><td>MgSO4 (5%), Sucrose (7.5%)</td><td> 6,20</td><td> 433</td>

<td>Levofloxacin</td><td> 50</td><td>MgSO₄ (5%), Glucose (7.5%)</td><td> 6,21</td><td> 625</td>

<td>Levofloxacin</td><td> 50</td><td>MgSO₄ (5%), Glucose (7.5%)</td><td> 5,40</td><td> 660</td>

<td>Levofloxacin</td><td> 50</td><td>MgSO₄ (5%), Lactose (5%)</td><td> 6,18</td><td> 387</td>

<td>Levofloxacin</td><td> 50</td><td>Control F- G (0.45% NaCI)</td><td> 6,5</td><td> 221</td>

<td>Levofloxacin</td><td> 50</td><td>Glucose (5%)</td><td> 6,5</td><td> 376</td>

<td>Levofloxacin</td><td> 50</td><td>sucrose (5%)</td><td> 6,5</td><td> 240</td>

<td>Levofloxacin</td><td> 50</td><td>Lactose (5%)</td><td> 6,62</td><td> 241</td>

<td>Levofloxacin</td><td> 50</td><td>Lactose (2.5%)</td><td> 6,55</td><td> 170</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5 %)</td><td> 6,10</td><td> 735</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (5%), Lactose (5%)</td><td> 6,21</td><td> 1037</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (2.5%), Lactose (5%)</td><td> 6,36</td><td> 565</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (2.5%), Lactose (2.5%)</td><td> 6,41</td><td> 370</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (1.25%), Lactose (2.5%)</td><td> 6,64</td><td> 227</td>

<td>Levofloxacin</td><td> 50</td><td>CaCI₂ (0.625%), Lactose (2.5%)</td><td> 6,06</td><td> 163</td>

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Table 18. Taste test scoring system.

<td>Punctuation</td><td>Taste</td><td>Tolerance</td>

<td> 1</td><td>Comparable to saline</td><td>No coughing sensation, no coughing</td>

<td> 1,25</td><td>Taste lighter than saline</td><td>Slight coughing sensation, no cough</td>

<td> 1,5</td><td>Bitter / mild metallic taste</td><td>Coughing sensation, slight cough</td>

<td> 1,75</td><td>Between 1.5 and 2.</td><td> -</td>

<td> 2</td><td>Moderate bitter / metallic taste</td><td>Coughing sensation, moderate cough</td>

<td> 2,25</td><td>between 2 and 2.5.</td><td> -</td>

<td> 2,5</td><td>Strong bitter / metallic taste</td><td> -</td>

<td> 2,75</td><td>between 2.5 and 3.</td><td> -</td>

<td> 3</td><td>Very strong bitter / metallic taste</td><td>Coughing and strong coughing sensation</td>

<td> 4</td><td>Very strong bitter / metallic taste and other unpleasant taste</td><td>Coughing sensation, strong cough and other irritation</td>

Table 19. Flavor and tolerance of Levofloxachine formulations containing sweeteners, divalent metal salts, and surfactant.

<td rowspan="3">Excipients</td><td colspan="8">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>Aspartame (0.1%)</td><td> 2</td><td> 1,25</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>Sucrulose (0.1%)</td><td> 2</td><td> 1</td><td> 1,75</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>Sucrose (7.5%); NaCI (0.225%)</td><td> 2</td><td> 1</td><td> 2,25</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>Glucose (5%)</td><td> 1,5</td><td> 2</td><td> 2,5</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>Gllcerlna (5%)</td><td> 2,25</td><td> 1</td><td> 2,25</td><td> 1</td><td> 2,5</td><td> 1</td><td> 2.3</td><td> 1</td>

<td>PS- 80 (0.1%)</td><td> 1,75</td><td> 1</td><td> 2,25</td><td> 1</td><td> 2,5</td><td> 1</td><td> 2.3</td><td> 1</td>

<td>CaCI₂ (5 %)</td><td> 1,25</td><td> 1</td><td> 1,5</td><td> 1,5</td><td> 2</td><td> 1</td><td> 1,5</td><td> 1</td>

<td>MgSO₄ (5 %)</td><td> 1,5</td><td> 1,5</td><td> 2,5</td><td> 2,5</td><td> 2,5</td><td> 1</td><td> 2,5</td><td> 1,5</td>

<td>Control -A (0.225% NaCl)</td><td> 3</td><td> 1</td><td> 3</td><td> 1</td><td> 2,5</td><td> 1</td><td> 3</td><td> 1</td>

Table 20. Flavor and tolerance of Levofloxachine CaCI formulations₂.

<td rowspan="3">Excipients</td><td colspan="8">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>CaCI₂ (5 %)</td><td> 1,75</td><td> 1</td><td> 2</td><td> 1</td><td> 2,75</td><td> 1</td><td> 2</td><td> 1</td>

<td>Sucrose (5%)</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>CaCI₂ (5 %)₂, Sucrose (7.5%)</td><td> 1,75</td><td> 1</td><td> 1,75</td><td> 1</td><td> 1,5</td><td> 1</td><td> 1,8</td><td> 1</td>

<td>CaCI₂(5%), Glucose (7.5%)</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1</td><td> 2</td><td> 1</td><td> 1,5</td><td> 1</td>

<td>CaCI₂ (5%), Lactose (5%)</td><td> 1</td><td> 1</td><td> 1,75</td><td> 1</td><td> 2</td><td> 1</td><td> 1,8</td><td> 1</td>

<td>Control BE (0.225% NaCI)</td><td> 3</td><td> 1</td><td> 2,5</td><td> 1</td><td> 3</td><td> 1</td><td> 3</td><td> 1</td>

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Test C: Taste test of Mono and Disaccharides in the Presence of Magnesium Chloride. As before, all the selected formulations in this experiment were well tolerated and tasted better than the control sample. Formulations containing both the magnesium salt and lactose appeared to perform slightly better than either alone. This experiment confirms that the combination of divalent metal salts and simple sugars is effective in improving flavor (Table 21).

Table 21. Flavor and tolerance of Levofloxacin MgCI formulations₂.

<td rowspan="3">Excipients</td><td colspan="8">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>MgCI₂ (5 %)</td><td> 1,5</td><td> 1</td><td> -</td><td> -</td><td> 1,75</td><td> 1</td><td> 1,6</td><td> 1</td>

<td>MgCI₂ (5%), Sucrose (7.5%)</td><td> 1,5</td><td> 1</td><td> 1,75</td><td> 1</td><td> 2</td><td> 1</td><td> 1,8</td><td> 1</td>

<td>MgCI₂(5%), Glucose (7.5%)</td><td> 1,25</td><td> 1</td><td> 2,25</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>MgCI₂ (5%), Lactose (5%)</td><td> 1</td><td> 1</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1</td>

<td>Control BE (0.225% NaCl)</td><td> 2,25</td><td> 1</td><td> -</td><td> -</td><td> 2,75</td><td> 1</td><td> 2,5</td><td> 1</td>

Test D: Mono- and Disaccharides taste test in the presence of magnesium sulfate. As with calcium and magnesium chloride, formulations containing magnesium sulfate and glucose, sucrose, or lactose tasted better than the control sample. This experiment confirms that the combination of divalent metal salts and simple sugars improves taste (Table 22).

Table 22. Flavor and tolerance of the formulations of Levofloxacin M2SO4.

<td rowspan="3">Excipients</td><td colspan="8">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>MgSO4, Sucrose</td><td> 1,5</td><td> 2</td><td> 1,5</td><td> 1,25</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1,3</td>

<td>MgSO4, Glucose</td><td> 1,5</td><td> 2,75</td><td> 2</td><td> 2,5</td><td> 1,5</td><td> 1,5</td><td> 1,5</td><td> 2,5</td>

<td>MgSO4, Lactose</td><td> 1,25</td><td> 2,25</td><td> 1,75</td><td> 1,25</td><td> 1,75</td><td> 1</td><td> 1,8</td><td> 1,3</td>

<td>Control BE (0.225% NaCl)</td><td> 2,25</td><td> 1</td><td> -</td><td> -</td><td> 3</td><td> 1</td><td> 2,6</td><td> 1</td>

Test E: Taste test 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 divalent cation salts was tested at low (&lt;5.5) and high (&gt; 6.0) pH. Small but consistent improvements were seen in flavor at higher pH (Table 23).

Table 23. Flavor and tolerance of Levofloxacin CaCl2 formulations at low versus high pH.

<td rowspan="3">Excipients</td><td colspan="8">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 2</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>CaCI₂ (5%), Glucose (7.5%), pH6.1</td><td> 1</td><td> 1</td><td> 1,5</td><td> 1</td><td> 2</td><td> 1</td><td> 1,5</td><td> 1</td>

<td>CaCI₂ (5%), Glucose (7.5%), pH 5.5</td><td> 1,25</td><td> 1</td><td> 1,75</td><td> 1</td><td> 2,5</td><td> 1</td><td> 1,8</td><td> 1</td>

<td>MgCI₂ (5%), Glucose (7.5%), pH6.0</td><td> 1,25</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td><td> 2</td><td> 1</td>

<td>MgCI₂ (5%), Glucose (7.5%), pH 5.0</td><td> 1,75</td><td> 1</td><td> 1,75</td><td> 1</td><td> 1,5</td><td> 1</td><td> 1,8</td><td> 1</td>

<td>MgSO₄ (5%), Glucose (7.5%), pH 6.2</td><td> 1,25</td><td> 2.25</td><td> 2.25</td><td> 1,75</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1,8</td>

<td>MgSO₄ (5%), Glucose (7.5%), pH 5.4</td><td> 1,5</td><td> 1,75</td><td> 1,75</td><td> 1,5</td><td> 2</td><td> 1</td><td> 1,8</td><td> 1,5</td>

<td>Control BE (0.225% NaCl)</td><td> 2</td><td> 1</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td>

IS 2 599 313 Τ3

Test F. Mono- and Disaccharide taste test. All formulations selected in this experiment were well tolerated and tasted better than the control sample. The three 5% sugars were better than the control, the 2.5% lactose tasted better than the control, but not as good as the 5%. This experiment confirms that simple sugars improve flavor (Table 24).

Table 24. Flavor and tolerance of the sugar formulations of Levofloxachine (described but not claimed).

<td rowspan="3">Excipients</td><td colspan="6">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>Glucose (5%)</td><td> 1,5</td><td> 1,5</td><td> 2</td><td> 1</td><td> 1,8</td><td> 1,3</td>

<td>Sucrose (5%)</td><td> 1,5</td><td> 1,5</td><td> 1,5</td><td> 1</td><td> 1,5</td><td> 1,3</td>

<td>Lactose (5%)</td><td> 1,75</td><td> 1,25</td><td> 2</td><td> 1</td><td> 1,9</td><td> 1,1</td>

<td>Lactose (2.5%)</td><td> 2,25</td><td> 1,5</td><td> 2</td><td> 1</td><td> 2,1</td><td> 1,3</td>

<td>Control FG (0.45% NaCI)</td><td> 2,5</td><td> 1</td><td> 2,5</td><td> 1</td><td> 2,5</td><td> 1</td>

Test G. Taste and Tolerance of Levofloxachine CaCI Formulations₂ Formulations in the presence of lactose. In this experiment, levofloxachine was formulated with varying concentrations of calcium chloride and lactose (Table 25). As noted throughout this series of experiments, all formulations containing divalent metal salts and sugar were improved with respect to taste and tolerance relative to the control formulation. Most importantly, 5% calcium chloride or 2.5% calcium chloride in the presence of 5% lactose were more effective in reducing the bitterness of levofloxachine. Additional decreases in the concentration of these excipients were less effective.

Table 25. Flavor and tolerance of Levofloxachine CaCI formulations₂ in the presence of lactose.

<td rowspan="3">Excipients</td><td colspan="6">Taster</td>

<td colspan="2"> 1</td><td colspan="2"> 3</td><td colspan="2">Median</td>

<td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td><td>Taste</td><td>Tol.</td>

<td>CaCI₂ (5 %)</td><td> 1,25</td><td> 1</td><td> 1,5</td><td> 1</td><td> 1,4</td><td> 1</td>

<td>CaCI₂ (5%), Lactose (5%)</td><td> 1,25</td><td> 1</td><td> 2</td><td> 1</td><td> 1,6</td><td> 1</td>

<td>CaCI₂ (2.5%), Lactose (5%)</td><td> 1,25</td><td> 1</td><td> 2</td><td> 1</td><td> 1,6</td><td> 1</td>

<td>CaCI₂ (2.5%), Lactose (2.5%)</td><td> 1,5</td><td> 1</td><td> 2,5</td><td> 1</td><td> 2</td><td> 1</td>

<td>CaCI₂ (1.25%), Lactose (2.5%)</td><td> 1,75</td><td> 1</td><td> 2</td><td> 1</td><td> 1,9</td><td> 1</td>

<td>CaCI₂ (0.625%), Lactose (2.5%)</td><td> 1,75</td><td> 1,25</td><td> 2</td><td> 1</td><td> 1,9</td><td> 1,1</td>

<td>Control FG (0.45% NaCI)</td><td> 3</td><td> 1</td><td> 2,5</td><td> 1</td><td> 2,8</td><td> 1</td>

Reference Example 3 - Characterization of aerosol levofloxachine in the PARI LC Plus jet fogger.

The following studies describe the potential for aerosolized delivery of levofloxachine delivered to a patient through a jet nebulizer. To accomplish this task, a simple formulation of levofloxachine was prepared and the aerosol was characterized in a jet fogger. The results of these studies are shown below in the summary.

Inhalation solution of levofloxachine (55 mg / mL) was evaluated using a PARI LC Plus Air jet nebulizer with ProNeb Compressor. The emitted dose, the particle size distribution and the fine particle fraction were measured by cascade compaction using a Marple Miller compactor. The parameters mentioned above were used to evaluate the in vitro performance of aerosolized drugs.

Marple Mlller Study

Objective. To determine the particle size distribution and estimate the amount of drug that is likely

ES 2 599 313 Τ3 that a patient inhales (breathable fraction). A secondary objective was to estimate the emitted dose, which is the amount of levofloxachine that escaped from the nebulization.

Methods. Formulation: 55 mg / ml of levofloxachine, 120 mM chloride, 70 mM sodium, pH 6.7. The formulation was established from the maximum solubility that allows a dosage of 300 mg in 6 mL and neutral pH. 5.5 mL of the levofloxachine formulation was added to a PARI LC Plus Jet Nebulizer with ProNeb Compressor. The nebulizer container contained a total of 302 mg of the levofloxachine. The nebulizer was connected in line with a Marple Mlller (MMI) compactor, which operated with an air flow rate of 60 L / ml. Each fogger (n = 2) ran to dryness (no aerosol is produced as judged by visual inspection for 15 minutes). Following aerosolization, the MMI was disconnected and the levofloxachine was quantitatively extracted with the mobile phase (90/10 ACN: water) from the USP inlet port, each of the compactor collection cups (stages) and the fiberglass filter. Any formulation remaining in the nebulizer after aerosolization (cup and nozzle) was quantified as well.

Results

As shown in Table 26, the total average amount recovered from the MMI experiments was 170.2 mg. The expected recovery was 302 mg. This represents a ~ 57% total recovery, which does not meet the generally accepted specifications for impact-based studies (85% -115% total recovery). This difference is due to the non-specific adherence of levofloxachine to the LC Plus nebulizer device. The average percentage of the drug exiting the nebulizer in fine particles was ~ 72%. Therefore, the emitted respillable dose was 89.7 mg. Assuming that ~ 50% is not Inhaled during normal tidal respiration, a total of ~ 40mg can be deposited in the lung at this 300mg dose. However, given the slow delivery time with this device, competition with lung clearance can likely prevent the accumulation of sufficient levofloxachine to meet the minimum concentration required for rapid delivery dosing, the high concentration required for maximum anti-microbial activity of the lung. fluoroqulnolone and prevention of resistance.

Table 26. Marple Mlller Impactor Data Set.

<td>Sample ID</td><td>TO Emitted dose (mg)</td><td>B Amount of drug that remains in the nebulizer cup (mg)</td><td>A + B Amount of drug recovered (mg)</td><td>Percent of the total drug in the nebulizer in the fine particle fraction (% &lt;5pm)</td>

<td>Levo cumshot 1</td><td> 134,70</td><td> 45,00</td><td> 179,70</td><td> 73,5</td>

<td>Cum Levo 2</td><td> 114,40</td><td> 46,30</td><td> 160,70</td><td> 70,4</td>

<td>Average</td><td> 124,6</td><td> 45,7</td><td> 170,2</td><td> 72,0</td>

Reference Example 4 - Animal Models and Evaluation of Fluoroqulnolones and Fluoroqulnolone Formulations.

Pharmacoclnetic Model

Six rats per study are given a single slow intravenous bolus dose of 10 mg / kg via the lateral tail vein or are given a single 10 mg / kg aerosol micro-spray dose using a 10 mg / kg micro-spray device. aerosol generation (PennCentury, Flladelfla, Pennsylvania). Blood samples were taken at different times of more than 3 hours to determine the pharmacokinetic parameters of the plasma. Two rats were sacrificed at 0.5, 1.5, and 3 hours after dosing to determine levels in lung, broncheoalveolar lavage (BAL), and epithelial lining fluid (ELF). Plasma and cell concentrations are determined by an HPLC method and data was then established using WlnNonlIn. The data is shown in Table 27.

Efficacy model

The PAM 1723 strain of P. aeruginosa is cultivated in Mueller-Hnton broth (MHB) at 35 ° C under constant aeration, after 16 hours, the inoculum is subcultured in fresh MHB and allowed to grow again at 35 ° C, under aeration. constant, for 4 hours. Inoculum was set to about 5 x 10⁶CFU / ml by correlation of absorbance at 600 nm with predetermined plate counts. Male CFW mice (4-6 weeks old, N = 4 / group) were rendered neutropenic by Intraperltoneal Injection of 150 mg / kg of clclophosphamide (Cytoxan, Mead Johnson, Prlnceton, NJ) on days 1 and 4 On day 5, mice were infected by intratracheal instillation of 0.05 mL of the

ES 2 599 313 Τ3 inoculum under anesthesia with fluurane (5% fluurane in oxygen running at 4 L / mln). Two hours after infection, mice were administered either intraperltoneal or intratracheal doses of each fluoroquinolone at a dose of 25 mg / kg. The mice were sacrificed 1 and 4 hours after treatment, the lungs were removed, homogenized, and seeded to determine colony counts. The data is shown in Table 28.

Table 27. Pharmacoclinical model.

<td>Drug</td><td>Route</td><td>Dose (mg / kg)</td><td>Serum AUC (O-lnf)</td><td>serum t1 / 2</td><td>ELF AUC (0.5-3h)</td><td>F,% Lung vs, IV</td>

<td>Levofloxacin</td><td>IV</td><td> 10</td><td> 3,8</td><td> 0,5</td><td> 10,5</td><td>NA</td>

<td>Levofloxacin</td><td>ITEM</td><td> 10</td><td> 3,28</td><td> 0,4</td><td> 12,07</td><td> 86 %</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Clprofloxachine</td><td>IV</td><td> 10</td><td> 2,56</td><td> 0,53</td><td>ND</td><td>NA</td>

<td>Clprofloxachine</td><td>ITEM</td><td> 3,3</td><td> 0,8</td><td> 0,93</td><td> 194</td><td> 82 %</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Cllnafloxaclna</td><td>ITEM</td><td> 10</td><td> 3,2</td><td> 0,74</td><td> 30,8</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Gatlfloxaclna</td><td>IV</td><td> 10</td><td> 5,31</td><td> 1,06</td><td> 5,32</td><td></td>

<td>Gatlfloxaclna</td><td>ITEM</td><td> 10</td><td> 5,83</td><td> 1,13</td><td> 54,7</td><td> 100 %</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Norfloxachine</td><td>IV</td><td> 10</td><td> 4,65</td><td> 1,21</td><td> 3,27</td><td></td>

<td>Norfloxachine</td><td>ITEM</td><td> 10</td><td> 4,46</td><td> 1,13</td><td> 41,7</td><td> 100 %</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Gemlfloxaclna</td><td>IV</td><td> 8</td><td> 4,54</td><td> 1,04</td><td> 3,72</td><td></td>

<td>Gemlfloxaclna</td><td>ITEM</td><td> 10</td><td> 5,86</td><td> 1,68</td><td> 536,5</td><td> 86 %</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Tobramlclna</td><td>IV</td><td> 10</td><td> 15,7</td><td> 0,5</td><td> 27,6</td><td>NA</td>

<td>Tobramlclna</td><td>ITEM</td><td> 10</td><td> 13,82</td><td> 1,0</td><td> 5152,0</td><td> 81 %</td>

In rat pharmacokinetic studies, aerosol administration of fluoroquinolones resulted in an increase in ELF AUCs of 0.5-3 hours for all fluoroquinolones tested, as well as for tobramlinol, suggesting that the aerosol route of administration will produce an increase in efficacy against lung infections.

In a mouse model of Lung Infection, increased efficacy was confirmed, at the suggestion of pharmacokinetic studies in rats. For all fluoroqunolones tested, the aerosol (intratracheal or IT) route of administration produced larger reductions in bacterial counts than the intraperltoneal (IP) route of administration, suggesting that the observed increase in efficacy was due to to the production of high local concentrations by direct administration of the aerosol.

Table 28 Efficiency model.

<td>Drug</td><td>Routa®</td><td>Dose (mg / kg)</td><td>DeltaLOG UFC 1 hr^b</td><td>DeltaLOG UFC 4 hr^b</td>

<td>Levofloxacin</td><td>IP</td><td> 25</td><td> -1,00</td><td> -0,52</td>

<td>Levofloxacin</td><td>ITEM</td><td> 25</td><td> -1,97</td><td> -1,28</td>

<td>Gemlfloxaclna</td><td>IP</td><td> 25</td><td> -0,28</td><td> -0,32</td>

<td>Gemlfloxaclna</td><td>ITEM</td><td> 25</td><td> -2,45</td><td> -1,81</td>

<td>Levofloxacin</td><td>IP</td><td> 25</td><td> -1,40</td><td> -1,14</td>

<td>Levofloxacin</td><td>ITEM</td><td> 25</td><td> -2,48</td><td> -1,45</td>

<td>Gemlfloxaclna</td><td>IP</td><td> 25</td><td> -0,74</td><td> -0,71</td>

IS 2 599 313 Τ3

<td>Gemifloxacin</td><td>ITEM</td><td> 25</td><td> -3,20</td><td> -2,28</td>

<td>Clinafloxacin</td><td>IP</td><td> 25</td><td> -1,32</td><td> -1,33</td>

<td>Clinafloxacin</td><td>ITEM</td><td> 25</td><td> -2,86</td><td> -2,47</td>

<td>Tobramycin</td><td>IP</td><td> 5</td><td> -0,70</td><td> 0,29</td>

<td>Tobramycin</td><td>ITEM</td><td> 5</td><td> -1,59</td><td> -0,94</td>

<td>Ciprofloxacin</td><td>IP</td><td> 25</td><td> -1,59</td><td> -0,41</td>

<td>Ciprofloxacin</td><td>ITEM</td><td> 25</td><td> -2,32</td><td> -1,45</td>

<td>Gatifloxacin</td><td>IP</td><td> 25</td><td> -0,34</td><td> -0,02</td>

<td>Gatifloxacin</td><td>ITEM</td><td> 25</td><td> -1,48</td><td> -2,11</td>

<td>Clinafloxacin</td><td>IP</td><td> 10</td><td> -0,96</td><td> -1,39</td>

<td>Clinafloxacin</td><td>ITEM</td><td> 10</td><td> -2,71</td><td> -2,40</td>

<td>Sparfloxacin</td><td>IP</td><td> 25</td><td> -0,85</td><td> 0,09</td>

<td>Sparfloxacin</td><td>ITEM</td><td> 25</td><td> -1,56</td><td> -0,81</td>

<td>Tosufloxacin</td><td>IP</td><td> 25</td><td> 0,00</td><td> 1,33</td>

<td>Tosufloxacin</td><td>ITEM</td><td> 25</td><td> -0,48</td><td> -0,24</td>

<td colspan="5">a, Route of drug administration, b, = Time post drug administration</td>

Reference Example 5 - Characterization of aerosol levofloxacin in the PARI eFlow nebulizer.

Laser Particle Size

The performance of the device was characterized by measuring the size of the emitted particles. By non-limiting example, the size of the aerosol emitted from the levofloxacin solution can be measured with a Malvern Spraytec particle meter under the following conditions. Environmental conditions are controlled to maintain an ambient temperature between 23.0 ° C and 24.0 ° C and relative humidity of 42% to 45%. Levofloxacin at 25 mg / mL was loaded into two fixed PARI eFlow nebulizers with 40 spray heads. The program for the Malvern Spraytec particle size meter was programmed to calculate the following information. A) Volume Mean Diameter (VMD), the mean volume of the particles that pass through the laser beam. B) Geometric Standard Deviation (GSD), percent of diameter 84^v° / percent diameter 50^vo C)% of the particles &lt;5 microns, the percentage of the number of particles smaller than 5 microns or the percentage of the particles&gt; 1 micron and &lt;7 microns, the percentage of the number of the particles between 1 and 7 microns.

The device was loaded with 2 mL of the 25 mg / mL levofloxacin. The device nozzle was positioned with the nozzle tip 2 cm from the center of the beam on the x-axis and as close to the optical lens of the laser as possible on the y-axis. The deviation of the conditioned ambient flow was provided through the nebulizer in an amount to obtain a total nebulizer flow of 20 LPM. Bypassing the conditioned ambient flow was provided through the nebulizer in an amount to obtain a total nebulizer flow of 20 LPM. The nebulizer was turned on and run continuously for 1 minute prior to measurement. The measurement sequence started after 1 minute and measurements were made continuously for 1 minute in 1 second intervals. At the end of the measurement phase, these 60 records were averaged for VMD, GSD and% &lt;5 microns and%&gt; 1 and &lt;7 microns. Finally, the nebulizer was weighed to determine the exit velocity.

Breathing Simulation Studies

Device performance was measured under conditions similar to natural inhalation using a PARI Compass respiration simulator programmed to use the European Standard pattern of 15 breaths per minute, with an inspiration to expiration rate of 1: 1. These measurements were made under environmental conditions that can be controlled to maintain an ambient temperature between 23.0 ° C and 24.0 ° C and relative humidity of 42% to 45%. For this experiment, the PARI eFlow device was loaded with 4 mL of 25 mg / mL levofloxacin solution.

The breathing simulation started, and the nebulizers started. The devices were run continuously until nebulization ceased. The duration was measured from the beginning of the nebulization. Following nebulization, the inspiration and expiration filters were individually washed in an amount

ES 2 599 313 Τ3 known solvent (dH₂OR). The nebulizer cup was also washed individually. For quantification, individual washes were tested by spectrophotometry at a wavelength of 290 nanometers, and the resulting concentration was converted into content. Using these quantitative data, the following analysis was performed. A) Inspired Dose (ID), the total amount of drug tested from the inspiratory filter. B) Residual dose (RD), the amount of drug in the nebulizer tested, at the end of nebulization. C) Fine Particle Dose (FPD), the ID multiplied by the respirable fraction (for example,% of particles &lt;5 microns VMD depending on the method used to determine the size of the particles emitted by the selected device). D) Duration, time from the beginning to the end of the nebulization. E) Delivered Respiratory Dose (RDD),% ID which is, for example, &lt;5 microns VMD.

The results in Table 29 indicate that a 100 mg dose of levofloxacin likely deposits ~ 34 mg of fluoroquinolone into the lung compartment in ~ 4 min using the PARI eFlow device (Table 29) compared to the 300 mg dose of PAR LC Plus device that delivers an equivalent dose in&gt; 15 min. From the rapid administration, high concentration the dosing and delivery model described in the present description, while the 15 min delivery time of the LC Plus will probably fail, a 4 min delivery time of 35-40 mg of levofloxacin may satisfy the criteria for maximum fluoroquinolone activity. However, increasing the drug concentration to allow for faster administration (for example, 50 mg / mL in a 2 mL dosage that delivers 35-40 mg of levofloxacin in ~ 2 min) will most likely meet the minimum requirements. . In addition, shorter administration times will improve patient compliance with dosing. Furthermore, it should be noted that hypotonic solutions of levofloxacin in concentrations greater than 10 mg / mL are poorly tolerated by inhalation.

Table 29. Properties of Levofloxacin in Aerosol (100 mg Loading Dose).

<td></td><td></td><td></td><td colspan="2">FPD (%)</td><td colspan="2">RDD (mg)</td><td>VMD</td><td>GS D</td><td>Osmo</td>

<td>Duration (minutes)</td><td>Dose residual</td><td>Dose inspired</td><td>&lt;5u</td><td>1-7u</td><td>&lt;5u</td><td>1-7u</td><td>um</td><td>um</td><td>mOs / kg</td>

<td> 3,9 ± 0,1</td><td> 24,8 ± 3,4</td><td> 61,1 ± 1,6</td><td> 54,9</td><td> 73,8</td><td> 33,5</td><td> 45,1</td><td> 4,7</td><td> 1,6</td><td> 67 ± 1,0</td>

Reference Example 6 - Levofloxacin Aerosol Tolerance in a Healthy Human Subject Methods

In a single individual, healthy volunteer, the viability of delivery of aerosol levofloxacin was established using either an Aerogen Clinical vibrating mesh device, which creates volumetric mean diameter (VMD) particles of 3.4 microns, or ~ 2 microns. from MMAD (hereafter Aerogen small), or using a PARI eFlow nebulizer that produces ~ 4.7 micron VMD particles (hereafter PARI large). Levofloxacin was tested at a concentration of 4.25 mg / mL or 18.75 mg / mL at doses of 10 mg, 35 mg, and 55 mg, in an isotonic solution.

Results

In the first test, 6 mL of the 4.25 mg / mL solution was inhaled using the Aerogen Small nebulizer. The estimated RDD based on in vitro separation characterization studies of devices using respiration simulation was estimated to be 10 mg. The delivery time was 22 minutes. No noticeable adverse effects on the throat, airways or lungs were observed during or after administration, including the sensation of coughing or coughing, and there was only a slight chemical taste during and after administration. No adverse effects or taste were observed during a 30 minute monitoring period following drug administration. At this low concentration and dose, and the slow rate of administration, levofloxacin was well tolerated.

In the second test, 4 ml of the 18.75 mg / mL solution was inhaled using the Aerogen Small nebulizer. The estimated RDD based on in vitro separation characterization studies of devices using respiration simulator was 35 mg. The delivery time for drug administration was 14 minutes. Despite the increased dose, the acute tolerance was very comparable to that of the first test, both during and after administration. The flavor, which was strongest, was the solution that had the most bitter / metallic chemical taste characteristic of levofloxacin. The taste was more discernible for a period of a few minutes after the end of the administration, again a characteristic of levofloxacin.

In the third test, 4 mL of the 18.75 mg / mL solution was inhaled using the PARI Grande device. The estimated RDD based on the in vitro separation characterization studies of the devices was ~ 55 mg (using FPD definition &lt;5 microns). The delivery time for drug administration was ~ 5 minutes. Although the particle size and drug delivery rate were significantly increased compared to test 2, no adverse effects were experienced in the throat, airways or lungs, apart from the acute taste effects that were noted. above, including the sensation of coughing or coughing, throughout the dosing period and during a 30 minute observation period after dose delivery. Urinary drug recovery, which is an accurate measure of exposure, confirms that the projected respirable dose of approximately 55 mg was delivered successfully.

ES 2 599 313 T3

These results demonstrate the feasibility of aerosol administration of levofloxacin in a human subject at the intermediate concentrations tested, and it is suggested that increased concentrations and doses, properly formulated for tolerability and taste are achievable.

Reference Example 8 - The Preformulation of the Levofloxacin Base.

The aim of this study was to characterize the levofloxacin base to understand the physicochemical capabilities and constraints of the levofloxacin base for various formulation approaches. The purpose of this study was to characterize the physicochemical properties of the levofloxacin base.

Preformulation

PH-Solubility Studies

The solubility of levofloxacin was determined as a function of pH. Buffers were first prepared in the pH 2-10 range. Small aliquots of each buffer (~ 200 to 250 pL) were saturated with drug and shaken to achieve equilibrium solubility. The samples were centrifuged and the supernatants were analyzed for dissolved drug by UV or HPLC. The buffers used in this study were shown to affect the solubility result (because different buffers counteract ions that can form different forms of levo salt in solution). Therefore, the pH-solubility will also be evaluated in the absence of the buffers (through titration). Determination of pKa

The pKa of levofloxacin was determined by titration. The pKa values ​​obtained were confirmed by UV spectrophotometry. This information was used to aid in salt selection for levofloxacin and to determine levofloxacin loading under pH conditions in the lung.

Preformulation for the Liquid System

The viability of a liquid formulation was investigated using (a) solubility and (b) surface tension as baseline parameters for the saline-only formulation.

Preformulation studies on levofloxacin

HPLC transfer method

Experimental Methodology

An HPLC method was used to evaluate the linearity, accuracy and precision of the levofloxacin assay. A 50mm X 4.6 column, C18 Monolithic Onyx (Phenomenex) was used at 30 ° C. The mobile phase consisted of 85% 0.1% TFA in water and 15% 0.1% TFA in acetonitrile. The flow rate was adjusted to 3 mL / min. The samples were injected into the chromatographic system and the eluent was monitored at 277 nm.

Results

The retention time for levofloxacin was approximately 0.82 min. The assay was found to be linear in a range of 5 to 15 pg / mL, with a correlation coefficient of 1,000. The RSD (relative standard deviation) was less than 0.5% and the precision was within 98-102%.

Solubility-pH studies

By degree

Experimental Methodology

A saturated solution of levofloxacin in 0.1N HCl was titrated with NaOH. After each addition of the base, the solution was vortexed. An aliquot of the sample solution was removed, centrifuged, and the supernatant analyzed by UV spectroscopy at 288 nm. The same solution was titrated again with HCl.

Results

The pH-solubility profile of levofloxacin is shown in Figure 12. By titration, levofloxacin exhibited a solubility of 25.4 mg / mL at pH 7.3. However, contrary to the results of the stirring experiments, the solubility by volumetry decreased below pH 6.5 which can be attributed to the common ion effect. Since a solution of levofloxacin was prepared in HCl, a hydrochloride salt of levofloxacin could be formed in the

ES 2 599 313 Τ3 solution. Furthermore, the addition of chloride ions in the form of hydrochloric acid can suppress the solubility of the hydrochloride salt.

Determination of pKa

By Volumetry

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 1N HCl. Measured aliquots of HCl were added and the pH was recorded after each addition. Titration was continued until pH 1.

To determine acidic 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 1N NaOH. Titration was continued until pH 6.55.

Results

Figure 13 shows a graph of pH versus volume of added titrant for titration of levofloxacin with HCL. This data was established in the following equation:

Vt [OH] - Kb. Vep - Kb.Vt where,

Vt = Volume of added titrant

V_ep= volume of titrant added to equivalence point

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

[H⁺] = hydronium ion concentration = 10^_pH

A graph of Vt [OH^-] Vs Vt generated a straight line (Figure 14). The data shown is from the region of the pre-equivalence point. From the slope, slope is obtained: Kb = 2.09X10 '⁸ pK, - - log K_b = 7.7 pKa = 14- pKb = 6.3

Figure 15 shows a graph of pH versus volume of added titrant for titration of levofloxacin with NaOH. Acidic pKa was difficult to calculate because it was quite low (&lt;2.0). However, a rough approximation of the pKa can be made with the pH in the middle of the equivalence point. From the representation of dpH / DV versus titrant volume (V_t) (Figure 16), the equivalence point is at 250 pl. The pH at half the equivalence point (that is, when V_t = 125 pl) is 1.6. So the acidic pKa is ~ 1.6.

By UV spectroscopy

Experimental Methodology

Diluted solutions of levofloxacin (0.013 mg / mL) were prepared in various buffers. The buffers that were used were HCL (pH 1.2), acetate (pH 4.5), phosphate (pH 6.7.8) and borate (9.10). Levofloxacin solutions were analyzed by UV spectroscopy at 257 nm.

Results

A graph of the pH versus absorbance of the levofloxacin solution at 257 nm is shown in Figure 17. These data were put into a modified Henderson Hasselbach equation:

Abs oewrvado - Abs ha l H] + Abs λ- [H ']

Ka + tlPj Ka + [ll *] where,

ES 2 599 313 T3

Observed Abs = absorbance of the levofloxacin solution Abs ha = absorbance of the levofloxacin solution pH 1.2;

Abs a- = absorbance of the levofloxacin solution at pH 7.8;

[H +] = hydronium ion concentration = 10^-pH

The established equation provides an estimate of pKa = 5.91.

Example 11 - Metal Ion Complexes of levofloxacin.

The objective of this study was to prepare levofloxacin from various forms of chelate salts to obtain an increase in taste masking properties, properties to improve the AUC profile throughout changes in solubility, dissolution and / or bioavailability. . These benefits may enhance the pharmacodynamic properties of levofloxacin after pulmonary administration using nanoparticle suspension, dry powder inhalation, or simple liquid formulations. These formulations can be optimized to create formulations to improve the AUC profile of levofloxacin from altered solubility or slow delivery or low bioavailability chelates. These properties can also be incorporated into other fluoroquinolone antibiotics, including, without limitation, gemifloxacin, gatifloxacin, norfloxacin, tosufloxacin, sarafloxacin, sitafloxacin, prulifloxacin, and pazufloxacin. In addition, studies are underway to characterize various chelates and chelated forms of gemifloxacin to mask taste, improve AUC profile, nanoparticle suspension, and dry powder inhalation administration.

Preparation of Metal Ion-Levofloxacin Complexes

Preliminary studies

A mixture of levofloxacin and a salt of an administered cation was solubilized in deionized water and titrated with sodium hydroxide. The titration curve was compared against that obtained for levofloxacin alone to assess the formation of the metal-levofloxacin complex as described in Physical Pharmacy (4th Edition) by Alfred Martin (pp. 261-263). Different metal cations (eg, Ca2 +, Mg2 +, etc.) were evaluated then evaluated to identify suitable candidate (s) for subsequent evaluations. In addition, different molar proportions of cations and levofloxacin were evaluated.

Complex Preparation

Levofloxacin solutions were titrated against aqueous solutions of selected metal salts. Titrations were carried out at constant pH. Complex formation was monitored by different methods, including titration, spectrofluorometry, solubility, etc., as appropriate. The end point of the complexation reaction depended on the method adopted.

Characterization of Levofloxacin Complexes

The metal cation-levofloxacin complexes were characterized by stoichiometry, the formation constants and kinetic dissociation using the appropriate methodology.

goals

Formulate and characterize the complexes of levofloxacin with metal cations (di- and trivalent).

Complexity Assessment

Preliminary research suggests that levofloxacin forms soluble complexes with metal cations. As a result, the evaluation of the precipitation complexation process was not possible. Other approaches that were tried are described below.

Volumetry

This approach is based on the assumption that the carboxylic acid portion of levofloxacin is involved in complexing with a given metal cation and that the results of complexation result in the release of a proton from levofloxacin. The concentration of protons released can therefore be proportional to the extent of complexation (depending on the binding constant) and the stoichiometry of the complex (Physical Pharmacy: 4th Edition by Alfred Martin; pp-261-263).

Experimental Methodology

Approximately 0.35 mmol of levofloxacin (in 16 mL of deionized water) were titrated with 6N NaOH in the presence and in the absence of the salt of a metal cation (equimolar). Levofloxacin solutions were acidified to pH values ​​below 2.0 with 6N HCl before titration with NaOH. Used metal cation salts

ES 2 599 313 Τ3 include calcium chloride, magnesium chloride, ferrous chloride, zinc chloride, aluminum sulfate and aluminum chloride.

Results

As shown in Figure 18, titrations performed in the presence of metal cations resulted in a positive change in the titration curves compared to that obtained with levofloxacin alone, suggesting that additional NaOH (titration reagent) is required to obtain a specific pH of the solution in the presence of a metallic cation. The magnitude of the change in the titration curve at any point can represent moles of protons released due to complexation and thus moles of the complexed levofloxacin.

The degree of complexation (binding and / or stoichiometry) appears to increase in the order of Ca⁺ &lt;Mg²⁺ &lt;Zn²⁺ = Faith²⁺ &lt;To³⁺, which is reasonably in accordance with the existing literature.

Note: It has been noted in the literature that aluminum chloride and aluminum sulfate have acid-like properties and can lower the pH of aqueous solutions. Consequently, the titration curves obtained with AICI3 and AI2 (SO4) 3 cannot provide conclusive information on complexation with levofloxacin.

Double degree

In this approach the levofloxacin solution was titrated with an administered metal cation solution to observe a drop in pH presumably due to proton release through complexation. NaOH was then added to revert to the initial pH of the levofloxacin solution (prior to the addition of the cation solution). This allows the fraction of levofloxacin in the complexed form to be determined at a given pH.

Experimental Methodology

Approximately 1.55 to 1.72 mmol of the levofloxacin was solubilized in deionized water and the resulting solution was acidified with 6N HCl to the desired initial pH. This solution of the acidified levofloxacin was titrated with a known volume of concentrated solution of a given metal cation (Ca²⁺, Mg²⁺, Faith²⁺ and Zn²⁺). The change in pH was neutralized (to initial pH) by the addition of 6N NaOH and the volume of the NaOH solution added was recorded. The addition of the metal cation solution followed by neutralization with NaOH was continued until another addition of the metal cation solution failed at a pH change of the levofloxacin solution, which may indicate the end point of the complexation. The cumulative amounts of added metal cation were plotted against the cumulative amounts of NaOH required to neutralize the pH change (Figures 42-45).

Results

From Figures 19-22, the plateau regions were extrapolated to obtain the total amount of NaOH required to neutralize the change in pH due to complexation. These values ​​also represent the amounts of levofloxacin in the complexed form (assuming that complexation of levofloxacin results in equimolar proton release). The amounts of levofloxacin in the Ca-complexed form²⁺, Mg²⁺, Faith²⁺and Zn²⁺they are 0.8, 1.0, 1.3, and 1.1 mmol, respectively. These represent 46.5, 64.5, 77.8 and 64.5% of the Ca complexes²⁺, Mg²⁺, Faith²⁺ and Zn²⁺, respectively. It should be noted that the percent complexation will depend on the total levofloxacin concentrations.

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

M + nA &lt;=&gt; MA_nK_b

Where M, A and MA_n represent the metal cation, levofloxacin, and the complex, respectively. Kb can be the union equilibrium constant. The above reaction assumes that n moles of levofloxacin react with one mole of metal to produce one mole of complex.

Kb AML „] / {| MJIA]} (Units M *)

Eq. 1

[MA_n] is the concentration of the complex formed, [M] and [A] are the concentrations of the unbound metal and unbound levofloxacin, respectively.

Reordering equation 1,

IS 2 599 313 Τ3

[MA_n]/[TO]ⁿ = --------------------------------------____ Eq. two

[A] - [Ajyotal - [Ajunido ^— [Ajyotal "[NaOHjusado [M] - [Mjfotal ~ [Mjunido ~ [Mjfotal" [NaOH] used / n [MAn] - [A] united / n ^— [NaOH] Used / n

Note: [Adjusted NaOH is the concentration of sodium hydroxide used at any given point to neutralize the change in pH caused by the addition of metal cation (presumably due to complexing).

Equation 2 can be modified to obtain,

[Joined / [A]ⁿ = ηλ '/, * ​​[Μ] ----------------------------------------- ---- Ec. 3

It is inferred from equation 3 that a graph of [M] versus [A]_a¡Do / [A]ⁿ will result in a straight line with a slope of nKb when, n = 1, for a 1: 1 complex n = 2, for a 2: 1 complex n = 3, for a 3: 1 complex etc.

The graphs for Ca are shown below in Figures 23-26.²⁺, Mg²⁺, Faith²⁺ and Zn²⁺, respectively.

As shown in Figures 23-26, for each of the evaluated cations a graph of [A] unldo / [A] n versus n / &lt;i, * [M] was linear when n = 2 (for Ca²⁺ n = 2 resulted in a better fit than n = 1). These results suggest that complexes of levofloxachine with Ca²⁺, Mg²⁺, Faith²⁺ and Zn²⁺ they are formed with a stoichiometry of 2 moles of drug per mole of cation (2: 1).

By using n = 2, the binding constants for the above complexes can be determined from the slopes of the respective linear representations.

The binding constants for 2: 1 complexes represented as log (Kb) (Kb) are as follows: Ca²⁺= 2.75, Mg²⁺= 3.69, Zn²⁺= 4.44, Fe²⁺= 4,54.

Solubility

This method allows a relatively simple way to determine the stoichiometry of the complex. The approach involved evaluation of the solubility of the drug (levofloxachine) in the presence of increasing concentrations of complexing agents (an administered metal cation). The total solubility of the drug (complexed + uncomplexed) was expected to increase completely due to the complexing and to reach the plateau corresponding to the saturation of the solubility of both the drug and the complex. Determination of the stoichiometry of such a solubility curve is explained in detail elsewhere (Physcal Pharmacy: 4th edition by Alfred Martin; pp. 265).

Experimental Methodology

Excess amounts of levofloxachine (amounts were recorded) were shaken, in the presence of increasing concentrations of MgCl2, with 25 mM MES buffer (pH 5.99) using a vortex mixer. The samples were then filtered and the filtrate was appropriately diluted and spectrophotometrically analyzed for levofloxachine concentrations (Figure 27).

Results

As shown in Figure 27, the solubility of levofloxachine increased with increasing concentrations of MgCl2. However, beyond the plateau solubility (~ 650mM levofloxachine), a further increase in solubility was observed, which is not consistent with the expected profile. This was attributed to the effect of ionic force on the solubility of levofloxachine. It is important to bear in mind that the final pH of all the solutions were constant, although greater than 5.99 (final pH -7.0).

Subsequently, the experiment was repeated at a constant ionic force of ~ 1.0M (adjusted with NaCl) and with MES 0.5 buffer (pH 5.99) to increase the buffer capacity of the solution (Figure 28).

Spectrofluorometry

This approach was adopted to evaluate the complex clone of levofloxachine based on the evidence from the existing literature.

ES 2 599 313 Τ3 that the complexation process is associated with a change in the fluorescence properties of fluoroquinolone. By monitoring the change in the fluorescence emission of levofloxacin in the presence of different concentrations of an administered metal cation, it was possible to determine the complexation binding constant, as well as the stoichiometry.

Experimental Methodology

The fluorescence emission of levofloxacin was evaluated at excitation and emission wavelengths of 298 nm and 498 nm, respectively. Studies were conducted at two different pH values, ie 5.0 (acetate) and 9.0 (hlstldlna). A series of solutions containing a constant concentration of levofloxacin, but increasing concentrations of a given cation were analyzed for the emission of fluorescence due to levofloxacin. Metallic salts studied included CaCl2, MgCl2, FeCl2, ZnCl2 and AI2 (SO4) 3.

Results

As shown in Table 34, significant data were obtained only for Fe²⁺ and Zn²⁺. For the remaining cations, the relative concentrations of levofloxacin and the cation need to be further optimized to observe a specific trend in the change in fluorescence of levofloxacin.

The influence of increasing concentrations of Fe2 + and Zn2 + on the fluorescence emission of levofloxacin is shown in Figures 29 and 30, respectively.

As described above, both Fe²⁺ as Zn²⁺ appear to form 2: 1 complexes with levofloxacin; however, their influence on the fluorescence of levofloxacin are different (Figures 29 and 30). The exact reason for this is unclear at this point.

Table 34. Fluorescence characteristics of levofloxacin in the presence of cations.

<td rowspan="2">Cation</td><td colspan="2">Levofloxacin fluorescence</td><td rowspan="2">Results</td><td rowspan="2">Comments</td>

<td>pH 5.0</td><td>pH 9.0</td>

<td>AC²⁺</td><td>Non-significant changes</td><td>Non-significant changes</td><td>N / A</td><td> -</td>

<td>Mg²⁺</td><td>Non-significant changes</td><td>Non-significant changes</td><td>N / A</td><td> -</td>

<td>Faith²⁺</td><td>Decrease in emission with increasing Fe²⁺</td><td>N / A</td><td>Figure 3.12 (PH 5.0)</td><td>FeCI2, insoluble at pH 9.0</td>

<td>Zn²⁺</td><td>Non-significant changes</td><td>Emission increase with increasing Zn²⁺</td><td>Figure 3.13 (PH 9.0)</td><td> -</td>

<td>To the³⁺</td><td>Non-significant changes</td><td>N / A</td><td>N / A</td><td>Ai₂(SW₄) 3 insoluble at pH 9.0</td>

Levofloxacin complex samples

Seven samples of levofloxacin complexes were evaluated in v / 'vo for efficacy and pharmacokinetics. Details of the samples tested are shown in Table 35 below.

IS 2 599 313 Τ3

Table 35. Molar Proportions of Levofloxachine Complexes.

<td>Identifier of the show</td><td>Cation</td><td>Molar ratio used</td><td>Total levofloxachine (mg / mL)</td><td>final pH of the solution</td>

<td>NB-049-001-06-066A</td><td>Mg²⁺</td><td> 1:1</td><td> 40,2</td><td> 6,24</td>

<td>NB-049-001-06-066B</td><td>Faith²⁺</td><td> 1:1</td><td> 40,1</td><td> 6,30</td>

<td>NB-049-001-06-066C</td><td>Mg²⁺</td><td> 1:1</td><td> 202</td><td> 5,98</td>

<td>NB-049-001-06-081A</td><td>AC²⁺</td><td> 1:1</td><td> 40,1</td><td> 6,53</td>

<td>NB-049-001-06-081B</td><td>AC²⁺</td><td> 1:1</td><td> 201</td><td> 6,04</td>

<td>NB-049-001-06-081C</td><td>Zn²⁺</td><td> 1:1</td><td> 40</td><td> 6,33</td>

<td>NB-049-001-06-081D</td><td>Zn²⁺</td><td> 1:1</td><td> 200</td><td> 5,69</td>

Conclusions and Next Stages

The results obtained from our double titer studies suggest that levofloxachine forms 2: 1 complexes with all dlvalent metal cations. The binding constants (log Kb) for the complex with Ca²⁺, Mg²⁺, Faith²⁺ and Zn²⁺ they are 2.75, 3.69, 4.44, and 4.54, respectively.

Reference Example 12 - Formulations of levofloxachine and gemlfloxachine with organic acids.

Experimental Methodology

The levofloxachine solution was prepared by dissolving either 50 or 100 mg of the water-based levofloxachine in 15-20 mL. The Initial pH of the levofloxachine solution in water was approximately 7.3. The pH of the solution was adjusted with approximately 10% acid solution prepared in water. The following acids were used to adjust the pH of the levofloxachine solution: acetic acid, ascorbic acid, citric, lactic, tartaric, and propyl acid. After increasing 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 with approximately 20% sodium chloride solution prepared in water. After adjusting the pH and osmolality the volume of the solution was made up to about 25 mL with water and its surface tension was measured. The pH and osmolality were measured after increasing the volume and are listed in Table 36. (The exact amounts of the heavy levofloxachine, the acid required to adjust the pH, the sodium chloride to adjust the osmolality, and the final volume of the solutions are listed in Table 36). The levofloxachine content in the solutions was determined by HPLC.

Results

Details about the organic acid formulations of levofloxachine are shown in Table 36. HPLC results are shown in Table 37.

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

Note: The solutions with acetic acid, citric acid and ascorbic acid were prepared again for analysis by HPLC and therefore the theoretical concentration of these solutions in Table 36 and Table 37 are different. Gemlfloxachine Formulations with Organic Bases

Methodology and Experimental Results

Formulation of Gemlfloxachine with Sodium Ascorbate.

50.30 mg of gemlfloxachine mesllate (equivalent to 40.37 mg of gemlfloxachine) was added to 1.5 mL of water. The resulting solution was cloudy. It was filtered through a 0.45 micron filter. 1.3 mL of solution were obtained after filtration with a pH of 4.28. The pH of this solution was adjusted to 5.48 with 400 uL of a 10% solution of sodium ascorbate prepared in water (amount of base required to adjust the pH = 0.04 g). The osmolality of this solution was 308 mOsm / kg, therefore, sodium chloride was not used to adjust the osmolality. The final volume of the solution was 1.7 mL. * The theoretical concentration of gemlfloxachine in this formulation can be 20.59 mg / ml.

IS 2 599 313 Τ3

Table 36. Formulations of Levofloxacin with Organic Acids.

<td>Weight of levo used (g)</td><td>9.94% acetic acid used (ml)</td><td>acetic acid used (g)</td><td>19.7% NaCI used (ml)</td><td>NaCI used (g)</td><td>Final volume of the measured solution (ml)</td><td>Cone Levo (mg / ml)</td><td>Osmolality final (mOsm / kg)</td><td>PH Final</td><td>tension superficial (mN / m)</td>

<td> 1,253</td><td> 1,05</td><td> 0,104</td><td> 0,681</td><td> 0,134</td><td> 25,105</td><td> 49,9</td><td> 312</td><td> 6,48</td><td> 63,2</td>

<td> 2,501</td><td> 2,05</td><td> 0,204</td><td> 0,326</td><td> 0,064</td><td> 25,935</td><td> 96,4</td><td> 300</td><td> 6,53</td><td> 62,5</td>

<td>weight of Levo used (g)</td><td>9.99% ascorbic acid used (ml)</td><td>used ascorbic acid (g)</td><td>19.7% from NaCI used (ml)</td><td>NaCI used (g)</td><td>Final vol of measured solution (ml)</td><td>Levo cone (mg / ml</td><td>Osmolality final (mOsm / kg)</td><td>PH final</td><td>tension superficial (mN / m)</td>

<td> 1,253</td><td> 3,400</td><td> 0,339</td><td> 0,550</td><td> 0,108</td><td> 25,135</td><td> 49,8</td><td> 297</td><td> 6,40</td><td> 64,4</td>

<td> 2,505</td><td> 7,400</td><td> 0,739</td><td> 0,300</td><td> 0,059</td><td> 25,135</td><td> 99,7</td><td> 298</td><td> 6,47</td><td> 62,5</td>

<td>Levo Weight used (g)</td><td>10.05% citric acid used (ml)</td><td>acid citric used (g)</td><td>21.54% of NaCI used (ml)</td><td>NaCI used (g)</td><td>Final vol of measured solution (ml)</td><td>Levo cone (mg / ml</td><td>Osmolality final (mOsm / kg)</td><td>PH final</td><td>tension superficial (mN / m)</td>

<td> 1,251</td><td> 1,25</td><td> 0,126</td><td> 1,005</td><td> 0,216</td><td> 25,12</td><td> 49,8</td><td> 299</td><td> 6,54</td><td> 61,5</td>

<td> 2,498</td><td> 2,6</td><td> 0,261</td><td> 0,918</td><td> 0,198</td><td> 25,82</td><td> 96,7</td><td> 301</td><td> 6,53</td><td> 61,4</td>

<td>Levo Weight used (g)</td><td>10% lactic acid used (ml)</td><td>acid lactic used (g)</td><td>21.54% of NaCI used (ml)</td><td>NaCI used (g)</td><td>Final vol of measured solution (ml)</td><td>Levo cone (mg / ml</td><td>Osmolality final (mOsm / kg)</td><td>PH final</td><td>tension superficial (mN / m)</td>

<td> 1,258</td><td> 2,1</td><td> 0,21</td><td> 0,745</td><td> 0,160</td><td> 25,135</td><td> 50,1</td><td> 297</td><td> 6,54</td><td> 59,4</td>

<td> 2,497</td><td> 4,2</td><td> 0,42</td><td> 0,392</td><td> 0,084</td><td> 25,605</td><td> 97,5</td><td> 301</td><td> 6,63</td><td> 57,5</td>

<td>Weight of Levo Used (g)</td><td>10 acid tartaric used (ml)</td><td> %</td><td>tartaric acid used (g)</td><td>21.54% NaCI used (ml)</td><td>NaCI used (g)</td><td>Final vol of measured solution (ml)</td><td>Cone of levo (mg / ml)</td><td>final osmolality (mOsm)</td><td>PH final</td><td>tension superficial (mN / m)</td>

<td> 1,252</td><td> 1,55</td><td></td><td> 0,155</td><td> 0,948</td><td> 0,204</td><td> 25,180</td><td> 49,7</td><td> 298</td><td> 6,51</td><td> 61,5</td>

<td>Weight of</td><td> 9,79</td><td> %</td><td>acid</td><td> 21,53%</td><td>NaCI</td><td>Final vol of</td><td>Cone of</td><td>osmolality</td><td>PH</td><td>tension</td>

<td>Levo used (g)</td><td colspan="2">acid propionic used (ml)</td><td>propionic used (g)</td><td>NaCI used (ml)</td><td>used (g)</td><td>measured solution (ml)</td><td>levo (mg / ml)</td><td>final (mOsm)</td><td>final</td><td>superficial (mN / m)</td>

<td> 1,25281</td><td> 1,310</td><td></td><td> 0,128</td><td> 0,737</td><td> 0,159</td><td> 25,045</td><td> 50,02</td><td> 298</td><td> 6,50</td><td> 58,1</td>

<td> 2,51342</td><td> 2,610</td><td></td><td> 0,256</td><td> 0,310</td><td> 0,067</td><td> 25,030</td><td> 100,42</td><td> 297</td><td> 6,57</td><td> 52,0</td>

* Theoretical concentration = Theoretical amount of gemifloxacin in the filtered solution (in this case 35 mg of filtered gemifloxacin in 1.3 mL) / Final volume of the solution (in this case 1.7 mL).

IS 2 599 313 Τ3

Table 37. Theoretical and actual / measured concentrations of Levofloxacin formulations.

<td>Acid</td><td>Theoretical Conc, (mg / mL)</td><td>Concentration measured (mg / ml) by HPLC</td>

<td>Acetic acid</td><td> 50,05</td><td> 51,45</td>

<td>Acetic acid</td><td> 99,9</td><td> 102,32</td>

<td>citric acid</td><td> 49,91</td><td> 50,31</td>

<td>citric acid</td><td> 99,86</td><td> 102,99</td>

<td>Ascorbic L-Acid</td><td> 49,95</td><td> 50,01</td>

<td>Ascorbic L-Acid</td><td> 100</td><td> 102,49</td>

<td>Lactic acid</td><td> 50,05</td><td> 50,07</td>

<td>Lactic acid</td><td> 97,54</td><td> 95,27</td>

<td>Tartaric acid</td><td> 49,74</td><td> 51,07</td>

<td colspan="3">Note: The solutions with acetic acid, citric acid and ascorbic acid were prepared again for analysis by HPLC and therefore the theoretical concentration of these solutions in Table 36 and Table 37 are different,</td>

Reference Example 13 - Toxic Inhalation in Rats.

In a non-GLP ascending dose study of aerosolized levofloxaxin in male and female Sprague-Dawley rats for 4 days, a 25 mg / mL solution of levofloxacin was administered for one hour on the first day and a 50 mg / mL solution mL of levofloxacin was administered for two hours per day on days 2 through 4. No clinical signs of toxicity were observed during the treatment period. The necropsy at 24 hours after the administration of the last dose showed no findings.

In a GLP study of aerosolized levofloxacin in male and female Sprague-Dawley rats, aerosolized levofloxacin was administered daily at an average dose of 6.92 mg / kg / day for males and 10.04 mg / kg / day. for females for 4 days using a nose-only spray delivery device. Total exposures were 29 and 42 mg / kg for males and females, respectively, during the study period. Each dose was given more than 2 hours a day. The dose for this study was selected based on the maximum solubility of levofloxacin that can be delivered in the device for more than 2 hours. No clinical signs of toxicity were observed, and all animals survived through the 4-day treatment period. The necropsy of the animals after the administration of the last dose showed no findings.

In a 28-day GLP study in Sprague-Dawley rats, animals were randomized to 3 dose levels of levofloxacin in aerosol or saline form. Additional recovery groups used vehicle control and were further treated with the highest dose and observed for a 14 day recovery period after the last dose. Average aerosol doses of levofloxacin were 1.49, 3.63, and 7.29 mg / kg / day for male rats and 2.20, 5.35, and 11.01 mg / kg / day for female rats. . Total exposures during the 28-day treatment period ranged from 41.7 to 204.1 mg / kg for males and 61.6 to 308.3 mg / kg for females. Each dose was given more than 2 hours a day. No dose-related clinical signs of toxicity were observed, and all animals survived through the 28-day treatment period. Necropsy of the animals after administration of the last dose showed dose-related squamous cell hyperplasticity of the larynx that reduced severity during a 14-day recovery period.