KR20070023761A - Composition comprising amphotericinb methods and systems - Google Patents

Abstract

The composition of the present invention comprises particles comprising at least about 95% by weight of amphotericin B, wherein the median diameter of the particles is from about 1.1 μm to about 1.9 μm. Other compositions also include particles comprising at least about 95 weight percent of amphotericin B, wherein at least about 80 weight percent of the particles have a geometric diameter of about 1.1 μm to about 1.9 μm. Another composition comprises particles comprising amphotericin B, wherein the median diameter of the particles is less than about 1.9 μm and the crystallinity level of amphotericin B is at least about 20%. Unit dosage forms, delivery systems, and methods may include similar compositions.

Amphotericin B, median mass, geometric diameter, crystallinity level, delivery system

Description

Compositions, methods and systems comprising amphotericin {{COMPOSITION COMPRISING AMPHOTERICINB METHODS AND SYSTEMS}

One or more embodiments of the present invention include a composition comprising amphotericin B, a method of making and using amphotericin B compositions, and a system for using amphotericin B compositions.

Pulmonary fungal infections, such as invasive filamentous pulmonary fungal infection (IFPFI), are a major cause of morbidity and mortality in immunocompromised patients. The individual's immune system may be compromised by several diseases, such as acquired immunodeficiency syndrome (AIDS) and / or may be deliberately compromised by immunosuppressive therapy. Immunosuppressive therapies are often administered to patients undergoing cancer treatment and / or to patients undergoing transplant procedures.

Immunocompromised patients have an increased risk of pulmonary and / or nasal fungal infections. In particular, patients with severe immunodeficiency, such as those with long-term neutropenia, or those requiring administration of at least 1 mg / kg / day of prednisone for at least 21 consecutive days, in addition to their other immunosuppressive agents, may be required for pulmonary and / or nasal fungal infection It is easy to get caught. Among immunocompromised patients, the total fungal infection rate is between 0.5 and 28%. At the Fred Hutchinson Cancer Center, 7.3% of autopsied bone marrow transplant patients with idiopathic pneumonia syndrome (IPS) had IFPFI. In another study by Vogeser et al., A 4% proportion of IFPFI was found in consistent necropsy of 1187 of European patients who died of any cause during the period 1993-1996. An absolute majority of these European patients include (1) high dose steroid medications; (2) malignant tumor therapies; (3) solid organ transplantation; Or (4) some form of bone marrow transplant.

The most common pulmonary and / or nasal fungal infection in immunocompromised patients is pulmonary and / or nasal aspergillosis. Aspergillosis is a disease caused by the Aspergillus fungal species ( Aspergillus spp.), Which first invades the body through the lungs. The incidence of aspergillosis is determined by the duration and severity of neutropenia, factors of the patient (eg, age, corticosteroid use, and prior pulmonary and / or nasal disease), environmental contamination levels, diagnostic criteria, and etiology Depends on persistence

Other filamentous and abnormal fungi can also induce pulmonary fungal infections. Such additional fungi are generally endemic and local, for example blastomycosis, disseminated candidiasis, coccidioidomycosis, cryptocococcosis, heath Histoplasmosis, mucormycosis and sporotrichosis. Although not typically affecting the pulmonary system, infections caused by Candida species are generally systemic and most often arise from infections through body detention or type IV catheters, wounds or infected solid organ implants. And accounts for 50 to 67% of the total fungal infection among immunocompromised patients.

Ampoterisin B is the only approved fungicidal compound currently being used to treat aspergillosis and is usually delivered intravenously. Amphotericin B is Streptomyces nodosus ) is a positive polyene macrolide obtained from the strain. In its commercial form, amphotericin B is present in both amorphous and crystalline forms. Ampoterisin B, formulated with sodium deoxycholate, was the first commercial parent formulation of amphotericin B. Systemic intravenous therapy is inhibited by dose-dependent toxicity, such as nephrotoxicity and liver toxicity, which interfere with the therapeutic effect and reduce the desirability of the prophylactic use of amphotericin B. Even with approved therapies, the incidence of aspergillosis is increasing and the mortality rate is estimated to exceed 50% of infected people treated.

There is a need in the art for safe and effective amphotericin B compositions, methods of making and using such compositions, and systems for using such compositions. For example, there remains a need for compositions and methods for the safe and effective treatment and / or prevention of patients with pulmonary and / or nasal fungal infections.

Summary of the Invention

Accordingly, one or more embodiments of the present invention include compositions comprising amphotericin B, methods of making and using amphotericin B compositions, and systems for using amphotericin B compositions. Other features and advantages of the invention will be set forth in the description of the invention which follows, and in part will be apparent from the description, or may be learned by practice of the invention. Embodiments of the invention will be realized and learned by the compositions and methods specifically pointed out in the written description and claims.

In one aspect, one or more embodiments of the invention relates to a composition comprising particles comprising at least about 95% by weight amphotericin B. The median mass diameter of the particles is from about 1.1 μm to about 1.9 μm.

In another aspect, one or more embodiments of the invention relates to a composition comprising particles comprising at least about 95% by weight amphotericin B. The geometric diameter of at least about 80% by weight of the particles is from about 1.1 μm to about 1.9 μm.

In another aspect, one or more embodiments of the invention relates to a composition comprising particles comprising amphotericin B. The median mass diameter of the particles is less than about 1.9 μm and the crystallinity level of amphotericin B is at least about 20%.

In another aspect, one or more embodiments of the invention relates to a pharmaceutical composition comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient. The pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of about 1.1 μm to about 1.9 μm.

In another aspect, one or more embodiments of the present invention are directed to pharmaceutical compositions comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient. The pharmaceutical composition is prepared from particles comprising amphotericin B, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is from about 1.1 μm to about 1.9 μm.

In another aspect, one or more embodiments of the invention relates to a pharmaceutical composition comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient. The crystallinity level of amphotericin B is about 20% to about 99%.

In another aspect, one or more embodiments of the invention relates to a dry powder comprising amphotericin B having a crystallinity level of at least about 20%. The dry powder comprises particles having a mass median aerodynamic diameter of less than about 10 μm.

In a further aspect, one or more embodiments of the invention relates to unit dosage forms comprising a container containing a pharmaceutical composition. The pharmaceutical composition comprises an effective amount of amphotericin B and a pharmaceutically acceptable excipient. The pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 1.9 μm.

In another aspect, one or more embodiments of the invention relate to unit dosage forms comprising a container containing a pharmaceutical composition. The pharmaceutical composition comprises an effective amount of amphotericin B and a pharmaceutically acceptable excipient. The pharmaceutical composition is prepared from particles comprising amphotericin B, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm.

In another aspect, one or more embodiments of the invention relates to unit dosage forms comprising a container containing a pharmaceutical composition. The pharmaceutical composition comprises amphotericin B and a pharmaceutically acceptable excipient. The crystallinity level of amphotericin B is about 20% to about 99%.

In another aspect, one or more embodiments of the invention relates to a delivery system comprising an inhaler and a pharmaceutical composition. The pharmaceutical composition comprises microparticles comprising amphotericin B and a pharmaceutically acceptable excipient. The microparticles are made from particles comprising amphotericin B having a median mass diameter of less than about 1.9 μm.

In a further aspect, one or more embodiments of the invention relate to a delivery system comprising an inhaler and a pharmaceutical composition. The pharmaceutical composition comprises microparticles comprising amphotericin B and a pharmaceutically acceptable excipient. The microparticles are made from particles comprising amphotericin B, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm.

In another aspect, one or more embodiments of the invention relates to a delivery system comprising an inhaler and a pharmaceutical composition. The pharmaceutical composition comprises microparticles comprising amphotericin B having a crystallinity level of about 20% to about 99% and a pharmaceutically acceptable excipient.

In another aspect, one or more embodiments of the present invention relates to a method for characterizing amphotericin B for the manufacture of a pharmaceutical composition. The method includes measuring the crystallinity level of the amphotericin B composition. The method also includes confirming that the crystallinity level is above a first predetermined level prior to releasing the amphotericin B composition for combination with a pharmaceutically acceptable excipient to form a pharmaceutical composition.

In another aspect, one or more embodiments of the present invention are directed to a method of characterizing amphotericin B for preparing a pharmaceutical composition. The method includes measuring the amorphousness of the amphotericin B composition. The method also includes confirming that the amorphousness is below a predetermined level prior to releasing the amphotericin B composition for combination with a pharmaceutically acceptable excipient to form a pharmaceutical composition.

In another aspect, one or more embodiments of the present invention are directed to methods of making and characterizing dry powder pharmaceutical compositions. The method includes combining the amphotericin B composition with a pharmaceutically acceptable excipient to form a dry powder pharmaceutical composition. The method also includes determining a mass median aerodynamic diameter of the dry powder pharmaceutical composition. The method further includes confirming that the mass median aerodynamic diameter is below a predetermined level prior to releasing the dry powder pharmaceutical composition for patient administration.

In another aspect, one or more embodiments of the present invention are directed to methods of making and characterizing dry powder pharmaceutical compositions. The method includes combining the amphotericin B composition with a pharmaceutically acceptable excipient to form a dry powder pharmaceutical composition. The method also includes measuring homogeneity of the dry powder pharmaceutical composition. The method further includes confirming that the homogeneity is above a predetermined level prior to releasing the dry powder pharmaceutical composition for patient administration.

In another aspect, one or more embodiments of the present invention are directed to a method of making spray dried particles. The method includes suspending particles comprising amphotericin B in a liquid to form a feedstock. The median mass diameter of the particles is less than about 3 μm. The method also includes spray drying the feedstock to produce spray dried particles.

In another aspect, one or more embodiments of the present invention are directed to a method for preparing spray dried particles. The method includes suspending particles comprising amphotericin B in a liquid to form a feed stock wherein the crystallinity level of amphotericin B is at least about 20%. The method further includes spray drying the feedstock to produce spray dried particles.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises inhaling an effective amount of a composition comprising amphotericin B to a patient in need thereof, wherein the composition comprises amphotericin B having a median mass diameter of about 1.1 μm to about 1.9 μm. It is prepared from the particles.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering inhaled an effective amount of a composition comprising amphotericin B to a patient in need thereof, wherein the composition is prepared from particles comprising amphotericin B and comprises amphotericin B. The geometric diameter of at least about 80% by weight of the particles to be about 1.1 μm to about 1.9 μm.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need thereof, wherein the crystallinity level of amphotericin B is at least about 20%.

In a further aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20%, and the lung solid tissue retention half-life of amphotericin B measured by lung tissue biopsy is at least about 1 week.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20% and the pulmonary epithelial endothelial fluid retention half-life of amphotericin B measured by bronchoalveolar lavage is at least about 10 hours.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering a composition comprising amphotericin B in an amount from about 0.01 mg / kg to 7.0 mg / kg to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20% and the plasma amphotericin B concentration is maintained at less than about 1000 ng / mL.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering a composition comprising amphotericin B in an amount from about 0.01 mg / kg to 7.0 mg / kg to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20% and the plasma amphotericin B concentration is kept low enough to avoid renal toxicity and / or liver toxicity.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering a composition comprising amphotericin B by inhalation to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20%. Lung amphotericin B concentration measured by bronchoalveolar lavage reaches at least about 5 times the minimum inhibitory concentration for at least some treatment periods. Plasma amphotericin B concentration is maintained at less than about 1000 ng / mL.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering a composition comprising amphotericin B by inhalation to a patient in need thereof. The crystallinity level of the amphotericin B is at least about 20%. The treatment period is from about 15 weeks to about 20 weeks. Lung amphotericin B concentration measured by bronchoalveolar lavage reaches at least about 5 times the minimum inhibitory concentration for at least some treatment periods.

In a further aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The present invention includes administering a composition comprising amphotericin B in an amount from about 2 mg to about 50 mg by inhalation to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20%. Administration is carried out in less than about 5 minutes.

In another aspect, one or more embodiments of the invention relate to methods of treating and / or preventing fungal infections. The method comprises administering an effective amount of a dry powder comprising amphotericin B by inhalation to a patient in need thereof. The crystallinity level of amphotericin B is at least about 20% and the dry powder comprises particles having a mass median aerodynamic diameter of less than about 10 μm.

Embodiments of the invention are further described in the detailed description of the invention with reference to a number of non-limiting figures mentioned below.

1 shows the predicted amphotericin B concentration in the lung after administration of the pharmaceutical composition according to the invention.

2 shows the predicted amphotericin B plasma concentrations after administration of the pharmaceutical composition according to the invention.

3A-3E are schematic side cross-sectional views showing the operation of a dry powder inhaler that can be used to aerosolize a pharmaceutical composition of the present invention.

FIG. 4 is a representative graph showing a plot of the flow rate dependence of deposition in the Anderson Cascade Impactor (ACI) for amphotericin B powder of the present invention.

Figure 5 is a representative graph that shows the stability of TSE (Trubospin) ^® DPI device of the present invention using a 60 L / minute amphotericin B powder emitted dose (ED) of the.

Figure 6 is a representative graph that shows plots of the stability of the aerosol performance of the turbo spin ^® DPI device the 28.3 L / min Terry ampo of the present invention using the new B powder.

7 is a representative graph showing a plot of aerosol performance of a pharmaceutical composition of the present invention comprising amphotericin B and various phosphatidylcholines.

FIG. 8 is a representative graph showing a plot of the aerosol performance of a pharmaceutical composition of the present invention comprising 70 wt% amphotericin B using various passive DPI devices at 56.6 L / min.

9 shows ED obtained from the powder of the invention per collector.

10 shows the average ED from the powder of the invention per lot.

11 shows the mass median aerodynamic diameter (MMAD) of the powder of the invention per collector.

12 shows the particulate dosage (% FPD <3.3 μm) of the powder of the invention per collector.

13 shows the ED (mg / capsule) of the powder of the invention per collector.

14 shows the average ED of the powder of the invention per collector and lot number.

15 shows the MMAD of the powder of the invention per collector.

16 shows% FPD <3.3 μm of the powder of the invention per collector

17 shows ED (mg / capsule) for two powders of the invention.

18 is a representative graph showing amphotericin B concentrations at various locations in the body of rats after intratracheal and intravenous administration.

19 is a representative graph showing the average amphotericin B concentration-time profile in the lungs of dogs after 14 days of pulmonary administration.

20 is a Kaplan-Meier Survival Curve showing the effects of one form of the present invention.

Figure 21 shows group mean amphotericin B concentrations in lung tissue, delivered by inhalation to rabbits.

22 shows group mean amphotericin B concentrations in lung tissue, delivered by inhalation to rabbits.

Figure 23 is Kaplan-Meier survival curves showing the effect of one form of the present invention.

FIG. 24 is a plot comparing gravimetric particle size distribution and drug-specific particle size distribution for two powders of the present invention. FIG.

Unless otherwise specified, it is to be understood that the invention is subject to change without being limited to particular formulation components, drug delivery systems, manufacturing techniques, administration steps, and the like. In this regard, unless stated otherwise, reference to a compound or component includes the compound or component itself, as well as a compound, for example a mixture of compounds, in combination with other compounds or components.

Before further discussion, the definitions of the following terms will aid in the understanding of embodiments of the present invention.

As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "phospholipid" includes not only one phospholipid but also a combination or mixture of two or more phospholipids, unless the context clearly dictates otherwise.

As used herein, "particulate" means a particle comprising amphotericin B and one or more pharmaceutically acceptable excipients. The microparticles can have various shapes and forms, such as hollow and / or porous microstructures. Hollow and / or porous microstructures may represent, define or include voids, pores, nicks, hollows, spaces, interstitial spaces, gaps, perforations, or holes, and may be particles that are spherical, collapsed, deformed, or broken.

When referring to an active agent, the term refers to the substance of the specified molecule, as well as salts, esters, amides, hydrazides, N-alkyl derivatives, N-acyl derivatives, prodrugs, conjugates, active metabolites and other such derivatives, analogs and Also included are pharmaceutically acceptable and pharmaceutically active analogs, including but not limited to related compounds. Thus, as used herein, the term “amphotericin B” refers to the aforementioned derivatives, analogs or related compounds, so long as amphotericin B itself, or such amphotericin B derivatives, analogs or related compounds exhibit antifungal activity. it means.

As used herein, the terms “treating” and “treatment” are intended to reduce the severity and / or frequency of symptoms, eliminate the symptoms and / or root cause, reduce the likelihood of developing symptoms and / or root causes, and improve or correct damage. it means. Thus, “treating” a patient with an active agent as set forth herein includes the treatment of individuals with clinical symptoms as well as the prevention of certain symptoms, diseases or disorders in susceptible individuals.

As used herein, “effective amount” means an amount encompassing both a therapeutically effective amount and a prophylactically effective amount.

As used herein, “therapeutically effective amount” means an amount effective to achieve the desired therapeutic result. The therapeutically effective amount of a given active agent will typically depend on the type and severity of the disorder or disease to be treated and factors such as the age, sex and weight of the patient.

As used herein, “prophylactically effective amount” means an amount effective to achieve the desired prophylactic result. Because a prophylactic dose is administered to a patient before the onset of the disease, the prophylactically effective amount is typically less than the therapeutically effective amount.

As used herein, "mass median diameter" or "MMD" typically refers to the median diameter of a plurality of particles in a population of polydisperse particles, ie in a population of varying particle sizes. MMD values as reported herein are determined by laser diffraction (Sympatec Helos, Klaustal-Gellerfeld, Germany) unless otherwise indicated in the text. Typically, the powder sample is added directly to the feed funnel of the Simpatec RODOS dry powder dispersion unit. This can be done manually or by stirring mechanically from the end of the VIBRI vibration feed member. The sample is dispersed into the initial particles through the application of pressurized air (2 to 3 bar) using a vacuum drop (suction) that is maximum for a given dispersion pressure. The dispersed particles are examined with a 632.8 nm laser beam that traverses the trajectory of the dispersed particles at the correct angle. Laser light scattered from the entire particle is imaged into a concentric array of photomultiplier detector elements using a reverse-Fourier lens assembly. Scattered light is obtained with a time-slice of 5 ms. The particle size distribution is inversely calculated from the scattering light space / intensity distribution using a proprietary algorithm.

As used herein, “geometry diameter” means the diameter of a single particle determined microscopically unless otherwise specified in the context.

"Mass median aerodynamic diameter" or "MMAD" as used herein typically means the central aerodynamic size of a plurality of particles or particulates in a polydisperse population. "Aerodynamic diameter" is generally the diameter of a unit density sphere having the same sedimentation velocity as the powder in the atmosphere, and therefore it is a useful means of characterizing aerosolized powder or other dispersed particles or particulate formulations in terms of their sedimentation behavior. Aerodynamic diameter includes particle or particulate shape, density, and physical size of the particle or particulate. As used herein, MMAD means the center of the aerodynamic particle or particulate size distribution of an aerosolized powder determined in continuous impact unless otherwise specified in the text.

As used herein, "crystallinity level" means the percentage of amphotericin B in crystalline form relative to the total amount of amphotericin B. Unless stated to the contrary, the crystallinity levels in this document are determined by wide-angle X-ray powder diffraction. The X-ray diffraction powder pattern is a Shimadzu with a holding time of 2 seconds (fixed time scan), a step size of 0.02 ° 2θ, a scan range of 3-42 ° 2θ, a 0.5 ° diverging slit, 1 ° scattering slit and 0.3 mm receiving slit It was measured with a (Shimadzu) X-ray diffractometer model XRD-6000, which is described in more detail in Example 1.

As used herein, “amorphousness” refers to the percentage of amphotericin B that is amorphous relative to the total amount of amphotericin B. Unless stated to the contrary, amorphous levels in the text are measured by wide-angle X-ray powder diffraction.

As used herein, the term "radiation dose" or "ED" is meant to indicate the delivery of dry powder from an inhaler device after operation or dispersion occurs from a powder unit or reservoir. ED is defined as the ratio of the dose delivered to the inhaler device relative to the nominal dose (ie the mass of powder per unit dose placed in a suitable inhaler device prior to foaming). ED is an experimentally determined amount and can be determined using an in vitro device facility that mimics patient administration. To determine the ED value as used herein, the turbospin described in U.S. Pat. ) (Registered trademark) DPI device (PH & T, Italy). The turbospin® DPI is activated to disperse the powder. The resulting aerosol cloud is then discharged from the device by vacuum (30 L / min) for 2.5 seconds after operation, which is a tarned glass fiber filter (Gelman, 47 mm attached to the device mouthpiece). Diameter). The amount of powder that reaches the filter constitutes the delivery capacity. For example, for a capsule containing 5 mg of dry powder placed in an inhaler device, when 4 mg of powder is recovered on the tared filter as described above due to the dispersion of the powder, the ED for the dry powder composition is 80 % [= 4 mg (delivered dose) / 5 mg (nominal dose)].

As used herein, “passive dry powder inhaler” relies on the patient's efforts to inhale to disperse and aerosolize the pharmaceutical composition contained within the device in a reservoir or unit dosage form, dispersing and aerosolizing the drug composition. By means of an inhaler device which does not include an energy supply means such as pressurized gas and an inhaler device comprising a vibrating or rotating member.

As used herein, an "active dry powder inhaler" does not rely solely on the patient's efforts to inhale to disperse and aerosolize the pharmaceutical composition contained within a reservoir or unit dosage form, dispersing the drug composition and aerosol Means an inhaler device comprising an energy supply means such as pressurized gas and an inhaler device comprising a vibrating or rotating member.

An overview of some embodiments of the invention is set forth in the Summary of the Invention herein. For simplicity, this summary is incorporated herein by reference in its entirety.

One or more embodiments of the invention relate to the surprising and unexpected finding that certain amphotericin B compositions have reduced toxicity. While not wishing to be bound by theory, it is believed that several factors influence amphotericin B toxicity. These factors include, but are not limited to, the crystallinity level of amphotericin B, the size of the amphotericin B particles, the homogeneity of the microparticles including the amphotericin B particles, and the size of the microparticles including the amphotericin B particles. . Depending on the situation, one or more of these factors are believed to affect toxicity.

In one or more embodiments of the invention, the composition comprises amphotericin B. Amphotericin B is a heptaene macrolide containing a 3-amino-3,6-dideoxymannose (mycosamine) moiety linked to the main ring by seven conjugated double bonds and glycosidic bonds in the trans position . The amphotericin B bulk drug material is available from Alpharma, Copenhagen, Denmark or Chemwerth, Woodbridge, Connecticut, USA.

In one or more embodiments of the present invention, the composition comprises amphotericin B derivatives having antifungal activity. The amphotericin B derivatives can be esters, amides, hydrazides, N-alkyls, and / or N-amino acyls. Examples of ester derivatives of amphotericin B include, but are not limited to, methyl esters, choline esters, and dimethylaminopropyl esters. Examples of amide derivatives of amphotericin B include, but are not limited to, primary, secondary and tertiary amides of amphotericin B. Examples of hydrazide derivatives of amphotericin B include, but are not limited to, N-methylpiperazine hydrazide. Examples of N-alkyl derivatives of amphotericin B include, but are not limited to, N ', N', N'-trimethyl and N ', N'-dimethylaminopropyl succininimidyl derivatives of amphotericin B methyl ester. Do not. Examples of N-aminoacyl derivatives of amphotericin B include N-ornithyl-, N-diaminopropionyl-, N-lysyl-, N-hexamethyllysyl-, and N-piperidine-propionyl- or N ', N'-methyl-1-piperazine-propionyl-amphotericin B methyl esters include, but are not limited to.

The composition comprising amphotericin B may comprise various amounts of amphotericin B. For example, the amount of amphotericin B is at least about 0.01 weight percent, for example at least about 1 weight percent, at least about 10 weight percent, at least about 50 weight percent, at least about 90 weight percent, at least about 95 weight percent, or Or at least about 98% by weight.

As mentioned above, it is believed that the crystallinity level of amphotericin B may be a factor that reduces toxicity. For example, when administered to the lung, the more crystalline form of amphotericin B is believed to dissolve more slowly and have a longer half-life than the more amorphous form of amphotericin B. While not wishing to be bound by theory, it is believed that slow dissolution and long half-life reduce toxicity. Conversely, without wishing to be bound by theory, it is believed that the amorphous develops into soluble aggregates that can be toxic to lung tissue. While not wishing to be bound by theory, it is believed that the above principles are not limited to the lungs.

The desired crystallinity level of amphotericin B will depend on factors such as dosage and treatment regimen. The crystallinity level of amphotericin B is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, for example at least about 80%. At least about 90%, at least about 95%, or at least about 99%. Thus, the crystallinity level is about 10% to 100%, for example about 20% to about 99%, about 50% to about 99%, about 70% to about 99%, about 70% to about 98%, about 80 % To about 98%, or about 90% to about 97%.

The crystallinity level can be determined by any of several known techniques. For example, the crystallinity level can be determined by X-ray diffraction, Raman and / or infrared spectroscopy, dynamic vapor adsorption, heat of solution calorimetry, or isothermal microcalorimetry. As mentioned above, unless stated to the contrary, the crystallinity level herein is measured by X-ray diffraction using the method described in Example 1.

In some cases, small diameter amphotericin B particles are used. In one form, the particles of amphotericin B have a mass median diameter of less than about 3 μm, such as less than about 2.5 μm, less than about 2 μm, less than about 1.9 μm or less than about 1.5 μm. For example, the particles of amphotericin B can be from about 0.5 μm to about 3 μm, such as from about 0.5 μm to about 1.8 μm, from about 0.8 μm to about 2.5 μm, from about 1.1 μm to about 1.9 μm, from about 1.2 μm to It may have a mass median diameter in the range of about 1.8 μm, or about 1 μm to about 2 μm. In some forms, at least about 20% of the amphotericin B particles have a size of less than about 3 μm in diameter, such as at least about 50% is less than about 3 μm, or at least about 90% is less than about 3 μm , At least about 95% is less than about 3 μm. For example, 60%, 70%, 80%, or 90% by weight of the particles can have a mass median diameter of about 1.1 μm to 1.9 μm.

In some forms, the crystallinity level of amphotericin B is high and the amphotericin B particle size is small. The crystallinity level can be any of those discussed above, and the particle size can be any of those discussed above. For example, in one form, the crystallinity level is at least about 50% and the median mass diameter is less than about 3 μm. In another form, the crystallinity level is at least about 70% and the median mass diameter is less than about 2.8 μm. In another form, the crystallinity level is at least about 80% and the median mass diameter is less than about 2.6 μm. In another form, the crystallinity level is at least about 90% and the median mass diameter is less than about 2.4 μm.

The crystallinity level can be the level discussed above and the amphotericin B particle size can be larger than the size discussed above. For example, the crystallinity level can be at least about 50% and the median mass diameter can be greater than about 3 μm. Conversely, the amphotericin B particle size may be within the size discussed above and the crystallinity level may be outside the levels discussed above. For example, the median mass diameter may be less than about 3 μm and the crystallinity level may be less than about 50%.

Pharmaceutical compositions according to one or more embodiments of the present invention may include amphotericin B and optionally one or more other active ingredients and / or pharmaceutically acceptable excipients. For example, the pharmaceutical composition may comprise pure particles of amphotericin B, and / or may comprise pure particles of amphotericin B in combination with other particles, and / or amphotericin B and one or more active ingredients. And / or microparticles comprising one or more pharmaceutically acceptable excipients.

Thus, pharmaceutical compositions according to one or more embodiments of the invention may contain a combination of amphotericin B and one or more other active ingredients, if desired. Examples of other active agents include, but are not limited to, actives that can be delivered through the lungs or nasal passages. For example, the other active agent (s) can be long-acting and / or active agents active against lung and / or nasal infections, such as antiviral, antifungal and / or antibiotics.

Examples of antiviral agents include, but are not limited to, acyclovir, gangcyclovir, azidothymidine, cytidine arabinoside, ribavirin, rifampacin, amantadine, iododeoxyuridine, poscarnet and trifluidine.

Examples of antifungal agents include azoles (eg imidazole, itraconazole, pozaconazole), mycafungin, caspafungin, salicylic acid, oxyconazole nitrate, cyclopyroxolamine, ketoconazole, myconazole nitrate and butoconazole nitrate Include, but are not limited to.

Examples of antibiotics include penicillin and penicillin-G, penicillin-V, penicillin, ampicillin, amoxacillin, cyclacillin, baccampillin, hetacillin, clocksacillin, dicloxacillin, methicillin, naphcillin Penicillin family drugs among antibacterial drugs including, but not limited to, oxacillin, azolocillin, carbenicillin, mezlocillin, piperacillin, ticaricillin and imipenim; Cephalosporins, and cephadroxyl, cefazoline, carpalexin, cephalotin, cephaphyrin, cepradine, cephachlor, cephamandol, cenisidide, cephacin, cepuroxime, celanide, cetethetan, cepineta Cephalosporin family drugs including but not limited to sol, cephaperazone, cefotaxime, ceftizone, ceftizone, moxalactam, ceftazidime and seppicsim; Aminoglycoside drugs, and aminoglycoside family drugs including but not limited to streptomycin, neomycin, kanamycin, gentamycin, tobramycin, amikacin, and netylmycin; Macrolides and macrolide family drugs exemplified by azithromycin, clarithromycin, roxythromycin, erythromycin, lincomycin and clindamycin; Tetracycline and tetracycline family drugs such as tetracycline, oxytetracycline, democlocycline, metacycline, doxycycline, and minocycline; Quinoline and quinoline-like drugs such as nalidixic acid, synoxacin, norfloxacin, ciprofloxacin, opfloxacin, enoxacin and pefloxacin; Antibacterial peptides, including but not limited to polymyxin B, colistin and bacatracin, as well as naturally occurring or genetically engineered defensins, margarine, cecro that make the peptides resistant to the action of pathogen-specific proteases Other antibacterial peptides and other inactivating enzymes such as pins and others; Other antimicrobial drugs, including but not limited to chloramphenicol, vancomycin, rifampicin, metronidazole, boriconazole, fluconazole, ethambutol, pyrazineamide, sulfonamide, isoniazid and erythromycin.

When a combination of active agents is used, the active agents may be provided in a combination of a single species of pharmaceutical composition or in a combination of separate species of pharmaceutical compositions individually. In addition, the pharmaceutical composition may be combined with one or more other active or bioactive agents that provide the desired dispersion stability or powder dispersibility.

The amount of active agent (s), amphotericin B in the pharmaceutical composition may vary. The amount of active agent (s) is typically at least about 5%, for example at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50% by weight relative to the total amount of the pharmaceutical composition. Or at least about 60% by weight, at least about 70% by weight or at least about 80% by weight. The amount of active agent (s) is generally from about 0.1% to 100% by weight, for example from about 5% to about 95% by weight, from about 10% to about 90% by weight, from about 30% to about 80% by weight, From about 40% to about 70%, or from about 50% to about 60% by weight.

As mentioned above, the pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include, but are not limited to, lipids, metal ions, surfactants, amino acids, carbohydrates, buffers, salts, polymers, and the like, and combinations thereof.

Examples of lipids include phospholipids, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; Lipids bearing polymer chains such as polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; Lipids with sulfonated monosaccharides, disaccharides and polysaccharides; Fatty acids such as palmitic acid, stearic acid and oleic acid; Cholesterol, cholesterol esters, and cholesterol hemisuccinate.

In one or more embodiments, the phospholipids comprise saturated phospholipids, for example one or more phosphatidylcholine. Exemplary acyl chain lengths are 16: 0 and 18: 0 (ie palmitoyl and stearoyl). Phospholipid content can be determined by active agent activity, mode of delivery, and other factors.

Phospholipids obtained from natural and synthetic sources can be used in various amounts. If phospholipid is present, the amount is typically sufficient to coat the active agent (s) with at least a single molecular layer of phospholipid. Generally, the phospholipid content is in the range of about 5% to about 99.9% by weight, for example about 20% to about 80% by weight.

In general, compatible phospholipids include those having a gel-liquid crystal phase transition of greater than about 40 ° C., for example greater than about 60 ° C. or greater than about 80 ° C. The included phospholipids can be relatively long chain (eg, C ₁₆ -C ₂₂ ) saturated lipids. Examples of phospholipids useful in the disclosed stabilized formulations include phosphoglycerides such as dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, diarachidoylphosphatidylcholine, dibehenoylphosphatidylcholine, diphosphatidyl glycerol, short-chain phosphatidylcholine, hydrogenated phosphatidylcholine , E-100-3 (Lipoid KG, Ludwigshafen, Germany), long chain saturated phosphatidylethanolamine, long chain saturated phosphatidylserine, long chain saturated phosphatidylglycerol, long chain saturated phosphatidyl inositol, phosphatidic acid, phosphatidylinositol, and Pingomyelins include, but are not limited to.

Examples of metal ions include, but are not limited to, divalent cations including calcium, magnesium, zinc, iron, and the like. For example, when using phospholipids, pharmaceutical compositions may also include polyvalent cations as described in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entirety. The polyvalent cation may be present in an effective amount to raise the melting temperature (T _m ) of the phospholipid so that the pharmaceutical composition exhibits a T _m that is at least about 20 ° C., for example at least about 40 ° C. above its storage temperature (T _s ). The molar ratio of polyvalent cation to phospholipid can be at least about 0.05: 1, for example from about 0.05: 1 to about 2.0: 1 or from about 0.25: 1 to about 1.0: 1. An example of a molar ratio of polyvalent cation: phospholipid is about 0.50: 1. If the polyvalent cation is calcium, it may be in the form of calcium chloride. Metal ions, such as calcium, are often included with phospholipids, but they are not necessary.

As mentioned above, the pharmaceutical composition may comprise one or more surfactants. For example, one or more surfactants may be present in the liquid phase in which one or more of these are associated with solid particles or particulates of the composition. “Associated” means that the pharmaceutical composition may comprise, adsorb, absorb, coat with, or be formed by a surfactant. Surfactants include, but are not limited to, fluorinated and nonfluorinated compounds such as saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations thereof. In addition to the surfactants mentioned above, it should be emphasized that suitable fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired formulations.

Examples of nonionic detergents include sorbitan esters including sorbitan trioleate (Span ^TM 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan mono Laurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and Sucrose esters, including but not limited to. Other suitable nonionic detergents can be readily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, New Jersey), which is incorporated herein by reference in its entirety.

Examples of block copolymers include polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic ^™ F-68), poloxamer 407 (Pluronic ^™ F-127), and poloxamer 338. Lock and triblock copolymers include, but are not limited to.

Examples of ionic surfactants include, but are not limited to, sodium sulfosuccinate and fatty acid soaps.

Examples of amino acids include, but are not limited to, hydrophobic amino acids. The use of amino acids as pharmaceutically acceptable excipients is known in the art, as described in WO 95/31479, WO 96/32096, and WO 96/32149, which are hereby incorporated by reference in their entirety.

Examples of carbohydrates include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. For example, monosaccharides such as dextrose (anhydrous and monohydrate), galactose, mannitol, D-mannose, sorbitol, sorbose, and the like; Disaccharides such as lactose, maltose, sucrose, trehalose and the like; Trisaccharides such as raffinose and the like; And other carbohydrates such as starch (hydroxyethyl starch), cyclodextrins and maltodextrins.

Examples of buffers include but are not limited to tris or citrate.

Examples of acids include, but are not limited to, carboxylic acids.

Examples of salts include but are not limited to sodium chloride, salts of carboxylic acids (e.g., sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.), ammonium carbonate, ammonium acetate, ammonium chloride, and the like. It doesn't work.

Examples of organic solids include, but are not limited to, camphors and the like.

Pharmaceutical compositions of one or more embodiments of the present invention may comprise biologically compatible polymers, copolymers, or blends or other combinations thereof, for example biodegradable. In this regard, useful polymers include polylactide, polylactide-glycolide, cyclodextrin, polyacrylates, methylcellulose, carboxymethylcellulose, polyvinyl alcohol, polyanhydrides, polylactams, polyvinyl pyrrolidones, polysaccharides ( Dextran, starch, chitin, chitosan, etc.), hyaluronic acid, proteins (albumin, collagen, gelatin, etc.). Those skilled in the art will understand that by selecting an appropriate polymer, one can optimize the effect of the active agent (s) by adjusting the delivery efficiency of the composition and / or the stability of the dispersion.

In addition to the pharmaceutically acceptable excipients mentioned above, it will be desirable to add other pharmaceutically acceptable excipients into the pharmaceutical composition to improve particulate stiffness, yield, radiation dose and deposition, shelf life, and patient acceptability. Such optimal pharmaceutically acceptable excipients include, but are not limited to, colorants, taste masking agents, buffers, hygroscopic agents, antioxidants and chemical stabilizers. In addition, various pharmaceutically acceptable excipients may be used to provide the structure and form (eg, latex particles) for the particulate composition. In this regard, it will be appreciated that post-preparation techniques, such as selective solvent extraction, may be used to remove the tonication component.

In addition, the pharmaceutical composition may comprise a mixture of pharmaceutically acceptable excipients. For example, mixtures of carbohydrates and amino acids are included within the scope of the present invention.

The compositions of one or more embodiments of the invention may be in various forms, for example dry powders, capsules, tablets, reconstituted powders, suspensions, or non-aqueous phases, for example propellants (eg, fluorocarbons, hydrofluoroalkanes) It may be a dispersion containing. The moisture content of the dry powder may be less than about 15 weight percent, for example less than about 10 weight percent, less than about 5 weight percent, less than about 2 weight percent, less than about 1 weight percent, or less than about 0.5 weight percent. Such powders are described in WO 95/24183, WO 96/32149, WO 99/16419, WO 99/16420, and WO 99/16422, which are hereby incorporated by reference in their entirety.

One or more embodiments of the present invention comprise a homogeneous composition of amphotericin B incorporated into a matrix material with some unincorporated amphotericin B particles, if present. For example, at least about 40 weight percent, at least about 50 weight percent, at least about 60 weight percent, at least about 70 weight percent, at least about 80 weight percent, at least about 90 weight percent, at least about 95 weight percent, or about 99 of the composition. The weight percent or more may include microparticles comprising both amphotericin B and the matrix material.

However, in some cases, heterogeneous compositions may be desirable to obtain the desired pharmacokinetic profile of amphotericin B to be administered, in which case large amphotericin B particles (eg, from about 3 μm to about median diameter in mass) 10 μm or more) may be used.

The homogeneous composition may comprise small amphotericin B particles. As discussed above, amphotericin B particles having a median mass diameter of less than about 3 μm may be dispersible and may facilitate the preparation of a homogeneous composition of amphotericin B incorporated into the matrix material. If the median diameter of amphotericin B is greater than about 3 μm, the result may be a heterogeneous composition comprising amphotericin B incorporated into the matrix material and particles comprising amphotericin B without any matrix material. Found. Such heterogeneous compositions often have poor powder flow and dispersibility, which can lead to capacity variations. Compositions made with larger amphotericin B particles also caused toxicity in rats. However, it should be noted that the heterogeneous distribution can be improved through a nebulization process, resulting in a homogeneous distribution for larger amphotericin B particles.

In view of the above, in some forms the pharmaceutical composition has a high homogeneity, the amphotericin B has a high crystallinity level and the size of the amphotericin B particles forming the composition is small. The degree of homogeneity, crystallinity level, and particle size may be any of those discussed above. For example, in one form, the crystallinity level is at least about 50% and the median mass diameter is less than about 3 μm. In another form, the crystallinity level is at least about 70% and the mass median diameter is less than about 2.8 μm. In another form, the crystallinity level is at least about 80% and the median mass diameter is less than about 2.6 μm. In another form, the crystallinity level is at least about 90% and the median mass diameter is less than about 2.4 μm.

However, in some cases, the degree of homogeneity is high and at least one of crystallinity level and amphotericin B is outside the range discussed above. Or, in some cases, the degree of homogeneity is low, and at least one of crystallinity levels and amphotericin B is within the ranges discussed above.

In one form, the pharmaceutical composition comprises amphotericin B incorporated into the phospholipid matrix. As described in the aforementioned WO 99/16419, WO 99/16420, WO 99/16422, WO01 / 85136, and WO 01/85137, which are hereby incorporated by reference in their entirety, said pharmaceutical composition is hollow and / or porous microparticles. It may comprise a phospholipid matrix that is in particulate form that is structural and comprises an active agent. Since the density, size, and aerodynamic properties of the hollow and / or porous microstructures facilitate transport to the deep lungs during inhalation of the user, the hollow and / or porous microstructures are useful for delivering amphotericin B to the lungs. . In addition, the phospholipid-based hollow and / or porous microstructures reduce the attractive force between the microparticles, making the pharmaceutical composition easier to deagglomerate during aerosolization, and improve the flowability of the pharmaceutical composition to make it easier to process.

In one form, the pharmaceutical composition has a bulk density of less than about 1.0 g / cm ^3, less than about 0.5 g / cm ^3, less than about 0.3 g / cm ^3, less than about 0.2 g / cm ³ , or less than about 0.1 g / cm ^3. It consists of hollow and / or porous microstructures. By providing particles or particulates of low bulk density, the minimum powder mass that can be filled into the unit dose container is reduced, thereby eliminating the need for carrier particles. That is, the relatively low density of the powder in one or more embodiments of the invention makes reproducible administration of relatively low doses of the pharmaceutical compound. Moreover, because large carrier particles, such as lactose particles, will irritate the throat and upper respiratory tract because of their size, removal of the carrier particles will potentially reduce throat deposition and any “gag” effect or cough.

In one form, the pharmaceutical composition is in the form of a dry powder and contained in a unit dose reservoir that can be inserted into or near an aerosolization apparatus that aerosolizes a unit dose of the pharmaceutical composition. Such forms are useful when the dry powder form can be stably stored in its unit dose reservoir for a long time. In some instances, pharmaceutical compositions of one or more embodiments of the present invention have been stable for at least about two years. In some forms, no refrigerator is required to achieve stability. In another form, stable storage is maintained using reduced temperatures, such as from 2 to 8 ° C. In many forms, aerosolization with an external power supply is allowed because of storage stability.

It will be appreciated that the pharmaceutical compositions disclosed herein may include structural matrices that represent, define, or include voids, pores, nicks, hollows, spaces, interstitial spaces, gaps, perforations, or holes. The absolute shape of the perforated microstructures (as opposed to morphology) is generally not important and any overall shape that provides the desired properties is considered to be within the scope of the present invention. Thus, some embodiments include shapes that are approximately spherical. However, disintegrated, deformed or crushed particulates are also compatible.

In one form, amphotericin B is incorporated into a matrix forming discrete particulates, and the pharmaceutical composition comprises a plurality of discrete particulates. The separate particulates can be sized to be effectively administered and / or available when needed. For example, in the case of an aerosolized pharmaceutical composition, the microparticles are sized such that the microparticles are aerosolized and delivered to the respirator of the user during inhalation by the user.

In some forms, the pharmaceutical composition comprises microparticles having a median mass diameter of less than about 20 μm, such as less than about 10 μm, less than about 7 μm, or less than about 5 μm. The microparticles can have a mass median aerodynamic diameter in the range of about 1 μm to about 6 μm, for example about 1.5 μm to about 5 μm, or about 2 μm to about 4 μm. If the particulates were too large, toxicity in rats was observed. If the particles are too small, a larger percentage of the particles can be released.

In view of the above, in some forms, the pharmaceutical composition comprises microparticles having a small mass median aerodynamic diameter, the pharmaceutical composition has a high homogeneity, the amphotericin B has a high crystallinity level, and the cancer cells forming the pharmaceutical composition. Tericin B particles are small in size. The mass median aerodynamic diameter, degree of homogeneity, crystallinity level, and amphotericin B particle size can be any of those discussed above. For example, in one form, the mass median aerodynamic diameter is less than about 20 μm, and at least about 60 weight percent of the pharmaceutical composition comprises both amphotericin B and the matrix material, and the crystallinity level is at least about 50% The median mass diameter is less than about 3 μm. In another form, the mass median aerodynamic diameter is less than about 10 μm, and at least about 70 weight percent of the pharmaceutical composition comprises both amphotericin B and the matrix material, the crystallinity level is at least about 70%, and the mass median diameter Is less than about 2.8 μm. In another form, the mass median aerodynamic diameter is less than about 7 μm, and at least about 80 weight percent of the pharmaceutical composition comprises both amphotericin B and the matrix material, the crystallinity level is at least about 80%, and the center of mass The diameter is less than about 2.6 μm. In another form, the mass median aerodynamic diameter is less than about 7 μm, and at least about 90 weight percent of the pharmaceutical composition comprises both amphotericin B and the matrix material, the crystallinity level is at least about 90%, and the center of mass The diameter is less than about 2.4 μm.

However, in some cases, the mass center aerodynamic diameter is small, and at least one of homogeneity, crystallinity level, and amphotericin B particle size is outside the range discussed above. Similarly, in other cases, the mass center aerodynamic diameter is large, and at least one of homogeneity, crystallinity level, and amphotericin B particle size is within the ranges discussed above.

In view of the above, the toxicity of amphotericin B particulates or particles inhaled in rats depends on several factors. Without wishing to be bound by theory, the factors include amphotericin B crystallinity levels, amphotericin B particle size used to prepare inhaled particles or particulates, homogeneity of inhaled particulates, and size of inhaled particles or particulates. Is considered to be included, but is not limited thereto.

The matrix material may include hydrophobic or partially hydrophobic materials. For example, the matrix material may comprise lipids such as phospholipids and / or hydrophobic amino acids such as leucine or tri-leucine. Examples of phospholipid matrices include WO 99/16419, WO 99/16420, WO 99/16422, WO 01185136, and WO 01/85137 and US Pat. No. 5,874,064; 5,855,913; 5,855,913; 5,985,309; 5,985,309; And US Pat. No. 10 / 750,934, co-pending co-owned, filed 6,503,480 and Dec. 31, 2003, all of which are incorporated herein by reference in their entirety. Examples of hydrophobic amino acid matrices are described in US Pat. Nos. 6,372,258 and 6,358,530, and US Application No. 10 / 032,239, filed December 21, 2001, all of which are incorporated herein by reference in their entirety.

When phospholipids are used as matrix materials, the pharmaceutical compositions may also include polyvalent cations as disclosed in WO 01/85136 and WO 01/85137, which are incorporated herein by reference in their entirety.

According to another embodiment, the release kinetics of the active agent (s) containing composition is controlled. According to one or more embodiments, the compositions of the present invention provide for immediate release of the active agent (s). Alternatively, to provide a desirable release rate of the antifungal agent, the compositions of other embodiments of the present invention may be provided as a heterogeneous mixture of active and unincorporated actives incorporated into the matrix material. According to this embodiment, the antifungal agent prepared using the emulsion-based manufacturing process of one or more embodiments of the present invention has utility in immediate release applications when administered to the respiratory tract. Rapid release is achieved by (a) a high specific surface area of the low density porous powder; (b) small size of drug crystals incorporated into the powder; And (c) low surface energy of the particulates.

Alternatively, it may be desirable to manipulate the particulate matrix to effect prolonged release of the active agent (s). This may be particularly desirable if the active agent (s) are to be cleaned quickly from the lungs or if sustained release is desired. For example, the phase behavior properties of phospholipid molecules are influenced by their chemical structure and / or methods of preparation in the spray-dried feedstock and the drying conditions and the nature of the other composition components used. When spray-drying the active agent (s) solubilized in Small Unilamellar Vesicle (SUV) or Multilamellar Vesicle (MLV), the active agent (s) are encapsulated in multiple bilayers and over extended time periods. Is released.

Conversely, spray-drying a feedstock consisting of emulsion droplets and dispersed or dissolved active agent (s) according to the teachings herein promotes rapid release by creating a phospholipid matrix with a smaller long-range order. . Without being bound by any particular theory, this is due in part to the fact that the active agent (s) are not formally encapsulated in phospholipids and the fact that phospholipids initially exist as monolayers (not bilayers as in the case of liposomes) on the surface of emulsion droplets. It is considered. Spray-dried particulates produced by the emulsion-based manufacturing process of one or more embodiments of the present invention often have a high degree of disorder. In addition, spray-dried particulates typically have low surface energy and values as low as 20 mN / m have been observed for spray-dried DSPC particulates (determined by reverse gas chromatography). Incineration X-ray scattering (SAXS) studies performed with spray-dried phospholipid particulates showed a high degree of disorder for lipids, the scattering peaks were not clear, and in some cases the length dimension extended only on the slightest neighbors .

It should be noted that a matrix having a high gel-liquid phase transition temperature by itself is not sufficient to achieve sustained release of the active agent (s). Having sufficient order in the bilayer structure is also important to achieve sustained release. To facilitate rapid release, high porosity (high surface area) emulsion-systems and minimal interactions between drug materials and phospholipids can be used. The pharmaceutical composition manufacturing process may also include the addition of other composition components (eg, small polymers such as Pluronic F-68; carbohydrates, salts, hydrotrope) to disrupt the bilayer structure. Consider.

In order to achieve sustained release, incorporation of a phospholipid in the form of a bilayer, in particular when the active agent is encapsulated therein, can be used. In this case, increasing the T _m of phospholipids may provide an advantage through the incorporation of divalent counterions or cholesterol. In addition, increasing the interaction between the phospholipid and the drug material (negatively charged activity + stearylamine, negatively charged activity + phosphatidylglycerol) through the formation of ion pairs can tend to decrease the dissolution rate. If the active agent is amphiphilic, the surfactant / surfactant interaction may also slow down the active agent dissolution.

The addition of divalent counterions (eg calcium or magnesium ions) to long chain saturated phosphatidylcholine results in an interaction between the negatively charged phosphate portion of the zwitterionic headgroup and the positively charged metal ions. This results in substitution of the hydrated water and condensation of the packing of the acyl chain and phospholipid lipid headgroups. In addition, this leads to an increase in the T _{m of} phospholipids. The reduction in headgroup hydration can have a significant impact on the spreadability of the spray-dried phospholipid particulates in contact with water. Fully hydrated phosphatidylcholine molecules will diffuse very slowly into dispersed crystals through molecular diffusion through the aqueous phase. This process is very slow because the solubility of phospholipids in water is very low (about 10 ^-10 mol / L for DPPC). Prior art attempts to overcome this phenomenon include homogenizing crystals in the presence of phospholipids. In this case, the high shear and curvature radii of the homogenized crystals facilitate the coating of the phospholipids on the crystals. In contrast, “dry” phospholipid powders according to one or more embodiments of the present invention spread quickly when in contact with an aqueous phase, so that dispersed crystals can be coated without the need to apply high energy.

For example, upon reconstitution, the surface tension of the spray-dried DSPC / Ca mixture at the air / water interface is reduced to an equilibrium value (about 20 mN / m) so fast that it can be measured. In contrast, liposomes of DSPC reduce the surface tension (about 50 mN / m) very slightly over time, which is likely due to the presence of hydrolytic degradation products such as free fatty acids in phospholipids. Single-tailed fatty acids can diffuse much faster at the air / water interface than hydrophobic parent compounds. Thus, the addition of calcium ions to phosphatidylcholine can result in rapid encapsulation of crystalline drugs more quickly with lower energy.

In another form, the pharmaceutical composition comprises low density particulates achieved with co-spray-dried nanocrystals using a water-in-perfluorocarbon emulsion. The nanocrystals may be formed by precipitation, for example, may range in size from about 45 μm to about 80 μm. Examples of perfluorocarbons include, but are not limited to, perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane.

According to the teachings herein, the particulate composition will preferably be provided in a "dry" state. That is, in one or more embodiments, the microparticles will have a moisture content that allows the powder to remain chemically physically stable and dispersible during storage at room temperature or at room temperature. In this regard, there is little or no change in major particle size, content, purity, and aerodynamic particle size distribution.

Thus, the moisture content of the microparticles is typically less than about 10 weight percent, for example less than about 6 weight percent, less than about 3 weight percent, or less than about 1 weight percent. The moisture content is determined at least in part by the composition and is controlled by the method conditions used, such as inlet temperature, feed concentration, pump speed and blowing agent type, concentration and postdrying. The reduction in bound water results in a significant improvement in the dispersibility and flowability of the phospholipid based powder and the potential for highly efficient delivery of the powdered waste surfactant or particulate composition comprising the active agent dispersed in the phospholipid. Due to the improved dispersibility, the powder can be effectively delivered using a simple passive DPI device.

Another form of pharmaceutical composition includes a particulate composition that may include a charged species that extends the residence time at the point of contact or enhances penetration through the mucosa, or may be partially or completely coated with the species. For example, cationic charges can be used to associate the formed particulates with negatively charged bioactive agents such as dielectric materials, while anionic charges are known to favor mucoadhesion. The charge can be imparted through the association or incorporation of polyanionic or polycationic materials such as polyacrylic acid, polylysine, polylactic acid and chitosan.

The unit dose pharmaceutical composition may be contained in a container. Examples of containers include, but are not limited to, capsules, blisters, vials, ampoules, or container closure systems made of metals, polymers (eg, plastics, elastomers), glass, and the like.

The container can be inserted into an aerosolization device. The container may be of a shape, size and material suitable for containing the pharmaceutical composition and for providing the pharmaceutical composition in a useful state. For example, the capsule or blister may comprise a wall comprising a material that does not react back to the pharmaceutical composition. In addition, the wall may comprise a material that opens the capsule to aerosolize the pharmaceutical composition. In one form, the wall comprises one or more of gelatin, hydroxypropyl methylcellulose (HPMC), polyethyleneglycol-compound HPMC, hydroxypropylcellulose, agar, aluminum foil, and the like. In one form, the capsule may comprise telescopically linked compartments, as described, for example, in US Pat. No. 4,247,066, which is incorporated herein by reference in its entirety. The size of the capsule can be selected to suitably contain the dose of the pharmaceutical composition. The size generally ranges from size 5 to size 000, the outer diameter ranges from about 4.91 mm to 9.97 mm, the height ranges from about 11.10 mm to about 26.14 mm, and the volume ranges from about 0.13 mL to about 1.37 mL. Suitable capsules are commercially available, for example, from Shionogi Qualicaps Co., Nara, Japan, and Capsugel, Greenwood, South Carolina, USA. After filling, the top can be placed above the bottom to form a capsule form and contain powder in the capsule, as described in US Pat. Nos. 4,846,876 and 6,357,490 and WO 00/07572, which are incorporated by reference in their entirety. . After the top is positioned above the bottom, the identification band can optionally be attached to the capsule.

In one form, the pharmaceutical composition comprising crystalline amphotericin B is aerosolized to be delivered to the patient's lungs while the patient is inhaling. In this way, amphotericin B in the pharmaceutical composition is delivered directly to the site of infection. This is advantageous over systemic administration. Because the active agent (s) have nephrotoxicity or other toxicity, it is typically desirable to minimize systemic exposure. Therefore, the amount of active agent (s) that can be delivered to the lungs is preferably limited to a minimum pharmacologically effective amount. By administering the active agent (s) directly to the lung, larger amounts can be delivered to the site where treatment is required while significantly reducing systemic exposure. In addition, by delivering amphotericin B primarily in its crystalline form, it is possible to maintain the desired concentration of amphotericin B at the site of infection over a period of time while reducing the likelihood of developing a toxic effect in the lung.

Pharmaceutical compositions of one or more embodiments of the invention are tasteless. In this regard, optionally, a taste masking agent is included in the composition, but the composition is often tasteless without the taste masking agent.

Particles, particulates, and compositions of one or more embodiments of the present invention may be prepared by any of a variety of methods and techniques known to those skilled in the art and available to those of ordinary skill in the art.

As mentioned above, the crystallinity level of amphotericin B affects performance. One skilled in the art can adjust the crystallinity level of amphotericin B by adjusting the crystallization conditions. For example, the crystallinity level may include solvent, amphotericin B concentration, pH, pH adjustment rate, purity level, temperature, cooling / heating rate, annealing time, use of seed crystals, solvent addition rate, stirring rate, It can be adjusted by changing the cosolvent type / concentration and the holding period during crystallization.

Alternatively, the crystallinity level can be adjusted through recrystallization. Recrystallization techniques are known in the art. Exemplary recrystallization techniques are described below.

During the recrystallization process, it is desirable to protect amphotericin B from light. Amphotericin B can be dissolved in a solvent. Examples of solvents include solvent systems such as methanol, dimethylformamide, and citric acid monohydrate. Once the solids are essentially dissolved, the solution is optionally filtered.

After filtration, additional solvent, such as methylene chloride, may be added to the filtrate. Thereafter, cooling water can be added while stirring.

Precipitation of amphotericin B can be accomplished by adding a base such as triethanolamine to adjust the pH of the solution, such as pH about 7. While not wishing to be bound by theory, the rate of precipitation affects the crystallinity level. Slow settling generally results in higher crystallinity.

Thus, in order to obtain more amorphous amphotericin B, the base can be added quickly, for example by pouring at a time with stirring. The resulting relatively amorphous amphotericin B can then be isolated. For example, amorphous amphotericin B can be isolated by centrifuging the slurry and decanting the supernatant. The product can be washed by resuspending the cake in, eg, cooled methanol, then centrifuging and decanting. The washing process may be repeated or may include additional washing steps, such as using acetone at room temperature.

In order to obtain a more crystalline amphotericin B, the base can be added slowly, for example with droplets, with stirring. The resulting slurry can then be heated, for example, to 44 to 46 ° C. for 90 minutes, and then cooled to 30 ° C., for example to room temperature for 60 minutes. The crystalline form may then be suitable for isolation. For example, the crystalline form of amphotericin B can be captured by vacuum filtration. The product can be washed at room temperature using, for example, cooled 40% methanol followed by acetone.

After amphotericin B is isolated and washed, it can be dried. For example, amphotericin B can be vacuum dried at room temperature, for example, for 1 to 3 days with the product shaded. Optionally, for example, a spatula may be used to break large aggregates during the drying process to promote evaporation of residual solvent.

As mentioned above, smaller amphotericin B particle sizes are often preferred. In many cases, the bulk median diameter of amphotericin B in bulk form is greater than about 3.0 μm and in many cases greater than about 10 μm. Thus, in one or more embodiments of the present invention, bulk amphotericin B is subjected to a size reduction process to reduce the median mass diameter to less than about 3 μm before use. Suitable size reduction processes are known in the art, including supercritical fluid processing methods, cryogenic milling, such as those described in WO 95/01221, WO 96/00610, and WO 98/36825, which are hereby incorporated by reference in their entirety. Wet milling, ultrasonication, high pressure homogenization, microfluidization, crystallization processes and processes described in US Pat. No. 5,858,410, which is incorporated herein by reference in its entirety.

In one or more embodiments of the present invention, a method of preparing a pharmaceutical composition comprises providing particles of amphotericin B as starting material, wherein at least about 50% of amphotericin B is in crystalline form. In another form, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% of amphotericin B is in crystalline form. The starting material amphotericin B particles may be suspended in a liquid feedstock optionally comprising one or more pharmaceutically acceptable excipients. The suspension feedstock is then dried, for example by spray drying, to produce microparticles comprising crystalline amphotericin B and one or more pharmaceutically acceptable excipients. In one form, in the microparticles comprising amphotericin B prepared, at least about 70% of amphotericin B in the microparticles produced is in crystalline form. In another form, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of amphotericin B is in crystalline form.

In one form, the crystallinity level of the starting material of the amphotericin B particles is determined. For example, prior to introduction into the feedstock, the X-ray powder diffraction pattern of the material can be measured. The crystallinity level can be determined from the data. Alternatively, as will be appreciated by those skilled in the art, infrared spectroscopy, Raman spectroscopy, dynamic vapor adsorption, heat of solution calorimetry, or isothermal microcalorimetry may be used to determine crystallinity levels. If the crystallinity level exceeds a predetermined amount, for example the above percentage, the starting material is used. If the crystallinity level is below a predetermined amount, the starting material may be discarded or recrystallized, for example.

In another form, determine the amorphousness of amphotericin B. Thus, in one form, a method of making a pharmaceutical composition comprises providing particles of amphotericin B as starting material, wherein less than about 50% of amphotericin B is in amorphous form. In other forms, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of amphotericin B is in amorphous form. The starting material amphotericin B particles may be suspended in a liquid feedstock optionally comprising one or more pharmaceutically acceptable excipients. The suspension feedstock is then dried, for example by spray drying, to produce microparticles comprising amphotericin B and one or more pharmaceutically acceptable excipients. In one form, in the microparticles comprising amphotericin B prepared, at least about 70% of amphotericin B in the microparticles produced is in crystalline form. In another form, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of amphotericin B in the prepared microparticles is in crystalline form.

Amorphousity can be determined using known techniques. For example, amorphousness can be determined using infrared spectroscopy, Raman spectroscopy, dynamic vapor adsorption, differential scanning calorimetry, and the like. If the amorphousness is less than the predetermined amount, for example the percentage, the starting material is used. If the amorphousness exceeds the predetermined amount, the starting material may be discarded or recrystallized.

In another form, instead of or in addition to crystallinity determination, the recrystallization process can be carried out as part of the preparation process. The recrystallization process can be carried out by dissolving amphotericin B in a suitable solvent, such as ethanol or other polar solvents, drying the solution slowly to generate crystalline amphotericin B or precipitating crystalline amphotericin B from solution. Can be. In one form, supercritical fluids can be used to simultaneously disperse and extract crystalline material, as disclosed in WO 95/01221, WO 96/00610, and WO 98/36825, which are incorporated by reference in their entirety. In another form, multiple spray-drying processes can be used to form crystalline amphotericin B, as disclosed in WO 01/00312, which is incorporated by reference in its entirety.

Another form includes determining the crystallinity level of the produced particles or particulates, which may include crystalline amphotericin B and optionally pharmaceutically acceptable excipients and / or other active ingredient (s). The crystallinity level in the particles or particulates produced can be above the predetermined percentage, for example greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99%. , To release particles or particulates prepared for administration to a patient. If the crystallinity level is below a predetermined amount, the prepared particles or particulates may be discarded or recrystallized.

Pharmaceutical compositions can be prepared using various known techniques. For example, the composition can be formed by spray drying, lyophilization, milling (eg, wet milling, dry milling), and the like.

In spray drying, the formulation or feedstock to be spray dried can be any solution, crude suspension, slurry, colloidal dispersion, or paste that can be atomized using a selected spray drying apparatus. In the case of an insoluble agent, the feedstock may comprise a suspension as described above. Alternatively, diluents and / or one or more solvents may be used in the feedstock solution. In one or more embodiments, the feed stock will comprise a colloidal system such as an emulsion, inverse emulsion, microemulsion, multiple emulsion, particle dispersion, or slurry.

In one form, amphotericin B and the matrix material are added to the aqueous feedstock to form a feedstock solution, suspension, or emulsion. The feedstock is then spray dried to produce dried particulates comprising the matrix material and crystalline amphotericin B. Suitable spray-drying processes are described, for example, in WO 99/16419 and US Pat. No. 6,077,543, which are incorporated herein by reference in their entirety; 6,051,256; 6,051,256; 6,001,336; 6,001,336; 5,985,248; 5,985,248; And 5,976,574, which are known in the art.

Whichever component is chosen, the first step in the production of the particulates typically involves preparing the feedstock. If the phospholipid-based microparticles are intended to act as a carrier for amphotericin B, the selected active agent (s) may be introduced into a liquid, for example water, to prepare a concentrated suspension. The concentration of amphotericin B and any active agent is typically determined by the amount of agent required in the final powder and the performance of the delivery device used (eg, fine particle capacity for quantitative inhalers (MDI) or dry powder inhalers (DPI)). Depends on.

Any additional active agent (s) may be introduced into a single feedstock formulation and spray dried to provide a single pharmaceutical composition species comprising multiple active agents. Conversely, each active agent can be added to separate raw materials and spray dried separately to provide multiple pharmaceutical composition species with different compositions. Each of these species may be added to the suspension medium or dry powder dispensing compartment in any desired ratio and placed in an aerosol delivery system as described below.

The polyvalent cation can be combined with amphotericin B suspension, with phospholipid emulsion, or with an oil-in-water emulsion formed in a separate container. Amphotericin B can also be dispersed directly in the emulsion.

For example, the polyvalent cations and phospholipids may be treated with hot distilled water (eg, 70 ° C.) for 2-5 minutes at 8000 rpm using a suitable high shear mechanical mixer (eg, Ultra-Turrax model T-25 mixer). ) Can be homogenized. Typically, 5-25 g of fluorocarbon are added dropwise with mixing to the dispersed surfactant solution. The polyvalent cation-containing perfluorocarbons in the resulting water emulsion can then be processed using a high pressure homogenizer to reduce the particle size. Typically, the emulsion proceeds in five separate routes at 12,000 to 18,000 PSI and is maintained at about 50 ° C to about 80 ° C.

When combining polyvalent cations with oil-in-water emulsions, blowing agents can be used to improve the dispersion stability and dispersibility of spray dried pharmaceutical compositions, as described in WO 99/16419, which is incorporated herein by reference in its entirety. This process typically forms an emulsion that is optionally stabilized by incorporated surfactants comprising submicron droplets of water-immiscible blowing agents dispersed in an aqueous continuous phase. The blowing agent may be a fluorinated compound (e.g., perfluorohexane, perfluorooctyl bromide, perfluorooctyl ethane, perfluorodecalin, perfluorobutyl ethane) that is evaporated during the spray-drying process and is generally aerodynamic Leave light hollow porous particulates. Other suitable liquid blowing agents include non-fluorinated oils, chloroform, Freon® fluorocarbons, ethyl acetate, alcohols, hydrocarbons, nitrogen, and carbon dioxide gases. The blowing agent may be emulsified with phospholipids.

The pharmaceutical composition may be formed using a blowing agent as described above, but in some cases no additional blowing agent is required and an aqueous dispersion of amphotericin B and / or pharmaceutically acceptable excipients and surfactant (s) may be used. It will be appreciated that it is spray dried directly. In such cases, the pharmaceutical composition may have certain physicochemical properties (eg, high crystallinity, elevated melting temperature, surface activity, etc.) that are particularly suitable for use in such techniques.

If desired, cosurfactants such as poloxamer 188 or span 80 may be dispersed into the accessory solution. It is also possible to add pharmaceutically acceptable excipients such as sugars and starch.

Thereafter, the feedstock (s) are fed to the spray dryer. Typically, the feedstock is sprayed with a stream of warm filtered air that evaporates the solvent and transfers the dried product to the collector. Thereafter, the air consumed is consumed with the solvent. Commercial spray dryers, manufactured by Buchi Ltd. or Niro Corp., can be operated for use in the manufacture of pharmaceutical compositions. Examples of spray drying methods and systems suitable for the preparation of the dry powders of one or more embodiments of the present invention are described in US Pat. No. 6,077,543; 6,051,256; 6,051,256; 6,001,336; 6,001,336; 5,985,248; 5,985,248; And 5,976,574, which are hereby incorporated by reference in their entirety.

The operating conditions of the spray dryer, such as inlet and outlet temperatures, feed rates, atomization pressures, drying air flow rates, and nozzle shapes can be adjusted to provide the required particulate size and the production yield of the resulting dry particulates. Selection of appropriate instruments and processing conditions is within the judgment of those skilled in the art in accordance with the teachings herein and can be accomplished without undue experimentation. Examples of settings are as follows: inlet temperature from about 60 ° C. to about 170 ° C .; Air outlet about 40 ° C. to about 120 ° C .; Feed rate from about 3 mL / min to about 15 mL / min; Air flow at intake of about 300 L / min; And atomizing air flow rate from about 25 L / min to about 50 L / min. The setting will of course vary depending on the type of device used. In any case, using this method and similar methods, aerodynamically light particulates having a diameter suitable for aerosol deposition into the lungs are formed.

Hollow and / or porous microstructures can be formed by spray drying, as disclosed in WO 99/16419, which is incorporated herein by reference. The spray-drying process can result in the formation of pharmaceutical compositions comprising particulates having relatively thin porous walls that define large internal voids. Spray-drying processes are also often advantageous over other processes in that the formed particulates are less likely to break during processing or during deagglomeration.

Alternatively, pharmaceutical compositions useful in one or more embodiments of the present invention may be formed by lyophilization. Lyophilization is a freeze-drying process in which water is frozen and then sublimed from the composition. Lyophilization processes are often used because relatively unstable biologics and pharmaceuticals in aqueous solutions can be dried without exposure to elevated temperatures, after which stability problems can be stored in a less dry state. In connection with one or more embodiments of the present invention, such techniques are particularly compatible with the incorporation of peptides, proteins, genetic material and other natural and synthetic macromolecules in pharmaceutical compositions without compromising physiological activity. Lyophilized cakes containing fine foam-like structures can be micronized using techniques known in the art to provide particulates of the desired size.

The compositions of one or more embodiments of the invention can be administered by known techniques, for example by inhalation, oral, intramuscular, intravenous, intratracheal, intraperitoneal, subcutaneous and transdermal.

For example, the pharmaceutical compositions of one or more embodiments of the invention are effective for treatment, including adjuvant treatment of pulmonary and / or nasal fungal infections. Amphotericin B acts as an antifungal agent that treats pulmonary and / or nasal fungal infections and / or prevents the onset of pulmonary and / or nasal fungal infections. It is believed that amphotericin B acts to slow down the growth and proliferation of susceptible fungi. If the concentration of amphotericin B is high enough, it can also destroy fungi. Amphotericin B appears to act on the fungal cell membrane to alter the integrity of the cell membrane.

In one form, the composition, upon inhalation, penetrates into the nasal and / or lung airways to achieve effective amphotericin B concentrations in, for example, infected secretions and lung tissue, including epithelial endothelial fluid, alveolar macrophages and neutrophils. do. Moreover, because the inhaled composition is efficiently targeted directly to the site of fungal infection, the dose of inhaled composition is typically much lower than the amount required to be administered by other routes to achieve antifungal effects.

In one or more embodiments of the invention, a pharmaceutical composition comprising amphotericin B, wherein the predominant amount of amphotericin B is present in crystalline form, is administered to the lungs of a patient in need thereof. For example, the patient may have been diagnosed as having a pulmonary and / or nasal fungal infection, or the patient may be determined to be susceptible to pulmonary and / or nasal fungal infection. Examples of pulmonary and / or nasal fungal infections include aspergillosis, blastomyces, disseminated candidiasis, coccidiosis fungi, cryptococcosis, histoplasmosis, fungi, sporotricumosis, candida Some infections caused by the species and others known in the art are included.

Accordingly, the pharmaceutical compositions of one or more embodiments of the present invention can be used to treat and / or prevent a wide range of patients. A patient suitable for receiving treatment and / or prophylaxis as described herein is any mammalian patient in need of such treatment and / or prophylaxis, preferably such mammal is a human. Examples of patients include, but are not limited to, pediatric patients, adult patients, and elderly patients. Patients are typically at risk of being infected with fungi.

In one form, the pharmaceutical compositions of one or more embodiments of the present invention may be used for example in a disease that adversely affects an immunocompromised patient, such as an individual undergoing chemotherapy or radiation therapy for cancer, organ transplant recipients, immune system, e.g. It is useful for preventing pulmonary and / or nasal fungal infections, for example, for patients with HIV or any other disease that causes the patient to develop pulmonary and / or nasal fungal infections. The pharmaceutical compositions may also contain active pulmonary and / or nasal fungal infections, for example aspergillosis (most commonly Aspergillus pumigatus, Aspergillus plabus, Aspergillus nager, Aspergillus Nidulans and Aspergillus tereus), coccidioide fungi, histoplasmosis, blastomyces and other fungal conditions.

In one form, the aerosolizable pharmaceutical composition comprising amphotericin B is administered to the lung and / or nasal cavity of the patient in a manner that results in amphotericin B concentrations greater than the minimum inhibitory concentration (MIC) of the fungus. MIC is defined as the minimum concentration of amphotericin B that inhibits fungal growth. MIC can be expressed as a specific concentration value or a range of concentrations. In a method according to one or more embodiments of the invention, an amount of a pharmaceutical composition is administered to achieve a target lung concentration of amphotericin B that is within the range of MIC values or above a certain MIC value. In another form, the target lung concentration of amphotericin B exceeds the MIC range. In another form, the target lung concentration of amphotericin B exceeds the minimum value of the MIC range. In other forms, the target amphotericin B concentration is above the MIC range and is at a concentration that is at least about 2 times the maximum of the MIC range, for example at least about 3 times, at least about 4 times, or at least about 5 times. The target amphotericin B concentration may be in the target lung concentration range. In one form, the target lung amphotericin B concentration range is above and below a value that is about 2 to about 20 times the median of the MIC range, for example about 3 to about 10 times the median or about 5 times the median. Fluctuate. In one form, amphotericin B concentration and MIC crystals are based on concentration in epithelial endothelium fluid. In another form, amphotericin B concentrations and MIC crystals are based on concentrations in solid lung tissue. Unless otherwise specified, a MIC value as used herein will take a particular value if a particular MIC value is determined and an intermediate value if a range of MIC values are determined. MIC determinations can be performed according to methods known in the art.

In one form, the pharmaceutical composition comprising amphotericin B is administered such that the target concentration is maintained over a desired period of time. For example, if the administration procedure maintains a target concentration of amphotericin B that is at least about 2 times, for example at least about 3 times, the determined MIC value is used to treat and / or prevent pulmonary and / or nasal fungal infections. Valid for. It is also possible in some patients to effectively treat pulmonary and / or nasal fungal infections by maintaining the amphotericin B concentration at the target lung concentration for a period of at least about 1 week, for example at least about 2 weeks, or at least about 3 weeks. It was decided. Additionally or alternatively, by maintaining the amphotericin B concentration at the target lung concentration during this period in immunocompromised patients, the likelihood of the patient developing a pulmonary and / or intranasal fungal infection may be reduced. In many cases, the treatment period and / or prophylaxis period can be extended to more than about 1 month, more than about 2 months, more than about 3 months (eg, 17 weeks), more than about 4 months, or longer.

In one form, the method of administering crystalline amphotericin B is advantageous in lung retention of pharmaceutical compositions comprising amphotericin B. In this regard, one or more embodiments of the present invention provide that amphotericin B of the pharmaceutical composition is determined by (1) bronchoalveolar lavage, at least about 10 hours, such as at least about 15 hours or about 20 hours in pulmonary epithelial fluid. And, and / or (2) the discovery that the lung tissue has an amphotericin B lung-retention half-life of at least about 1 week, eg, at least about 2 weeks, as measured by lung tissue homogenization.

Because the lung-retention half-life of amphotericin B is long and low doses may be used in one or more embodiments, systemic exposure (blood / plasma amphotericin B concentration) is low enough to avoid renal toxicity and / or liver toxicity. maintain. For example, after 5 mg administration of inhaled amphotericin B, the blood level of amphotericin B is less than about 1000 ng / mL, eg, less than about 750 ng / mL, less than about 500 ng / mL, about 250 It may be maintained below ng / mL, below about 100 ng / mL, below about 80 ng / mL, below about 60 ng / mL, or below about 40 ng / mL.

With regard to the long residence half-life, once the target lung tissue amphotericin B concentration is reached, a limited dose is required to maintain lung tissue amphotericin B concentration. For example, pharmaceutical compositions may be administered once per week to maintain lung amphotericin B concentrations within the target.

Dosages and frequency of administration necessary to maintain amphotericin B concentrations within target concentrations depend on the composition and the concentration of amphotericin B in the composition. In each dosing regimen, the dosage and frequency are determined such that pulmonary amphotericin B concentration is maintained within a specific target range. In one form, amphotericin B can be administered weekly. In this form, the weekly dosage of amphotericin B is about 2 mg to about 75 mg, for example about 2 mg to about 50 mg, about 4 mg to about 25 mg, about 5 mg to about 20 mg, and about 7 mg to about 10 mg.

The dose may be administered during a single inhalation or may be administered during multiple inhalations. Variation in pulmonary amphotericin B concentration may be reduced by administering the pharmaceutical composition more frequently, or may be increased by administering the pharmaceutical composition less frequently. Therefore, the pharmaceutical compositions of one or more embodiments of the invention may be from about three times a day to about once a month, for example about once a day to once every about two weeks, once to about every two days It can be administered once a week and once a week.

In one form, the pharmaceutical composition is administered prophylactically to patients susceptible to immunocompromise. For example, prophylactic treatment of patients receiving drug immunosuppressive therapy, eg, patients with bone marrow transplantation, with a pharmaceutical composition comprising crystalline amphotericin B reduces the likelihood of developing a fungal infection during the period of immunocompromised risk. You can. In this form, amphotericin B administration commences long enough for the patient to be immunocompromised to allow the lung amphotericin B concentration to reach the target concentration at or before the time of immunocompromise. If the dose is administered once a week, the duration of prophylaxis may vary from about 1 week to about 20 weeks, depending on the composition and dosage. However, in one or more embodiments of the invention, the time to an effective prophylactic amphotericin B concentration provides a high dose of amphotericin B during the initial prophylactic period and / or a dosage (eg, loading dose) during the initial prophylactic period. ) Is shortened by more frequent administration. In this form, an additional dose is administered during the first week of therapy. For example, the dose may be administered on days 1, 2, 3, and 4 (or 4 doses on day 1), followed by every 7 days thereafter. This early loading results in faster target lung amphotericin B concentrations. Thus, the time to achieve effective prevention is reduced, and the patient can start his immunodeficiency period sooner. In some instances, the patient may be immunocompromised after 1-4 days with a significantly reduced likelihood of developing pulmonary and / or nasal fungal infections. Additionally or alternatively, the dose administered during the period prior to immunosuppression can be higher than the dose administered (eg, loading dose) to maintain the target lung amphotericin B concentration. For example, in one form, once the target lung amphotericin B concentration is achieved, the first dose may be at least about twice the steady-state dose.

Thus, in one form, amphotericin B is administered in a loading dose followed by a maintenance dose. The loading dose of amphotericin is, for example, in the range of about 5 mg to about 75 mg, for example about 10 mg to about 50 mg, about 15 mg to about 40 mg, or about 20 mg to about 30 mg, for example about 25 mg. Can be. The maintenance dose may be administered regularly, eg weekly, after the loading dose. The maintenance dose is typically in the range of about 2 mg to about 20 mg, for example about 3 mg to about 15 mg or about 4 mg to about 10 mg, for example about 5 mg.

Early loading may also be desirable when treating patients diagnosed with pulmonary and / or nasal fungal infections. By early loading, the target lung amphotericin B concentration is achieved faster than without early loading. Therefore, treatment of pulmonary and / or nasal fungal infections can be initiated or provided more quickly.

In one particular method of treatment, the patient receiving immunosuppressive therapy provides for the prevention of pulmonary and / or nasal fungal infections. According to this form, the patient is administered at least about 5 mg, such as about 5 mg to about 10 mg of amphotericin B, which is aerosolized during inhalation of the patient at least about twice per week during the initial period. Aerosolized amphotericin B is administered at least about three times per week during the initial period. In one form, the initial period may last from about 1 week to about 3 weeks. After the initial period, the patient is administered the same dose less frequently. For example, aerosolized amphotericin B can be administered once every two weeks, more preferably once a week. After the initial period or at the end of the initial period, immunosuppressive therapy can begin. The second dosing period continues if necessary or if pulmonary and / or nasal fungal infection develops, so that the target lung amphotericin B concentration is maintained for at least the risk of immunodeficiency and longer. Additionally or alternatively, the dose administered during the first time period may be greater than the dose administered during the second time period. For example, about 10 mg to about 20 mg of amphotericin B can be administered during a first period of time and a smaller amount, eg, about 5 mg to about 10 mg, is administered during a second period of time. Optionally, a third dosing period may be provided in which the dose is administered less frequently and / or in lesser amounts than in the second period. The third dosing period may begin at the end of the immunocompromise period, for example if the immunosuppressive therapy has ended or has been reduced in severity.

In one form, for example, as described in US Application No. 10 / 751,342, filed December 31, 2003, which is incorporated by reference in its entirety, the amphotericin B concentration is at a concentration above the determined minimum inhibitory concentration. Maintained for a period of time. The pulmonary amphotericin B concentration may be the amphotericin B concentration in the epithelial lining or the amphotericin B concentration in solid lung tissue, preferably the latter. In some forms, the pulmonary amphotericin B concentration is at least about 4 μg / g, such as at least about 9 μg / g, and from about 4.5 μg / g to about 20 μg / g, for example from about 9 μg / g About 15 μg / g.

For prophylaxis, the amount per dose of amphotericin B can be an amount effective to prevent pulmonary and / or intranasal infection by fungi and is generally from about 0.01 mg / kg to about 5.0 mg / kg, for example about 0.4 mg / kg to about 4.0 mg / kg, or about 0.7 mg / kg to about 3.0 mg / kg.

The pharmaceutical composition may be administered to the patient in any regimen effective to prevent pulmonary infection by fungi. Exemplary prophylactic regimens include administration of an antifungal dry powder as described herein once to 21 times a week over a period of 1 to 6 weeks, followed by once or twice a week if necessary. have.

An example of an embodiment of the present invention for the administration of predominantly crystalline aerosolized amphotericin B is shown in FIG. 1 showing prophylactic loading. In this form the MIC values for amphotericin B were determined in the range of about 0.5 μg / g to about 4 μg / g as shown in block 300. The median MIC value (300 ') is about 2.25 μg / g. Curve 301 represents the predicted lung amphotericin B concentration according to the particular dosing regimen. As can be seen, the amphotericin B concentration reaches the target lung amphotericin B concentration range 302 above the MIC range 300, which is at least two times greater than the median MIC value 300 ′. In this form, the target lung amphotericin B concentration range 302 may range from 4 μg / g to 50 μg / g, more preferably 4.5 μg / g to 20 μg / g. In the specific form shown, the target lung amphotericin B concentration range 302 is in the range of 9 μg / g to 15 μg / g, approximately at a concentration value of about five times the midpoint value 300 ′ of the MIC range 300. Is changed.

Maintaining pulmonary amphotericin B concentrations within the target pulmonary amphotericin B concentration ranges according to one or more embodiments of the present invention is advantageous in terms of their effectiveness in the treatment and / or prevention of fungal infections, and also conventional treatments. More secure. 2 shows the predicted plasma amphotericin B concentration 400 obtained during administration of amphotericin B in accordance with one or more embodiments of the present invention. As can be seen, the amphotericin B concentration is significantly less than the plasma amphotericin B concentration minimum toxicity concentration 401, thus increasing the safety of administration.

To treat patients suffering from pulmonary and / or nasal fungal infections, the amount per dose of amphotericin B administered can be an amount effective to treat the infection. The amount of amphotericin B for the treatment of an infection will generally be higher than the amount used for prophylaxis, typically from about 0.01 mg / kg to 7.0 mg / kg, for example from about 0.2 mg / kg to about 6.0 mg / kg Or from about 0.8 mg / kg to about 5.0 mg / kg. In one exemplary treatment regimen, the antifungal powder according to one or more embodiments of the invention is about 1 to about 8 times per day, preferably about 2 to about 6 per day over a period of about 7 to about 28 days. May be administered once.

In treating the respiratory fungal disease, pharmaceutical compositions are typically administered at a dose of about 3 to about 10 times or more of the MIC of the causative fungal pathogen. In general, the dose of amphotericin B delivered to a patient will range from about 2 mg to about 400 mg per day, for example from about 10 mg to 200 mg per day, depending on the disease to be treated, the age and weight of the patient, and the like.

Without wishing to be bound by theory, by providing amphotericin B in accordance with one or more embodiments of the present invention, by reducing the rate of dissolution of amphotericin B and / or increasing deposition throughout the lung and local toxicity in the upper lung By avoiding the local toxicity of amphotericin B can be reduced. Thus, administration of crystalline amphotericin B is believed to be safer than administration in the mainly amorphous form of amphotericin B. In addition, administration of smaller inhaled particulates formed from smaller amphotericin B particles tends to improve safety.

When administering larger doses, safety is a more important issue. Thus, for high doses, higher crystallinity amphotericin B, smaller amphotericin B particle sizes, and smaller inhaled particle or particulate sizes are proposed. For example, for doses greater than 50 mg, those skilled in the art will appreciate that the amphotericin B crystallinity level is at least about 90%, the amphotericin B mass median diameter is less than about 2.8 μm, and the MMAD of the inhaled particles or particulates is It may be desirable to use a composition that is less than about 2.8 μm.

When solid particles or particulates comprising amphotericin B are administered to the lungs, it has been determined that it is desirable to control the rate of dissolution of the solid particles or particulates in the lungs. If the rate of dissolution is undesirably high, soluble supramolecular aggregates are formed at topical sites in the lungs. The soluble aggregates may in some cases be toxic to lung tissue. However, if the rate of dissolution is sufficiently low, the probability of developing soluble toxic aggregates is low. While not wishing to be bound by theory, it is believed that providing solid particles or particulates comprising crystalline amphotericin B lowers the rate of dissolution compared to amphotericin B in its amorphous form. It has also been found that the pulmonary amphotericin B concentration in crystalline form can be maintained at a concentration high enough to provide a therapeutic effect against pulmonary and / or nasal fungal infections while significantly reducing or preventing toxic soluble aggregates.

Thus, in one form, the pharmaceutical composition can be delivered to the patient's lungs in the form of dry powder. Thus, the pharmaceutical composition comprises a dry powder that can be effectively delivered deep into the lung or to another target site. The pharmaceutical composition is in the form of a dry powder comprising particles or particulates having a size selected to be permeated into the alveoli of the lung.

In some instances, it is desirable to deliver a unit dose, eg, at least 5 mg or 10 mg of amphotericin B to the lungs in a single inhalation. The phospholipid hollow and / or porous dry powder particles described above deliver a dose of at least about 5 mg, often greater than about 10 mg, sometimes greater than about 25 mg in a single inhalation and advantageous manner. Alternatively, the dose may be delivered via two or more inhalations. For example, a 10 mg dose can be delivered by providing two unit doses of 5 mg each, and the two unit doses can be inhaled separately.

Dispersions or powder pharmaceutical compositions can be administered using an aerosolization device. The aerosolization device may be a nebulizer, a metered dose inhaler, a liquid dose dropping device, or a dry powder inhaler. Powder pharmaceutical compositions are filed as a nebulizer as described in WO 99/16420, a quantitative inhaler as described in WO 99/16422, a liquid dose infusion device as described in WO 99/16421, and June 22, 2001. Dry powder inhaler as described in US application Ser. No. 09 / 888,311, WO 99/16419, WO 02/83220, US Pat. No. 6,546,929, and US application Ser. No. 10 / 616,448, filed July 8,2003. Which is hereby incorporated by reference in its entirety. Accordingly, the inhaler may comprise a canister containing particles or particulates and a propellant, wherein the inhaler comprises a metering valve connected to the interior of the canister. The propellant may be hydrofluoroalkane.

Pharmaceutical compositions of one or more embodiments of the present invention typically have improved radiation dose efficiency. Thus, high dose pharmaceutical compositions can be delivered using various aerosolization devices and techniques.

The spinning capacity (ED) of the powder may be greater than about 30%, for example greater than about 40%, greater than about 50%, greater than about 60% or greater than about 70%.

An example of a dry powder aerosolization apparatus particularly useful for aerosolizing pharmaceutical compositions 100 according to one or more embodiments of the present invention is shown schematically in FIG. 3A. The aerosolization apparatus 200 includes a housing 205 that defines a chamber 210 having one or more air inlets 215 and one or more air outlets 220. Chamber 210 is sized to contain a capsule 225 containing an aerosolizable pharmaceutical composition comprising crystalline amphotericin B. The perforator 230 includes a perforation member 235 that is movable within the chamber 210. An end 240, which may be sized and shaped to be administered to the user's mouth or nose, is close to the outlet 220 so that the user can inhale through the opening 245 at the end 240 which is connected to the outlet 220. Or adjacent to each other.

The dry powder aerosolization apparatus 200 uses the air flow through the chamber 210 to aerosolize the pharmaceutical composition in the capsule 225. For example, FIGS. 3A-3E illustrate the operation of one form of aerosolization apparatus 200, where the aerosolized pharmaceutical composition using air flow through inlet 215, the aerosolized pharmaceutical composition exits Flow through 220 may be delivered to the user through opening 245 at end 240. The initial state of the dry powder aerosolization apparatus 200 is shown in FIG. 3A. Capsule 225 is located in chamber 210, and the pharmaceutical composition is contained in capsule 225.

In order to use the aerosolization apparatus 200, the pharmaceutical composition in the capsule 225 is exposed and aerosolized. In the form of FIGS. 3A-3E, perforator 230 advances into chamber 210 by applying force 250 to perforator 230. For example, as shown in FIG. 3B, the user presses the surface 255 of the perforator 230 so that the perforator member 235 contacts the capsule 225 in the chamber 210. 205) to slide in. As shown in FIG. 3C, by continuously applying the force 250, the perforation member 235 advances through the wall of the capsule 225. Having the puncture member include one or more sharp tips 252 may facilitate the advancement through the walls of the capsule 225. The perforator 230 is then inserted into the position shown in FIG. 3D, whereby an opening 260 remains in the wall of the capsule 225 to expose the pharmaceutical composition in the capsule 225.

Thereafter, air or other gas flows through the inlet 215, as indicated by arrow 265 in FIG. 3E. The flow of air causes the pharmaceutical composition to be aerosolized. When the user inhales through the end 240 (270), the aerosolized pharmaceutical composition is delivered to the user's respirator. In one form, the air flow 265 can be caused by the inhalation 270 of the user. In other forms, compressed air or other gas may be blown out to the inlet 215 to aerosolize the air stream 265.

Certain forms of dry powder aerosolization apparatus 200 are described in US Pat. Nos. 4,069,819 and 4,995,385, the entire contents of which are incorporated herein by reference. In this arrangement, the chamber 210 generally comprises a longitudinal axis extending in the suction direction, and the capsule 225 is of a length insertable into the chamber 210 such that the longitudinal axis of the capsule can be parallel to the longitudinal axis of the chamber 210. . The chamber 210 is sized to receive a capsule 225 containing the pharmaceutical composition in a manner that allows the capsule to move into the chamber 210. Inlet 215 includes a plurality of tangential slots. When the user inhales through the end, the outside air flows through the tangential slot. This airflow creates a vortex airflow in chamber 210. The vortex airflow causes the capsule 225 to contact the compartment and then causes the capsule 225 to move into the chamber 210 in such a way that the pharmaceutical composition exits the capsule 225 and is incorporated into the vortex airflow. This form is particularly effective for consistently aerosolizing high dose pharmaceutical compositions. In one form, the capsule 225 rotates in the chamber 210 in such a way that the longitudinal axis of the capsule remains at an angle of less than 80 degrees, preferably less than 45 degrees, from the longitudinal axis of the chamber. Movement of the capsule 225 in the chamber 210 can be caused because the length of the chamber 210 is less than the length of the capsule 225. In one particular form, the chamber 210 includes a tapered region ending at one edge. While the vortex air in the chamber 210 flows, the front end of the capsule 225 remains in contact with the partition and the sidewall of the capsule 225 slides in contact with the edge and / or rotates along the edge. do. This movement of the capsule is particularly effective when pushing a large amount of pharmaceutical composition through one or more openings 260 behind the capsule 225.

In another passive dry powder inhaler form, the dry powder aerosolization apparatus 200 may be of a different shape than that shown in FIGS. 3A-3E. For example, as described in US Pat. No. 3,991,761, which is incorporated by reference in its entirety, chamber 210 may be sized and shaped to receive capsule 225 such that capsule 225 is perpendicular to the direction of suction. . As also described in US Pat. No. 3,991,761, perforator 230 may perforate both ends of capsule 225. In another form, as described, for example, in US Pat. Nos. 4,338,931 and 5,619,985, the chamber may receive the capsule 225 in such a way that air flows through the capsule 225. In another form, aerosolization of the pharmaceutical composition is described, for example, in US Pat. 5,785,049; US Pat. And by flowing gas through an inlet as described in US Pat. No. 6,257,233 or through a propellant as described in WO 00/72904 and US Pat. No. 4,114,615, which is incorporated by reference in its entirety. Included as. Dry powder inhalers of this type are generally referred to as active dry powder inhalers.

The pharmaceutical compositions disclosed herein may also be administered via aerosolization, for example, in the lung and / or nasal air route of a patient using a quantitative inhaler. As disclosed in WO 99/16422, which is incorporated by reference in its entirety, the use of such stabilized formulations provides good dose reproducibility and improved lung deposition. MDI is well known in the art and can be used for the administration of amphotericin B. Those including respiratory activated MDI as well as other types of improvements that have been developed or will be developed are also compatible with the pharmaceutical compositions of one or more embodiments of the present invention.

Nebulizers are known in the art and can be readily used to administer the claimed dispersions without undue experimentation. Respiratoryly activated nebulizers as well as those comprising other types of improvements to be developed or to be developed are also compatible with stabilized dispersions, which are considered to be within the scope of one or more embodiments of the invention. In combination with the above-mentioned embodiments, the stabilized dispersions of one or more embodiments of the present invention may also be used in the lung and / or nasal air of a patient in need of administration, as disclosed in WO 99/16420, which is incorporated herein by reference in its entirety. It may be used with a nebulizer to provide an aerosolized medicament that can be administered by the passage.

Stabilized dispersions of one or more embodiments of the present invention, in conjunction with DPI, MDI, and Nebulizers, may be employed in liquid dose infusion or LDI techniques, for example, as disclosed in WO 99/16421, which is incorporated herein by reference in its entirety. It will be appreciated that it can be used with. Liquid dose infusion involves the direct administration of a stabilized dispersion into the lungs. In this regard, direct pulmonary and / or intranasal administration of biofunctional compounds is particularly effective in the treatment of disorders in which poor vascular circulation of the lung disease part reduces the effect of intravenous drug delivery. In the context of LDI, stabilized dispersions are preferably used with partial liquid ventilation or total liquid ventilation. Moreover, one or more embodiments of the present invention may further comprise introducing into the pharmaceutical microdispersion a therapeutically effective amount of a physiologically acceptable gas (such as nitric oxide or oxygen) during or after administration.

Dosing time is typically short. For single capsules (eg 5 mg doses), the total administration time is usually less than about 1 minute. Two capsule administration (eg 10 mg) usually takes about 1 minute. 5 capsule administration (eg, 25 mg) may take about 3.5 minutes to the manager. Thus, the administration time may be less than about 5 minutes, for example less than about 4 minutes, less than about 3 minutes, less than about 2 minutes or less than about 1 minute.

The description will be more fully understood with reference to the following examples. However, these examples are merely representative of how one or more embodiments of the invention may be practiced and should not be construed as limiting the scope of the invention.

Example 1

Different Lot number  Interstitial Amphotericin  Variability of Crystallinity Levels in B

This example involves determining the crystallinity percentage values of amphotericin B of various lot numbers by quantitative X-ray powder diffraction. This example shows a range of crystallinity levels for amphotericin B of different lot numbers.

Equipment and materials

equipment

Sample holder

Shimadzu X-ray Diffractometer Model XRD-6000

matter

Silicon Reference Standard, NIST (National Institute of Standards and Technology) 640c

Lithium fluoride, particle size <5 μm

Amphotericin B crystalline standard, sieve <48 μm

Amphotericin B "amorphous", sieve <48 μm

Vehicle Powder (DSPC / CaCl _{2 in} 2: 1 Molar Ratio)

Way

Alignment confirmation of the Shimadzu X-ray diffractometer model XRD-6000 was performed as a silicon reference standard. During the alignment check, the dispersion slits were set at 1 °, the scattering slits were set at 1.0 °, and the receiving slits were set at 0.15 mm.

The diffractometer was calibrated to obtain X-ray powder diffraction data for calibration standards and lot numbers of unknown crystallinity (sample size of about 60 mg). The following settings were used for this data acquisition.

Retention time: 2 seconds (fixed time scan)

Step size: 0.02 ° 2θ

Scanning Range: 3-42 ° 2θ

Slits: 0.5 ° dispersion slits, 1 ° scattering slits and 0.3 mm receiving slits.

Internal standard methods were used to prepare calibration plots for determining the crystallinity of amphotericin B in the samples. The internal standard was LiF. Physical mixtures of crystalline and wide-angle X-ray amorphous standards were prepared. In the present embodiment, later, the wide-angle X-ray amorphous standard will be referred to as "amorphous". These physical mixtures were then doped with LiF at 20% by weight.

The crystalline standard was selected from several lot numbers of high crystalline amphotericin B. Based on the qualitative comparison of the X-ray powder diffraction pattern, the material with the least amorphous background was selected. Amorphous standards were prepared by freezing milling of crystalline material (ie, amphotericin B of the same lot number was used for both amorphous and crystalline amphotericin B standards). Both standards were sieved less than 48 μm before preparation of the physical mixture. Physical mixtures were prepared at 40 ± 5% RH (20-25 ° C.). All gravimetric measurements were performed with an analytical balance with a resolution of at least 0.01 mg.

Data analysis

The ratio of the integrated intensity of the diffraction peak of crystalline amphotericin B to the peak of LiF was calculated from the diffraction pattern of each LiF-doped physical mixture. The integrated intensity ratio IIR is

_Wherein I _{AmB, Cr} and I _LiF are the integrated intensities of the selected reference peaks of crystalline amphotericin B and LiF, respectively.

Single diffraction peaks (38 to 39.5 ° 2θ) were chosen for LiF and a narrow angular range was chosen for amphotericin B (12.75 to 16.25 ° 2θ). Only a pair of amphotericin B peaks are used because this eliminates the need for creation / interpolation of the curve baseline, and these peaks are sensitive to initial crystallinity. Peaks were integrated using Jade software (MDI Inc.) version 7.1.2.

Results and discussion

Table 1 shows the crystallinity results for amphotericin B of various lot numbers. The maximum degree of change of the drug substance method is about ± 10% crystalline and depends on the percent crystallinity. Without wishing to be bound by theory, the uncertainty of the result due to the very different bulk density of the selected standard is due to the variability of the compression used to fill the sample holder. Improvements in precision can be achieved by using lower sample weights (as a result of lower need for sample compression).

Example  2

Preparation of Spray-Dried Ampoterisin B Fine Particles

Ampotericin B microparticles were prepared by a two-step process. In the first step, 10.52 g of amphotericin B (Alpharma, Copenhagen, Denmark), distearoyl phosphatidylcholine (DSPC) (10.12 g, Genzyme, Cambridge, Mass., USA and calcium chloride (US) 0.84 g of JT Baker, Phillipsburg, NJ, was heated using an Ultra-Turrax mixer (Model T-25) for 2-5 minutes at 10,000 rpm (T =). 70 ° C.) 1045 g The DSPC and amphotericin B were mixed until visually dispersed.

Thereafter, 381 g of perfluorooctyl ethane (PFOE) was added slowly at a rate of approximately 50-60 mL / min. After the addition was complete, the emulsion / drug dispersion was mixed at 12,000 rpm for a further period of at least 5 minutes. The crude emulsion was then passed three times at 12,000 to 18,000 psi through a high pressure homogenizer (Avestin, Ottawa, Canada) and then twice at 20,000 to 23,000 psi.

The resulting fine emulsion was used as feedstock for the second step, spray-drying on Niro Mobile Minor. The following spray conditions were used: total flow rate = 70 SCFM, inlet temperature = 110 ° C., outlet temperature = 57 ° C., feed pump = 38 mL / min, atomizer pressure = 105 psig, atomizer flow rate = 12 SCFM.

Free-flowing pale yellow powder was collected using a cyclone separator. The collection efficiency was 60%. The geometric diameter of the amphotericin B particles was confirmed by laser diffraction (Sympatec Helos, Klaustal-Cellerfeld, Germany) with a volume weight average diameter (VMD) of 2.44 μm. Scanning electron microscopy (SEM) analysis showed that the powder was small porous particulates with high surface roughness. There was no evidence of unincorporated amphotericin B crystals in the 5 SEM observations of the powder from each collector. Differential scanning calorimetric analysis of dry particulates showed that the T _m of distearoyl phosphatidylcholine in the powder was 78 ° C., similar to that observed with spray-dried pure distearoyl phosphatidylcholine.

Example 3

Aerosol Performance for Spray-Dried Amphotericin B Particles

The resulting dried amphotericin B microparticles prepared in Example 2 were manually filled into # 2 HPMC capsules (Shionogi, Japan), which reached equilibrium at ambient temperature overnight at 15-20% RH. A fill weight of approximately 10 mg was used, which represented approximately one half of the fill volume of the # 2 HPMC capsule.

Aerodynamic particulate size distribution was determined gravimetrically on Anderson Cascade Impactor (ACI). 28.3 L / min the fine particle size distribution (i.e., comfortable inhalation effort) and 56.6 L / min (that is, the strong suction effort), flow rates in TSE described in the entirety incorporated by reference US Patent 4,069,819 and 4,995,385 herein ^® DPI of It measured using the apparatus. A total volume of 2 liters was taken through the apparatus. At higher flow rates, two ACIs were used equilibrated at the calibrated flow rate of 28.3 L / min and the total flow rate of 56.6 L / min through the device. In both cases, the setting indicates the condition under which the ACI impactor plate was calibrated. Good aerosol characteristics were observed, evidenced by MMAD _< 2.6 μm and FPF _{<3.3 μm} 72%. In addition, the effect of flow rate on performance was evaluated by using a turbo spin ^® (PH & T, Italy) DPI device works in two ACI that is used to equilibrate at 56.6 L / min (Fig. 4). No significant difference was observed at higher flow rates in the deposition profile, indicating minimal flow dependent performance. This example mentioned above indicates that the aerosol performance of the powder is independent of flow rate, resulting in more reproducible patient administration.

Example 4

Storage effect on aerosolization of amphotericin B particulates

The obtained dry amphotericin B microparticles prepared in Example 2 were manually filled into # 2 HPMC capsules (Shionogi, Japan), which reached equilibrium overnight at 15-20% RH. A fill weight of approximately 10 mg was used, which represented approximately one half of the fill volume of the # 2 HPMC capsule. The filled capsules were placed in individually indexed glass vials packaged in laminated foil-sealed pouches and then stored at 25 ° C./60% RH or 40 ° C./75% RH.

A radiation dose (ED) measurement was used as the total volume of two liters and performed using a turbo spin ^® (PH & T, Italy) DPI device described in US Patent 4,069,819 and US Patent 4,995,385 which is operating at optimal sampling flow rate of 60 L / min. A total of 10 measurements were determined for each storage variant.

Aerodynamic particulate size distribution was determined gravimetrically on Anderson Cascade Impactor (ACI). The particle size distribution using a total volume of 2 liter was measured using a turbo spin ^® DPI device with 28.3 L / min flow rate of.

Good aerosol characteristics were observed, evidenced by mean ED 93% ± 5.3%, MMAD = 2.6 μm and FPF _{<3.3 μm} = 72% (FIGS. 5 and 6). No significant change in aerosol performance (ED, MMAD or FPF) after storage at elevated temperature and humidity exhibiting good stability characteristics was observed. The current USP for ED performance requires that> 90% of the delivered dose be within ± 25% of the label claim with less than 10% of the dose within ± 25% and greater than ± 25%. A recent draft sample published by the FDA strictly suggests a limit that states that> 90% of delivered doses should be within ± 20% of the label claim with less than ± 25%. Statistically, 6% RSD is required to meet the requirements of the proposed FDA details.

The results of this embodiment are not only within current guidelines, but also within the limits of the proposed guidelines. Thus, the composition has good dispersibility, aerosol characteristics and stability.

Example 5

Spray-dried amphotericin B particulates with various phosphatidylcholines

Spray-dried particulates comprising approximately 50% amphotericin B were prepared using various phosphatidylcholines (PCs) as surfactants according to the two-step process described in Example 2. Dipalmitoyl phosphatidylcholine (DPPC) (genzyme, Cambridge, Mass.), Distearoyl phosphatidylcholine (DSPC) (genzyme, Cambridge, Mass.) And SPC-3 (Lipoid, Ludwigshafen, Germany) KG)) to prepare the composition. The feed solution was prepared using the same equipment and process conditions as described herein. The 50 wt% amphotericin B composition is as follows:

Amphotericin B 0.733 g

PC 0.714 g

CaCl ₂ 60 mg

PFOB 32 g

75 g deionized water

The resulting multi-particulate emulsion was used as feedstock for the second step, spray-drying on a B-191 mini spray-dryer (Buechi, Flavil, Switzerland). The following nominal spray conditions were used: suction = 100%, inlet temperature = 85 ° C., outlet temperature = 60 ° C., feed pump = 1.9 mL / min, atomizer pressure = 60-65 psig, atomizer flow rate = 30-35 cm . The suction flow rate (69-75%) was adjusted to maintain the exhaust bag pressure 30-31 mbar. Free flowing yellow powder was collected using a standard cyclone separator. The geometric diameter of the amphotericin B microparticles was measured by laser diffraction (Simpatech Helos, Klaustal-Cellerfeld, Germany) and the volume weight average diameter (VMD) ranged from 2.65 to 2.75 μm. Scanning electron microscope (SEM) analysis indicated that the powder was small porous particulates with high surface roughness.

Aerodynamic particulate size distributions were determined by gravimetric analysis on Anderson Cascade Impactor (ACI) (see FIG. 7). The particle size distribution was measured using a turbo spin ^® DPI device at a flow rate of 56.6 L / min (that is, the strong suction effort). A total volume of 2 liters was taken through the apparatus. Two ACIs were used equilibrated at the calibrated flow rate of 28.3 L / min and the total flow rate of 56.6 L / min through the device. Similar aerosol characteristics were observed in amphotericin B made of three types of phosphatidylcholine, with MMAD _< 2.5 μm and FPF _{<3.3 μm} 72%. This example mentioned above indicates that the adaptability of the compositional technique to prepare amphotericin B powder is independent of the type of phosphatidylcholine used.

Example 6

Preparation of 70% by weight Ampoterisin B spray-dried particulates

Ampotericin particulates were prepared according to the two-step process described in Example 2. Feed solutions were prepared using the same equipment and process conditions as described herein. The 70% by weight amphotericin B composition is as follows:

0.70 g of amphotericin B

DSPC 0.265 g

CaCl ₂ 24 mg

PFOB 12 g

Deionized water 35 g

The resulting multiparticulate emulsion was used as feedstock for the second step, spray-drying on a B-191 mini spray-dryer (Busy, Flavil, Switzerland). The following spray conditions were used: suction = 100%, inlet temperature = 85 ° C., outlet temperature = 60 ° C., feed pump = 1.9 mL / min, atomizer pressure = 60-65 psig, atomizer flow rate = 30-35 cm. The suction flow rate (69-75%) was adjusted to maintain the exhaust bag pressure 30-31 mbar. Free flowing yellow powder was collected using a standard cyclone separator. The geometric diameter of the amphotericin B microparticles was measured by laser diffraction (Simpatech Helos, Klaustal-Cellerfeld, Germany) with a volume weight average diameter (VMD) of 2.96 μm. Scanning electron microscopy (SEM) analysis The silver powder was shown to be small porous particulates with high surface roughness This example illustrates the adaptability of powder engineering techniques to produce a high amphotericin B content using the multi-particulate approach described herein.

Example 7

Aerosol Performance of Spray-Dried Ampoterisin B Particulates

The obtained dry amphotericin B microparticles prepared in Example 6 were manually filled into # 2 HPMC capsules (Sionogi, Japan) or # 3 HPMC (Capsugel, Greenwood, SC) capsules. This allowed to equilibrate overnight at 15-20% RH. A fill weight of approximately 10 mg was used, representing approximately 1/2 of the fill volume of the # 2 HPMC capsule or 5/8 of the # 3 HPMC capsule. The aerosol characteristics were tested using the TSE ^® (PH & T, Italy Material), Eclipse (Eclipse) ^® (Aventis (Aventis) in the United Kingdom) and cyclo Haller ^® (No-Par Tees (Novartis) in Switzerland material) DPI device. Cyclohaler ^® uses # 3 HPMC capsules, while TurboSpin ^® and Eclipse ^® use size # 2 HPMC capsules.

Aerodynamic particulate size distributions were determined by gravimetric analysis on the Anderson Cascade Impactor (ACI) (see FIG. 8). Particulate size distribution was measured at a flow rate of 56.6 L / min, with TurboSpin ^® and Eclipse ^® DPI devices exhibiting strong suction effort and Cyclohaler ^® devices showing comfortable suction effort. A total volume of 2 liters was taken through the apparatus. Two ACIs were used equilibrated at the calibrated flow rate of 28.3 L / min and the total flow rate of 56.6 L / min through the device. Similar aerosol characteristics were observed in all devices, evidenced by less than 2.5 μm MMAD and more than 71% FPF _{<3.3 μm} . This example mentioned above is that the aerosol performance of the powder is independent of the medium and the device design with low resistance, independent of the capsule size, which indicates the dispersibility of the amphotericin B powder tested.

Example 8

Spray-drying Amphotericin  Aerosol Performance of B Particles

This example further tested the aerosol performance of amphotericin B microparticles of development lot number.

The following amphotericin B microparticles were formed using the method of Example 2, where amphotericin B was obtained from alphama.

-4000T

-4001T

9 and 10 respectively show the radiation dose (ED) per mg (mg / capsule) and the average ED per lot number. In order to determine ED, U.S. Pat. An inhaler device as shown in application number 10 / 298,177 was used.

11 and 12 respectively show the mass median aerodynamic diameter (MMAD) and fine particle capacity (% FPD <3.3 μm) per collector.

The following amphotericin B microparticles were formed using the method of Example 2, wherein amphotericin B was obtained from Chemwerth.

-4024T

-4025T

-4026T

-4027T

-4028T

-4029T

-4030T

13 and 14 respectively show the radiation dose (ED) (mg / capsule) and average ED per collector and lot number.

15 and 16 respectively show the mass median aerodynamic diameter (MMAD) and% FPD _{<3.3 μm} per collector.

As described in more detail below, the following amphotericin B microparticles were formed using processed amphotericin B.

N020226 (from highly crystalline API)

N020227 (from rubber amorphous API)

These amphotericin B microparticles were formed using a modification of the method of Example 2, where amphotericin B was originally produced by Alfama. However, prior to spray drying, amphotericin B is processed to (i) rubber shaping; Or (ii) a high crystalline API batch (as determined by X-ray powder diffraction).

Specifically, the reprocessing procedure of amphotericin B is provided below. As shown in the flow chart and description below, both crystalline and amorphous forms were prepared from the same starting material.

Amphotericin  B drug substance ( API Description of reprocessing procedure

Solubilization  And filtration

Approximately 20 g of amphotericin B was added to 1130 mL of methanol with stirring at room temperature. Approximately 450 mL of dimethylformamide was then added 39 g of citric acid monohydrate to the amphotericin B suspension with continuous stirring. Once all solids had dissolved essentially, the solution was cleared by filtration.

Approximately 280 mL of methylene chloride was added to the filtrate. The vessel containing the product was protected from light during the rework period. Approximately 450 mL of cold water (2-8 ° C.) was added and the solution stirred for 15 minutes.

pH control and precipitation / crystallization

Ampoterisin B was precipitated by adjusting the pH of the solution to about 7 with 40% triethanolamine (about 140 mL). For the amorphous form, the base was added in one portion with stirring. Thereafter, an amorphous form was prepared for isolation as described below.

For the crystalline form, the base was added dropwise over 30 minutes with stirring. The slurry was then heated to 44-46 ° C. for 90 minutes and then cooled to room temperature for 30 minutes. Finally, the slurry was cooled at 2-8 ° C. for 60 minutes. Thereafter, the crystalline form was prepared for isolation.

Collection and washing of crystalline forms

The crystalline form of amphotericin B was captured by vacuum filtration using a large stainless steel suction filter fitted with a paper filter inside. The product was washed with 170 mL of cold 40% methanol (2-8 ° C.) followed by 100 mL of acetone (room temperature).

Collection and cleaning of amorphous form

The slurry was centrifuged to capture the amorphous form of amphotericin B and then the supernatant was decanted. The cake was resuspended in 600 mL of cold 40% methanol (2-8 ° C.), then the product was washed by centrifugation and decantation. The washing process was repeated using 600 mL of acetone (room temperature).

Drying in crystalline and amorphous forms

The filter paper containing the washed crystalline amphotericin B was placed in a stainless steel tray in a vacuum chamber. Similarly, washed amorphous amphotericin B was removed from the centrifuge bottle and the paper filter in the chamber was spread on a stainless steel tray pasted inside. The drying process was run at room temperature for 1-3 days while protecting the product from light. Occasionally, during the drying process, large particles were ground using a spatula to facilitate the evaporation of the residual solvent. The final amphotericin B product was transferred to an amber glass pail for storage at 2-8 ° C.

As indicated above, amphotericin B microparticles were then formed using the method of Example 2. 17 shows the radiation dose (ED) (mg / capsule) for both N020226 and N020227. The mass median aerodynamic diameter (MMAD) for N020226 and N020227 was 2.4 μm and 2.6 μm, respectively. The% FPD _{<3.3 μm} for N020226 and N020227 was 55% and 47%, respectively.

Example 9 and Comparative Example 1

Comparison of Intratracheal and Intravenous Administration

This example includes determining the concentration of amphotericin B in plasma, pulmonary lavage fluid and lung tissue following intratracheal administration of amphotericin B solution or intravenous administration of a commercially available amphotericin B deoxycholate solution.

Substances and Methods

Pre-intubated (jugular intubation [JVC]) Sprague-Dawley rats (Hilltop Lab Animals Inc, Scottsdale, Pa.) Weighing 334-366 g were used in this example. Ampoterisin B was administered by intratracheal instillation or intravenous injection. The study design is shown in Table 2 below.

group Route of administration Animal count / gender Volume Volume of Volume (mL) Blood, Lung Tissue, BAL Collection (Days) Amphotericin B powder Intratracheal (IT) 32 M 4.0 mg (about 11 mg / kg) 0.3 0.5, 1, 2, 4, 6, 8, 11 and 14 Ampotericin B Deoxycholate Formulation Intravenous (IV) 24 M 0.5 mg (about 1.4 mg / kg) 1.0 0.5, 1, 2, 4, 6, 8, 11 and 14 BAL = bronchoalveolar lavage M = male

Ampotericin B solution for endotracheal instillation was prepared at a concentration of 13.3 mg / mL amphotericin B in phosphate buffered saline (PBS). Amphotericin B deoxycholate intravenous formulation was provided as 50 mg of amphotericin B lyophilized cake and reconstituted to prepare a 1 mg / mL containing solution.

Study endpoints and procedures include collection of blood, BAL, and tissue for body weight, drug concentration analysis, and visual necropsy observations prior to dose administration.

Blood, BAL and lung tissue samples were collected 12 hours, 1, 2, 4, 6, 8, 11 and 14 days after dosing. At each sampling time, four animals of the IT group and three animals of the IV group were sacrificed and blood, lung lavage and lung tissue samples were taken.

Plasma and wash samples were analyzed for amphotericin B concentrations by MDS Pharma Services, Montreal, Canada. Lung tissue samples were analyzed for amphotericin B concentrations by MedTox Laboratories, St. Paul, MN.

result

Visual autopsy

Rats administered amphotericin B by intratracheal instillation showed gross symptoms of pulmonary toxicity. Observations included pulmonary edema, bleeding in the lungs and discoloration of lung tissue.

BAL In lung tissue and plasma Amphotericin  B concentration

Plasma, BAL and lung tissue amphotericin B concentrations and putative ELF amphotericin B concentrations after delivery of amphotericin B trachea 100 and intravenous 200 are shown in FIG. 18. As shown, amphotericin B was rarely systemically present when administered to the airways. In contrast, high concentrations of amphotericin B were present in the blood for a period up to 4 days after intravenous administration. In addition, as shown in FIG. 18, the lung concentration of amphotericin B was higher in intratracheal administration 100 than intravenous administration 200. In the experiments performed, the plasma concentration of amphotericin B was lower in intratracheal administration, while the lung concentration of amphotericin B was several times higher in intratracheal administration than in intravenous administration.

Conclusion and discussion

Rats to which amphotericin B was administered by intratracheal liquid instillation showed macroscopic symptoms of toxicity. Observations included pulmonary edema, bleeding in the lungs and discoloration of lung tissue.

Ampoterisin B could not be measured in plasma samples taken from animals that received test articles via intratracheal administration. Concentrations were measurable in BAL and lung tissue from animals to which amphotericin B was administered by intratracheal instillation. Drugs were measured in all animals for 2 days after IT administration and in 1 BAL of 4 animals for 8 days post-dose. Drugs were measured in lung tissues of all animals 14 days after IT administration.

In animals administered amphotericin B intravenously, the drug was measured in plasma up to 2 days post-dose, but at no point in time there was no animal measurable in the lungs, and only one animal was 12 hours post-dose. Had a measurable concentration of amphotericin B in BAL.

In conclusion, endotracheal delivery of amphotericin B 11 mg / kg in a solution of 13.3 mg / mL resulted in local lung toxicity. In addition, intratracheal administration had a limit of quantitation for 14 days after dose administration at this concentration. In contrast, the concentration of amphotericin B was below the limit of quantification in lung tissue of animals administered amphotericin B intravenously.

Example  10 and comparison Example  2

Aerosolization  And Intravenous  Comparison of Dosages

Aerosolable amphotericin B powder was administered to dogs for 14 days into the lungs. Ampoterisin B was delivered at a daily dose of up to 11.5 mg / kg. A dose of amphotericin B powder was delivered to the conscious dog using a facial mask containing a one-way valve to evacuate exhaled air and a suction line connected to a central holding chamber containing aerosolized powder. A respirable dry powder aerosol of formulated amphotericin B powder was prepared by dispersing the powder with a rotary brush generator and delivering it to the central chamber for 30 minutes of inhalation exposure.

19 shows mean pulmonary amphotericin B concentration-time profiles in 101 dogs after administration. As shown, amphotericin B, which remained in the lungs for several days after administration, had an observed half-life of approximately 19 days. In contrast, intravenous administration 201 with an observed half-life of about 28 hours was not well maintained.

Example  11

Preventive  By administration Increased  Survival rate

summary

This example includes determining whether amphotericin B prophylactically administered via inhalation is potent against Aspergillus fumigatus in an immunosuppressed rabbit model. The purpose of this study was to determine whether a single dose of amphotericin B by inhalation prior to inoculation of Aspergillus fumigatus increased survival time and / or reduced overall mortality.

The study included three groups of 10 animals per group. Group 1 received intravenous cytarabine and methyl prednisolone immunosuppressive therapy and sham saline. Group 2 received immunosuppressive therapy and Aspergillus fumigatus of 5 × 10 ⁷ conidia. Group 3 received immunosuppressive therapy and an estimated dose of amphotericin B of 1.5 mg / kg via inhalation one day prior to inoculation of Aspergillus pumigatus of 5 × 10 ⁷ conidia. Amphotericin B dry powder formulation and Aspergillus pumigatus were administered to anesthetized rabbits through an endotracheal tube. Immunosuppressants were administered by intravenous injection. Antibiotics to prevent bacterial infections were administered by intravenous injection or in drinking water.

Assessments performed in this study included mortality and morbidity, daily clinical observations, body weight, clinical pathology (hematology and clinical chemistry), gross necropsy, lung weight, yield of visible pulmonary infarction, fungal colonization in BAL, blood and lung tissue samples Forming unit (CFU) measurements and histopathological evaluation of lung tissue.

By 14 days the planned survival of the animals was 60% in group 1, 20% in group 2 and 70% in group 3. The mean survival time of the animals in groups 1 and 3 was 13 days, and the median day of death or sacrifice in both groups was 14 days. In contrast, the average survival of animals in group 2 was 10 days, and the median day of death or sacrifice was 10 days. Kaplan-Meier (KM) analysis showed survival of animals treated with amphotericin B prior to aspergillus fumigatus (group 3) compared to animals receiving only Aspergillus fumigatus (group 2). This significantly increased (p <0.05). Survival of group 1 (control) animals was significantly increased compared to group 2. There was no significant difference when comparing groups 1 and 3.

Clinical symptoms of respiratory effects, including shortness of breath, wheezing, or blistering, were observed in groups 2 and 3 animals. These symptoms were not observed in group 1 animals. Seven animal groups in group 2 started on day 7 of the study and showed rapid breathing, wheezing or bullous sounds. All animals in group 2 who exhibited symptoms of respiratory effect died or died in the first day of symptoms. In group 3, five animals were observed to have wheezing, rapid breathing or bullous sound. However, although the initial onset of respiratory symptoms also occurred on day 7, only two of the animals that exhibited symptoms of respiratory effects had died. The other three animals survived until two of the three (both bully) stopped showing symptoms of respiratory effects by 14 days with their planned autopsy.

Effect on body weight was observed in all groups. Body weight was reduced at approximately 8 days in all groups. All groups showed a decrease of approximately 6% by 8 days. After the initial decrease, the weights of the remaining animals in Group 1 and Group 2 remained stable during the study. In contrast, the mean group weight of the animals in Group 3, which continued to decrease by 14 days, was 18% less than their starting weight and 10% less than the mean weight of Group 1.

The effect was shown in hematology and clinical chemistry parameters measured in all groups. The primary effects shown on hematology parameters were reduced levels of leukocytes (WBC), including reduced lobe neutrophils (ANS) and platelets (PLC). These effects were seen in all groups.

Further effects are on the levels of erythrocytes, hemoglobin, erythrocyte volume, total protein, albumin, aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, cholesterol, globulin and triglycerides in groups 2 and 3 Appeared on. Reduced levels of red blood cells (RBC), hemoglobin (HGB) and red blood cell volume (HCT) shown in groups 2 and 3 are signs of anemia and are consistent with microscopic bleeding in lung tissue. Total with increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBI) and direct bilirubin (DBI) and cholesterol (CHO) shown in groups 2 and 3 Reduced levels of protein (TPR), albumin (ALB) are consistent with liver effects. Increased globulin (GLO) will be a microscopic finding of the response to fungal infection and / or inflammation observed in the lungs. Increased triglyceride (TRI) levels are consistent with the fat mobilization shown with the reduced food consumption and body weight observed in this study.

Visual necropsy was performed on day 14 in surviving animals as well as in sacrificed animals after dying. The number of lung infarctions per animal was counted during gross necropsy. There was no visible infarction in group 1. Seven of the eight animals in Group 2 available for autopsy (88%) had a visible lung infarction and six of these eight animals (75%) had more than 20 visible infarctions with the naked eye. In group 3, 8 out of 10 animals (80%) had visible pulmonary infarction, whereas group 2 had by contrast 2 out of 10 animals (20%) visually having at least 20 visible lung infarctions.

Microbiological evaluation of bronchoalveolar lavage fluid, blood and lung tissue samples taken at necropsy was performed. No colony forming units (CFUs) were detected in any of the samples from group 1. In group 2, culture samples were obtained from 8 out of 10 animals. There were no samples available from the two dead animals. Six of the animals evaluated (75%) had one or more tissues positive for fungi. In group 3, culture samples were obtained from 9 of 10 animals and 4 of the animals (44%) had one or more tissues positive for fungi.

Absolute lung weight as well as lung-to-weight increased in groups 2 and 3 compared to group 1.

Lung histopathological evaluation indicated that fungal mycelium was not present anywhere in the lung tissue evaluated from control rabbits. Fungal hyphae was present in 44% of Group 2 animals and 50% of Group 3 animals evaluated. There was no microscopic difference between the tissues of the group. Lung cross-sections from rabbits with fungal infections are also associated with secondary changes, including edema, necrosis, bleeding, alveolar macrophage proliferation, interstitial inflammation and / or perivascular inflammation. The changes present were similar in severity and prevalence. Microscopic evaluation of the available tissue cross sections revealed fungal infections in approximately half of the evaluated animals. There was no microscopic difference between tissue or fungal hyphae in groups 2 or 3.

In conclusion, based on mortality data, along with supporting clinical observations, microbiological data, and pulmonary infarction, amphotericin B is directed against Aspergillus fumigatus inducing mortality in the immunosuppressed rabbit model used in this study. Prophylactic effect was provided.

Study design

The experiment included three groups of 10 animals per group. Group 1 received intravenous cytarabine and methyl prednisolone immunosuppressive therapy and sham saline. Group 2 received immunosuppressive therapy and Aspergillus fumigatus of 5 × 10 ⁷ conidia. Group 3 received immunosuppressive therapy and an estimated lung dose of 1.5 mg / kg of amphotericin B one day prior to inoculation of Aspergillus fumigatus of 5 × 10 ⁷ conidia. General designs are provided in Table 3 below.

group Immunosuppressive therapy Target dose of amphotericin B (mg / kg) Aspergillus pumigatus (conidia / animal) One Yes none 0 ^a 2 Yes none 5 x 10 ⁷ 3 Yes 1.5 ^b 5 x 10 ^{7 a} inoculated with dilution (0.025% Tween 20 in 0.9% sodium chloride) ^b treated with amphotericin B on day 1

Mammal Testing Systems and Livestock

New Zealand White SPF rabbits, double intubated into the vascular passage port (VAP) and weighing 2.5-4.0 kg, were used early in the study. Rabbits were randomly assigned to treatment groups using a computerized body weight stratification procedure.

Per group  Number / gender

10 / female per group; 3 groups

Immunosuppression and Neutropenia

Immunosuppression was induced in rabbits by mimicking the conditions of human patients as shown in Table 4 below.

drug purpose Capacity level Route of administration frequency Research Cytarabine Immunosuppression 44 mg / kg IV sid -1, 1, 2, 3, 4 Cytarabine Maintaining Neutropenia 40 mg / kg IV sid 7, 8, 12, 13 Methylprednisolone Inhibit macrophage formation 5 mg / kg IV sid -1,1 Ceftazidime Bacterial infection prevention 75 mg / kg IV bid 3 to 13 Gentamicin Bacterial infection prevention 5 mg / kg IV qod 3, 5, 7, 9, 11, 13 Vancomycin Bacterial infection prevention 15 mg / kg IV sid 3 to 13 Vancomycin Bacterial infection prevention 50 mg / L Drinking water sid 3 to 13 iv = intravenous sid = once daily dose bid = twice daily dose qod = every other day

Small animal aerosol delivery system In vitro  Determination of Transmission Efficiency

U.S., incorporated herein by reference in its entirety, connected to a small animal ventilator. Dry powder amphotericin B was delivered to anesthetized rabbits via the inhalation (IH) route using an endotracheal tube connected to a small animal aerosol delivery system comprising an aerosol delivery device as disclosed in patent 6,257,233. In this regard, the powder from the blister pack was operated from the inhaler into the chamber. The small animal ventilator pushed the dispersed powder into the endotracheal tube. Delivery efficiency to animals was determined by measuring the gravimetric weight of the powder collected on a filter connected to the end of the endotracheal tube. The weight collected on the filter was normalized to the actual blister pack fill weight. Delivery efficiency was used to calculate the estimated dose (mg) for the in vivo experiment.

Processing group

The treatment regimens are summarized in Table 5 below.

Group number Test article Route of administration Average number of blister packs Target dose of AmB (mg / kg) Estimated delivered dose (mg / kg) Number / gender of animals One NA NA 0 0 0 10 / F 2 NA NA 0 0 0 10 / F 3 AmB inhale 20 1.5 0.6 10 / F

20 shows Kaplan-Meier survival curves from rabbits with neutropenia. Of the rabbits that were immunosuppressed and actually exposed to Aspergillus pumigatus 600, only 50% survived beyond 9 days. In contrast, 100% of rabbits that were immunosuppressed, exposed to Aspergillus fumigatus, and administered amphotericin B 601 survived beyond 9 days. Curve 602 represents the control group of only immunosuppressed rabbits. In the long term, less than 25% of untreated and exposed rabbits 600 survived beyond 14 days, while about 70% of treated and exposed rabbits 602 survived beyond 14 days.

Example  12

In the rabbit  Delivered by suction Amphotericin  Pharmacokinetics of B

This example includes the determination of amphotericin B concentrations in rabbit plasma, bronchoalveolar lavage (BAL) fluid and lung tissue after a single administration of amphotericin B powder by inhalation. This example describes only the results obtained in plasma and lung tissue.

Substances and Methods

Ampoterisin B (AmB) was formulated as an inhalable powder containing 50% by weight active ingredient using the method described in Example 2. The lot number of the formulation was N020042. The test powder was manually filled into the blister pack at an approximate fill weight of approximately 1.5 mg per blister pack and contact sealed for 1.0 seconds at 340 ° F.

U.S., incorporated herein by reference in its entirety, connected to a small animal ventilator. Test articles were delivered to anesthetized rabbits via an inhalation (IH) route using an endotracheal tube connected to a small animal aerosol delivery system comprising an aerosol delivery device as disclosed in Patent 6,257,233. Test inhalations were administered in a single inhalation exposure.

The study included two groups of 48 animals per group. Groups 1 and 2 each received a target dose of amphotericin B 0.25 and 1.5 mg / kg IH. Four animals were sacrificed at 0.16, 0.3, 1, 1.3, 2, 3, 4, 5, 14, 21, 28 and 45 days respectively after administration. Plasma and lung tissues were collected and analyzed for amphotericin B content using LC / MS. The limit of quantitation (LOQ) of the assay was 5 ng / ml in plasma and 20 ng / g in lung tissue.

Discussion and conclusion

The purpose of this example is to determine the concentration of amphotericin B in plasma, BAL fluid and lung tissue of rabbits after a single administration of amphotericin B powder by inhalation. Two target lung doses of 0.25 and 1.5 mg / kg were tested. This example describes only the results obtained in plasma and lung tissue.

The limit of quantitation in plasma (LOQ) was 5 ng / ml. Plasma amphotericin B concentrations of all animals at all doses tested at all time points were below LOQ. Thus, there was little systemic exposure after IH administration of amphotericin B.

However, IH administration of amphotericin B resulted in high and sustained concentrations of the drug in the lungs. Drug concentration was achieved quickly. Group mean concentrations of 4.2 and 24.2 μg / g were achieved at the first sampling time of 4 hours. T _max was observed at 32 and 24 hours after 0.25 and 1.5 mg / kg dose administration for groups 1 and 2 respectively. Group mean C _max values were 8.0 and 46.6 μg / g for 0.25 and 1.5 mg / kg doses, respectively. C _max values were proportional to dose.

Drug concentrations were maintained for several weeks after IH administration. As shown in Figures 21 and 22, the group mean amphotericin B concentration continued to rise after the last sampling time, i.e. 45 days of administration. Half-life of the new B ampo Terry removed from the lungs (t _1/2) was 254 hours with respect to 292 hours, 1.5 mg / kg dose relative to the 0.25 mg / kg dose.

The results of this example indicate that IH administration of amphotericin B to rabbits results in plasma amphotericin B concentrations that are uniformly below LOQ (<5 ng / ml). In addition, it indicates that high and sustained concentrations of amphotericin B are achieved in lung tissue.

Example  13

In the rabbit  Inhaled for prevention Amphotericin  Efficacy of B

This example includes: (1) determining whether amphotericin B (AmB) concentrations in epithelial endothelium fluid (ELF) or lung parenchyma are more appropriate in protection against Aspergillus fumigatus morbidity; And (2) establishing an effective prophylactic dose.

Substances and Methods

The amphotericin B inhalable powder lot No. 020042 of Example 14 was used. The test powder was manually filled into the blister pack at an approximate fill weight of approximately 1.5 mg per blister pack and contact sealed for 1.0 seconds at 340 ° F.

U.S., incorporated herein by reference in its entirety, connected to a small animal ventilator. Test articles were delivered to anesthetized rabbits via an inhalation (IH) route using an endotracheal tube connected to a small animal aerosol delivery system comprising an aerosol delivery device as disclosed in Patent 6,257,233. Test inhalations were administered in a single inhalation exposure.

New Zealand white SPF rabbits, double intubated into the femoral vein with a vascular passageway port and weighing about 2.5-4.0 kg, were used early in the study and were obtained from Convan's Research Products, Denver, Pa.

Aspergillus pumigatus, NIH strain 4215, was obtained from American Type Culture Collection (ATCC). Inoculation of Aspergillus fumigatus was performed on a Sabro dextrose flask. Conidia were harvested with a solution of 0.025% Tween 20 in 0.9% sodium chloride, transferred to conical tubes, washed and counted. The concentration was adjusted to the same solution to give 5 × 10 ⁷ conidia in 300 μl. Animals of the tested groups were inoculated in the same volume. Inoculation was performed on anesthetized rabbits. After rabbits were intubated using endotracheal tubes, the inoculation was administered intratracheally with a tuberculin syringe attached to a catheter introduced through the endotracheal tube.

The study included six groups of 10 rabbits per group. These groups are listed in Table 6 below. All groups were immunosuppressed and treated with antibiotics to prevent bacterial infection. Immunosuppressive and antibiotic cover therapies are described in detail in Table 7 below. Group 1 was not inoculated with Aspergillus fumigatus and was not treated with amphotericin B and served as a control for immunosuppressive therapy. Group 2 was inoculated with Aspergillus fumigatus but was not treated with amphotericin B. Group 3 was treated with amphotericin B 1.5 mg / kg 12 days prior to inoculation of Aspergillus fumigatus. Group 4 was treated with 0.15 mg / kg of amphotericin B 1 day prior to inoculation of Aspergillus fumigatus. Group 5 was treated with amphotericin B 0.5 mg / kg 1 day prior to inoculation of Aspergillus fumigatus. Group 6 was treated with amphotericin B 1.5 mg / kg 1 day prior to inoculation of Aspergillus fumigatus.

group Explanation Immunosuppression Aspergillus fumigatus vaccination AmB treatment (mg / kg) AmB Treatment (Days) One Immunosuppression Non-Vaccination Yes no 0 NA ^* 2 Immunosuppression / Non-treatment Yes Yes 0 NA 3 Immunization vaccination / 12 days high dose Yes Yes 1.5 12 days 4 Immunosuppression vaccination per day low dose Yes Yes 0.15 1 day 5 Immunosuppression vaccination / 1 day medium dose Yes Yes 0.5 1 day 6 Immunosuppression vaccination per day high dose Yes Yes 1.5 1 day ^* NA = not available

drug purpose Dose level (mg / kg) Route of administration frequency Study day ^B Cytarabine Immunosuppression 44 mg / kg iv sid -1,1,2,3,4 Cytarabine Maintaining Neutropenia 40 mg / kg iv sid 7,8,12,13 Methylprednisolone Inhibiting macrophage function 5 iv sid -1,1 Ceftazidime Bacterial infection prevention 75 iv bid 3 13 Gentamicin Bacterial infection prevention 5 iv qod 3,5,7,9,11,13 Vancomycin Bacterial infection prevention 15 iv sid 3 13 Vancomycin Bacterial infection prevention 50 mg / L ^A Drinking water sid 3 13 Amphotericin Treatment treatment Low, medium, high capacity inhale sid -12 or -1 A: mg / liter of drinking water B: Study 1 day = day inoculated with Aspergillus fumigatus

result

Survival data is shown in FIG. 23. Group 1 was immunosuppressed but not treated with amphotericin B and not inoculated with Aspergillus fumigatus. All rabbits except one of the group 1 rabbits survived, and a 14-day study concluded a 90% survival rate.

Group 2 was immunosuppressed but not inoculated with amphotericin B but inoculated with Aspergillus fumigatus. All rabbits in group 2 died and the survival rate was 0%. Median survival time was 6 days.

Group 3 was immunosuppressed and treated with amphotericin B 1.5 mg / kg at −12 days and inoculated with Aspergillus fumigatus. All rabbits in group 3 died and the survival rate was 0%. Median survival time was 10 days.

Group 4 was immunosuppressed, treated with 0.15 mg / kg of amphotericin B on day −1 and inoculated with Aspergillus fumigatus. Nine rabbits in group 4 died and had a 10% survival rate. Median survival time was 9 days.

Group 5 was immunosuppressed, treated with amphotericin B 0.5 mg / kg on day −1 and inoculated with Aspergillus fumigatus. Nine rabbits in group 5 died and had a 10% survival rate. Median survival time was 10.5 days.

Group 6 was immunosuppressed, treated with amphotericin B 1.5 mg / kg on day −1 and inoculated with Aspergillus fumigatus. Nine rabbits in group 6 died and had a 10% survival rate. Median survival time was 10 days.

A statistical comparison of the paired group of 2 vs 3, 2 vs 4, 2 vs 5 and 2 vs 6 using the logrank test showed that inhaled amphotericin B was significantly (P) inoculated with Aspergillus fumigatus. <0.001) to impart protection. The comparison of paired groups 3, 4, 5 and 6 using the same test did not show any differences in the survival curves obtained from the different treatments.

Discussion and conclusion

This example tested the protective effect of inhaled amphotericin B on immunosuppressed rabbits inoculated with Aspergillus pumigatus spores. Comparison of the survival curves of group 2 treated with aspergillus fumigatus but not treated group 2 showed that treatment by inhalation of amphotericin B had a protective effect in immunosuppressed rabbits. All doses were tested (0.15, 0.5 and 1.5 mg / kg). Rabbits were treated 1 or 12 days prior to Aspergillus fumigatus inoculation.

Example  14

In the rat  1 month inhalation toxicity study

This example includes evaluating the toxicity of a dry powder formulation of amphotericin B in rats after inhalation administration and the reversibility assessment of any effects after approximately one month recovery period. This example includes studies of locally induced toxicity and systemic toxicity in the airways.

Ampoterisin B powder containing 50% by weight of active ingredient was formulated using the method described in Example 2. The obtained powder has the characteristics as shown in Table 8 below.

API lot number Mfg. API, Form% API particle size, x50 (μm) Powder Lot Number Powder, Crystalline% Powder MMAD, Gravity (μm) Powder MMAD, Drug Specificity (μm) 101677 Khemwet 90 1.25 4001T 95 2.2 2.3

Rats (198 M and 198 F) were divided into six dose groups and treated as shown in Table 9 below.

Capacity group / processing Daily target formulation dose level (mg / kg / day) Target Agent Dose Levels at 8, 15, 22, and 29 (mg / kg / day) Number of animals cock female Air control 0 0 Main research toxicology 101-110 111-128 129-138 139-156 Vehicle control 80 80 Main study recovery toxicology 201-210 211-216 217-234 235-244 245-250 251-268 Low dose 2* 0.4 * Main study recovery toxicology 301-310 311-316 317-334 335-344 711, 346-550 351-368 Medium capacity I 10 * 2* Main study recovery toxicology 401-409,701 411-416 417-434 435, 712, 437-444 445-450 451-468 Medium capacity II 30 * 6 * Main study recovery toxicology 501-510 511-516 517-534 535-540, 713, 542-544 545-550 551-568 High capacity 80 * 80 * Main study recovery toxicology 601-610 611-616 617-634 635-644 645-650 651-668 * Dry powder formulation recovery animals containing about 50% by weight amphotericin B were maintained for approximately one month recovery period after administration. Animal 410M died prematurely at −1 days and was replaced with 701M. After preliminary optoscopic examination, animals 345F, 436F and 541F were replaced with 711F, 712F and 713F, respectively.

Animals were administered weekly in 5 runs for about 60 minutes using the technique of exposing the nose only. Recovery animals were maintained for approximately one month recovery period.

The following investigations were performed: clinical observation, body weight, food consumption, ophthalmology, hematology, clinical chemistry, urinalysis, histopathology and toxicology.

All group average exposure chamber concentrations of the vehicle formulation aerosol were 2.35 mg / L. All group mean exposure chamber concentrations of amphotericin B formulations were 0.06, 0.39 and 0.91 mg / L per day for groups 3, 4 and 5, respectively, and 0.02, 0.06 and 0.16 mg / L the next day. All group mean exposure chamber concentrations (analytically determined) of amphotericin B alone were 0.0290, 0.152 and 0.389 mg / L per day for groups 3, 4 and 5, respectively, and 0.00847, 0.0214 and 0.0519 mg / L the following day. It was. For group 6, all group average exposure chamber concentrations of amphotericin B formulation were 2.22 mg / L or 0.694 mg / L for amphotericin B alone.

All (gender combined) group mean estimated dose of vehicle formulation achieved was 73.56 mg / kg / dose. All (gross) group mean estimated doses of amphotericin B preparation achieved are 1.89, 12.27 and 28.45 mg / kg per day for groups 3, 4 and 5, respectively, 0.528, 1.74 and 4.87 mg / day kg / day. All (gross) group mean estimated doses (analytically determined) of amphotericin B alone were 0.914, 4.78 and 12.36 mg / kg per day for groups 3, 4 and 5, respectively, 0.256, 0.646 and 1.58 mg / kg. For group 6, all (gross) group mean estimated doses of amphotericin B preparations were 68.24 mg / kg / dose or 21.64 mg / kg / dose for amphotericin B alone.

The particle size distribution data shows that 64.7% of the vehicle aerosol particles are less than 3.5 μm, have a mass median aerodynamic diameter (MMAD) (± geometric standard deviation (GSD)) of 1.61 μm (3.244), and 81.2 of the amphotericin B formulation microparticles. %, 60.6% and 70.7% are less than 3.5 μm per day, 68.2%, 59.0% and 83.6% are less than 3.5 μm the next day and MMAD (GSD) is 1.35 per day for groups 3, 4 and 5 respectively. It was shown that they were 1.82 μm (2.382), 1.46 μm (4.508) and 1.43 μm (2.560), and the following day were 1.42 μm (3.572), 1.89 μm (2.710) and 0.81 μm (4.329).

For amphotericin B alone (analytically determined), 68.1%, 58.1% and 68.6% of the particulates were less than 3.5 μm per day, 66.2%, 55.6% and 71.1% were less than 3.5 μm the next day, group 3 MMAD (GSD) is 1.69 μm (2.755), 1.71 μm (3.351) and 1.55 μm (2.652) per day for, 4 and 5 respectively, and 1.47 μm (3.599), 2.29 μm (2.525) and 1.06 μm the next day (6.077). For group 6, particle size data showed that 57.8% of the amphotericin B preparation microparticles were less than 3.5 μm and the MMAD (± GSD) was 2.11 μm (3.089). For amphotericin B alone (analytically determined), 54.3% of the microparticles were less than 3.5 μm and MMAD (± GSD) was 2.29 μm (2.966).

Particulates were considered respirable to the test species.

There were no adverse effects on body weight, food consumption, ophthalmology, hematology, clinical chemistry or organ weight.

There were 181 deaths during the study, most of which were associated with signs of clinical symptoms of dyspnea, including wheezing, rupture and shortness of breath, which was observed in animals in all amphotericin B treated groups. One died before the treatment, so it was not considered to be related to the treatment.

Autopsy findings associated with administration of amphotericin B include weight, adhesions, abnormal contents, elevated areas / lesions, light lesions, hardening, as well as dark lesions and dark, discolored or cavernous lungs. Abnormal contents appeared in the trachea or bronchus of two Group 6 (high dose) animals. After approximately one month recovery period, there was lung adhesion in one female group 3 (low dose) female animal.

Microscopically, findings in the lungs of amphotericin B-administered animals include pneumonia, pleurisy, lung lobe collapse, luminal exudates in bronchial / bronchioles, bronchial mucosa, increased inflammatory cell infiltration, alveolar inflammation and eosinophil infiltration, Includes congestion / bleeding. Pneumonia / pleuritis, collapse or alveolar inflammation was found in animals 17/110 of groups 3 to 6 in the main study.

Autopsy and histological findings in the lung, especially adhesions and pleurisy; There was a correlation between weight, cure, elevated area / lesion or abnormal contents and pneumonia. Secondary to pneumonia, other findings include inflammation-associated necrosis in the spleen and liver, increased granulocytosis in the sternum and femur, atrophy of lymphoid tissue, peripheral hepatocellular fears and diffuse adrenal cortex hypertrophy.

There was no significant difference in the incidence or severity of these findings between the amphotericin B treated groups.

After approximately one month recovery period, minimal or mild organ mucosal hypertrophy was still present in some animals administered amphotericin B. In the recovery study, the lungs of one of 18 animals in groups 3 and 4, ie animal 344, had chronic inflammation and pleurisy. Inflammation was characterized by the accumulation of lymphocyte lesions and pigmented macrophages.

Toxicity analysis indicated that the maximum plasma concentration generally increased with the dose and generally reached within 4 hours of discontinuation. No quantifiable levels of amphotericin B were detected in the air or vehicle control samples.

In conclusion, nasal administration of amphotericin B only at doses up to 21.64 mg / kg / dose is a severe clinical symptom of respiratory distress in many animals in all amphotericin B treated groups, including all animals in groups 5 and 6. Symptoms have led to signs and premature discontinuation. Inhaled administration of amphotericin B can be performed at all doses by organ mucosal hypertrophy associated with luminal exudate and / or diffuse inflammatory cell infiltration; And pneumonia, pleurisy, lung lobe collapse, luminal exudates in bronchioles, bronchial mucosal hypertrophy and increased inflammatory cell infiltration, alveolar inflammation and eosinophil infiltration, as well as lungs with congestion / bleeding. After approximately one month recovery period, the incidence and severity of tracheal mucosal hypertrophy and lung inflammation was reduced.

Example  15

Daily unit  or Main unit  By administration In the rat  Dry Powder Toxicology Research

This example involves determining the toxicity of two different dry powder batches of amphotericin B in rats after inhalation administration using two different dosing regimens. The information obtained was used to select dose levels and / or dose regimens for subsequent toxicity studies.

Ampoterisin B powder containing 50% by weight of active ingredient was formulated using the method described in Example 2. The powder obtained has the characteristics as shown in Table 10 below. In addition, as shown in Figure 24, the powder is U. S., incorporated herein by reference in its entirety. Application No. Inhaler devices as shown in 10 / 298,177 were used to make heterogeneity as measured in the drug-specific aerosol particle size versus gravity particle size analysis.

API lot number Mfg. API, Form% API particle size, x50 (μm) Powder Lot Number Powder, Crystalline% Powder MMAD, Gravity (μm) Powder MMAD, Drug Specificity (μm) 100370 Alfama 11 NA 4001T NA NA 2.8 100581 Alfama 48 3.53 4019T 56 2.3 2.8 NA = not available

Ninety male rats were divided into ten dose groups and treated as shown in Table 11 below.

Capacity group / process Daily target agent dose level (mg / kg) 2-day and 3-day formulation dose levels (mg / kg) 8- and 15-day formulation dose levels (mg / kg) Number of animals Vehicle Control 80 80 N / A 1-9 2. Low volume ^A 30 * 6 * N / A 10-18 3. High capacity ^A 80 * 80 * N / A 19-27 4. Low volume 2 ^B 30 * 6 * N / A 28-36 5. High capacity 2 ^B 80 * 80 * N / A 37-45 6. Vehicle Control 2 80 N / A 80 46-54 7. Low volume 3 ^A 30 * N / A 6 * 55-63 8. High capacity 3 ^A 80 * N / A 80 * 64-71,91 9. Low volume 4 ^B 30 * N / A 6 * 73-81 10. High capacity 4 ^B 80 * N / A 80 * 82-90 * = Dry powder formulation containing 50% by weight amphotericin B A = amphotericin B administered using lot number 4001T B = amphotericin B administered using lot number 4019T N / A = not available None Animal 72 (Group 9) was replaced with Animal 91 before initiation of administration.

Animals were administered once per day for 1 hour according to Table 11 using a technique to expose the nose only.

The following investigations were performed: clinical observation, body weight, hematology, clinical chemistry, organ weight, histopathology, toxicology and bioanalytical chemistry.

All group mean exposure concentrations of the vehicle formulation aerosol were 2.23 and 2.11 mg / L for groups 1 and 6, respectively. All group mean exposure chamber concentrations of amphotericin B formulations were 1.01, 0.88, 1.01 and 0.88 mg / L per day for groups 2, 4, 7 and 9 respectively, and 0.18, 0.17, 0.17 and 0.18 mg / day the following day. It was L. All exposure chamber concentrations of amphotericin B formulations were 2.25, 2.33, 2.51 and 2.41 mg / L for groups 3, 5, 8 and 10, respectively.

All group mean exposure chamber concentrations (analytically determined) of amphotericin B alone were 0.353, 0.306, 0.353 and 0.306 mg / L per day for groups 2, 4, 7 and 9, respectively, 0.0684, 0.0598, 0.0615 and 0.0783 mg / L. All group mean exposure chamber concentrations (analytically determined) of amphotericin B alone were 0.856, 0.927, 0.963 and 0.987 mg / L for groups 3, 5, 8 and 10 respectively.

All group mean estimated doses of vehicle formulation were 72.79 and 66.34 mg / kg / day for Groups 1 and 6, respectively. All group mean estimated doses of amphotericin B preparations were 33.44, 29.14, 33.44 and 28.95 mg / kg per day for groups 2, 4, 7 and 9, respectively, 2.25, 5.55, 5.22 and 5.49 the following day mg / kg. All group mean estimated doses of amphotericin B preparations were 74.85, 75.00, 77.81 and 77.41 mg / kg / dose for groups 3, 5, 8 and 10 respectively.

All group mean estimated doses (analytically determined) of amphotericin B alone were 11.69, 10.13, 11.69 and 10.07 mg / kg per day for groups 2, 4, 7 and 9, respectively, and 2.25, the following day. 1.96, 1.89 and 2.38 mg / kg. All group mean estimated doses (analytically determined) of amphotericin B alone were 24.45, 29.97, 29.91 and 30.60 mg / kg for groups 3, 5, 8 and 10, respectively.

Particle distribution data shows that 69.7% and 77.6% of vehicle aerosol particles for groups 1 and 6 are less than 3.5 μm, and the mass median aerodynamic diameter (MMAD) (± geometric standard deviation (GSD)) is 1.38 μm (2.880). And 1.43 μm (2.281). 63.0%, 69.2%, 63.0% and 69.2% of the amphotericin B preparation microparticles per day were less than 3.5 μm and MMAD (GSD) was 1.36 μm (4.603), 1.54 for groups 2, 4, 7 and 9 respectively. Μm (2.502), 1.36 μm (4.603) and 1.54 μm (2.502). 77.2%, 69.8%, 71.3%, and 61.3% of the amphotericin B preparation microparticles were less than 3.5 μm and MMAD (GSD) was 0.79 μm (4.541), 1.75 μm the next day for groups 2, 4, 7 and 9 respectively. (2.455), 1.56 μm (3.618) and 2.16 μm (3.228). 69.2%, 63.4%, 64.7% and 61.8% of the amphotericin B preparation microparticles were less than 3.5 μm, MMAD (GSD) was 0.95 μm (4.344), 1.44 μm (4.312) for groups 3, 5, 8 and 10, respectively. ), 1.10 μm (4.759) and 1.52 μm (4.233).

For amphotericin B alone (analytically determined), 56.4%, 61.9%, 56.4% and 61.9% of aerosol particles per day for Groups 2, 4, 7 and 9 were less than 3.5 μm and MMAD (GSD ) 1.58 μm (3.563), 1.71 μm (3.309), 1.58 μm (3.563), and 1.71 μm (3.309). 66.8%, 57.4%, 62.0% and 63.0% of the aerosol particles were less than 3.5 μm, MMAD (GSD) was 1.09 μm (4.181), 1.95 μm (3.386), the following day for groups 2, 4, 7 and 9 respectively, 1.42 μm (3.372) and 1.83 μm (3.075).

For groups 3, 5, 8, and 10, respectively, 61.9%, 57.6%, 63.8%, and 61.5% of amphotericin B alone (analytically determined) was less than 3.5 μm, and the MMAD (GSD) was 1.30 μm (4.208) , 1.77 μm (3.782), 1.20 μm (4.608) and 1.58 μm (4.097).

Autopsy findings related to inhalation of amphotericin B include abnormal contents of the dark lesions of the lungs, dark, red or spongy lungs, trachea, and hypertrophy bronchus and cervical lymph nodes. In addition, the swollen stomach and intestines indicated that the animal died prematurely.

Histological evaluation showed diffuse mucosal hypertrophy in organs in most animals treated by inhalation with both lot numbers of amphotericin B in both dose regimens. The severity of mucosal hypertrophy was increased by the second dose regimen (amphotericin B inhalation on days 1, 8 and 15) compared to the first dose regimen (amphotericin B inhalation on days 1, 2 and 3). There was no significant difference between lot number 4001T (A) and lot number 4019T (B) in terms of incidence and severity of this finding.

Submucosal inflammatory cell infiltration and luminal exudates in the trachea were also expected by administration of amphotericin B by inhalation. These findings were mostly observed in the trachea of animals treated using the second dose regimen (amphotericin B inhalation on days 1, 8 and 15). The lower incidence of these symptoms in the first dose regimen group and the absence of an apparent dose-responsive relationship are thought to be due to the above short-term therapy (inhalation of amphotericin B on days 1, 2 and 3, removal of animals on day 4). It became.

Bronchial lumen exudates, bronchial pneumonia, and peribronchial / peribronchial inflammation or inflammatory cell infiltration were present in the lung, which was also expected by administration of amphotericin B. In animals with bronchial pneumonia, bronchial exudate was also present, and it was thought that bronchial pneumonia was caused by a secondary infection resulting from a larger airway coloration. The lower incidence of bronchial luminal exudates in the lungs in the first dose regimen was thought to be due to the above short-term therapy. The incidence of pulmonary findings was generally higher in the high dose group than in the low dose group.

Toxicity analyzes indicated that plasma concentrations increased with dose. No quantifiable level of amphotericin B was detected in the vehicle control sample.

In conclusion, nasal administration of amphotericin B only at doses up to 30.60 mg / kg / dose resulted in clinical signs of shortness of breath and / or irritation and premature death in three animals. Inhaled administration of amphotericin B is associated with diffuse mucosal hypertrophy in the trachea, submucosal inflammatory cell infiltration, luminal exudates in the trachea and bronchus, bronchial pneumonia and peribronchial / perbronchial inflammation, or inflammatory cell infiltration in the lungs at all doses. have. The severity of these findings was increased in the second dose regimen (amphotericin B inhalation on days 1, 8 and 15). This pulmonary finding was associated with an apparent dose-related increase in lung weight, which was more significant than in animals administered using the second therapy. The increase was most significant in animals treated with amphotericin B lot number 4001T.

Example  16

two  Of dry powder Daily unit  or Main unit  By administration In the rat  toxicity

This example involves determining the toxicity of two different dry powder batches of amphotericin B in rats after inhalation administration using two different dosing regimens.

Ampoterisin B powder containing 50% by weight of active ingredient was formulated using the method described in Example 8. The powder obtained has the characteristics as shown in Table 12 below.

API, Form% API particle size, x50 (μm) ABIP lot number ABIP, Crystalline% ABIP MMAD, Gravity (μm) ABIP MMAD, drug specificity (μm) 94 NA N020226 100 2.3 2.3 6 3.05 N020227 4 2.5 3.2 NA = not available

120 male rats were divided into 10 dose groups and treated as shown in Table 13 below.

Capacity group / process Daily target agent dose level (mg / kg) 2-day and 3-day formulation dose levels (mg / kg) 8- and 15-day formulation dose levels (mg / kg) Animal number / designation Vehicle Control 30 6 N / A Main research toxicology 1-6 7-12 2. Low volume ^A 10 * 2* N / A Main research toxicology 13-18 19-24 3. High capacity ^A 30 * 6 * N / A Main research toxicology 25-30 31-36 4. Low volume 2 ^B 10 * 2* N / A Main research toxicology 37-42 43-48 5. High capacity 2 ^B 30 * 6 * N / A Main research toxicology 49-54 55-60 6. Vehicle Control 2 30 N / A 6 Main research toxicology 61-66 67-72 7. Low volume 3 ^A 10 * N / A 2* Main research toxicology 73-78 79-84 8. High capacity 3 ^A 30 * N / A 6 * Main research toxicology 85-90 91-96 9. Low volume 4 ^B 10 * N / A 2* Main research toxicology 97-102 103-108 10. High capacity 4 ^B 30 * N / A 6 * Main research toxicology 109-114 115-120 * = Dry powder formulation A containing 50% by weight amphotericin B A = amphotericin B administered using lot number N020226 (solid crystal form) B = amphotericin B administered using lot number N020227 (rubber amorphous) N / A = not available. Extra animals were numbered 121-126.

Animals were administered once per day for 1 hour according to Table 13 above using techniques to expose the nose only.

The following investigations were performed: clinical observation, body weight, respiratory measurements, hematology, clinical chemistry, histopathology, toxicology and bioanalytical chemistry.

All group mean exposure chamber concentrations of the vehicle formulation were 0.91 mg / L per day and 0.22 and 0.17 mg / L the next day for groups 1 and 6, respectively. All group mean exposure chamber concentrations of amphotericin B formulations were 0.31, 0.36, 0.31 and 0.36 mg / L per day for groups 2, 4, 7 and 9, respectively, 0.056, 0.078, 0.073 and 0.088 mg / day L, 1.04, 0.94, 1.04 and 0.94 mg / L per day for groups 3, 5, 8 and 10, respectively, and 0.18, 0.21, 0.18 and 0.19 mg / L the next day.

All group mean exposure chamber concentrations (analytically determined) of amphotericin B alone were 0.125, 0.124, 0.125 and 0.124 mg / L per day for groups 2, 4, 7 and 9, respectively, 0.0166, 0.0249, 0.0277 and 0.0278 mg / L, 0.455, 0.330, 0.455 and 0.330 mg / L per day for groups 3, 5, 8 and 10 respectively and 0.0686, 0.0717, 0.0724 and 0.0687 mg / L the next day.

 All group mean estimated doses of vehicle formulation were 29.29 and 29.40 mg / kg per day for Groups 1 and 6, respectively, and 6.87 and 4.98 mg / kg the following day. All group mean estimated doses of amphotericin B preparations were 10.03, 11.63, 9.93 and 11.65 mg / kg per day for groups 2, 4, 7 and 9 respectively, and 1.80, 2.50, 2.19 and 2.66 the following day mg / kg, 33.48, 30.54, 33.83 and 30.58 mg / kg per day for groups 3, 5, 8 and 10, respectively, and 5.75, 6.76, 5.47 and 5.81 mg / kg the next day.

All group mean estimated doses (analytically determined) of amphotericin B alone were 4.04, 4.01, 4.00 and 4.01 mg / kg per day for groups 2, 4, 7 and 9, respectively, 0.53, 0.79, 0.83 and 0.84 mg / kg, 14.65, 10.72, 14.80 and 10.74 mg / kg per day for groups 3, 5, 8 and 10, respectively, and 2.19, 2.31, 2.20 and 2.11 mg / kg the next day.

Particle distribution data shows that 70.8% of vehicle aerosol particles are less than 3.5 μm per day, 77.1% and 75.6% are less than 3.5 μm the next day for groups 1 and 6, respectively, and have a mass median aerodynamic diameter (MMAD) (± Geometric Standard Deviation (GSD)) is 1.56 μm (2.832) per day and 0.84 μm (3.446) and 1.24 (2.948) the next day for groups 1 and 6, respectively, and 1 for group 2, 4, 7 and 9 respectively. 65.1%, 75.7%, 65.1% and 75.7% of the amphotericin B formulation aerosol particles were less than 3.5 μm on the following day, and 52.2%, 79.4%, 65.8% and 75.2% were less than 3.5 μm on the following day, MMAD (GSD) 1.77 μm (3.787), 2.06 μm (2.805), 1.77 μm (3.787) and 2.06 μm (2.805) per day, 2.88 μm (2.269), 1.81 μm (2.540), 1.97 μm (2.719) and 2.14 the following day It was shown that the micrometer (2.288). For groups 3, 5, 8 and 10 respectively, 72.0%, 78.9%, 72.0% and 78.9% of the amphotericin B preparation microparticles were less than 3.5 μm per day, and 68.6%, 76.0%, 72.4% and 77.5% Less than 3.5 μm the next day, MMAD (GSD) is 2.07 μm (2.368), 1.87 μm (2.464), 2.07 μm (2.368) and 1.87 μm (2.464) a day, and 1.67 μm (3.466), 1.77 μm the next day (2.378), 1.42 μm (2.965) and 1.40 μm (2.911).

For amphotericin B alone (analytically determined), 70.4%, 72.6%, 70.4% and 72.6% of the particulates were less than 3.5 μm per day, 68.0%, 75.3 for groups 2, 4, 7 and 9 respectively. %, 77.3% and 63.0% are less than 3.5 μm the next day, MMAD (GSD) is 1.80 μm (2.563), 2.06 μm (2.702), 1.80 μm (2.563) and 2.06 μm (2.702) per day, 2.05 μm (2.230), 2.01 μm (2.752), 1.99 μm (2.005) and 2.60 μm (2.463). For groups 3, 5, 8 and 10 respectively, 55.4%, 69.9%, 68.0% and 69.6% of the particulates were less than 3.5 μm per day and 67.9%, 63.6%, 67.8% and 63.3% were less than 3.5 μm the next day MMAD (GSD) is 2.74 μm (2.116), 2.03 μm (2.788), 2.05 μm (2.230) and 2.03 μm (2.788) per day, and 1.81 μm (2.428), 1.94 μm (3.310), 1.92 the following day Μm (2.402) and 2.04 μm (3.236).

Toxicity analysis showed that the plasma concentration of amphotericin B increased with dose. No quantifiable level of amphotericin B was detected in the vehicle control sample.

There was no significant autopsy finding associated with inhalation of amphotericin B.

Histological evaluation showed diffuse mucosal hypertrophy in many animals treated with both dry powder batches of amphotericin B in both dose regimens. The incidence and severity of hypertrophy caused by amphotericin B N020227 (rubber amorphous) was increased compared to amphotericin B N020226 (solid crystal). There was little increase in the incidence and severity of hypertrophy with the second dose regimen (Days 1, 8 and 15) compared to the first dose regimen (Days 1, 2 and 3).

Diffuse organ inflammation was also expected by inhalation of amphotericin B. The incidence and severity of these lesions was increased by amphotericin B N020227 (rubber amorphous) compared to amphotericin B N020226 (solid crystal). The incidence of lesions was reduced by the second dose regimen (weekly) compared to the first dose regimen (daily).

Tracheal and bronchial lumen exudates were found only in animals treated with amphotericin B N020227 (rubber amorphous).

In conclusion, the administration of amphotericin B (alone) only nasal at doses up to 14.80 mg / kg resulted in clinical signs of dyspnea. Inhalation of amphotericin B dry powder formulations N020226 (high crystalline) and N020227 (amorphous) on days 1, 2 and 3 or 1, 8 and 15 may cause tracheal and bronchial mucosal hypertrophy, tracheal inflammation, bronchial mucosal inflammatory cell infiltration and trachea And bronchial lumen exudate. The incidence and severity of these findings was increased by amphotericin B N020227 (amorphous) compared to amphotericin B N020226 (high crystalline). The incidence and severity of hypertrophy was little increased by the second dose regimen (day 1, 8 and 15) compared to the first dose regimen (day 1, 2 and 3), but the severity and incidence of organ inflammation decreased. There was little difference between the high and low dose groups treated with the first or second therapy of amphotericin B N020227 (amorphous). There was little effect of reducing severity and incidence in the low dose group treated with amphotericin B N020226 (high crystalline) in both dose regimens.

Example  17

two  Of dry powder Daily unit  or Main unit  Toxicity in Dogs by Administration

This example involves determining the toxicity of two different dry powder batches of amphotericin B in beagle dogs after weekly administration by inhalation route.

Ampoterisin B powder containing 50% by weight of active ingredient was formulated using the method described in Example 8. The obtained powder has the characteristics as shown in Table 14 below.

API, Form% API particle size, x50 (μm) Powder Lot Number Powder, Crystalline% Powder MMAD, Gravity (μm) Powder MMAD, Drug Specificity (μm) 94 NA N020226 100 2.3 2.3 6 3.05 N020227 4 2.5 3.2 NA = not available

Four male and four female beagle dogs were divided into four dose groups and treated as shown in Table 15 below.

Capacity group / process Daily target agent dose level (mg / kg) 8- and 15-day formulation dose levels (mg / kg) Animal count / gender Vehicle Control 30 6 1M, 5F 2. Low volume ^A 10 * 2* 2M, 6F 3. High capacity ^A 30 * 6 * 3M, 7F 4. High capacity ^B 30 * 6 * 4M, 8F * = Dry powder formulation A containing 50% by weight amphotericin B A = amphotericin B administered using lot number N020226 (solid crystal form) B = amphotericin B administered using lot number N020227 (rubber amorphous) box

Animals were administered for 30 minutes per day according to Table 15 above using a fitted face mask (fit to oral tube) system.

The following investigations were performed: clinical observations, body weight, food consumption, hematology, clinical chemistry, respiratory measurements (tidal volume, respiratory rate, minute tidal volume), histopathology, toxicology and bioanalytical chemistry.

All group average exposure chamber concentrations of the vehicle formulation were 1.34 mg / L per day and 0.31 mg / L the next day. All group average exposure chamber concentrations of amphotericin B formulations were 0.73, 1.80 and 1.58 mg / L per day for groups 2, 3 and 4, respectively, and 0.13, 0.33 and 0.33 mg / L the next day. All group mean exposure chamber concentrations (analytically determined) of amphotericin B alone were 0.236, 0.521 and 0.769 mg / L per day for groups 2, 3 and 4, respectively, 0.0427, 0.120 and 0.154 mg / L the following day It was.

All (gender combined) group mean estimated dose levels of the vehicle formulation were 29.3 mg / kg per day and 6.9 mg / kg the next day. All (gross) group mean estimated dose levels achieved for amphotericin B formulations are 16.3, 45.1 and 33.0 mg / kg per day for groups 2, 3 and 4, respectively, and 2.9, 8.1 and 6.7 mg / day kg / day. All (gross) group mean estimated dose levels (analytically determined) of amphotericin B alone were 5.3, 13.0 and 16.0 mg / kg per day for groups 2, 3 and 4, respectively, 0.96, 3.0 and 3.2 mg / kg.

Particle distribution data shows that 81.1% of vehicle aerosol particles are less than 5 μm per day, 78.9% are less than 5 μm the next day, and the mass median aerodynamic diameter (MMAD) (± geometric standard deviation (GSD)) is 1 day. Was 0.84 mu m (4.553), and the next day was 1.41 mu m (3.104). For groups 2, 3 and 4 respectively, 69.0%, 83.1% and 43.3% of the amphotericin B preparation microparticles are less than 5 μm per day, 85.9%, 67.6% and 57.3% are less than 5 μm the next day and MMAD (GSD) was 1.96 μm (2.406), 1.17 μm (2.725) and 3.26 μm (2.789) per day and 0.69 μm (4.623), 1.62 μm (3.137) and 2.85 μm (2.692) the next day. For amphotericin B alone (analytically determined), 57.7%, 73.6% and 46.2% of the microparticles were less than 5 μm per day and 72.6%, 63.6% and 62.8% for groups 2, 3 and 4 respectively. The next day is less than 5 μm, the MMAD (GSD) is 2.20 μm (2.644), 1.79 μm (2.378) and 3.32 μm (2.482) per day, the next day 1.96 μm (2.507), 2.00 μm (2.526) and 2.48 μm (2.168).

No significant changes were observed in body weight, hematology, clinical chemistry, food consumption or respiratory measurement parameters.

All autopsy findings were background findings that occurred spontaneously in dogs of this age. There was no autopsy finding by the administration of amphotericin B.

All histopathological findings were background findings that occurred spontaneously in dogs of this age. There was no histopathologic finding by amphotericin B administration.

Toxicity analysis revealed that quantifiable levels of amphotericin B were detected in the plasma of all treated animals (except for 4M of animals in group 4). Levels detected were comparable across all treated dose groups. No quantifiable level of amphotericin B was detected in the vehicle control sample.

In conclusion, inhalation of amphotericin B preparations (or analytically determined amphotericin B) once a week to beagle dogs at doses of up to 8.2 (3.2) mg / kg was found in all dose groups (including vehicle control). Related to saliva secretion. Ancillary clinical symptoms, including red gums, were also observed in individual animals in groups 2 and 4, and red ears were observed in one animal in group 2. Vomiting and reflux of food was observed in one animal in group 3. There were no changes observed in body weight, food consumption, hematology, clinical chemistry or respiratory profile. There was no autopsy or histopathologic finding by amphotericin B administration. Particle size distribution measurements indicated that high dose levels of amphotericin B lot number N020227 (rubber amorphous) had a significantly larger particle size than amphotericin B lot number N020226 (high crystalline).

Example  18

In the rat  14-day inhalation toxicity study

This example includes a 14-day toxicology study of amphotericin B preparations prepared using the method of Example 2. 80 rats are administered using the technique of nasal administration for 60 minutes per day for at least 14 days at a target dose of 0 mg / kg (only air control or vehicle), 2.5 mg / kg, 10 mg / kg or 25 mg / kg It was.

Restorative animals were maintained during the recovery period after 14-day administration. On days 1 and 14 of the study, blood samples for toxicology analysis were obtained from animals that were only vehicle administered and animals of the high dose (25 mg / kg) group before, after 2 hours and after 4 hours of dosing. In addition, toxicological blood samples were obtained during the 14-day recovery period on days 7 and 14 of recovery for males and only 14 days of recovery due to less body weight for females.

All study animals were necropsied at the completion date of the 14-day treatment period (15 days) or at the completion date of the 14-day recovery period (29 days). During necropsy, lung tissue samples for toxicology analysis were obtained from each animal. Ampotericin B concentration in plasma was determined using the liquid chromatography-serial mass spectrophotometer (LC-MS / MS) method (lower limit of quantitation [LLQ] = 10 ng / mL) and amphotericin in lung tissue. B concentration was determined using high performance liquid chromatography (HPLC) method and visible light detection (LLQ = 4 μg / g).

Amphotericin B was detectable in plasma and lung tissue of all sampled animals that received amphotericin B powder. Concentration-time profiles in plasma or lung were similar in both genders and were dose independent.

Ampotericin B did not accumulate somewhat in plasma after 14 days of administration and was reduced as expected by published values for amphotericin B clearance half-life after intravenous (IV) administration. Plasma C _max values were 1/1000 to 1/3000 of the comparable IV dose of liposome amphotericin B.

Lung concentration increased with increasing dose but less than proportionally. The mean lung concentration at the end of dosing was 10-30 times higher than the concentration reported for amphotericin B in the lungs after IV administration of comparable doses of liposome amphotericin B. Lung tissue exposure in this example was greater than previously reported after prolonged IV administration. During the recovery period, lung concentrations elimination half-life of 22 and 34 days (t _1/2) was decreased in proportion to the 1.1 and 12.4 mg / kg dose level, representing the value, respectively, which after IV and inhalation administration of liposomal amphotericin B Similar to that reported for the concentration of amphotericin B in the lungs.

Example  19

14-day inhalation toxicity study in dogs

This example shows pulmonary and systemic toxicity after 14 consecutive days of inhalation administration in a total of 28 dogs (14 M, 14 F) of amphotericin B preparations prepared using the method of Example 2, and 14-day Analysis of the reversibility of any effect at high dose levels after the recovery period.

The active drug was administered for 30 minutes per day using a closed face mask system at doses of 1.4, 5.6 and 11.5 mg / kg. On days 1 and 14 of the study, blood samples for toxicology analysis were taken from vehicle and high dose groups of animals daily for 2, 4, 8, 12 and 24 hours and 14-day recovery period before and after exposure. The main study animals were anesthetized and anesthetized on the 15th, and the recovered animals were anesthetized and anesthetized on the 29th, at which time the right anterior lobe of each animal was obtained for toxicology analysis. Ampotericin B concentration in plasma was determined using the liquid chromatography-serial mass spectrophotometer (LC-MS / MS) method (lower limit of quantitation [LLQ] = 10 ng / mL) and amphotericin in lung tissue. B concentration was determined using high performance liquid chromatography (HPLC) method and visible light detection (LLQ = 4 μg / g).

Ampoterisin B accumulated in plasma 14 days after administration and decreased as expected by the plasma clearance half-life values observed in this example. The mean plasma C _max value of dogs receiving 11.5 mg / kg is 1/25 of the value reported for dogs receiving IV amphotericin desoxycholate and is reported for dogs receiving IV liposome amphotericin B daily for the same period. 1/1000 of the calculated value.

Lung concentration increased with increasing dose but less than proportionally. Mean amphotericin B lung concentrations at the end of the exposure period were 22-44 times higher than reported concentrations after IV administration of amphotericin B desoxycholate or liposome amphotericin B. Lung tissue exposure in this study was greater than previously reported after prolonged IV administration. During the recovery period, lung concentration decreased at a rate that showed an apparent elimination half-life of 18.8 days, which was similar to that reported after IV and inhalation administration of liposome amphotericin B to rats.

Example  20

Drug Product Stability Study

The stability of amphotericin B powder was evaluated as shown in Table 16 below. In each case, the powder was included in the HPMC capsule. Capsules were placed in 25 cc HDPE bottles. Each bottle was placed in a double-pouch with a desiccant in the outer pouch.

Lot number Strength (% by weight) Lot Number Target Fill Weight (mg of Powder per Capsule) Data available at 2-8 ° C. (mos.) 10017 5 9.43 18 10029 50 9.52 18 10247 50 10.62 9

A summary and conclusions of these stability observations are provided below.

Exterior

All three lot numbers met the acceptance criteria for appearance at all test points and under all conditions.

content

5 wt% amphotericin B: For lot number 10017, the initial results were 0.053 mg of amphotericin B per mg of powder. Over a period of 18 months, the results ranged from 0.049 to 0.053 mg of amphotericin B per mg of powder at both storage conditions.

50 wt.% Amphotericin B: For lot number 10029, the initial results were 0.507 mg of amphotericin B per mg of powder. Over a period of 18 months, the results ranged from 0.469 to 0.508 mg of amphotericin B per mg of powder under all storage conditions.

The initial result for lot number 10247 was 0.470 mg of amphotericin B per mg of powder. After 9 months storage at 2-8 ° C., the result was 0.448-0.446 mg of amphotericin B per mg of powder. After 1 month, 3 months and 6 months storage at 25 ° C./60%RH, they were 0.441, 0.399 and 0.385 mg of amphotericin B per mg of powder, respectively.

humidity

Initial results for three lot numbers ranged from 3 to 5 weight percent. The water content ranged from 3 to 6 weight percent after storage at both normal and accelerated storage conditions.

Radiation capacity

Initial mean ED for all lot numbers ranged from 89 to 94% (8.9 mg / capsule to 10.0 mg / capsule). The results after storage at normal and accelerated conditions ranged from 88 to 96% (8.8 to 10.2 mg / capsule).

Residue Purple Flubron  ( PFOB )

PFOB analysis was performed on the bulk powder before filling into capsules. The result for bulk powders from all lot numbers tested was <0.1 wt%. PFOB was tested at lots 12 months for lot numbers 10017 and 10029. The level was <0.05 to 0.1 wt%.

Aerobic modulus and specific pathogens

The microbial limit test was performed at the initial test point for all lot numbers. In addition, tests were performed after 12 months storage at 25 ° C./60%RH for Lot No. 10017 and 10029 and met acceptance criteria.

water

Initial region normalized main peak purity for all lot numbers ranged from 93.6 to 96.4%. During storage at normal and accelerated conditions, the main peak purity ranged from 94.0 to 96.5%.

Aerosol granularity

MMAD: Initial results of the mass median aerodynamic diameter (MMAD) for lot numbers ranged from 2.7 to 3.0 μm. During storage at normal and accelerated conditions, MMAD ranged from 2.6 to 3.1 μm.

conclusion

The stability results presented herein indicate that 5% by weight amphotericin B powder (10 mg) and 50% by weight amphotericin B powder (10 mg) are stable, appearance upon packaging and storage at 2-8 ° C., amphotericin B It has been shown to exhibit acceptable product properties including content, purity, water content, microbial properties and aerosol performance.

Example  21

Performance after exposure to high humidity

This example includes defining a suitable time period during which the capsule can be removed from the pouch at 70% RH and still meet the radiation dose acceptance criteria (% ED ≧ 85% and ED ≧ 8.5 mg).

Two formulations were tested: 5 wt% amphotericin B powder (lot no. X1429) and 50 wt% amphotericin B powder (lot no. N020242). The formulation is contained in a capsule contained in the pouch. In order to determine ED, U.S. Pat. An inhaler device as shown in application number 10 / 298,177 was used. The device reached equilibrium at 25 ° C./70% RH.

Capsules were exposed to 25 ° C./70% RH prior to specific intervals and runs. ED was measured at 10 time points for a total of 10 ED runs per formulation, as shown in Table 17 below.

The results for lot number N020242 are shown in Table 18 below.

The results for lot number X1249 are shown in Table 19 below.

The data indicate no correlation between increased exposure to high humidity and ED performance. Average ED performance decreases under high humidity environments but still meets acceptance criteria. The average ED performance of lot No. 020042 (50 wt% formulation) met the acceptance criteria, but all individual EDs were not ≧ 85% and ≧ 8.5 mg.

selected Example  summary

A summary of the selected examples is shown in Table 20 below.

Example Species (number / group) Average inhaled dose level of the formulation dose of the powder formulation (dose of active drug) Significant Findings 14 Rat lOM / lOF 5M / 5F (recovery) 18M / 18F (TK) Daily dose level 8, 15, 22 and 29 days 0 (air control) 0 (air control) none 73.6 (O; Vehicle) mg / kg 73.6 (O; Vehicle) mg / kg None [Recovery: None] 1.9 (0.9) mg / kg [low dose] 0.5 (0.26) mg / kg About 35% morbidity or mortality by day 28. Clinical symptoms: Respiratory effect (wheezing, shortness of breath). Visual autopsy observation: Lung: adhesions, lesions (dark), discoloration or spongy lungs. Histopathology: organ; Minimal-to-moderate mucosal hypertrophy, inflammatory cell infiltration. lungs; Inflammatory cell infiltration, congestion / bleeding. [Recovery: Minimal tracheal mucosa] 12.3 (4.8) mg / kg [mid-1 dose] 1.7 (0.65) mg / kg About 26% morbidity or mortality by 28 days. Clinical symptoms: Respiratory effect (wheezing, shortness of breath). Visual autopsy observation: Lung: focal (dark), sclerotic, discolored or cavernous lung. Histopathology: organ; Minimal-to-moderate mucosal hypertrophy, inflammatory cell infiltration and luminal exudates. lungs; Moderate bronchial mucosa, luminal exudate, congestion / bleeding, pleurisy and pneumonia. [Recovery: Histopathology: Minimal tracheal mucosa]

Example Species (number / group) Average inhaled dose level of the formulation dose of the ABIP formulation (dose of active drug) Significant Findings 14 Rat lOM / lOF 5M / 5F (recovery) 18M / 18F (TK) Daily dose level 8, 15, 22 and 29 days 28.5 (12.4) mg / kg [mid-2 dose] 4.9 (1.6) mg / kg 100% morbidity or mortality by day 19. Clinical symptoms: Respiratory effect (wheezing, shortness of breath). Visual autopsy observation: Lungs: abnormal contents, lesions (light or dark), discoloration and cavernous lungs. Mucous membrane in the trachea. Histopathology: organ; Minimal-to-moderate mucosal hypertrophy. lungs; Minimal-to-moderate bronchial mucosa, lumen exudate, inflammatory cell infiltration, congestion / bleeding, pleurisy and pneumonia. [Recovery: No Surviving Animal] 68.2 (21.6) mg / kg [high dose] 68.2 (21.6) mg / kg 100% morbidity or mortality by 20 days. Clinical symptoms: Respiratory effect (wheezing, shortness of breath). Visual autopsy observation: Lungs: abnormal contents, lesions (light or dark), discoloration and cavernous lungs. Histopathology: organ; Minimal-to-moderate mucosal hypertrophy. Inflammatory cell infiltration and luminal exudates. lungs; Minimal-to-moderate bronchial mucosa, lumen exudate, inflammatory cell infiltration, congestion / bleeding, pleurisy and pneumonia. [Recovery: No Surviving Animal]

Example Species (number / group) Average inhaled dose level of the formulation dose of the ABIP formulation (dose of active drug) Significant Findings 15 Rat lOM Co., Ltd. Daily dose level 2 and 3 days (daily) or 8 and 15 days (weekly) 72.8 (O; Vehicle) 72.8 (O; Vehicle) none Lot number 4001T; 33.4 (11.7) mg / kg Lot number 4001T; 5.6 (2.1) mg / kg One animal in the daily X3 dose group was dying (day 2). Clinical symptoms of respiratory effect (mild-to-severe wheezing or lung sounds). Inflated stomach and intestines in dying animals. Abnormal contents of the trachea. Lungs with dark lesions, red lungs. Histopathology: organ; Minimal-to-mild mucosal hypertrophy with submucosal inflammatory cell infiltration and luminal exudates. lungs; Peribronchial / peribronchial inflammation, alveolar macrophage accumulation and congestion / bleeding. Lot number 4001T; 73.4 (27.2) mg / kg Lot number 4001T; 73.4 (27.2) mg / kg One animal in the daily X3 dose group was dying (day 2) and one animal in the X3 group was found dead each day (day 8). Clinical symptoms of respiratory effect (mild-to-severe wheezing or lung sounds). Inflated stomach and intestines in dying animals. Lungs with dark lesions. Histopathology: organ; Minimal-to-moderate diffuse mucosal with submucosal inflammatory cell infiltration and luminal exudates. lungs; Bronchial exudate, peribronchial / peribronchial inflammation, alveolar macrophage accumulation, congestion / bleeding and bronchial pneumonia.

Example Species (number / group) Average inhaled dose level of the formulation dose of the powder formulation (dose of active drug) Significant Findings 15 Rat lOM Co., Ltd. Daily dose level 2 and 3 days (daily) or 8 and 15 days (weekly) Lot number 4019T; 29.1 (10.9) mg / kg Lot number 4019T; 5.6 (2.2) mg / kg Clinical symptoms of respiratory effect (mild-to-severe wheezing or lung sounds). Abnormal contents of the trachea. Lungs with dark lesions. Histopathology: organ; Minimal-to-mild mucosal hypertrophy with submucosal inflammatory cell infiltration and luminal exudates. lungs; Peribronchial / peribronchial inflammation, alveolar macrophage accumulation and congestion / bleeding. Lot number 4019T; 76.2 (30.3) mg / kg Lot number 4019T; 76.2 (30.3) mg / kg Clinical symptoms of respiratory effect (mild-to-severe wheezing or lung sounds). Abnormal contents of the trachea. Lungs with red lesions, reddened or spongy lungs. Histopathology: organ; Minimal-to-mild mucosal hypertrophy with submucosal inflammatory cell infiltration and luminal exudates. lungs; Peribronchial / peribronchial inflammation, alveolar macrophage accumulation and congestion / bleeding and bronchial pneumonia.

Example Species (number / group) Average inhaled dose level of the formulation dose of the powder formulation (dose of active drug) Significant Findings 16 Rat 6M 6M (TK) Daily dose level 2 and 3 days (daily) or 8 and 15 days (weekly) Vehicle; 29.4 (0) mg / kg Vehicle; 29.4 (0) mg / kg none Lot number N020226; 10.0 (4.0) mg / kg Lot number N020226; 2 (0.7) mg / kg None (weekly therapy) Lot number N020226; 33.7 (14.8) mg / kg Lot number N020226; 5.7 (2.2) mg / kg Trachea, minimal diffuse mucosa and minimal inflammation (daily and weekly therapy) Lot number N020227; 11.7 (4.0) mg / kg Lot number N020227; 2.6 (0.8) mg / kg Bronchobronchial, minimal-to-mild diffuse mucosal hypertrophy, minimal-to-mild inflammation and exudate (daily and weekly therapy) Lot number N020227; 30.6 (10.7) mg / kg Lot number N020227; 6.3 (2.2) mg / kg Bronchobronchial, minimal-to-mild diffuse mucosal hypertrophy, minimal-to-mild inflammation and exudate (daily and weekly therapy) NOEL for lot number N020226 = 10 mg / kg per day and 2 mg on days 8 and 15 / kg single dose regimen

Example Species (number / group) Average inhaled dose level of the formulation dose of the powder formulation (dose of active drug) Significant Findings 17 Beagle 4F Daily dose level 8 and 15 days Vehicle; 29.3 (0) mg / kg Vehicle; 6.9 (0) mg / kg none Lot number N020226; 16.3 (5.3) mg / kg Lot number N020226; 2.9 (1.0) mg / kg none Lot number N020226; 45.1 (13.0) mg / kg Lot number N020226; 8.1 (3.0) mg / kg none Lot number N020227; 33.0 (16.0) mg / kg Lot number N020227; 6.7 (3.2) mg / kg none NOEL; Lot number N020226; = Single dose therapy NOEL of 45.1 (13.0) mg / kg on day 1 and 8.1 (3.0) mg / kg on days 8 and 15; Lot number N020227; = Single dose regimen of 33.0 (16.0) mg / kg per day and 6.7 (3.2) mg / kg on days 8 and 15

While various embodiments of the invention have been described in considerable detail with respect to certain preferred variations thereof, other variations are possible and alternatives, modifications, and equivalents of the variations shown will become apparent to those skilled in the art upon reading the specification and studying the drawings. . For example, the relative position of the components of the aerosolization device can be changed, and the hinges that mimic the action of the flexible parts can be replaced by flexible or movable stiff parts. In addition, the path need not be substantially linear as shown in the figure and may be curved or oblique, for example. In addition, the various features of the modifications herein can be combined in various ways to provide further embodiments of the invention. Also, specific terms have been used for clarity of description and are not intended to limit the invention. Therefore, the appended claims submitted for this disclosure should not be limited to the description of the preferred variations contained herein, but should include all such alternatives, modifications, and equivalents falling within the spirit and scope of the invention.

Claims (355)

At least about 95% by weight of a particle comprising amphotericin B, wherein the particle has a median diameter of about 1.1 μm to about 1.9 μm. The composition of claim 1 wherein the median mass diameter is from about 1.2 μm to about 1.8 μm. The composition of claim 1, wherein the geometric diameter of at least about 80% by weight of the particles is from about 1.1 μm to about 1.9 μm. The composition of claim 1, wherein the geometric diameter of at least about 90% by weight of the particles is from about 1.1 μm to about 1.9 μm. The composition of claim 1, wherein the crystallinity level of amphotericin B is at least about 20%. The composition of claim 1, wherein the crystallinity level of amphotericin B is at least about 70%. The composition of claim 1, wherein the crystallinity level of amphotericin B is at least about 90%. The composition of claim 1, wherein the crystallinity level of amphotericin B is from about 50% to about 99%. At least about 95 weight percent of particles comprising amphotericin B, wherein at least about 80 weight percent of the particles have a geometric diameter of from about 1.1 μm to about 1.9 μm. The composition of claim 9, wherein the geometric diameter of at least about 90% by weight of the particles is from about 1.1 μm to about 1.9 μm. The composition of claim 9 wherein the median mass diameter of the particles is from about 1.1 μm to about 1.9 μm. The composition of claim 9, wherein the crystallinity level of amphotericin B is at least about 20%. The composition of claim 9, wherein the crystallinity level of amphotericin B is at least about 70%. The composition of claim 9, wherein the crystallinity level of amphotericin B is at least about 90%. The composition of claim 9, wherein the crystallinity level of amphotericin B is from about 50% to about 99%. A particle comprising amphotericin B, wherein the particle has a median diameter of less than about 1.9 μm and the crystallinity level of amphotericin B is at least about 20%. The composition of claim 16 wherein the median mass diameter is from about 0.5 μm to about 1.8 μm. The composition of claim 16, wherein the geometric diameter of at least about 80% by weight of the particles is less than about 3 μm. The composition of claim 16, wherein the geometric diameter of at least about 90% by weight of the particles is less than about 3 μm. The composition of claim 16, wherein the crystallinity level of amphotericin B is at least about 70%. The composition of claim 16, wherein the crystallinity level of amphotericin B is at least about 90%. The composition of claim 16, wherein the crystallinity level of amphotericin B in the particles is about 50% to about 99%. A pharmaceutical composition comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of about 1.1 μm to about 1.9 μm. The pharmaceutical composition of claim 23, wherein the composition comprises amphotericin B having a median mass diameter of about 1.2 μm to about 1.8 μm. The composition of claim 23, wherein the geometric diameter of at least about 80% by weight of the particles is from about 1.1 μm to about 1.9 μm. The composition of claim 23, wherein the geometric diameter of at least about 90% by weight of the particles is from about 1.1 μm to about 1.9 μm. The pharmaceutical composition of claim 23, wherein the crystallinity level of amphotericin B is at least about 50%. The pharmaceutical composition of claim 23, wherein the crystallinity level of amphotericin B is at least about 70%. The pharmaceutical composition of claim 23, wherein the crystallinity level of amphotericin B is at least about 80%. The pharmaceutical composition of claim 23 comprising the microparticles. The pharmaceutical composition of claim 30, wherein the microparticles have a median mass aerodynamic diameter of about 1 μm to about 6 m. The pharmaceutical composition of claim 23, wherein at least about 80% by weight comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 23 comprising microparticles having a mass median aerodynamic diameter of less than about 10 μm, wherein the crystallinity level of amphotericin B is at least about 70%. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable excipient comprises phospholipids. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable excipient comprises metal ions. The pharmaceutical composition of claim 23, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. The pharmaceutical composition of claim 23 comprising hollow and / or porous particulates. The pharmaceutical composition of claim 23 comprising microparticles comprising amphotericin B in a matrix formed by a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 39, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. The pharmaceutical composition of claim 23 comprising a dry powder. The pharmaceutical composition of claim 23 further comprising a propellant. The pharmaceutical composition of claim 42, wherein the propellant comprises one or more hydrofluoroalkanes. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition comprises particulates having a mass median aerodynamic diameter of about 1 μm to about 6 μm, wherein the crystallinity level of amphotericin B is at least about 70% and at least about 80% by weight. The composition comprises a microparticle comprising amphotericin B in a matrix comprising a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. A pharmaceutical composition comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B, wherein at least about 80% by weight of the particles comprising amphotericin B Pharmaceutical compositions having a geometric diameter of about 1.1 μm to about 1.9 μm. The composition of claim 45, wherein the geometric diameter of at least about 90% by weight of the particles is from about 1.1 μm to about 1.9 μm. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of about 1.2 μm to about 1.8 μm. 46. The pharmaceutical composition of claim 45, wherein the crystallinity level of amphotericin B is at least about 50%. 46. The pharmaceutical composition of claim 45, wherein the crystallinity level of amphotericin B is at least about 70%. 46. The pharmaceutical composition of claim 45, wherein the crystallinity level of amphotericin B is at least about 80%. 46. The pharmaceutical composition of claim 45, comprising microparticles. The pharmaceutical composition of claim 51, wherein the particulate median aerodynamic diameter is from about 1 μm to about 6 m. 46. The pharmaceutical composition of claim 45, wherein at least about 80% by weight comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition comprises particulates having a mass median aerodynamic diameter of less than about 10 microns, wherein the crystallinity level of amphotericin B is at least about 70%. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutically acceptable excipient comprises phospholipids. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutically acceptable excipient comprises metal ions. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 46. The pharmaceutical composition of claim 45, comprising hollow and / or porous particulates. 46. The pharmaceutical composition of claim 45 comprising microparticles comprising amphotericin B in a matrix formed by a pharmaceutically acceptable excipient. 61. The pharmaceutical composition of claim 60, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. 46. The pharmaceutical composition of claim 45, comprising a dry powder. 46. The pharmaceutical composition of claim 45, further comprising a propellant. 64. The pharmaceutical composition of claim 63, wherein the propellant comprises one or more hydrofluoroalkanes. 46. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of about 1 μm to about 6 μm, wherein the crystallinity level of amphotericin B is at least about 70% and at least about 80% by weight. A pharmaceutical composition comprising microparticles comprising amphotericin B in a matrix comprising this pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises at least one phospholipid. Effective amount of amphotericin B; And a pharmaceutically acceptable excipient, wherein the crystallinity level of amphotericin B is from about 20% to about 99%. 67. The pharmaceutical composition of claim 66, wherein the crystallinity level of amphotericin B is from about 70% to about 98%. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 3 μm. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 2 μm. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B and has a geometric diameter of at least about 80% by weight of the particles comprising amphotericin B of less than about 3 microns. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B and has a geometric diameter of at least about 90% by weight of the particles comprising amphotericin B of less than about 3 μm. 67. The pharmaceutical composition of claim 66 comprising microparticles. 73. The pharmaceutical composition of claim 72, wherein the median mass aerodynamic diameter of the particulates is less than about 10 microns. 73. The pharmaceutical composition of claim 72, wherein the particulate median aerodynamic diameter is from about 1 μm to about 6 μm. 67. The pharmaceutical composition of claim 66, wherein at least about 80% by weight comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutically acceptable excipient comprises phospholipids. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutically acceptable excipient comprises metal ions. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 67. The pharmaceutical composition of claim 66, comprising hollow and / or porous particulates. 67. The pharmaceutical composition of claim 66, comprising microparticles comprising amphotericin B in a matrix comprising pharmaceutically acceptable excipients. The pharmaceutical composition of claim 81, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. 67. The pharmaceutical composition of claim 66, comprising a dry powder. 67. The pharmaceutical composition of claim 66, further comprising a propellant. 85. The pharmaceutical composition of claim 84, wherein the propellant comprises at least one hydrofluoroalkane. 67. The pharmaceutical composition of claim 66, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of about 1 μm to about 6 μm, wherein at least about 80% by weight of the pharmaceutical composition comprises amphotericin B in the matrix comprising a pharmaceutically acceptable excipient. A pharmaceutical composition comprising microparticles, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. A dry powder comprising amphotericin B having a crystallinity level of at least about 20%, wherein the powder comprises particles having a mass median aerodynamic diameter of less than about 10 μm. 88. The dry powder of claim 87, wherein the crystallinity level of amphotericin B is from about 70% to about 99%. 88. The dry powder of claim 87, wherein the dry powder comprises particles having a mass median aerodynamic diameter of about 1 μm to about 6 μm. 88. The dry powder of claim 87, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 3 μm. 88. The dry powder of claim 87, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B, and wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 3 μm. 88. The dry powder of claim 87, wherein at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 88. The dry powder of claim 87, further comprising a pharmaceutically acceptable excipient. 95. The dry powder of claim 93, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 95. The dry powder of claim 93, wherein the pharmaceutically acceptable excipient comprises phospholipids. 95. The dry powder of claim 93, wherein the pharmaceutically acceptable excipient comprises metal ions. 95. The dry powder of claim 93, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 88. The dry powder of claim 87 comprising hollow and / or porous particles. 88. The dry powder of claim 87 comprising microparticles comprising amphotericin B in a matrix comprising pharmaceutically acceptable excipients. A container containing a pharmaceutical composition made from particles comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient and comprising amphotericin B having a median mass diameter of less than about 1.9 μm. Unit dosage form comprising a. 101. The unit dosage form of claim 100, wherein the container comprises a capsule. 101. The unit dosage form of claim 100, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of about 0.5 μm to about 1.8 μm. The unit dosage form of claim 100, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 101. The unit dosage form of claim 100, wherein the geometric diameter of at least about 90% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 101. The unit dosage form of claim 100, wherein the crystallinity level of amphotericin B is at least about 50%. 101. The unit dosage form of claim 100, wherein the pharmaceutical composition comprises microparticles. 107. The unit dosage form of claim 106, wherein the median mass aerodynamic diameter of the particulates is less than about 10 microns. 107. The unit dosage form of claim 106, wherein the median mass aerodynamic diameter of the microparticles is from about 1 micron to about 6 microns. 101. The unit dosage form of claim 100, wherein at least about 80% of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 101. The unit dosage form of claim 100, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of less than about 10 microns and the crystallinity level of amphotericin B is at least about 70%. 101. The unit dosage form of claim 100, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 101. The unit dosage form of claim 100, wherein the pharmaceutically acceptable excipient comprises phospholipids. 101. The unit dosage form of claim 100, wherein the pharmaceutically acceptable excipient comprises metal ions. 101. The unit dosage form of claim 100, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 101. The unit dosage form of claim 100, wherein the pharmaceutical composition comprises hollow and / or porous particulates. 101. The unit dosage form of claim 100, wherein the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 101. The pharmaceutical composition of claim 100, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of about 1 μm to about 6 μm, wherein the amphotericin B crystallinity level is at least about 70% and at least about 80% by weight A unit dosage form comprising microparticles comprising amphotericin B in a matrix comprising this pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. A container comprising a pharmaceutical composition comprising an effective amount of amphotericin B and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B and comprises the amphotericin B Unit dosage forms having a geometric diameter of at least about 80% by weight of less than about 1.9 μm. 118. The unit dosage form of claim 118, wherein the container comprises a capsule. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of about 0.5 μm to about 1.8 μm. 119. The unit dosage form of claim 118, wherein the geometric diameter of at least about 90% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 119. The unit dosage form of claim 118, wherein the geometric diameter of at least about 95% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 118. The unit dosage form of claim 118, wherein the crystallinity level of amphotericin B is at least about 50%. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition comprises microparticles. 125. The unit dosage form of claim 124, wherein the median mass aerodynamic diameter of the particulates is less than about 10 microns. 126. The unit dosage form of claim 124, wherein the median mass aerodynamic diameter of the particulates is from about 1 μm to about 6 μm. 118. The unit dosage form of claim 118, wherein at least about 80% of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of less than about 10 micrometers and wherein the crystallinity level of amphotericin B is at least about 70%. 118. The unit dosage form of claim 118, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 118. The unit dosage form of claim 118, wherein the pharmaceutically acceptable excipient comprises phospholipids. 118. The unit dosage form of claim 118, wherein the pharmaceutically acceptable excipient comprises metal ions. 118. The unit dosage form of claim 118, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition comprises hollow and / or porous particulates. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 134. The unit dosage form of claim 134, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. 118. The unit dosage form of claim 118, wherein the pharmaceutical composition comprises a dry powder. 118. The pharmaceutical composition of claim 118, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of about 1 μm to about 6 μm, wherein the crystallinity level of amphotericin B is at least about 70% and at least about 80% by weight A unit dosage form comprising microparticles comprising amphotericin B in a matrix comprising this pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. A container containing a pharmaceutical composition comprising amphotericin B having a crystallinity level of about 20% to about 99%, and a pharmaceutically acceptable excipient Unit dosage form comprising a. 138. The unit dosage form of claim 138, wherein the crystallinity level of amphotericin B is from about 70% to about 98%. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 3 microns. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition is prepared from particles comprising amphotericin B and wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 3 microns. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition comprises microparticles. 142. The unit dosage form of claim 142, wherein the median mass aerodynamic diameter of the particulates is less than about 10 microns. 142. The unit dosage form of claim 142, wherein the median mass aerodynamic diameter of the particulates is from about 1 μm to about 6 μm. 138. The unit dosage form of claim 138, wherein at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 138. The unit dosage form of claim 138, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 138. The unit dosage form of claim 138, wherein the pharmaceutically acceptable excipient comprises phospholipids. 138. The unit dosage form of claim 138, wherein the pharmaceutically acceptable excipient comprises metal ions. 138. The unit dosage form of claim 138, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition comprises hollow and / or porous particulates. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 151. The unit dosage form of claim 151, wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. 138. The unit dosage form of claim 138, wherein the pharmaceutical composition comprises a dry powder. 137. The amphotericin B of claim 138, wherein the pharmaceutical composition comprises microparticles having a mass median aerodynamic diameter of about 1 μm to about 6 μm, and wherein at least about 80% by weight of the pharmaceutical composition comprises a pharmaceutically acceptable excipient in a matrix. And wherein said pharmaceutically acceptable excipient comprises at least one phospholipid. inspirator; And A pharmaceutical composition comprising microparticles comprising amphotericin B and pharmaceutically acceptable excipients and particles made from particles comprising amphotericin B having a median mass diameter of less than about 1.9 μm. Delivery system comprising a. 155. The delivery system of claim 155, wherein the inhaler comprises a dry powder inhaler. 155. The delivery system of claim 155, wherein the inhaler comprises a metering valve comprising a canister containing particulate and propellant and in communication with the interior of the canister. 155. The delivery system of claim 155, wherein the inhaler comprises a nebulizer and the particulates are suspended in a liquid. 155. The delivery system of claim 155, wherein the particulate is made from particles comprising amphotericin B having a median mass diameter of about 0.5 μm to about 1.8 μm. 155. The delivery system of claim 155, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 156. The delivery system of claim 155, wherein the crystallinity level of amphotericin B is at least about 50%. 156. The delivery system of claim 155, wherein the crystallinity level of amphotericin B is from about 70% to about 99%. 155. The delivery system of claim 155, wherein the mass median aerodynamic diameter of the particulates is less than about 10 microns. 156. The delivery system of claim 155, wherein the mass median aerodynamic diameter of the particulates is from about 1 micron to about 6 microns. 155. The delivery system of claim 155, wherein at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 155. The delivery system of claim 155, wherein the particulate median aerodynamic diameter is less than about 10 microns and the crystallinity level of amphotericin B is at least about 70%. 155. The delivery system of claim 155, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers and salts. 155. The delivery system of claim 155, wherein the pharmaceutically acceptable excipient comprises phospholipids. 155. The delivery system of claim 155, wherein the microparticles comprise amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 156. The method of claim 155, wherein the microparticles have a mass median aerodynamic diameter of about 1 μm to about 6 m and at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising a pharmaceutically acceptable excipient. , Wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. inspirator; And A pharmaceutical composition comprising microparticles comprising amphotericin B and a pharmaceutically acceptable excipient, wherein the microparticles are prepared from particles comprising amphotericin B and at least about 80% by weight of the particles comprising amphotericin B Geometric diameter is less than about 1.9 μm) Delivery system comprising a. 172. The delivery system of claim 171, wherein the inhaler comprises a dry powder inhaler. 171. The delivery system of claim 171, wherein the inhaler comprises a metering valve comprising a canister containing particulate and propellant and in communication with the interior of the canister. 172. The delivery system of claim 171, wherein the inhaler comprises a nebulizer and the particulates are suspended in a liquid. 172. The delivery system of claim 171, wherein the microparticles are made from particles comprising amphotericin B having a median mass diameter of about 0.5 μm to about 1.8 μm. 171. The delivery system of claim 171, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 172. The delivery system of claim 171, wherein the crystallinity level of amphotericin B is at least about 50%. 171. The delivery system of claim 171, wherein the crystallinity level of amphotericin B is from about 70% to about 99%. 172. The delivery system of claim 171, wherein the mass median aerodynamic diameter of the particulates is less than about 10 microns. 172. The delivery system of claim 171, wherein the median mass aerodynamic diameter of the particulates is from about 1 μm to about 6 μm. 172. The delivery system of claim 171, wherein at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 172. The delivery system of claim 171, wherein the particulate median aerodynamic diameter is less than about 10 microns and the crystallinity level of amphotericin B is at least about 70%. 172. The delivery system of claim 171, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers, and salts. 172. The delivery system of claim 171, wherein the pharmaceutically acceptable excipient comprises phospholipids. 172. The delivery system of claim 171, wherein the microparticles comprise amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 171. The microorganism of claim 171, wherein the microparticles have a mass median aerodynamic diameter of about 1 μm to about 6 m and at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising a pharmaceutically acceptable excipient. , Wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. inspirator; And Pharmaceutical composition comprising amphotericin B having a crystallinity level of about 20% to about 99% and microparticles comprising a pharmaceutically acceptable excipient Delivery system comprising a. 187. The delivery system of claim 187, wherein the inhaler comprises a dry powder inhaler. 187. The delivery system of claim 187, wherein the inhaler comprises a metering valve comprising a canister containing particulate and propellant and in communication with the interior of the canister. 187. The delivery system of claim 187, wherein the inhaler comprises a nebulizer and the particulates are suspended in a liquid. 187. The delivery system of claim 187, wherein the crystallinity level of amphotericin B is from about 70% to about 98%. 187. The delivery system of claim 187, wherein the microparticles are made from particles comprising amphotericin B having a median mass diameter of less than about 3 microns. 192. The delivery system of claim 187, wherein the microparticles are made from particles comprising amphotericin B having a median mass diameter of less than about 2 microns. 187. The delivery system of claim 187, wherein the microparticles are prepared from particles comprising amphotericin B, and wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 3 microns. 187. The delivery system of claim 187, wherein the particulate median aerodynamic diameter is less than about 10 microns. 187. The delivery system of claim 187, wherein the particulate median aerodynamic diameter is between about 1 μm and about 6 μm. 187. The delivery system of claim 187, wherein at least about 80% by weight of the pharmaceutical composition comprises both amphotericin B and a pharmaceutically acceptable excipient. 187. The delivery system of claim 187, wherein the microparticles have a mass median aerodynamic diameter of less than about 10 micrometers and the microparticles are made from particles comprising amphotericin B having a median mass diameter of less than about 3 micrometers. 187. The delivery system of claim 187, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers, and salts. 187. The delivery system of claim 187, wherein the pharmaceutically acceptable excipient comprises phospholipids. 187. The delivery system of claim 187, wherein the microparticles comprise amphotericin B in a matrix comprising pharmaceutically acceptable excipients. 187. The microorganism of claim 187, wherein the microparticles have a mass median aerodynamic diameter of about 1 μm to about 6 μm and at least about 80% by weight of the pharmaceutical composition comprises microparticles comprising amphotericin B in a matrix comprising a pharmaceutically acceptable excipient. , Wherein the pharmaceutically acceptable excipient comprises one or more phospholipids. Measuring the crystallinity level of the amphotericin B composition; And Prior to releasing the amphotericin B composition for combination with a pharmaceutically acceptable excipient to form a pharmaceutical composition, determining that the crystallinity level is above a first predetermined level Comprising a method for characterizing amphotericin B for the manufacture of a pharmaceutical composition. 203. The method of claim 203, wherein determining the crystallinity level comprises performing X-ray powder diffraction of at least some amphotericin B composition. 203. The method of claim 203, wherein determining the crystallinity level comprises performing infrared spectroscopy on at least some amphotericin B compositions. 203. The method of claim 203, wherein determining the crystallinity level comprises performing dynamic vapor sorption for at least some amphotericin B composition. 203. The method of claim 203, wherein the first predetermined level is at least about 50%. 203. The method of claim 203, wherein the first predetermined level is at least about 70%. 203. The method of claim 203, wherein the first predetermined level is at least about 90%. 203. The method of claim 203, further comprising recrystallizing the amphotericin B composition when the crystallinity level is below the first predetermined level. 203. The method of claim 203, further comprising removing the amphotericin B composition when the crystallinity level is below the first predetermined level. 203. The method of claim 203, further comprising combining the amphotericin B composition with a pharmaceutically acceptable excipient to form a pharmaceutical composition. 214. The method of claim 212, wherein combining the amphotericin B composition with a pharmaceutically acceptable excipient to form the pharmaceutical composition comprises spray drying. 213. The method of claim 212, further comprising measuring the crystallinity level of the pharmaceutical composition. 214. The method of claim 214, further comprising releasing the pharmaceutical composition for patient administration when the crystallinity level is below the second predetermined level. 215. The method of claim 215, wherein the second predetermined level is at least about 50%. Measuring the amorphicity of the amphotericin B composition; And Before releasing the amphotericin B composition for combination with a pharmaceutically acceptable excipient to prepare the pharmaceutical composition, confirming that the amorphousness is below a predetermined level Comprising a method for characterizing amphotericin B for the manufacture of a pharmaceutical composition. 228. The method of claim 217, wherein the amorphousness measuring step comprises performing X-ray powder diffraction of at least some amphotericin B composition. 228. The method of claim 217, wherein the amorphous measuring step comprises performing infrared spectroscopy on at least some amphotericin B compositions. 217. The method of claim 217, wherein the amorphousness measuring step comprises performing dynamic vapor sorption on at least some amphotericin B compositions. 217. The method of claim 217, wherein the first predetermined level is less than about 50%. 217. The method of claim 217, wherein the first predetermined level is less than about 30%. 217. The method of claim 217, wherein the first predetermined level is less than about 10%. 228. The method of claim 217, wherein combining the amphotericin B composition with a pharmaceutically acceptable excipient to form the pharmaceutical composition comprises spray drying. 226. The method of claim 217, further comprising measuring the amorphousness of the pharmaceutical composition. 225. The method of claim 225, further comprising releasing the pharmaceutical composition for patient administration when the amorphousness is above the second predetermined level. 226. The method of claim 226, wherein the second predetermined level is less than about 50%. Combining the amphotericin B composition with a pharmaceutically acceptable excipient to form a dry powder pharmaceutical composition; Measuring the mass median aerodynamic diameter of the dry powder pharmaceutical composition; And Before releasing the dry powder pharmaceutical composition for patient administration, confirming that the mass median aerodynamic diameter is below a predetermined level. Comprising, a method of preparing and characterizing a dry powder pharmaceutical composition. 228. The method of claim 228, wherein the mass median aerodynamic diameter is measured by cascade impaction. 228. The method of claim 228, wherein the first predetermined level is less than about 10 microns. 228. The method of claim 228, wherein the first predetermined level is less than about 5 microns. Combining the amphotericin B composition with a pharmaceutically acceptable excipient to form a dry powder pharmaceutical composition; Measuring the homogeneity of the dry powder pharmaceutical composition; And Confirming that the homogeneity is above a predetermined level before releasing the dry powder pharmaceutical composition for patient administration Comprising, a method of preparing and characterizing a dry powder pharmaceutical composition. 234. The method of claim 232, wherein the measuring homogeneity comprises determining what percentage by weight of the dry powder pharmaceutical composition comprises particulates comprising both amphotericin B and a pharmaceutically acceptable excipient. 234. The method of claim 232, wherein the dry powder pharmaceutical composition is released for administration when the weight percentage exceeds a level that is greater than 80% by weight. 234. The method of claim 232, wherein the dry powder pharmaceutical composition is released for administration when the weight percentage exceeds a level above 90% by weight. Suspending particles comprising amphotericin B having a median mass diameter of less than about 3 μm in a liquid to form a feed stock; And Spray drying the feedstock to produce spray dried particles Comprising, spray dried particle production method. 235. The method of claim 236, wherein the median diameter of the particles comprising amphotericin B is from about 1 μm to about 2 μm. 236. The method of claim 236, wherein the crystallinity level of the particles comprising amphotericin B is at least about 50%. 236. The method of claim 236, wherein the median diameter of the spray dried particles is less than about 10 microns. 236. The method of claim 236, wherein at least about 80% by weight spray dried particles comprise amphotericin B and a pharmaceutically acceptable excipient. 236. The method of claim 236, wherein the spray dried particles have a mass median aerodynamic diameter of less than about 10 microns and the crystallinity level of the particles comprising amphotericin B is at least about 50%. 236. The method of claim 236, wherein the feedstock further comprises a pharmaceutically acceptable excipient. 236. The method of claim 236, wherein the spray drying step of the feedstock further comprises spray drying the additional feedstock including a pharmaceutically acceptable excipient. 236. The method of claim 236, wherein the spray dried particles further comprise a pharmaceutically acceptable excipient. 236. The method of claim 236, further comprising the step of collecting spray dried particles. 236. The method of claim 236, further comprising adding an emulsifier to the feedstock solution. 246. The method of claim 246, wherein the emulsifier comprises phosphatidylcholine. 247. The method of claim 247, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine. 236. The method of claim 236, further comprising adding a blowing agent to the feedstock solution. 236. The method of claim 236, wherein the spray dried particles comprise amphotericin B crystals in the phospholipid matrix. 236. The method of claim 236, wherein the spray dried particles are hollow and / or porous. 251. The method of claim 251, wherein the feedstock further comprises hydrophobic amino acids. Suspending particles comprising amphotericin B having a crystallinity level of at least about 20% in a liquid to form a feed stock; And Spray drying the feedstock to produce spray dried particles Comprising, spray dried particle production method. 253. The method of claim 253, wherein the crystallinity level of amphotericin B is at least about 50%. 253. The method of claim 253, wherein the median diameter of the particles comprising amphotericin B is less than about 3 μm. 253. The method of claim 253, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 3 μm. 253. The method of claim 253, wherein the median diameter of the spray dried particles is less than about 10 microns. 253. The method of claim 253, wherein at least about 80% by weight spray dried particles comprise amphotericin B and a pharmaceutically acceptable excipient. 255. The method of claim 253, wherein the spray dried particles have a mass median aerodynamic diameter of less than about 10 μm, wherein the crystallinity level of the particles comprising amphotericin B is at least about 50%. 253. The method of claim 253, wherein the feedstock further comprises a pharmaceutically acceptable excipient. 253. The method of claim 253, wherein the feedstock spray drying step further comprises spray drying of the additional feedstock comprising a pharmaceutically acceptable excipient. 253. The method of claim 253, wherein the spray dried particles further comprise a pharmaceutically acceptable excipient. 253. The method of claim 253, further comprising the step of collecting spray dried particles. 253. The method of claim 253, further comprising adding an emulsifier to the feedstock solution. 264. The method of claim 264, wherein the emulsifier comprises phosphatidylcholine. 265. The method of claim 265, wherein the phosphatidylcholine comprises distearoyl phosphatidylcholine. 253. The method of claim 253, further comprising adding blowing agent to the feedstock solution. 253. The method of claim 253, wherein the spray dried particles comprise amphotericin B crystals in the phospholipid matrix. 253. The method of claim 253, wherein the spray dried particles are hollow and / or porous. 270. The method of claim 269, wherein the feedstock further comprises hydrophobic amino acids. An effective amount of a composition comprising amphotericin B prepared from particles comprising amphotericin B having a median mass diameter of about 1.1 μm to about 1.9 μm is inhaled by a patient in need of treatment and / or prevention of fungal infections. A method of treating and / or preventing a fungal infection comprising administering. 280. The method of claim 271, wherein a sufficient amount of the composition is administered to maintain the target lung amphotericin B concentration for at least about 5 times the predetermined minimum inhibitory concentration for at least about 4 weeks. 272. The method of claim 272, wherein the minimum inhibitory concentration is the minimum inhibitory concentration of the lung epithelial lining. 272. The method of claim 272, wherein the minimum inhibitory concentration is a minimum inhibitory concentration of lung solid tissue. 272. The method of claim 272, wherein the target lung amphotericin B concentration is maintained for at least about 8 weeks. 272. The method of claim 272, wherein the target lung amphotericin B concentration is at least about 4 μg / g. 272. The target lung amphotericin B concentration is about 4.5 μg / g to about 20 μg / g, and the pharmaceutical composition is delivered periodically so that the lung amphotericin B concentration is within the target amphotericin B lung concentration range. Administering to maintain. 272. The method of claim 272, comprising administering to deliver one dose of the composition during the first week of administration. The method of claim 271, comprising administering to deliver two doses of the composition during the first week of administration. 280. The method of claim 271, wherein the administration comprises a first administration period and a second administration period, and wherein the composition is administered more frequently or at a higher dosage in the first administration period than during the second administration period. 278. The method of claim 271, wherein the median diameter of the particles comprising amphotericin B is from about 1.2 μm to about 1.8 μm. 278. The method of claim 271, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 1.9 μm. 280. The method of claim 271, wherein the crystallinity level of amphotericin B is at least about 50%. 272. The method of claim 271, wherein the composition comprises microparticles. 290. The method of claim 284, wherein the mass median aerodynamic diameter of the particulates is less than about 10 microns. 303. The method of claim 284, wherein the particulate median aerodynamic diameter is between about 1 μm and about 6 μm. 280. The method of claim 271, wherein at least about 80% by weight of the composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 280. The method of claim 271, wherein the composition comprises microparticles having a mass median aerodynamic diameter of less than about 10 μm, wherein the crystallinity level of amphotericin B is at least about 50%. 280. The method of claim 271, wherein the composition comprises particles composed primarily of amphotericin B. 290. The method of claim 289, wherein the composition further comprises particles comprising a pharmaceutically acceptable excipient. 280. The method of claim 271, wherein the composition comprises microparticles comprising amphotericin B and a pharmaceutically acceptable excipient. 303. The method of claim 291, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers, and salts. The method of claim 291, wherein the pharmaceutically acceptable excipient comprises phospholipids. 303. The method of claim 291, wherein the pharmaceutically acceptable excipient comprises metal ions. 303. The method of claim 291, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 272. The method of claim 271, wherein the composition comprises hollow and / or porous particles. 280. The method of claim 271, wherein the composition comprises microparticles comprising crystalline amphotericin B in the matrix material. 297. The method of claim 297, wherein the matrix material comprises one or more phospholipids. 278. The method of claim 271, wherein administering comprises delivering the composition in dry powder form using a dry powder inhaler. 272. The method of claim 271, wherein the composition comprises a propellant, and wherein administering comprises releasing the composition by opening the valve to aerosolize the composition. 309. The method of claim 300, wherein the propellant comprises at least one hydrofluoroalkane. 278. The method of claim 271 comprising treating a fungal infection. 278. The method of claim 271 comprising prophylaxis against fungal infections. 278. The method of claim 271 wherein the fungal infection comprises a pulmonary fungal infection. 280. The method of claim 271, wherein the fungal infection comprises a nasal fungal infection. 278. The method of claim 271 wherein the administration comprises pulmonary administration. 278. The method of claim 271, wherein the administration comprises intranasal administration. 272. The method of claim 271. The administration comprises pulmonary administration, Administering a sufficient amount of the composition to maintain the target lung amphotericin B concentration at least about 5 times the predetermined minimum inhibitory concentration for at least about 8 weeks, The composition is prepared from particles comprising amphotericin B having a median mass diameter of about 0.5 μm to about 1.8 μm, The crystallinity level of the amphotericin B is at least about 50%, The composition comprises particulates having a mass median aerodynamic diameter of about 1 μm to about 6 μm, At least about 80% by weight of the composition comprises microparticles including both amphotericin B and phospholipids Methods that include prevention against pulmonary fungal infections. Prepared from particles comprising amphotericin B, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is from about 1.1 μm to about 1.9 μm, A method of treating and / or preventing fungal infections comprising administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need of treatment and / or prevention of fungal infections. Treatment and / or prophylaxis of said fungal infection comprising administering an effective amount of a composition comprising amphotericin B having a crystallinity level of at least about 20% by inhalation to a patient in need of treatment and / or prevention of fungal infection. Way. 309. The method of claim 310, wherein a sufficient amount of the composition is administered to maintain the target lung amphotericin B concentration at about 5 times or more of the predetermined minimum inhibitory concentration for at least about 4 weeks. 338. The method of claim 311, wherein the minimum inhibitory concentration is a minimum inhibitory concentration of the lung epithelial lining. 338. The method of claim 311, wherein the minimum inhibitory concentration is a minimum inhibitory concentration of lung solid tissue. 338. The method of claim 311, wherein the target lung amphotericin B concentration is maintained for at least about 8 weeks. 338. The method of claim 311, wherein the target lung amphotericin B concentration is at least about 4 μg / g. 335. The target lung amphotericin B concentration is about 4.5 μg / g to about 20 μg / g, and the pharmaceutical composition is delivered periodically to bring the lung amphotericin B concentration into the target lung amphotericin B concentration range. Administering to maintain. 338. The method of claim 311, comprising administering to deliver one dose of the composition during the first week of administration. 309. The method of claim 310, comprising administering to deliver two doses of the composition during the first week of administration. 309. The method of claim 310, wherein the administration comprises a first administration period and a second administration period, and wherein the composition is administered more frequently in the first administration period or at a higher dosage than during the second administration period. 309. The method of claim 310, wherein the composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 3 μm. 309. The method of claim 310, wherein the composition is prepared from particles comprising amphotericin B, wherein the geometric diameter of at least about 80% by weight of the particles comprising amphotericin B is less than about 3 μm. 309. The method of claim 310, wherein the crystallinity level of amphotericin B is at least about 70%. 309. The method of claim 310, wherein the composition comprises microparticles. 323. The method of claim 323, wherein the particulate median aerodynamic diameter is less than about 10 microns. 323. The method of claim 323, wherein the median mass aerodynamic diameter of the particulates is from about 1 μm to about 6 μm. 323. The method of claim 323, wherein at least about 80% by weight of the composition comprises microparticles comprising both amphotericin B and a pharmaceutically acceptable excipient. 309. The method of claim 310, wherein the composition comprises microparticles having a mass median aerodynamic diameter of less than about 10 microns and the crystallinity level of amphotericin B is at least about 70%. 309. The method of claim 310, wherein the composition comprises particles composed primarily of amphotericin B. 328. The method of claim 328, wherein the composition further comprises particles comprising a pharmaceutically acceptable excipient. 309. The method of claim 310, wherein the composition comprises microparticles comprising amphotericin B and a pharmaceutically acceptable excipient. 330. The method of claim 330, wherein the pharmaceutically acceptable excipient comprises one or more components selected from carbohydrates, lipids, amino acids, buffers, and salts. 330. The method of claim 330, wherein the pharmaceutically acceptable excipient comprises phospholipids. 330. The method of claim 330, wherein the pharmaceutically acceptable excipient comprises metal ions. 330. The method of claim 330, wherein the pharmaceutically acceptable excipient comprises phospholipids and metal ions. 309. The method of claim 310, wherein the composition comprises hollow and / or porous particles. 309. The method of claim 310, wherein the composition comprises microparticles comprising crystalline amphotericin B in the matrix material. 336. The method of claim 336, wherein the matrix material comprises one or more phospholipids. 309. The method of claim 310, wherein administering comprises delivering the composition in dry powder form using a dry powder inhaler. 309. The method of claim 310, wherein the composition comprises a propellant and administering comprises releasing the composition by opening the valve to aerosolize the composition. 340. The method of claim 339, wherein the propellant comprises one or more hydrofluoroalkanes. 309. The method of claim 310 comprising treating a fungal infection. 309. A method according to claim 310, comprising prevention against fungal infections. 309. The method of claim 310, wherein the fungal infection comprises a pulmonary fungal infection. 309. The method of claim 310, wherein the fungal infection comprises a nasal fungal infection. 309. The method of claim 310, wherein the administration comprises pulmonary administration. 309. The method of claim 310, wherein the administration comprises intranasal administration. 309. The method of claim 310, The administration comprises pulmonary administration, Administering a sufficient amount of the composition to maintain the target amphotericin B lung concentration at least about 5 times the predetermined minimum inhibitory concentration for at least about 8 weeks, The composition is prepared from particles comprising amphotericin B having a median mass diameter of less than about 3 μm, The crystallinity level of amphotericin B is at least about 70%, The composition comprises particulates having a mass median aerodynamic diameter of about 1 μm to about 6 μm, At least about 80% by weight of the composition comprises microparticles including both amphotericin B and phospholipids Methods comprising prevention of pulmonary fungal infections. The crystallinity level of amphotericin B is at least about 20%, Lung solid tissue retention half-life of amphotericin B measured by lung tissue biopsy is at least about 1 week, A method of treating and / or preventing fungal infections comprising administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need of treatment and / or prevention of fungal infections. The crystallinity level of amphotericin B is at least about 20%, Pulmonary epithelial endothelial fluid retention half-life of amphotericin B, measured by bronchoalveolar lavage, is about 10 hours or more, A method of treating and / or preventing fungal infections comprising administering an effective amount of a composition comprising amphotericin B by inhalation to a patient in need of treatment and / or prevention of fungal infections. The crystallinity level of amphotericin B is at least about 20%, Plasma amphotericin B concentration is maintained at less than about 1000 ng / mL, Treating and / or treating a fungal infection comprising administering a composition comprising amphotericin B in an amount from about 0.01 mg / kg to 7.0 mg / kg to a patient in need of treatment and / or prevention of a fungal infection Or preventive measures. The crystallinity level of amphotericin B is at least about 20%, The plasma amphotericin B concentration is kept low enough to avoid renal toxicity and / or liver toxicity, Treating and / or treating a fungal infection comprising administering a composition comprising amphotericin B in an amount from about 0.01 mg / kg to 7.0 mg / kg to a patient in need of treatment and / or prevention of a fungal infection Or preventive measures. The crystallinity level of amphotericin B is at least about 20%, Lung amphotericin B concentration measured by bronchoalveolar lavage reaches at least about 5 times the minimum inhibitory concentration for at least some treatment periods, Plasma amphotericin B concentration is maintained at less than about 1000 ng / mL, A method of treating and / or preventing a fungal infection comprising administering a composition comprising amphotericin B by inhalation to a patient in need of treatment and / or prevention of a fungal infection. The crystallinity level of amphotericin B is at least about 20%, The treatment period is from about 15 weeks to about 20 weeks, The lung amphotericin B concentration measured by bronchoalveolar lavage reaches at least about 5 times the minimum inhibitory concentration for at least some treatment periods, A method of treating and / or preventing a fungal infection comprising administering a composition comprising amphotericin B by inhalation to a patient in need of treatment and / or prevention of a fungal infection. The crystallinity level of amphotericin B is at least about 20%, Administration is carried out in less than about 5 minutes, A method of treating and / or preventing fungal infection comprising administering a composition comprising about 2 mg to about 50 mg of amphotericin B by inhalation to a patient in need of treatment and / or prevention of fungal infection. The crystallinity level of amphotericin B is at least about 20%, The dry powder comprises particles having a mass median aerodynamic diameter of less than about 10 μm, A method of treating and / or preventing a fungal infection comprising administering an effective amount of a dry powder comprising amphotericin B to a patient in need thereof by treating and / or preventing a fungal infection.

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