DRY POWDER FOR INHALATION

专利摘要:
methods for preparing a dry powder for inhalation, for treating or preventing a disease or condition, formulating dry powder, dry powder for inhalation, and, use of oxytocin and/or a derivative thereof. this invention relates to the delivery of medicament and in particular the delivery of biologically active agents in the form of dry powders for inhalation. the invention also relates to methods of preparing such dry powder formulations and methods of using them.
公开号:BR112014002646B1
申请号:R112014002646-7
申请日:2011-11-07
公开日:2021-09-14
发明作者:Michelle McIntosh；David Morton；Tomas Sou；Livesey Olerile；Richard Prankerd
申请人:Monash University；
IPC主号:

专利说明:

[0001] This invention relates to the release of medicine and in particular the release of biologically active agents in the form of dry powders for inhalation. The invention also relates to methods for preparing such dry powder formulations and methods for their use.
[0002] Each year, more than 150,000 women, mainly in developing countries, die from postpartum hemorrhage. This condition is largely preventable with the administration of uterotonic drugs, such as oxytocin, in the third stage of labor. The World Health Organization has approved oxytocin as the most effective therapy in the treatment of postpartum hemorrhage. The use of oxytocin in developing countries presents several difficulties. Oxytocin is a peptide with relatively poor stability in solution. This then requires refrigerated storage, for example at 2 to 8 °C, which is problematic in developing countries. An additional consideration with the use of oxytocin is that it requires sterile needles and syringes and trained administration personnel, another requirement that cannot be guaranteed in developing nations. Consequently, a problem to be resolved is the development of a system for the effective and cheap release of oxytocin for the appropriate use in developing countries, in order to prevent newborn deaths due to PPH. A possible solution to this problem may be to create a dry powder pulmonary delivery system suitable for the effective and affordable delivery of oxytocin, or other biologically active peptides or proteins, that is suitable for use in remote and environmentally demanding regions. of developing nations.
[0003] Pulmonary delivery has been proposed to be a suitable systemic pathway for biological agents such as agents based on peptides, proteins, vaccines, and nucleic acid. The inoculations in the release of such large macromolecules are substantial and result in a wide and greater uncertainty and risk as to the possibility of success. Inoculations begin with the generation of an appropriate dose aerosol that is suitable for efficient and consistent deep lung delivery. If such adequate release can be obtained, the material must dissolve or become available in such a form that it can be transported across biological membranes and other barriers such as surfactant and mucous interfaces. Dissolution kinetics will determine the degree of solubilization, and this would be expected to be prevented by hydrophobic formulations and hence the material will undergo the pulmonary clearance runtime before being systemically absorbed, as outlined by O'Donnel and Smythe in “Controlled Pulmonary Drug Delivery”, published by Springer 2011, ISBN 978-1-44199744-9. Before a macromolecule can be transported into the systemic circulation, it must then be able to survive an attack by the body's defense mechanisms, including the mucociliary escalator, peptidases, and macrophages. These inoculations are clearly reviewed and outlined in the recent expert text, "Controlled Pulmonary Drug Delivery", published by Springer 2011, ISBN 978-1-4419-9744-9. For example, it is concluded by Olsson et al that “for systemic targets of peptides and proteins, inoculation appears to be achieving absorption at a rate that is competitive with clearance rate mechanisms in order to ensure sufficient bioavailability” and “ what constitutes optimal characteristics in each particular case and since these can be predicted from the abstracted properties remains far from clear with our present understanding”. The rapid onset of action for pulmonary delivery relative to oral delivery of selected small molecules, such as smaller than 781 Da, which are primarily hydrophilic, has been proposed. However, for macromolecules such as peptides and proteins of molecular weight typically on the order of 1000 Da and above, the onset of action is highly uncertain and likely to be hampered by a need for paracellular diffusion mechanisms. The half-lives for removal from the pulmonary epithelium into the blood via such membranes are highly variable and uncertain, but typically appear to require several minutes and even more than hundreds of minutes, as outlined by Sakagami and Gumbleton in “Controlled Pulmonary Drug Delivery”, published by Springer 2011, ISBN 978-1-44199744-9. Consequently, clearly a rapid pharmacokinetic absorption and pharmacodynamic onset of the order of one or two minutes for a peptide of the order of 1000 Da and greater would not be assumed but would be surprising from a pulmonary released macromolecule. It would be especially surprising if the pulmonary delivery system comprised a powder that required dissolution, and even more so if this particle had a hydrophobic surface.
[0004] Additional inoculations in this context include producing a physically and chemically stable product that is maintained during production, storage and transportation, and in use. It is also important for agents not to cause bronchoconstriction. A peptide such as oxytocin, which causes uterine contractions, would interact with receptors present in the pulmonary system and consequently cause bronchoconstriction. It is also important for agents not to be metabolized in the lung. If these multiple and very significant inoculations can be achieved, pulmonary delivery can provide unique advantages, which include avoiding the use of needles, avoiding first-pass metabolism via the oral route, and the extra stability offered by powder formulations.
[0005] Spray drying has been widely used for many years to produce powdered products - in areas such as food, detergent and industrial chemicals. In many cases, relatively large, free-flowing particles are produced. To a lesser degree, the technique has been adapted to generate fine and ultrafine particles, and innovation here has been driven by inorganic materials industries such as technical ceramic producers (TT Kodas, Adv. Mater., 1989, 6, 180) . This method of generating droplets from often complex solutions to form an aerosol, and then drying these as isolated units into particles, has been recognized as providing enhanced control over particle morphology, stoichiometry, purity, size as well as structure. The advantages can be seen as a hybrid between “top-down” and “bottom-up” methods of particle engineering. However, the pharmaceutical industry has only relatively recently recognized the advantages of this pathway to engender fine particles (R. Vehrig, Pharm Res., 2008, 25(5), 999), and recently spray dried materials have appeared in inhaler systems commercially available dry powder. More attention in this area has been directed towards the impact of shape, density and roughness on aerosolization.
[0006] It is challenging, however, to release drug molecules within the respiratory tract and even within the lower lung to provide a therapeutic effect, especially drugs that are solid at the administration temperature. In this regard, although dry powders present an attractive means of drug delivery, generating micronized particles suitable for highly efficient aerosolization remains a very significant technical challenge.
[0007] On inhalation, larger aerosolized drug particles tend to be deposited by impaction and gravitational sedimentation in the back of the throat and upper respiratory tract where they are prone to mucociliary clearance within the gastrointestinal tract and subsequent metabolism. Also, larger drug particles cannot advance deep into the lower lung due to the narrowing of the bronchioles. It is believed that for effective local aerosol transport and delivery to the respiratory system that includes the trachea, bronchi and alveoli, particles of less than 5 μm in aerodynamic diameter are preferred, whereas for the deep lung, bronchioles and alveoli, particles of less than 3 µm are preferred — especially for systemic absorption.
[0008] Although delivery by inhalation is a desirable means, a significant technological barrier to this remains the practicality of engineering a suitable aerosol for highly efficient delivery, (eg: >50% dose delivery to the treatment site), reproducible release (eg having a dose release coefficient of variation (CV %) <10 %) and high payload (eg >1 mg powder release to the treatment site) in a practical and cost-effective format that comprises a device and a formulation. Dry powder release provides an attractive release format. However, generating micronized particles suitable for highly efficient aerosolization remains a very significant technical challenge. In addition to the problems mentioned above, any practical inhalation delivery system for dry powders would need to avoid or minimize particle agglomeration at the time of inhalation, have low variation in the released dose due to poor flow properties or incompatible agglomeration, and avoid or minimize incomplete removal of dust from the delivery device caused by dust adhering to the device walls.
[0009] In recent years a new generation of "relatively large" powdered formulations for the inhalation of biomolecules has been proposed, often formed through spray drying. In many cases, these formulations are designed empirically, and comprise a cocktail of excipients each of which is proposed to provide one or more functional roles in the solid phase. One of these excipients used in this context has been the amino acid L-leucine. Potential advantageous properties provided by addition of L-leucine, co-milling or condensation/precipitation were first demonstrated by Staniforth and Ganderton et al. (See for example WO 96/23485 and WO 00/33811). This work indicated that the peculiar physical properties of this amino acid provided its performance-enhancing behavior. Several groups have since studied the benefit that L-leucine provides for powder aerosolization, especially when co-pulverized with actives and excipients, however, the true nature of the structure-performance relationships in such systems remains uncertain. More broadly, it also appears that certain peptides/proteins in the spray dried particle structures, e.g. albumins, isoleucine or tri-leucine, may also impart improved aerosolization performance in use. Alternatively, lipid and fatty acid materials may also provide some benefit in this regard, such as phospholipids (for example DPPC), lecithins or fatty acid salts (for example sodium or magnesium stearate).
[00010] It has now surprisingly been found that L-leucine can be used to advantage in processes for preparing dry powders for inhalation by spray drying, and can result in the formation of suitable dry powders in circumstances where, in the absence of L- leucine, suitable particles would not form at all or would immediately coalesce or agglomerate irreversibly.
[00011] Consequently, in a first aspect the present invention provides a method for preparing a dry powder for inhalation comprising: preparing an aqueous solution and/or suspension comprising a biologically active protein or peptide, one or more mono-, di- or polysaccharides and/or amino acids capable of forming an amorphous glassy matrix, and L-leucine; and spray drying the aqueous solution or suspension to produce a dry powder suitable for inhalation.
[00012] Without wishing to be bound by theory, it is believed that L-leucine concentrates on the surface of the formed particles, possibly due to its hydrophobicity, in such a way that the particles are stabilized and such that agglomeration is inhibited. It was also found that the particles thus formed are protected from degradation, with a decrease in the glass transition temperature and recrystallization from atmospheric or other moisture, such that they do not require the use of a moisture-free packaging environment to the same degree as current dry powder formulations. This also enables the dry powders of the present invention to be used in remote and environmentally demanding regions of developing nations. Inoculations in the production of a powder that contains a biological macromolecule that is suitable for inhalation are well recognized and have been extensively reported following the market failure of the Exubera inhaled insulin product. Although some of the technical inoculations were addressed in the development of the Exubera product, it was necessary to provide a very complex, expensive and impractical inhaler device, plus the powder was extremely sensitive to moisture having to be handled in very low humidity environments. Many additional factors have contributed to its failure, including bioavailability and uncertainty in its fate in the lungs. This product failure has highlighted the extreme complexity of developing a solution to the problem outlined here.
[00013] As used herein the term "amorphous glassy matrix" refers to a matrix in which the biologically active protein or peptide is dispersed which is substantially non-crystalline, or has no substantial region of crystallinity or structural molecular order of regular repetition .
[00014] The solution/dispersion may include other components, including other amino acids, albumins and amino acid derivatives such as tri-leucine, which may also aid in the formation and stabilization of the dry powder formulation. Depending on the final use of the formulation, other drugs may be incorporated, including non-peptide drugs. The biologically active protein or peptide, other drugs or other components can be in solution or suspension, and additional excipients such as stabilizing agents, surfactants and the like can also be included.
[00015] Consequently, as used herein, a reference to a "solution and/or suspension" or "solution/suspension" indicates a mixture of water and other components in which some components may be dissolved (i.e., in solution) and some components can be in suspension, or be in the form of a nano-suspension, emulsion or micro-emulsion. The aqueous solution can include other co-solvents in some embodiments.
[00016] The aqueous liquid may include other co-solvents in some embodiments. The term "aqueous" will be understood to refer to a liquid that is made up at least in part of water, but may include other water-miscible liquids such as an alcohol (eg, ethanol, isopropanol). In any event the skilled person will recognize that the aqueous liquid must be suitable for spray drying in accordance with the methods of the invention.
[00017] The dry powder formulations of the present invention can be used for the treatment or prevention of diseases or conditions, which depend on the biologically active peptides or proteins incorporated in the formulations. When the biologically active peptide or protein is an antigen the dry powder formulation can be used as a vaccine.
[00018] The biologically active agent can be any protein or peptide, or combination thereof. The present invention is specifically adapted for formulations of the peptide, oxytocin, its derivatives (including analogues and agonists) as well as other similar agents such as vasopressin and desmopressin. Formulations containing oxytocin and/or its derivatives can be used in the treatment or prevention of postpartum hemorrhage (PPH). In such circumstances the formulation may also include other components suitable for treating or preventing PPH, such as ergometrine and related medications. Formulations containing oxytocin and/or its derivatives may also be useful in treating anxiety and autism, as well as in inducing behavior modification. For example, psychiatric illnesses or conditions including autism, schizophrenia, anxiety, stress and depression which includes postnatal depression, cancer which includes breast, ovarian and endometrial carcinoma, as a lifestyle medication which involves lack of confidence, intimacy and treatment of sexual dysfunction, treatment of pain such as chronic headache, lactation and fertility, male or female.
[00019] The components used to form the amorphous vitreous matrix in the final spray dried powder can be any suitable mono-, di- or polysaccharide and/or amino acid. For example, this component can comprise D-mannitol and glycine. These components will generally be dissolved in the water of the aqueous solution/suspension.
[00020] In another embodiment this component may comprise trehalose or inulin. A person skilled in the art would be well aware of suitable saccharides and/or amino acids for this purpose. Sugar alcohols, for example, can include xylitol and sorbitol. Monosaccharides for example can include, but are not limited to glucose (dextrose), fructose (levulose), galactose, xylose and ribose, and can include any combination of stereoisomers. Disaccharides for example may include but are not limited to lactose, sucrose, trehalose, maltose. Alternatively they may include trisaccharides such as raffinose, tetrasaccharides such as stachyose, and pentasaccharides such as verbascose.
[00021] The micronized particles, optionally containing one or more other physiologically active agents, can further include one or more pharmaceutically acceptable carriers, diluents or excipients. Other excipients may include, but not be limited to, bulking agents, buffering agents and stabilizers such as sodium citrate, absorption enhancers, protease and peptidase inhibitors, taste or aroma modifying agents, adhesion modifiers, flow agents , dissolution modifiers, or mucolytics.
[00022] Powders can be further formulated by combining with any known carrier particle, or other additive excipients such as taste, aroma or organoleptic sense modifiers. Some improvements can also be achieved by pelletizing the powder into soft pellets with improved powder flow, and appropriate selection of dry powder inhaler accordingly.
[00023] The dry powder formulation particles typically have a mass average aerodynamic diameter of less than 10 microns, more preferably less than 5 µm and most preferably less than 3 µm. Preferably L-leucine will represent between 5 and 50% by weight of the dry ingredients of the formulation. More preferably, the L-leucine will comprise between 10 and 40% by weight of the dry ingredients of the formulation.
[00024] As used herein the term "aerodynamic diameter" (Dae) is defined as the diameter of a sphere of unit density equivalent volume with the same terminal sedimentation velocity as the actual particle in question. Pulmonary deposition of pharmaceutical powders is generally expressed in terms of the aerodynamic behavior of the particle. Particles under the influence of gravity will sediment to the base at a certain speed. In the aerodynamic diameter, this velocity is assumed to be measurable and takes into account the unit particle density (po), particle density (pp), unit density of an equivalent sphere (Deq), Dynamic form factor - (X). For particles larger than about 1 µm the following equation applies to relate aerodynamic diameter and unit density equivalent volume sphere.

[00025] The term "mass mean aerodynamic diameter" ("MMAD") is a statistical representation of the particle size distribution classified according to aerodynamic diameter, defined herein as the mean aerodynamic diameter expressed on a mass-weighted basis , and is a widely accepted parameter used by aerosol scientists. Mass Mean Aerodynamic Diameter (MMAD) can be measured by a pharmacopeial impactor method as defined by the US Pharmacopeia, by the use of an Andersen Cascade Impactor, or by the Next Generation Impactor (NGI). In this regard, so that the dry powder is highly aerosolizable, the particles will generally have a mass average aerodynamic diameter of less than 10 µm, but preferably less than 6 µm, preferably less than 5 µm, more preferably less than than 3.5 µm or most preferably less than 2 µm.
[00026] The emitted dose (ED) is the total mass of active agent emitted from the device following actuation. It does not include material left on the internal or external surfaces of the device, or in the measurement system that includes, for example, the capsule or blister. ED is measured by collecting the total mass emitted from the device. It can be conducted in a device often identified as a dose uniformity sampling device (DUSA), and recovering this by a validated quantitative wet chemical assay (a gravimetric method is possible, but this is less accurate). Alternatively, where an impactor or booster is used, the ED is measured by combining the dose collected through all stages of the respective impactor or booster system.
[00027] The fine particle dose (FPD) is the total mass of active agent that is emitted from the device following actuation that is present at an aerodynamic particle size smaller than a defined threshold. This limit is generally considered to be 5 µm if not expressly stated to be an alternative limit, such as 3 µm, 2 µm or 1 µm, etc. FPD is measured using an impactor or booster, such as a dual stage booster (NI), multiple stage booster (MSI), Andersen Cascade Impactor (ACI) or Next Generation Impactor (NGI). When using a TSI, the FPD is generally assumed to be 6.4 µm as this booster has only one cutoff that is estimated at this value. Each impactor or booster has a predetermined aerodynamic particle size of collection cut-off points for each stage. The FPD value is then obtained by interpreting the active agent recovery at each quantified stage by a validated quantitative wet chemical assay (a gravimetric method is possible, but this is less accurate) where a single stage cutoff is used to determine FPD or a more complex mathematical interpolation of the deposition at each stage is used.
[00028] The fine particle fraction (FPF) is usually defined as the FPD divided by the ED and expressed as a percentage. Here, the FPF of ED is referred to as FPF(ED) and is calculated as FPF(ED) = (FPD/ED) x 100%.
[00029] The fine particle fraction (FPF) can also be defined as the FPD divided by the MD and expressed as a percentage.
[00030] Spray drying can be carried out using spray drying equipment well known to a person skilled in the art. It has now been found that the use of L-leucine in the solution for spray drying allows spray drying to be achieved at temperatures lower than the temperatures generally required for this purpose. Since the temperatures used in the spray drying process can cause the active agent to decompose this is a particular advantage of the present invention. For example, spray drying of the solutions/suspensions of the present invention can be achieved at temperatures of less than 80°C, preferably less than 60°C, more preferably less than 40°C and most preferably less than 40°C. less than 30 °C or at ambient temperatures. Depending on the configuration of the spray dryer, these temperatures may refer to the internal temperature or external temperature of the dryer, but preferably will refer to the temperature experienced by the droplets under drying, which due to the evaporative refrigerant effect is often the external temperature of the system.
[00031] The dry powder prepared by the present invention is new and presents another aspect of the present invention. In accordance with this aspect a dry powder formulation is provided which comprises:
[00032] a biologically active protein or peptide,
[00033] an amorphous glassy matrix comprising one or more mono-, di- or poly-saccharides and/or amino acids, and
[00034] L-leucine.
[00035] In a preferred embodiment the amorphous vitreous matrix comprises D-mannitol and glycine, trehalose and/or inulin. Alternatively, it may comprise a polymer such as a dextran, or PVA or PVP, or any known glass-forming material such as those known in freeze-drying which freeze-dry.
[00036] The dry powder formulation particles will have at least a portion of the L-leucine located on the surface. In a preferred embodiment the surface will comprise at least 50% coverage by L-leucine, more preferably more than 75% and most preferably more than 90%. The assessment of the presence of L-leucine on the surface can be measured directly using a technique such as ToFSIMS (Time-of-Flight Secondary Mass Spectrometry) or XPS (X-ray photoelectron spectroscopy). Alternatively, it can be evaluated by reversed-phase gas chromatography. A preferred method of evaluating is the indirect method outlined below by measuring the cohesion of the powder.
[00037] As mentioned above, it is believed that the concentration of L-leucine on the surface acts to protect the dry particles from agglomeration and moisture ingress.
[00038] The dry powders of the present invention can be administered using equipment and techniques known in the art. In this regard there are many inhalation devices described in the art for the purpose of allowing a patient to inhale a dry powder and this equipment can be used for administering the dry powders of the present invention.
[00039] Dry powder inhaler devices (DPIs) are well known in the art and there are a variety of different types. In general, the dry powder is stored within the device and is extracted from the storage place in the actuation of the device, as a result of which the powder is expelled from the device in the form of a cloud of powder that must be inhaled by the individual. In most DPIs, the powder is stored in a unitary manner, for example in blisters or capsules that contain a predetermined amount of the dry powder formulation.
[00040] Some DPIs have a powder reservoir and powder doses are measured inside the device. These reservoir devices may be less favored when treatment is likely to be one or a small number of doses in a single treatment.
[00041] Dry powder inhalers can be passive or active. Passive inhalers are those through which the powder is aerosolized using the flow of air drawn through the device by patients breathing in, and active devices are those through which the powder is aerosolized by a separate source of energy, which , for example, may be a source of compressed gas such as Nektar's Exubera device or Vectura's Aspirar device, or a form of mechanical energy such as vibration (such as the Microdose device) or impact.
[00042] Dry powder inhaler devices suitable for use in the present invention include "single dose" devices, for example the Rotahaler (trademark), the Spinhalcr (trademark) and the Diskhaler (trademark) in which individual doses of the powder composition are introduced into the device, for example, in single dose capsules or blisters. Devices can be presented as pre-measured eg with powder in a blister strip (as with the GSK Diskus device) where the pre-measured format comprises multiple doses) or where the patient inserts a pre-measured external dose form , such as a capsule containing the medicine (eg Handihaler by Boehringer Ingelheim, or Single-dose by Miat).
[00043] Alternatively, the device may be a reservoir device, where the powder dose is measured into the device from a powder reservoir during patient handling (eg Astra's Turbuhaler). Any of these types of inhaler device can be used.
[00044] The device preferably may be a single use device, or one that is intended for use with a small number of doses, and may be disposable. For example, the Twineer device, the Direct Haler device, the TwinCaps device or the Puff-haler. An advantage of these devices is their simplicity, small number of components and low cost. Preferably a device with less than 10 independent components is preferred. More preferably 5 or less, most preferably 3 or less.
[00045] There are several factors associated with delivery devices that will affect the efficiency of the dosage obtained. First, there is dose extraction. Additionally, the dynamics of the generated dust cloud will also affect the dosage release. Preferably, the device will allow high emitted dose, and high deagglomeration efficiency. High deagglomeration efficiency is often associated with high levels of powder impaction in the actuation. The device may have a low to medium or high airflow resistance.
[00046] It should be appreciated, that the compositions of the present invention can be administered with passive or active inhaler devices.
[00047] The release of biologically active protein and peptides into the pulmonary pathway using an inhalable dry powder formulation requires the solubilization of the particles when they come into contact with the mucous membrane of the lungs and the subsequent release and absorption of the protein or peptide. Although L-leucine is believed to provide a form of hydrophobic coating to spray-dried particles sufficient to provide improved stability and shelf life, particularly in hot and humid environments, it has surprisingly been found that this does not interfere with the ability of the powders to release proteins and peptides for lung absorption. Previously, it has been shown that coating particles with hydrophobic excipients can delay dissolution for significant periods, as in WO 01/76575. In fact it has been found for a spray dried formulation comprising oxytoxin that the oxytoxin absorption and onset time is particularly fast, and considerably faster than current methods and formulation used to administer oxytoxin during parturition. Consequently, it has surprisingly been found that the release of oxytoxin via inhalation into the lung provides significant advantages over current routes of administration.
[00048] Accordingly, in another aspect of the present invention there is provided a dry powder for inhalation comprising oxytoxin and/or a derivative thereof, and a pulmonary acceptable carrier, such as an amorphous glassy matrix for the oxytoxin, wherein more than that 40%, preferably more than 50%, more preferably more than 60% and most preferably more than 65% of the dry powder particles on inhalation have an aerodynamic diameter of less than 5 µm, more preferably less than 3 µm. Preferably the mass median aerodynamic diameter (MMAD) of the generated aerosol mist is less than 5 µm, more preferably less than 3 µm, more preferably still less than 2.5 µm, and most preferably less than 2 µm .
[00049] Dry powder formulations according to this aspect of the invention are especially suitable for use in the treatment or prevention of postpartum hemorrhage (PPH). The invention also provides the use of oxytocin and/or an oxytocin derivative in the manufacture of a dry inhalation powder for the treatment or prevention of PPH. The invention also provides a method of treating or preventing PPH which comprises administering to a subject in need thereof by inhaling an effective amount of a dry powder comprising oxytocin or an oxytocin derivative. Preferably the dry powder formulation is in a form described above.
[00050] In a preferred embodiment of this aspect of the invention the amorphous glassy matrix comprises one or more mono-, di- or polysaccharides and/or amino acids, and most preferably the matrix will include L-leucine, preferably in amounts and proportions such as described above. However, in other embodiments the amorphous vitreous matrix comprises an inert polymer suitable for pulmonary delivery, such as a polyvinylpyrrolidone or polyvinyl alcohol or polyethylene glycol or propylene glycol polymer or copolymers. Matrices composed of these polymers can also include components such as leucine, isoleucine or trileucine to improve the stability of the particles and to ensure that they have an appropriate aerodynamic diameter.
[00051] According to this embodiment the dry powder may further include one or more other physiologically active agents and further include one or more pharmaceutically acceptable and pulmonary acceptable components such as those described above. In a preferred embodiment the pulmonary acceptable carrier includes sodium citrate, or a stabilizer for the oxytocin component.
[00052] Micronized particles according to this aspect of the invention are typically prepared by spray drying as described above under suitable conditions which can be determined by the skilled person. The term "spray drying" is intended to encompass any process in which a solution or suspension of one or more solutes is formed in a liquid, whereby the liquid is physically atomized into individual droplets which are then dried to form a powder dry particulate. It may encompass any form of a droplet-to-particle formation process, and may encompass related processes such as spray-freeze drying, spray cooling and flash spray drying. Droplets can be formed by any known atomization process, including but not limited to pressure atomization, pneumatic atomization, dual or multiple fluid atomization, rotating disk atomization, electrohydrodynamic atomization, ultrasonic atomization, and any variant of such processes of atomization. Atomization can occur from one spray source or multiple sources. The liquid carrier spray may be aqueous or non-aqueous and may optionally comprise co-solvents plus additional components dissolved or suspended. The liquid can include a material that is a vapor or solid under ambient conditions but exists as a liquid under selected process conditions. The droplets formed can be dried by applying heat in the form of a heated drying gas, or heat can be applied in other ways, for example, radiatively from the walls of the drying chamber or as microwaves. Once collected from this drying process, the particles can be further dried or conditioned to a controlled moisture level through a process such as vacuum drying or freeze drying. Alternatively drying can be achieved by freezing followed by drying or applying a vacuum.
[00053] It will be recognized that any other means of obtaining such particles are also considered herein, for example supercritical fluid synthesis, emulsion synthesis and any other form of controlled precipitation which forms substantially spherical particles. Alternatively, any form of size reduction, friction, grinding and grinding can be used to obtain properly sized particles.
[00054] Particles can be obtained and engineered in any known particle engendering system, such as but not limited to the following: Pulmosphere® or Pulmosol® technologies developed by Nektar, AIR® Beveled porous particle technology by Alkermes, Technosphere® technology developed by Mannkind, Powderhalem technology developed by Vectura, particles created by Prosonix sound crystallization methods, particles created by wet or dry nanogrinding technologies for example developed by Elan, Hovione or Savara.
[00055] The micronized particles of dry powder for inhalation are of a suitable size for aerosolization and inhalation, having a physical size of less than 15 µm, such as less than 10 µm, or less than 6 µm, or less than 5 µm, or less than 3 µm or less than 2 µm. Particles according to this embodiment will have a mass median aerodynamic diameter of less than 10 µm, but preferably less than 5 µm, or less than 3 µm.
[00056] Typically, in addition to the size equivalents discussed above, 90% of the particles by volume may have an aerodynamic diameter of less than 10 μm, less than 8 μm, or less than 6 μm or less than 5 μm or less than 3 µm. Mass mean aerodynamic diameter can be measured by a pharmacopeial impactor method as defined by the United States Pharmacopoeia, using an Andersen Cascade Impactor, or by the Next Generation Impactor (NGI). Particles according to this embodiment may have a mass median diameter of less than 5 µm, or less than 3 µm, which can be measured by a laser light scattering method, such as using a Mastersizer instrument 2000 from Malvern.
[00057] In order to obtain a high efficiency in aerosolization it is also advantageous for the particles to exhibit a low level of cohesion. Typically, cohesion can be measured using a powder shear cell test, such as the Freeman FT4 powder rheometer shear cell. Advantageously, a powder could exhibit an average cohesive value of less than 2, more preferably less than 1.5 and most preferably less than 1.
[00058] In another embodiment, respiratory release of oxytocin and/or its derivatives may also include nasal release. Nasal delivery involves inspiration through the nose but where dust is primarily collected in the nasal cavity and nasal turbinates, and where absorption into the systemic circulation also occurs. Nasal inhalation is similar to pulmonary administration as it provides a non-invasive route of delivery to the systemic circulation. Nasal release avoids needles and allows for repeated administration from a single device.
[00059] Dose ranges can be easily calculated and administered without concern for fluid volume (not volume dependent). Delivery devices for nasal powder delivery may differ from those required for pulmonary delivery. Examples include devices from Optinose, the ViaNase (Kurve), the Direct-Haler, The Monopowder (Valois) or the Bespack nasal powder systems. The mass average particle size for nasal delivery is preferably in excess of 5 µm and more preferably greater than 10 µm as this reduces the passage of material through the nasal cavity and maximizes its deposition in nasal turbinates. Nasal formulations will include nasally acceptable carriers and may include additional excipients such as bioadhesive polymers and penetration enhancers, for example chitosan, HPMC or carbopols. It is an especially preferred embodiment that oxytocin or one of its analogues or derivatives can be delivered as a formulated powder comprising a glassy matrix via the nose, in a mass average particle size greater than 10 µm for treatment of PPH, or other maternally related conditions such as postnatal depression, and preferably have a rapid onset of action. Dry powders prepared for pulmonary delivery can also be inhaled via the nasal passage into the pulmonary system, for example where a patient has difficulty inhaling the powder through the mouth, although this is not a preferred mode of delivery of such powders. .
[00060] Oxytocin is the uterotonic agent of choice according to the World Health Organization for use in the active control of the third stage of labor due to the speed of onset of action, minimal side effect profile and lack of contraindications. According to the clinical practice guidelines of the Royal Women's Hospital (Melbourne, Aus) prophylactic oxytocin should be administered to the woman with the birth of the anterior shoulder or within one to two minutes of the birth of the baby. Timing of administration is critical in preventing uterine atony, and there is a clear benefit in providing a more rapid onset form following such a birth to reduce the risk of onset of blood loss.
[00061] In addition to the prevention of PPH, oxytocin is used as a treatment for PPH at a dosage of 30 to 40 IU as an intravenous infusion. In well-controlled clinical settings, low dose oxytocin (0.5 to 1 mU/ml, IV infusion) is indicated for the initiation or improvement of uterine contractions, where this is desirable and considered appropriate for reasons of fetal or maternal concerns, so to obtain vaginal release.
[00062] More recently the scientific and medical literature has reported a link between low endogenous oxytocin levels and postnatal depression, and there may be an advantage in developing a practical, cost-effective, non-invasive form of oxytocin in its derivatives therapeutically effective for systemic delivery in or around this area of indication.
[00063] In developing countries the parenteral administration of oxytocin the practice encounters several obstacles including the need for cold chain storage to prevent chemical degradation, the need for trained medical personnel to administer IV or IM injections, or the potential for syringes to be reused in an effort to reduce costs which increases the likelihood of bloodborne virus transmission and a general lack of access to high quality oxytocin products for the prevention and/or treatment of PPH. In an effort to increase access to oxytocin, skilled birth attendants in some countries are trained to administer IM injections of oxytocin, however there are numerous reports of inappropriate use of the parenteral product to increase labor. An inhaled oxytocin formulation would overcome many of these limitations associated with parenteral formulations. A single unit dosage form system would eliminate the potential for use in increasing labor as it would not be possible to deliver the low dosage required. Providing oxytocin in an inhalable dry powder form also provides more flexibility with dosing in view of the absence of a liquid or gas carrier and the lack of volume dependence. It also avoids the need for needles, minimizes the risk of contamination, and allows the flexibility for repeated administration of a single device.
[00064] The rapid onset of action obtained through the dry powder formulations of the present invention provides the ability to rapidly titrate the dosage during PPH treatment where the usual therapeutic dose is between 30 and 40 IU. Midwives/birth attendants will typically feel the uterus to assess when the magnitude of contraction is sufficient to control PPH. The rapid absorption and onset of action (as demonstrated in a sheep model) indicates that it would be possible for multiple inhaled doses to be administered to obtain the necessary plasma levels required for effective uterine contractions. Oxytocin's broad therapeutic index is also beneficial for inhaled therapy when variability in delivery or absorption efficiency would not cause serious side effects in patients.
[00065] According to the present invention the onset of action following inhalation of a dry powder comprising oxytocin and/or an oxytocin derivative within the pulmonary system, as measured by uterine contraction can be obtained within 200 seconds of inhalation , preferably within 150 seconds of inhalation, more preferably within 100 seconds of inhalation, and most preferably within 60 seconds of inhalation. Preferably the time between pulmonary administration of a dry powder formulation according to the invention and the onset of uterine contractions compared to the onset of action following IM injection of a solution of oxytocin and/or an oxytocin derivative is less than 80%, preferably less than 60% and most preferably less than 40% of the time between IM injection and onset of uterine contractions.
[00066] A wide range of peptides and proteins can be formulated according to the present invention. The present invention is particularly suitable for the administration of oxytocin or derivatives, and similar peptides such as vasopressin and desmopressin. Examples of oxytocin derivatives include desamino-oxytocin, those described in Endocrinologica Experimentalis Vol 14, p 151, 1980, and oxytocin agonists such as carbetocin which have similar uses to oxytocin, focusing on controlling bleeding after release. Other oxytocin derivatives include desamino-1-monocarba-(2-O-methyltyrosine)-oxytocin, syntometrine and atosiban [d(CUMOT)]. However, the invention can also be used to provide dry powder formulations of other proteins that include insulin and vaccines such as an influenza vaccine. In the case of vaccines, the formulation may also require additional immunostimulatory components. Examples of proteins and peptides that can be formulated in accordance with the present invention and released via the pulmonary route include cytokines, hormones, clotting factors, vaccines, and monoclonal antibodies.
[00067] The following is a list of proteins that can be used as the active agent in the compositions and processes according to the present invention. Calcitonin, erythropoietin (EPO), Factor IX, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), growth hormone, type I insulin, interferon alpha, interferon beta, interferon gamma, interleukin-2, luteinizing hormone releasing hormone (LHRH), somatostatin analogue, vasopressin analogue, follicle-stimulating hormone (FSH), amylin, ciliary neurotrophic factor, growth hormone releasing factor (GRF), hormone insulin-like growth, insulinotropin, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-6, macrophage colony stimulating factor (M-CSF), nerve growth factor, parathyroid hormone, analogue of somatostatin, thymosin alpha I, IIb/IIIa inhibitor, antitrypsin alpha, relaxin, anti-RSV antibody, cystic fibrosis transmembrane regulator (CFTR), deoxyribonuclease (DNase), bactericidal/permeability enhancing protein ( BPI), anti-CMV antibody and interleukin-1 receptor.
[00068] The invention will now be described with reference to some specific examples and drawings. However, it is to be understood that the feature of the description that follows is not to replace the generality of the invention as described above.
[00069] Referring to the drawings:
[00070] Figure I is a schematic representation of an apparatus for spray drying.
[00071] Figure 2 is a schematic representation of a dual stage booster for measuring in vitro aerosol deposition of dry powders.
[00072] Figure 3 provides X-ray diffractograms of crude trehalose, Trehalose (crude) and spray dried trehalose (SD) after spray drying under the specified conditions.
[00073] Figure 4 provides X-ray diffractograms of spray-dried trehalose and spray-dried trehalose with 10% and 20% w/w leucine after spray drying under the specified conditions.
[00074] Figure 5 is a scanning electron microscope image of trehalose after spray drying under specified conditions.
[00075] Figure 6 provides scanning electron microscope images of trehalose with 10% w/w leucine (left) and 20% w/w (right) after spray drying under specified conditions.
[00076] Figure 7 is an EMG trace showing uterine contraction following inhalation of oxytocin. The arrow indicates the delay between administration and contraction. The initial overflow is surrounded with a dotted line. The black line represents a random thirty-minute sample of oxytocin-induced activity. The dashed line represents the total time of oxytocin-induced uterine activity taken before returning to baseline.
[00077] Figure 8 is a series of graphs that provide an analysis of the behavior of Uterine EMG during the immediate postpartum period and following intrapulmonary (IP) and intramuscular (IM) oxytocin release. (a) Delay between oxytocin administration and EMU response; (b) Duration of first EMG burst; (c) Number of EMG bursts in the first 30 minutes; (d) Total duration of EMG activity. Data expressed as mean ± SEM. P > 0.05 RM. THE NEW; n = 5. Green bars represent uterine activity immediately after delivery, blue bars represent uterine activity after dry powder delivery, and red bars represent uterine activity after intramuscular administration.
[00078] Figure 9 shows bronchoscope video images of a sheep trachea before (a) and after (b) powder release.
[00079] Figure 10 shows non-polar surface energy distributions at finite dilution in reverse gas chromatography.
[00080] Figure 11 shows surface polar energy distributions at finite dilution in reverse gas chromatography.
[00081] Figure 12 shows total surface energy distributions at finite dilution in reverse gas chromatography.
[00082] Figure 13 shows the work of surface cohesion energy distributions determined in finite dilution in inverse gas chromatography.
[00083] Figure 14 shows surface energy at infinite dilution in reverse gas chromatography.
[00084] Figure 15a is an EMG trace showing uterine contraction following inhalation of the oxytocin released by formulation 1 of example 10 containing mannitol; glycine and leucine in equal amounts. The dashed line shows the formulation release time and the solid line the start of uterine contraction. The x-axis is in hh:mm:ss and the y-axis is in mV.
[00085] Figure 15b is an EMG trace showing uterine contraction following inhalation of oxytocin released by formulation 2 of example 10 which contains 90% trehalose and 10% leucine. The dashed line shows the formulation release time and the solid line the start of uterine contraction. The x-axis is in hh:mm:ss and the y-axis is in mV.
[00086] Figure 16a is an EMG trace showing uterine contraction following inhalation of oxytocin released by formulation 3 of example 10 which contains 90% PVP(30) and 10% leucine. The dashed line shows the formulation release time and the solid line the start of uterine contraction. The x-axis is in hh:mm:ss and the y-axis is in mV.
[00087] Figure 16b is an EMG trace showing uterine contraction following intramuscular release of oxytocin (formulation 4 of example 10). The dashed line shows the formulation release time and the solid line the start of uterine contraction. The x-axis is in hh:mm:ss and the y-axis is in mV. EXAMPLES.Example 1 Spray drying
[00088] Spray drying is a single step process involving the formation of powders from a starting solution of the desired dissolved material. By definition, it is the transformation of the feed from a fluid state to a dried form by spraying the liquid feed into a hot drying medium. The four key stages in the spray drying process are: (i) atomization of the feed through the nozzle, (ii) spray-contact the air between the liquid droplets and the drying gas, (iii) drying of the particles through evaporation of liquid, and (iv) collection of the final powder.
[00089] Referring to the schematic in Figure 1, air is collected within system 1 and heated by the provided heater 2 before measuring the inlet temperature 3. The liquid feed is pulled up separately into the nozzle 4 where the droplets are formed and dispersed within the drying chamber 5 which are mixed with hot air. At this point, a dry particle is formed. The exit temperature is measured 6 as the particles move inside the cyclone 7 where the dust is separated from the air. The powder is trapped in the collection vessel 8 to be recovered while the air becomes filtered from any fine particles that may have remained in the air stream in the bag filter 9. The air circulation in spray drying is continued for the work of the vacuum 10.
[00090] Atomization is a very crucial part in defining the droplets, and hence the subsequent particle size and distribution. It involves the formation of a spray of droplets from the raw liquid as the feed is pumped through a small orifice in the nozzle. In the case of a dual fluid nozzle, the supplied gas collides with the liquid volume in the nozzle at high speeds. This high velocity gas creates high frictional forces on liquid surfaces, causing the liquid to disintegrate and form spray droplets, which project into the drying chamber.
[00091] Dissolved material properties and drying conditions will influence final powder characteristics. With evaporation of the liquid solvent from the droplet surface (water in this case) the precipitation of the solute takes place. Often as the particle is forming, a scab can form and the scab can be porous, semi-porous or non-porous which allows moisture removal at different rates and with varying effects. Particles of variable morphology can therefore form. Controlling drying conditions is therefore an important consideration.
[00092] According to the experiments carried out, the formulations of the powders and their spray drying parameters were varied. In all powder formulations, mannitol was used as the baseline material, with variable amino acids added. The spray drying parameters that remained constant throughout were the aspirator setting, setting in full flow and the atomizer air flow rate (800 L/hour).
[00093] For each formulation, mannitol, glycine and oxytocin were spray dried in fixed amounts with varying amounts of leucine. The spray drying conditions were fixed by adjusting the outlet temperature to 70°C.
[00094] The parameters that were used are shown in Table I below. The percentages of amino acids shown were calculated relative to those of the amount of mannitol only, not the total powder content. The percentage of oxytocin shown was that of the total powder content.

[00095] The powders were weighed and dissolved in the appropriate amount of Milli-Q water to obtain the desired feed concentration. The solutions were then spray dried to produce dry powders using the 190 Mini Spray Dryer from Buchi (Buchi, Switzerland). Example 2 In Vitro Aerosol Deposition
[00096] The in vitro aerosol deposition of the powders was measured using the Double Stage Booster (TSI) (Copley Scientific Ltd., Nottingham, UK). The TSI methods and adjustments were performed according to the British Pharmacopoeia 2011 as shown in Figure 2. In the glassware of part D and part H, 7 ml and 30 ml of water respectively were added during the assembly of the TSI.
[00097] The TSI is a simple model of the respiratory tract; with the upper (stage 1) and lower (stage 2) chambers representing the upper and lower airways respectively. The aerodynamic cutting diameter in the first stage is 6.4 µm. Particles larger than 6.4 µm should ideally be collected in the 7 ml of liquid; smaller particles (6.4 µm) that are not collected will proceed to the lowest stage, which contains 30 ml of liquid. Most of the particles will be collected in the lower stage due to excess liquid, however if the particle size is too small for collection in the lower stage, it will be withdrawn at the exit.
[00098] Measurements for each powder sample were made in four replicates. For each replicate, five size 3 HPMC capsules were manually filled with 20.4 ± 0.24 mg with the sample powder and placed in five Monodose inhalers (Miat. Italy). A vacuum pump was attached to part F and the air flow rate was calibrated to 60 L/min and was adjusted to 5 seconds. The capsule was pierced in the device and placed in the adapter (part A) ready to be activated by the vacuum pump. When the pump was turned on, the powder was loaded from the inhalation device into the TSI device.
[00099] All five capsules were activated in the same TSI. The used capsules and inhalers were then washed with Milli-Q water in a 100 ml volumetric flask and made up to volume. This was called the 'residual' stage. The parts that make up Stage 1 (parts A. B, C and D) were washed in a 200 ml volumetric flask and the parts that make up Stage 2 (parts E, F, G and H) were washed in a volumetric flask of 50 ml with Milli-Q water and called 'stage 1' and 'stage 2' respectively. The amounts of oxytocin at each stage of the TSI were determined by the LC/MS assay. The fine particle fraction (FPF) was calculated as the amount of powder that reached stage 2 of the TSI apparatus divided by the total amount of drug that was tested. This test was the most important measure as it can determine whether a powder containing oxytocin can be formulated with adequate aerosol deposition, and consequent absorption from the lung.

[000100] Particles passing into the lower portion of the TSI device ie stage 2 were considered to be respirable, so the higher the fine particle fraction (FPF), the higher the chance of the drug reaching the alveoli and become absorbed into the bloodstream, which is ideal in an IPD. The FPF of the five tests shown in Table 2 was high compared to an average FPF of powders formulated with traditional carriers (~10 to 20%).
[000101] The results showed that the FPF can reach between 55 and 75% which means that very efficient levels of aerosolization were obtained and high amounts of oxytocin in the formulations were delivered as the required therapeutically active dose. Oxytocin stability
[000102] Peptides can potentially be denatured due to extreme heat. From these tests that were conducted in this study, the only indication as to the stability of oxytocin was the content of the LC/MS assay that follows the TSI experiments. When oxytocin content was assayed from all stages of the TSI apparatus, capsules, and inhalation devices, an average of 90.23 ± 5.41% of the initial capsule dose was recovered, suggesting that oxytocin was not degraded from temperatures used in the spray drying process or handling processes. Example 3 Trehalose/Leucine
[000103] Trehalose is a non-reducing sugar with a high glass transition temperature (Tg) of 117 °C that has been used as an excipient in several studies for protein stabilization in dry solid state formulations. Sugar molecules are generally used as stabilizing excipients in this context as they contain carboxyl groups which are able to form hydrogen bonds with the protein of interest and therefore stabilize the bio-macromolecule with the replacement of the hydrogen bond in the dry solid state. . Spray drying has been used successfully in several studies for the manufacture of inhalable dry powder formulations as the process is capable of producing fine particles with a particle size range that is suitable for pulmonary delivery.
[000104] In an attempt to formulate inhalable pharmaceutical proteins for pulmonary delivery, spray-dried trehalose is produced at a relatively low exit temperature of 70 °C in order to minimize the impact of heat stress on the processing stability of the relevant protein. Conditions were otherwise the same as the examples described with mannitol and amino acids.
[000105] Although trehalose is relatively crystalline as a raw material, the spray-dried trehalose under the specified spray-drying conditions appears to be entirely amorphous (see Figure 3). This resulting formulation can further stabilize the protein of interest with stabilization in the glassy state by providing an amorphous matrix that reduces the molecular mobility of the bio-macromolecule in the formulation.
[000106] The resulting trehalose-only formulation is, however, composed of primary fused structures with large particle size that are unlikely to be suitable for pulmonary delivery (Figure 5). Incorporating leucine into the formulation at a concentration of 10% w/w substantially improves the particle size and morphology of the spray dried formulation under the specified conditions (Figure 6). Leucine incorporation appears from this to be able to prevent fusion of the otherwise highly hygroscopic primary particle structures of trehalose from the spray drying process, and therefore retains the fine particle size range suitable for pulmonary delivery. . In addition, the presence of leucine is also able to improve these particle properties during longer term storage by providing a protection against moisture from the internal amorphous matrix of spray dried trehalose formulations (Figure 4). Example 4 Materials
[000107] D-Manitol was obtained from VWR International Ltd. (Poole, BH15 LTD, England). L-leucine (LEU), glycine (GLY) and L-alanine (ALA) were obtained from Sigma-Aldrich Chemicals (Castle Hill, NSW, Australia). Preparation of spray dried powders
[000108] Aqueous solutions containing mannitol and selected amino acids (LEU, GLY, ALA) in various compositions as shown in Table I were dissolved in 200 ml, of Milli-Q water. A small amount of methylene blue (10 mg) was incorporated into each formulation to allow simple powder quantification by UV-VIS spectrometric analysis as described below. The prepared formulations were subsequently spray dried using a Buchi 190 mini spray dryer with a 0.5 mm dual fluid nozzle, using the following standard operating conditions: air flow rate, 800 L/h; pump adjustment, 5 (6.67 ml/min); vacuum adjustment, 20; outlet temperature, 75 °C. Particle size distribution analysis
The particle size distribution of the powders was determined by laser light scattering using the Malvern Mastersizer 2000 (Malvern Instruments Ltd, Worcestershire. UK) equipped with a Scirocco cell and a Scirocco 2000 dry powder dispersion unit. The powders were dispersed in air at a shear pressure of 3.0 to 4.0 bar which was selected to obtain adequate deagglomeration. Average particle size was measured in three replicates for each sample. The volume mean diameter (D50) was derived from the diffraction data using the built-in software for each sample. Powder aerosolization and in vitro particle deposition
[000110] The performance of powder aerosolization and in vitro particle deposition was evaluated using a dual stage booster (TSI, Apparatus, A; British Pharmacopoeia, 2000) with the single-dose inhaler (Miat SpA, Milan, Italy) as the aerosol dispersing device. The flow rate was adjusted to 60 L/min using a Model TPK 2000 Critical Flow Controller & Model DFM 2000 Flowmeter (Copley Scientific Limited, Nottingham, UK). Approximately 20 mg of each powder was filled into size 3 HPMC capsules (Capsugel, Peapack, NJ, USA) for the tests that were performed in an air-conditioned laboratory (20 ± 2 °C, 50 ± 5% relative humidity) . Each capsule was actuated from the inhaler within 4 seconds for each measurement (n = 5). The amount of powder deposited at different stages was determined using a UV-VIS light spectrophotometer as described below. The cutoff diameter for the TSI at 60 L/min is approximately 6.3 µm (Hallworth and Westmoreland, 1987).
[000111] The total amount of powder deposited in the inhaler, stage I (S1) and stage 2 (S2) was the recovered dose (RD). The amount of powder deposited in stages 1 and 2 was the emitted dose (ED) and was calculated as the percentage of DR (Eq. 1). The fine particle fraction (FPF) was defined as the percentage of DR deposited in stage 2 (Eq. 2).
Scanning Electron Microscopy (SEM)
[000112] The morphology of the particles was visualized under a scanning electron microscope (Phenom®, HI company, USA). The powder samples were gently poured onto a double-sided carbon tape mounted on a sample holder for examination under the SEM. Excessive dust has been removed to leave a thin layer of particles on the tape surface. The samples were ejection coated with gold using an electrical potential of 2.0 kV at 25 mA for 6 minutes with an ejection coater (K550X, EMITECH). SEM micrographs were captured using the built-in image capture software. Results
[000113] The volume mean particle size (D50) of all formulations measured using Mastersizer 2000 are listed in Table 4. Spray drying of mannitol alone produced small particles with a D50 of 1.87 µm. However, this powder was fully crystalline and lacked the amorphous glassy structure required to stabilize the biomolecules.
[000114] Leucine is an excipient that can be used to improve the aerosolization of spray dried particles, but also leucine helps in the formation of suitable small size particles. However glycine and alanine, while being structurally similar to leucine, were not able to achieve similar effects as they significantly increase the particle size of the formulations. It is noteworthy that although the initial concentration in the fed solution is a known determinant of particle size, the solid charge range used within the study design space does not appear to have a strong influence on the geometric particle size as measured by laser diffraction. The total solid charge in the fed solution ranged from 2.50% to 3.72% in the present study. It is proposed that the change in particle size within its relatively small solid loading range was negligible compared to the effects of formulation excipients on cohesion and shape. Furthermore, considering the particle sizes produced from the mixed amino acids, it is possible that the use of combinations of these amino acids with leucine in appropriate concentrations may also influence the particle size in contrast to that obtained by leucine alone. Dispersibility and deagglomeration of powder
[000115] Spray-dried mannitol produced particles with a D50 of 2.83 µm which appear to indicate satisfactory dispersibility for inhalable dry powder formulations, but it also produces the lowest emitted dose (ED). The retention of powder in the device after the experiment was visually evident, and suggests a more cohesive powder than the other formulations here. The presence of amino acids in all combinations resulted in the improved ED (Table 4). The beneficial effect of leucine was evident in its ability to offset the effect of the other two amino acids on D50 and to improve both deagglomeration and ED. Aerosolization and in vitro particle deposition
[000116] The TST was used as a preliminary screening of this range of formulations to provide aerodynamic aerosol information.
[000117] Fine particle fraction (FPF) results showing formulations containing leucine, with D50 below 5 µm demonstrate the highest FPF of more than 68% (Table 4). Powders containing amino acids without leucine, with D50 above 5 µm show significantly lower FPF as demonstrated by formulations containing 30/30% glycine/alanine, 30% alanine and 30% glycine, with 2.96% FPF, 9 .11% and 34.62%, respectively. Although mannitol alone shows a reasonable FPF of 66.20%, this formulation also demonstrates the lowest ED). The combined amino acids at 15% were most effective in improving FPF (Table 4). These results suggest that the inclusion of glycine and alanine with leucine at the appropriate concentrations can improve the aerosolization performance of the formulation. surface morphology
[000118] Spray dried mannitol as a foundation material alone has been observed to form small spherical particles which are heavily agglomerated. The result matches the Mastersizer particle size distribution data. Upon addition of amino acids, the spherical particles were preserved in all formulations containing leucine regardless of the presence of glycine and/or alanine. Other formulations containing glycine and/or alanine without the addition of leucine formed much larger irregularly shaped particles with rough surfaces.
[000119] The result suggests that the presence of leucine aids in the formation of spherical particles by coating the surface of the particle on drying, and therefore providing a protective shell that preserves the individual particles as they come together, preventing any melting, while the presence of glycine and alanine did not prevent this effect. The above results indicated that a relatively high concentration of leucine (i.e., >5% w/w) tends to lead to corrugated particles. The morphology of the leucine-containing particles in the present study appears to behave differently. The leucine concentrations used within the study design space (15 to 30 mol%), which correspond approximately to 10 to 18% w/w, did not form corrugated particles. It is therefore speculated that the presence of glycine and/or alanine altered the core structure of the spherical drying particles, whereas leucine tended to reside on the particle surface, providing a coating to reduce surface cohesiveness and prevent melting in the drying process.
[000120] In the present study, leucine was able to enhance the aerosolization performance of mannitol formulations without requiring the formation of corrugated particles. Furthermore, the FPF results suggest that this may be advantageous.

[000121] Abbreviation: ED, emitted dose; SD, spray drying; TSI, dual stage booster.
a Indicates the central point of planning. Abbreviation: n/a, not available. Example 5 In vivo test
[000122] On day 135 of gestational age, pregnant ewes (n = 5) were anesthetized with thiopentone in preparation for surgery. Isufloran (2.5% in oxygen) was used to maintain anesthesia and depomycin, procaine and penicillin, and dihydrostreptomycin were given for pain relief and to reduce the risk of infection. Each sheep was shaved and a 10 cm incision was made in the abdominal skin at the midline below the umbilicus to expose the uterine wall, with care taken to avoid large blood vessels.
[000123] Three sterile stainless steel wires to measure electromyographic (EMG) activity (0.07 mm in diameter, inside a 2 mm catheter) were embedded in the smooth muscle layer of the myometrium surrounding the uterus and held there for two points. The electrodes were passed through a catheter and out of the sheep through a small (2 cm) incision through the right flank. A catheter was inserted into the right jugular vein to allow blood samples and to induce labor. Ewes were returned to metabolic cages and given 3 to 5 days to recover from surgery.
[000124] Labor was induced with two intravenous injections of 5 ml dexamethasone (consisting of 5 mg dexamethasone phosphate and 10 mg dexamethasone phenylpropionate) 24 hours separately. Labor occurred 54 ± 2 hours after the first dexamethasone injection.
[000125] Oxytocin administrations, as detailed above, were performed within 15 hours of release. Each sheep received an intratracheal dose of oxytocin dry powder formulation, an intratracheal instillation of oxytocin in solution, and an intramuscular injection of oxytocin. There was at least a one-and-a-half hour break period between each treatment.
[000126] For intratracheal administration; an endoscope was passed through the nasal passage into the trachea and positioned close to the first bronchial bifurcation, where a 1 ml aliquot of oxytocin in solution (10 IU) was delivered or 10 mg (average) of dry powder was delivered through a modified PennCentury powder release device.
[000127] The dry powder formulation comprised a spray-dried composition as described in Example 1. This powder contained 13 units of oxytocin per mg by mass, co-spray dried with equal mass proportions of mannitol, glycine and leucine.
[000128] During this procedure, bronchoscope video images were captured using a Linvatec IM3301 Pal Video Camera attached to an endoscope (Pentax FU-16X), which were saved as a digital file on a computer using Video Capture Software. Examples of images are provided in Figures 9a and 9b. The image in Figure 9a shows the sheep trachea before powder release, and Figure 9b shows the image approximately 30 seconds from release. Figure 9b shows clear evidence of white undissolved powder, as white spots not present prior to release, indicating that immediate dissolution did not occur within the pulmonary system.
[000129] A cyberamp 380 in conjunction with MACLAB hardware (400 Hz sample rate) and Chart 4 software (10V input range) was used to demonstrate and record the action potential originating from smooth muscle cells within the uterus. Cyberamp 380 used probe A1401 with positive input set on AC and negative input set on Earth. The AC cutoff was at 10 Hz and the prefilter gain set at 100 mV. The low pass filter was set at 300 Hz, the slit filter set turned off, the output gain set at 5 and the total gain left at 500. Two repeated path ANOVA measurements were performed to determine the statistical significance of our Dice. See Figure 7.
[000130] Various properties of recorded activity were analyzed. Considering the elapsed time (delay) from oxytocin release to the initial burst of EMG activity, release via the lungs results in a faster onset time for the first contraction in contrast to IM release (Figure 8a). No difference was seen in the duration of the initial burst of EMG activity (Figure 8b) nor was there a difference in the number of bursts that occurred during the first 30 minutes following the initial burst of activity (Figure 8). However, the total duration of EMG activity was significantly longer for MI of oxytocin compared to airway release and normal activity observed immediately after delivery (Figure 8).
[000131] These in vivo studies demonstrate that uterine contractile responses to oxytocin administered via pulmonary release occur on average after approximately 120 seconds in contrast to IM release which occurred on average after approximately 250 seconds. Surprisingly, the onset of action of pulmonary powder release was significantly faster compared to intramuscular release, and was also consistent with plasma versus time profiles. The mean onset of action was approximately 50% less than for IM. This is despite the fact that the dry powder particles contain approximately 30% of leucine which is an insufficiently soluble and hydrophobic amino acid, which would be expected to delay dissolution. Furthermore, a substantial proportion of the leucine is expected to be present on the surface of the powder. Image 9b supports this concept that rapid dissolution of such powder is not expected in this environment. The data also demonstrate that uterine contractile responses to pulmonary dry powder oxytocin mimics the activity naturally observed in the immediate postpartum period, as seen with the duration of the initial burst of uterine activity and the total number of recorded EMG activity bursts for the next thirty minutes. Example 6 Influenza antigen dry powder formulation
[000132] The influenza antigen haemagglutinin (HA) powder sample was first dissolved with other excipients (ie mannitol 45 % w/w, glycine 45 % w/p and leucine 10 % w/w) in a solution aqueous to produce a final formulation with an antigen loading of 5 µg HA per mg powder. This solution was then spray dried in a Buchi 190 laboratory spray dryer at a relatively low temperature ie 70 degrees C outside temperature to minimize the effect of thermal stress on antigen integrity under spray drying conditions which follow: pump adjustment 6.7 ml/min; aspirator, 20 (100%); air flow, 800 L/hour. The spray-dried influenza antigen formulation was then collected from the collection vessel for storage. Example 7 Method for Testing Biological Activity of Influenza Antigen Dry Powder Formulation
[000133] The following describes the method for testing the biological activity of the spray-dried influenza antigen dry powder formulation which contains hemagglutinin (HA) as the active protein. The hemagglutination assay (HA assay) is used to test the integrity of the HA protein in the dry powder formulation. The influenza antigen dry powder formulation of interest is first reconstituted with phosphate buffered saline (P135) immediately prior to testing in a standard HA protein concentration solution. A small amount of this reconstituted solution is then placed in the first column of a 96-well plate. The solution is then diluted 1:2 with PBS across the 96-well plate by serial dilution. A standard amount of chicken red blood cell solution with a standard red blood cell concentration (i.e. 1%) is then added to each well on the plate. The plate is incubated at room temperature for 30 minutes immediately after addition of red blood cells. Since intact HA protein will cause hemagglutination of red blood cells, the level of dilution that the HA-containing solution is able to sustain before it is no longer able to cause hemagglutination within 30 minutes will indicate the amount of intact HA protein in the formulation. The antigen was found to be in excess of 95% active, within the limits of this procedure. Example 8 Measurement of particle cohesion and preferred cohesion values. Apparatus and materials
[000134] The apparatus used was the 1 ml shear cell module, and ventilated piston, as part of the FREEMAN FT4 Rheometer unit (Freeman Technology, UK) and computer user interface and freeman shear cell conditioning module. 1 ml. The materials used were spray dried powders of 1:99% w/w leucine/mannitol, 3:97% w/w leucine/mannitol, 5:95% w/w leucine/mannitol, and 10:90% w/w leu/mannitol. These powders were produced following the conditions of Example 1 above.
[000135] A powder sample was loaded into the cell and conditioned. During conditioning the 1 ml shear cell conditioning module was used to gently disturb the powder as it moved throughout the entire sample. The purpose of this was to homogenize the powder by removing excess air and isolated pre-compacted powder particles. After conditioning the powder was compressed. This was accomplished by the flat surface ventilated piston to ensure uniform particle-particle interactions. Compression was followed by shear. During shear, a 24 mm shear cell (a unitary component of: base, blade, dividing wedge and shear cell module) was used. The shear tip comprising 18 blades, moved vertically downwards to induce normal shear stresses while the shear tip blades pierced the surface of the powder. Shear stress was then measured and was at a maximum when the powder failed to withstand the shear stress. The shear stress versus normal stress graph was generated by the built-in FT4 FREEMAN software. From the graph, the extrapolated y-intercept gives the cohesiveness of the powder at zero consolidation. Data for ffc [the ratio of major principal stress (consolidation stress), o1, for unconfined production force, oc] was also recorded.

[000136] From 10 to 10 % leucine content, an increase in leucine content decreases cohesion and improves the fluidity parameter (ffc).
[000137] This experiment was then repeated using spray-dried leucine and PVP, produced and tested under similar conditions. The results were as follows:,
Example 9 Surface Energy MeasurementsSamples
[000138] Two batches of powder were spray dried from water using conditions as described in example i, but where the powders comprised compositions of pure mannitol or mannitol with 10 % w/w L-Leucine added. surface by reverse gas chromatography
[000139] The surface energies of these powders were determined using Inverse Gas Chromatography (IGC, Surface Measurement Systems Ltd, and London, UK). Approximately 0.33 g of each powder was packed into pre-silanized glass columns (300 mm x 3 mm inner diameter) which were loosely capped with silanized glass wool at both ends. The powder-filled columns were conditioned for 2 h at 303 K before each measurement in order to remove surface impurities. Probes were loaded into the column by helium at a gas flow rate of 10 sccm (standard cubic centimeter per minute) and retention times were detected by a flame ionization detector. The dead volume was calculated based on the methane elution time which was conducted at a concentration of 0.1 p/p0 (where p indicates the partial pressure and p0 the vapor pressure). Determination of surface energy in infinite dilution:
[000140] Hexane, heptane, octane, nonane, and GC grade decane (all from Sigma-Aldrich GmbH, Steinheim, Germany) for non-polar surface energy (YNP), and two polar probes (ie, dichloromethane and ethyl acetate) for the polar surface energy (YP) were used at a concentration of 0.03 p/p0. Detailed calculation of YP has been described elsewhere (Thielmann et al., Investigation of the acid-base properties of a MCM-supported ruthenium oxide catalyst by reverse gas chromatography and dynamic vapor sorption. Jackson, SD, Hargreaves, JSJ, Lennon. D., editors, Catalysis in application Great Britain, Royal Soc. Chem., p 237 (2003), and Traini et al., Drug Development and Industrial Pharmacy 34: 992-1001 (2008) Figure 1 shows the contributions of surface energy in infinite dilution:
[000141] Total surface energy (YT) was the additive result of non-polar (YNP) and polar (YP) contributions (Grimsey et al., Journal of Sciences 91: 571-583 (2002). The work of cohesion ( Wco) was calculated (see Vanoss et al., Langmuir 4:884-891 (1988) and Tay, et al., International Journal of Pharmaceutics (Kidlington) 383:62-69 (2010).These experiments were conducted in triplicate. Surface energy distributions and surface area determination in finite dilution:
[000142] Non-polar surface energy distribution profiles (YNP profile) were determined according to the method described elsewhere ( F. Thielmann et al., Drug Development and Industrial Pharmacy 33: 1240-1253 (2007) and Ila-Maihaniemi, et al., Langmuir 24: 9551-9557 (2008)). This is shown in Figure 10. The non-polar surface energies in all coatings are clearly reduced for the leucine-containing powder and in this case shows a reduction of more than 30% at a 1% coating level. The polar surface energy distribution is shown in Figure 11.
[000143] The Brunauer-Emmet-Teller (BET) surface area was calculated from hexane adsorption isotherms. By dividing the adsorbed amount (n) by the monolayer capacity (nm, the number of moles of probe adsorbed for monolayer coverage), the surface coverage (n/nm) was calculated. At each surface coverage, the liquid retention volume (VN) was calculated for each probe. Non-polar surface energy (YNP) was calculated from the slope (2 NΔ VYNP) of a plot of RTlnVN against an alkanes v/NP. The YP and YT were calculated on each surface coverage and their distribution profiles were then constructed (as described in Das et al. Langmuir 27: 521-523 (2011a)).
[000144] Figures 12 and 13 show that the total surface energy distributions and calculated cohesion work are also substantially reduced in the case where leucine is added, for example by 20% or more at a surface coverage of approximately 1% . Example 10 In vivo testing of other formulations
[000145] As for example 5 a single pregnant sheep was prepared by surgery to implant the EMG electrodes and a catheter to allow blood collection. Induction of labor started on the same day as surgery as well as by the method shown in example 5. Labor and release occurred 2 days after the start of the induction period.
[000146] Oxytocin administrations, as detailed below, were started within 22 hours of release. There was an interval of one and a half hours between each treatment.
[000147] For intratracheal administration, an endoscope was passed through the nasal passage into the trachea and positioned close to the first bronchial bifurcation, where a dose of dry powder (as detailed below) was delivered through a PennCentury powder delivery device modified. In addition to delivering dry powder to the lung, an intramuscular injection of oxytocin (as per example 5) was also delivered.
[000148] The dry powder formulations comprised spray dried compositions manufactured as described in example 1. The compositions of these powders together with the nominal dose released are shown in Table 7. The elapsed time (delay) from the release of oxytocin to the first burst of EMG activity. The delay, the elapsed time from oxytocin release to the initial burst of EMG activity is also shown in Table 7. The EMG traces for these four doses are shown in Figures 15 and 16.

[000149] Surprisingly, the onset of action of pulmonary powder release was significantly faster compared to intramuscular release, and was also consistent with plasma versus time profiles. The data also show that rapid response can be achieved with dry powder formulations made with a range of excipients such as polyols, sugars, amino acids and polymers. This is despite the fact that the dry powder particles contain between 10 to 30% of Leucine which is an insufficiently soluble and hydrophobic amino acid, which would be expected to retard dissolution. Furthermore, a substantial proportion of the leucine is expected to be present on the surface of the powder.
[000150] The invention has been described only by way of non-limiting example and many modifications and variations can be made thereto without departing from the spirit and scope of the described invention.
[000151] Throughout this descriptive report and the claims that follow, unless the context otherwise requires, the word "understand", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a number established integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
[000152] Reference in these descriptive reports to any previous publication (or information derived therefrom), or to any matter that is known, is not, and should not be construed as an acknowledgment or admission or any form of suggestion that this publication prior (or information derived therefrom) or known matter forms part of the common general knowledge in the field of diligence to which this descriptive report refers.

权利要求:
Claims (15)
[0001]
1. Dry inhalation powder, characterized in that it is for use in the treatment or prevention of postpartum hemorrhage, comprising: spray dried particles comprising oxytocin or carbetocin, and an amorphous glassy matrix comprising one or more mono-, di- or polysaccharides and/or amino acids, eL-leucine.
[0002]
2. Dry powder for inhalation according to claim 1, characterized in that the dry powder for inhalation is for nasal administration.
[0003]
3. Dry powder for inhalation according to claim 1, characterized in that said dry powder for inhalation is for inhalation into the pulmonary system through the mouth.
[0004]
4. Dry powder for inhalation, according to any one of claims 1 to 3, characterized in that at least a portion of L-leucine is located on the surface of the dry powder particles.
[0005]
5. Dry powder for inhalation according to any one of claims 1 to 4, characterized in that more than 40% of the dry powder particles at the time of inhalation have an aerodynamic diameter of less than 5 µm.
[0006]
6. Dry powder for inhalation according to any one of claims 1 to 5, characterized in that the dry powder for inhalation is prepared by a process comprising: preparing an aqueous solution/suspension comprising oxytocin or carbetocin, one or more mono -, di- or polysaccharides and/or amino acids capable of forming an amorphous glassy matrix, and L-leucine; spray dry the aqueous solution/suspension to produce a dry powder suitable for inhalation.
[0007]
7. Dry powder for inhalation according to claim 6, characterized in that L-leucine is present in an amount of 5 to 50% by weight based on the dry weight of the powder.
[0008]
8. Dry powder for inhalation according to claim 7, characterized in that L-leucine is present in an amount of 10 to 40% by weight.
[0009]
9. Dry powder for inhalation according to any one of claims 1 to 8, characterized in that the one or more mono-, dior polysaccharides and/or amino acids capable of forming an amorphous glassy matrix comprise D-mannitol and glycine .
[0010]
10. Dry powder for inhalation according to any one of claims 6 to 9, characterized in that spray drying is carried out at a temperature below 80 °C.
[0011]
11. Dry powder for inhalation according to any one of claims 1 to 10, characterized in that the amorphous vitreous matrix comprises trehalose.
[0012]
12. Dry powder for inhalation according to any one of claims 1 to 10, characterized in that the onset of action after inhalation of the dry powder, measured by uterine contraction, is reached within 200 seconds of inhalation.
[0013]
13. Dry powder for inhalation according to claim 12, characterized in that the onset of action is reached within 150 seconds of inhalation.
[0014]
14. Dry powder for inhalation according to claim 12, characterized in that the onset of action is reached within 100 seconds of inhalation.
[0015]
15. Dry inhalation powder according to any one of claims 1 to 14, characterized in that said inhalation powder further includes one or more pulmonary and pharmaceutically acceptable components, preferably including a pulmonary acceptable carrier, preferably a pulmonary acceptable carrier, including a stabilizer for the oxytocin component.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-07-09| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-06-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/11/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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AU2011903049|2011-08-01|

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AU2011903049A|AU2011903049A0|2011-08-01|Method and formulation for inhalation|

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PCT/AU2011/001430|WO2013016754A1|2011-08-01|2011-11-07|Method and formulation for inhalation|

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