TW200916126A - Organic compounds - Google Patents

Abstract

A pharmaceutical composition that comprises: (A) one or more drug substances; (B) a lipid; (C) a co-lipid; and (D) a flowability enhancer, wherein the co-lipid and the flowability enhancer together form a liposomal dispersion that comprises lipid vesicles that encapsulate the one or more drug substances. The pharmaceutical composition is optionally dried to form a dry powder formulation that is free-flowing and preferably suitable for inhalation or nasal administration. Processes for preparing the composition and the dry powder formulation are also described.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel pharmaceutical compositions and dry powder formulations containing one or more pharmaceutical substances encapsulated in lipid vesicles. The invention is also directed to methods of making such compositions and formulations, and methods of treating such diseases or conditions with such compositions and formulations. Other aspects, objects, and advantages of the invention will be apparent from the description. [Prior Art] The active noodle ingredient [API] or drug substance can only have a pharmacological effect in a patient if it is in a therapeutically effective amount to reach a therapeutic site. Treatment of potentially active drugs is sometimes ineffective in vivo because of its instability, its absorption by non-target systems, or its inability to enter relevant cells. It is known to improve the delivery of certain drugs to their therapeutic targets by encapsulating the drug in liposomes. This ancient -1, J, ' can be used to reduce macrophage phagocytosis and bioavailability rate / seven points, | use sheep and / or reduce side effects. The low-burial efficiency of the 0 music is encapsulated in the liposome, and can not be repeated to the following AW including AH λ 7jc ^ . Also encapsulates sufficient API, instability (especially in the water-based nuclear environment) County u in the storage period of the door ...), sensitivity to mechanical stress, during the storage period from the vesicle osmosis, processing reckless use of physico-chemically unsuitable fat due to uneconomic large-scale manufacturing capacity. b is to be replaced by a lipid capsule that overcomes one or more of the above problems: a pharmaceutical composition of a musical substance. There is also a need for a pharmaceutical composition for the preparation of a lipid-containing compound which provides at least a suitable and effective composition for the known compositions. 133847.doc 200916126 SUMMARY OF THE INVENTION The present invention provides a pharmaceutical composition comprising: (a) or a drug substance; a lipid; (C) a co-lipid; and (D) a fluidity enhancer The pharmaceutical composition is characterized by a lipid, a co-lipid, and an increased fluidity to form a lipid dispersion comprising lipid vesicles encapsulating the or the drug substance. In a second aspect, the present invention provides a dry powder formulation comprising: (A) or a drug substance; a lipid; a co-lipid; and (1) a fluidity enhancer; the formulation characterized by a lipid, a co-lipid and The fluidity enhancer functions to form a lipid dispersion comprising lipid vesicles encapsulating the or the drug substance f and the lipid dispersion is dried to form a free flowing dry powder formulation. [Embodiment] The average diameter of the moon mussel vesicles is preferably between 7〇11111 and 55〇, more preferably between 100 nm and 450 nm, and even more preferably between 1〇〇11111 and 25〇. It is better to use a dry powder formulation for inhalation. When the quinone compound is an inhalable powder, the average aerodynamic particle size is preferably not more than 1 Å, more preferably not more than 5 μm, and even more preferably not more than 3 μm. Alternatively, the dry powder formulation is suitable for nasal administration in the form of a reconstituted liquid or in the form of a dry powder. When the formulation is a nasal powder, the average aerodynamic force and particle size are preferably greater than 1 〇 microns. In a third aspect, the invention provides a method of preparing a pharmaceutical composition comprising the steps of: 133847.doc 200916126 (a) mixing lipids and co-lipids under high shear; (b) mixing one or more drugs And a method of sealing the formulation of the drug substance, the (C) mixing fluidity enhancer to form a lipid dispersion comprising a lipid vesicle of the capsule. In a fourth aspect, the invention provides a method of preparing a dry process comprising the steps of: (a) mixing a lipid with a co-lipid under high shear; (b) mixing one or more drug substances; (c) enhancing port mobility The agent is used to form a lipid dispersion comprising a menstrual vesicle that encapsulates the drug substance; (d) diluting the lipid dispersion as appropriate; and 0) drying the lipid dispersion to form a powder formulation. The terms used in this specification have the following meanings: "Carrls Index" as used herein is an indication of powder fluidity. It is calculated as follows: Carr index (%) = (knock tightness _ bulk density) * 丨 00 / knock tightness as used herein, · co-lipids, substances that stabilize lipids, such as lipid vesicles stabilizer. As used herein, "dsRNA" refers to a modified or unmodified ribonucleotide or polyribonucleotide, and fragments or portions thereof, which are genomic or synthetic sources or derived from a vector, which may be Partially or completely double-stranded and which may be pure ends or contain 5'-overhangs and/or 3,_overhangs, and may also have a hairpin form comprising a single oligoribonucleotide, the single oligoribonucleoside The acid is folded back on itself to obtain a double-stranded region. The dsRNA may also contain a nucleotide residue that has been modified by 133847.doc 200916126. The π-emission dose or "ED" as used herein is actuated The total mass of the drug substance emitted from the device. It does not include the material remaining inside or on the surface of the device. The total emission quality is collected by a device from a suitable device, such as a dose uniformity sampling device (DUSA). The ED is measured by a confirmed quantitative wet chemical assay. The "encapsulation efficiency" as used herein is generally defined as the amount of drug substance buried in the lipid structure and expressed as a percentage. "fine" used in The particle dose" or "FPDn is the total mass of the drug substance emitted from the device after actuation, the drug substance being present at an aerodynamic particle size less than the defined limit. If not explicitly stated as an alternative limit (such as 1 μιη) Or 3 μηι, etc., this limit is generally considered to be 5 μηη. Use inertial impactors or impactors such as two-stage striker (TSI), multi-stage liquid impactor (MSLI), Anderson cascade impactor (Andersen Cascade Impactor, ACI) or Next Generation Impactor (NGI) to measure FPD. Each impactor or striker has a pre-defined aerodynamic grain collection cut-off point for each stage at a defined flow rate. Clarify the FPD value obtained by confirming the quantified active agent recovery quantified by quantitative wet chemical assays in which simple cuts are used to determine FPD or use stepwise More complex mathematical interpolation of deposition. As used herein, "fine particle fraction" or "FPF" is generally defined as FPD divided by ED and expressed as a percentage. Here, the FPF of ED is called FPF (ED) And Calculated as FPF (ED) = (FPD / ED) x lOO%. "fine particle portion" can also be defined as FPD divided by MD and expressed as a percentage. Here, the FPF of MD is called 133847.doc 200916126 is FPF (MD) And calculated as FPF (MD) = (FPD / MD) x 1 〇〇%. As used herein, "fluidity enhancer" is a substance that enhances the fluidity of a pharmaceutical composition. As used herein, "geometry standards The difference " or "GSD, is a measure of the aerosol polydispersity from the laser diffraction particle size distribution data and is calculated as follows: GSD = ^ VX10 where X90 and Χίο are 90% and 10% of the particles in this size The following granularity. "Ion balance agent" as used herein is a substance that neutralizes a drug substance ion and is encapsulated in a lipid. As used herein, "lipid" means a pharmaceutically acceptable lipid, including, for example, neutral lipids, pegylated lipids, cationic lipids, zwitterionic lipids (such as helper lipids), and anionic lipids. As used herein, "lipid dispersion" means a structure consisting of a spherical vesicle comprising a bilayer membrane, which may comprise a natural or synthetic phospholipid or a pure surfactant component. These vesicles are dispersed, such as water, In a suitable dispersion medium of organic solvent or oil-based medium. As used herein, "mass median aerodynamic diameter" or "μμα〇" is a spherical unit dense with the same sedimentation velocity. The median size of the particles. The average particle size of the particles as measured by laser light diffraction is an average of χ90 average particle size - average particle size, and 9% of the sample particles have a lower average particle size below the average particle size. The average particle size of Χ50 is an average granule and 0/〇 test 篆 granules have an average granule at that average 纟 Τ 133 133 847 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Χίο The average particle size is an average particle size below the average particle size. The "metering dose" or " of the dry powder formulation as used herein is presented by the relevant inhaler device as the initial amount of the musical substance present in the form of straw Φ. For example, MD may be present in the capsule of a special dry powder inhaler or in the presence of a substance suitable for a particular dry powder inhaler. The mercy of the armor, the "parenteral administration" used in the text means that it is controlled by intravenous, subcutaneous, ancient, temporal, intramuscular, intra-articular, intra-articular, etc. Intraocular, intracranial, intrathecal injection for administration: intravenous, αλ_ Hanguangpi·intra, intramuscular, intra-articular, intraocular, intracranial, intrathecal, sputum to any other body part or tissue 0” "Pharmaceutically acceptable" means a compound, material, composition, and/or dosage form that is within the scope of sound medical judgment. It is suitable for contact with tissues of a mammal (especially: class) without excessive toxicity, irritation, or allergies. Reaction: Other problematic complications commensurate with a reasonable benefit/risk ratio. As used herein, "RNA" means a single or double-stranded nucleic acid molecule that mediates ribonucleic acid interference, including but not limited to double-stranded nucleic acids ( "dsNA"), double-stranded RNA ("dsRNA,,), microRNA (,, miRNA)), short hairpin rna ("shRNA"), short interfering nucleic acid ("siNA"), and short interfering ribonucleic acid ( "siRNA"). As used herein, "siRNA" refers to short interfering RNA and refers to short double-stranded ribonucleic acid suitable for RNA interference. Such siRNAs have, for example, from (7) to 50 nucleotides, especially, for example, 丨5 to Length between 25 nucleotides. 133847.doc -10- 200916126 "Volume average diameter" or "vmd" as used herein means measured and correlated using laser diffraction or any other suitable technique. The particles have the same size as the average size of the spherical unit dense particles. In the scope of the patent application and the patent application scope thereof, unless the context of the text, 3 3 I & include " is considered to include the integer or step or number or group of Y steps but not Exclude any other integer or step or group of integers or steps. In the broadest sense of the art, the present invention relates to pharmaceutical compositions. The composition 匕3. (A) one or more drug substances; (B) lipids; (C) co-lipids; and, M) kinetics. The pharmaceutical composition is characterized in that the lipid and the co-lipid 2 are increased in mobility to form a lipid dispersion comprising lipid vesicles encapsulating the drug substance or the drug substance. The pharmaceutical composition can be dried to form a dry powder formulation which is preferably free of fluidity and which is preferably suitable for inhalation and nasal administration. The drug substance is embedded in a lipid bilayer to form a lipid vesicle. Several lipid vesicles are further coated by the lipid layer to form a lipid dry powder capsule. The capsule is further coated by a fluidity enhancer of a preferred = crystalline material to form particles having an average particle size. The final particle structure forms a free-flowing lipid powder due to the small contact area (4) (4). By encapsulating the drug substance 'may increase local and systemic bioavailability, reduce macrophage phagocytosis of macromolecules and negatively charged molecules, thus reducing side effects (eg, nasal stimulation and damage to the nasal epithelial layer) and The hygroscopic characteristic of the drug substance is reduced, whereby the drug substance is a pharmaceutical substance comprising a small molecular weight compound: a molecule. Suitable small molecular weight compounds include, but are not limited to, (example 133847.doc 200916126 eg) P2_adrenoceptor agonist (p2-adrenoceptor agonist), scorpion venom inhibitor, glucocorticosteroid, non-steroidal glucocorticoid Hormone receptor agonist, Aza agonist, A2B antagonist, antihistamine, caspase inhibitor, LTB4 antagonist, LTD4 antagonist, phosphodiesterase inhibitor (especially PDE4 inhibitor or PDE5) Inhibitors), mueolytics, antibiotics, matrix metalloproteinase inhibitors (MMpi), leukotriene receptor antagonists (LTRA), IgE synthesis inhibitors, antibiotics, interferons, clock channel inhibitors, immunity Modulators, antineoplastic agents, elastase inhibitors, prostaglandin D2 (pGD2) antagonist active agents at the CRTH2 receptor, and prostatin inhibitors. Suitable macromolecules include, but are not limited to, peptides, proteins, oligonucleotides, RNA (including dsNA, dsRNA, miRNA, shRNA, siNA, and siRNA), DNA, plastids, insulin, interleukin, growth hormone, heparin, Estradiol, GLP-1, antibiotics, antitumor agents and antibodies. Each drug substance is present in a therapeutically effective amount or concentration. This therapeutically effective amount or concentration is known to those of ordinary skill' because the amount or concentration will vary with the therapeutic agent employed and the indication being treated. In certain preferred embodiments, the drug substance is an oligonucleotide or rNa (especially siRNA). In certain preferred embodiments, the or the drug substance is suitable for administration by inhalation or nasal administration. Lipids are pharmaceutically acceptable lipids including neutral lipids, cationic lipids, zwitterionic lipids, and anionic lipids. Examples of neutral lipids are, but are not limited to, phospholipid choline (which may or may not be Hydrogenation or polyethylene II 133847.doc • 12- 200916126 alcoholation) * or synthetic linoleum ethanolamine, linoleic acid, glycerin, linoleic acid, in certain preferred embodiments, The genus is squamous choline (eg, one-myristyl ketone (10) coffee (1) phospholipid choline (DMPC)), one-lipidylphospholipid choline (Dppc), hydrogenated phospholipid guanidine test ( For example, lipid S PC_3) or soybean linoleum (for example, lipid π or large denier phosphatase). The sorghum is preferably di-myristyl phospholipid choline, di-lipid ruthenium Lipid sputum test, husband from ^ > Occupational ugly lecithin or hydrogenated phospholipid choline. Examples of cationic enamel include (but not limited to, 2 - dioleyl trimethylammonium propylene (DOTAP) ' N-[l-2(2,3-dioleyloxy)propyl]_N,N,N_s methyl ammonium hydride (DOTMA); 2,3-dioleyloxy group N_called spermine曱Amidino)ethyl]-N,N-methyl+propanol (D〇spA); bis (18 glysine diol (giycu) spermine (DOGS); and 3,[n_n1,N -Dimethylethylenediamine)-aminomethylmercapto]-cholesterol (D-cholesterol). Examples of zwitterionic lipids include (C is not limited to) 1,2-monooleyls.sn.-glyceryl_3_acidic ethanolamine (D〇pE) and cholesterol. Auxiliary moon mussels (which are substances that stabilize lipids, especially those that stabilize lipid vesicles) can be (not limited to) cholesterol, PEGylated phospholipids, phospholipid glycerol, polysorbate, medium 7 t Yizhizhi (PEG), polyvinylpyrrolidine (PVP) or cholesterol. In some cases, #香& y, in a preferred embodiment, the co-lipid is a polyacetylidene phosphate test or cholesterol. Examples of lipid/co-lipid combinations include (but T is limited to) di-lipid oxime-based discs (four) biliary assays [DPPC]/N-(severyl-decyloxy polyethylene glycol-2000)-i, 2_di-hard Sodium sulfhydryl-sn-glycerol _3_phosphoethanolamine, sodium salt [MpEG_a〇〇_SPE] _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Polyethylene glycol-2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, sodium salt [MPEG-2000-DSPE]/cholesterol, bis-lipidyl phospholipid choline [ DPPC]/N-(carbonyl-decyloxy polyethylene glycol-2000)-1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, sodium salt [MPEG-750-DSPE], two- Peptidyl phospholipid choline [DPPC]/N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt [ MPEG-750-DSPE]/cholesterol, bis-lipidylphosphatidylcholine [DPPC]/N-(carbonyl-methoxypolyethylene glycol-5000)-1,2-dimyristyl-sn - glycerol-3-phosphoethanolamine, sodium salt [MPEG-5000-DMPE], bis-lipidylphosphatidylcholine [DPPC]/N-(carbonyl-methoxypolyethylene glycol-5000)-1, 2-dimyristyl-sn-glycerol-3-phosphoethanolamine, Salt [MPEG-5000-DMPE]/cholesterol, bis-lipidylphosphatidylcholine [DPPC]/N-(carbonyl-decyloxy polyethylene glycol-2000)-1,2-dimyristyl -sn-glycerol-3-phosphoethanolamine, sodium salt [MPEG-2000-DMPE] and di-pipylphosphatidylcholine choline PPPC]/N-(carbonyl-decyloxy polyethylene glycol-2000)-1 , 2-dimyristyl-sn-glycerol-3-phosphoethanolamine, sodium salt [MPEG-2000-DMPE]/cholesterol 0 fluidity enhancer (which is a substance that enhances the fluidity of a pharmaceutical composition) can be ( But not limited to) hydrophobic non-hygroscopic substances such as amino acids, mannitol, inulin, sucrose, lactose, L-leucine, D-leucine, isoleucine, serum albumin, right Rotose, maltose, glycine, maltitol, calcium stearate, magnesium stearate, erythritol or mixtures thereof. In certain preferred embodiments, the flow enhancer is crystalline. Crystalline fluidity enhancers tend to precipitate on the outer surface of lipid vesicles in lipid dispersions. 133847.doc 200916126 増...The strongener is maltose, mannose, sugar maltitol 'glycine, hard at some preferred In the examples, alcohol, inulin, sucrose, lactose, calcium or calcium stearate. When the drug substance is an ion, it is often necessary to have a pharmaceutical composition containing an early weighing agent and/or a buffer system. Ion Ping 3 has an ion thousand (which is a substance that reduces the ion of the drug substance and is encapsulated in the neutral lipid), but is not limited to calcium stearate, calcium telluride, tricalcium citrate, and sour acid. _Technology - calcium 'sodium sorbate, acetaminophen, chlorinated or magnesium stearate. In certain preferred embodiments, the ion balance agent is a chlorine storage, Wei San 33s cut fat. The buffer system regulates the enthalpy of the lipid dispersion and modulates the ionic charge on the drug substance. It can be, but is not limited to, lactic acid, #酸酸, acetate buffer, glycine, sulphate buffer or tris buffer. In certain preferred embodiments, the buffer is lactic acid (pH 4.0) and phosphate buffer (pH 65). As indicated above, the pharmaceutical compositions of the present invention may be dried to form a dry powder formulation which is free flowing and which is preferably suitable for inhalation and/or nasal administration. According to the present invention, there is provided a method of improving the drug substance-induced inflammation in a human subject (e.g., a patient), the method comprising providing a dry powder formulation as described herein. In a preferred embodiment, the dry powder formulation comprises a lipid dispersion. The lipid dispersion comprises a neutral lipid such as DPCC. We therefore also provide a dry powder formulation comprising one or more drug substances for administration to a human patient, wherein once the formulation is administered to the human patient, the inflammation induced by the drug substance is improved, the dry powder formulation comprising such as DPCC Sexual enamel, co-lipids, and fluidity enhancers (eg, crystal flow enhancer 133847.doc -15-200916126). The dry powder formulation can be administered systemically and locally by drug delivery via the respiratory tract as described herein. The route of drug administration. Compared with API (especially for bio-invasive ^ as an attractive enteral drug, it is currently a relatively simple self-injection compared to the conventional non-skin or parenteral route through the respiratory tract," The branch-mouth route provides a larger viscosity than the bypass of the liver, which reduces the enzymatic and deuterium degradation of the drug.

Stomach inhalation delivery of the drug substance also enables topical application to the lungs, reducing side effects and improving the effectiveness of the drug at higher local concentrations. A dry powder formulation for inhaling in the treatment of respiratory diseases is typically formulated by mixing micronized active pharmaceutical ingredients with coarse carrier particles to provide an ordered mixture. However, ultrafine particles tend to have poor fluidity and aerosolization, resulting in a relatively low respirable portion' that is, an aerosol portion deposited on the periphery of the lung. Another concern is particle-particle interaction forces, such as hydrophobic binding, electrostatic and capillary. Tube-water interactions, which can result in the formation of aggregates and agglomerates. These factors can lead to significant efficiencies in conventionally prepared dry powder systems. This can make the dry powder for the manufacture of expensive macromolecules uneconomical and cause long-term stability concerns. In broad terms, intranasal routes of administration are only preferred to deliver conventional drugs to treat local discomfort, such as nasal congestion and sinus infections. However, recent clinical trials have shown that intranasal routes are also applicable when treating severe respiratory diseases such as asthma, allergies, cystic fibrosis, severe acute respiratory syndrome, and infections caused by respiratory syncytial and influenza viruses. - see Hussain Α., Jc/v. Drwg £) by 7?ev_ (1998) 29·· 3 9-49; 133847.doc •16- 200916126

Nyce and Metzger, iVa/wre (1997) 385:721-725; Finotto et al., V: Φ·Md. (2001) 193: 1247-1260; Allakhverdi et al., J. Respir. Crit. Care Med. (2002 ) 165: 1015-1021, Aurora J., Drwg Fantasy; (2007) 85. Nasal administration can be an attractive method for systemic administration of a drug because it allows for non-invasive administration while at the same time achieving improved patient compliance. The nasal cavity provides a large surface area for absorption. It is covered by a surface active lipoprotein that acts as a surfactant and thus can contribute to the intracellular uptake of drug substances, including anti-inflammatory agents, immunostimulants, hormones, peptides. , proteins, antibodies, nucleotides, vaccines, pDNA, RNA, DNA and anti-tumor agents. Liquid nasal sprays and droplets are the most commonly used pharmaceutical formulations for intranasal administration. However, 'dry powder formulations can be compared to liquid rafts.

The femoral hernia will be regional and provide sustained release. Continued release. In addition, the nasal delivery system can be designed to target specific areas of the nasal cavity

Deposition, wherein the permeability is generally high, and the reticulated ligand can be targeted to the posterior portion of the nose, thereby providing a short retention in the nose, and thus systemic absorption, and the dry powder forms the present invention. Although the use of a liquid nasal spray delivery system to target a particular nasal area is challenging due to the wide range of droplets produced; the lack of distribution and dependence on the spray pattern, the nasal dry powder of the present invention can be accurately and efficiently The drug is delivered to the relevant area in the nose. Dry powder of the present invention for free flowing dry powder for nasal and/or pulmonary administration

Formulations offer a variety of applicable benefits. For example, it tends to be more stable than: the plastisate formulation is more stable, m effectively delivers the drug substance and allows for a reduction in the dose to be administered, which enables the drug substance to target a specific area of the nasal cavity, upper trachea or alveolar deposition _ In order to apply locally to the whole body and it does not require the use of preservatives. Anti-adhesives such as magnesium stearate have been used to treat some of these problems in dry powder formulations containing low molecular weight pharmaceutical materials. Then, when blending large knives with peptides, proteins, oligonucleotides, and rna*dna, these drugs are not suitable. In addition, it does not help to target the API to the cells or to arrest the natural clearance mechanism of the lungs until the drug has been effectively delivered. Lipid-encapsulated dry granules generally have poor flow and high viscosity, which cause problems in downstream processing including filling and packaging. n. The dry powder formulation of the present invention is substantially free flowing and has low viscosity. Sex. The dry powder formulation of the present invention also has good physicochemical powder characteristics, such as a contact area of the particles/particles of the granules and a reduced contact area of the granules/packaging material and an adhesive/internal: fr, which is reduced by humans. This ensures the desired powder aerosolization characteristics and less powder retention in the primary packaging or inhalation devices. The dry powder formulation may also contain pharmaceuticals, adjuvants, stabilizers, preservatives, flaking agents, such as other agents well known in the art of carrying the art (considering: agents such as citrate, citrate, succinic acid , sexual; buffer acid or its salt; antioxidants, such as ascorbic acid; 夂 = he organic such as polyvinylpyrrolidone; monosaccharide, _ 'D thing' furnace to go to seven sugar / one sugar and other carbohydrates, including = U-bio, glucose, mannose or dextrin such as bribe A; sugar alcohols such as mannitol or sorbitol; balance family such as and / or non-ionic surfactants, such as poly-Γ =: 剞 pull ~ move ^ The technologist can select the _ or more of the above-mentioned Fu (4) 藉 by routine experiment without any undue burden. The use of each of the morphings j In the broad term, the present invention is prepared by the method of the pharmaceutical composition comprising the following steps: (a) mixing the lipid with the co-lipid under high shear; (b) mixing one or a plurality of drug substances; and (4) mixing a fluidity enhancer to form an inclusion a lipid dispersion of lipid vesicles encapsulating the drug substance or substances. In the material, with or without supersonic assistance such as c〇iieu#: cut:: Heyi (Niro Pharmasystems, (10) one) The high-shear mixed mixture is mixed with lipid and co-lipid to form lipid particles. It is preferred to have lipid particles 133847.doc -19· 200916126 2 knots at a constant temperature above the temperature of the sylvestre. Lipids and co-lipids can be combined with the package' Or the solvent system of the glutinous glutinous glutinous rice is mixed together, and the lipid, co-lipid, and music substances are dissolved in the solvent system. This is usually an organic solvent such as an alcohol (for example, ethanol or isopropanol). The drug substance and the lipid may be mixed by any method known in the art for the drug substance to be used. However, it is preferably carried out slowly in a continuous mixed sigma manner to promote sufficient and homogeneous mixing and drug substance in the month of mussel. Buried in the vesicles but avoids degradation or denaturation of the drug substance. In a preferred embodiment, a suction system is used (for a batch size between 1 〇... and (7) ❹L, for example, the flow rate is (M „ 11/„1丨11 to 55 〇ml/min) The drug substance is slowly injected into the lipid particle dispersion at a controlled temperature in a continuous manner. When the drug substance is an ion, it is often required that the pharmaceutical composition contains an ion balance agent and/or a buffer system. The substance may be mixed with the drug substance in step (b). In step (c), any lipid dispersion known in the art to be suitable for forming the drug substance or substances encapsulated in the lipid vesicle is used. The method of mixing a fluidity enhancer (with or without a buffer salt) with a mixture of lipid particles and a drug substance, such as by using Master ZetaSiZer (Malvem Inst]«uments

The dynamic light scattering technique carried out by Ltd, UK) determines that the average diameter of the vesicles is preferably between 70 nm and 550 nm, more preferably between 10 〇 nm and 45 〇 nm 2 , and even more preferably at 1 〇〇 nm. Between 250 nm. The pharmaceutical compositions of the present invention can be further processed to prepare dry powder formulations. Additional steps include: (d) diluting the lipid dispersion as appropriate; and 133847.doc • 20- 200916126 (4) drying the lipid dispersion to form a dry powder formulation. In the step (4), when dilution is required, the lipid dispersion is preferably diluted to a final rolling degree, preferably 2% to 5% and most preferably 2, using water or a buffer solution. /. . However, when preparing a dry powder formulation for nasal administration, the lipid dispersion is preferably diluted with water or a buffer solution to a final concentration of 2% to 20% 'preferably 5% to 10%. /Step (4) The lipid dispersion is dried using any method known and suitable in the art (the preferred method is described in more detail below). Preferably, the lipid dispersion is spray dried. The resulting dry powder formulation is substantially free flowing. According to the present invention, there is provided a dry scalpel formulation for producing a lipid dispersion comprising lipid vesicles encapsulating one or more drug substances (e.g., siRNA and/or nucleotides) (e.g., having an average particle size greater than 2 Å μηη A method for delivering a powder to the base of the nose or, for example, having an average particle size between 1 〇 1 ^ 111 and 2 〇 μηη for delivery to the posterior portion of the nose, the method comprising: (a) at high shear (eg 5 〇) Mixing the lipid and the co-lipid at rpm 45 rpm or higher; (b) mixing the one or more drug substances; and (c) mixing the fluidity enhancer to form a lipid comprising a lipid vesicle encapsulating the drug substance or the drug substance Dispersing solution; (d) Dilute the lipid dispersion to between 5% and 10% of the final concentration, optionally with water or a buffer solution; (e) Dry the lipid dispersion (eg by at about 13 (TC or 130 ° C) Spray drying at the above temperature to form a powder formulation. 133847.doc -21 - 200916126 In some embodiments, the method is followed by a smoulding step at 13 (rCn_u() to () Go to... The invention extends to the method by the invention &Amp; dry powder formulations.

:: The drying method of step (4) is carried out by optimizing the spray drying technique, and the bypass spray nozzle is used to atomize the dispersion liquid, which is operated by using a pressurized gas such as air or nitrogen to form The size is 5 droplets. These droplets are suspended in a deuterium such as air or nitrogen for drying. During this dry process, the water is evaporated based on the rate of expansion; All of the hydrophilic compositions of the same day's compound including lipid vesicles move toward the center of the droplet, while the hydrophobic composition such as the additional lipid hydrophobic tail 1) moves toward the gas/droplet interface. Adjusting this kinetic process by tide controlling through a diffusion process can effectively seal lipid dry powder particles of one or more drug substances. In addition, the fluidity reducing agent precipitates on the surface of the powder capsules, so that the particle-particle contact area is lowered, which significantly improves the powder fluidity and aerosolization efficiency. Accordingly, a method of making a dry powder formulation comprising a lipid dispersion of a lipid vesicle encapsulating one or more drug substances (eg, siRNA and/or nucleotides) is provided, the method comprising: (a) Shearing lipids and co-lipids under shear (eg, 5 jaws or above rpm); '(b) mixing one or more drug substances; and (c) mixing fluidity enhancers (eg, in crystalline form), such as Maltose, mannitol, _, sucrose, lactose, dextrose, maltitol, glycine 133847.doc -22- 200916126 acid, calcium stearate or magnesium stearate to form a capsule or the like a lipid dispersion of a lipid vesicle; (sequentially diluting the lipid dispersion; (e) drying the lipid dispersion to form a powder formulation, wherein the drying method is included at (about) 13 〇 t: or above 130 ° C The lipid dispersion is spray dried at a temperature to form a droplet of dispersion between 5 μm and 50 μm suspended in a gaseous atmosphere (eg, air, oxygen, nitrogen). In some embodiments, the method is In the order 0) to proceed. In some preferred embodiments, the dry powder formulation may be inhaled at a towel aerodynamic diameter (MMAD) of no greater than 1 micron but preferably no greater than 5 microns. For certain powder compositions, the average mmad is no greater than丨 to 3 microns, which ensures deep deposition of the lungs, and a volume average diameter (viyiD) of no more than 35 microns, but preferably no more than 25 microns. In some preferred embodiments, when the dry powder has a mass greater than 10 microns The dry powder formulation is suitable for nasal administration when the median aerodynamic diameter (MMAD) or average particle size is used. As described above, the dry powder formulation of the present invention having a powder average particle size greater than about 20 Å can be targeted to the nasal anterior portion. The drug is deposited and produces a local effect. The dry powder formulation of the present invention in which the average particle size of the powder (4) and 20_ can be targeted to the drug deposition absorption in the posterior part of the nose. /, soil versus 〇J s To produce any desired area having a *7 dry-direction lung tract. The pharmaceutical compositions and dry powder formulations of the present invention can be used to treat a variety of diseases and conditions, which are defined by the choice of drug substance. Administered by any suitable route, for example, orally, for example, in the form of a lozenge or capsule; parenterally, such as intravenously; topically applied to the skin (eg, in the treatment of psoriasis); Oral; or preferably by inhalation. Oral dosage forms may include lozenges and capsules. Formulations for topical administration may be in the form of a cream, ointment, gel or a transdermal delivery system such as a patch. The composition for inhalation may comprise an aerosol or other atomizable formulation or dry powder formulation. However, in a preferred embodiment of the invention, the pharmaceutical composition is in the form of a dry powder formulation suitable for inhalation administration. In a preferred embodiment of the invention, the pharmaceutical composition is a lipid dispersion suitable for inhalation administration. In another preferred embodiment of the invention, the pharmaceutical composition is in the form of a dry powder formulation suitable for nasal administration. In a preferred embodiment of the invention, the pharmaceutical composition is a lipid dispersion suitable for nasal administration. The inhalable dry powder formulations of the present invention can take a variety of forms commonly used in the pharmaceutical industry. For example, it can be packaged in a capsule, foam or any other packaging system and applied to the respiratory or nasal cavity as a dry powder using various inhalation/nasal devices. Suitable devices for delivering dry powder in encapsulated form are for example, but not limited to, those described in US 3,991,761 (including AEROLIZERTM devices) or WO 05/1 13042, while suitable DPI devices are included in WO 97/20589 ( Includes CERTIHALERTM devices), WO 97/30743 (including TWISTHALERTM devices), WO 05/14089 (including GEMINITM devices), WO 05/37353 (including GYROHALERtm devices), and w〇99/64095 (MicroDoseTM) Those who are. Suitable unit dose devices for delivering dry powder to the nasal cavity are included in WO 96/22802 (including DIRECTHALERtm devices), us 6626379 (including 133847.doc -24- 200916126

Pfeiffer dry powder nasal delivery device). Suitable multi-dose DPI devices for nasal administration include WO 04/33009 (including POWERJETTM devices), WO 06/90149 (including OPTINOSETM dry powder nasal applicators), and WO 90/13328 and WO 96/16687 ( These include those described in the TURBOHALERTM device. The lipid dispersions and respirable dry powder formulations of the present invention may also be reconstituted in the vehicle immediately prior to administration and aerosolized using a nebulizer, an aqueous droplet inhaler or a nasal applicator. Suitable nebulizers include conventional nebulizers such as jet nebulizers' such as the PARI LC series (PARI GmbH) and vibrating membrane nebulizers such as OMRON Micro A-I-R, (OMRON),

Aeroneb® (Nektar Therapeutics Inc.) ' PARI eFlow® (PARI GmbH), PARI eFlow®rapid, iNeb inhaler (Respironics) and soft or soft spray inhalers such as AERx® (Aradigm Corp., US),

Mystic (Ventaira Pharmaceuticals Inc.), Aria (Chrysalis

Techn〇logles Inc.) or Respimat® (Boehringer Ingelheim). The inhalable dry powder formulations of the present invention may also be dispensed in a suitable propellant system such as, but not limited to, a hydrofluoroalkane (hfa) such as HFA 134a or d HFA 227 with or without other excipients Aerosolization is carried out using a suitable pressurized metered dose inhaler (PMDI). Thus in accordance with the invention there is provided a pharmaceutical composition for inhaled and/or nasal delivery of one or more drug substances, such as oligonucleotides and/or siRNA, comprising a lipid dispersion, a lipid vesicle of a drug substance . According to another aspect of the invention, the lipid dispersion comprises an encapsulated one or more 'provided for inhalation and/or nasal delivery 133847.doc -25- 200916126 one or more drug substances (such as oligonucleotides and/or Dry powder formulation of siRNA) The composition comprises a lipid dispersion comprising an encapsulation having an average diameter between 70 nm and 550 nm, preferably between 1 〇〇 nm and 250 nm. Lipid vesicles such as drug substances. Thus according to the invention 'providing a pharmaceutical composition for inhaled and/or nasal delivery of one or more drug substances, such as nucleotides and/or siRNAs, comprises a lipid dispersion comprising A lipid vesicle encapsulating one or more drug substances, wherein the dispersion is formed by a method comprising the steps of: (a) mixing the lipid with a co-lipid (such as a pegylated phospholipid, or cholesterol) from the south to the cut. (b) mixing one or more drug substances; and (0 removing the fluidity enhancer to form a lipid dispersion comprising lipid vesicles encapsulating the or the drug substance. According to another aspect of the invention Providing a dry powder formulation for inhaled and/or nasal delivery of one or more drug substances, such as nucleotides and/or siRNAs, the composition comprising a lipid dispersion comprising an encapsulated or π a lipid vesicle of a drug substance such as hai, wherein the formulation is formed by a method comprising the steps of: (a) mixing the lipid with the co-lipid under high shear; (b) mixing one or more drug substances; (c) Mixing fluidity To form a lipid dispersion comprising lipid vesicles encapsulating the or the drug substance; (d) diluting the lipid dispersion as appropriate; and 133847.doc • 26- 200916126 (e) drying the lipid dispersion to form Powder formulation. According to another aspect of the invention 'providing a free-flowing dry powder formulation for intranasal administration of one or more drug substances (such as oligonucleotides and/or siRNA) via the nasal anterior portion, the formulation The lipid dispersion comprises a lipid vesicle encapsulating the or the drug substance, wherein the average vesicle size distribution as measured by laser diffraction is: (a) 10°/. Below X 1 ( value (a) 50% below χ5〇 value (c) 90% below χ9〇 value. In another aspect of the invention, one or more drug substances (such as nucleotides) are provided And/or siRNA) a pharmaceutical composition substantially as described in any one of compositions a, Β, c or D. According to another aspect of the invention, one or more inhaled or nasal delivery drugs are provided Substance treatment for human patients (by, for example, asthma, allergic rhinitis, COPD, pulmonary fibrosis) A method of pulsing blood pressure in a pulmonary circulation and/or erosive fibrosis, the method comprising providing a pharmaceutical composition or a dry formulation as described above. In another wicking, as provided above The invention relates to a pharmaceutical composition or a dry powder formulation for medical treatment of a human patient. The invention also provides a pharmaceutical product comprising a pharmaceutical composition as described above in combination with one or more delivery devices. In another aspect, the invention The invention provides a delivery device or a package of two or more delivery devices comprising a pharmaceutical composition as described above. Example 133847.doc • 27· 200916126 The invention is illustrated by the following examples. EXAMPLES Example 1 Preparation of a pharmaceutical composition of the invention

Preparation of pharmaceutical compositions A, B, C and D from the following: Composition A Component amount (%) Functional oligonucleotide A 1.5 Drug substance hydrogenated PC (lipid SPC-3) 70 Lipid material for encapsulation Pegylated phospholipid choline 8 co-lipid (lipid vesicle stabilizer) calcium phosphate 5 anion compensation lactic acid, pH 4.0 0.1 buffer mannitol 10 fluidity enhancer bovine serum albumin 2 diluent phosphate buffer salt , pH 6.5 3.4 buffer

Composition B Component Amount (%) Functional Oligonucleotide A 1.5 Drug substance Di-pipylphosphatidylcholine choline (DPPC) 60 Lipid material for encapsulation Pegylated phospholipid choline 8 Co-lipid ( Lipid vesicle stabilizer) CaCl2.2H20 5 Negative ion compensated lactic acid, pH 4.0 0.1 Buffer glycine 5 Flow enhancer Bovine serum albumin 17 Thinner phosphate buffered saline, pH 6.5 3.4 Buffer

Composition C Component (%) Functional SiRNA A 3 Drug substance DPPC 60 Lipid material for encapsulation Pegylated phospholipid choline 8 Co-lipid (lipid vesicle stabilizer) CaCl2.2H20 5 Negative ion compensated lactic acid 'pH 4.0 0.1 Buffer Maltose 5 Fluidity Enhancer Bovine Serum Albumin 15.5 Thinner Phosphate Buffer Salt, pH 6.5 3.4 Buffer 133847.doc -28- 200916126

Composition D Component (%) Functional SiRNA A 3 Drug substance hydrogenation PC (lipid S PC-3) 60 Lipid material for encapsulation Pegylated phospholipid choline 10 Co-lipid (lipid vesicle stabilizer) Calcium Stearate 3 Negative Ion Compensating Lactic Acid, pH 4.0 0.1 Buffer Glycine 5 Fluidity Enhancer Bovine Serum Albumin 15.5 Thinner Phosphate Buffered Salt, pH 6.5 3.4 Buffer Oligonucleotide A is TLR9-Efficient An immunomodulatory oligonucleotide or "immunomer 1" suitable for the treatment of allergic inflammatory diseases, including allergic rhinitis. Its structure can be expressed as follows: 5'-TCR2AACR2TTCR2-Y-TCTTRlCTGTCT-5 '(SEQ.IDNO:l) wherein C is cytosine, T is thymine, G is guanine, Y is a propylene glycol linker and R1/R2 is a synthetic guanosine derivative as follows: OH ΟΗ

Rl = Arabinoguanosine 〇

瞧R2=2'-deoxy-7_deazaguanine 133847.doc -29- 200916126 describes oligonucleotide A and in International Patent Application WO 2006/002038 (see SEQ ID NO 22, elsewhere) Its preparation method. Immunological substances (generally) and methods for their manufacture are also described in the International Patent Application WO 2003/035836 and WO 2003/057822. The contents of all three documents are incorporated herein by reference. Oligonucleotide A is water soluble, so it is dissolved in a 0.1% lactic acid solution (pH 4) to neutralize the negative charge on the molecule. siRNA A is an siRNA for Flu. It is a chemically synthesized RNA that silences Flu-mRNA in the treatment of pandemic influenza. The SiRNA in use is a short double-stranded oligosaccharide (MW: 14 kDa) containing 21 nucleotides per share. Its structure can be expressed as follows: Sense strand sequence: 5,-GAGCCUAUGUGGAUGGAUUTsT-3, (SEQ.IDNO: 2) Antisense strand sequence: 5'-AAUCCAUCCACAUAGGCUCTsT-3' (SEQ.IDNO: 3) where single letter nucleus The glycoside is encoded as: A is adenosine, C is a cytosine nucleoside, G is guanosine, U is uridine, sT is thymidine and the hyphen indicates a 3'-5' phosphodiester bond. The drug substance is also negatively charged and water soluble, so it is dissolved in a 0.1% lactic acid solution (pH 4) to reduce the negative charge on the molecule. In each case, the lipid, co-lipid and solvent system was poured into a high shear mixer (Collette Micro Gral high shear mixer, Niro Pharmasystems, Denmark) and at 50-60 rpm at 50-150 rpm. Mix the impeller speed and the chopper speed of 200-600 rpm for about 20 minutes straight to 133847.doc -30-200916126 to form lipid particles. A pumping system providing a flow rate of 0 5_丨〇 mL/min was used to inject the drug substance, diluent, lactic acid (pH 4.0) and ion balance agent into the lipid granules by ISMATEC SA (Switzerland). A fluidity enhancer and a buffer (pH 6.5) are added to form a lipid dispersion of the drug substance or the drug substance encapsulated in the lipid vesicle. This is evident by the fact that the turbidity of the lipid dispersion does not significantly deposit over two hours. The lipid vesicles in the lipid dispersion have an average diameter between 70 nm and 5 50 nm as determined by dynamic light scattering techniques using a Master Zetasizer (Malvern Instruments Ltd, UK). Example 2 Preparation of the dry powder formulation of the present invention The pharmaceutical compositions A, b, c and D prepared in Example 1 were further processed to prepare a dry powder formulation of the present invention. In the case of A and B, the nanosuspensium formed in the example crucible was diluted to have a solids weight of 2% per volume, while in the case of Example 1 + C&D, it was diluted to have a volume per volume. 5% by weight solids to give a lipid dispersion with low viscosity which can be easily atomized in a spray dryer and form small respirable free-flowing powder particles (less than 1 〇μηι), followed by a Biichi 191 spray dryer (Biich lab〇r Technick Α(},

Each of the diluted lipid dispersions was spray dried, and the dispersion was atomized in a hot air of 9 G ° C in a spray dryer to obtain a free-flowing dry powder composition. The dry powder has an average geometric particle size of about 4 -1 3 μηη as measured by a HEL〇s laser diffraction instrument (Sympatech GmbH, Germany). 133847.doc 31 200916126 Example 3 Flowability of the dry powder formulation of the present invention The dry powder formulation prepared in Example 2 t was tested using STAV j 、 (j Engelsmann AG, Germany). In each case, the 丨〇g dry powder formulation was placed in a graduated cylinder. Measure the volume of the powder and beat the dry powder as follows in g/cm3 & units of the same bulk density or "BD": BD [g/cm3] = ^ > end weight (g) / powder bed volume (8) 丨 or cy) 1250 times and the volume of the powder bed was measured. Calculate the knock tightness or "TBD": TBD [g/cm3] = powder weight (g) / knock the powder bed volume (ml or cm3) as follows to calculate the Carr index: Carr index [%] = (Knock tight Degree-volume density*100/knock tightness The Carr index of the four dry powder formulations is given in Table 1 below: Dry powder Karl index (%) A 22 B 29 —~-—c 30 D 17 These results indicate dry powder blending The powder is free flowing. Example 4 Particle Size Characteristics of the Dry Powder Formulation of the Invention The particle size characteristics of the dry powder formulations prepared in Example 2 were determined by a HEL〇S laser diffraction apparatus [SymPateeh GmbH, Germany]. The results are given in Table 2 below (n=3): 133847.doc • 32- 200916126 Table 2 Dry Powder VMDium) GSD "^Ί A 12,90 ±2.74 5.10 ±0.37 B 7.58 ±0.05 2.20 ±0.01 ' C 4.18 ± 0.03 1.95 ±0.01 D 4.46 ±0.01 1·92±0.00 In the table, 'VMD is the volume average diameter and GSD is the geometric standard deviation. These particle size characteristics indicate that the dry powder formulation should target the desired area of the lung. Example 5

Further Analysis of Dry Powder Formulation Prepared from Pharmaceutical Composition C The dry powder formulation prepared from Pharmaceutical Composition C of Example 2 was further analyzed. The characteristics of this powder are summarized in Table 3 below: Table 3 Parameter results ΧΙΟ (μιη) 1.88 ±0.02 —~ Χ50 (μιη) 3.66 ±0.04 Χ90 (μηι) 7.17 ±0.03 Volume osmotic concentration (mOsmo) 6.33 ±0.58 —~ Powder density (g/cm3) 0.1015 Carr index Γ 30% VMD ("m) 4.18 ± 0.03 GSD 1.95 ±0.01 "1 degree, 50% of the particles have an average particle size below the X50 value and 9% of the particles have The X90 value measures the average particle size below. The volumetric osmotic concentration as measured by a micro-osmometer (Advanced Inst, lnc, US) indicates that the siRNA system is effectively encapsulated. The VMD is the volume average diameter and the GSD is the geometric standard deviation. The median aerodynamic diameter [MMAD or daer] can be calculated from the volume diameter [dv] by the following formula: 133847.doc -33- 200916126 d〇er where P is the powder density in g/cm3. The low density ensures low MMAD, which produces a high-fine fine particle fraction [FPF<5 μηι]. The particle size distribution of the lipid dispersion as measured by laser diffraction is shown in Figure 1 of the accompanying drawings. The particle size distribution measured by the shot is shown in Figure 2 The average particle size of the dispersion was 517 5 nm. The lipid dispersion had a dispersion index of 0.47. 'These results show that the dry powder formulation comprises monodisperse particles suitable for inhaled administration and which are substantially free flowing. Example 6 Self-medicinal composition D Further analysis of the prepared dry powder formulation further analyzed the dry powder formulation prepared from Pharmaceutical Composition D of Example 2. The characteristics of this powder are summarized in Table 4 below: ΎΚ " Results Χ10 (um) 2.01 ±0.00 X50 (um) 3.97 ±0.00 X90 (um) 7.42 ± 0.02 Grain concentration (mOsmo) 0 Powder density (g/cm3) 0.07 Carr index 19% VMDium) 4.46 ±0.01 GSD 1_92±0.00 In this table, 10% The particles have an average particle size below the χ10 value metric, 50% of the particles have an average particle size below the χ5〇 value metric and 90% of the 133847.doc -34- 200916126 particles have an average particle size below the X90 value metric. The volumetric osmotic concentration measured by a micro-osmometer (Advanced Inst. Inc, US) indicates that the siRNA is effectively encapsulated. The VMD is the volume average diameter and the GSD is the geometric standard deviation. The mass median aerodynamic diameter (MMAD or daer) can be calculated from the volume diameter [dv] by the following formula: daer = where is the density of the powder in g/cm3. The low density of this powder ensures low MMAD, It produces a high-fine particle fraction (FPF < 5 μιη). The particle size distribution of the lipid dispersion as measured by laser diffraction is shown in Figure 3 of the accompanying drawings. The particle size distribution measured by laser diffraction (Malvern Zetasizer, UK) is shown in Figure 4. The average particle size of the lipid dispersion was 229.5 nm. The lipid dispersion has a dispersion index of 0.261. These results demonstrate that dry powder formulations are suitable for inhalation administration and are generally free of 'flow. Example 7 Preparation of Other Pharmaceutical Compositions of the Invention Pharmaceutical compositions E, F, G and oxime were prepared from the following: Composition Ε Component amount (g/i) Functional nucleus A 0.075 Drug substance bis-lipid 醯Phospholipid choline (DPPC) 3.000 Lipid material for encapsulation PEG- scorpion sputum test 0.250 Co-lipid (lipid vesicle stabilizer) 133847.doc -35- 200916126 Component (g/i) Function CaCl2. 2H20 0.250 Negative ion compensated lactic acid, pH 4.5 0.005 Buffer Na2HP04.H20 > pH 6.5 0.170 Buffer bovine serum albumin 1.150 Thinner inulin 0.100 Fluidity enhancer / particle surface modifier

Composition F Component Amount (g/i) Functional Oligonucleotide A 0.075 Drug Substance Di-pipylphosphatidylcholine choline (DPPC) 3.000 Lipid material for encapsulation PEG-phospholipid choline 0.250 Co-lipid ( Lipid vesicle stabilizer) CaCl2.2H20 0.250 Negative ion compensated lactic acid, pH 4.5 0.005 Buffer Na2HP04.H20, pH 6.5 0.170 Buffer D, L-leucine 1.150 Thinner inulin 0.100 Fluidity enhancer / particle surface modification Agent

Composition G component amount (g/1) Function siRNAB 0.140 Drug substance di-myristyl phospholipid choline (DMPC) 2.000 Lipid material for encapsulation PEG-filled fat test 0.250 Co-lipid (lipid vesicle) Stabilizer) Calcium phosphate 0.150 Negative ion compensated lactic acid, pH 4.5 0.005 Buffer Na2HP04.H20, pH 6.5 0.170 Buffer bovine serum albumin 1.785 Thinner glycine 0.500 Fluidity enhancer composition Η Component amount _ Functional siRNAB 0.140 Drug substance Di-myristylphospholipid choline (DMPC) 3.500 Lipid material for encapsulation cholesterol 0.250 Co-lipid (lipid vesicle stabilizer) CaCl2.2H20 0.250 Negative ion-compensated lactic acid, pH 4.5 0.005 Buffer Na2HP04.H20, pH 6.5 0.170 Buffer Bovine Serum Albumin 0.435 Diluent Magnesium Stearate 0.250 Fluidity Enhancer These lipid lipid dispersions were prepared as described in Example 1, 133847.doc -36- 200916126 where oligonucleosides Acid A is the same immunomodulatory oligonucleotide with TLR9-agonist activity, whereas the oligonucleotide is negatively charged and has a -45 mV (using Master Zetasizer, Ma) Lvern Instruments Ltd, UK measures the g(Zeta) potential, and after encapsulation, the zeta potential increases to a neutral value of (0 mV to -6 mV). Reducing the negative charge on the oligonucleotide in this manner avoids irritation in the nasal mucosa and allows the drug substance to exist in an unionized form suitable for absorption. siRNA B is an siRNA designed against luciferase. It is a chemically synthesized RNA which hydrolyzes luciferine to produce a light firefly protein. The SiRNA in use is a short double-stranded oligosaccharide (MW: 13486 Da) containing 21 nucleotides per share. Its structure can be expressed as follows: Sense strand sequence: 5'-CUUACGCUGAGUACUUCGAdTsdT-3' (SEQ.IDNO: 4) Antisense strand sequence: 5'-UCGAAGUACUCAGCGUAAGdTsdT-3' (SEQ.IDNO: 5) where single letter nucleus The glycoside is encoded as: A is adenosine, C is a cytosine nucleoside, G is guanosine, U is a uridine nucleoside, sT is thymidine and the hyphen indicates a 3·-5' phosphodiester bond. The drug substance is also negatively charged and water soluble, so it is dissolved in a 0.1% lactic acid solution (pH 4) to reduce the negative charge on the molecule. The solid concentration of the composition in the lipid dispersion was set to 5% w/w. Example 8 Preparation of a dry powder formulation of the present invention for intranasal administration The pharmaceutical compositions E, F, G and hydrazine prepared in Example 7 were spray-dried to 133847.doc -37-200916126 for intranasal application. The dry powder formulation of the present invention administered. Using a Biichi 191 spray dryer (Biich lab〇r Techniek AG,

Switzerland) Spray drying to obtain a free flowing dry powder. A 2 mm spray nozzle was used to prepare a dry powder formulation for nasal administration with an average particle size greater than 2 〇 μηι for nasal deposition prior to target. A 〇.7 mm spray nozzle was used to prepare a dry powder formulation for nasal administration with an average particle size between 1 μm and 20 μm for nasal deposition after targeting. Example 9 Characterization of Dry Powder Formulations for Intranasal Administration of the Invention Master Zetasizer (Malvern) by Dynamic Light Scattering Technique

Instruments Ltd, UK) was used to determine the average vesicle size of one of the compositions E, F, G and sputum of Example 7. Therefore, the polydispersity index (PDI) of each composition was determined. Is it an indication of the width of the particle distribution, low? ;^ can indicate a narrow distribution. The results are shown in Table 5 below: Table 5 Lipid dispersion vesicle size (rnn) Polydispersity index Ε 220.7 0.291 F 352.6 0.251 G 207.3 0.205 Η 379.6 0.208 Example 10 Encapsulation efficiency of dry powder formulations for intranasal administration The encapsulation efficiency of each of the formulations E v F, G and Η was determined by high performance liquid chromatography (HPLC) by gel electrophoresis and by capillary gel electrophoresis (CGE). In the HPLC method, each dry powder formulation was resuspended in phosphate buffered saline (PBS), simply mixed, and the non-133847.doc • 38 - 200916126 encapsulated nucleotides were extracted by centrifugation, and analyzed. The presence of free oligonucleotides in the serum. Using this method, the encapsulation efficiency of nucleotide A and siRNA A was calculated to be >95°/. . In the gel electrophoresis method, each of the pharmaceutical compositions £, ρ, 〇, and η is loaded on a 4% agarose-gel (In vitro genTM), and a negatively charged free oligo is loaded thereon. The glycoside moves toward the positively charged cathode. The uv transilluminator was used to determine the qualitative detection of oligonucleotides. In capillary gel electrophoresis, a liposome sample is injected into a coated capillary containing a replaceable gel for screening. A BECKMAN PA800 capillary electrophoresis instrument equipped with a fixed length detector has been used. Examples of compositions E and F are presented in Figure 5. All methods demonstrate consistent results for liposome and encapsulation efficiency in powders. Example 11 Particle size of dry powder formulations for intranasal administration The particle size of each of the dry powder formulations e, F, G and mash was measured using a HELAS laser diffraction with a wet dispersion (from Synipateh GmbH, Germany). The results are not shown in Table 6 below: Table 6 Average diameter of dry powder reservoir (μηι) Target νΜΐ) (μιη) E 16.98 ± 1.16 >10 “m _ F 30.03 ± 0.26 > 10 um “ G 11.01 ± 0.19 > 10 HXYi Η 10.53 ±0.06 >10 um In each case, the volume mean diameter (VMD) was found to be optimal for nasal deposition. The mass median aerodynamic diameter (MMAE^tdaer) can be directly controlled from volume [dv ] Calculated by: 133847.doc •39- 200916126 daer=dv^[p where p is the choice of the powder density particle size in g/cm3 to dry to a specific area in the nasal cavity. For example, The dry powder formulation E has a drug deposition into the anterior portion of the nose as shown by the laser diffraction measurement shown in Figure 6 and summarized in Table 7, wherein the permeability is generally lower than elsewhere in the nose: 7 δδ Ί Χ10 (ixm) 16.75 ±0.49 _Χ50 (μιη) 31.25 ±0.18 " Χ90 (μιη) 43.53 ±0.85 GSD 1·16±〇·〇3 10% of the particles have an average particle size below the X10 value 50% of the particles have an average particle size below the X5G value and 9G% of the particles have an X90 value The following average particle size is measured, in each case measured by ELOS field diffraction as a wet dispersion (from GmbH, Germa (4), and GSD is the geometric standard deviation of the previously measured particle size. The particle size distribution is limited by the first three-thirds of the skin-like epithelium deposited on the nose—providing longer than the usual nasal retention time, which limits absorption' and is particularly useful for providing local therapeutic effects. 'Dry powder formulation F has the particle size distribution shown in Figure 7 and summarized in Table 8 by the & laser diffraction measurement to dry the nose in the nose of the nose, which is thin Covered by laminar mucosa and dense vascular network, the permeability is generally higher than other parts of the nose: 133847.doc -40- 200916126 Table 8 Parameter results ΧΙΟ(μηι) 3.40 ±0.05 Χ50(μιη) 9.60 ±0.19 Χ90(μηι) 20.83 ± 0.36 GSD 2.48 ± 0.00 where ΧΙΟ, Χ5〇, χ9〇 and GSD have the same meaning as before and the particle size distribution shown in Figure 7 and Table 8 is therefore shorter than usual due to the dense vascular network in this region Nasal retention In particular, and therefore particularly suitable for providing systemic absorption.Example 12 pH of dry powder formulations for intranasal administration The pH of each dry powder formulation is acidic, ie between about pH 4.5 and pH 6.5 The pH of the nasal cavity of each formulation is sufficiently acidic to avoid inactivation of the lysozyme present in the nose and to combat any bacteria that pass into the nose. For example, the dry powder formulations E, F, G and strontium have pH values of 5.3, 5.5, 6.8 and 5.5, respectively. Example 13 Flowability of a dry powder formulation of the present invention for intranasal administration refers to the volumetric bulk density (BD), knock tightness (TBD), and especially the Karl index (using a STAVII densitometer (J Engelmann AG, Germany)). The measured values were used to determine the fluidity of the dry powder formulations E, F, G and hydrazine. In each case, 1 g of dry powder formulation was placed in a graduated cylinder. Measure the volume of the powder and calculate the bulk density in g/cm3 as follows or "BD·,: BD [g/cm3] = powder weight (g) / powder bed volume (mi or cm3) 133847.doc •41 - 200916126 The dry powder was tapped 1250 times and the powder volume bed was measured. Calculate the knock density or "TBD" as follows: TBD [g/cm3] = powder weight (g) / tighten the powder bed volume or cm" Calculate the Carl index as follows: Carr index [°/. ] = (knock tightness - bulk density) * 100 / knock tightness results are the average of the three measurements and are shown in Table 9 below: Table 9 Dry powder formulation bulk density (g / cm3) knock tightness ( g/cm3) Carr index (%) E 0.150 0.177 16 F 0.123 0.166 26 G 0.166 0.200 17 Η 0.222 0.250 11 ------- 决 I 1Χ· cur. Jo, 7' 2.936>), this 4 powder Has good fluidity. Although not intended to be limited by theory, this good fluidity can be attributed to the formation of m m enhancer nanocrystals in the upper part of the powder particles as by scanning electron microscopy (p(8) coffee red 20, PhiUips Bv, The (4) Heart) and X-ray powder diffraction analysis (Stoe & Cie GmbH, Gemany) detected, for example, dry powder formulation G (see Figure 8). Example 14 For the spray drying of the dry powder formulation for intranasal administration, the cut point (cuUp〇int) of the cyclone of the spray dryer was optimized to optimize the spray drying method. Yield. The antistatic coating is further coated with spin 铷, KW, 纮, Shi Xi Shi Yan Feng poly (3,4-extended ethylenedioxythiophene) 汆 (this vinyl sulfonate) or amphiphilic 133847.doc -42 - 200916126 Sex surfactant to coat) to increase the yield. For example, the pharmaceutical compositions E, F, G and hydrazine, which are lipid dispersions, are spray dried in Β. γ 191 spray dried n(10)Chi AG, Switzerland). A high efficiency glass cyclone having an internal enthalpy of 6 to 3 cm is coated by an antistatic surfactant such as, but not limited to, hydroxypropylmethylcellulose. Setting the inner diameter of the cyclone to 6 3 improves the separation of smaller particles that can cause loss of product on the post filter (four). The drying of each of the above pharmaceutical compositions is given in Table 10 below. The yield of each dry powder formulation prepared. Table 10 Dry powder formulation method Yield E 68 F 62 G 74 Η 75 The yield was above 6〇% at laboratory scale, indicating a further scale-up step Higher yield. Female Example 15: In this example, a formulation composition was used. By custom preparation, a free-flowing lipid powder having two different particle sizes can be prepared. The dry powder granules are small enough to target alveolar deposition. The granules prepared using Method 2 are larger to target upper tracheal deposition. Both vesicles have a narrow particle size distribution that ensures efficient local deposition in the lungs. Doc -43· 200916126 Composition example i Component amount [g/L] Functional siRNAB 0.300 Drug substance bis-lipidyl phospholipid choline (DPPC) 3.500 Lipid material PEG-phospholipid choline for sputum seal 0.200 Co-lipid (lipid vesicle stabilizer) Cholesterol 0.500 Co-lipid (lipid vesicle stabilizer) CaCl2.2H20 0.250 Negative ion-compensated lactic acid, pH 4.5 0.006 Buffer Na2HP04.H20 'pH 6.5 0.017 Buffer glycine 0.227 Fluidity enhanced Agent/Particle Surface Modifier As described above, the preparation method comprises 2 steps. Step I is kept constant, and Step II is a spray drying method tailored to target a specific dry powder particle size distribution. Step 1 (in high shear Solvent injection technique with cut mixing): • Dissolve Formulation A (DPPC, PEG-PC and cholesterol) in 100 ml of ethanol and place in a high shear mixer and adjust to a temperature of 45 ± 5 ° C. • Formulation B (siRNA B, CaCl2.2H20) was dissolved in 800 ml of lactate buffer (pH 4.5) and slowly injected into Formulation A at a flow rate of 3 ml/min using a suction system while continuously mixing. Formulation C (glycine dissolved in 100 ml of phosphate buffer (pH 6.5)) was mixed with Formulation A+B at a flow rate of 10 ml/min. • The resulting lipid dispersion had a 〇. 5% w Solid content of /w. Step 11 (spray drying): • Use Btichi B-191 laboratory scale spray dryer (Biichi AG, Flawil, Switzerland) spray dried the prepared lipid dispersion to form a custom aerodynamic diameter for pulmonary application to 133847.doc -44 - 200916126 Free-flowing particles that target the alveolar region (Method 1) or the upper trachea (Method 2). A complete description of the setup parameters for both methods is presented in Table 11: Table 11

Parameter Method 1 Method 2 Target Alveolar Deposition Upper Airway Deposition Spray Nozzle Two Fluid Parallel Flow Nozzles Two Fluid Parallel Flow Nozzle Nozzle Diameter 0.5 mm1 0.7 mm1 Feed Air Pressure 7 Bar 1 5 Bar Aspirator Airflow 120 L/min1 90 L /min Liquid feed rate 2.5 mL/min 6 mL/min1 Inlet temperature 115±5〇C 130±5〇C - Outlet temperature 50 ± 5°C 50 ± 5°C *Main factors affecting particle size. The following findings can be derived from this experiment: • Since the formulation composition and step I are the same for both batches, a lipid dispersion with the same characteristics (vesicle size and zeta potential) was prepared using Master Zetasizer (Malvern Instruments) Ltd, UK) determined these characteristics [Table 12]. ί, • The encapsulation efficiency of the siRNA in the lipid dispersion was measured by the capillary gel electrophoresis method previously described. The data is presented in Table 12. Table 12 Lipid Dispersion Results Particle Size 423 ± 20 nm Polydispersity Index (PDI) 0.28 ± 0.01 ζ Potential 0·0 ± 3.1 mV Encapsulation Efficiency [%] 47.0 ± 0.08 133847.doc • 45· 1 Using HELOS Laser Diffraction Particle size based on powder volume measured by wet dispersion (Sympatech GmbH, Germany) [Table 13]: 200916126 Table 13 Dry powder method 1 Method 2 Target alveolar deposition upper tracheal deposition XI0 [μιηΐ 1.61 ±0.02 2.71 ±0·04 Χ50 Γμιηΐ 3.09 ±0.06 10.62 ±0.07 Χ90 ίμιηΐ 5.29 ±0.03 1 5.52 ±0.02 νΜϋ*Γμιη1 3.73 ± 0.04 12.03 ±0.03 GSD** 1.81 ±0.01 2.39 ±0.08 *νΜϋ=volume average diameter**GSD=geometric standard deviation •Use Next Generation The aerodynamic particle size distribution was measured at 60 L/min by a cascade impactor [NGI] (Copley Scientific Inc., UK). 5 mg of powder was aerosolized in the impactor using a DP4 PennCentury dry powder inhaler (PennCenury Inc., USA (n=2)) [Table 14, Figure 9, Figure 10]. Table 14 Dry powder method 1 Method 2 Target alveolar deposition upper tracheal deposition marker siRNA dose [μΕ] 300 300 Injection number per inhaler 1 1 Flow rate [L/min] 60 60 Total recovery from NGI [%LC* * * 93.3 ±3.1 95.7 ±2.0 Fine particle dose <5 μηι [jig] 198.3 ±8.3 94.6 ±4.8 Fine particle fraction <5 μηι [°/〇] 70.8 ± 0.6 33.1 ± 2.4 MMAD*_1 1.5 ±0.0 7.7 ±0.4 GSD** 2.4 ±0.1 4.1 ±0.7 Encapsulation efficiency [%] 78 ±0.6 80 ±0.8 *MMAD=mass median aerodynamic diameter**GSD=geometric standard deviation* * *LC=Labelle Claim=300 μ§ siRNA • Method 1 produces particles of MMAD of 1.5 μηι, which ensure effective alveolar 133847.doc -46- 200916126 deposition, and method 2 produces rivers into 1) 7.7 μηι particles, which are mainly deposited in the upper trachea. • Method 1 produced 71% fine particle fraction (FPF) < 5 μιη particles, which represents about 71% deep lung deposition. This high deposition efficiency can be attributed to the good flow and aerodynamic characteristics of this powder. Based on this high efficiency, a reduction in the therapeutic dose administered can be achieved, which is desirable in the case of expensive macromolecules. • Conversely, using Method 2, an FPF with 66% particles > 5 4 claws can be produced which ensures upper tracheal deposition. • The particle size distribution [PSD] presented in Figures 9 and 10 emphasizes that both methods produce particles with a single mode distribution. Method 1 produces particles smaller than Method 2, however it also produces more agglomerates than Method 2, which can be inferred from higher throat deposition. This is attributable to the higher surface area of the 1-2 μηη particles compared to the 7_8 μηη particles. Method 1 produced particles with high deposition in stages 4 and 5, which had a truncated diameter of 2.82 μηη and 1_66 μηι at 6 〇 L/min, respectively. In contrast, Method 2 produced particles having a high deposition in the first and second stages, which had a cut-off diameter of > 8.06 μηη and 8.06 μηη at 60 L/min, respectively. Example 16 • Composition Example 1 (Example 15) was used to encapsulate a double-stranded siRNA that stimulates IF? and TNFa. • by using Malvern Zetasizer (Malvern Instruments Ltd., UK) or using the Helos Sympatech particle classifier (Sympatech I33847.doc -47- 200916126

GmbH, Germany) was used to measure the particle size distribution and zeta potential to characterize the lipid formulation. • Capillary gel electrophoresis was used to measure the encapsulation efficiency of siRNA in liposomes and powders. • Dynamic vapor adsorption (SMS Inc., UK) was used to measure the water absorption of drug substance powders and free-flowing lipid powders. Table 15 Parameter siRNA Liposomes Powder Average Particle Size N/A 423 ± 20 run 3.09 ± 0 06 Polydispersity Index (PDI) N/A 0.28 ± 0.01 31.25 ± 0 18 ζ Potential -45,3 ± 4.0 mV 0.0 ±3.1 mV N /A . ~ Encapsulation efficiency N/A 33 ± 0.1% 82 ±3 6 Maximum suction in SO% KH 30% w/w N/A 1.9% w/w

From the data presented in Table 15, it can be inferred that when the zeta potential is observed to decrease from _45 mV to 〇mV, the formulation can effectively encapsulate the siRNA. In addition, the powder has a much higher encapsulation efficiency than the lipid formulation, which emphasizes that each powder particle is a complete capsule in which the lipid vesicle and free 311 are encapsulated. This can be confirmed by SEM data [Fig. Xiq. In addition, free flowing powders produce particles that encapsulate siRNAs therein and thus reduce their hygroscopic characteristics and thus allow for better stability. However, this can be illustrated by the SEM shown in FIG. 10, which is not a porous space having an empty space such as pollen grains in its interior, and the volume average diameter of the specific particles and the amount of 2 aerodynamics The difference in value diameter is emphasized. These particles have a side of 3.73 _, at the same time MMAD, which means that the particle volume is more than twice the size of its aerodynamics 133847.doc -48- 200916126, which indicates that one-half of the space in these pockets is *. In addition, the actual density of these particles can be calculated as follows: ^aer ~ ^ ν λ[ρ where Ρ is the actual density in g/cm3, daer is the aerodynamic particle size in μηι and dv is in μηι The volume diameter of the unit. The density can be calculated as follows: /?-(l.5/3.7)2 The density of these particles = 0.16 g/cm3 The empty space in the particle can be calculated as follows: Space into the empty space in the capsule % = [1_ ( Knock tightness/particle density)]X 100=[1-(0.25/0.16)]χ 1〇〇] = 56% • This siRNA is encapsulated in a neutral lipid such as DPPC, which is a biological fluid of the lungs. Naturally, neutralizes and reduces possible side effects such as inflammatory reactions caused by IF? and TNFa [Figure 12]. • In addition, because the PennCentury DP4 endotracheal inhalation device (PennCentury lnc, USA) was administered in two doses at a dose of |mg/kg per dose to 80 atmospheres, no side effects were observed, so s The weekly ligand is well tolerated in vivo. Example 17 - Scale Up Test • Example of composition! (See Example 15) for scale-up experiments. • This method uses the UltimaGral high shear mixer (GEA pr〇cess)

Engineering, Denmark) from i [batch size scaled up to 1 〇 L batch size. The method parameters are the same for both batches 133847.doc -49- 200916126. The only exception was the solvent feed rate, which was set to 3 ml/min for the 1 L batch and 30 ml/min for the 10 L batch. • Scale the spray drying process from 5 g to 100 g using a Niro Mobile Minor spray dryer (GEA process Engineering, Denmark). The method parameters are illustrated in Table 16: Table 16 Parameters Powder Spray nozzle 0.5 mm bypass nozzle Feed nitrogen pressure 5.-7 bar Aspirator gas flow 80-110 m3/hr Inlet temperature 110-150. . Outlet temperature 45-65 〇C Feed liquid stream 0.5-3 kg/hr • A full description of the scale-up method is presented in Figure 12. • When the data obtained from the pilot scale batch material is comparable to the lean material obtained using the laboratory scale batch material, the method test was successfully performed. Proportional enlargement [Table 17]. Table 17

Laboratory scale trial size liposome batch size 1 L 100 L powder batch size 5 g 1〇〇g particle size 423 ± 20 nm 445 ± 6 nm polydispersity index (PDI) 0.28 ±0.01 0·20±0_02 zeta potential _ 0·0±3·1 mV 0.0 ±1.6 mV. ΧΙΟ [μιηΐ 1.61 ±0.02 2.23 ± 0.02 Χ50 [μιηΐ 3.09 ± 0.06 3.69±0_03 Χ90 [μιηΐ 5.29 ±0.03 6.09 ±0.06 VMD*_1 3.73 ± 0.04 , 3.84 土0_03 GSD** 1.81 ±0.01 1.77±0.02 . Carr index [%], 25% 30% Method yield [%] 70% 65% . Powder flow good flow powder Good flow powder 133847.doc -50- 200916126 *VMD= Volume average diameter "GSDs geometric standard deviation Example 18 - Accelerated stability data • Example of composition] (see Example 15) was stored in a white glass vial closed with untied rubber stopper (open storage stability) in the following Two different conditions lasted two months: 5. 〇, 25t: /6〇0/〇RH• and 40t/75% RH·. Distribute the powder with 5 mg of each powder, contributing 300 pg of siRNA per vial B. • The data shown in Table 18 indicates the siRNA according to the present invention. Stability. The only exception is a decrease in encapsulation efficiency and an increase in particle size distribution after storage for two months at 40T: and 75% RH. This can be attributed to a high relative humidity of 75% RH after 2 months. The lower CaCl2 is recrystallized from the formulation, which is expected when the packaging system used herein is released. This is confirmed by the SEM data [see Figure 14]. It is extrapolated from this data and stabilized at 5 °C. Stable for at least 8 months and stable for 2 months at 25 ° C / 60% RH. In addition, reduce the diffusion of water vapor into the powder by packaging the powder in a tidal packaging system (such as Alu / Alu foaming) Achieving a significant improvement in long-term stability at 4 (TC/70% rh.) Table 18 Stability data 0 time 2 months 5°C _25°C/60% R, H. _ 40°Γ /7S% R目 Visual white powder white powder white powder white powder <LOD* degradation product [%] <LOD* <LOD* <LOD* ΧΙΟ [μη] powder 1.61 ±0.02 1.61 ±0.03 1.66 ± 0.03 3 81 ± 0 03 Χ50 [μιη] powder 3.09 ±0.06 3.10 ±0.05 _ 3.30 ±0.09 15.91 ± 0.04 133847.doc -51 - 200916126 X90 [μπι] powder 5.29 ±0.03 5.69 ±0.01 6.26 ± 0.04 29.40 ± 0.10 VMD* [μιη] powder 3.73 ± 0.04 3.62 ± 0.03 3.71 ± 0.04 17.84 ± 0.20 GSD" powder 1.81 soil 0.01 1.88 ±0.01 1.94 ±0.02 2.78 ± 0.09 siRNA encapsulation efficiencyΓ %1 82.0 ± 3.6% 80.0 ± 0.8% 82.0 ± 2.1% 72.0 ± 1.0% Water content [°/. ]**** 1.49% 1.56% 1.69% 1.80% Powder Flow Good Flowing Powder Good Flowing Powder Good Flowing Powder Good Flowing Powder *LOD = Detection Limit ^VMDz Volume Average Diameter + s|t*GSD = geometric standard deviation "" using a thermogravimetric analyzer (Perkin Elmer, UK) to measure Example 19 • Fill 5 mg of Composition I powder containing 300 pg of siRNA B in 2 DIRECTHALER inhaling devices (DireetHaler A/ S, Denmark). The emission dose uniformity of free-flowing powders from these devices was tested at 12 L/min and the powder was collected on a glass fiber filter. • The average emission dose was equal to 4.14 soil 0.35 mg (n=20) and The residual amount of powder entering the device was 17.5%. All individual values and average values are in the EU Pharmacopoeia instructions. See Figure 14. Example 20 • Testing of siRNA via biological fluids using a specific release model for simulating biological conditions in the lungs Diffusion release profile • A 20 μm thick artificial lung surfactant (ALS) layer was placed in 6 plates on top of a PET filter with a 4 μm pore diameter. Use 0.05% (w/w) DPPC Phosphate Flush (pH 7〇) as the receptor medium. 133847.doc -52- 200916126 • 5 mg powder (containing 300 pg siRNA B) or 0.5 ml liposome (containing 300 pg) of the composition example I (see example 15) siRNA B) was dispensed on top of the ALS layer and the amount of siRNA diffused into the receptor medium via ALS and PET membrane filters at 37 ° C using Rp_HPLC over 24 hrs. • Entered phosphate buffer (pH as a control) A 0.5 ml of 600 pg/ml siRNA B solution in 7.0 ml was dispensed on top of the ALS layer and tested as described above. Compared to the siRNA solution and the lipid dispersion, the powder exhibited a faster siRNA release profile since after 2 hr siRNA was about 660/. Release from powder' compared to 53% and 32% release of siRNA solution and lipid dispersion, respectively (see Figure 15). [Simplified illustration] Figure 1: Measurement with Malvern Zetasizer Particle size distribution of the lipid dispersion of Composition C. Figure 2: Powder particle size distribution data for Composition c as measured using a Sympatech laser diffractometer. Figure 3: Lipid dispersion particle size of Composition d measured using a Malvern Zetasizer Distribution Figure 4: Using Symp Particle size distribution data of composition d measured by atech laser diffractometer. Figure 5. Evaluation of encapsulation efficiency of nucleotides a in formulation compositions E and F using capillary gel electrophoresis' which exhibited an encapsulation efficiency greater than 9%. Figure 6: Powder of Composition E as measured using a Sympatech laser diffractometer 133847.doc -53- 200916126 Particle size distribution data that can be deposited prior to targeting into the nasal cavity. Figure 7. Powder particle size distribution data for Composition E, measured using a Sympatech laser diffractometer, which can be deposited after implantation into the nasal cavity. Figure 8: X-ray diffraction of the powder of composition G in combination with a scanning electron microscope image. It emphasizes the presence of fluidity enhancer nanocrystals in the upper portion of the powder particles, which improves the powder flow characteristics. Figure 9: Aerodynamic particle size distribution of the composition measured using a PennCentury DP4 inhalation device at 60 L/min using a Next Generation Impactor (NGI). Method 1 shows a higher deposition compared to Method 2 in the lower stage. Figure 1 : Cumulative aerodynamic particle size distribution of the composition measured using a PennCentury DP4 inhalation device at 60 L/min using a Next Generation Impactor (NGI). Compared with the 4 5 μπι of the method 2, 50% of the particles produced by the method 1 are below 1.1. Figure 11: Scanning electron microscope (Sem) image of the powder of Composition I, showing each powder particle as a complete capsule. Figure 12: Use of the present invention to encapsulate the effect of inflammatory-triggered siRNA on cytokine expression. Although naked siRNA triggered INF? and TNFa, the siRNA encapsulated in the liposomes of the invention and the placebo liposomes of the powder of the invention did not trigger any cytokine-mediated inflammation. Figure 13: A scaled-up manufacturing process diagram of SuperSomesTM technology. Figure 14: At 40. 〇/75〇/〇 R.H. Scanning electron microscopy data of composition I for two months of open storage. Figure 15. The dose of composition e using DirectHaler nasal devices 133847.doc • 54- 200916126 Uniformity data. Figure 16. Comparison between siRNA release profiles via artificial lung surfactants for three different formulations: siRNA dissolved in pH 7.0 buffer, siRNA encapsulated in lipid dispersion composition I and capsule siRNA encapsulated in the powder of Composition I. ί.. 133847.doc -55- 200916126 Sequence Listing <H0>Swiss Business Novartis <120>Organic Compound<130> 52227-EP-EPA2 <140> 097132233 <141> 2008-08-22 <lt;;150> EP 07114981.9 <151> 2007-08-24 <150> EP 07123163.3 <151> 2007-12-17 <160> 6 <170> Patentln version 3.3 <210> 1 <211> 11 <212> DNA <213> artificial sequence <220><223> synthetic sequence: oligonucleotide A, immunoregulatory oligonucleotide or 11 immunogen having TLR9 agonist activity (i leg The portion of the "unomer)" <220>221> misc_feature <222> (3,7jl) <223> η is 2'_deoxy_7_deodorant <220><221> Misc_feature <222> (11).7(11) <223> is linked to SEQ ID NO: 2 via a propylene glycol linker <400> 1 tcnaacnttc η 11 <210> 2 <211> 11 <212> DNA <;213> artificial sequence <220><223> synthetic sequence: oligonucleotide A, immunomodulatory oligonucleotide or 11-immunity (i leg unomer) having TLR9 agonist activity <221> misc_feature <222> (7)..(7) <223> n is arabinose <220><221>misc-feature<222> (11).. (11 <223> linked to SEQ ID NO: 1 via a propylene glycol linker <400> 2 tctgtcnttc t Π <210> 3 <211> 21 133847 - Sequence Listing.doc 200916126 <212> DNA <213><220><223> Synthesis sequence: siRNA <220><221> misc_feature <222> (20).7(21) <223> phosphorothioate linkage <400> 3 gagccuaugu ggauggauut t 21 <210>4 <211> 21 <212> DNA <213> artificial sequence<220><223> synthetic sequence: siRNA <220><221> misc_feature <222> (20) .7(21) <223> phosphorothioate linkage <400> 4 aauccaucca cauaggcuct t 21 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220>223>Synthetic sequence: siRNA <220><221> misc_feature <222> (20).7(21) <223>Thylic phosphate linkage <400> 5 cuuacgcuga guacuucgat t 21 <210><211> 21

\ <212> DNA <213> artificial sequence <220><223> synthetic sequence: siRNA <220><221> misc_feature <222> (20)..(21) <223> Phospholipid bond, <400> 6 ucgaaguacu cagcguaagt t 21 -2- 133847 - Sequence Listing.doc

Claims (1)

200916126 X. Patent Application Range: 1. A pharmaceutical composition comprising: (A) - or a plurality of drug substances; (B) a lipid; (C) a co-lipid; and (D) a fluidity enhancer; the pharmaceutical composition Characterized in that the lipid, the co-lipid, and the flow enhancer together form a lipid dispersion comprising lipid vesicles encapsulating the or the drug substance. 2. The composition of claim 1 wherein the lipid vesicles have an average diameter between 70 nm and 550 nm. 3. The composition of claim 1 or 2, wherein the or each drug substance is a β2-adrenergic receptor agonist, a muscarinic antagonist, a glucocorticosteroid, a non-steroidal glucocorticoid receptor agonist , Αζα agonist, α2β antagonist, antihistamine, caspase (CASPASE) inhibitor, lTB4 antagonist, LTD4 antagonist, phosphodiesterase inhibitor, mucolytic drug s / (muc〇lytic), Matrix metalloproteinase inhibitors, leukotriene receptor antagonists, growth hormone, heparin, estradiol, prostaglandins! 2 antagonists, CRTH2 receptor antagonists, sodium cromoglycate, ig^ synthesis inhibitors, antibiotics, interferons, potassium channel inhibitors, immunomodulators, cytostatics, elastase inhibitors, antitumor agents, columns PROSTATIN inhibitors, peptides, oligonucleotides, rna (such as siRNA), DNA, plastids, insulin, interleukins, GLp], anti-tumor agents or antibodies. 133847.doc 200916126 4. As described in the request, any of the technical sulphurs, h, H, wherein the lipid is a sirloin-based sulphuric acid, _n_a _ S-soft moon phosphatase choline, Soybean egg yolk or bismuth phospholipid choline. PW 5. The diol phosphatide "16" co-lipid according to any of the preceding claims is polyethyl guanidine choline or cholesterol. 6. As stated in the above request, "@ Λ ...., , , , · And 5, wherein the fluidity enhancer is maltose, mannitol, inulin (4) to sugar sugar, dextrose, wheat =, glycine, serum albumin, L-leucine, D-amine Acid, diamine, stearic acid, stearic acid, erythritol or mixtures thereof. The composition of any of the claims, further comprising an ion balance agent and/or a buffer system The composition of any of the preceding claims, in the form of a dry powder formulation. 9. A dry powder formulation comprising: (Α) one or more drug substances; (β) lipids; (C) a co-lipid; and (D) a fluidity enhancer; the formulation characterized in that the lipid, the co-lipid, and the fluidity enhancer together form a lipid dispersion comprising a lipid vesicle encapsulating the drug substance or the drug substance and The enamel dispersion is dried to form a free-flowing dry powder formulation. 1 〇·If requested A dry powder formulation of the powder wherein the powder has an average particle size of no greater than about 10 microns for inhalation. 1 1. A dry powder formulation according to claim 9 wherein the powder has an average particle size of no greater than about 10 microns for nasal administration. The dry powder formulation of claim 11, wherein the average particle size is between 1 〇μηι 133847.doc 200916126 and 20 μπι called drug deposition in the posterior portion of the nose. 13·% of the dry powder formulation of claim 11, wherein The average particle size is greater than the deposition of the drug into the anterior portion of the nose. The method of preparing a pharmaceutical composition comprises the steps of: (a) mixing the lipid with the co-lipid under sputum shear; (b) Mixing one or more drug substances; and (4): combining a fluidity enhancer to form a lipid dispersion containing lipid vesicles encapsulating It or the drug substance. 15. A method for preparing a dry powder formulation, the method The method comprises the steps of: (a) mixing the lipid and the co-lipid under high shear; (b) mixing one or more drug substances; (4) mixing the fluidity enhancer to form a lipid comprising the drug or the drug substance Vesicle lipid Dispersing solution; (d) diluting the lipid dispersion as appropriate; and (4) drying the lipid dispersion to form the dry powder formulation. 133847.doc