Process for preparing submicron sized particles via dispersion and solvent or liquid phase removal

ϊυ»’» πίκα in vtttt ruvnov’T β>υηρ>» jm o>p>pbn vbflji

Process for preparing submicron sized particles via dispersion and solvent or liquidphase removal

Baxter International Inc. C.189868 WO 03/026611 PCT/US02/30447 196631/2

Processes for preparing frozen dispersed system of particles in a solid state 5

Technical Field

The present invention is a divisional application from IL Patent Application No. 10 160570 and pertains to a process for preparing submicron sized particles that may be used inpharmaceutical compositions. / 15 Background of .the Invention

There are an ever increasing number of pharmaceutical drugs being formulated that arepoorly soluble or insoluble in aqueous solutions. Such drugs provide challenges to deliveringthem in an injectable form such as through parenteral administration. Drugs that are insolublein water can have significant, benefits when formulated as a stable suspension of submicron sized 20 particles. Accurate control of particle size is essential for safe and efficacious use of theseformulations.

Particles must be less than seven microns in diameter to safely pass through capillarieswithout causing emboli (Allen et al., 1987; Davis and Taube, 1978; Schroeder et al., 1978; Yokelet al., 1981). One solution to this problem is the production of extremely small particles of the 25 insoluble drug candidate and the creation of a microparticulate or nanoparticulate suspension.In this way, drugs that were previously unable to be formulated in an aqueous based system canbe made suitable for parenteral administration. Suitability for parenteral administration includes 2 196631/2 small particle size (< 7 micron), low toxicity (as from toxic formulation components or residualsolvents), and bioavailability of the drug particles after administration.

The parenteral administration of such poorly water soluble pharmaceutical agents has beenachieved in the past using emulsions composed of a hydrophobic solvent (e.g., oil) and a 5 stabilized drug dispersed within an aqueous medium, such as a buffer solution or normal salinesolution. These liquid/liquid emulsions may be injected intravenously.

One example of this approach utilized the anesthetic, propofol (2,6 diisopropylphenol), in whichthe pharmacological agent was dissolved within a vegetable oil emulsion to enable intravenousadministration. See, e.g., US Pat. Nos. 4,056,635; 4,452,817 and 4,798,846, all to Glen et al. Such 10 emulsions, however, tend to be unstable given the predominance of the oil phase and the absenceof antimicrobial agents. In other instances, even where the pharmacological agent is successfullyincorporated into an oil-free formulation, particles containing the pharmacological agent maycause irritation at the site of delivery because of their size or form.

Furthermore, many insoluble drugs of interest do not show appreciable solubility within 15 traditional oil emulsion systems. One reason for this is that solubility is not strictly defined bypolarity, but also includes hydrogen bonding, dipole-dipole interactions, ionic stabilization andatom to atom interactions. WO 98/14174 and WO 99/00113 teach formation of nanoparticles inthe absence of any conventional surfactants. US Pat. No. 4,073,943, issued to Wretlind et al.,discloses a method of administering a water-insoluble pharmaceutically active agent by dissolving 20 the agent in oil and emulsifying the solution with water in the presence of surfactants (eggphosphatides, pluronics, polyglycerol oleate, etc.) to form stable lipoid particles of the agentdispersed in the aqueous phase. US Pat. No. 4,540,602, issued to Motoyama et al., discloses a process for the preparation of anactivated pharmaceutical composition containing a water insoluble drug for oral administration, i 25 one procedure of the invention (see Examples 4 to 10), the process is carried out by dissolving thedrug in hydrophobic organic solvents, and the resulting solution is emulsified in water. Thedispersing medium is then removed rapidly by spray drying, resulting in particles ranging inparticle size of from about 0.1 to about 3.0 micron. A variety of approaches have been explored for developing stable formulations of a substantially 30 water-insoluble pharmacologically active agent for in vivo delivery. One approach is directed to the production of suspended particles coated with protein. US Pat. 5,916,596, issued to Desai et al, discloses the application of high shear to a mixture of an organic phase having a pharmacologically active agent dispersed therein and an aqueous medium containing WO 03/026611 PCT/US02/30447 -3- a biocompatible polymer. The mixture is sheared in a high pressure homogenizer at a pressurein the range of from about 3,000 to 30,000 psi. The ‘596 patent provides that the mixture mustcontain substantially no surfactants because the combined use of a surfactant with a proteinresults in the formation of large, needle-like crystalline particles that increase in size during 5 storage. See columns 17-18, example 4. The biocompatible polymer may be crosslinked as theresult of exposure to the high shear conditions in a high pressure homogenizer. In theembodiment in which protein containing sulfhydryl or disulfide groups is used (e.g. albumin),the protein forms a crosslinked shell around droplets of non-aqueous medium. See Column 8,lines 35-48. In Examples 1, 2, 5, 6,9,10,11, and 12, toe organic phase is removed rapidly by 10 rotary evaporation at 40°C and at a reduced pressure of 30 mm Hg, resulting in an aqueousdispersion of particles coated with crosslinked protein. The aqueous dispersion may further belyophilized to remove the aqueous phase. The ‘596 patent discloses other alternative methodsof removing the solvent, including falling film evaporation, spray drying, and freeze-drying.Example 2 discloses that toe crude emulsion may be sonicated to produce nanoparticles ranging 15 from 350-420 nanometers. Example 5 discloses a method to prepare sterile-filterablenanoparticles of less than 200 nm. This method requires that the pharmaceutical agent is initiallydissolved in a mixture of substantially water immiscible organic solvent (e.g., chloroform) anda water miscible organic solvent (e.g. ethanol). U.S. Pat. No. 5,560,933, issued to Soon-Shiong et al., discloses the formation of a 20 polymeric shell around the water-insoluble oil (containing the drug) for in vivo delivery. Themethod discloses the application of sonication to a mixture comprising a polymer-containingaqueous medium and a dispersing agent (oil) having a substantially water-insoluble drugdispersed therein. In this reference, sonication is used to drive the formation of disulfide bondsin the polymer, causing it to crosslink so as to produce a polymeric shell around the drug. 25 Sonication is conducted for a time sufficient for the disulfide bonds to form.

In U.S. Pat. No. 5,665,383, Grinstaff et al. discloses the application of ultrasound to a single-phase, i.e., an aqueous medium, to encapsulate an immunostimulating agent within apolymeric shell for in vivo delivery. The ultrasound promotes crosslinking of toe encapsulatingagent by disulfide bonds to form toe shell. 30 Another approach to preparing a water-insoluble drug for in vivo delivery centers on reducing the size of toe particles that deliver toe drug. In one such series of patents, whichinclude U.S. Pat. Nos. 6,228,399; 6,086,376; 5,922,355; and 5,660,858, Parikh et al. discloses WO 03/026611 PCT/US02/30447 -4- that sonication may be used to prepare microparticles of the water-insoluble compound. Of these patents, U.S. Pat. No. 5,922,355 discloses an improvement to a method that uses sonication for making the smaller particles. The improvement comprises mixing an active pharmacological agent with a phospholipid and surfactants in a single-phase aqueous system and applying energy to the system to produce the smaller particles. U.S. Pat. No. 5,091,188, issued to Haynes, also discloses reducing the size of particlesof a pharmacologically active water-insoluble drug and employing a lipid coating on the particlesto confer a solid form. The patent is directed to a pharmaceutical composition of an aqueoussuspension of solid particles of the drug having a diameter of about 0.05 to about 10 microns.The lipid coating affixed to the surface of the particles contributes to their solid form. Thecomposition is produced by adding the drug to water and then reducing the particle size withinthe aqueous suspension. Example 6 of this reference discloses the use of a pharmacologicallyacceptable oil, which is selected for its inability to dissolve the crystalline drug. See column 16,lines 8-12.

Still another approach for preparing microparticles of a pharmacological agent focuseson the use of phase inversion principles. U.S. Pat. Nos. 6,235,224 Β1 and 6,143,211, both issuedto Mathiowitz et al., disclose the use of phase inversion phenomena to precipitatemicroencapsulated microparticles. The method includes mixing a polymer and a drug with asolvent. This mixture is introduced into an effective amount of a miscible nonsolvent, therebycausing spontaneous formation of the microencapsulated product.

Microprecipitation by pH shifting is another technology used to prepare dispersions ofa nanoparticulate pharmaceutical agent See, e.g., U.S. Pat. Nos. 5,665,331; and 5,662,883. Thistechnology involves dissolving a pharmaceutical in an aqueous base that is then neutralized toform a dispersion.

In yet another approach, such as that disclosed in U.S. Pat. No. 5,766,635, issued toSpenlenhauer et al., nanoparticles have been prepared by dissolving a polyethylene) oxide and/orpolypropylene) oxide in an organic solvent, mixing tire organic solution so formed with anaqueous solution to cause nanoparticles to precipitate out of solution, and microfluidizing theprecipitated solution without the use of surfactants.

The commonly assigned and co-pending U.S. Application Serial Nos. 09/874,499;09/874,799; 09/874,637; 09/953,979; and 10/021,692, which arc incorporated herein by referenceand made a part hereof, disclose a process for preparing submicron particles by 5 196631/2 microprecipitation. The process disclosed includes the steps of; (1) dissolving an organiccompound in a water miscible first organic solvent to create a first solution; (2) mixing the firstsolution with a second solvent of water to precipitate the organic compound to create apresuspension; and (3) adding energy to the presuspension in the form of high-shear mixing or 5 heat to provide a stable form of the organic compound having the desired size ranges. One ormore optional surface modifiers can be added to the first organic solvent or the second aqueoussolution.

The commonly assigned and co-pending U.S. Application Serial No. 09/964,273, which isincorporated herein by reference and made a part hereof, discloses a process for preparing 10 submicron particles by sonicating a crude emulsion of a multiphase phase system having anorganic phase and an aqueous phase, the organic phase having a pharmaceutically activecompound therein. The organic phase is evaporated from the emulsion under specific sonicationconditions in order to generate the drug particles. The particle formed by this process typically hasan average effective particle size of less than 2 micro m. Because of the difficulties posed by 15 poorly soluble drags in drug therapy, the need for new technologies continues to expand foraddressing these problems. SUMMARY OF THE INVENTION:

The present invention is directed to a process for preparing a frozen dispersed system ofparticles in a solid state comprising the steps of: 20 providing a crude dispersion of a multiphase system having an organic phase and an aqueous phase, the organic phase having a pharmaceutically active compound therein; adding at least first and second surface active compounds to the multiphase system;providing energy to the crude dispersion to form a fine dispersion; and freezing the fine dispersion to obtain a frozen dispersed system of particles in a solid state, 25 the frozen dispersed system comprising the organic phase, the aqueous phase, and the particles,the particles having a mean particle size of less than 500 nm, wherein the first surface activecompound is selected from the group consisting of anionic surfactants, cationic surfactants, andnonionic surfactants, and the second surface active compound comprises a surface activebiological molecule selected from the group consisting of albumin, casein, heparin, and hirudin. 30 The present invention is directed to a process for preparing a frozen dispersed system of particles in a solid state comprising the steps of: 01898683\87-01 6 196631/2 providing an organic phase of a pharmacologically active compound dissolved in a waterimmiscible solvent; providing an aqueous phase; adding at least first and second surface active compounds to the organic phase, the 5 aqueous phase, or both the organic phase and the aqueous phase; combining the organic phase with the aqueous phase to form a crude dispersion; providing energy to the crude dispersion to form a fine dispersion; and freezing the fine dispersion to obtain a frozen dispersed system of particles in a solid state,the frozen dispersed system comprising the organic phase, the aqueous phase, and the particles, 10 the particles having a mean particle size of less than 500 nm, wherein the first surface active compound is selected from the group consisting of anionic surfactants, cationic surfactants, andnonionic surfactants, and the second surface active compound comprises a surface activebiological molecule selected from the group consisting of albumin, casein, heparin, and hirudin. BRIEF DESCRIPTION OF THE DRAWINGS: 15 Fig. 1 is a schematic representation of a process of preparing submicron sized particles in amultiphase system by sonicating the system to evaporate a portion of the organic phase;

Fig. 2A is a schematic representation of a process of of preparing submicron sized particles in amultiphase system by freezing and lyophilizing a dispersion; Fig. 2Bis a schematic diagram of thesteps of freezing and lyophilizing the dispersion to obtain submicron sized particles of the present 20 invention.

Fig. 3 is a high magnification SEM photograph of itraconazole particles;

Fig. 4 is a low magnification SEM photograph of itraconazole particles; and 01898683\87-01 WO 03/026611 PCT/US02/30447 -7- FIG. 5 is an x-ray powder diffraction spectrum of itraconazole nanoparticles and rawmaterial.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While this invention is susceptible of embodiment in many different forms, there is 5 shown in the drawing, and will be described herein in detail, specific embodiments thereof withthe understanding that the present disclosure is to be considered as an exemplification of theprinciples of the invention and is not intended to limit the invention to the specific embodimentsillustrated.

The present invention relates to a process for preparing submicron sized particle 10 dispersions which includes the steps of: (1) providing a multiphase system having a liquid phasecomprising an organic phase and an aqueous phase, the organic phase having a pharmaceuticallyactive compound herein, and (2) removing a portion of the organic phase or the liquid phase ofthe multiphase system to form submicron sized particles of the compound.

In one aspect, the present invention provides a process for preparing submicron-sized 15 particle dispersions. The process comprises the steps of: (1) providing a multiphase systemhaving an organic phase and an aqueous phase, the organic phase having a pharmaceuticallyactive compound therein; and (2) sonicating the system to evaporate a portion of the organicphase to cause precipitation of the compound in the aqueous phase and having an averageeffective particle size of less than about 2 pm. 20 In another aspect, the present invention provides a process for preparing submicron-sized nanoparticles. The process includes the steps of: (1) providing a crude dispersion of a multiphasesystem having an organic phase and an aqueous phase, the organic phase having apharmaceutically active compound with or without surface active compounds dissolved therein;(2) providing energy conditions to the crude dispersion to form a fine dispersion having an 25 average effective oil droplet size of less than about 2 pm; (3) freezing the fine dispersion so thatthe bulk phase is minimally in a frozen state; and (4) lyophilizing the dispersion to obtainnanoparticles having a mean particle size of less than 500 nm, and preferably less than 200 nm.

The preferred step of providing the multiphase system includes the steps of: (1) mixinga water immiscible solvent with the pharmaceutically active compound to define an organic 30 solution, (2) preparing an aqueous based solution, and (3) mixing the organic solution with theaqueous solution to form, the multiphase dispersion system. The water immiscible solvent used WO 03/026651 PCT/USO2/30447 -8- may be partially water miscible and the organic solution in the system may further include a co-solvent. The multiphase system may also include one or more surface active compounds whichcan be added to the aqueous based solution, or to the organic solution, or to the both the aqueoussolution and the organic solution, or to the mixture of the aqueous solution and the organicsolution. The multiphase system can be agitated or mixed to form a crude dispersion. The crudedispersion can also be formed by any other low-energy input processes known in the art, such asshaking, vortexing, agitating, mixing (e.g. Ultraturrax) or stirring. In a preferred embodiment ofthe invention, the crude dispersion is formed by mixing the multiphase system with anUltraturrax-T25 mixer for 10 seconds. The crude dispersion will have oil droplets in the waterof a size of approximately less than 2 μιη in diameter. The crude dispersion is subjected toaddition of energy to define a microdispersion or submicron oil in water suspension. Examplesof methods for providing energy to the crude dispersion include sonication, homogenization,microfluidization or other appropriate high shear techniques.

What is meant by the term “multiphase system” is a dispersion having at least one organicphase and at least one aqueous phase and in a preferred form of the invention is an oil in water(O/W) emulsion where the water phase forms the continuous phase and the oil phase forms thedispersed phase. The organic phase is preferably a water immiscible or a partially water miscibleorganic solvent. The organic phase may also include a co-solvent for the pharmaceutically activecompound. A preferred co-solvent is a water miscible organic solvent, such as ethanol,methanol, and acetone. The ratio by weights ofthe organic phase to the aqueous phase is fromabout 1:99 to about 99:1, more preferably from 1:99 to about 40:60, and most preferably fromabout 2:98 to about 30:70, or any range or combination of ranges therein. The present inventionfurther contemplates utilizing reverse emulsions or water in oil emulsion (W/O) where the oilphase forms the continuous phase and water the dispersed phase. The present invention furthercontemplates utilizing emulsions having more than two phases such as an oil in water in oilemulsion (O/W/O) or a water in oil in water emulsion (W/O/W). The present invention isintended in forming a liquid in liquid dispersion multiphase system. Submicron sized particlesare formed when the liquid phases of the multiphase system are removed by, for example,lyophilization. Furthermore, such a dispersion system can be sterile filtered. However, solidparticles may be formed during the process of forming the crude dispersion or the fine dispersion.These solid particles may be dispersed in the organic phase and/or the aqueous phase of themultiphase system. WO 03/026611 PCT/US02/30447 -9-

What is meant by the term “pharmaceutically active compound” is any compound that has therapeutic effect and more particularly to such compounds that are insoluble or slightly soluble in water with a solubility of preferably less than 10 mg/ml, and more preferably less than 8 mg/mi Such compounds can be found in the Physicians* Desk Reference. Particularly suitable 5 pharmaceutically active compounds include, but are not limited to, antibyperlipidemics;antimicrobials, e.g., antibacterials such as sulfadiazine, antifungals such as itraconazole; non-steroidal anti-inflammatory drugs, e.g., indomethacin; antihypercholesteremic agents, e.g.,probucol; and steroidal compounds, e.g., dexamethasone; immunosuppresants, e.g., cyclosporinA, tacrolimus, and mycophenolate mofetil. Or the organic compound might be from the group 10 used as adjuvants or excipients in pharmaceutical preparations and cosmetics, such as, but notlimited to, preservatives, e.g., propylparaben.

The pharmaceutically active compound can be present in a concentration to the extent itis soluble in the organic phase. In a preferred form of the invention the pharmaceutically activecompound can be present in an amount from less than 1% to about 40%, more preferably from 15 about 1% to about 25%, and most preferably from about 1% to about 10% by weight of theorganic phase, or any range or combination of ranges therein.

What is meant by the term “water immiscible solvent” are those solvents which form aninterfacial meniscus when combined with an aqueous solution in a 1:1 ratio (o/w). hi a preferredform of the invention the water immiscible solvent will have a vapor pressure higher than that 20 of water when both the solvent and water are measured at room temperature. Suitable waterimmiscible solvents include, but are not limited to, substituted or unsubstituted, linear, branchedor cyclic alkanes with a carbon number of 5 or higher, substituted or unsubstituted, linear,branched or cyclic alkenes with a carbon number of 5 or higher, substituted or unsubstituted,linear, branched or cyclic alkynes with a carbon number of 5 or higher; aromatic hydrocarbons 25 completely or partially halogenated hydrocarbons, ethers, esters, ketones, mono-, di- or tri-glycerides, native oils, alcohols, aldehydes, acids, amines, linear or cyclic silicones,hexamethyldisiloxane, or any combination of these solvents. Halogenated solvents include, butare not limited to carbon tetrachloride, methylene chloride, chloroform, tetrachloro ethylene,trichloroethylene, trichloroethane, hydro fluorocarbons, chlorinated benzene (mono, di, tri), 30 trichlorofluoromethane. Particularly suitable solvents arc methylene chloride, chloroform, diethylether, toluene, xylene and ethyl acetate. What is meant by “partially water miscible solvents” arethose solvents which are water immiscible at one concentration, and water miscible at another WO 03/026611 PCT/US02/30447 -10- lower concentration. These solvents are of limited water miscibility and capable of spontaneous emulsion formation. Examples of partially water miscible solvents are tetrahydrofuran (THF), propylene carbonate, benzyl alcohol, and ethyl acetate.

What is meant by the term “fine dispersion” is a system where one liquid is dispersed into 5 a second liquid (bulk phase) that may or may not contain emulsifying agents and the disperseddroplets have an average droplet size less than 1 micron. Such fine dispersion systems may ormay not be thermally stable. During the formation of the fine dispersion, solid particles may beformed. These solid particles may be dispersed in one or more phases in the system.

What is meant by the term “surface active compounds” are compounds such as an anionic 10 surfactant, a cationic surfactant, a zwitterionic surfactant, a nonionic surfactant or a biologicalsurface active molecule. The surface active compound can be added to the organic phase, theaqueous phase or to both the organic phase and the aqueous phase. The surface active compoundshould be present in an amount by weight of the aqueous phase or the organic phase, whateverthe case may be, from less than about 1% to about 30%, more preferably from about 1% to about 15 20%, or any range or combination of ranges therein.

Suitable anionic surfactants include but are not limited to: potassium laurate, sodium lauryl sulfate, sodium dodecylsulfate, alkyl polyoxyethylene sulfates, sodium alginate, dioctylsodium sulfo succinate, phosphatidyl choline, phosphatidyl glycerol, phosphatidyl inosine,phosphatidylserine, phosphatidic acid and their salts, glyceryl esters, sodium 20 carboxymethylcellulose, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid,glycocholic acid, taurocholic acid, glycodeoxycholic acid) and salts thereof (e.g., sodiumdeoxycholate, etc.).

Suitable cationic surfactants include, but are not limited to, quaternary ammoniumcompounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, 25 lauryldimethylbenzylammonium chloride, acyl carnitine hydrochlorides, or alkyl pyridinium halides. As anionic surfactants, phospholipids may be used. Suitable phospholipids include, forexample phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidyl glycerol, phosphatidic acid, lysophospholipids, egg or soybeanphospholipid or a combination thereof. The phospholipid may be salted or desalted, hydrogenated 30 or partially hydrogenated or natural, semi synthetic or synthetic.

Suitable nonionic surfactants include: polyoxyethylene fatty alcohol ethers (Macrogol and

Brij), polyoxyethylene sorbitan fatty acid esters (Polysorbates), polyoxyethylene fatty acid esters WO 03/026611 PCT/US02/30447 - 11 - (Myrj), sorbitan esters (Span), glycerol mono stearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxomers), polaxamines, methylcellulose, hydroxycellulose, hydroxy propylcellulose, hydroxy propylmethylcellulose, noncrystalline 5 cellulose, polysaccharides including starch and starch derivatives such as hydroxyethylstarch(HES), polyvinyl alcohol, and polyvinylpyrrolidone. In a preferred form of the invention, thenonionic surfactant is a polyoxyethylene and polyoxypropylene copolymer and preferably a blockcopolymer of propylene glycol and ethylene glycol. Such polymers are sold under the tradenamePOLOXAMER also sometimes referred to as PLURONIC®, and sold by several suppliers 10 including Spectrum Chemical and Ruger. Among polyoxyethylene fatty acid esters is includedthose having short alkyl chains. One example of such a surfactant is SOLUTOL® HS 15,polyethylene-660-hydroxystearate, manufactured by BASF Aktiengesellschaft.

Surface active biological molecules include such molecules as albumin, casein, heparin,hirudin, hetastarch or other appropriate biocompatible agents. 15 In a preferred form of the invention, the aqueous phase includes a protein as the surface active compound. A preferred protein is albumin. The protein may also function as an excipient.In embodiments in which protein is not the surface active compound, other excipients may beincluded in the multiphase system. Suitable excipients include, but are not limited to,saccharides, disaccharides, and sugar alcohols. A preferred disaccharide is sucrose, and a 20 preferred sugar alcohol is mannitol.

Removing a portion of the organic solvent in the multiphase system by sonication to form submicron sized particles

One method to form submicron sized particles in tlie present invention is to sonicate themultiphase system to evaporate a portion of the organic phase to cause precipitation of the 25 compound as a suspension of particles in the aqueous phase (FIG. 1). The step of sonicating canbe carried out with any suitable sonication device (Branson Model S-450A or Cole-Parmer500/750 Watt Model). Such devices are well known in the industry. Typically the sonicationdevice has a sonication horn or probe that is inserted into the multiphase system of interest toemit sonic energy into the solution. The sonicating device, in a preferred form of the invention, 30 is operated at a frequency of from about 1 kHz to about 90 kHz and more preferably from about20 kHz to about 40 kHz or any range or combination of ranges therein. The probe sizes can vary WO 03/026611 PCT/US02/30447 -12- and preferably is in distinct sizes such as !6 inch or % inch or the like. It may also be desirable to cool the solution during sonication to temperatures below room temperature. It may also be desirable to employ other mixing devices such as homogenizers, blenders or other stirring devices to assist in the process. 5 Exposing the dispersion droplets to shear energy can reduce the droplet sizes. Sonication provides a source of shear energy that effectively reduces the diameters of the dispersion droplets.Shear from sonication results from the compression and rarefaction of the propagation mediumof the sound waves. In pure liquids this oscillation between compression and rarefaction issufficiently energetic to cause cavitation, which is the tearing of the liquid to cause bubble 10 formation. In a dispersion, the analogous process results in tearing the dispersed liquid particlesinto smaller particles. Cavitation and the warming of the dispersion during sonication alsoappear to effect removal of the water immiscible solvent. As the solvent is removed the solubilityof the water-insoluble compound in the dispersion decreases, eventually allowing precipitationof the compound. Under appropriate conditions, the precipitation of the insoluble compound 15 occurs in a manner which retains the original particle size of the sonicated dispersion.

The sonicating step is effective to remove nearly all solvent in the system to provide a particle suspension essentially free ofthe organic phase.

The present invention further contemplates additional processing of the resulting dispersion including removal of any residual solvent that may exist by means such as evaporation 20 by the addition of heat or under reduced pressure, or through diafiltration. The solvent-freesuspension can then be filtered through an appropriate 0.2 gm filter, resulting in a sterilesuspension. This suspension is then amenable to further processing including freezing orlyophilization.

The particles of the pharmaceutically effective compound should be less than about 2 pm 25 in diameter as determined by light scattering (HORIBA) or microscopic measurements. Morepreferably the particles should be less than about 1 pm, even more preferably less than about 400nm and even more preferably less than about 200 nm and most preferably less than about 100 nmor any range or combination of ranges therein.

The particles have a generally spherical shape. Further, in a preferred form of the 30 invention the particles will be amorphous. What is meant by amorphous is an X-ray crystal studyof the particles shows virtual absence of x-ray peaks. See example 8 and FIG. 5. WO 03/026611 PCT/US02/30447 -13-

Fonnation of the fine dispersion

Another method to form submicron sized particles in the present invention is to remove the liquid phase of the multiphase system by freezing and lyophilizing a fine dispersion of the multiphase system. Fine dispersions can be formed from crude dispersions by energy addition. 5 While various energy addition methods can be used to form the fine dispersion from the crudedispersion, the preferred methods.are sonication and homogenization. In the methods usingsonication, any suitable sonication device can be used. Examples of suitable sonication deviceinclude Branson Model S-450A or Cole-Parmer 500/750 Watt Model. Such devices are wellknown in the industry. Typically the sonication device has a sonication horn or probe that is 10 inserted into the multiphase system of interest to emit sonic energy into the solution. Thesonicating device, in a preferred form of the invention, is operated at a frequency of from about1 kHz to about 90 kHz and more preferably from about 20 kHz to about 40 kHz or any range orcombination of ranges therein. The probe sizes can vary and preferably is in distinct sizes suchas 1 inch , ’/2 inch or % inch or the like. It may also be desirable to cool the solution during 15 sonication to temperatures below room temperature. In the methods using homogenization, any suitable homogenization device can be used. One example of such a device is the AvestinEmulsiflex-C5 homogenizer operating at about 5,000 psi to about 30,000 psi, and preferably fromabout 10,000 to 20,000 psi. In Example 11, an Avestin Emulsiflex-C5 homogenizer is used toform the fine dispersion. In this example, the crude dispersion is homogenized at 10,000 to 20 15,000 psi for 5 minutes in the temperature range of 23°C to 30°C. Other suitable energy addition methods to form the fine dispersion include, but are not limited to, high speed mixing,mechanical agitation, extrusion, microfluidization and other appropriate high shear techniquessufficient to provide dispersed droplets less than 2 microns.

Exposing the crude dispersion droplets to shear energy can reduce the droplet sizes to 25 form a fine dispersion. Addition of energy to the crude dispersion by methods such as sonicationor homogenization provides a source of shear energy that effectively reduces the diameters of thedispersion droplets. Shear forces from sonication or homogenization results from thecompression and rarefaction of the propagation medium of the sound waves as well asmechanical shear* from components of such systems. In pure liquids this oscillation between 30 compression and rarefaction is sufficiently energetic to cause cavitation. In a dispersion, theanalogous process results in tearing the dispersed liquid particles into smaller particles. WO 03/026611 PCT/US02/30447 - 14-

Removing of the liquid phase of the fine dispersion

The present invention further removes the bulk liquid phase of the fine dispersion,including the remaining water immiscible organic solvent, to obtain the submicron sizedparticles. The sonicated or homogenized dispersion is first frozen to form a solid bulk state,which is then followed by lyophilization (e.g., using a Vertis Sentry bench model lyophilizer).(See FIG. 2). The solid product obtained upon lyophilization results in nanoparticles having amean particle size of less than 500 nm in diameter as determined by light scattering (HORIBA)or microscopic measurements, and preferably less than 200 nm. The dry nanoparticles preparedin the present invention are preferably substantially free of any residual organic solvent from theorganic phase of the multiphase system. The term “substantially free” means that the residualorganic solvent present in the dry particles is substantially less than the level of solvent regardedas acceptable from a toxicological standpoint, for example, 5 ppm or less.

The particles generally have a spherical shape. Furthermore, in a preferred form of theinvention the particles will be amorphous. What is meant by amorphous is an X-ray crystal studyof the particles shows virtual absence of x-ray peaks.

Sterilization of the fine dispersion and redispersing of the nanoparticles

In another preferred form of the invention, the fine dispersion is sterilized before beingfrozen. The preferred method of sterilization is sterile filtration using a 0.22 pm membrane filter.The nanoparticles obtained from lyophilization may further be redispersed in an appropriateaqueous dispersing medium, such as water, normal saline, buffered solutions, buffered saline, andthe like. The redispersed nanoparticle suspension is now suitable for in vivo delivery byparenteral administration. Modes of parenteral administration include intravenous, intra-arterial,intrathecal, intraperitoneal, intraocular, intra-articular, intramuscular, subcutaneous injection, andthe like. The preferred mode of parenteral administration is intravenous.

Example 1: Preparation of a 0.5% itraconazole suspension using a 1:10 ratio of O/W A 5% lecithin/glyco cholate surfactant solution was prepared (100mL) and combined with10 mL of a chloroform solution containing itraconazole (0.5 grams). The resulting mixture wasmanually shaken to generate a crude emulsion and set in an ice bath to chill. After cooling for5 minutes the emulsion was sonicated every other minute for 10 minutes (5 minutes totalsonication time at 40% power using a probe at 20kHz) and then rotovapped at -120 Torr (no WO 03/026611 PCT/US02/30447 - 15- heat) to remove the chloroform. The resulting solid particle dispersion was analyzed by lightscattering detection (HORIBA) which revealed particles having a mean diameter of 97.78 nm.

Example 2: Preparation of a 1.0% itraconazole suspension using a 1:5 ratio of O/W A 5% lecithin/glycocholate surfactant solution was prepared (50 mL) and combined with 5 5 mL of a chloroform solution containing itraconazole (0.5 grams). The resulting mixture was i manually shaken to generate a crude emulsion and set in an ice hath to chill. After cooling for5 minutes the emulsion was sonicated every other minute for 10 minutes (5 minutes totalsonication time) and then rotovapped at -100 Torr (no heat) to remove the chloroform. Theresulting solid particle dispersion was analyzed by light scattering detection (HORIBA) which 10 revealed particles having a mean diameter of 135 nm.

Example 3: The process described in example 1 was repeated with the resulting particles havinga mean diameter of 139 nm. This suspension was further analyzed by scanning electronmicroscopy to reveal solid spherical particles less than 200 nm in size. FIG. 3 reveals thespherical nature of the particles produced. The sample was prepared by filtration of a small 15 portion o the suspension through a 80 nm filter and using standard SEM sample preparationtechniques. Analysis of particles produced by this process revealed the particles to be completelyamorphous as determined by x-ray powder diffraction (FIG. 4).

Example 4: Preparation of a 1.0% itraconazole suspension using a 2:5 ratio of OZW A 5% lecithin/sodium glycocholate solution was prepared (50 mL) and combined with 20 20 mL of chloroform containing itraconazole (0.5 grams). The resulting mixture was manually shaken to generate a crude emulsion and set in ice bath to chill. After cooling for 5 minutes theemulsion was sonicated every other minute for 8 minutes and than sonicated for 30 seconds(giving 4 minutes and 30 seconds of total sonication time) using a 1/2" probe at 40% amplitude.The sonicated dispersion was solvent evaporated at -100 Torr (no heat) to remove chloroform. 25 10 mL of the final solution was filtered through a 0.2 micron filter. Both filtered and unfiltered solid particle dispersions were analyzed by light scattering detection (HORIBA), which revealedparticles having a mean diameter of 110 nm and 106 nm respectively.

Example 5: A 5% lecithin/sodium glycocholatc solution was prepared (50 mL) and combinedwith 10 mL of methylene chloride containing itraconazole (0.5 grams). The resulting mixture WO 03/026611 PCT/US02/30447 -16- was manually shaken to generate a crude emulsion and set in ice bath to chill. After cooling for 5 minutes the emulsion was sonicated every other minute for 6 minutes (giving 3 minutes of total sonication time) using a 1/2" probe at 40% amplitude. The sonicated dispersion was solvent evaporated at ~ 100 Torr (no heat) to remove methylene chloride. The resulting solid particle 5 dispersion was analyzed by light scattering detection (HORIBA) which revealed particles havinga mean diameter of 144 nm.

Example 6: A 5% lecithin/sodium glycocholate solution was prepared (50 mL) and combinedwith 5 mL of methylene chloride containing itraconazole (0.5 grams). The resulting mixture wasmanually shaken to generate a crude emulsion and set in ice bath to chill. After cooling for 5 10 minutes the emulsion was sonicated every other 30 seconds for 6 minutes (giving 3 minutes oftotal sonication time) using a 1/4" probe at 20% amplitude. The sonicated solution wasevaporated using rotavapor at -100 Torr (no heat) to remove methylene chloride. Resulting solidparticle dispersions was analyzed by light scattering detection (HORIBA) which revealedparticles having a mean diameter of 109 nm. 15 Example 7: Determination of solid particle size and morphology directly after sonication

The process described in example 6 was repeated except that no solvent removal was performed after sonication. Instead the sample was submitted for particle size determination byHORIBA analysis and scanning electron microscopy. HORIBA results indicated a mean particlediameter of 156 nm. The SEM pictures revealed solid spherical particles under 200 nm in size. 20 Example 8: Determination of Amorphous Nature of Drug Particles A crude itraconazole emulsion was prepared by combining 50 mL of a surfactant solution (2.2% lecithin, 0.5% sodium glycocholate, 1.0% polyvinylpyrrolidone) with 5 mL of a methylenechloride solution containing 0.5 grams of itraconazole. The mixture was then manually shakento disperse the oil droplets into the surfactant matrix. 25 The crude emulsion was sonicated every other 30 seconds for 6 minutes using 1/4" probe at 20% amplitude and 20 kHz (temperature ~5°C using an ice bath). The sonicated solution wasthen rotovapped under house vacuum (lOOtorr) for 15-20 minutes followed by 10 minutes undera high vacuum (<20 Torr). Part of the solution was stored at -70 degrees Celsius for about anhour, and subsequently lyophilized (>48 hours). Particle size of the remaining suspension was 30 determined to be 168 nm by light scattering analysis (HORIBA). Inspection of the freeze-dried WO 03/026611 PCT/US02/30447 -17- nanoparticles after lyophilization by visible light microscopy did not reveal any crystals present.Spherical particle halos were barely observable indicating that the itraconazole nanoparticleswere intact.

The lyophilized itraconazole nanoparticles were assessed by X-ray powder diffraction and5 determined to be completely amorphous (virtual absence of x-ray peaks). In the raw material scan(the lower curve in FIG. 5) many peaks are observable revealing the crystalline nature of the compound in its original state.

Example 9: Nanoparticles of itraconazole using sonication to create the fine dispersion10 Itraconazole (0.5 grams) was dissolved in 3mL of methylene chloride and combined with 50 mL of a 5% albumin solution. The combined solutions were manually shaken to effectdispersion of the two liquids. The crude dispersion was than sonicated at 5°C for 6 minutes(sonicating every other 30 seconds using a 1/4" probe at 20% amplitude). The sonicated solutionwas frozen at -80°C and subsequently lyophilized. The lyophilized product was analyzed by light 15 scattering detection (HORJBA), which revealed particles having a mean diameter of 187 nm.

Example 10: Nanoparticles of cyclosporin using sonication to create the fine dispersion

The general process described in example 1 was repeated except cyclosporin was usedin place of itraconzole. Final particle size was 185 nm (HORTBA light scattering analysis).

Example 11: Dispersion lyophilization (using homogenization as the energy addition step) 20 Itraconazole (0.5 grams) was dissolved in 5.0 mL of dichloromethane and mixed with 50 mL of a 5% albumin solution. This was made into a crude dispersion by treatment with anUltraturrax-T25 mixer for 10 seconds. The crude dispersion was homogenized at 10,000-12,000psi using an Avestin Emulsiflex C5 homogenizer for 6 passes in the temperature range of 18°Cto 27°C to form a fme dispersion. The fine dispersion was immediately frozen at -80°C and 25 lyophilized without temperature control to produce a fluffy powder. The powder wasreconstituted with water and the solid particle dispersion analyzed by light scattering detection(HORTBA) which revealed itraconazole particles having a mean diameter of 122 nm.

Example 12: Sterile Filtration incorporated into the dispersion/lyophilization process

Itraconazole (0,5 grams) was dissolved in 5 mL of dichloromethane and combined with 30 50 mL of a 5% albumin solution. The contents were then made into a crude dispersion by WO 03/026611 PCT/US02/30447 -18- treatment with an Ultratuirax-T25 mixer for 10 seconds. Homogenization of the crude dispersion at 10,000 to 15,000 psi (Avestin Emulsiflex C5 homogenizer) for 5 minutes in the temperature range of 23 °C to 30°C resulted in a microdroplet or fine dispersion. This microdroplet dispersion was easily filtered through a 0.22 pm membrane filter. Inspection of the filter did not reveal the 5 presence of any drug material. The filtered dispersion was then frozen at -80° C. After severalhours the frozen dispersion system was lyophilized without temperature control to produce afluffy powder. The resulting lyophilized cake, containing solid itraconazole particles, wasreconstituted with water and the dispersion analyzed by light scattering detection (HORIBA) toreveal itraconazole particles with a mean diameter of 144 nm. 10 Example 13: Incorporation of sodium deoxycholate into the process 50 mL of 5% albumin solution, 2 mL of a 2.75 % sodium deoxycholate solution and 3.5 mL of a solution of itraconazole in dichloromethane containing 0.5 grams of itraconazole weremixed in a 100 mL beaker and treated with an Ultraturrax-T25 mixer for 10 seconds at 11,000rpm. The crude dispersion was homogenized at 15,000 to 20,000 psi at room temperature to 15 form a fine dispersion. The fine dispersion was transferred to a 250 mL glass bottle and frozenimmediately at -80°C. The frozen sample was lyophilized to produce submicron particles. Thelyophilized product was reconstituted with water and analyzed by light scattering detection(HORIBA), which revealed particles having a mean diameter of 207 nm.

From the foregoing, it will be observed that numerous variations and modifications may 20 be effected without departing from the spirit and scope of the invention. It is to be understoodthat no limitation with respect to the specific apparatus illustrated herein is intended or shouldbe inferred. It is, of course, intended to cover by the appended claims all such modifications asfall within the scope of the claims. + 4 + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Passages of the description, which are not within the scope of the claims, do not consist part of the claimed invention. □νΰ2'\ΓΏ7ΐ TPUZa , οτίχώπ rw:n οϊό inia^o pnow pnszn irn nr “|»oa,Ρ’ηη ηχΰπ ηοοοπο πα&amp;amp;’πο notrmaa np’ioo .zruwan rwao mpnan ρπίΛ oxnmοιηπη Pi?

<img img-format="tif" img-content="drawing" file="IL196631AD00021.tif" id="idf0001" />

.(moia παΊηπ) cra&amp;amp;’an -roa