Indian Patents. 269752:METHOD FOR PREPARING NANO-SCALE PARTICLE OF ACTIVE MATERIAL

Title of Invention	METHOD FOR PREPARING NANO-SCALE PARTICLE OF ACTIVE MATERIAL
Abstract	ABSTRACT The present invention relates to a method lor preparing nanoscale particles of etive materia] with using a gas of a supercritical fluid, by dissolving active material mo n solvent which is in solid phase at room temperature, to produce nanoscale particles if the active materials which can he advantageous!v used in medicine, cosmetics, imctional foods or the like.

Full Text	METHOD FOR PREPARING NANO-SCALE PARTICLE OF ACTIVE MATERIAL TECHNICAL FIELD OF THE INVENTION The present invention relates lo a method for preparing nanoscale panicles of active material with using a gas of a supercritical fluid, by dissolving ai: active material into a solvent which is in soiid phase at room temperature, to produce nanoscale particles of the active materials which can be advantageously used in medicine, cosmetics. functioiui] foods or (he like. BACKGROUND OF THE INVENTION A demand for a technique of an effective and rapid preparation of very fine particles in regular size has been constantly required in various industrial fields. Such fine panicles in regular size have many advantages, particularly among which good flowability and little deviation in particle interaction are very advantageous in industrial application. In medical field, the particle size of a therapeutic agent greatly affects to the dissolution rate, bioavailability, formulation and the like, and for example, the smaller the deviation in the interaction between the particles of a therapeutic agent is. the better the whole stability of the therapeutic agent becomes. When the panicle of a therapeutic agent is made into nanoscale size in medicinal products, following advantages may be obtained. First of all. in a drug having a small enteral absorption rate in oral administration, one having a smaller size can be absorbed more than one having a bigger size, thereby increasing the bioavailability of the therapeutic agent. Further, the dosage-form of drugs can be varied, for instance a drug being possibly administered only via oral route can be administered by inhalation. In a conlrolled-rcleasc drug formulation, the release rate of a therapeutic agent is a very important factor. When the particle size of (he therapeutic agent is formed to be in nanoscalc. the particle size becomes relatively more uniform, thus the release rale can become more expectable, thereby being possible to provide more effective therapeutic agent. In order to take various advantages of regular nanoparticles as described above, many attempts have been made to prepare an active ingredient as a nanop;.rticle. For this object, mechanical techniques such as crushing, grinding, milling and the like have been conventionally empioved to make relatively large panicles smaller. In the pharmaceutical industry, a method of milling a mass amount of drugs t:i the si/.c range being suitable for the medicinal or pharmaceutical use with an air-je. mill has been commonly used. However, such mechanical process involves the risk c-f contamination and had a limitation on decreasing the particle size to about tens of micrometers. US patent No. 5.145.684 discloses a method for preparing particles of poorly water-soluble drugs in the size of hundreds of nanometers by wet milling the poorly water-soluble drugs in the presence of a surface modifier. This technique should be applied after a preparation of the drugs in the particle size of not more than 100 micrometer by using a conventional milling process. Generally in (his method, the time taken for the preparation of particles having a targeted size range depends on the particular mechanical device used thereto. For example, when using a ball mill, processing times of up to 5 days or longer may be required, however, when using a high shear media mill. I da}' would be enough to provide particles of a desired size. However, in connection with the use of a high shear media mill, contamination associated with die high corrosion of grinding media and grinding vessel should be concerned. Further, a drying process such as spray or freeze drying should be conducted for getting powder form, because the resulted nanoparticles from the wet milling method are in liquid pha-ic. During the drying process, coagulation of the particles is occurred due lo iiilerparlicle attraction forces, hence it is substantially difficult to obtain a dispersion of particles in a nanometer scale by redispersing the resulted powder into a liquid. In order to solve such problem, US Patent No. 5.302.401 describes an anti-coagulating agent employed during lyophilization. Additionally. US Patent No. 6.592.903 B2 describes an invention comprising a stabilizer, a surfactant and an anti-coagulating agent used during a spray dry process. Further, US Patent No. 20(13/01 85869 Al describes an application of a wet milling technique for some poorly soluble drugs, with using lysozymc as a surface stabilizer. However, such protein surface stabilizer used therein has many restrictions in a drying process, accordingly it only describes the preparation in liquid phase. Other conventionally available methods include a recrystallization technique which provides fine particles of an active ingredient by changing the environment of a solution containing dissolved active ingredient to cause the precipitation or crystallization of solutes. The recrystallization technique can be practiced in two different ways; the one being comprised of dissolving a therapeutic agent in a suitable solvent and lowering the temperature, thereby changing the solubility of the therapeutic agent lo precipitate particles; and the other being comprised of adding antisolvent lo a solution containing dissolved therapeutic agent, thereby decreasing the solubility of the solute to precipitate particles. However, the recrystallization technique usually requires the use of toxic organic solvent and often causes flocculation or coagulation of the p; rticles during a drying process in wet condition, following after the filtration of the precipitated particles. As a result, the final particles may be irregular in their sue. US Patent No. 2003/0104068 Al discloses a method for preparing fine particles comprising: dissolving polymers into an organic solvent: dissolving or dispersing a proteineous drug thereto; then rapidly cooling the solution to ultra-low temperature for solidification; and lyophilizing the resulted product to provide a fine powder. In this ease, however, there are concerns for the denaturation of a proteineous drug by the contact with an organic solvent and the process economy owing to the rapid cooling jnd lyophilizing process. Other techniques for reducing particle size include emuls'fication. The emu! si lying: method is common!) used in cosmetic field, which comprise^ melting poorly water soluble substances with heat or dissolving them in an organic solvent, and then adding the melted or dissolved substances to an aqueous solution containing a surfactant dissolved therein, with stirring at high speed or with sonication to disperse the added substances and provide fine particles. However, in this emulsification method, a step for removing water is required for providing the fine particles in a powdered form, and the step gives variously restrictions to the process. Further, when using an oiganic solvent to dissolve the poorly walcr-solubie substance, there always has been a concern for residual toxic organic solvent. US Patent No. 2004/0067251 Al discloses a method for preparing fine particles by dissolving active ingredients into an organic solvent and spraying the resaltcd solution to an aqueous solution containing a surfactant dissolved therein. The invent ion involves the use of an organic solvent, and requires a drying process for removing the water used, to provide the particles as a powdered form, since the resulted particles are present in aqueous phase. During the drying process, the coagulation of the particles is likelv to be occurred, hence (he coagulated particles arc hardly redispcrsed with maintaining the particle size to a nanoscale. Recently, many attempts have been made to use a supercritical fluid in the amorphous or nanoscale particle preparation. Supercritical fluid is a lluid existing in liquid form at a temperature higher than its critical temperature and undei pressure higher than its critical pressure. Commonly used supercritical fluid is carbon dioxide. As one of techniques involving the use of supercritical fluids in a nanopaiticle preparation, the rapid expansion of a supercritical solution (hereinafter. RESS) is known from the following literatures: Tom ct al. Biotechnol. Proi;. 7(51:403-411. (199!) US Patent No. 6.3I6.C30 Bl: US Patent No. 6.352.737 Bl: and US Patent No. 6.368.620 B2. According to RESS. an object solute is firstly dissolved in a supercritical lluid. and then the supercritical solution is rapidly sprayed into a relatively low-pressure condition via nozzle. Then, the density of the supercritical fluid rapidly falls down. As a result, the ability of the supercritical fluid to solubilize the solute is also rapidly reduced, and the solutes are formed into very minute particles or crystallines. Other techniques using a supercritical fluid include a gas-antisolvent rccrystailization (hereinafter. GAS) (Debenedetti el al. ./. Control. Rt lease 24:27-44. (1993J: WO 00/37169). The method comprises dissolving a therapeutic agent in a conventional organic solvent to prepare a solution and spraying the resulted solution into a supercritical fluid served as an anlisolvent. through a nozzle. Then, the i olumc becomes rapidly expanded upon the contact between the solution and the supercritical fluid. As a result, the density and capacity of the solvent become so much lower to cause excessive supersaturation, hence the solutes form seeds or particles. US Patent No. 6,630.121 describes a method for preparing fine particles by nebulizing a solution containing active ingredients to provide fine particlei with the use of a supercritical fluid, and drying the resulted particles with a dry gas. The method can be used regardless of the solubility of the active ingredients lo the supercritical fluid. WO 02/3X127 \2 describes a method using SEDS (Solution Enhanced Dispersion by Supercritical fluids] technique for preparing fine particles of active ingredients and coating the resulted fine particles with an additive such as a polymer. Further US Patent No. 6.596.206 B2 describes a technique of preparing fine particles of active ingredients by dissolving the active ingredients in an organic solvent and focusing acoustic energy to the resulted solution so thai the solution can be ejected into a supercritical fl.iid as a form of fine particles. Those above-mentioned prior arts propose a method for prodjeing very fine particles with relatively uniform size, but have several disadvantages. The first disadvantage is likely to occur in a tube for transferring a solution and a nozzle. In a preparation method of fine particles using a supercritical fiuid. the particle size generally determined by the diameter of a nozzle used in the method, accordingly the diameter of a nozzle ought to be very fine and precise. However, upon the repeated use of a nozzle, the diameter of the nozzle becomes changed, hence the particle size becomes irregular as time elapses. Moreover, due to the use of a nozzle having an ultra-fine diameter for the preparation of ultra-fine particles, the clogging of the nozzle is likely to occur very often. Further, during unclogging of the nozzle, caking of the particles remained in the tube is frequently occurred. The second disadvantage of the prior arts is that the species of solutes applicable and solvents available are very limited- The RESS technique can be suitably applied only provided that the solutes are well dissolved in a supercritical fluid. Depending on I lie solutes, the solubility thereof is possibly increased with the use of a co-solvent, however, if the amount of co-solvent increases, the existence of the residual solvent after the particle generation would cause the growth of crystals, which obstruct:, the preparation o( the particles in regular size. In the GAS technique, a solvent should be selected with great concern. Only provided that the solvent containing the solutes dissolved therein is rapidly diffused into the supercritical fluid as being contacted together, f ne panicles can be generated. Further, the growth of particles can be prevented, provided that the amount of solvent remained between the particles during filtration is minimized. In addition, the GAS technique requires a special filtration device for filtering the resulted fine particles from the sol\ent. The third disadvantage of the prior arts is that there are many restrictions in commercial scale production of nanopariicles by those conventional methods using a supercritical fluid. For the commercial scale use of RESS. solutes usee should he very soluble in a supercritical fluid, which are very rare. Further, the preparation of nanoscaie fine particles of a single species of material involves the coagulation of the particles, hence an anti-coagulating material such as an emulsifier. cellulose or lipids shoald be dissolved together, and the mixture thereof should be made into fine panicles in nanoscaie. However, most of the anti-coaaulating materials would not be soluble ir carbon dioxide which is mainly used as a supercritical fluid. In preparing nanopariicles using GAS. the solution containing solutes dissolved therein is injected into a reaction vessel containing a supercritical fluid, but the injection rate is so slow that the preparation of uniform-sized particles is difficult. However, when increasing the injection rate, the particle sizes become irregular and further problems would be occurred in a filtration process. Moreover, the resulted particles with the composition ratio, which was not originally intended, would be obtained instead of particles with desired composition ratio, due io the differences between ihc solubility of the solutes to the solvent and the solubility of the ami-coagulating material added thereto, for preventing the coagulation of particles. Korean paient application No. 2004-90832 describes a method for preparing nanoscale particles b} using a fat maintaining solid phase at 30. or less, as a solvent, such as saturated fatty acids, esters and alcohols with C10-C22, and us ng a gas of a supercritical fluid at the temperature of 20-40._! under the pressure of 10-200aUr\. SUMMARY OF THE INVENTION The object of the present invention is to provide a method for preparing nanoscale panicles of active ingredients by using a subcritical or supercritical fluid at low temperature under low pressure, to prepare nanoscale particles with good efficiency. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, provided is a method for preparing nanoscale particles of active ingredient, comprising (he steps of: (I) preparing a mixture comprising one or more active ingredients and solid solvent. (2) pressurizing the mixture comprising one or more active ingredients and solid solvent to 40 to 400 atmosphere. prcfcrabK 50 to 200 atmosphere at 10 to 40 G. preferably 10 to 30 '_ by adding the gas of a supercritical fluid into a reaction vessel containins the mixture, and (3) removing the solid solvent from the mixture by releaMng out the solid solvent together with the gas of the supercritical fluid, with maintaining the temperature and pressure in the reaction vessel at 10 to 40 '". preferably 10 to 30 , and 40 to 400 atmosphere. prcfcrabK 50 to 100 atmosphere, respectively. The term "gas of a supercritical fluid" used herein, refers 10 an inert gas. which has no reactivity such as a carbon dioxide gas or a nitrogen gas. but can be a supercritical fluid under specific temperature and pressure conditions, i.e. beyond their critic: I point. The terms, "critical temperature" and "critical pressure" used herein, refer to specific temperature and pressure, respectively, under which the gas of a supercritical fluid can be liquefied as a supercritical fluid. The terms, "sub-critical temperature" and "subcritical pressure" u.-.ed herein, refer to temperature and pressure around the critical temperature and pressure, respectively. For example, in case that the gas of a supercritical fluid is carbon dioxide gas. the subcritical temperature and pressure conditions may mean, but no limited to. a temperature condition of 32 or less and a pressure condition of 70 atmosphere or less, respectively. The active ingredients useful in the method for preparing nanoscaie or amorphous particles (hereinafter, referred as •"iianoparticies") according to the present invention include, lor example, organic compounds, organometallic compounds, natural extracts, peptides, proteins, polysaccharides or the like, which exhibit specific physiological activities in medicinal products, functional foods, cosmetics or the like, and there is no specific icstriclioii on their phase al room temperature such as solid or liquid phase and electrical properties such as being neutral or ionic. The term, -'nanoparticles" used herein, refers to particles wherein 90Vr or more of the particles have a size of 5 The solid solvent, which may also be referred as "solid fat," useful in the method Cor preparing nanoparlicles according to the present invention, is a compound or a mixture thereof maintaining solid phase at room temperature, i.e. at 30 or less, having a relatively low melting point as being 30 to 150". preferably 30 to a0.. and showing a large solubility in supercritical fluid. For example, the solid solvent ued in Korean patent application No. 2004-90832 may also be used in the present invention. For instance, the solid solvent may be one or more selected from the group consisting of saturated fatty acids, esters and alcohols with C10-C22; mono- or di-g]ycerides having saturated fatly acid group with C10-C22; hydrocarbons with C16 or more: compounds having reduced fatly acid of tri-giyeerides with CI0-C22; linear oi branched diol compounds with C6-C22. preferably C6-C10 such as 1.6-hexanedio': and mixtures thereof. According to one preferred embodiment of the present invention, when a mixture of diol compound and other solid solvent than diol is used as the solid solvent, the amount of the other solid solvent used is preferably, but not limited to. 1-1000 paits by weight per 100 parts by weight of the diol compound used, in terms of production efficiency of nanoparticles. In the method for preparing nanoparticles of the present invention, the mixture comprising one or more active ingredients and solid solvent in the step (I) may further comprise a surfactant. Preferably, the surfactant may be one or more selected from the group consisting of synthetic surfactants, natural surfactants, lipids and polymers. Also, in the method for preparing nanoparticles of the present invention, the mixture comprising one or more active ingredients and solid solvent in the step (!) may further comprise a non-surfactant rype anti-coagulating (or ami-aggregating or ami-crystallizing) agent. Preferably, the non-surfactant type anti-coagulating agent may be one or more selected from the group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins. in the method for preparing nanoparliclcs of the present invention, the nanopartieles may be prepared by using the active ingredients as a single component. Optionally, an anti-coagulating agent may be further used for preventing the coagulation of the resulted nanopartieles. Such anti-coagulating agents useful in the present invention may be classified into a surfactant type and a non-surfactant type. As the surfactant lypc anti-coagulating agent, \arious synthetic and natural surfactants, lipids, polymers and the like mav be used. As the non-surfactant type anti-coagulating agent, monosaccharides, polysaccharides, dietary fibers, gums, proteins and the like may be used. Phospholipids such as lecithin, lysolecilhin. phosphatidyl choline, phosphatidyl ethylamine and the like are referred herein as a surfactant, though it may be classified as lipids in general. Surfactants may be generally divided, upon their affinity to water, into a bydrophilic type and a lipophilic type, which are determined by the HLB (hydrophilic-lipophilic balance) value. Upon the functional groups, theie are four types of surfactants such as cationic. anionic, neutral and zwitlerionic. \ surfactant or non-surfactant type anti-coagulating agent useful in the present invention is not specifically restricted to a certain type or species, as long as it prevents the coagulation of the active ingredients, and it is well dissolved in the solid solvent and is not readily removed by a supercritical fluid. According to one preferred embodiment of the present inversion, when diol compound is used as the solid solvent, some materials showing low solubility in a tieneral solid solvent other than diol. for example, polymeric surfactant or ami-coagulating agent such as Eudragh or hydroxypropyl methyl cellulose, may be dissolved well. Thus, such surfactant or anli-coagulaling agent can be utilized to prepare nanoparticles of some active ingredients which arc hard to prepare in nanoscalc particles by using general solid fat. In case that active ingredients can be prepared in nanoscalc particles by using general solid fat. the production efficiency can be improved. Also, if active ingredient is sensitive to heat, the activity loss of the active ingredient due to heat during the production of nanoparticles can be reduced since diol compound can dissolve active ingredient or the like relatively low temperature, compared with general .solid fat. Further, when sufficient dissolution of the active ingredients and surfactants is not achieved by using only solid solvent, one or more co-solvents selected from the group consisting of alcohol, water and mixtures thereof may be further used in the method of the present invention. For the alcohol as the co-solvent, lower alcohol with C2-C6 is preferred, and ethanol is the most preferred. When a mixture solution of alcohol and water is used as the co-solvent, a mixture solution of 70-80 \v\c/c of alcohol and 20-30 wtfS of water is preferred. Also, active ingredients may be dissolved together with anti-coagulating agent such as sucrose, lactose and xylitol. by using the cr-solvent such as alcohol or water. In the step (2) of the method for preparing nanoparticles according to the present invention. Ihe mixture comprising one or more active ingredients and solid solvent is pressurized to 40 to 400 atmosphere, preferably 50 to 200 atmosphere at 10 to 40 ;. preferably 10 to 30 '.'. by adding the gas of a supercritical fluid into a reaction vessel containing the mixture. In the step (3- of the method for preparing nanoparticles accordirg to the present invention, the solid solvent is removed from the mixture by releasing out .he solid solvent together with the gas of the supercritical fluid, with maintaining the icmpcralure and pressure in the reaction vessel at 10 to 40 .. preferably It) to 30 '. and 40 to 400 atmosphere, preferably 50 lo 200 atmosphere, respectively. In the steps (2) and (3) ol the method for preparing nanoparticles according lo the present invention, when the Temperature condition is less than 10 or (he pressure condition is less than 40 atmosphere, the productivity of whole process becomes worse since the solid solvent is not removed easily. To the contrary, when the temperature condition is greater than 40 !_'■ or the pressure condition is greater than 400 atmosphere, the loss of active'ingredients may happen. In the steps (2) and (3) of the method for preparing nanoparticles according lo the present invention, the temperature and pressure conditions may he selected appropriately, according to the specific kinds of active ingredient or solid solvent, or in order to improve the efficiency of nanoparticle production. For example, in case that a low temperature - low pressure condition is needed to improve the efficiency of nanoparticle production, the temperature and pressure in the steps (2) and (3) of the present method may be set to. respectively. 10 to .15 '. . preferably 10 lo 22 ~, more preferably 10 to 20 " and 40 to 90 atmosphere, preferably 50 lo 80 atmosphere, more preferably 50 lo 70 atmosphere. Such low temperature - low pressure condition is particularly useful in case that the active ingredients are released out together with the gas of the supercritical fluid or the solid solvent in which the active ingredients are dissolved, and so the resulting particle size distribution becomes broad and the average particle size becomes too large. Also, when the active ingredients are sensitive to heat. the low lemperature - low pressure condition can reduce the loss of activity due to the hear during the procedure for producing nanoparticles. Further, since the solid solvent is removed under lower pressure condition than conventional ones, high-pressure equipments are not required and thus the costs for the equipments and operation can he reduced. When a diol compound is selected as the solid solvent, the temperature and pressure in the steps (2) and \|3) may he set to. respectively. 10 to 40 ".. preferably 15 to 30 ', and 50 to 400 armosphere. preferably 70 to 200 atmosphere. Such temperature and pressure conditions can prevent situations of melting of the diol compound or a mixture of the diol compound and other solid fat due to high temperature in the reaction vessel, thereby a crystal growth ol active ingredient, surfactant, anti-coagulatmg agent or the like homogeneously dispersed in the mixture, and a final failure lo obta n uniform fine particles in nanoscale. Hereinafter, the method for preparing nanoparticles of the present invention is now illustrated step by step with more details. In the step (1) of the method for preparing nanoparticles accordirg to the present invention, a mixture comprising one or more active ingredients and solid solvent is prepared. The details thereof are now described as follows. According lo one preferred embodiment of the present invention, the step (I) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction \essel and melt-mixing them homogeneously. According to other preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel and melt-mixing them homogeneously: rapidly coolina the mixture for solidification: pulverizing the solidified mixture: adding one or more surfactants and/or one or more non-surfactant type ami-coagulating agents or aqueous solution thereof to the pulverized powder and mixing them homogeneously; and diving the mixed product at room lempcralure. According to another preferred embodiment of the present invention, the step (11 comprises: adding one or more surfactants and solid solvent into a reaction vessel, and melt-mixing them homogeneously; rapidly cooling the mixture fo' solidification; pulverizing the solidified mixture; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents together with one or more active ingredients or aqueous solution thereof, to the pulverized powder and mixing them homogeneously: and drying the mixed product ai room temperature. According to another preferred embodiment of the present invent ion. the step 11t comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, further adding die gas of a supercritical fluid, and then me It-mixing the mixture by heating. According to another preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, pressurizing the mixture by adding the gas of a supercritical fluid into the mixture and then melt-mixing the mixture, and spraying the melted mixture to the atmospheric pressure. According to another preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, pressurizing the mixture by adding the gas of a supercritical fluid and then melt-mixing the mixture, and pulverizing the melted mixture by spraying it to the atmospheric pressure; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the pulverized mixture and mixing them homogeneously: and drying the mixture at room temperature. According to another preferred embodiment of the present invention, the step 11 > comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, and melt-mixing the mixture homogeneously: adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the melted mixture and mixing them homogeneously: rapidly cooling the mixture for solidification; and pulverizing and drying the solid fied mixlurc. According to another preferred embodiment of the present invention, the step (11 comprises: adding solid solvent and optionally one or more surfactants mlo a reaction vessel, and melt-mixing the mixlurc homogeneously: adding one or more aclive ingredients and one or more surfactants and/or one or more non surfactant type anti-coagulating agents or aqueous solution thereof to the melted mixture and mixing them homogeneously; rapidly cooling the mixture for solidification: and pulverizing and drying the solidified mixture. According to one preferred embodiment of the present invention, one or more aclive ingredients and solid fal are added into a reaction vessel wherein the amount of the solid solvent is 0.1-1000 parts by weight per 1 part by weight of the aclive ingredients. At this siage. when necessary. 0.001-10 parts by weight of surfactant, or 0.001-if.) parts bv weight of lower alcohol or a mixture solution of 70 to SO wt% of the alcohol and 20 to 30 wtl of water as co-solvent, or a mixture of 0.001-10 parts by weight of surfactant and 0.001-10 pans hy vveielu of lower alcohol ov a mixture solution of 70 to SO wt'iv of the alcohol and 20 to 30 wtvr of water as co-solvent, based on 1 pan by weight of the active ingredients may he optionally added to the reaction vessel. Also, prclerably. 0.01-50 parts hy weight of an aqueous solution of one or more non-surfactant type anli-coaeuialina agents selected from ihe group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins may be added, based on 100 parts by weight oi' the solid solvent. The optionally added surfactant should have relatively large solubility to the solid solvent so as to form a homogeneous solution when being dissolved together with the active ingredients in solid solvent, or in solid solvent containing a lower alcohol described above. Further, different surfactants may be selected, depending on the properties of the active ingredients and the use or the purpose of use of the resulted nanoparlicles. When the resulted nanoparlicles are used finally in the form of a water dispersion, a surfactant with a high HLB value is preferably selected, and when the purpose is in increase the internal absorption rale, a surfactant with a relatively low HLB value is preferably selected. As mentioned above, the active ingredients and solid solvent are added to a reaction vessel and when being necessary, surfactant or lower alcohol or a mixture solution of 70 to 80 wt% of the alcohol and 20 to 30 wt% of water as co-solvent, is further added to the reaclion vessel, and then the mixture in the reaction vessel is gradually melted as being healed. If necessary, the surfactant and co-solvent may be added after the active ingredients and solid solvent arc melted clearly. As the temperature inside the reaction vessel rises, the solid solvent becomes melt, and the active ingredients and surfactant or the like are dissolved therein. The temperature is raised until a homogeneous solution is formed. It is preferred to start stirring from the point when it becomes possible, since it will make riie solution of the mixture more homogeneous and reduce the working time. The poirt when stirring becomes possible depends on the specific kinds of the active ingredients surfactant and soiid solvent used in the method, however the determination of the starting point of stirring will be easily made at the working site by the skilled person in this field. According to other preferred embodiment of Ihc present invention, as it has been mentioned above, a mixture comprising one or more active ingredients and solid solvent is prepared by; adding the one or more active ingredients, solid solvent and optionally one or more surfactants to a reaction vessel; melt-mixing them together homogeneously: rapidly cooling the resulted mixture for solidification; pulverizing the solidified mixture; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the resulted powder, and mixing them homogeneously; and drying the resulted rnixlure at room temperature. In the above processes, the drying process is not particularly restricted to a certain method, but it should be conducted below the melting point of the solid solvent used. The term, "melting point of the solid solvent" used herein, refers to the temperature at which the melting of the surfjce of the solid solvent is observed first as the temperature rises. According to another preferred embodiment of the present invention, when the active ingredients are those sensitive to the temperature or soluble ir water such as peptides, proteins or polysaccharides, the mixture comprising the active igents and solid solvent is prepared by: firstly, adding one or more surfactants and solid solvent into a reaction vessel and melt-mixing them homogeneously; rapidly cooling the melted mixture for solidification: pulverizing the solidified mixture; then adding the active ingredients together with one or more surfactants and/or one or more non- surfactant type anti-coagitlating agents or aqueous solution thereof, to the resulted pow.ler. and mixing them homogeneously; and drying the resulted mixture at room temperature, hi the above processes, the drying process is not particularly restricted to a certain method, but it should be conducted belou the melting point of the solid solvent used. In the solidification of the mixture by rapid cooling, it is preferred to rapidly decrease the temperature of the solution of the melted mixture to the temperature of 10 or less. When cooling is conducted slowly, crystal growth of the active ingredients may occur, and under such circumstances, the nanoparticles of the active ingredients are hardly achieved and the obtained particles are likely to have a broad particle distribution. The solid product obtained from the rapid cooling, is conventionally milled by, for example, dry milling and the like. The smaller the size of the milled particles is. i.e. the larger the surface area o\ ihe particles is. the more it is advantageous ii later processes such as a fat removal process. The particle size after the milling process is preferably 100 micrometer or less, but not limited thereto. According to another preferred embodiment of the present invention, the mixture comprising one or more active ingredients and solid solvent is prepared by: adding the one or more active- ingredients, solid solvent and optionally one or more surfactants to a reaction vessel; further adding the gas of a supercritical fluid(for instance. CO? gas) to the mixture, preferably so as to form subcrilical or supercritical conditions; ?nd then melting the resulted mixture by heating. According to another preferred embodiment of the present invention, the mixture comprising one or more active ingredients and solid solvent is prepared b\: adding the one or more active ingredients, solid solvent and optionally one or more surfactants to a reaction vessel: adding thereto the gas of a supercritical fluid, preferably uo to the pressure <.ner the critical pressure and melting mixture then spraying melted to atmospheric pressure.> According to another preferred embodiment of the present invention, a mixture comprising one or more active ingredients and solid solvent is prepared b\: adding the one or more active ingredients, solid fat and optionally one 01 more sutfacta its to a reaction vessel: adding thereto the gas of a supercritical fluid, preferably up to the pressure over the critical pressure and melting the mixture: then spraying the melted mixture to the atmospheric pressure for pulverization: adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the resulted mixture and mixing homogeneously: and drying the mixture at room temperature. In the above processes, the drying process is not particularly restricted to a cerla.n method, but it should be conduclcd below the melting point of the solid solvent used. In the case of using a supercritical fluid in the step (1) of the piescnl invention, after the components of the mixture are completely melted and homogeneously mixed, a supercritical fluid such as CO: i slowly added into a reaction vessel t:> pressurize the mixture, preferably up to the pressure under which the gas of a supe-critical fluid is liquefied as a supercritical fluid, i.e. the critical pressure (for CCK 70 atm) or more. The pressure inside the reaction vessel at this stage depends on the reaction vessel size and the amount of the mixture, but generally preferred is 50-200 atm. The temperature at this stage is a temperature that can provide the sufficient fluidity to the solution of the mixture for stirring. Once the critical pressure or more is achieved by raising the pressure inside the reaction vessel with the gas of a supercritical fluid, it is preferred to carry out stirring for additional 10 minutes or more at that condition, so that the supercritical fluid may be sufficiently permeated into the solution of the mixture. In completing the additional stirring, while slowh adding thereto the gas of the supercritical fluid further, (he exhaust port, which is connedcd to anothe - reaction vessel under atmospheric pressure, is opened to the full for spraying the resulted solution of the mixture into the reaction vessel under atmospheric pressure. At this moment, the supercritical fluid is instantly vaporized, thereby rapidly cooling down the surroundings and causing the solidification of the resulted solution of the mixture in an instant. The solidification of the solution of the mixture is so instantaneous that it becomes short of energy imd time demanded for crystal growth, therefore it is possible to obtain solid products in which the solutes including the active ingredients, surfactant and the like and the solid solvent are homogeneously mixed in the form of very fine particles. In the solid products obtained therefrom, the very fine nanoscale particles of the activo ingredients arc dispersed uniformly. Further, since the surfactant is also uniformly mixed with the active ingredients, the dispersability and stability of the finally produced fine particles become significantly improved. The purpose of this step is To make The particles of active ingredients he finer and more uniform in the solid product Therefore, as long as the particle size of the solid product containing the active ingredients is in the range that does not cause any problem to the workability in subsequent processes, it is not necessary to specifically adjust the panicle size of the solid product itself. Accordingly, it is not necessary to adjust the spray nozzle diameter or the spraying rale, in order to adjust the particle size ol the solid product itself produced by spraying into the atmospheric pressure condition. Therefore, die risk of deformation or clogging of the spray nozzle does not need to bs concerned any more. In spraying tiie solution of the mixture into another reaction \ essel under the atmospheric pressure condition, a conical supporting plate is preferably placed inside the reaction vessel under the atmospheric pressure condition, at a distance from the spray outlet such as nozzle, in order to solidify the sprayed solution into the form oi finer powders. By doing ->o. the solids can be formed into finer particles, and in the next step, the solid solvent can be more easily removed with the supercritical fluid. According to another preferred embodiment of the present invention, a homogeneous mixture comprising active ingredient and other additives can be obtained by cooling and pulverizing the melted mixture of active ingredient, surfactant and solid solvent, if necessary, after further adding surfactant and/or non-surfactant type ami-coagulating agcni or aqueous solution thereof to the mixture. According lo die preferred embodiment of the present invention, .0 the powdered mixture obtained bv using a supercritical fluid or milling, when being necessary, one or more surfactants and/or one or more non-surfactant type anti-coagulating agent* or aqueous solution thereof can be added, or alternatively when the active ingredients are those temperature sensitive or water soluble such as peptides, proteins or polysaccharides, the surfactant and/or the non-surfactant type anti-coagulating agent together with the active ingredients or aqueous solution thereof can be added. The resulted mixture may be homogeneously mixed by using a general mixer. In the above, when necessary, the non-surfactant type anti-coagulating agcnl is added in the amount of 0.001-10 parts by weight per I part by weight of the active ingredients. When the aqueous solution of surfactant or the non surfactant type anti-coagulating agent is added, the physical state of the resulted mixture may be varied upon the amount of water used and the kinds of the surfactant and anti-coagulating agent, but it the amount of water added is generally 30f; Tiic details of the steps (2) and (3) in the present method for preparing nanopailicles are described as follows. While maintaining the temperature of the reaction vessel contaii ing the mixture obtained from the preceding steps including the step (1) in the range of I0~40ij. preferably 10-30! ,. the gas of a supercritical fluid is added to the reaction vessel to pressurize il to 40-400 aim. preferably 50-200 atm. Then, maintaining the reaction vessel under said temperature and pressure by controlling an input valve and an output valve for the gas of a supercritical fluid such as carbon dioxide, the gas of a supercritical fluid is gradually released out. Along with the release of the gas o^ the supercritical fluid, the solid solvent is also released out. i.e. removed from the reaction vessel. At this stage. by maintaining said temperature and pressure conditions, dissolution and releasing out of the active ingredient together with the supercritical fluid and The solid solvent can he prevented, and the growth of panicles of the active ingredient caused by melting and recryslalli/.ation ol" the active ingredient, can be suppressed. When the supercritical fluid and the solid solvent arc removed, the pressure of inside of the reaction vessel is preferably maintained in a range wherein ihc solid solvent is readily dissolved in the supercritical lluid bul the active ingredient is ha-dlv dissolved in the supercritical fluid or solid solvent which is dissolved in the supercriti:al fluid. Most active ingredients are not dissolved in the supercritical fluid, but some may be dissolved and in such case, the pressure of inside of the reaction vessel during the removal of the supercritical fluid and the solid solvent is preferably maintained to about 50 aim to prevent the dissolution and releasing out of the active ingredient together with the supercritical lluid and the solid solvent. The time taken for removing the solid solvent with a supercritical fluid is quite dependent on the kinds and amount of the solid solvent used. In ord?r to obtain the particles of active ingredients with higher purity, it is preferred to take time in removing the solid solvent as long as possible, thereby minimizing the residual amount of the solid solvent. The solid solvent preferably used in the present invention is non-toxic to a human body, therefore the residual amount is not particularly limited to a specific range. However, considering the purity of the resulted active ingredients, the residual amount is preferably not more than 10 wt9 The solid solvent removed from the mixture by the method described above, can be collected in a separate reaction vessel and then used again in future. Hereinafter, the present invention is illustrated in detail with a reference to the examples as follows, however the present invention is by no means limited to those example*- Example i 30g of myristyl alcohol as a solid solvent was placed into a 250m 1 volume beaker and slowly heated to 100." . and then 1 g of polyvinylpyrrolidone (K 30) as a surfactant and I g of paclilaxel as an active ingredient were added thereto. The resulting mixture was melted completely and then cooled slowly at room temperature to obtain solid product. 5 g of the resulted solid product was charged into a pressure-resistant reaction \essel. Then, while maintaining the temperature inside the reaction vessel in 15-201. a carbon dioxide gas was added lo elevate the pressure inside ihe reaction vessel to 60-90 aim. While maimaining said lemperaiure and pressure, mvristyl alcohol was removed by continuously adding the carbon dioxide gas for 8 hours. As a result. 0.31 g of mixed powder of paclilaxel and polyvinylpyrrolidone was obtained. The obtained mixed powder was dispersed into distilled water, and the particle size thereof was determined with using a particle size analyzer (Horiha LA910SI. and the result was shown in Table 1. Example 2 10 g of the solid product obtained in Example 1 was charged into the pressure-resistant reaction vessel. Then, while maintaining the temperature inside the reaction vessel in 25-32- :, a carbon dioxide gas was added to elevate the pressure inside the reaction vessel to about 100 ami. While maintaining said temperature and pressure, myristyl alcohol was removed by continuously adding the carbon dioxide gas for 10 hours. As a result. 0.62 » of mixed powder of paclitaxel and polv\ lnvlpyrrohdone was obtained. The obtained mixed powder was dispersed into distilled water, and the panicle size thereof was determined with using a particle size analyzer (Horiba LA9I0S1. and the residl was shown in Tabic 1. From the resuiT shown in Table 1. it can be known that under high temperature and high pressure, some active ingredients are melted into the solid solvent which is dissolved in the supercritical fluid, and recryslallized. and thus the particle size distribution becomes broad. Example 3 21g of cetyl alcohol as a solid solvent was placed into a 250ml vo.ume beaker and slowly heated to 100i j, and then 0.56 g of polyvinylpyrrolidone (K 30) as a surfactant and 0.7 g of itraconazole as an active ingredient were added thereto. The resulting mixture was melted completely and then cooled slowly to 70 .:. 3.85 ml of a solution, which was prepared by dissolving 400 mg of hydroxypropyl methyl cellulose in a nixed solvent of 80% of elhano! and 20 product. The obtained solid product was dried for 24 hours at room temperature under reduced pressure. 1.6 g ol ihc resulted solid product was charged into the pressure-resistant read ion vessel. Then, while maintaining the temperature inside the reaction vessel in 22-28C,. a carbon dioxide gas was added to elevate liic pressure inside the reaction vessel to 60-90 aim. While maintaining said temperature and pressure, cclvl alcohol \>.as removed by continuously adding the carbon dioxide gas tor 8 hours. As a result. 0.1 g of mixed powder of itraconazole, hydroxypropyi methyl cellulose and polyvinylpyrrolidone was obtained. The obtained mixed powder was dispersed into distilled water, and the particle size lliercol was determined vvilh using a particle size analyzer (Horiba LA910S), and Ihe result was shown in Table 2. Example 4 3g of 1.6-hexanediot was placed into a container and slowly heated to ]00 ". and then 50 mg of hydroxypropyi methyl cellulose as a surfactant was added thereto. The resulting mixture was melted completely and then cooled to 80'.. 100 mg of itraconazole as an active ingredient was added and stirred to melt it completely lo form a transparent liquid. Then. 0.4 g of aqueous solution of lactose (]g/3mll was slowly added dropwise and the mixture was stirred iiifficiently for fi minutes. Then, the melted mixture was poured into a stainless steel plate at room temperature for solidifying. The obtained solid mixture was dried under reduced pressure to give a solid iroducl wherein line particles of the active ingredient were dispersed uniformly in solid phese diol. 1.66 g of the resulted solid product was charged into the prcssure-iesistanl reaction vessel. Then, while maintaining the temperature inside the reaction vessel in 18-27 _.. a carbon dioxide gas was added to elevate the pressure inside the reaction vessel to 70-100 atm. While maintaining said temperature and pressure. 1.6-hexanediol was removed by continuously adding the carbon dioxide gas for 8 hours. As a result. 0.12 g of mixed powder of itraconazole and hydroxypropyl methyl cellulose was obtained. The obtained mixed powder was dispersed into distilled water, and the particle size [hereof was determined with using a particle size analyzer (Horiba LAP I OS i. and the result was shown in Table 3. Example 5 3g of 1.6-hexanediol was placed into a container and slowly heated to 100' . and then 100 mg of polyvinylpyrrolidone (K 30) as a surfactant was added thereto. The resulting mixture was melted completely and then cooled to 80'..". 10(1 mg of itraconazole as an active ingredient was added and stirred to melt it completely lo form a transparent liquid. Then. 0.4 g of aqueous solution of lactose (fg/3ml) was slowly added dropwise and the mixture was stirred sufficiently for 5 minutes. Then, the melted mixture was poured into a stainless steel plate at room temperature for solidifying. The obtained solid mixture was dried under reduced pressure to give a solid product wherein fine particles of the active ingredient were dispersed uniformly in solid pfuse diol. 1.7 g of the resulted solid product was charged into the pressure-1 esistant reaction vessel. Then, while maintaining the temperature inside the reaction vessel m !8-27_>. a carbon dioxide gas was added to elevate the pressure inside the reaction \ essel to 70-100 aim. While maintaining said temperature and pressure. 1.6-hexanediol was removed bv continuously adding the carbon dioxide gas for 8 hours. As a result. (1,14 g of mixed powder of itraconazole and polyvinylpyrrolidone was obtained. The obtained mixed powder was dispersed into distilled water, and the particle size thereof was determined with using a particle size analyzer (Horiba LA^IOS). and the result was shown in Table 3. Example 6 0.1 g of mixed powder of itraconazole and Eudragit L-100 was obtained by the same process as in Example 4 excepting that 100 mg of Eudragit L-100 was used as a surfactant. Eudragit L-100 in the obtained mixed powder was not dissolved in neutral solvent such as distilled water, and so the particle size of the obtained mixed powder could not be determined. However, the state of the final mixed powder was very similar to those of Example 4. Example 7 0.1 g of mixed powder of itraconazole and Endragil S-KK) was obtained by the same process as in Example 4 excepting thai 1(10 mg ol' Eudrugit 5-100 was used as a surfactant. Endragil S-100 in the obtained mixed powder was not dissolved in neutral solvent such as distilled wate:\ and so the particle size of the obtained mixed powder could not be determined. However, the state of the final mixed powder was very similar to those of Example 4. INDUSTRIAL APPLICABILITY According to the present invention, the loss of active ingredients and regrowth of particles can he prevented significantly, and thus finer nanoparticles of acti\e ingredients can be prepared with good efficiency. Also, the loss of activity by the heal during the procedure for preparing nanoparticles can be reduced. The nanoparticles prepared by the present invention may be suitably used in medicinal products, functional or general foods, cosmetics and the like, due to their excellent dispersability. absorbing property, physiological activity and the like. CLAIMS 1. A method for preparing nanoscalc panicles of active ingred cnt. comprising the steps of: (1) preparing a mixture comprising one or more active ingredients and solid solvent. (2) pressurizing the mixture comprising one or more active ingredients and solid solvent to 4(1 to 4(H) atmosphere at 10 to 40 '_..' by adding the gas of a supercritical fluid into a reaction vessel containing the mixture, and (3) removing die solid solvent from the mixture by releasing out die solid solvent together with the gas of the supercritical fluid, with maintaining the temperature and pressure in the renction vessel at 10 to 40 and 40 to 400 atmosphere. 2. The method according to claim I. wherein the active ingredient is one or more physiologically active materials selected from the group consisting of organic compounds, organometallic compounds, natural extracts, peptides, proteins and polysaccharides. 3. The method according to claim 1, wherein the solid solvent is selected from the group consisting of saturated fatty acids, esters and alcohols with C10-C22: mono- or di-glyceridcs having saturated fatly acid group with C10-C22: hydrocarbons with C16 or more; compounds having reduced fatty acid of tri-glycerides with C10-C22: linear or branched did compounds with C6-C22: and mixtures thereof. 4. The method according to claim 1. wherein the solid solvent is diol compound. 5. The method according to claim 1. wherein the solid solvent is .1 mixture of diol compound and other solid solvent than diol. 6. The method according to claim 1. wherein the mixture comprising one or more acli\e ingredients and solid solvent in the sicp (1) furlher comprises one or more surfactants. 7. The method according to claim 6. wherein the surfactant is one or more selected from the group consisting of synthetic surfactants, natural surfactants, lipids and polymers. 8. The method according to claim 1. wherein the mixture compri^ ing one or more aelive ingredients and solid solvcnl in the step further comprises one or more non-surfactant type anti-coagulaling agents. 9. The method according to claim 8. wherein the non-surfactant type unti-coagulating agent is one or more selected from the group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins. 10. The method according to claim 1. wherein a co-solvent is further used in the step (I), 11. The method according to claim 10, wherein the co-solvent is one or more selected from The group consisting of alcohols, water, and mixtures thereof. 12. The method according to claim 11. wherein the co-solvcni is one or more alcohols wild C2-C6. 13. The method according to claim 11, wherein the co-solvent is one or more mixture solutions of 70-80 wt^r of alcohol and 20-30 \v\% of water. 14. The method according to any one of claims 1 to 13. wherein the temperature inside the reaction vessel in the step (3) is ]0~25i~ . 15. The method according to any one of claims 1 to 13. wherein the pressure inside the reaction vessel in the step (3) is 50-80 aim.

Full Text

METHOD FOR PREPARING NANO-SCALE PARTICLE OF ACTIVE
MATERIAL
TECHNICAL FIELD OF THE INVENTION
The present invention relates lo a method for preparing nanoscale panicles of active material with using a gas of a supercritical fluid, by dissolving ai: active material into a solvent which is in soiid phase at room temperature, to produce nanoscale particles of the active materials which can be advantageously used in medicine, cosmetics. functioiui] foods or (he like.
BACKGROUND OF THE INVENTION
A demand for a technique of an effective and rapid preparation of very fine particles in regular size has been constantly required in various industrial fields. Such fine panicles in regular size have many advantages, particularly among which good flowability and little deviation in particle interaction are very advantageous in industrial application. In medical field, the particle size of a therapeutic agent greatly affects to the dissolution rate, bioavailability, formulation and the like, and for example, the smaller the deviation in the interaction between the particles of a therapeutic agent is. the better the whole stability of the therapeutic agent becomes.
When the panicle of a therapeutic agent is made into nanoscale size in medicinal products, following advantages may be obtained. First of all. in a drug having a small enteral absorption rate in oral administration, one having a smaller size can be absorbed

more than one having a bigger size, thereby increasing the bioavailability of the therapeutic agent. Further, the dosage-form of drugs can be varied, for instance a drug being possibly administered only via oral route can be administered by inhalation. In a conlrolled-rcleasc drug formulation, the release rate of a therapeutic agent is a very important factor. When the particle size of (he therapeutic agent is formed to be in nanoscalc. the particle size becomes relatively more uniform, thus the release rale can become more expectable, thereby being possible to provide more effective therapeutic agent.
In order to take various advantages of regular nanoparticles as described above, many attempts have been made to prepare an active ingredient as a nanop;.rticle. For this object, mechanical techniques such as crushing, grinding, milling and the like have been conventionally empioved to make relatively large panicles smaller. In the pharmaceutical industry, a method of milling a mass amount of drugs t:i the si/.c range being suitable for the medicinal or pharmaceutical use with an air-je. mill has been commonly used. However, such mechanical process involves the risk c-f contamination and had a limitation on decreasing the particle size to about tens of micrometers.
US patent No. 5.145.684 discloses a method for preparing particles of poorly water-soluble drugs in the size of hundreds of nanometers by wet milling the poorly water-soluble drugs in the presence of a surface modifier. This technique should be applied after a preparation of the drugs in the particle size of not more than 100 micrometer by using a conventional milling process. Generally in (his method, the time taken for the preparation of particles having a targeted size range depends on the particular mechanical device used thereto. For example, when using a ball mill, processing times of up to 5 days or longer may be required, however, when using a high shear media mill. I

da}' would be enough to provide particles of a desired size. However, in connection with the use of a high shear media mill, contamination associated with die high corrosion of grinding media and grinding vessel should be concerned. Further, a drying process such as spray or freeze drying should be conducted for getting powder form, because the resulted nanoparticles from the wet milling method are in liquid pha-ic. During the drying process, coagulation of the particles is occurred due lo iiilerparlicle attraction forces, hence it is substantially difficult to obtain a dispersion of particles in a nanometer scale by redispersing the resulted powder into a liquid. In order to solve such problem, US Patent No. 5.302.401 describes an anti-coagulating agent employed during lyophilization. Additionally. US Patent No. 6.592.903 B2 describes an invention comprising a stabilizer, a surfactant and an anti-coagulating agent used during a spray dry process. Further, US Patent No. 20(13/01 85869 Al describes an application of a wet milling technique for some poorly soluble drugs, with using lysozymc as a surface stabilizer. However, such protein surface stabilizer used therein has many restrictions in a drying process, accordingly it only describes the preparation in liquid phase.
Other conventionally available methods include a recrystallization technique which provides fine particles of an active ingredient by changing the environment of a solution containing dissolved active ingredient to cause the precipitation or crystallization of solutes. The recrystallization technique can be practiced in two different ways; the one being comprised of dissolving a therapeutic agent in a suitable solvent and lowering the temperature, thereby changing the solubility of the therapeutic agent lo precipitate particles; and the other being comprised of adding antisolvent lo a solution containing dissolved therapeutic agent, thereby decreasing the solubility of the solute to precipitate particles. However, the recrystallization technique usually requires the use of toxic

organic solvent and often causes flocculation or coagulation of the p; rticles during a drying process in wet condition, following after the filtration of the precipitated particles. As a result, the final particles may be irregular in their sue.
US Patent No. 2003/0104068 Al discloses a method for preparing fine particles comprising: dissolving polymers into an organic solvent: dissolving or dispersing a proteineous drug thereto; then rapidly cooling the solution to ultra-low temperature for solidification; and lyophilizing the resulted product to provide a fine powder. In this ease, however, there are concerns for the denaturation of a proteineous drug by the contact with an organic solvent and the process economy owing to the rapid cooling jnd lyophilizing process.
Other techniques for reducing particle size include emuls'fication. The emu! si lying: method is common!) used in cosmetic field, which comprise^ melting poorly water soluble substances with heat or dissolving them in an organic solvent, and then adding the melted or dissolved substances to an aqueous solution containing a surfactant dissolved therein, with stirring at high speed or with sonication to disperse the added substances and provide fine particles. However, in this emulsification method, a step for removing water is required for providing the fine particles in a powdered form, and the step gives variously restrictions to the process. Further, when using an oiganic solvent to dissolve the poorly walcr-solubie substance, there always has been a concern for residual toxic organic solvent.
US Patent No. 2004/0067251 Al discloses a method for preparing fine particles by dissolving active ingredients into an organic solvent and spraying the resaltcd solution to an aqueous solution containing a surfactant dissolved therein. The invent ion involves the use of an organic solvent, and requires a drying process for removing the water used, to

provide the particles as a powdered form, since the resulted particles are present in aqueous phase. During the drying process, the coagulation of the particles is likelv to be occurred, hence (he coagulated particles arc hardly redispcrsed with maintaining the particle size to a nanoscale.
Recently, many attempts have been made to use a supercritical fluid in the amorphous or nanoscale particle preparation. Supercritical fluid is a lluid existing in liquid form at a temperature higher than its critical temperature and undei pressure higher than its critical pressure. Commonly used supercritical fluid is carbon dioxide. As one of techniques involving the use of supercritical fluids in a nanopaiticle preparation, the rapid expansion of a supercritical solution (hereinafter. RESS) is known from the following literatures: Tom ct al. Biotechnol. Proi;. 7(51:403-411. (199!) US Patent No. 6.3I6.C30 Bl: US Patent No. 6.352.737 Bl: and US Patent No. 6.368.620 B2. According to RESS. an object solute is firstly dissolved in a supercritical lluid. and then the supercritical solution is rapidly sprayed into a relatively low-pressure condition via nozzle. Then, the density of the supercritical fluid rapidly falls down. As a result, the ability of the supercritical fluid to solubilize the solute is also rapidly reduced, and the solutes are formed into very minute particles or crystallines.
Other techniques using a supercritical fluid include a gas-antisolvent rccrystailization (hereinafter. GAS) (Debenedetti el al. ./. Control. Rt lease 24:27-44. (1993J: WO 00/37169). The method comprises dissolving a therapeutic agent in a conventional organic solvent to prepare a solution and spraying the resulted solution into a supercritical fluid served as an anlisolvent. through a nozzle. Then, the i olumc becomes rapidly expanded upon the contact between the solution and the supercritical fluid. As a result, the density and capacity of the solvent become so much lower to cause excessive

supersaturation, hence the solutes form seeds or particles.
US Patent No. 6,630.121 describes a method for preparing fine particles by nebulizing a solution containing active ingredients to provide fine particlei with the use of a supercritical fluid, and drying the resulted particles with a dry gas. The method can be used regardless of the solubility of the active ingredients lo the supercritical fluid. WO 02/3X127 \2 describes a method using SEDS (Solution Enhanced Dispersion by Supercritical fluids] technique for preparing fine particles of active ingredients and coating the resulted fine particles with an additive such as a polymer. Further US Patent No. 6.596.206 B2 describes a technique of preparing fine particles of active ingredients by dissolving the active ingredients in an organic solvent and focusing acoustic energy to the resulted solution so thai the solution can be ejected into a supercritical fl.iid as a form of fine particles.
Those above-mentioned prior arts propose a method for prodjeing very fine particles with relatively uniform size, but have several disadvantages.
The first disadvantage is likely to occur in a tube for transferring a solution and a nozzle. In a preparation method of fine particles using a supercritical fiuid. the particle size generally determined by the diameter of a nozzle used in the method, accordingly the diameter of a nozzle ought to be very fine and precise. However, upon the repeated use of a nozzle, the diameter of the nozzle becomes changed, hence the particle size becomes irregular as time elapses. Moreover, due to the use of a nozzle having an ultra-fine diameter for the preparation of ultra-fine particles, the clogging of the nozzle is likely to occur very often. Further, during unclogging of the nozzle, caking of the particles remained in the tube is frequently occurred.
The second disadvantage of the prior arts is that the species of solutes applicable

and solvents available are very limited- The RESS technique can be suitably applied only provided that the solutes are well dissolved in a supercritical fluid. Depending on I lie solutes, the solubility thereof is possibly increased with the use of a co-solvent, however, if the amount of co-solvent increases, the existence of the residual solvent after the particle generation would cause the growth of crystals, which obstruct:, the preparation o( the particles in regular size. In the GAS technique, a solvent should be selected with great concern. Only provided that the solvent containing the solutes dissolved therein is rapidly diffused into the supercritical fluid as being contacted together, f ne panicles can be generated. Further, the growth of particles can be prevented, provided that the amount of solvent remained between the particles during filtration is minimized. In addition, the GAS technique requires a special filtration device for filtering the resulted fine particles from the sol\ent.
The third disadvantage of the prior arts is that there are many restrictions in commercial scale production of nanopariicles by those conventional methods using a supercritical fluid. For the commercial scale use of RESS. solutes usee should he very soluble in a supercritical fluid, which are very rare. Further, the preparation of nanoscaie fine particles of a single species of material involves the coagulation of the particles, hence an anti-coagulating material such as an emulsifier. cellulose or lipids shoald be dissolved together, and the mixture thereof should be made into fine panicles in nanoscaie. However, most of the anti-coaaulating materials would not be soluble ir carbon dioxide which is mainly used as a supercritical fluid. In preparing nanopariicles using GAS. the solution containing solutes dissolved therein is injected into a reaction vessel containing a supercritical fluid, but the injection rate is so slow that the preparation of uniform-sized particles is difficult. However, when increasing the injection rate, the particle sizes

become irregular and further problems would be occurred in a filtration process. Moreover, the resulted particles with the composition ratio, which was not originally intended, would be obtained instead of particles with desired composition ratio, due io the differences between ihc solubility of the solutes to the solvent and the solubility of the ami-coagulating material added thereto, for preventing the coagulation of particles.
Korean paient application No. 2004-90832 describes a method for preparing nanoscale particles b} using a fat maintaining solid phase at 30. or less, as a solvent, such as saturated fatty acids, esters and alcohols with C10-C22, and us ng a gas of a supercritical fluid at the temperature of 20-40._! under the pressure of 10-200aUr\.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a method for preparing nanoscale panicles of active ingredients by using a subcritical or supercritical fluid at low temperature under low pressure, to prepare nanoscale particles with good efficiency.
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, provided is a method for preparing nanoscale particles of active ingredient, comprising (he steps of: (I) preparing a mixture comprising one or more active ingredients and solid solvent. (2) pressurizing the mixture comprising one or more active ingredients and solid solvent to 40 to 400 atmosphere. prcfcrabK 50 to 200 atmosphere at 10 to 40 G. preferably 10 to 30 '_ by adding the gas of a supercritical fluid into a reaction vessel containins the mixture, and (3) removing the solid solvent from

the mixture by releaMng out the solid solvent together with the gas of the supercritical fluid, with maintaining the temperature and pressure in the reaction vessel at 10 to 40 '". preferably 10 to 30 , and 40 to 400 atmosphere. prcfcrabK 50 to 100 atmosphere, respectively.
The term "gas of a supercritical fluid" used herein, refers 10 an inert gas. which has no reactivity such as a carbon dioxide gas or a nitrogen gas. but can be a supercritical fluid under specific temperature and pressure conditions, i.e. beyond their critic: I point.
The terms, "critical temperature" and "critical pressure" used herein, refer to specific temperature and pressure, respectively, under which the gas of a supercritical fluid can be liquefied as a supercritical fluid.
The terms, "sub-critical temperature" and "subcritical pressure" u.-.ed herein, refer to temperature and pressure around the critical temperature and pressure, respectively. For example, in case that the gas of a supercritical fluid is carbon dioxide gas. the subcritical temperature and pressure conditions may mean, but no limited to. a temperature condition of 32 or less and a pressure condition of 70 atmosphere or less, respectively.
The active ingredients useful in the method for preparing nanoscaie or amorphous particles (hereinafter, referred as •"iianoparticies") according to the present invention include, lor example, organic compounds, organometallic compounds, natural extracts, peptides, proteins, polysaccharides or the like, which exhibit specific physiological activities in medicinal products, functional foods, cosmetics or the like, and there is no specific icstriclioii on their phase al room temperature such as solid or liquid phase and electrical properties such as being neutral or ionic.
The term, -'nanoparticles" used herein, refers to particles wherein 90Vr or more of

the particles have a size of 5 The solid solvent, which may also be referred as "solid fat," useful in the method Cor preparing nanoparlicles according to the present invention, is a compound or a mixture thereof maintaining solid phase at room temperature, i.e. at 30 or less, having a relatively low melting point as being 30 to 150". preferably 30 to a0.. and showing a large solubility in supercritical fluid. For example, the solid solvent u*ed in Korean patent application No. 2004-90832 may also be used in the present invention. For instance, the solid solvent may be one or more selected from the group consisting of saturated fatty acids, esters and alcohols with C10-C22; mono- or di-g]ycerides having saturated fatly acid group with C10-C22; hydrocarbons with C16 or more: compounds having reduced fatly acid of tri-giyeerides with CI0-C22; linear oi branched diol compounds with C6-C22. preferably C6-C10 such as 1.6-hexanedio': and mixtures thereof.
According to one preferred embodiment of the present invention, when a mixture of diol compound and other solid solvent than diol is used as the solid solvent, the amount of the other solid solvent used is preferably, but not limited to. 1-1000 paits by weight per 100 parts by weight of the diol compound used, in terms of production efficiency of nanoparticles.
In the method for preparing nanoparticles of the present invention, the mixture comprising one or more active ingredients and solid solvent in the step (I) may further comprise a surfactant. Preferably, the surfactant may be one or more selected from the group consisting of synthetic surfactants, natural surfactants, lipids and polymers.
Also, in the method for preparing nanoparticles of the present invention, the

mixture comprising one or more active ingredients and solid solvent in the step (!) may further comprise a non-surfactant rype anti-coagulating (or ami-aggregating or ami-crystallizing) agent. Preferably, the non-surfactant type anti-coagulating agent may be one or more selected from the group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins.
in the method for preparing nanoparliclcs of the present invention, the nanopartieles may be prepared by using the active ingredients as a single component. Optionally, an anti-coagulating agent may be further used for preventing the coagulation of the resulted nanopartieles. Such anti-coagulating agents useful in the present invention may be classified into a surfactant type and a non-surfactant type. As the surfactant lypc anti-coagulating agent, \arious synthetic and natural surfactants, lipids, polymers and the like mav be used. As the non-surfactant type anti-coagulating agent, monosaccharides, polysaccharides, dietary fibers, gums, proteins and the like may be used. Phospholipids such as lecithin, lysolecilhin. phosphatidyl choline, phosphatidyl ethylamine and the like are referred herein as a surfactant, though it may be classified as lipids in general. Surfactants may be generally divided, upon their affinity to water, into a bydrophilic type and a lipophilic type, which are determined by the HLB (hydrophilic-lipophilic balance) value. Upon the functional groups, theie are four types of surfactants such as cationic. anionic, neutral and zwitlerionic. \ surfactant or non-surfactant type anti-coagulating agent useful in the present invention is not specifically restricted to a certain type or species, as long as it prevents the coagulation of the active ingredients, and it is well dissolved in the solid solvent and is not readily removed by a supercritical fluid.
According to one preferred embodiment of the present inversion, when diol

compound is used as the solid solvent, some materials showing low solubility in a tieneral solid solvent other than diol. for example, polymeric surfactant or ami-coagulating agent such as Eudragh or hydroxypropyl methyl cellulose, may be dissolved well. Thus, such surfactant or anli-coagulaling agent can be utilized to prepare nanoparticles of some active ingredients which arc hard to prepare in nanoscalc particles by using general solid fat. In case that active ingredients can be prepared in nanoscalc particles by using general solid fat. the production efficiency can be improved. Also, if active ingredient is sensitive to heat, the activity loss of the active ingredient due to heat during the production of nanoparticles can be reduced since diol compound can dissolve active ingredient or the like relatively low temperature, compared with general .solid fat.
Further, when sufficient dissolution of the active ingredients and surfactants is not achieved by using only solid solvent, one or more co-solvents selected from the group consisting of alcohol, water and mixtures thereof may be further used in the method of the present invention. For the alcohol as the co-solvent, lower alcohol with C2-C6 is preferred, and ethanol is the most preferred. When a mixture solution of alcohol and water is used as the co-solvent, a mixture solution of 70-80 \v\c/c of alcohol and 20-30 wtfS of water is preferred. Also, active ingredients may be dissolved together with anti-coagulating agent such as sucrose, lactose and xylitol. by using the cr-solvent such as alcohol or water.
In the step (2) of the method for preparing nanoparticles according to the present invention. Ihe mixture comprising one or more active ingredients and solid solvent is pressurized to 40 to 400 atmosphere, preferably 50 to 200 atmosphere at 10 to 40 ;. preferably 10 to 30 '.'. by adding the gas of a supercritical fluid into a reaction vessel containing the mixture.

In the step (3- of the method for preparing nanoparticles accordirg to the present invention, the solid solvent is removed from the mixture by releasing out .he solid solvent together with the gas of the supercritical fluid, with maintaining the icmpcralure and pressure in the reaction vessel at 10 to 40 .. preferably It) to 30 '. and 40 to 400 atmosphere, preferably 50 lo 200 atmosphere, respectively.
In the steps (2) and (3) ol the method for preparing nanoparticles according lo the present invention, when the Temperature condition is less than 10 or (he pressure condition is less than 40 atmosphere, the productivity of whole process becomes worse since the solid solvent is not removed easily. To the contrary, when the temperature condition is greater than 40 !_'■ or the pressure condition is greater than 400 atmosphere, the loss of active'ingredients may happen.
In the steps (2) and (3) of the method for preparing nanoparticles according lo the present invention, the temperature and pressure conditions may he selected appropriately, according to the specific kinds of active ingredient or solid solvent, or in order to improve the efficiency of nanoparticle production.
For example, in case that a low temperature - low pressure condition is needed to improve the efficiency of nanoparticle production, the temperature and pressure in the steps (2) and (3) of the present method may be set to. respectively. 10 to .15 '. . preferably 10 lo 22 ~, more preferably 10 to 20 " and 40 to 90 atmosphere, preferably 50 lo 80 atmosphere, more preferably 50 lo 70 atmosphere. Such low temperature - low pressure condition is particularly useful in case that the active ingredients are released out together with the gas of the supercritical fluid or the solid solvent in which the active ingredients are dissolved, and so the resulting particle size distribution becomes broad and the average particle size becomes too large. Also, when the active ingredients are sensitive to heat.

the low lemperature - low pressure condition can reduce the loss of activity due to the hear during the procedure for producing nanoparticles. Further, since the solid solvent is removed under lower pressure condition than conventional ones, high-pressure equipments are not required and thus the costs for the equipments and operation can he reduced.
When a diol compound is selected as the solid solvent, the temperature and pressure in the steps (2) and |3) may he set to. respectively. 10 to 40 ".. preferably 15 to 30 ', and 50 to 400 armosphere. preferably 70 to 200 atmosphere. Such temperature and pressure conditions can prevent situations of melting of the diol compound or a mixture of the diol compound and other solid fat due to high temperature in the reaction vessel, thereby a crystal growth ol active ingredient, surfactant, anti-coagulatmg agent or the like homogeneously dispersed in the mixture, and a final failure lo obta n uniform fine particles in nanoscale.
Hereinafter, the method for preparing nanoparticles of the present invention is now illustrated step by step with more details.
In the step (1) of the method for preparing nanoparticles accordirg to the present invention, a mixture comprising one or more active ingredients and solid solvent is prepared. The details thereof are now described as follows.
According lo one preferred embodiment of the present invention, the step (I) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction \essel and melt-mixing them homogeneously.
According to other preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more

surfactants into a reaction vessel and melt-mixing them homogeneously: rapidly coolina the mixture for solidification: pulverizing the solidified mixture: adding one or more surfactants and/or one or more non-surfactant type ami-coagulating agents or aqueous solution thereof to the pulverized powder and mixing them homogeneously; and diving the mixed product at room lempcralure.
According to another preferred embodiment of the present invention, the step (11 comprises: adding one or more surfactants and solid solvent into a reaction vessel, and melt-mixing them homogeneously; rapidly cooling the mixture fo' solidification; pulverizing the solidified mixture; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents together with one or more active ingredients or aqueous solution thereof, to the pulverized powder and mixing them homogeneously: and drying the mixed product ai room temperature.
According to another preferred embodiment of the present invent ion. the step 11t comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, further adding die gas of a supercritical fluid, and then me It-mixing the mixture by heating.
According to another preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, pressurizing the mixture by adding the gas of a supercritical fluid into the mixture and then melt-mixing the mixture, and spraying the melted mixture to the atmospheric pressure.
According to another preferred embodiment of the present invention, the step (1) comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, pressurizing the mixture by adding the gas of a

supercritical fluid and then melt-mixing the mixture, and pulverizing the melted mixture by spraying it to the atmospheric pressure; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the pulverized mixture and mixing them homogeneously: and drying the mixture at room temperature.
According to another preferred embodiment of the present invention, the step 11 > comprises: adding one or more active ingredients, solid solvent and optionally one or more surfactants into a reaction vessel, and melt-mixing the mixture homogeneously: adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the melted mixture and mixing them homogeneously: rapidly cooling the mixture for solidification; and pulverizing and drying the solid fied mixlurc.
According to another preferred embodiment of the present invention, the step (11 comprises: adding solid solvent and optionally one or more surfactants mlo a reaction vessel, and melt-mixing the mixlurc homogeneously: adding one or more aclive ingredients and one or more surfactants and/or one or more non surfactant type anti-coagulating agents or aqueous solution thereof to the melted mixture and mixing them homogeneously; rapidly cooling the mixture for solidification: and pulverizing and drying the solidified mixture.
According to one preferred embodiment of the present invention, one or more aclive ingredients and solid fal are added into a reaction vessel wherein the amount of the solid solvent is 0.1-1000 parts by weight per 1 part by weight of the aclive ingredients. At this siage. when necessary. 0.001-10 parts by weight of surfactant, or 0.001-if.) parts bv weight of lower alcohol or a mixture solution of 70 to SO wt% of the alcohol and 20 to 30 wtl of water as co-solvent, or a mixture of 0.001-10 parts by weight of surfactant and

0.001-10 pans hy vveielu of lower alcohol ov a mixture solution of 70 to SO wt'iv of the alcohol and 20 to 30 wtvr of water as co-solvent, based on 1 pan by weight of the active ingredients may he optionally added to the reaction vessel. Also, prclerably. 0.01-50 parts hy weight of an aqueous solution of one or more non-surfactant type anli-coaeuialina agents selected from ihe group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins may be added, based on 100 parts by weight oi' the solid solvent.
The optionally added surfactant should have relatively large solubility to the solid solvent so as to form a homogeneous solution when being dissolved together with the active ingredients in solid solvent, or in solid solvent containing a lower alcohol described above. Further, different surfactants may be selected, depending on the properties of the active ingredients and the use or the purpose of use of the resulted nanoparlicles. When the resulted nanoparlicles are used finally in the form of a water dispersion, a surfactant with a high HLB value is preferably selected, and when the purpose is in increase the internal absorption rale, a surfactant with a relatively low HLB value is preferably selected.
As mentioned above, the active ingredients and solid solvent are added to a reaction vessel and when being necessary, surfactant or lower alcohol or a mixture solution of 70 to 80 wt% of the alcohol and 20 to 30 wt% of water as co-solvent, is further added to the reaclion vessel, and then the mixture in the reaction vessel is gradually melted as being healed. If necessary, the surfactant and co-solvent may be added after the active ingredients and solid solvent arc melted clearly.
As the temperature inside the reaction vessel rises, the solid solvent becomes melt, and the active ingredients and surfactant or the like are dissolved therein. The temperature is raised until a homogeneous solution is formed. It is preferred to start

stirring from the point when it becomes possible, since it will make riie solution of the mixture more homogeneous and reduce the working time. The poirt when stirring becomes possible depends on the specific kinds of the active ingredients surfactant and soiid solvent used in the method, however the determination of the starting point of stirring will be easily made at the working site by the skilled person in this field.
According to other preferred embodiment of Ihc present invention, as it has been mentioned above, a mixture comprising one or more active ingredients and solid solvent is prepared by; adding the one or more active ingredients, solid solvent and optionally one or more surfactants to a reaction vessel; melt-mixing them together homogeneously: rapidly cooling the resulted mixture for solidification; pulverizing the solidified mixture; adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the resulted powder, and mixing them homogeneously; and drying the resulted rnixlure at room temperature. In the above processes, the drying process is not particularly restricted to a certain method, but it should be conducted below the melting point of the solid solvent used. The term, "melting point of the solid solvent" used herein, refers to the temperature at which the melting of the surfjce of the solid solvent is observed first as the temperature rises.
According to another preferred embodiment of the present invention, when the active ingredients are those sensitive to the temperature or soluble ir water such as peptides, proteins or polysaccharides, the mixture comprising the active igents and solid solvent is prepared by: firstly, adding one or more surfactants and solid solvent into a reaction vessel and melt-mixing them homogeneously; rapidly cooling the melted mixture for solidification: pulverizing the solidified mixture; then adding the active ingredients together with one or more surfactants and/or one or more non- surfactant type

anti-coagitlating agents or aqueous solution thereof, to the resulted pow.ler. and mixing them homogeneously; and drying the resulted mixture at room temperature, hi the above processes, the drying process is not particularly restricted to a certain method, but it should be conducted belou the melting point of the solid solvent used.
In the solidification of the mixture by rapid cooling, it is preferred to rapidly decrease the temperature of the solution of the melted mixture to the temperature of 10 or less. When cooling is conducted slowly, crystal growth of the active ingredients may occur, and under such circumstances, the nanoparticles of the active ingredients are hardly achieved and the obtained particles are likely to have a broad particle distribution.
The solid product obtained from the rapid cooling, is conventionally milled by, for example, dry milling and the like. The smaller the size of the milled particles is. i.e. the larger the surface area o\ ihe particles is. the more it is advantageous ii later processes such as a fat removal process. The particle size after the milling process is preferably 100 micrometer or less, but not limited thereto.
According to another preferred embodiment of the present invention, the mixture comprising one or more active ingredients and solid solvent is prepared by: adding the one or more active- ingredients, solid solvent and optionally one or more surfactants to a reaction vessel; further adding the gas of a supercritical fluid(for instance. CO? gas) to the mixture, preferably so as to form subcrilical or supercritical conditions; ?nd then melting the resulted mixture by heating.
According to another preferred embodiment of the present invention, the mixture comprising one or more active ingredients and solid solvent is prepared b\: adding the one or more active ingredients, solid solvent and optionally one or more surfactants to a reaction vessel: adding thereto the gas of a supercritical fluid, preferably uo to the pressure

<.ner the critical pressure and melting mixture then spraying melted to atmospheric pressure.> According to another preferred embodiment of the present invention, a mixture comprising one or more active ingredients and solid solvent is prepared b\: adding the one or more active ingredients, solid fat and optionally one 01 more sutfacta its to a reaction vessel: adding thereto the gas of a supercritical fluid, preferably up to the pressure over the critical pressure and melting the mixture: then spraying the melted mixture to the atmospheric pressure for pulverization: adding one or more surfactants and/or one or more non-surfactant type anti-coagulating agents or aqueous solution thereof to the resulted mixture and mixing homogeneously: and drying the mixture at room temperature. In the above processes, the drying process is not particularly restricted to a cerla.n method, but it should be conduclcd below the melting point of the solid solvent used.
In the case of using a supercritical fluid in the step (1) of the piescnl invention, after the components of the mixture are completely melted and homogeneously mixed, a supercritical fluid such as CO: i* slowly added into a reaction vessel t:> pressurize the mixture, preferably up to the pressure under which the gas of a supe-critical fluid is liquefied as a supercritical fluid, i.e. the critical pressure (for CCK 70 atm) or more. The pressure inside the reaction vessel at this stage depends on the reaction vessel size and the amount of the mixture, but generally preferred is 50-200 atm. The temperature at this stage is a temperature that can provide the sufficient fluidity to the solution of the mixture for stirring.
Once the critical pressure or more is achieved by raising the pressure inside the reaction vessel with the gas of a supercritical fluid, it is preferred to carry out stirring for additional 10 minutes or more at that condition, so that the supercritical fluid may be

sufficiently permeated into the solution of the mixture.
In completing the additional stirring, while slowh adding thereto the gas of the supercritical fluid further, (he exhaust port, which is connedcd to anothe - reaction vessel under atmospheric pressure, is opened to the full for spraying the resulted solution of the mixture into the reaction vessel under atmospheric pressure. At this moment, the supercritical fluid is instantly vaporized, thereby rapidly cooling down the surroundings and causing the solidification of the resulted solution of the mixture in an instant. The solidification of the solution of the mixture is so instantaneous that it becomes short of energy imd time demanded for crystal growth, therefore it is possible to obtain solid products in which the solutes including the active ingredients, surfactant and the like and the solid solvent are homogeneously mixed in the form of very fine particles. In the solid products obtained therefrom, the very fine nanoscale particles of the activo ingredients arc dispersed uniformly. Further, since the surfactant is also uniformly mixed with the active ingredients, the dispersability and stability of the finally produced fine particles become significantly improved.
The purpose of this step is To make The particles of active ingredients he finer and more uniform in the solid product Therefore, as long as the particle size of the solid product containing the active ingredients is in the range that does not cause any problem to the workability in subsequent processes, it is not necessary to specifically adjust the panicle size of the solid product itself. Accordingly, it is not necessary to adjust the spray nozzle diameter or the spraying rale, in order to adjust the particle size ol the solid product itself produced by spraying into the atmospheric pressure condition. Therefore, die risk of deformation or clogging of the spray nozzle does not need to bs concerned any more.

In spraying tiie solution of the mixture into another reaction \ essel under the atmospheric pressure condition, a conical supporting plate is preferably placed inside the reaction vessel under the atmospheric pressure condition, at a distance from the spray outlet such as nozzle, in order to solidify the sprayed solution into the form oi finer powders. By doing ->o. the solids can be formed into finer particles, and in the next step, the solid solvent can be more easily removed with the supercritical fluid.
According to another preferred embodiment of the present invention, a homogeneous mixture comprising active ingredient and other additives can be obtained by cooling and pulverizing the melted mixture of active ingredient, surfactant and solid solvent, if necessary, after further adding surfactant and/or non-surfactant type ami-coagulating agcni or aqueous solution thereof to the mixture.
According lo die preferred embodiment of the present invention, .0 the powdered mixture obtained bv using a supercritical fluid or milling, when being necessary, one or more surfactants and/or one or more non-surfactant type anti-coagulating agent* or aqueous solution thereof can be added, or alternatively when the active ingredients are those temperature sensitive or water soluble such as peptides, proteins or polysaccharides, the surfactant and/or the non-surfactant type anti-coagulating agent together with the active ingredients or aqueous solution thereof can be added. The resulted mixture may be homogeneously mixed by using a general mixer.
In the above, when necessary, the non-surfactant type anti-coagulating agcnl is added in the amount of 0.001-10 parts by weight per I part by weight of the active ingredients. When the aqueous solution of surfactant or the non surfactant type anti-coagulating agent is added, the physical state of the resulted mixture may be varied upon the amount of water used and the kinds of the surfactant and anti-coagulating agent,

but it the amount of water added is generally 30f; Tiic details of the steps (2) and (3) in the present method for preparing nanopailicles are described as follows.
While maintaining the temperature of the reaction vessel contaii ing the mixture obtained from the preceding steps including the step (1) in the range of I0~40ij. preferably 10-30! ,. the gas of a supercritical fluid is added to the reaction vessel to pressurize il to 40-400 aim. preferably 50-200 atm. Then, maintaining the reaction vessel under said temperature and pressure by controlling an input valve and an output valve for the gas of a supercritical fluid such as carbon dioxide, the gas of a supercritical fluid is gradually released out. Along with the release of the gas o^ the supercritical fluid, the solid solvent is also released out. i.e. removed from the reaction vessel. At this stage. by maintaining said temperature and pressure conditions, dissolution and releasing out of

the active ingredient together with the supercritical fluid and The solid solvent can he prevented, and the growth of panicles of the active ingredient caused by melting and recryslalli/.ation ol" the active ingredient, can be suppressed.
When the supercritical fluid and the solid solvent arc removed, the pressure of inside of the reaction vessel is preferably maintained in a range wherein ihc solid solvent is readily dissolved in the supercritical lluid bul the active ingredient is ha-dlv dissolved in the supercritical fluid or solid solvent which is dissolved in the supercriti:al fluid. Most active ingredients are not dissolved in the supercritical fluid, but some may be dissolved and in such case, the pressure of inside of the reaction vessel during the removal of the supercritical fluid and the solid solvent is preferably maintained to about 50 aim to prevent the dissolution and releasing out of the active ingredient together with the supercritical lluid and the solid solvent.
The time taken for removing the solid solvent with a supercritical fluid is quite dependent on the kinds and amount of the solid solvent used. In ord?r to obtain the particles of active ingredients with higher purity, it is preferred to take time in removing the solid solvent as long as possible, thereby minimizing the residual amount of the solid solvent. The solid solvent preferably used in the present invention is non-toxic to a human body, therefore the residual amount is not particularly limited to a specific range. However, considering the purity of the resulted active ingredients, the residual amount is preferably not more than 10 wt9 The solid solvent removed from the mixture by the method described above, can be collected in a separate reaction vessel and then used again in future.
Hereinafter, the present invention is illustrated in detail with a reference to the examples as follows, however the present invention is by no means limited to those

example*-
Example i
30g of myristyl alcohol as a solid solvent was placed into a 250m 1 volume beaker and slowly heated to 100." . and then 1 g of polyvinylpyrrolidone (K 30) as a surfactant and I g of paclilaxel as an active ingredient were added thereto. The resulting mixture was melted completely and then cooled slowly at room temperature to obtain solid product.
5 g of the resulted solid product was charged into a pressure-resistant reaction \essel. Then, while maintaining the temperature inside the reaction vessel in 15-201. a carbon dioxide gas was added lo elevate the pressure inside ihe reaction vessel to 60-90 aim. While maimaining said lemperaiure and pressure, mvristyl alcohol was removed by continuously adding the carbon dioxide gas for 8 hours. As a result. 0.31 g of mixed powder of paclilaxel and polyvinylpyrrolidone was obtained.
The obtained mixed powder was dispersed into distilled water, and the particle size thereof was determined with using a particle size analyzer (Horiha LA910SI. and the result was shown in Table 1.
Example 2
10 g of the solid product obtained in Example 1 was charged into the pressure-resistant reaction vessel. Then, while maintaining the temperature inside the reaction vessel in 25-32- :, a carbon dioxide gas was added to elevate the pressure inside the reaction vessel to about 100 ami. While maintaining said temperature and pressure, myristyl alcohol was removed by continuously adding the carbon dioxide gas for 10 hours.

As a result. 0.62 » of mixed powder of paclitaxel and polv\ lnvlpyrrohdone was obtained.
The obtained mixed powder was dispersed into distilled water, and the panicle size thereof was determined with using a particle size analyzer (Horiba LA9I0S1. and the residl was shown in Tabic 1.

From the resuiT shown in Table 1. it can be known that under high temperature and high pressure, some active ingredients are melted into the solid solvent which is dissolved in the supercritical fluid, and recryslallized. and thus the particle size distribution becomes broad.
Example 3
21g of cetyl alcohol as a solid solvent was placed into a 250ml vo.ume beaker and slowly heated to 100i j, and then 0.56 g of polyvinylpyrrolidone (K 30) as a surfactant and 0.7 g of itraconazole as an active ingredient were added thereto. The resulting mixture was melted completely and then cooled slowly to 70 .:. 3.85 ml of a solution, which was prepared by dissolving 400 mg of hydroxypropyl methyl cellulose in a nixed solvent of 80% of elhano! and 20
product. The obtained solid product was dried for 24 hours at room temperature under reduced pressure.
1.6 g ol ihc resulted solid product was charged into the pressure-resistant read ion vessel. Then, while maintaining the temperature inside the reaction vessel in 22-28C,. a carbon dioxide gas was added to elevate liic pressure inside the reaction vessel to 60-90 aim. While maintaining said temperature and pressure, cclvl alcohol \>.as removed by continuously adding the carbon dioxide gas tor 8 hours. As a result. 0.1 g of mixed powder of itraconazole, hydroxypropyi methyl cellulose and polyvinylpyrrolidone was obtained.
The obtained mixed powder was dispersed into distilled water, and the particle size lliercol was determined vvilh using a particle size analyzer (Horiba LA910S), and Ihe result was shown in Table 2.

Example 4
3g of 1.6-hexanediot was placed into a container and slowly heated to ]00 ". and then 50 mg of hydroxypropyi methyl cellulose as a surfactant was added thereto. The resulting mixture was melted completely and then cooled to 80'.. 100 mg of itraconazole as an active ingredient was added and stirred to melt it completely lo form a transparent liquid. Then. 0.4 g of aqueous solution of lactose (]g/3mll was slowly added

dropwise and the mixture was stirred iiifficiently for fi minutes. Then, the melted mixture was poured into a stainless steel plate at room temperature for solidifying. The obtained solid mixture was dried under reduced pressure to give a solid iroducl wherein line particles of the active ingredient were dispersed uniformly in solid phese diol.
1.66 g of the resulted solid product was charged into the prcssure-iesistanl reaction vessel. Then, while maintaining the temperature inside the reaction vessel in 18-27 _.. a carbon dioxide gas was added to elevate the pressure inside the reaction vessel to 70-100 atm. While maintaining said temperature and pressure. 1.6-hexanediol was removed by continuously adding the carbon dioxide gas for 8 hours. As a result. 0.12 g of mixed powder of itraconazole and hydroxypropyl methyl cellulose was obtained.
The obtained mixed powder was dispersed into distilled water, and the particle size [hereof was determined with using a particle size analyzer (Horiba LAP I OS i. and the result was shown in Table 3.
Example 5
3g of 1.6-hexanediol was placed into a container and slowly heated to 100' . and then 100 mg of polyvinylpyrrolidone (K 30) as a surfactant was added thereto. The resulting mixture was melted completely and then cooled to 80'..". 10(1 mg of itraconazole as an active ingredient was added and stirred to melt it completely lo form a transparent liquid. Then. 0.4 g of aqueous solution of lactose (fg/3ml) was slowly added dropwise and the mixture was stirred sufficiently for 5 minutes. Then, the melted mixture was poured into a stainless steel plate at room temperature for solidifying. The obtained solid mixture was dried under reduced pressure to give a solid product wherein fine particles of the active ingredient were dispersed uniformly in solid pfuse diol.

1.7 g of the resulted solid product was charged into the pressure-1 esistant reaction vessel. Then, while maintaining the temperature inside the reaction vessel m !8-27_>. a carbon dioxide gas was added to elevate the pressure inside the reaction \ essel to 70-100 aim. While maintaining said temperature and pressure. 1.6-hexanediol was removed bv continuously adding the carbon dioxide gas for 8 hours. As a result. (1,14 g of mixed powder of itraconazole and polyvinylpyrrolidone was obtained.
The obtained mixed powder was dispersed into distilled water, and the particle size thereof was determined with using a particle size analyzer (Horiba LA^IOS). and the result was shown in Table 3.

Example 6
0.1 g of mixed powder of itraconazole and Eudragit L-100 was obtained by the same process as in Example 4 excepting that 100 mg of Eudragit L-100 was used as a surfactant.
Eudragit L-100 in the obtained mixed powder was not dissolved in neutral solvent such as distilled water, and so the particle size of the obtained mixed powder could not be determined. However, the state of the final mixed powder was very similar to those of Example 4.

Example 7
0.1 g of mixed powder of itraconazole and Endragil S-KK) was obtained by the same process as in Example 4 excepting thai 1(10 mg ol' Eudrugit 5-100 was used as a surfactant.
Endragil S-100 in the obtained mixed powder was not dissolved in neutral solvent such as distilled wate:\ and so the particle size of the obtained mixed powder could not be determined. However, the state of the final mixed powder was very similar to those of Example 4.
INDUSTRIAL APPLICABILITY
According to the present invention, the loss of active ingredients and regrowth of particles can he prevented significantly, and thus finer nanoparticles of acti\e ingredients can be prepared with good efficiency. Also, the loss of activity by the heal during the procedure for preparing nanoparticles can be reduced. The nanoparticles prepared by the present invention may be suitably used in medicinal products, functional or general foods, cosmetics and the like, due to their excellent dispersability. absorbing property, physiological activity and the like.

CLAIMS
1. A method for preparing nanoscalc panicles of active ingred cnt. comprising
the steps of:
(1) preparing a mixture comprising one or more active ingredients and solid solvent.
(2) pressurizing the mixture comprising one or more active ingredients and solid solvent to 4(1 to 4(H) atmosphere at 10 to 40 '_..' by adding the gas of a supercritical fluid into a reaction vessel containing the mixture, and
(3) removing die solid solvent from the mixture by releasing out die solid solvent together with the gas of the supercritical fluid, with maintaining the temperature and pressure in the renction vessel at 10 to 40 and 40 to 400 atmosphere.

2. The method according to claim I. wherein the active ingredient is one or more physiologically active materials selected from the group consisting of organic compounds, organometallic compounds, natural extracts, peptides, proteins and polysaccharides.
3. The method according to claim 1, wherein the solid solvent is selected from the group consisting of saturated fatty acids, esters and alcohols with C10-C22: mono- or di-glyceridcs having saturated fatly acid group with C10-C22: hydrocarbons with C16 or more; compounds having reduced fatty acid of tri-glycerides with C10-C22: linear or branched did compounds with C6-C22: and mixtures thereof.
4. The method according to claim 1. wherein the solid solvent is diol compound.

5. The method according to claim 1. wherein the solid solvent is .1 mixture of diol compound and other solid solvent than diol.
6. The method according to claim 1. wherein the mixture comprising one or more acli\e ingredients and solid solvent in the sicp (1) furlher comprises one or more surfactants.
7. The method according to claim 6. wherein the surfactant is one or more selected from the group consisting of synthetic surfactants, natural surfactants, lipids and polymers.
8. The method according to claim 1. wherein the mixture compri^ ing one or more aelive ingredients and solid solvcnl in the step further comprises one or more non-surfactant type anti-coagulaling agents.
9. The method according to claim 8. wherein the non-surfactant type unti-coagulating agent is one or more selected from the group consisting of monosaccharides, polysaccharides, dietary fibers, gums and proteins.

10. The method according to claim 1. wherein a co-solvent is further used in the step (I),
11. The method according to claim 10, wherein the co-solvent is one or more

selected from The group consisting of alcohols, water, and mixtures thereof.
12. The method according to claim 11. wherein the co-solvcni is one or more
alcohols wild C2-C6.
13. The method according to claim 11, wherein the co-solvent is one or more
mixture solutions of 70-80 wt^r of alcohol and 20-30 \v\% of water.
14. The method according to any one of claims 1 to 13. wherein the temperature
inside the reaction vessel in the step (3) is ]0~25i~ .
15. The method according to any one of claims 1 to 13. wherein the pressure
inside the reaction vessel in the step (3) is 50-80 aim.

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Patent Number

269752

Indian Patent Application Number

5955/CHENP/2008

PG Journal Number

45/2015

Publication Date

06-Nov-2015

Grant Date

04-Nov-2015

Date of Filing

03-Nov-2008

Name of Patentee

KIM, Kab, Sig

Applicant Address

311, SOGANG BUSINESS INCUBATOR, 1-1, SINSU-DONG, MAPO-GU, SEOUL 121-854,

Inventors:

#	Inventor's Name	Inventor's Address
1	KIM, KAB, SIG,	311, SOGANG BUSINESS INCUBATOR, 1-1, SINSU-DONG, MAPO-GU, SEOUL 121-854
2	CHO, YOUNG, TAI	#71-36, SHINSU-DONG, MAPO-GU, SEOUL 121-854,

PCT International Classification Number

B82B 3/00

PCT International Application Number

PCT/KR07/2172

PCT International Filing date

2007-05-03

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	10-2006-0040416	2006-05-04	Republic of Korea
2	10-2006-0040317	2006-05-04	Republic of Korea