Protein stabilized pharmacologically active agents and their use

The present invention relates to a method for producing a granular vehicle for intravenous administration of a pharmacologically active agent and a novel composition produced by this method. In certain embodiments, the present invention relates to a method for in vivo release of a substantially water-insoluble pharmacologically active agent [eg, the anti-cancer drug taxol]. In another embodiment, a dispersible colloidal system containing a water-insoluble pharmacologically active agent is provided. The suspended particles are encapsulated in a polymer shell composed of a biocompatible polymer and have a diameter of less than about 1 micron. The colloidal system according to the invention is produced without the use of conventional surfactants or any polymer core matrix. In a particularly preferred embodiment of the present invention, there is provided a method for producing exceptionally small particles that can be sterilized and filtered . The polymer shell contains particles of the pharmacologically active agent and optionally a bioadaptive dispersant, wherein the pharmacologically active agent can be dissolved or suspended therein. Accordingly, the present invention provides a pharmaceutical release system in liquid or redispersible powder form. Both forms immediately provide both bioeffective pharmaceutical molecules (ie, pharmaceutical molecules that are molecularly bound to proteins) and protein-covered pharmaceutical particles.

Intravenous drug release allows for rapid, direct equilibrium with the bloodstream that carries the drug to the rest of the body. To avoid reaching peak serum levels within a short period of time after intravenous infusion, administration of a drug carried in a stable carrier after intravenous infusion of therapeutic nanoparticles. , Allows gradual release of the drug inside the venous chamber. Injectable controlled release microparticles can provide a pre-planned duration of action over a range of days to a week with a single injection. They can also provide several outstanding benefits to the entire habitually administered drug, which are automatically guaranteed patient compliance according to the dosing regimen and to specific tissues or organs. Includes the administration of drugs that set the goals of (Non-Patent Document 1 and Non-Patent Document 2).

Microparticles and foreign substances present in the blood are generally purified from the circulatory system by "hemofiltration organs", namely the spleen, lungs and liver. Particulate matter contained in normal whole blood includes red blood cells (typically having a diameter of 8 microns), white blood cells (typically having a diameter of 6 to 8 microns), and platelets (typically 1 to 1 to 1). (Has a diameter of 3 microns) is included. The microcirculatory system of most organs and tissues allows these blood cells to pass freely. The presence of microthrombus (blood coagulation) larger than 10-15 microns in the circulatory system poses a risk of capillary infarction or blockage, resulting in ischemia or oxygen depletion and possibly tissue death. Therefore, injection of particles larger than 10-15 microns into the circulatory system must be avoided. However, suspensions with diameters smaller than 7-8 microns are relatively safe and have been used to supply pharmacologically active agents, nutritional supplements, and photographic contrast agents in the form of liposomes and emulsions. The size of the particles and their release mode determine their biological behavior. Strand et al. Disclosed that the fate of particles depends on their size (Non-Patent Document 3). Particles in the size range of a few nanometers (nm) to 100 nm may enter the lymphatic capillaries after intrastitial injection and phagocytosis within the lymph nodes. After intravenous / interstitial injection, particles smaller than about 2 microns are rapidly purified from the blood stream by the reticuloendotheliatic system (RES). It is also known as the mononuclear phagocytosis system (MPS). Particles larger than about 7 microns are captured by lung capillaries after venous influx. After intra-arterial injection, the particles are trapped in the first capillary bed reached. The surviving particles are captured by alveolar macrophages.

Drugs that are water-insoluble or sparingly soluble in water and that are sensitive to the acidic environment in the stomach cannot be administered habitually (eg, by intravenous or oral administration). Parenteral administration of such drugs is achieved by emulsifying the oil-dissolved drug in the presence of a surfactant or emulsion stabilizer with an aqueous liquid (eg, saline) to form a stable microemulsion. Has been done. These emulsions can be injected intravenously as long as the components of the emulsion are pharmacologically inert. Patent Document 1 describes pharmacology in which it is dissolved in oil and then emulsified with water in the presence of surfactants such as egg phosphatide, pluronics (copolymer of polypropylene glycol and polyethylene glycol), and polyglycerol oleate. It is described to administer a target active agent. Patent Document 2 describes pharmaceutical microdroplets of an anesthetic coated with a phospholipid such as dimeristylphosphatidylcholine having dimensions suitable for intramuscular or intravenous injection.

An example of a water-insoluble drug is taxol. Taxol is a natural product first isolated from the Pacific yew tree [Taxus brevifolis] [Non-Patent Document 4]. Among the anti-mitotic agents, taxol having a diterpene carbon skeleton exhibits a specific mode of action on microtubule proteins involved in the formation of the mitotic spindle. Unlike other anti-mitotic agents such as vinblastine or cortisin, which prevent the assembly of microtubules, taxol is known to prevent microtubule depolymerization, ie, the cell replication process. It is the only plant product that exists. Taxol, a naturally occurring diterpenoid, has been shown to have exceptional anti-neoplasmic and anti-cancer effects in drug-resistant ovarian cancer. Taxol showed excellent antitumor activity in a wide variety of tumor models, such as B16 melanoma, L1210 leukemia, MX-1 breast cancer and C6-1 colon tumor heterologous implants. Several recent reports have named taxol as a great new anti-cancer drug. To be sure, Taxol has recently been a Federal Drug Approved by the Administration for the treatment of ovarian cancer. However, taxol poses a problem in human administration due to its poor water solubility. Certainly, the release of a drug that is inherently insoluble or sparingly soluble in water in an aqueous medium can be a serious problem when oral administration is not effective. Therefore, recently used taxol formulations require cremahole to make the drug soluble. The dose range for human medicine is 200-500 mg. This dose is dissolved in a 1: 1 solution of ethanol-cremahole and then diluted to 1 liter of intravenous liquid. The recently used cremahole is polyethoxylated castor oil.

In Faith 1 clinical trials, taxol itself does not show excessive toxic effects, but several allergic reactions are caused by the emulsifiers used to solubilize the drug. Recent dosing regimens include treating patients with antihistamines and steroids prior to administration of the drug to reduce allergic side effects of cremahole. In studies of improving the water solubility of taxol, several researchers have modified its chemical structure with functional groups that impart increased water solubility. Among these are sulfonated derivatives (Patent Document 3 by Kingston et al.) And amino acid esters (Non-Patent Document 5), which have shown considerable biological activity. Modifications to produce water-soluble derivatives facilitated the intravenous supply of taxol dissolved in a non-toxic carrier such as saline. However, such modifications may induce unwanted side reactions and / or allergic reactions, and / or reduce the efficacy of the drug, in addition to the manufacturing price of the drug. Protein microspheres have been reported in publications as carriers of pharmacological or diagnostic agents. Albumin microspheres are produced by thermal denaturation or chemical cross-linking. Heat-denatured microspheres are produced from an emulsified mixture (eg, albumin, the drug to be formulated and a suitable oil) at a temperature of 100 ° C to 150 ° C. The microspheres are then washed with a suitable solvent and then stored. Leucuta et al. Disclose the production of heat-denatured microspheres (Non-Patent Document 6).

Methods for producing chemically crosslinked microspheres include treating the emulsion with glutaraldehyde, crosslinking the protein, then washing and storing. Lee et al. (Non-Patent Document 7) and Patent Document 4 teach this manufacturing method. The above techniques for producing protein microspheres as carriers for pharmacologically active agents are suitable for the release of water-soluble agents, but cannot capture water-insoluble agents. This limitation is unique to manufacturing techniques that rely on cross-linking or heat denaturing protein components in the aqueous phase of water-in-oil emulsions. Water-soluble agents dissolved in the protein-containing aqueous phase may be trapped in the resulting crosslinked or heat-denatured protein matrix, but poorly water-soluble or oil-soluble agents are these techniques. It cannot be incorporated into the protein matrix formed by. One of the conventional methods for producing pharmaceuticals containing microparticles is polylactic acid (or other bioadaptive, water-insoluble polymer) with a water-immiscible solvent (eg, methylene chloride or other chlorine). Dissolve in an aliphatic or aromatic solvent), then dissolve the pharmacologically active agent in this polymer solution, add the surfactant to this oily or aqueous phase, and use appropriate means to prepare the oil-in-water emulsion. It involves forming and then evaporating this emulsion under reduced pressure. If the oil droplets are small enough and stable during evaporation, a polymer suspension in water is obtained. Since the drug is present in the polymer solution from the beginning, this method allows the composition in which the drug molecules are trapped inside the particles of the polymer matrix. The formation of microspheres and microparticles by using this solvent evaporation method has been reported by several researchers (eg, using various pharmaceuticals, Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 9; Non-Patent Document 9; See Patent Document 10; and Patent Document 5).

Bazile et al. Report in Non-Patent Document 11 and Spenlehauer et al. Report in Patent Document 6 the formation of ultrafine particles by using two types of biocompatible polymers. One (eg, polylactide) dissolves in the organic phase along with active ingredients such as pharmaceuticals, and the other polymer, such as albumin, is used as a surfactant. After emulsification and then removal of the solvent, microparticles in which the drug resides inside the polymer matrix of polylactide particles are formed. The nature of the polymer solution from which the polymer matrix is formed is very important for obtaining a suitable emulsion in the first step. For example, polylacti (this polymer is commonly used in the production of injectable microparticles) has surface activity, causing its rapid absorption in dichloromethane-water, resulting in reduced interfacial tension ( For example, see Non-Patent Document 12 by Boury et al.), Then improve the emulsification process. Furthermore, the same researchers found that bovine serum albumin (BSA) interacts with polylactide and then penetrates into the polylactide monolayer present at the oil-water interface. Therefore, based on the above reference publications, it is expected that the presence of a surfactant (polylactide) is extremely preferable for emulsification in a conventional solvent evaporation method. In fact, the presence of polylactide is not only a sufficient condition, but is actually necessary for the formation of microparticles of appropriate size.

Another method based on the solvent evaporation method is to dissolve the drug in a hydrophobic solvent (eg, toluene or cyclohexane) without dissolving any polymer in an organic solvent and emulsify the mixture with a conventional surfactant. To form an oil-in-water emulsion, which then evaporates the solvent to obtain dry particles of the pharmaceutical (see, eg, Non-Patent Document 13). Separation of the non-polar solvent causes precipitation of the drug inside the solvent droplets, resulting in quasi-micron particles. It has been found that the size of these particles is largely controlled by the initial dimensions of the emulsion droplets. Furthermore, it is important to note that the size of the final particles has been reported to decrease as the drug concentration in the organic phase decreases. This finding contradicts the results of the above report, which does not use conventional surfactants in the production of microparticles. Furthermore, the authors of this Sjostrom paper are aware that the drug used, cholesteryl acetate, is surfactant in toluene and can be oriented at the oil-water interface. Therefore, the higher the drug concentration at this interface, the greater the precipitating power. The formation of quasi-micron particles is also achieved by the precipitation method, as disclosed in Non-Patent Document 14. This method dissolves a drug (eg, indomethacin) and a polymer (polycaprolactone) in methylene chloride and acetone, then pours the solution into an aqueous phase containing a detergent [Poloxamer 188]. Produces quasi-micron particles (216 nm). However, this method is carried out at a solvent concentration that does not form an emulsion.

<p><patcit num="1"><text>U.S. Patent No. 4,073,943</text></patcit><patcit num="2"><text>PCT International Release No.WO85 / 00011</text></patcit><patcit num="3"><text>U.S. Patent 5,059,699 (1991)</text></patcit><patcit num="4"><text>U.S. Patent No. 4,671,954</text></patcit><patcit num="5"><text>WO94 / 10980</text></patcit><patcit num="6"><text>Fr patent 2,660,556</text></patcit></p>

<p><nplcit num="1"><text>Journal of Controlled Release by Tice and Gilley, 2: 343-352 (1985)).</text></nplcit><nplcit num="2"><text>Journal of Controlled Release by Gilley, 2: 343 ~ 352 (1985)).</text></nplcit><nplcit num="3"><text>Microspheres-Biomedical Applications, edited by A. Rembaum, pp. 193-227, CRC Publisher (1988)</text></nplcit><nplcit num="4"><text>J.Am.Chem.Soc., 93: 2325 (1971)].</text></nplcit><nplcit num="5"><text>J. Med, Chem., 35: 145 ~ 151 (1992) by Mathew et al.</text></nplcit><nplcit num="6"><text>International Journal of Pharmaceutics, 41,213 ~ 217 (1988)</text></nplcit><nplcit num="7"><text>Science, 213: 233 ~ 235 (1981)</text></nplcit><nplcit num="8"><text>Journal of Controlled Release by Tice and Gilley, 2: 343 ~ 352 (1985)</text></nplcit><nplcit num="9"><text>Int. J. Pharmaceutics, 43: 179 (1988) by Bodmeier and McGinity</text></nplcit><nplcit num="10"><text>J. Pharm. Pharmacol., 38: 249 (1985) by Cavalier et al.</text></nplcit><nplcit num="11"><text>Biomaterials, 13: 1093 (1992)</text></nplcit><nplcit num="12"><text>Lanqmuir by Boury et al., 11: 1636 (1996)</text></nplcit><nplcit num="13"><text>J. Dispersion Science and Technology by Sjostrom et al., 15: 89 ~ 117 (1994)</text></nplcit><nplcit num="14"><text>J. Pharm. Sci., 85: 530 (1996) by Calvo et al.</text></nplcit></p>

<p> Therefore, an object of the present invention is to prepare a composition that does not cause an allergic reaction due to the presence of an additive emulsifier and a solubilizer currently used in a pharmaceutical release system in an unmodified form with a pharmacologically active agent (eg, taxol, taxane). , Taxotere, etc.). Another object of the present invention is to supply a pharmacologically active agent to a composition of microparticles or microparticles that may be suspended in a suitable biocompatible liquid. Yet another object of the present invention is solvent evaporation from oil-in-water emulsions that use proteins as stabilizers in the absence of any conventional surfactants and also in the absence of any polymeric core material. The technique is to provide a method of forming quasi-micron particles (microparticles) of a pharmacologically active agent.</p>

<p> These and other purposes of the present invention are deemed to be apparent by looking at this specification and the claims. In accordance with the present invention, we have found that a substantially water-insoluble pharmacologically active agent can be supplied in the form of microparticles or microparticles suitable for parenteral administration as an aqueous suspension. This feeding scheme eliminates the need for administration of a substantially water-insoluble pharmacologically active agent (eg, taxol) in an emulsion containing, for example, ethanol and polyethoxylated castor oil, which has been diluted in physiological saline. Obvious (see, for example, Abstracts of the 2nd National Cancer Institute Workshop on Taxol &amp; Taxus by Norton et al., September 23-24, 1992). The drawback of such known compositions lies in their nature of causing allergic side effects.</p><p> Therefore, in accordance with the present invention, under high shear conditions (sonication, high pressure homogenization) in the absence of any conventional surfactant and also in the presence of no polymer core material in the formation of the matrix of microparticles. A method for forming ultrafine particles of a pharmacologically active agent by solvent evaporation technology from an oil-in-water emulsion produced in (such as) is provided. Instead, a protein (eg, human serum albumin) is used as a stabilizer. Furthermore, the present invention provides a method for forming reproducibility of specifically small microparticles (diameter smaller than 200 nm), which can be sterilized and filtered through a 0.22 micron filter. This is achieved by adding a water soluble solvent (eg, ethanol) to the organic phase and carefully selecting the type of organic phase, phase fraction and drug concentration in the organic phase. The possibility of forming microparticles of a size that can be filtered by a 0.22 micron filter is exceptionally important and outstanding. The reason is that a composition containing a considerable amount of any protein (for example, albumin) cannot be sterilized by a conventional method such as an autoclave because the protein is thermally coagulated.</p><p> Following another aspect of the invention, we have developed a composition useful for in vivo release of a substantially water-insoluble pharmacologically active agent. The compositions according to the invention include a substantially water-insoluble pharmacologically active agent contained within a polymer shell (eg, as a solid or liquid). This polymer shell is a crosslinked biocompatible polymer. The polymer shell, which contains a substantially water-insoluble pharmacologically active agent, can then be suspended in a biocompatible aqueous liquid for administration. The present invention also provides a pharmaceutical release system in which some of the molecules of a pharmacologically active agent are bound to a protein (eg, human serum albumin) and are therefore bioutilized as soon as administered to a mammal. .. The rest of the pharmacologically active agent is contained within the microparticles covered with protein. The microparticles containing this pharmacologically active agent are present as pure active ingredients without dilution by any polymer matrix.</p><p> The majority of conventional pharmacologically active agents bind to carrier proteins (via hydrophobic or ionic interactions) and circulate in the blood stream. The most commonly used example of this carrier protein is serum albumin. The method according to the invention and the compositions produced thereby provide a pharmacologically active agent that is pre-bonded to a protein (via hydrophobic or ionic interactions) prior to administration. This description demonstrates both of the above bioeffectiveness schemes for taxol [Faclitaxel], an anticancer drug that can bind to human serum albumin (eg, Kumar et al.). Research Communications in Chemical Pathology and Pharmacology, 80: 337 (1993)). The concentration of albumin in the particles of the invention, which is higher compared to taxol, provides a significant amount of the drug in the form of albumin-bound molecules. This albumin is also a natural carrier of medicine in the blood stream. Furthermore, the ability of human serum albumin to bind taxol and other drugs is a consideration consideration, which increases the ability of taxol to adsorb on the particle surface. Since albumin is present on colloidal pharmaceutical particles (formed after removal of the organic solvent), the formation of a colloidal dispersion that is stable over an extended period of time is responsible for electrical repulsion and steric stabilization. Promoted by combination.</p><p> According to the present invention, quasi-micron particles are provided in powder form which can be easily reconstituted in water or salt solutions. This powder is obtained after removing water by lyophilization. Human serum albumin acts as a structural element of microparticles according to the present invention, and also serves as an antifreeze agent and a reconstitution agent. The production of particles that can be filtered through a 0.22 micron filter by following the method according to the invention described herein and then drying or lyophilizing produces sterile solid formulations useful for intravenous infusion. The present invention provides, in a particular embodiment, the composition of an anti-cancer drug, eg taxol, in the form of microparticles in a liquid dispersion or as a solid that can be easily reconstituted for administration. Due to the special properties of certain pharmaceuticals, such as taxol, such compositions cannot be obtained by conventional solvent evaporation methods that rely on the use of surfactants. In the presence of various surfactants, very large (eg, about 5 to hundreds of microns in size) pharmaceutical crystals are produced during storage within minutes of the manufacturing process. The size of such crystals is typically significantly larger than the size allowed for intravenous infusion.</p>

<p> It is recognized that the particles produced according to the present invention can be either crystalline, amorphous or a mixture thereof, but in general it is preferred that the pharmaceutical be present in the composition in amorphous form. This facilitates dissolution and absorption and provides good bioavailability.</p>

<figref num="1">FIG. 1 shows the results of intravenous administration of paclitaxel microparticles to tumor-bearing mice [5 animals in each group (n = 5)], and the treatment group compared with the control group () ingesting the salt solution. It shows the complete regression of the tumor in (). Almost uncontrolled tumor growth is seen in the control group. The dose in the treatment group was paclitaxel 20 mg / kg, which was administered intravenous nodules for 5 consecutive days.</figref><figref num="2">FIG. 2 shows the results of intraperitoneal administration of paclitaxel microparticles to rats suffering from arthritis that developed in their limbs after intradermal injection of collagen. Measure the volume of the limb to indicate the severity of the disease. The volume of this limb shall be 100% at the beginning of the procedure. Day 0 indicates the start date of treatment. Group 3, the control group ingesting the salt solution (n = 2, indicated by a thin line, labeled "untreated" in this figure), 1 mg / kg paclitaxel microparticles Dose of first treatment group (n = 4, shown by thick line, labeled "paclitaxel microparticles, 1.0 mg / kg" in this figure) and 0.5 mg / kg Second treatment group treated with a combination of doses of paclitaxel microparticles and 0.2 mg / kg dose of prednisolone (n = 4, shown by thick line, in this figure, "prednisolone 0.2 mg / kg + paclitaxel" (Marked as "ultrafine particles 0.5 mg / kg") is used. The two treatment groups showed a dramatic decrease in limb volume over time, while the control group showed an increase in limb volume over the same period.</figref>

In the present invention, a method for producing a substantially water-insoluble pharmacologically active substance for in vivo delivery. The organic phase in which the above pharmacologically active substance is dispersed, and A mixture containing an aqueous medium containing a biocompatible polymer, which is substantially free of surfactants. A method is provided consisting of subjecting to a high pressure homogenizer with a pressure in the range of about 3,000 to 30,000 psi. After being subjected to high shear conditions, the organic and / or aqueous phase is then removed from the mixture at any time. Further, in the present invention, the composition produced by the above method is provided. In a further embodiment of the invention, a drug delivery system comprising particles of a solid or liquid substantially water-insoluble pharmacologically active substance coated with a protein. The protein coating contains the free protein bound therein and Some of the pharmacologically active substances are contained in the protein coating, and some of the pharmacologically active substances are associated with free proteins, and The average diameter of the particles is less than about 1 micron, The system is provided.

The compositions are particularly effective as they have been observed to provide very low toxic forms of various pharmacologically active substances, eg, a combination of taxol and albumin (as a biocompatible polymer) thereof. It is a suitable combination of the present invention due to its low toxicity. The combination of taxol and albumin also has the added advantage of being substantially non-myelosuppressive. In a preferred embodiment, the average diameter of the particles is about 200 nm or less. Such particles are particularly effective because they can be aseptically filtered and therefore do not require further vigorous treatment to sterilize the solution containing the desired pharmacologically active substance. As used herein, the term "in vivo delivery" refers to routes of administration such as oral, intravenous, subcutaneous, intraperitoneal, submucosal, intramuscular, inhalation, topical, transdermal, suppository (rectal), pessary (vaginal), etc. Means delivery of the pharmacologically active substance by. As used herein, the term "micron" means one thousandth of a millimeter. As used herein, the term "biocompatibility" refers to a substance that does not significantly alter or affect the introduced biological system in a detrimental manner. The major differences between the pharmacologically active substances contained in the polymer shells of the present invention and the prior art protein microspheres are the nature of particle formation, the final state of the protein after formation, and low water solubility. It has the ability to carry substances or substances that are substantially water-insoluble. In the present invention, polymers (eg, proteins) are crosslinked as a result of exposure to high shear conditions in a high pressure homogenizer. The high shear force optionally disperses a dispersant containing a pharmacologically active substance dissolved or suspended in an aqueous solution of a biocompatible polymer (eg, albumin) having sulfhydryl or disulfide groups, thus microfluidic in a non-aqueous medium. Used to form a shell of crosslinked polymer around the drops. High shear conditions cause cavities in the liquid, which cause very large local heating, thus oxidizing (and / or breaking existing disulfide bonds), for example, sulfhydryl residues to create new cross-linked disulfide bonds. It forms superoxide ions that can crosslink the polymer that can form.

Prior art, the glutaraldehyde cross-linking method is non-specific to the methods of the invention and essentially reacts with all nucleophilic groups (eg amines and hydroxyls) present in the protein structure. Prior art teaches that heating causes large and irreversible changes in protein structure. In contrast, the intended disulfide bond formation of the present invention does not substantially denature the protein. Further, since the polymer shell produced by the present invention is relatively thin compared to the diameter of the coated particles, the particles of the substantially water-insoluble pharmacologically active substance contained in the shell are crosslinked or heat-denatured by the prior art. It is different from the protein microspheres. The "shell thickness" of the polymer coating has been measured (by transmission electron microscopy) to be about 25 nanometers for coated particles with a diameter of 1 micron (1000 nanometers). In contrast, the prior art microspheres do not have a protein shell, but rather the proteins are dispersed in the volume of the microspheres.

That is, in the present invention, the pharmacologically active substance is a suitable solvent (for example, chloroform, methylene chloride, ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, methylpyrrolidone, etc., and any two of them. Or a mixture of more) is dissolved. Additional solvents intended for practice in the present invention are soybean oil, coconut oil, olive oil, benibana oil, cottonseed oil, sesame oil, orange oil, limonene oil, C1-C20 alcohol, C2-C20 ester, C3-C20. There are ketones, polyethylene glycols, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and combinations thereof. Unlike conventional methods of nanoparticle formation, polymers (eg, polylactic acid) are insoluble in solvents. The oil phase used in the production of the compositions of the present invention contains only pharmacologically active substances dissolved in a solvent. A protein (eg, human serum albumin) is then added (to the aqueous phase) to act as a stabilizer for the formation of stable nanodroplets. The protein is added at a concentration in the range of about 0.05-25% (w / v), more preferably in the range of about 0.5-5% (w / v). Unlike conventional methods of nanoparticle formation, no surfactant is added to the mixture (eg, sodium lauryl sulfate, lecithin, Tween 80, Pluronic F-68, etc.).

The emulsion is then formed by homogenizing under high pressure and high shear. Such homogenization is conveniently carried out in a high pressure homogenizer, typically at pressures in the range of about 3,000 to 30,000 psi. Preferably such a method is carried out at a pressure in the range of about 6,000 to 25,000 psi. The resulting emulsion has very small nanodroplets of a non-aqueous solvent (containing a dissolved pharmacologically active substance) and very small nanodroplets of a protein stabilizer. Acceptable homogenization methods include methods that provide high shear and cavity formation (eg, high-pressure homogenization, high-shear mixers, sonication, high-shear rotors, etc.).

Finally, the solvent is distilled off under reduced pressure to obtain a colloidal system consisting of protein-coated nanoparticles of a pharmacologically active substance and a protein. Acceptable evaporation methods include the use of rotary evaporators, falling film evaporators, spray dryers, freeze dryers and the like. After distilling off the solvent, the suspension is dried to obtain a powder containing a pharmacologically active substance and a protein. The resulting powder can be any suitable aqueous medium at any time (eg, saline solution, buffered saline, water, buffered aqueous medium, amino acid solution, vitamin solution, carbohydrate solution, etc.), as well as any of these. Redisperse in a combination of two or more of these to give a suspension that can be administered to a mammal. The methods intended to obtain this powder include freeze-drying, spray-drying and the like.

In a specific embodiment of the invention there is provided a method for the formation of very small submicron particles (nanoparticles), i.e. particles less than 200 nanometers in diameter. Such particles can be sterile filtered before being used in the form of liquid suspensions. Since it is not possible to sterilize dispersions containing high concentrations of protein (eg, serum albumin) by conventional methods (eg, autoclaving), the final product of the formulation process of the invention (ie, drug particles) The ability to perform sterile filtration is very important. In order to obtain sterile filterable particles (ie, particles &lt;200 nm), a pharmacologically active substance is first mixed in a high concentration and substantially immiscible with water in an organic solvent (eg, a solvent having a solubility in water of about 5% or less, eg. It dissolves in chloroform), thus producing an organic phase containing a pharmacologically active substance. Suitable solvents are those mentioned above. Unlike conventional methods of nanoparticle formation, polymers (eg, polylactic acid) are insoluble in solvents. The oil phase used in the method of the present invention contains only pharmacologically active substances dissolved in a solvent. Next, an organic solvent to be mixed with water (for example, a solvent having a solubility in water of about 10% or more, for example, ethanol) has a final concentration of about 1% to 99% v / v, more preferably about 5% of the total organic phase. Add to the oil phase in the range of ~ 25% v / v. The organic solvent to be mixed with water can be selected from solvents such as ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, methylpyrrolidone and the like. Alternatively, a mixture of a solvent that is immiscible with water and a solvent that is miscible with water is first prepared, and then the pharmacologically active substance in the mixture is dissolved. Human serum albumin or any other suitable stabilizer is then dissolved in an aqueous medium as described above. This component acts as a stabilizer for the formation of stable nanodroplets. A sufficient amount of the first organic solvent (eg, chloroform) is dissolved in the aqueous phase from time to time to bring it closer to the saturated concentration. Another measured amount of organic phase, which here contains the pharmacologically active substance, the first organic solvent and the second organic solvent, is added to the saturated aqueous phase to phase the organic phase. Minutes are about 0.5% to 15% v / v, more preferably 1% to 8% v / v.

Next, a mixture of microdroplets and nanodroplets is formed with low shear. This is done in a variety of ways, as will be readily appreciated by those skilled in the art, for example, conventional laboratory homogenizers operating in the range of about 2,000 to about 15,000 rpm are used. It is then homogenized under high pressure (ie, in the range of about 3,000 to 30,000 psi). The resulting mixture comprises an aqueous protein solution (eg, human serum albumin), a water-insoluble pharmacologically active substance, a first solvent, and a second solvent. Finally, the solvent is rapidly distilled off under vacuum to give a colloidal dispersion (pharmacologically active substances and proteins) in the form of extremely small nanoparticles (ie, particles in the range of about 10 nm to 200 nm in diameter). Therefore, it can be sterilized. The preferred size range of particles is from about 50 nm to 170 nm, depending on the formulation and parameters used. Colloidal systems prepared according to the present invention are further converted to powder form by removing water from them, for example by lyophilization at a suitable temperature-time profile. The protein itself (eg, human serum albumin) acts as a cryoprotectant, and the powder requires the use of conventional cryoprotectants (eg, mannitol, sucrose, glycine, etc.) by adding water, saline or buffer. There is no, and it is easily restored. Although not particularly necessary, of course, conventional cryoprotectants may be added to the formulations of the present invention if desired.

Polymer shells containing solid or liquid cores of pharmacologically active substances allow delivery of large amounts of pharmacologically active substances in relatively small amounts. This reduces discomfort in patients receiving large amounts of fluid and reduces hospital stays. In addition, the polymer shell or coating wall is generally completely degraded in vivo by proteolytic enzymes (eg, when the polymer is a protein), completely eliminating side effects from the delivery system as in the formulations of the present invention.

In embodiments of the present invention, particles of a substantially water-insoluble pharmacologically active substance have a cross-sectional diameter of about 10 microns or less. A cross-sectional diameter of less than 5 microns is more preferred, and a cross-sectional diameter of less than 1 micron is most preferred for intravenous administration. The substantially water-insoluble pharmacologically active substance intended in the practice of the present invention contains a pharmacologically active substance, a diagnostic substance, a substance having nutritional value, and the like. Examples of pharmacologically active substances include:

Analgesics / antipyretics (eg, aspirin, acetaminophen, ibuprofen, naproxene sodium, buprenorphine hydrochloride, propoxyphene hydrochloride, propoxyphenapsilate, meperidine hydrochloride, hydromorphone hydrochloride, morphine sulfate, oxycodone hydrochloride, codeine phosphate, barbitur Dihydrocodeine acid, pentazocine hydrochloride, hydrocodone barbiturate, revorphanol tartrate, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mephenamic acid, butorphanol tartrate, choline salicylate, butarbital, phenyltroxamine citrate, diphenhydramine citrate, metotrimeprazine, Synamedrin hydrochloride, meprobamate, etc.); Anesthetics (eg cyclopropane, enflurane, halothane, isoflurane, methoxyflurane, nitric oxide, propofol, etc.); Anti-asthma drugs (eg azelastine, ketotifen, traxanox, etc.); Antibiotics (eg neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline, etc.); Antidepressants (eg nephopam, oxypertin, doxepin hydrochloride, amoxapine, trazodon hydrochloride, amitriptyline hydrochloride, maprotyline hydrochloride, phenergine sulfate, desipramine hydrochloride, nortriptyline hydrochloride, tranylcypromin sulfate, fluoxetine hydrochloride, doxepin hydrochloride, imipramine hydrochloride, imipramine hydrochloride , Nortriptyline, amitriptyline hydrochloride, isocarboxazide, desipramine hydrochloride, trimipramine maleate, protryptrin hydrochloride, etc.);

Antidiabetic drugs (eg, buguanide agents, hormones, urea sulfonate derivatives, etc.); Antifungal drugs (eg griseofulvin, keroconazole, amphotericin B, nystatin, candicidine, etc.); Antihypertensive drugs (eg propranolol, propaphenol, oxyprenolol, nifedipine, reserpine, trimetaphan cansylate, phenoxybenzamine hydrochloride, pargulin hydrochloride, deserpidin, diazoxide, guanethidine monosulfate, minoxidil, rescinamine, sodium nitroprusside, lau Wolfia serpentina, alceroxylone, fenthramine mesylate, reserpine, etc.); Anti-inflammatory drugs (eg, (non-steroidal) indomethacin, naproxen, ibuprofen, laminphenazone, piroxicam, (steroidal) cortisone, dexamethasone, fluazacort, hydrocortisone, prednisone, prednisone, etc.);

Anti-neoplastic agents (eg, adriamycin, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferon, camptothecin and Its derivatives, phenesterin, taxol and its derivatives, taxotere and its derivatives, vincristine, vincristine, tamoxyphene, etoposide, piposulfane, etc.); Anxiolytics (eg, lorazepam, buspirone hydrochloride, plazepam, chlorodiazepoxide hydrochloride, oxazepam, dipotassium chlorazepam, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, harazepam, chlormezanone, dantrolene, etc.);

Immunosuppressive drugs (eg, cyclosporine, azathioprine, mizoribine, FK506 (tacrolimus), etc.); Anti-tonsil pain drugs (eg, ergotamine tartrate, propanolol hydrochloride, isometheptene mucate, dichloroalphenazone, etc.); Sedatives / hypnotics (eg, barbiturates (eg, pentobarbital, sodium pentobarbital, sodium secobarbital), benzodiazapines (eg, flurazepam hydrochloride, triazolam, tomazepam, midazolam hydrochloride, etc.); Antianginal drugs (eg, beta-adrenaline blockers, calcium channel blockers (eg, nifedipine, diltiazem hydrochloride, etc.), nitrates (eg, nitroglycerin, isosorbide dinitrate, pentaerythritol tetranitrate, erythritol tetranitrate, etc.));

Antipsychotics (eg haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride, thiothixene, fluphenazine hydrochloride, fluphenazine decanoate, fluphenazine enanoate, trifluoperazine hydrochloride, chloropromazine hydrochloride, perphenazine, quen Lithium acid acid, prochlorperazine, etc.); Antidepressants (eg lithium carbonate); Antiarrhythmic agents (eg, brethylium tosylate, esmorol hydrochloride, verapamil hydrochloride, amiodarone, encainide hydrochloride, digoxin, digitoxin, mexiletine hydrochloride, disopyramide phosphate, prokineamide hydrochloride, quinidine sulfate, quinidine gluconate, quinidine polygalacturonate, flecainide acetate , Tocainide hydrochloride, lidocaine hydrochloride, etc.); Anti-arthritis drugs (eg, phenylbutazone, slindac, penicillamine, salsalate, piroxicam, azathioprine, indomethacin, mecrophenamate sodium, sodium aurothiomalate, ketoprofen, auranophin, aurothioglucose, sodium tolmethin, etc.);

Anti-ventilators (eg, corhicin, allopurinol, etc.); Anticoagulants (eg, heparin, sodium heparin, sodium warfarin, etc.); Thrombolytic drugs (eg, urokinase, streptokinase, altoprase, etc.); Anti-fibrinolytic agents (eg aminocaproic acid); Blood rheology drugs (eg, pentoxifylline); Antiplatelet drugs (eg, aspirin, empyrin, aspryptin, etc.); Anticonvulsants (eg, valproic acid, sodium divalproate, phenytoin, phenytoin sodium, chronazepam, primidone, phenobarbital, phenobarbital sodium, carbamazepine, amobarbital sodium, metoscusmid, metalbital, mefobalbital, mephenytoin, fensuximide) , Parameter dione, etotoin, phenytoin, secobarbital, chlorazepet dipotassium, trimetadione, etc.); Antiparkinsonian drugs (eg, ethosuximide); Antihistamines / antipruritic agents (eg, hydroxyzinc hydrochloride, diphenhydramine hydrochloride, chlorophenylamine maleate, bromopheniramine maleate, cyproheptazine hydrochloride, terphenazine, clemastine fumarate, triprolidine hydrochloride, carbinoxamine maleate, diphenylpyraline hydrochloride, tartaric acid Phenindamine, azatazine maleate, tryperenamine hydrochloride, dexchloropheniramine maleate, methidirazine hydrochloride, trimprazine tartrate, etc.); Substances that are effective in regulating calcium (eg, calcitonin, parathyroid hormone, etc.); Antibacterial agents (eg, cephalosporins (eg, cepazoline sodium, cephalazine, cefacloa, cepapilin sodium, ceftyzoxim sodium, cefoperazone sodium, cefotetandisodium, ceftoximeazotyl, cefotaxim sodium, cefadoroxyl) Water salt, ceftazim, cephalexin, cephalotin sodium, cephalexin monohydrate, cefamandol nafate, cefoxitin sodium, cephonicid sodium, cephoranide, ceftriaxone sodium, ceftazim, cefadoroxyl, cefrazin, cephlixim sodium, etc. ), Penicillins (eg, ampicillin, amoxylin, penicillin G-benzazine, cyclacillin, ampicillin sodium, penicillin G-potassium, penicillin V-potassium, piperacillin sodium, oxacillin sodium, baccampicillin hydrochloride, cloxacillin sodium, ticarcillin sodium, azurosylin sodium, Carbenicillin indanyl sodium, penicillin G potassium, penicillin G procaine, methicillin sodium, naphthylin sodium, etc.), erythromycins (erythromycin ethylsuccinate, erythromycin, erythromycin estrate, erythromycin lactobionate, erythromycin sialate, ethylsuccinate erythromycin, etc.) Tetracyclins (tetracycline hydrochloride, doxicillin hisiclate, minocycline hydrochloride, etc.);

Anti-infective drug (eg GM-CSF); Bronchial dilators (eg, sympathomimetics (eg, epinephrine hydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetaline, isoetaline mesylate, isoetaline hydrochloride, albuterol sulfate, albuterol, bitorterol, isoproterenol hydrochloride, terbutamol sulfate, Epinephrine barbiturate, metaproterenol sulfate, epinephrine, epinephrine ditartate), anticholinergic agents (eg, ipratropium bromide), xanthin (eg, aminophyllin, diphyllin, metaproterenol sulfate, aminophilin), obesity cell stabilizers For example, chromolin sodium), inhalable corticosteroids (eg, flurisolidebecromethasone dipropionate, beclomethasone dipropionate monohydrate), salbutamol, beclomethasone dipropionate (BDP). , Ipratropium bromide, budesonide, ketotiphen, salbutamol, xinafoate, terbutaline sulfate, triamsinolone, theophylline, sodium nedochromyl, metaproterenol sulfate, albuterol, flunisolide, etc.); Hormones (eg, danazol, testosterone cypionate, fluorimesterone, ethyltostosterone, testosterone enanihet, methyltestosterone, fluorimesterone, testosterone cypionate), estrogens (eg, estradiol, estropeate, etc. Conjugated estrogen), progestin (eg, methoxyprogesterone acetate, noretindron acetate), cortisone steroids (eg, triamsinolone, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisolone, methylprednisolone acetate suspension Thyroids, triamsinolone acetonide, methylprednisolone, sodium prednisolone phosphate, sodium methylprednisolone succinate, sodium hydrocortisone succinate, sodium methylprednisolone succinate, triamsinolone hexacatonide, hydrocortisone, hydrocortisone cypionate, prednisolone, fluorocortisone acetate Parameterzone, prednisolone tebrate, prednisolone acetate, prednisolone sodium phosphate, hydrocortisone sodium succinate, etc.), thyroid hormone (eg, levothyrosin sodium), etc.;

Hypoglycemic agents (eg, human insertion, purified bovine insulin, purified porcine insulin, glybrid, chlorpropamide, glipizide, tolbutamide, tolazamide, etc.); Hyperlipidemic drugs (eg, clofibrate, dextrothyrox sodium, probucol, lovastatin, niacin, etc.); Proteins (eg DNase, arginase, superoxide dismutase, lipase, etc.); Nucleic acid (eg, sense or antisense nucleic acid encoding a therapeutically effective protein, including any of the above proteins); Drugs useful for stimulating erythropoiesis (eg, erythropoietin); Anti-ulcer / anti-reflex drugs (eg famotidine, cimetidine, ranitidine hydrochloride, etc.);

Antiemetics / antiemetics (eg, meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, scopolamine, etc.); Fat-soluble vitamins (eg vitamins A, D, E, K, etc.); As well as other drugs such as mitotane, visadine, halonitrosourea, anthrocyclin, ellipticin.

Examples of diagnostic substances intended for use in the practice of the present invention include ultrasonic contrast agents, radioactive contrast agents (eg, iodo-octane, halocarbons, renographynes, etc.), magnetic contrast agents (eg, fluorocarbons, etc.). There are diagnostic substances that cannot be easily delivered without physical and / or chemical modifications of some of them to allow their substantial water insolubility, such as fat-soluble paramagnetic compounds). Examples of nutritionally valuable substances intended for use in the practice of the present invention include amino acids, sugars, proteins, carbohydrates, fat-soluble vitamins (eg, vitamins A, D, E, K, etc.) or fats, or There are any two or more combinations of these. Many biocompatible polymers can be used in the practice of the present invention for the formation of polymer shells that substantially surround water-insoluble pharmacologically active substances. Basically all natural or synthetic polymers, which optionally have sulfhydryl groups or disulfide bonds in their structure, are used to prepare disulfide-crosslinked shells around particles of substantially water-insoluble pharmacologically active substances. To. Sulfhydryl groups or disulfide bonds may be pre-existing in the polymer structure or may be introduced by appropriate chemical modification. For example, natural polymers such as proteins, peptides, polynucleics, polysaccharides (eg starch, cellulose, dextran, alginate, chitosan, pectin, hyaluronic acid, etc.), proteoglycans, lipid proteins, etc. are candidates for such modifications. Is.

Proteins intended for use as stabilizers in accordance with the present invention include albumin (with 35 cysteine residues), immunoglobulins, casein, insulin (with 6 cysteine residues), hemoglobin (with 6 cysteine residues). Includes lysoteam (having 9 cysteine residues), immunoglobulins, α-2-macroglobulin, fibronectin, bitronectin, fibrinogen, lipase and the like. Proteins, peptides, enzymes, antibodies, and combinations thereof are common classes of stabilizers intended for use in the present invention. A suitable protein of the invention used to form polymer shells is albumin. Proteins such as the known opsonin α-2-macroglobulin enhance the uptake of particles of substantially water-insoluble pharmacologically active substances by shelled macrophage-like cells, or into the liver and spleen. It could be used to enhance the uptake of particles in the shell of the liver. Specific antibodies are also used to target nanoparticles at specific locations.

Synthetic polypeptides containing cysteine residues as well are also good candidates for the formation of shells around substantially water-insoluble pharmacologically active substances. In addition, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylic acid, polyethoxyoxazoline, polyacrylamide, polyvinylpyrrolidone, etc. can be chemically modified (eg, by introducing sulfhydryl groups and / or disulfide bonds) and shell-forming (crosslinking thereof). A good candidate (by causing). For example, those intended to be practiced in the present invention are synthetic polyamino acids containing cysteine residues and / or disulfide groups; polyvinyl alcohols modified to contain free sulfhydryl groups and / or disulfide groups; free sulfhydryl groups and / Or polyhydroxyethyl methacrylate modified to contain a disulfide group; polyacrylic acid modified to contain a free sulfhydryl group and / or a disulfide group; modified to contain a free sulfhydryl group and / or a disulfide group Polyethyloxazoline; polyacrylamide modified to contain free sulfhydryl and / or disulfide groups; polyvinylpyrrolidone modified to contain free sulfhydryl and / or disulfide groups; free sulfhydryl and / or disulfide groups Polyalkylene glycol modified to contain; polylactide, polyglycolide, polycaprolactone, or copolymers thereof modified to contain free sulfhydryl and / or disulfide groups; and any two or theirs thereof. There are the above combinations.

In the production of the compositions of the present invention, a wide variety of organic media can be used to suspend or dissolve a substantially water-insoluble pharmacologically active substance. Any non-aqueous medium that is intended for use in the practice of the present invention can suspend or dissolve the pharmacologically active substance, but does not chemically react with the polymer used to make the shell. Contains liquids or pharmacologically active substances themselves. Examples include vegetable oils (eg soybean oil, olive oil, etc.), coconut oil, benzene oil, cottonseed oil, sesame oil, orange oil, limonene oil, aliphatics with 4 to 30 carbon atoms, alicyclic, Or aromatic hydrocarbons (eg, n-dodecane, n-decane, n-hexane, cyclohexane, toluene, benzene, etc.), aliphatic or aromatic alcohols with 2-30 carbon atoms (eg, ethyl caprilate (eg, ethyl caprilate) Octanoate), alkyl i, aryl, or cyclic ethers with 2 to 30 carbon atoms (eg, diethyl ether, tetrahydrofuran, etc.), alkyl or aryl halides with 1 to 30 carbon atoms (and optionally 2). Having one or more halogen substituents, eg CH₃Cl, CH₂Cl₂, CH₂Cl-CH₂There are ketones with 3 to 30 carbon atoms (eg, acetone, methyl ethyl ketone, etc.), polyalkylene glycols (eg, polyethylene glycol, etc.), or any combination of two or more of these.

A particularly preferred combination of organic media intended for use in the practice of the present invention typically has a boiling point of about 200 ° C. or less and is a volatile liquid such as dichloromethane, chloroform, ethyl acetate, benzene (ie, ie). It contains a solvent having a high degree of solubility in a pharmacologically active substance and being soluble in other organic media used) together with a high molecular weight (low volatility) organic medium. When added to other organic media, these volatile additives facilitate the dissolution of the pharmacologically active substance in the organic medium. This is preferable because this step is usually time consuming. After dissolution, the volatile components can be removed by distilling off the solvent (at any time under vacuum).

Particles of the pharmacologically active substance associated with the polymer shell prepared as described above are delivered as a suspension in a biocompatible aqueous liquid. The liquid is selected from water, saline solution, solutions containing suitable buffers, solutions containing nutrients (eg, amino acids, sugars, proteins, carbohydrates, vitamins or fats, etc.). Those skilled in the art will readily appreciate that some modifications are possible within the scope and spirit of the invention. In the formation of the walls of the polymer shell, the organic medium within the polymer shell is diverse, a variety of pharmacologically active substances are available, and a wide range of proteins as well as other natural and synthetic polymers can be used. The applications are also widespread. Besides biomedical applications such as delivery of drugs, diagnostic substances (in imaging applications), blood replacers and parenteral nutrients, the polymer shell structures of the present invention can be used in cosmetics such as skin creams or hair care products. Introduced in applications such as perfume, pressure sensitive ink, etc.

The present invention will be described in more detail with reference to the following non-limiting examples.

<u style="single">Example 1</u><u style="single">Manufacture of nanoparticles by high-pressure homogenization</u> Dissolve 30 mg paclitaxel in 3.0 ml ethylene chloride. This solution was added to 27.0 ml of human serum abumin solution (1% w / v). To form a crude emulsion, the mixture was homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000 to 18,000 psi while the emulsion was recirculated for at least 5 cycles. The resulting system was transferred into a Rotaxy evaporator and methylene chloride was rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was translucent and the typical diameter of the resulting paclitaxel particles was 160-220 (Z-average, Malvern Zetasizer). The dispersion was lyophilized for an additional 48 hours without the addition of cryoprotectant. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 2</u><u style="single">Manufacture of nanoparticles by sonication</u> The purpose of this example is to demonstrate the formation of paclitaxel nanoparticles by using cavitation and high shear during the sonication process. Therefore, 20 mg of paclitaxel is dissolved in 1.0 ml of ethylene chloride. Add this solution to 4.0 ml of human serum abmin solution (5% w / v). To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a 40 KHz sonicator cell. Operate the sonicator at 0 ° C for 1 minute at 60-90% output (550 Sonic Dismembrator). The mixture is transferred to a Rotary evaporator and the methylene chloride is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 20-30 minutes. The typical diameter of the obtained paclitaxel particles was 350-420 nm (Z-average, Malvern Zetasizer). The dispersion was lyophilized for an additional 48 hours without the addition of cryoprotectants. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 3</u><u style="single">Conventional surfactant and protein use results in the formation of large crystals</u> The following examples demonstrate the effect of adding the surfactants used in conventional solvent evaporation methods. A series of experiments were performed using a method similar to that described in Example 1, but a surfactant such as Tween 80 (1% -10%) was added to the organic solvent. After removal of methylene chloride, light microscopy and observation under polarized light, it was found that a large number of paclitaxel crystals with an average size of 1-2 microns were obtained. These crystals grow within a few hours to form very large needle-like crystals with sizes in the range of about 5-15 microns. Similar phenomena are observed with other commonly used surfactants such as Pluronic F-68, Pluronic F 127, Cremophor EL and Brij 58.

From these results, a submicron drug without a polymer core, where the conventional solvent vaporization method uses a polar solvent (eg, methylene chloride), using a conventional surfactant in combination with a protein such as albumin, for example. It can be concluded that it is not suitable for the formation of particles (eg paclitaxel).

<u style="single">Example 4</u><u style="single">Use of conventional surfactants alone results in large crystals</u> In this example, for a pharmacologically active agent that is soluble in a polar water-immiscible solvent (eg, chloroform), conventional surfactants are used without a polymer core material to form nanoparticles. Demonstrate that it is impossible to do. Dissolve 30 mg Taxol in 0.55 ml chloroform and 0.05 ml ethanol. This solution is added to 29.4 ml of Tween 80 solution (1% w / v) presaturated with 1% chloroform. Low RPM (Vitris Homogenizer, Model: Tempest) of this mixture to form a crude emulsion Homogenize with IQ) for 5 minutes and then transfer to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 6 cycles. The resulting system was transferred into a Rotaxy evaporator and chloroform was rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 15-30 minutes. The resulting dispersion was opaque and contained large acicular crystals of the drug. The initial size of the crystals (also observed by polarization) was 0.7-5 microns. When this dispersion was stored at room temperature for several hours, the crystal size further increased and eventually settled.

<u style="single">Example 5</u><u style="single">Aseptic below 200 nm-Manufacture of filterable nanoparticles</u> This example describes how sterile-filterable drug particles can be obtained. Therefore, 30 mg of Taxol is dissolved in 0.55 ml of chloroform and 0.05 ml of ethanol. This solution is added to 29.4 ml of human serum abmin solution (1% w / v) presaturated with 1% chloroform. To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 6 cycles. The resulting system is transferred into a Rotaxy evaporator and chloroform is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 15-30 minutes. The resulting dispersion is translucent and the typical diameter of the resulting Taxol particles is 140-160 nm (Z-average, Malvern Zeta). Sizer). The dispersion is passed through a 0.22 micron filter (Millipore) and filtered without significant changes in turbidity or particle size. HPLC analysis of Taxol content revealed that over 97% of Taxol was recovered after filtration, thus resulting in a sterile Taxol dispersion.

The sterile dispersion was lyophilized for an additional 48 hours without the addition of cryoprotectants. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 6</u><u style="single">Aseptic below 200 nm-Manufacture of filterable nanoparticles</u> This example describes how sterile-filterable drug particles can be obtained. Therefore, 225 mg of Taxol is dissolved in 2.7 ml of chloroform and 0.3 ml of ethanol. This solution is added to 97 ml of human serum abmin solution (3% w / v). To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 6 cycles. The resulting system is transferred into a Rotaxy evaporator and chloroform is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 15-30 minutes. The resulting dispersion is translucent and the typical diameter of the resulting taxol particles is 140-160 nm (Z-average, Malvern Zeta Sizer). This dispersion is filtered by a 0.22 micron filter (Sartorius, Sartobran). Through 300), filter without significant changes in turbidity or particle size. HPLC analysis of Taxol content typically revealed that 70-100% taxol could be recovered after filtration, depending on the conditions used. Therefore, a sterile Taxol dispersion was obtained.

This sterile dispersion was aseptically filled in sterile glass vials and lyophilized without the addition of cryoprotectants. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 7</u><u style="single">Effect of organic solvent on phase fraction particle size</u> The following examples demonstrate the importance of having an unusually low phase fraction of the organic solvent in the system. Therefore, a series of experiments was performed according to the same method as described in Example 5, except that the phase fraction of the organic solvent was changed to maintain the ethanol content in the organic phase at 10% v / v. Increasing the phase fraction results in a significant increase in particle size: at a phase fraction of 4% v / v (above saturation concentration or 5% v / v total chloroform concentration), the resulting particles have a diameter of 250 nm; 3 In the% v / v phase fraction, the particles have a diameter of 200 nm; in the 2% v / v phase fraction, the particles were found to have a diameter of 150 nm. Obviously, only particles produced with a very low phase fraction could be sterile-filtered.

<u style="single">Example 8</u><u style="single">Effect of drug concentration on particle size</u> The role of drug concentration in the organic phase will be demonstrated in the following examples. Two experiments were performed in which the Taxol concentration in the organic phase was 50 mg / ml or 75 mg / ml and all other parameters were the same as those described in Example 3. It was found that low drug concentrations yield particles with a diameter of about 150 nm, and particles produced at high drug loads are smaller, i.e. 130-138 nm. Similar trends were observed when similar experiments were performed with an ethanol concentration of about 50% in the organic phase, i.e., for drug concentrations of 25 mg / ml and 50 mg / ml, the particles were 210 nm and 156 nm in diameter, respectively. Met.

These findings directly deny the results reported by Sjostrom et al. Regarding the formation of nanoparticles in the presence of surfactants in the above literature.

<u style="single">Example 9</u><u style="single">Nanoparticle formation of model drug</u> Dissolve 30 mg Isoresorpine (model drug) in 3.0 ml methylene chloride. Add this solution to 27.0 ml of human serum abmin solution (1% w / v). To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 5 cycles. The resulting system is transferred into a Rotaxy evaporator and the methylene chloride is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was translucent and the typical diameter of the resulting paclitaxel particles was 120-140 nm (Z-average, Malvern Zetasizer). The dispersion was filtered through a 0.22 micron filter (Millipore).

The sterile dispersion was lyophilized for an additional 48 hours without the addition of cryoprotectants. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 10</u><u style="single">Formation of tiny particles by model drug</u> The effect of ethanol addition on particle size reduction will be demonstrated for Isoreser pine. Therefore, 30 mg Isoresorpine is dissolved in 2.7 ml methylene chloride and 0.3 ml ethanol. Add this solution to 27.0 ml of human serum abmin solution (1% w / v). To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 5 cycles. The resulting system is transferred into a Rotaxy evaporator and the methylene chloride is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was translucent and the typical diameter of the resulting paclitaxel particles was 90-110 nm (Z-average, Malvern Zetasizer). The dispersion was filtered through a 0.22 micron filter (Millipore).

The sterile dispersion was lyophilized for an additional 48 hours without the addition of cryoprotectants. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 11</u><u style="single">Use of only water-miscible solvents supersaturated by drugs</u><u style="single">-Not suitable for the method of the present invention</u> Disperse 30 mg Taxol in 0.6 ml ethanol. At this concentration (50 mg / ml), Taxol does not dissolve completely, forming a supersaturated dispersion. This dispersion is added to 29.4 ml of human serum abmin solution (1% w / v). To form a crude emulsion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 6 cycles. The resulting system is transferred into a Rotaxy evaporator and the ethanol is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 15-30 minutes. The particle size of the resulting dispersion is extremely wide, ranging from about 250 nm to a few microns.

Microscopic observation revealed the presence of large Taxol particles and typical acicular crystals. These particles were too large for intravenous injection. This experiment has a very wide particle size distribution when a solvent such as ethanol, which is freely miscible in water, is used in the method of the present invention, and as such, it can be used alone in the method of the present invention. Demonstrate the formation of large particles that cannot. Therefore, the method of the present invention specifically eliminates the use of water-miscible solvents when used alone for dissolution or dispersion of drug components. In the method of the present invention, when such a solvent is used, such a solvent must be mixed with an essentially water-immiscible solvent in order to enable the production of the nanoparticles of the present invention. To request.

<u style="single">Example 12</u><u style="single">Use of only water-miscible solvents containing dissolved drugs</u><u style="single">-Not suitable for the method of the present invention</u> Disperse 30 mg Taxol in 1.3 ml ethanol. At this concentration (about 24.5 mg / ml), Taxol is completely soluble in ethanol. Add this solution to 28.7 ml of human serum abmin solution (1% w / v). To form a coarse dispersion, the mixture is homogenized with low RPM (Vitris homogenizer, model: Tempest IQ) for 5 minutes and then transferred to a high pressure homogenizer (Avestin). Emulsification is performed at 9000 to 18,000 psi, with the emulsion being recirculated for at least 6 cycles. The resulting system is transferred into a Rotaxy evaporator and the ethanol is rapidly removed at 40 ° C. under reduced pressure (30 mmHg) for 15-30 minutes. The particle size of the resulting dispersion is extremely wide, ranging from about 250 nm to a few microns.

In addition to Example 11 above, this example has a very wide particle size distribution when a solvent such as ethanol, which is freely miscible in water, is used in the method of the invention. As a result, it is demonstrated that the formation of large particles that cannot be used in the method of the present invention alone occurs. Therefore, the method of the present invention specifically eliminates the use of water-miscible solvents when used alone for dissolution or dispersion of drug components. When such a solvent is used, the method of the present invention requires that such a solvent be essentially mixed with a water immiscible solvent in order to enable the production of the nanoparticles of the present invention. To do.

<u style="single">Example 13</u><u style="single">Measurement of the physical state of paclitaxel in the nanoparticulate form by X powder diffraction</u> Paclitaxel feedstocks typically exist as acicular crystals of various sizes, typically 5 to 500 microns. The presence of crystals in drug formulations for intravenous injection is clearly disadvantageous due to the potential for obstruction of capillaries when the crystals are present in sizes greater than a few microns. In addition, the solubility of drug crystals is generally lower than that of amorphous drugs, thus reducing the bioavailability of the drug after intravenous administration. It is also known that as the drug loading in the formulation increases, so does the crystallization tendency. Therefore, it is advantageous for the formulation to contain an essentially amorphous drug.

X-ray powder diffraction was used to determine if paclitaxel in the lyophilized powder formulation was crystalline or amorphous. Sample 1-paclitaxel powder; sample 2-freeze-dried serum albumin; sample 3-physical mixture of paclitaxel and albumin; and sample 4-formulated paclitaxel. Each sample was sampled from 2 ° to 70 using CuK α-rays, 40 KeV / 30 mA acceleration voltage, 0.05 ° 2θ step size and 2.0 seconds / step data acquisition time. X-ray analysis was performed up to the 2θ angle. Sample 1 showed a strong peak typical of crystalline samples. The strongest peak was at 5.1 ° 2θ. Sample 2 showed a wide hump typical of amorphous materials. Sample 3 roughly showed the wide hills of Sample 2, but in addition, paclitaxel 5.1. A peak at 2θ was also seen. Sample 4, formulated paclitaxel does not show the crystalline signs characteristic of paclitaxel and appears to be the same as sample 2, which is a substantially amorphous pharmacology in the formulated sample. It suggested the presence of an activator.

The amorphous nature of the nanoparticles produced by the present invention is in direct contrast to the products produced by other methods described in the art with respect to the production of nanoparticles. For example, as described in US Pat. No. 5,145,684 (Liversidge et al.), And by Liversidge-Merisko et al.<u style="single">Pharmaceutical Research 13 (2)</u>The use of grinding methods as described in: 272 ~ 278 (1996) results in substantially crystalline products.

<u style="single">Example 14</u><u style="single">Treatment of tumors with paclitaxel nanoparticles in animal models</u> Nanoparticles of paclitaxel (taxol) were produced as described above in Example 1. This formulation of the drug was tested in a mouse MX-1 human breast cancer xenograft model. MX-1 human breast cancer was subcutaneously transplanted into mice, and treatment was started when the tumor size reached about 150-300 mg. This occurred by day 12, so treatment was started 13 days after the first inoculation.

Tumor-bearing mice were treated with paclitaxel nanoparticles at a dose of 20 mg / kg given for 5 consecutive days by intravenous bolus injection as a suspension in saline solution. Five mice were included in the treatment group. The five control tumor-carrying groups received saline alone on the same schedule. Tumor size was monitored as a function of time. The control group showed a marked increase in tumor weight. All mice in this group were sacrificed between days 28 and 39. On the other hand, the treatment group showed remarkable therapeutic efficacy, and all the mice had no measurable tumor by day 25. All mice in this group were sacrificed on day 39, at which point they had no evidence of recurrence or tumor. The results are shown in Figure 1.

<u style="single">Example 15</u><u style="single">Treatment of rheumatoid arthritis with paclitaxel nanoparticles in animal models</u> A collagen-induced arthritis model of Louvain rat was used to test the therapeutic effect of paclitaxel nanoparticles on arthritis. The paw size of this laboratory animal was monitored to assess the severity of arthritis. After complete onset of arthritis (usually ~ 9-10 days after injection of collagen), experimental animals were treated with paclitaxel nanoparticles 1 mg / kg qod for 6 doses / week and then 1 dose / week for 3 weeks. Or they were divided into different groups receiving either paclitaxel nanoparticles 0.5 mg / kg + Prednison 0.2 mg / kg qod (combination therapy) intraperitoneally. Foot size was measured at the start of treatment (day 0) and each time the drug was injected. One group received only standard saline as a control. By the end of the experiment, the group receiving paclitaxel nanoparticles achieved a 42% reduction in foot size, the combination therapy group showed a 33% reduction in foot size, while the control group showed about 20% reduction in foot size. There was an increase of%. The original foot size before the induction of arthritis was 50%. The result is shown in figure 2.

In conclusion, paclitaxel-containing nanoparticles have been shown to have a therapeutic effect on arthritis. To avoid the side effects of long-term use of both paclitaxel and steroids, similar effects can be achieved, but it is probably better to choose a combination therapy where the dose of each drug is only half.

<u style="single">Example 16</u><u style="single">In vivo targeting of nanoparticles</u> It is possible to target specific parts of the body by incorporating certain specific target components such as proteins, antibodies, enzymes, peptides, oligonucleotides, sugars, polysaccharides, etc. into the protein coating of nanoparticles. .. This targeting ability can be used for therapeutic or diagnostic purposes.

<u style="single">Example 17</u><u style="single">Intravenous delivery system prescribed from various materials</u> The material used in the manufacture of the venous delivery system can be a polymeric material (eg, polyethylene, polyvinyl, polypropylene tubing material, etc.) or glass. Standard medical grade tube materials are known to contain hydrophobic components on their inner surface. These ingredients are therefore available for contact with the injectable solution. In fact, such tubing materials are custom made to allow the hydrophobic component to be present in contact with the therapeutic solution, as is the case with catheters, thus reducing the absorption of aqueous material into the tubing material. However, in therapeutic solutions any hydrophobic component can bind to both the catheter tube material and other components of the delivery system. As a result, a substantial portion of the pharmacologically active hydrophobic reagent may be trapped in the inner wall of the catheter and delivery vessel of the tubing material. After all, administration of the active reagent is irregular, as a substantial portion of the pharmacologically active hydrophobic reagent can become adsorbed on the walls of the tube material. In critical therapeutic treatments in which pharmacologically active hydrophobic reagents are used to treat disease, significant reductions in the effective dose of active reagents can lead to treatment failure. This failure is particularly pronounced when using therapeutic ingredients that require the active reagent to be present above a certain level and have a narrow therapeutic window.

A novel method for intraarterial introduction of pharmacologically active hydrophobic reagents has now been developed here. By protecting the hydrophobic component of this active reagent through association with the hydrophobic component of a biocompatible coating (eg albumin), the propensity of the active reagent to become bound to the tube material is dramatically reduced. .. Thus, the present invention allows the combination of highly hydrophobic agents with standard medical grade polymers and hydrophobic glasses that are agent protected and thus not adsorbed on the surface. The method of the present invention comprises placing a protective coating of a biocompatible polymer (eg, albumin) around a hydrophobic agent and placing the resulting composition in a hydrophobic polymer delivery system. .. The methods of the present invention are therefore capable of improving the deliverability of various hydrophobic therapeutic agents.

<u style="single">Example 18</u><u style="single">Intravenous administration of therapeutic agents</u> Intravenous administration of a therapeutic agent, such as a drug, imaging agent, etc., causes the therapeutic agent to pass through the liver at least once in advance. Because the drug is filtered by the liver, a significant portion of the drug is absorbed and captured by the liver, thus making it useless for systemic distribution. Moreover, once absorbed in the liver, it can be metabolized and the resulting metabolic by-products often have systemic general toxicity. Encapsulation of the drug, or other therapeutic agent, in a coating according to the invention (eg, using a protein such as albumin) alleviates hepatic capture during intravenous administration. Albumin is known to pass, for example, through the liver and become generally distributed throughout the patient. Thus, hepatic capture of albumin does not occur to the same extent as toxic compounds or agents that have hepatic receptors (or other mechanisms) and initiate the process of removing those receptors from the bloodstream. By protecting the therapeutic agent with a coating of biocompatible polymer (eg, coating of human albumin), the agent then bypasses the liver and is generally distributed through the system of any organ. According to one aspect of the invention, there is provided a novel liver bypass method comprising encapsulating a drug in human liver albumin (essentially a physiological component). Thus, more of the drug becomes available for systemic treatment. In addition to increasing drug availability, hepatocyte production of drug-degrading and metabolic by-products is reduced. Increased hepatic diversion and decreased metabolic by-products of the drug synergistically improve the overall efficacy of the drug. This improved potency extends to any drug or substance encapsulated in human albumin.

<u style="single">Example 19</u><u style="single">Myelocyte depressant effect of the drug and reduction of general toxicity</u> Some chemotherapeutic agents have dose-limiting toxicity due to their myelocyte-suppressing effects. Taxol (paclitaxel) is a standard example of such a drug. When administered as the currently approved cremaphor / ethanol formulation, taxol limits repeated doses of the drug to allow the patient's blood count to return to normal, and the patient's Produces a myelocyte-suppressing effect that prevents retreatment for at least 3 weeks. It has been hypothesized that due to the non-toxic compatibility of the drug carriers of the invention, human albumin, the toxic side effects of myelocyte suppression can be significantly reduced. Sprague dawley rat with over-the-counter formulation (Bristol Myers) Paclitaxel produced as (available as cremaphore / ethanol from Squibb: BMS) or as nanoparticles containing albumin by the method of the invention was given. Both formulations were administered by intravenous tail injection. A single dose level of 5 mg / kg was administered for the BMS formulation, whereas two dose levels of 5 mg / kg and 12 mg / kg were administered for the formulation of the present invention [Capxol]. Was done. The white blood cell count of rats was monitored daily after administration as a myelocyte suppression index.

For the BMS preparation (5 mg / kg), the WBC number decreased by 47.6% and 63.5%, respectively, 1 day and 2 days after administration, whereas for the 5 mg / kg level capxol preparation, the WBC number was 1 day. On the second day, it increased by 14.7% and 2.4%, respectively. At higher doses of capxol at the 12 mg / kg level, WBC numbers increased by 6.5% and 3.6% on days 1 and 2, respectively. These results indicate that administration of the drug with the formulations of the present invention significantly reduces short-term myelocyte inhibition.

Another indicator of general toxicity is animal weight. Rat body weight was also monitored following paclitaxel administration. At a dose of 5 mg / kg, the BMS formulation lost 10.4% body weight 3 days after administration, whereas the same dose of paclitaxel administered with the formulation of the present invention (Capxol) lost only 3.9% body weight. It was shown that the formulation of the present invention had significantly reduced toxicity.

<u style="single">Example 20</u><u style="single">Bolas dose administration of nanoparticle preparation</u> Paclitaxel, an anticancer drug as a commercial BMS preparation with cremaphore / ethanol, cannot be administered as an intravenous bolus. This is due to the widespread toxicity of excipients that cause severe anaphylactic reactions, and it is necessary to pre-administer steroids, antihistamines, etc. to patients receiving the drug. This BMS preparation is administered everywhere as an intravenous infusion lasting 1 to 24 hours. In contrast, the formulations according to the invention are readily available to patients as an intravenous bolus (ie, 1) due to the use of non-toxic carriers, without the characteristic problems found in BMS formulations that are clinically used today. Can be administered (in less than an hour).

The effective dose of paclitaxel for a patient is typically 200-500 mg, depending on the patient's weight or body surface. BMS formulations should be administered at a final dose concentration of 0.6 mg / mL, which requires a large infusion volume (typically in the range of about 300-1000 mL). On the other hand, the preparation of the present invention (for example, Capxol) does not have these restrictions and can be administered at a desired concentration. This allows clinicians to treat patients with a rapid intravenous bolus that can be administered in as little as a few minutes. For example, if the formulations of the invention were reconstituted to a dosing concentration of 20 mg / mL, the infusion volume for a total dose of 200-500 mg would be only 10-25 mL each. This is a great advantage in clinical practice.

<u style="single">Example 21</u><u style="single">Compared to commercial cremaphore / ethanol preparations,</u><u style="single">Reduced toxicity of paclitaxel in nanoparticles</u> The anti-cancer drug paclitaxel has widespread toxicity in commercial BMS preparations containing cremaphore / ethanol, and the toxicity causes a severe anaphylactic reaction, and it is often necessary to pre-administer steroids, antihistamines, etc. to patients receiving the drug. Known. The toxicity of BMS formulations was compared to the nanoparticle formulations of the present invention. Thus, these formulations were intravenously injected through the tail vein of C57BL mice at various dose levels and monitored by general observation of the mice after the injection.

For BMS preparations, the dose of 30 mg / kg was uniformly lethal within 5 minutes of intravenous administration. For the same dose, the nanoparticle formulations according to the invention showed no apparent toxic effects. A dose of 103 mg / kg nanoparticle formulation showed some reduction in mouse body weight, but this high dose was not lethal. The doses of about 1000 mg / kg, 800 mg / kg and 550 mg / kg were all lethal doses, but the time to case fatality rate varied and ranged from a few hours to 24 hours. The lethal dose of the formulation of the present invention is greater than 103 mg / kg but less than 550 mg / kg. Thus, the lethal dose of the paclitaxel preparation of the present invention is substantially higher than the lethal dose of the commercially available BMS preparation. This has great significance in clinical practice where higher doses of chemotherapeutic agents will be administered for the purpose of more effective tumor cell disruption activity with significantly lower toxicity.

<u style="single">Example 22</u><u style="single">Production of cyclosporine nanoparticles by high-pressure homogenization</u> Dissolve 30 mg of cyclosporine in 3.0 mL of methylene chloride. The solution is then added to 27.0 mL of human serum albumin solution (1% w / v). The mixture was homogenized with a low RPM [Vitris homogenizer, model; Tempest IQ] for 5 minutes to form a crude emulsion, followed by a high pressure homogenizer [Avenstin]. Move to. Emulsification was performed at 9000-18,000 psi and the emulsion was recirculated for at least 5 cycles at the same time. The resulting system was transferred to Rotavap and the methylene chloride was rapidly removed at 40 ° C. under reduced pressure (30 mm Hg) for 20-30 minutes. The resulting dispersion was translucent and the typical diameter of the resulting cyclosporine particles was 160-200 [Z-average, Malvern Zetasizer].

The dispersion was further lyophilized for 48 hours without the addition of any cryoprotectant. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline. The particle size after reconstruction was the same as before freeze-drying.

<u style="single">Example 23</u><u style="single">Cyclosporine nanodroplets by high pressure homogenization</u><u style="single">Manufacture of [Capsorine Oral]</u> Dissolve 30 mg of cyclosporine in 3.0 mL of suitable oil (sesame oil containing 10% orange oil). The solution is then added to 27.0 mL of human serum albumin solution (1% w / v). The mixture is homogenized with a low RPM [Vitris homogenizer, model; Tempest IQ] for 5 minutes to form a crude emulsion, and then transferred to a high pressure homogenizer (Avenstin). Emulsification is performed at 9000-18,000 psi and the emulsion is recirculated for at least 5 cycles at the same time. The typical diameter of the resulting dispersion was 160-200 (Z-average, Malvern Zetasizer).

The dispersion could be used directly for private use by adding the appropriate cryoprotectant, if desired, or could be lyophilized for 48 hours. The resulting cake could be easily reconstituted into the original dispersion by the addition of sterile water or saline.

<u style="single">Example 24</u><u style="single">Cyclosporine nanoparticles following intravenous administration</u><u style="single">(Capsoline IV) Drug Dynamics (PK) Data</u><u style="single">Sandimmune IV</u><u style="single">Comparison with [Products currently marketed by Sandoz]</u> Cyclosporine nanoparticles (Capsoline IV) prepared as described above (Examples 22 and 23) were reconstituted in saline and administered intravenously to the first group of three sprag dolay rats. The second group of 3 rats was fed Sandimune IV with cremaphore / ethanol after dilution with saline. Each group received the same dose of 2.5 mg / kg. Blood samples were taken at 0, 5, 15, 30 (minutes), 1, 2, 4, 8, 24, 36 and 48 (hours). Cyclosporine levels in blood were assayed by HPLC and typical PK parameters were determined. The PK curve showed typical damping behavior over time as follows:

<u style="single"> Attenuation over elapsed time </u> AUC, mg-hours / mL C_maximum, ng / mL Capsolin IV 12,228 2,853 Capsolin IV) 7,791 2,606

In addition, due to the toxicity of Sandimune IV preparations, 2 of 3 rats in the group died within 4 hours after dosing. Thus, the nanoparticle formulation according to the invention (Capsoline IV) exhibits a higher AUC and is non-toxic compared to the commercially available formulation (Capsoline IV).

<u style="single">Example 25</u><u style="single">Cyclosporine nanodroplets following oral administration</u><u style="single">(Capsoline Oral) Drug Dynamics (PK) Data</u><u style="single">Comparison with Neoral (a formulation currently marketed by Sandts)</u> The cyclosporine nanodroplets produced above were placed in orange juice and administered to the first group of three sprag dolay rats by oral gastrointestinal feeding. The second group of three rats was similarly given neoral containing an emulsifier after dilution with orange juice by oral gastrointestinal feeding. Each group received the same dose of 12 mg / kg in the same volume of orange juice. Blood samples were taken at 0, 5, 15, 30 (minutes), 1, 2, 4, 8, 24, 36 and 48 (hours). Cyclosporine levels in blood were assayed by HPLC and typical PK parameters were determined. The PK curve showed typical damping behavior over time as follows:

<u style="single"> Attenuation over elapsed time </u> AUC, mg-hours / mL C_maximum, ng / mL Capsoline Oral 3,195 887 Neoral 3,213 690

Thus, the nanodroplet formulation (Capsoline Oral) of the present invention exhibits PK behavior similar to that of a commercially available formulation (Neoral). Although the present invention has been described in detail above with reference to certain preferred embodiments, various modifications and modifications are also described herein and are within the spirit and scope of the claimed invention. Will be understood.