MXPA00000294A - Novel formulations of pharmacological agents, methods for the preparation thereof and methods for the use thereof - Google Patents

Abstract

In accordance with the present invention, there are provided compositions and methods useful for the in vivo delivery of substantially water insoluble pharmacologically active agents (such as the anticancer drug paclitaxel) in which the pharmacologically active agent is delivered in the form of suspended particles coated with protein (which acts as a stabilizing agent). In particular, protein and pharmacologically active agent in a biocompatible dispersing medium are subjected to high shear, in the absence of any conventional surfactants, and also in the absence of any polymeric core material for the particles. The procedure yields particles with a diameter of less than about 1 micron. The use of specific composition and preparation conditions (e.g., addition of a polar solvent to the organic phase), and careful selection of the proper organic phase and phase fraction, enables the reproducible production of unusually small nanoparticles of less than 200 nm diameter, which can be sterile-filtered. The particulate system produced according to the invention can be converted into a redispersible dry powder comprising nanoparticles of water-insoluble drug coated with a protein, and free protein to which molecules of the pharmacological agent are bound. This results in a unique delivery system, in which part of the pharmacologically active agent is readily bioavailable (in the form of molecules bound to the protein),and part of the agent is present within particles without any polymeric matrix therein.

Description

NEW FORMULATIONS OF PHARMACOLOGICAL AGENTS. METHODS FOR PREPARATION AND METHODS FOR USE THEREOF.

FIELD OF THE INVENTION The present invention relates to methods for the production of particulate carriers for the intravenous administration of pharmacologically active agents, as well as with novel compositions produced therefrom. In a particular aspect, the invention relates to a method for the in vivo delivery of pharmacologically active agents substantially insoluble in water (for example, the anti-cancer drug paclitaxel). In another aspect, dispersible colloidal systems containing water insoluble pharmacologically active agents are provided. The suspended particles may be formed of 100% of an active agent, or they may be encapsulated in a polymeric shell formulated from the biocompatible polymer, and generally have a diameter of at least about 1 miera. The colloidal systems of the invention can be prepared without the use of conventional surfactants, or any polymeric core matrix. In a presently preferred aspect of the invention, a method is provided for preparing extremely small particles, which can be sterile-filtered. The polymeric shell contains particles of pharmacologically active agent, and optionally a biocompatible, dispersible agent in which the pharmacologically active agent can be either dissolved or suspended. Thus, the invention provides a drug delivery system, either in the liquid form or in the form of a redispersible powder. Any form provides both immediately bioavailable drug molecules (ie, drug molecules that molecularly bind to the protein), and particles of pure drug coated with a protein. The invention is also related to the method of use and preparation of the compositions (formulations) of drugs such as the anti-cancer agent paclitaxel. In one aspect, the paclitaxel formulation, known as Capxol ™, is significantly less toxic and more effective than Taxol®, a commercially available formulation of paclitaxel. In another aspect, the novel formulation Capxol ™ is localized in certain tissues after parenteral administration, thereby increasing the efficiency of the treatment of cancers associated with such tissues.

BACKGROUND OF THE INVENTION The intravenous drug delivery allows a rapid and direct balance with the bloodstream and transports the drug to the rest of the body. To avoid peak serum levels that are achieved in a short period of time after intravascular injection, drug administration i ~ 2 £ 'Si¡B8B? ¡lBÍ »* r ~~ ^ .JtAi. transported within stable carriers allows the gradual release of drugs within the intravascular compartment, after an intravenous bolus injection of therapeutic nanoparticles. Injectable nanoparticles with controlled release can provide a pre-programmed duration of action, which varies from days to weeks to months from a single injection. They may also offer several important advantages compared to conventionally administered medications, including the automatically assured compatibility of the patient with the dose regimen, as well as the targeting of the drug to specific tissues or organs (Tice and Gilley, Journal of Controlled Relay, 2 : 343-352 (1985)). Microparticles and foreign bodies present in the blood are generally eliminated from the circulation by means of "blood filtering organs", mainly the spleen, lungs and liver. The particulate matter contained in normal whole blood comprises cells of red blood cells (typically 8 microns in diameter), white blood cells (typically 6-8 microns in diameter), and platelets (typically 1-3 microns in diameter). The microcirculation in most organs and tissues allows the free passage of these blood cells. When microthrombi (blood clots) larger than 10-15 microns are present in the circulation, it results in a risk of infarction or blockage of the capillaries, leading to ischemia or oxygen deprivation and possible tissue death. The injection of particles within the circulation greater than 10-15 microns in diameter, therefore should be avoided. A suspension of particles of less than 7-8 microns is, however, relatively safe and has been used for the delivery of pharmacologically active agents in the form of liposomes and emulsions, nutritional agents, and contrast media for imaging applications . The particle size and its mode of supply determines its biological behavior. Strand et al. (in Microspheres-Biomedical Applications, De. A. Rembaum, pp. 193-227, CRC Press (1988)) has described the fate of particles as dependent on their size. Particles in the size range of a few nanometers (nm) to 100 nm enter the lymphatic capillaries after interstitial injection and phagocytosis can occur within the lymph nodes. After intravenous / intra-arterial injection, particles of less than about 2 microns will be rapidly eliminated from the bloodstream by the reticuloendothelial system (RES), also known as the phagocytosis mononuclear system (MPS). Particles larger than about 7 microns will, after intravenous injection, be trapped in the pulmonary capillaries. After intra-arterial injection, the particles are trapped in the first capillary bed ^^^^^ reached. The inhaled particles are trapped by the alveolar macrophages. Drugs that are insoluble in water or poorly soluble in water and sensitive to acidic media in the stomach can not be conventionally administered (for example, by intravenous injection or oral administration). Parenteral administration of such drugs has been achieved by emulsifying the medicament solubilized in oil with an aqueous liquid (such as a normal saline solution) in the presence of surfactants or emulsion stabilizers to produce stable microemulsions. These emulsions can be injected intravenously, taking into account that the components of the emulsion are pharmacologically inert. The North American Patent No.

No. 4,073,943, describes the administration of pharmacologically active agents insoluble in water dissolved in oil and emulsified in water in the presence of surfactants such as, for example, phosphatides, pluronic (copolymers of propylene glycol and polyethylene glycol), polyglycerol oleates, etc. The PCT International Publication No. WO95 / 00011 discloses pharmaceutical microarrays of an anesthetic coated with a phospholipid such as dimiristol phosphatidylcholine having dimensions suitable for intradermal or intravenous injection. An example of a drug insoluble in water is paclitaxel, a natural product first isolated from the tree Pacific Yew, Taxus brevifolia, in Wani et al. (J. Am. Chem. Soc, 93_: 2325 (1971)). Among the antimitotic agents, paclitaxel, which contains a carbon skeleton of diterpene, exhibits a unique mode of action in microtubule proteins responsible for the formation of mitotic use. Compared with other antimitotic agents such as vinblastine or colchicine, which prevent the assembly of tubulin, paclitaxel is the only known plant product that can inhibit the tubulin depolymerization process, thus avoiding the _? or cell replication process. It has been shown that paclitaxel, a naturally occurring diterpenoid, has anti-neoplastic and anti-cancer effects in ovarian cancer refractor drug. Paclitaxel has demonstrated excellent antitumor activity in a broad range of variety of tumor models such as melanoma B16 xenografts, L1210 leukemias, MX-1 mammary tumors, and CS-1 colon tumor. Several releases of recent presses have called paclitaxel as the wonderful new anticancer drug. In fact, paclitaxel has recently been approved by the Federal Drug Administration for the treatment of ovarian cancer. The poor aqueous solubility of paclitaxel, however, presents a problem for human administration. In fact, the delivery of drugs that are inherently insoluble or poorly soluble in an aqueous medium can be seriously hindered if the oral supply is not effective. Accordingly, currently used paclitaxel formulations (ie, Taxol®) require a creamfor to solubilize the drug. The variation of human clinical dose is 200-500 mg. This dose is dissolved in a 1: 1 solution of ethanoLcremafor and diluted with a saline solution to approximately 300-100 ml of fluid provided intravenously. The cream currently used is polyethoxylated castor oil. The presence of cremafor in this formulation has been linked to severe hypersensitivity reactions in animals (Lorenz et al., Agents Actions 7: 63-67 (1987)) and in humans (Weiss et al., J. Clin. Oncol. : 1263-68 (1990)) and consequently requires a premedication of patients with corticosteroids (dexamethasone) and antihistamines. The wide dilution results in large volumes of infusion (typical dose 175 mg / nr or until 1 liter), and the infusion times vary from 3 hours to 24 hours. Thus, there is a need for an alternative less toxic formulation for paclitaxel. In phase I of the clinical trials, paclitaxel itself did not show excessive toxic effects, but severe allergic reactions were caused by the emulsifiers used to solubilize the drug. The current regimen of administration involves the treatment of patients with antihistamines and steroids before the injection of the drug to reduce the allergic side effects of cremafor. - * - * ~ - i - - fnrpimr i • ** "" - - ****** »* - * In an effort to improve the water solubility of paclitaxel, several researchers have modified their chemical structure with groups functionalities that impart improved water solubility. Among these are sulfonated derivatives (Kingston et al., U.S. Patent 5,059,699, (1991)), and amino acid esters (Mathew et al., J. Med. Chem. 35: 145-151 (1992)) which show an activity biologically significant. Modifications to produce a water-soluble derivative facilitate the intravenous delivery of paclitaxel dissolved in a harmless carrier such as a normal saline solution. However, such modifications add to the cost of drug preparation, and may induce undesirable side reactions and / or allergic reactions and / or may decrease drug efficiency. Protein microspheres have been reported in the literature as carriers of pharmacological or diagnostic agents. The albumin microspheres have been prepared either by heat denaturation or chemical cross-linking. The heat denatured microspheres are produced from an emulsified mixture (for example albumin, the agent to be incorporated, and a suitable oil) at temperatures between 100 ° C and 150 ° C. The microspheres are then washed with a suitable solvent and stored. Leucuta et al. (International Journal of Pharmaceutics, 41: 213-217 (1988)) describes the method of preparation of denatured microspheres.

The process for preparing the chemically crosslinked microspheres involves treating the emulsion with a glutaraldehyde to cross-link the protein, followed by washing and storage. Lee et al. (Science, 213: 233-235 (1981)) and U.S. Patent No. 4,671,954 teach this method of preparation. The prior techniques for the preparation of protein microspheres as carriers of pharmacologically active agents, while being suitable for the delivery of water-soluble agents, are incapable of retaining insoluble in water. This limitation is inherent in the preparation technique which is based on the cross-linking or heat denaturation of the protein component in the aqueous phase of a water-in-oil emulsion. Any water-soluble agent dissolved in the protein-containing aqueous phase can be retained within the resulting heat-denatured or cross-linked protein matrix, but an agent soluble in oil or poorly soluble in water can not be incorporated into a protein matrix formed by these techniques. A conventional method for making the nanoparticles containing the drug comprises dissolving polylactic acid (or other biocompatible water insoluble polymers) in a water immiscible solvent (such as methylene chloride or other aliphatic, or chlorinated aromatic solvents), dissolving the agent pharmaceutically active in ^^ g ^^^^^ &Sagj ^ SB | 5! eSH ^ .. ^^^^^^^^ & ^^^^^ polymer solution, add a surfactant to the oil phase or phase aqueous, forming an emulsion of oil in water by suitable means, and evaporating the emulsion slowly under vacuum. If the oil droplets are sufficiently small and stable during evaporation, a suspension of the polymer in water is obtained. Since the drug is initially present in the polymer solution, it is possible to obtain by this method, a composition in which the drug molecules are retained within the composite particles within a polymer matrix. The formation of microspheres and nanoparticles using the solvent evaporation method has been reported by several investigators (see, for example, Tice and Gilley, in Journal of Controlled Relay, 2: 343-352 (1985); Bodmeier and McGinity, in Int. J. Pharmaceutics, 43: 179 (1988), Cavalier et al., In J. Pharm. Pharmacol., 38: 249 (1985): and D'Souza et al., WO 94/10980) while several are used drugs. Bazile et al., In Biomaterials, 13: 1093 (1992); and Spenlehauer et al., in French Patent 2 660 556, have reported the formation of nanoparticles using two biocompatible polymers, one (eg, polylactide) is dissolved in the organic phase, together with an active component such as the drug, and the other polymer such as albumin is used as the surface active agent. After emulsification and solvent removal, nanoparticles are formed, in which the drug is present within the polymer matrix of the polylactide particles. The properties of the polymer solution from which the polymer matrix is formed are very important to obtain the proper emulsion in the first stage. For example, polylactide (the polymer commonly used in the preparation of injectable nanoparticles) has a surface activity which causes its rapid adsorption at the dichloromethane-water interface, causing reduced interfacial tension (see, for example, Boury et al., In Langmuir, 11: 1636 (1995)), which in turn improves the emulsification process. In addition, the same researchers found that Bovine Serum Albumin (BSA) interacts with poly lactide and penetrates into the polylactic monolayer present at the oil-water interface. Therefore, it is expected based on the above references, that the emulsification during the conventional solvent evaporation method is widely favored by the presence of the surface active polymer (polylactide) in the non-aqueous organic phase. In fact, the presence of polylactide is not only a sufficient condition, but is currently necessary for the formation of nanoparticles of an adequate size. Another process which is based on the evaporation method of the solvent, comprises dissolving the drug in the hydrophobic solvent (for example, toluene or cyclohexane), without any polymer dissolved in the organic solvent, adding a *? j ** ~, ^ ati ^ fc .. ^. ^^ «* ^. conventional surfactant to the mixture as an emulsifier, forming an oil-in-water emulsion using sound application equipment or high-speed cutting, and then evaporating the solvent to obtain dry particles of the drug (see, for example, Sjostrom et al. al., in J. Dispersion Science and Technology, 15: 89-117 (1994)). By removing the non-polar solvent, precipitation of the drug occurs within the solvent droplets, and submicron particles are obtained. It has been found that the size of the particles is mainly controlled by the initial size of the emulsion droplets. Furthermore, it is interesting to note that the final particle size is reported to decrease with a decrease in the concentration of the drug in the organic phase. This discovery is contrary to the results reported here, where no conventional surfactant is used for the preparation of the nanoparticles (in some embodiments of the invention). In addition, it has been noted by the authors of the Sjostrom document that the drug used, cholesteryl acetate, has active surface in toluene, and thus can be oriented at the oil-water interface; therefore the concentration of drug in the interface is higher, thus increasing the potential for precipitation. The formation of submicron particles has also been achieved by a precipitation process, as described in Calvo et al., In J. Pharm. Sci., 85: 530 (1996). The process is ^^^ ¡^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ based on the dissolution of the drug (eg, indomethacin) and the polymer (poly-caprolactone) in methylene chloride and acetone, and then pouring the solution into an , containing a surfactant (Poloxamer 188), to produce particles of submicron size (216 nm). However, the process is carried out at concentrations of solvent to which no emulsion is formed. Paclitaxel is a naturally occurring compound which has shown great promise as an anticancer drug. For example, paclitaxel has been found to be an active agent against ovarian cancer, refractor to the drug or by McGuire et al. see "Taxol: A Unique Anti-Neoplastic Agent With Significant Activity Against Advanced Ovarian Epithelial Neoplasms." Ann. Int. Med., 111: 273-279 (1989). All patents, scientific articles and other documents mentioned herein are incorporated by reference, as will be reproduced in full hereafter. Unfortunately, paclitaxel has an extremely low solubility in water, which makes it difficult to provide an adequate dosage form. In fact, in Phase I of clinical trials, severe allergic reactions were caused by emulsifiers administered in conjunction with paclitaxel to compensate for their low water solubility; at least the death of a patient was caused by a reaction induced by the emulsifiers. The dose-limiting toxicities include the ^ ^ "^ gfe Bgjil" and * aa3, neutraopenia, peripheral neuropathy, and hypersensitivity reactions Brown et al., in "A Phase I Trial of Taxol Given by A 6-Hour Intravenous Infusion." J. Clin Oncol., 9 (7): 1261-67 (July, 1991), reports on the Phase I Tests in which Taxol was provided as an IV infusion of 6 hours every 21 days without premedication. 31 patients received 64 evaluable courses of Taxol. One patient had a severe (or acute) hypersensitivity reaction, which required the discontinuation of infusion and immediate treatment to save the patient's life. Another patient experienced a hypersensitivity reaction, but was not so severe as to require the discontinuation of the infusion. Myelosuppression was the dose limiting, with two fatalities due to sepsis. Non-haematological toxicity was grade 1 and 2, except for one Grade 3 mucositis patient and two patients with Grade 3 neuropathy. The neuropathy consisted of reversible painful paresthesia, requiring the discontinuation of Taxol in two patients. Four partial responses were seen (3 in patients with non-small cell lung cancer, and one in a patient with adenocarcinoma of an unknown primary). The maximum tolerated dose reported was 275 mg / m2, and the recommended initial dose of Phase II was 225 mg / m2. The incidence of hypersensitivity reaction was reported as dependent on schedule, with 6 infusions of 6 to 24 hours of drug having an incidence of hypersensitivity reactions of 0% to 8%. It was also reported that hypersensitivity reactions persisted with or without premedication despite the prolongation of infusion times. Since these Phase I studies were conducted in terminally ill patients suffering from a variety of cancers, the efficacy of Taxol treatments could not be determined. In a study by Kris et al., Paclitaxel formulated with Cremafor EL in dehydrated alcohol was given as an IV infusion of 3 hours every 21 days, with the administered dose varying from 15 to 230 mg / m2 in nine escalation steps. Kris et al. concluded that "with the severity and unpredictability of hypersensitivity reactions, the additional use of Taxol is not indicated with this drug formulation at this time of administration". See Cancer Treat. Rep., Vol. 70, No. 5, May 1986. Since initial tests using a bolus injection or short infusions (1-3 hours) induced anaphylactic reactions or other hypersensitivity responses, additional studies were carried out in the Taxol was administered only after premedication with steroids (such as dexamethasone), antihistamines (such as diphenylhydramine), and H2 antagonists (such as cimetidine or ranitidine), and the infusion time was extended to 24 hours in an attempt to eliminate the most serious allergic reactions. Various ^ -rSBteua. - 5S & Results of Phase I and Phase II studies have been published using 24-hour infusions of Taxol with maximum total doses of 250 mg / m2, generally with the course being repeated every 3 weeks. Patients were pre-treated with dexamethasone, diphenhydramine, and cimetidine to compensate for allergic reactions. See Einzig, et al., "Phase II Trial of Taxol in Patients with Metastatic Renal Cell Carcinoma," Cancer Investigation, 9 (2) 133-136 (1991), and AB Miller et al., "Reporting Results of Cancer Treatment, "Cancer, 47: 207-214 (1981). Koeller et al., In "A Phase I Pharmacokinetic Study of Taxol Given by a Prolonged Infusion Without Premedication," Proceedings of ASCO, Vol. 8 (March, 1989) recommends routine premedication in order to avoid the significant number of allergic reactions that It is believed that they are caused by the cremophor (polyethoxylated castor oil) carrier used for infusions of paclitaxel. Patients received doses ranging from 175 mg / m2 to 275 mg / m2. Wiernik et al. in "Phase I Clinical and Pharmacokinetic Study of Taxol," Cancer Research, 47: 2486-93 (May 1, 1987) also reports the administration of paclitaxel in a cremophor carrier by an IV infusion over a period of 6 hours in a study of Phase I. Hypersensitivity reactions of grade 3-4 occurred in 4 of 13 courses. The initial dose for the study was 15 mg / m2 (one third of the lowest toxic dose in dogs). The doses were escalated and a minimum of 3 patients were treated until the dose level until the toxicity was identified and then 4-6 patients were treated at each subsequent level. The study concluded that neurotoxicity and leukopenia were dose-limiting, and the recommended Phase II trial dose was 250 mg / m2 with premedication. Other exemplary studies in paclitaxel include: Legha et al., "Phase II Trial of Taxol in Metastatic Melanoma," 65: 2478-81 (June, 1990); Rowinsky et al., "Phase I and Pharmacodynamic Study of Taxol in Refractory Acute Leukemias," Cancer Research, 49: 4640-47 (August 15, 1989); Grem et al., "Phase I Study of Taxol Administered as a Short IV Infusion Daily for 5 Days," Cancer Treatment Reports, Vol. 71, No. 12, (December, 1987); Donehower et al., "Phase I Trial of Taxol in Patients With Advanced Cancer," Cancer Treatment Reports, Vol. 71, No. 12, (December, 1987); Holmes et al., "Phase II Study of Taxol in Patients (PT) with Metastatic Breast Cancer (MBC)," Proceedings of the American Society of Clinical Oncology, 10:60, (March, 1991). See also Suffness, "Development of Antitumor Natural Products at the National Cancer Institute," Gann Monograph of Cancer Research, 31: 21-44 (1989) (which recommends that Taxol® only be delivered as a 24-hour infusion). Weiss et al., In "Hypersensitivity Reactions from Taxol," Journal of Clinical Oncology, 8 (7): 1263-68 (July 1990) reported that it is difficult to determine a reliable overall incidence of hypersensitivity reactions (HSR), due to the > -, wide variations of paclitaxel and oral doses used, and the unknown degree of influence that changes the infusion schedule and the use of premedication in HSR incidences. For example, of five patients who received Taxol in a 3-hour infusion at more than 190 mg / m2 without any pre-medication, three had reactions, whereas only one of 30 patients, administered with even higher doses during an infusion of 6 hours without any pre-medication had a reaction. Therefore, this suggests that prolonging the infusion beyond 6 hours is sufficient to reduce HSR incidents. However, Weiss et al. Found that patients who received 250 mg / m2 of Taxol administered by means of a 24-hour infusion still had definitive HSR. Thus, while the prolongation of the infusion of drugs at 6 or 24 hours can reduce the risk of an acute reaction, this conclusion can not be confirmed, since it is 78% of the HSR reactions that occurred during the ten minutes after the initiation of the Taxol infusion, which indicates that the planned length of time for the total infusion would not have sense. In addition, the concentration of paclitaxel in the infusion may also not make a difference, since substantial numbers of patients had reactions to several small doses of Taxol. Finally, not only is the mechanism of Taxol HSR unknown, but it is also unclear if paclitaxel itself induces HSR, or if - h - ^ - -,., .. ^ ÉHfc. ^ rf ^ M, - ^ HSRs occur because of the excipient (Cremafor EL, Badische Anilin und Soda Fabrik AG (BASF), Ludwigshafen, Federal Republic of Germany). Despite the uncertainty as to whether the pre-medications had or not influence to reduce the severity or the number of HSRs, prophylactic therapy was recommended, since there is no known danger of its use. Conflicting recommendations in the prior art related to the pre-medicament should be used to avoid hypersensitivity reactions, when prolonged infusion durations are used, and the lack of effective data for infusions made over a period of six hours or taken to the use of an infusion of 24 hours of higher doses (above 170 mg / m2) of paclitaxel in an emulsion of Cremafor EL as an acceptable cancer treatment protocol. Although it seems possible to minimize the side effects of the administration of paclitaxel in an emulsion by the use of a long duration of infusion, the long duration of infusion is inconvenient for patients and is expensive due to the need to monitor patients throughout the duration of infusion from 6 to 24 hours. In addition, the long duration of infusion requires patients to spend at least one night in a hospital or treatment clinic. Higher doses of paclitaxel have also been described in the literature. To determine the maximum tolerated dose (MTD) of paclitaxel in combination with a cyclophosphamide and high-dose cisplatin followed by a support of autologous hematopoietic progenitor cells (AHPCS), Stemmer et al. ("High-dose paclitaxel, cyclosphosphamide, and cisplatin with autologous hematopoietic progenitor-cell support: A phase I trial," J. Clin. Oncol., 14: 1463-1472 (1996)) have conducted a Phase I trial in forty and nine patients with breast cancer of poor prognosis in non-Hodgkins lymphoma (NHL) or ovarian cancer with escalating doses of paclitaxel with infusions for 24 hours, followed by cyclophosphamide (5.625 mg / m2) and cisplastin (165 mg / m2). ) and in AHPCS. The dose-limiting toxicity was found in two patients at 825 mg / m2 of paclitaxel; one patient died of multiple organ failure and the other developed renal toxicity, CNS, and respiratory grade 3 which resolved. Grade 3 polyneuropathy and grade 4 CNS toxicity were also observed. The MTD of this combination was determined to be paclitaxel (775 mg / m2), cyclophosphamide (5,625 mg / m2), and cisplatin (165 mg / m2), followed by the AHPCS. Sensory polyneuropathy and mucositis were prominent toxicities, but both were reversible and tolerable. Eighteen of 33 patients (54%) with breast cancer achieved a partial response. Responses were also observed with patients in NHL (four out of five patients) and ovarian cancer (two out of two patients). US Pat. No. 5,641,803 reports the use of Taxol at doses of 175 to 135 mg / m2 administered in a 3-hour infusion. Infusion protocols require the use of premedications and report hypersensitivity reactions in 35% of patients. Neurotoxicity was reported in 51% of patients with 66% of patients experiencing neurotoxicity in the high dose group and 37% in the low dose group. In addition, it was noted that 48% of patients experienced neurotoxicity during infusion periods longer than 24 hours, while 54% of patients experienced neurotoxicity during a shorter infusion of 3 hours. There is evidence in the literature that higher doses of paclitaxel result in a higher response rate. The optimal doses and schedules for paclitaxel are still under investigation. To assess the possibility that dose intensity of paclitaxel may be important in inducing a response to disease, Reed et al., Of NCI (Reed E, Bitton R, Sarosy G, and Kohn E, "Paclitaxel dose intensity," Journal of Infusional Chemotherapy, 6: 59-63 (1996)) analyzed the data from the phase II trial available in the treatment of ovarian cancer and breast cancer. Their results suggest that the removal between the objective disease response and the dose intensity of paclitaxel in recurrent ovarian cancer is high and statistically significant with a double side p value of 0.022. The relation in breast cancer is even stronger, with a double side p value of 0.004. At 135 mg / m2 / 21 days, the objective response rate was 13.2% and at 250 mg / m2 / 21 days, the objective response rate was 35.9%. The response rate seen at the intermediate dose of 175 mg / m2 was linear with the results of 135 mg / m2 and 250 mg / m2 and the linear regression analysis shows a correlation coefficient for this data of 0.946 (Reed et al. , nineteen ninety six). In a study by Holmes et al. ("Phase II trial of Taxol, an active drug in the treatment of metastatic breast cancer," J. Nati, Cancer, Inst., 83: 1797-1805, (1991)), and MSKCC (Reichman BS et al., " Paclitaxel and recombinant human granulocyte colony-stimulating factor as initial chemotherapy for metastatic breast cancer, "J. Clin. Oncol., 11: 1943-1951 (1993)), it has been shown that the highest doses of paclitaxel, up to 250 mg / m2, produced greater responses (60%) than the 175 mg / m2 dose (26%) currently approved for paclitaxel. However, these results have not been reproduced due to the highest toxicity at these higher doses. These studies, however, create the potential increase in the rate of response to increased doses of paclitaxel. Since the pre-medication is required for Taxol, which very often requires a patient to spend the night in the hospital, it is highly desirable to develop a paclitaxel formulation that does not need a premedication. Since premedications are required for Taxol, due to HSRs associated with the administration of the drug, it is highly desirable to develop a paclitaxel formulation that do not provoke hypersensitivity reactions. It is also desirable to develop a paclitaxel formulation that does not cause neurotoxicity. Since Taxol infusions are usually preceded by premedication and require post-infusion monitoring and maintenance, which very often requires the patient to spend the night in the hospital, it is highly desirable to develop a paclitaxel formulation. which allows the recipients to be treated on an inpatient basis. Since it has been demonstrated that higher doses of Taxol achieve improved clinical responses, although with higher toxicity, it is desirable to develop a paclitaxel formulation that can achieve these doses without this toxicity. Since taxol dose-limiting toxicity has been shown to be cerebral and neurotoxic, it is desirable to develop a combination of paclitaxel that decreases such toxicity. It is also desirable to eliminate premedications as this increases the patient's discomfort and increases the cost and duration of treatment. It is also desirable to shorten the duration of infusion of paclitaxel, currently administered in 3-24 hours, to minimize the patient's stay in the hospital or clinic. Since Taxol is currently approved for administration in concentrations between 0.6-1.2 mg / ml, and a ^^^ Stój ^ - ^ ^^^^^^^^^^ g ^^^? Typical dose in humans is approximately 250-350 mg, this results in infusion volumes typically greater than 300 ml. It is desirable to reduce these infusion volumes by developing paclitaxel formulations that are stable at higher concentrations to reduce the time of administration. Since the infusion of Taxol is limited to the use of tubes and bags, or special bottles I.V. Due to the extraction of plasticizers by cremafor in the Taxol® formulation it is also desirable to develop a formulation of paclitaxel that does not have cremafor and that does not extract potentially toxic materials from the tubes or plastic bags conventionally used for intravenous infusion. Thus, it is an object of the invention to provide pharmacologically active agents (e.g., paclitaxel, taxane, taxotere, and the like) in an unmodified form in a composition that does not cause allergic reactions due to the presence of added emulsifiers and solubilizing agents, as currently employed in a drug supply. It is another object of the present invention to provide pharmacologically active agents in a microparticle or nanoparticle composition, optionally suspended in a suitable biocompatible liquid. It is still another object of the present invention to provide methods for the formation of submicron particles (nanoparticles) of pharmacologically active agents by a solvent evaporation technique of an oil in water emulsion. Some methods use proteins as stabilizing agents. Some methods are performed in the absence of any conventional surfactant agents, in the absence of any polymeric core material. These and other objects of the invention will be obvious when reviewing the specifications and claims. In accordance with the present invention, it has been discovered that pharmacologically active agents substantially insoluble in water can be supplied in the form of microparticles or nanoparticles which are suitable for parenteral administration in an aqueous suspension. This mode of delivery avoids the need to administer pharmaceutically active agents substantially insoluble in water (eg, paclitaxel) in an emulsion containing, for example, ethanol and polyethoxylated castor oil, diluted in normal saline (see for example, Norton et al., in Abstracts of the 2nd National Cancer Institute Workshop on Taxol / Taxus, September 23-24, 1992). A disadvantage of such known compositions is that they are prone to produce allergic side effects. In this way, according to the present invention, methods are provided for the formation of nanoparticles of pharmacologically active agents by a solvent evaporation technique of an oil in water emulsion prepared under a variety of conditions. For example, high speed cutting forces (e.g., sound application, high pressure homogenization, or the like) can be used in the absence of any conventional surfactant, and without the use of any polymeric core material to form the matrix of the nanoparticle. Instead, proteins (eg, human serum albumin) are used as a stabilizing agent. In an alternative method, nanoparticles can be formed without the need for high-speed shear forces, simply by selecting materials that spontaneously form microemulsions. The invention further provides a method for the reproducible formation of unusually small nanoparticles, (less than 200 nm in diameter), which can be sterile filtered through a 0.22 micron filter. This is achieved by the addition of a water-soluble solvent (eg, ethanol) to the organic phase and carefully selecting the type of organic phase, the phase fraction, and the concentration of drug in the organic phase. The ability to form nanoparticles of a size that can be filtered on 0.22 micron filters is of great importance and significance, since formulations containing a significant amount of any protein (eg, albumin), can not be sterilized by conventional means such as in autoclaves, due to the heat coagulation of the protein.

In accordance with another embodiment of the present invention, we have developed compositions useful for the in vivo delivery of pharmacologically active agents substantially insoluble in water. The compositions of the invention comprise pharmacologically active agents substantially insoluble in water (such as a solid or liquid) contained within a polymeric shell. The polymeric shell is a cross-linked biocompatible polymer. The polymeric shell containing the pharmacologically active agents, substantially insoluble in water, within it, can then be suspended in a biocompatible aqueous liquid for administration. The invention further provides a drug delivery system in which part of the pharmacologically active agent molecules bind to the protein (e.g., human serum albumin), and therefore, are immediately bioavailable when administered to the mammal. The other portion of the pharmacologically active agent is contained within nanoparticles coated by protein. The nanoparticles containing the pharmacologically active agent are present as a pure active component, without dilution by any polymer matrix. A large number of conventional pharmacologically active agents circulate in the bloodstream bound to the carrier proteins (through hydrophobic interactions or ionic), of which the most common example is serum albumin. The methods and compositions of the invention produced herein provide a pharmacologically active agent that is "pre-linked" to a protein (through hydrophobic or ionic interactions) prior to administration. The present disclosure demonstrates both the previously described modes of bioavailability for paclitaxel, an anti-cancer drug capable of binding to human serum albumin (see, for example, Kumar et al., In Research Communications in Chemical Pathology and Pharmacology; 80: 337 (1993)). The high concentration of albumin in the particles of the invention, compared to Taxol® (Bristol-Myers Squibb's paclitaxel formulation), provides a significant amount of the drug in the form of molecules bound to albumin, which is also a natural carrier of the drug in the bloodstream. In addition, it takes advantage of the ability of human serum albumin to bind to paclitaxel, as do other drugs, which improves the ability of paclitaxel to absorb on the surface of the particles. Since albumin is present in the colloidal drug particles (formed by removing the organic solvent), the formation of a colloidal dispersion which is stable for extended periods is facilitated, due to the combination of electrical repulsion and spherical stabilization.

In accordance with the present invention, submicron particles are also provided in the powder form, which can be easily reconstituted in water or saline. The powder is obtained after removing the water by lyophilization. Human serum albumin serves as the structural component of some nanoparticles of the invention, and also as cryoprotectant and reconstitution aid. The preparation of particles that can be filtered through a 0.22 micron filter according to the method of the invention as described herein after drying or lyophilization, produces a sterile solid formulation useful for intravenous injection. The invention provides, in a particular aspect, a composition of anti-cancer drugs, for example, paclitaxel in the form of nanoparticles in a liquid dispersion or as a solid which can be easily reconstituted for administration. Due to the specific properties of certain drugs, for example, paclitaxel, such compositions can not be obtained by conventional solvent evaporation methods which are based on the use of surfactants. In the presence of several surfactants, crystals of very large drugs (eg, size from about 5 microns to several hundred microns) are formed during a few minutes of storage, after the process of preparation. The size of such crystals is normally much larger than the size allowed for intravenous injection. While it is recognized that the particles produced according to the invention may already be crystalline, amorphous, or a mixture thereof, it is generally preferred that the drug be present in the formulation in an amorphous form. This will lead to greater ease of dissolution and absorption, resulting in better bioavailability. The anti-cancer agent paclitaxel has a surprising clinical activity in a number of human cancers including cancers of the ovary, breast, lung, esophagus, head and neck region, bladder and lymphomas. It has now been tested for the treatment of ovarian carcinoma, where it is used in combination with cisplatin and for metastatic breast cancer that has failed in previous treatments with a combination chemotherapy regimen. The major limitation of Taxol® is its deficient solubility and consequently the formulation in BMS contains 50% of Cremafor EL and 50% of ethanol as a solubilizing carrier. Each container of this formulation contains 30 mg of paclitaxel dissolved at a concentration of 6 mg / ml. Prior to intravenous administration, this formulation should be diluted 1:10 in saline to obtain a final dosage solution containing 0.6 mg / ml paclitaxel. This formulation has been linked to severe hypersensitivity reactions in animals (Lorenz et al., Agents G ^^^^ | g ^ gg | ^ j | ¡Actions, 7: 63-67 (1987) and in humans (Weiss et al., J. Clin. Oncol., 8: 1263-68 (1990)) and consequently requires the premedication of patients with corticosteroids (for example, dexamethasone) and antihistamines. The wide dilution results in large volumes of infusion (typical doses of 175 mg / m2 or up to 1 liter) and infusion times ranging from 3 hours to 24 hours. Thus, there is a need for an alternative less toxic formulation for paclitaxel. Capxol ™ is a novel cremofor free formulation of the anti-cancer drug paclitaxel. The inventors, based on animal studies, believe that a cremofor-free formulation will be significantly less toxic and will not require patient premedication. Premedication is necessary to reduce the hypersensitivity and anaphylaxis that occurs as a result of cremophor in the commercial and currently approved Taxol® formulation of paclitaxel. Capxol ™ is a lyophilized powder for reconstitution and intravenous administration. When reconstituted with a suitable aqueous medium such as a 0.9% injection of sodium chloride or a 5% dextrose injection, Capxol ™ forms a stable colloidal solution of paclitaxel. The size of the colloidal suspension can vary from 20 nm to 8 microns with a preferred range of approximately 20-400 nm. The two main components of Capxol ™ are unmodified paclitaxel and human serum albumin (HSA). Since HSA is freely soluble in water, Capxol ™ can be reconstitute at any desired concentration of paclitaxel, limited only by the solubility limits for the HSA. In this way Capxol ™ can be reconstituted in a wide range of concentrations ranging from diluted (0.1 mg / ml paclitaxel) to concentrated (20 mg / ml paclitaxel). This can result in rather small volumes of administration. In accordance with the present invention, useful compounds and methods are provided for the in vivo delivery of biologicals, in the form of nanoparticles which are suitable for parenteral administration in an aqueous suspension. The compositions of the invention are stabilized by means of a polymer. The polymer is a biocompatible material, as is the albumin of the protein. The use of the compositions of the invention for the supply of biologicals facilitates the need for the administration of biologicals in toxic diluents or carriers, for example, ethanol and polyethoxylated castor oil, diluted in a normal saline solution (see, for example, Norton et al., in Abstracts of the 2nd National Cancer Institute Workshop on Taxol / Taxus, September 23-24, 1992). A disadvantage of such known compositions is that they are prone to produce severe allergic side effects and other side effects. It is known that the supply of biologics in the form of a particulate suspension allows them to be directed to organs such as the liver, lungs, spleen, lymphatic circulation and the like, due to the absorption in these organs, of the particles by the reticuloendothelial system (RES ) of the cells. Directing the RES that contains organs can be controlled through the use of variable size particles, and through the administration of different routes. But, when administered to rats, it was unexpectedly and surprisingly found that Capxol ™ accumulated in tissues to those containing the RES as the prostate, pancreas, testes, seminiferous ducts, bone, etc., at a significantly higher level. greater than Taxol® at similar doses. Thus, it is very surprising that the formulation of the invention of paclitaxel, Capxol ™ a nanoparticle formulation, is concentrated in tissues such as the prostate, pancreas, testes, seminiferous ducts, bone, etc., that is, in organs that do not they contain the RES, at a significantly higher level than a non-particulate formulation of paclitaxel such as Taxol®. In this way, Capxol ™ can be used to treat cancers of these tissues with a higher efficacy than Taxol®. However, the distribution to many other tissues is similar for Capxol ™ and Taxol®. Therefore, Capxol ™ is expected to maintain anti-cancer activity at least equal to that of Taxol® in other tissues. The basis for localization within the prostate could be a result of the particle size of the formulation (20-400 nm), or the presence of protein albumin in the formation, which can cause localization within the prosthetic tissue to through specific membrane receptors (gp 60, gp18, gp 13 and similar). It is also likely that other biocompatible, biodegradable polymers other than albumin, may show specificity to certain tissues such as the prostate, resulting in a high local concentration of paclitaxel in these tissues as a result of the properties described above. Such biocompatible materials are contemplated within the scope of this invention. A preferred embodiment of a composition to achieve high local concentrations of paclitaxel in the prostate is a formulation containing paclitaxel and albumin with a particle size in the range of 20-400 nm, and free of cremophor. It has also been shown that this modality results in higher levels of paclitaxel in the pancreas, kidney, lung, heart, bone, and spleen compared to Taxol® at equivalent doses. These properties provide novel applications for this formulation of paclitaxel including methods to lower testosterone levels, achieve medical orchiectomy, and provide high local concentrations in the coronary vasculature for the treatment of restenosis. It has also been very surprisingly that paclitaxel when administered as Capxol ™ is metabolized in its metabolites and at a much slower rate than when administered as Taxol®. This represents increased anticancer activity for longer periods with similar doses of paclitaxel.

It is also very surprising when Capxol ™ and Taxol® are administered to rats at equivalent doses of paclitaxel, a much higher degree of myelosuppression results for the Taxol® group compared to the Capxol ™ group. This can result in lower incidences of infections and episodes of fever (eg, febrile neutropenia). You can also reduce the cycle time between treatments, which is currently 21 days. In this way, the use of Capxol ™ can provide a substantial advantage over Taxol®. Surprisingly it was found that the Taxol® carrier (cremophor / ethanol diluted in saline solution) alone caused a strong myelosuppression and caused severe hypersensitivity reactions and death in several groups of dosed mice. No such reactions were observed in the groups with Capxol ™ at equivalent or higher doses. Thus, Capxol ™, a formulation of paclitaxel that is free of Taxol® carrier is substantially advantageous. It is also very surprising that when Capxol ™ and Taxol® were administered to rats at equivalent doses of paclitaxel, a much lower toxicity is seen for Capxol ™ as compared to Taxol® as evidenced by the significantly higher LD50 values. . This may allow higher therapeutically effective doses of paclitaxel to be administered to patients. There is evidence in the literature that shows the increased rates of dose response more high of paclitaxel. The Capxol ™ formulation can allow the administration of these higher doses due to lower toxicity and therefore exploit the full potential of this drug. It is also surprising that Capxol ™, a substantially water-insoluble drug formulation, paclitaxel, is stable when reconstituted in an aqueous medium at various different concentrations ranging from, but not limited to, 0.1-20 mg / ml. This offers a substantial advantage over Taxol® during drug administration, as it results in smaller infusion volumes, resolves the known instability issues for Taxol® (such as precipitation), and avoids the use of a filter in line in the infusion line. In this way, Capxol ™ greatly simplifies and improves the administration of paclitaxel to patients. It is also surprising that Capxol ™ when administered to rats at equivalent doses of paclitaxel as Taxol® shows no signs of neurotoxicity while Taxol® at a low dose shows neurotoxic effects. The formulation of the invention further allows the administration of paclitaxel, and other pharmacologically active agents substantially insoluble in water, employing a much smaller volume of liquid and requiring greatly reduced administration time in relation to the volumes of administration and the times required by the prior art supply systems.

In combination with a biocompatible polymer matrix, the inventive formulation (Capxol ™) enables local sustained delivery of paclitaxel with lower toxicity and longer activity. The surprising encounters mentioned above for Capxol ™ offer the potential to substantially improve the quality of life of patients receiving paclitaxel. Capxol ™ is a lyophilized powder that contains only paclitaxel and human serum albumin. Due to the nature of the colloidal solution formed during the reconstitution of toxic emulsifiers of lyophilized powder such as cremophor (in the BMS formulation of paclitaxel), or polysorbate 80 (as used in the docetaxel formulation of Rhone Poulenc) and solvents such as ethanol to solubilize the drug are not required. The removal of toxic emulsifiers will reduce the incidence of severe hypersensitivity reactions and anaphylactic reactions that are known to occur in TAXOL products. In addition, no pre-medication with steroids and antihistamines is anticipated before administration of the drug. Due to the reduced toxicities, as evidenced by the LD10 / LD50 studies, a higher dose can be employed for greater efficacy.

The reduction in myelosuppression (compared to the BMS formulation) is expected to reduce the period of treatment cycle (currently 3 weeks) and improve treatment outcomes. The Capxol ™ can be administered at much higher concentrations (up to 20 mg / ml) compared to the BMS (0.6 mg / ml) formulation, allowing infusions much lower volume and with an intravenous bolus administration. Taxol® has the additional disadvantage that it can be delivered by infusion only with nitroglycerin polyolefin infusion sets due to the removal of plasticizers from standard infusion tubes within the formulation. Capxol ™ does not show extraction and can be used with any standard infusion tube. In addition, only polyolefin or glass containers should be used to store all solutions containing cremophor. The formulation of Capxol ™ has no such limitations. Another problem recognized with Taxol® is the precipitation of paclitaxel in internal catheters. This results in erratic and poorly controlled dosing. Due to the inherent stability of the colloidal solution of the new formulation, Capxol®, the problem of precipitation is solved. Due to this precipitation problem, the administration of Taxol® requires the use of online filters to - ^ - - - * - - '- ^^^^^ - • "> *" • - .. - «^ tttfJE». remove precipitates and other particulate matter. Capxol ™ has no such requirement due to its inherent stability. The literature suggests that particles in the size range of below one hundred nanometers, are preferably divided into tumors through the permeable blood vessels at the tumor site. Colloidal particles of paclitaxel in the formulation Capxol ™ therefore can show an effect of preferential direction, greatly reducing the side effects of paclitaxel administered in the formulation of Taxol®. Therefore, it is a primary object of the present invention to provide a new paclitaxel formulation that provides the aforementioned desirable characteristics. It is another object of the present invention to provide a new formulation of paclitaxel that localizes paclitaxel in certain tissues, thereby providing a higher anticancer activity in these places. It is another object of the invention to administer paclitaxel at concentrations greater than about 2 mg / ml in order to reduce the infusion volumes. It is another object of the present invention to provide a formulation of paclitaxel that is free of the Taxol® carrier. ugly-, .z ^ - j & ie &áékiSietQ: It is yet another object of the invention to provide a formulation of paclitaxel that improves the quality of life of patients receiving paclitaxel for the treatment of cancer.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents the results of intravenous administration of paclitaxel nanoparticles to mice with tumors (n = 5 in each group), showing a complete regression of the tumor in the treatment group (), compared to the control group that received saline (•). The virtually uncontrolled growth of the tumor can be seen in the control group. The dose for the treatment group is 20 mg / kg of paclitaxel administered as an intravenous bolus for five consecutive days. Figure 2 presents the results of an intraperitoneal administration of paclitaxel nanoparticles in rats that have developed arthritis in their legs after an intradermal injection of collagen. The volumes of the legs were measured and indicated the severity of the disease. The volumes of the legs were normalized to 100% at the beginning of the treatment. Day 0 represents the initiation of treatment. There are 3 groups - control group that received saline solution (n = 2, shown as a thin line and labeled in the figure as "without treatment"); a first treatment group received paclitaxel nanoparticles in a dose of 1 mg / kg (n = 4, showed as a thick line and labeled in the figure as "paclitaxel nanoparticles") 1. 0 mg / kg "), and a second treatment group that received a combination therapy of paclitaxel nanoparticles at a dose of 0.5 mg / kg and prednisone at a dose of 0.2 mg / kg (n = 4, shown as a line thick and labeled in the figure as "prednisone 0.2 mg / kg + paclitaxel nanoparticles 0.5 mg / kg"). The two treatment groups showed a dramatic reduction in the volume of the legs over time, indicating a regression of arthritis, while that the control group showed an increase in the volume of the legs during the same period.Figure 3 presents the results of a study of myelosuppression in rats.A first group of 3 rats was supplied with Capxol ™, and a second group of 3 rats were supplied with Taxol®, both at a dose of 7 ml of a formulation per kg of body weight, where each formulation contained paclitaxel at 5 mg / kg.All doses were delivered by IV bolus through the tail vein. Figure 3 shows the% of the change in the white blood cell count (WBC) as an indicator of myelosuppression. Figure 4 presents a pilot study with Capxol ™ to determine the ranges and efficacy of targeted doses. Mice (n = 10) were implanted subcutaneously with breast tumor MX-1 and treatment was initiated when the tumor reached approximately 150-300 mg in size. This happened on day 12 and the treatment started on day 13 after the initial culture. Capxol ™ was reconstituted in saline to obtain a colloidal solution of paclitaxel nanoparticles. Tumor bearing mice (n = 5) were treated with reconstituted Capxol ™ at a dose of 20 mg / kg (denoted by VIV-1), delivered by a tail vein bolus injection each day for five consecutive days . The control control group, (n = 5) received only one saline solution at the same time. The size of the tumors was monitored as a function of time. The control group showed a tremendous increase in tumor weight at an average of more than 4500 mg and all animals in this group were sacrificed between day 28 and day 39. The treatment group on the other hand showed surprising efficacy and all the animals did not have measurable tumors when they arrived at day 25. The animals in that group were all slaughtered on day 39 in which they showed no evidence of recurrence or evidence of tumor. In accordance with the present invention, methods are provided for reducing the toxicity of paclitaxel in a subject undergoing treatment with paclitaxel, such method comprising systematically administering such paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 175 mg / m2. during a period of administration not greater than two hours.

In accordance with the present invention, a method for administering paclitaxel to a subject in need thereof is also provided, without the need for pre-medication prior to the administration of paclitaxel, such method comprises systematically administering such paclitaxel to such subject in a pharmaceutically acceptable formulation at a dose of at least 135 mg / m2 for a period of administration of not more than 2 hours. According to yet another alternative embodiment of the present invention, there is provided a method for administering paclitaxel to a subject in need thereof, such method comprising systematically administering such paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least mg / m2 during a period of administration of no more than 2 hours, with a treatment cycle of less than 3 weeks. According to yet another embodiment of the present invention there is provided a method for the administration of paclitaxel to a subject in need, such method comprises systematically administering paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 250 mg / m2. According to yet another embodiment of the present invention, there is provided a method for administering paclitaxel to a subject in need, such method comprises systematically administering paclitaxel to a subject in the formulation that can be safely administered using medical equipment made of materials that contain components that can be extracted. According to yet another embodiment of the present invention, there is provided a method for administering paclitaxel to a subject in need thereof, such method comprising systematically administering paclitaxel to a subject in a formulation that can be administered safely without the use of an online filter. According to another embodiment of the present invention, there is provided a method for administering paclitaxel to a subject in need thereof, such method comprising systematically administering a full dose of paclitaxel to a subject in a volume of less than 250 ml. According to another embodiment of the present invention, there is provided a method for administering paclitaxel to a subject in need thereof, such method comprising systematically administering paclitaxel to a subject at a rate of at least 50 mg / m2 / hour. According to yet another embodiment of the present invention, a formulation of paclitaxel having reduced haematological toxicity is provided to a subject undergoing treatment with paclitaxel, the formulation comprising paclitaxel in a pharmaceutically acceptable formulation suitable for systemic delivery at a dose of at least 175 mg / m2 during a period of administration of not more than 2 hours. According to yet another embodiment of the present invention, a formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof is provided, in need of premedication prior to administration of such paclitaxel, the formulation comprises paclitaxel in a pharmaceutically acceptable formulation suitable for systemic administration at a dose of at least 135 mg / m2 for a period of administration of not more than 2 hours. According to yet another embodiment of the present invention, a formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof is provided with a treatment cycle of less than 3 weeks, the formulation comprising paclitaxel in a suitable pharmaceutically acceptable formulation. for systemic administration at a dose of at least 135 mg / m3, for a period of administration of no more than 2 hours. According to yet another embodiment of the present invention there is provided a formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof, such formulation comprising paclitaxel in a pharmaceutically acceptable formulation free of cremafor. According to another embodiment of the present invention, a lyophilized formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof after reconstitution is provided. According to another embodiment of the present invention, a frozen formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof after thawing is provided. In accordance with another embodiment of the present invention, a liquid formulation of paclitaxel comprising water and paclitaxel at a concentration of at least 2.0 mg / ml is provided. According to another embodiment of the present invention, there is provided a drug delivery system comprising particles of a solid or liquid, a pharmacologically active agent substantially insoluble in water, coated with a protein, wherein the protein coating has free protein associated therewith, wherein a portion of such pharmacologically active agent is contained within the protein coating and a portion of such pharmacologically active agent is associated with the free protein, and wherein the average diameter of the particles is not greater than about 1 miera. According to yet another embodiment of the present invention, a drug formulation suitable for the administration of drug to a subject in need thereof is provided by inhalation, such formulation comprises protein microparticles having a size of about 1-10 μm, wherein the protein microparticles comprise drug nanoparticles having a size of about 50-1000 nm, more optionally an excipient. In accordance with the present invention, methods are also provided for the preparation of pharmacologically active agents substantially insoluble in water for in vivo delivery, the method comprising: a) combining i) an organic solvent having an active solvent dissolved therein; ii) an aqueous or water solution; iii) a surfactant; and iv) a co-surfactant agent that spontaneously forms a microemulsion; and b) removing such organic solvent to produce a suspension of nanoparticles of the active agent in the water. According to yet another embodiment of the present invention, there is provided a method for making nanoparticles containing an active agent, the method comprising combining a non-volatile phase, a volatile phase, and a surfactant which spontaneously forms a microemulsion, wherein the volatile phase contains the active agent, and remove the volatile phase and with g a-aja-te This allows obtaining a suspension of solid nanoparticles in the non-volatile phase, where the nanoparticles contain the active agent and have an average diameter of less than 100 nm. According to another embodiment of the present invention there is provided a method for making nanoparticles containing an active agent, the method comprising combining a non-volatile phase and a volatile phase that spontaneously form a microemulsion, wherein the non-volatile phase contains the active agent , and remove the non-volatile phase and thereby obtain solid nanoparticles in the volatile phase, where the nanoparticles contain the active agent and have an average diameter of less than 100 nm. The compositions produced by the methods described above are particularly advantageous since they have been found to provide a very low form of toxicity of a variety of pharmacologically active agents. Also described herein are other methods for making low toxicity forms of pharmacologically active agents, for example, paclitaxel. In a preferred embodiment, the average diameter of the particles described above is not greater than about 200 nm. Such particles are particularly advantageous since they can be subjected to sterile filtration, thereby avoiding the need for more vigorous treatment to achieve sterilization of solutions containing the desired pharmacologically active agent. As used herein, unless otherwise specified, the term "paclitaxel" includes all forms, modifications and derivatives of paclitaxel, eg, taxotere, and the like. Capxol ™ is a registered trademark for the formulation of paclitaxel to be marketed by the proxies of the applicant. As used herein, Capxol ™ is simply a short means to refer to paclitaxel nanoparticles coated with proteins produced by the method of Example 1. Capxol ™ is a cream-free formulation with a new owner of the anti-cancer drug paclitaxel. The inventors, based on animal studies, believe that a cream-free formulation will be significantly less toxic and will not require pre-medication of patients. Pre-medication is necessary to reduce the hypersensitivity and anaphylaxis that occurs as a result of cremafor in commercially available and currently approved paclitaxel Taxol® formulations. Capxol ™ is a lyophilized powder that is reconstituted and administered intravenously. Each Capxol ™ container contains 30 mg paclitaxel of approximately 400 mg human serum albumin. When reconstituted with a suitable aqueous medium such as a 0.9% injection of sodium chloride or a 5% dextrose injection, Capxol ™ forms a stable colloidal solution of paclitaxel. The size of the colloidal nanoparticles is typically less than 400 nm. The nanoparticles are prepared by high pressure homogenization of a solution of human serum albumin USP and a solution of paclitaxel in an organic solvent. The solvent is then removed to generate the colloidal suspension or paclitaxel solution in human albumin. This suspension is sterile filtered and lyophilized to obtain Capxol ™. The formulation does not contain any other added excipient or stabilizer. The sterility of the product is ensured by an aseptic manufacturing process and / or by sterile filtration. The two major components of Capxol ™ are unmodified paclitaxel and human serum albumin (HSA). Since HSA is freely soluble in water, Capxol ™ can be reconstituted at any desired concentration of paclitaxel limited only by the solubility limits of HSA. In this way, Capxol ™ can be reconstituted in a wide range of concentrations ranging from diluted (0.1 mg / ml paclitaxel) to concentrated (20 mg / ml paclitaxel). This can result in rather small volumes of administration. As used herein, the term "in vivo delivery" refers to the delivery of a pharmacologically active agent through those routes of administration such as oral, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, inhalation, topical, transdermal, suppository (rectal) , pessary (vaginal), as ^ MfesafeM, tife »^ á | BßjH§ g ^^ í intrauretal, intraportal, intrahepatic, intra-arterial, intrahumoral, and the like. As used herein, the term, "miera" refers to a unit of measurement of one thousandth of a millimeter. As used herein, the term "biocompatible" describes a substance that does not appreciably affect, in any adverse way, the biological system within which it is introduced. The pharmacologically active agents substantially insoluble in water contemplated for the use of the practice of the present invention include pharmaceutically active agents, diagnostic agents, nutritional value agents and the like. Examples of pharmaceutically active agents include: analgesics / antipyretics (e.g., aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine hydrochloride, propoxyphene hydrochloride, propoxyphene napsylate, meperidine hydrochloride, hydromorphone hydrochloride, morphine sulfate, hydrochloride oxycodone, codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride, hydrocodone bitartrate, levorphanol tartrate, diflunisal, trolamine salicylate, nalbuphine hydrochloride, mefenamic acid, butorphanol tartrate, choline salicylate, butabital, phenyltoloxamine, citrate of diphenhydramine, methotrimeprazine, cinamedrin hydrochloride, meprobamate, and the like: V A ~ anesthetics (eg, cyclopropane, enflurane, halothane, soflurane, methoxyflurane, nitrous oxide, propofol, and the like); antiasthmatics (eg Azelastine, Ketotifen, Traxanox, Amlexanox, Cromolin, Ibudilast, Montelukast, Nedocromil, Oxatomide, Pranlukast, Seratrodast, Suplastate tosylate, Tiaramide, Zafirlukast, Zileuton, Beclomethasone, Budesonide, Dexamethasone, Flunisolide, Trimcinolone acetonide and the like, antibiotics (e.g., neomycin, streptomycin, chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline and the like; antidepressants (e.g., nefopam, oxypertin, doxepin hydrochloride, amoxapine, trazodone hydrochloride, amitriptyline hydrochloride, maprotiline, phenelzine sulfate, desipramine hydrochloride, nortriptyline hydrochloride, tranylcypromine sulfate, fluoxetine hydrochloride, doxepin hydrochloride, impiramine hydrochloride, impramine pamoate, nortriptyline, amitriptyline hydrochloride, isocarboxazide, desipramine hydrochloride, trimipramine maleate , protriptyline hydrochloride and the like. r example, biguanides, hormones, sulfonylurea derivatives, and the like. antifungal agents (for example, griseofulvin, keloconazole, amphotericin B, Nystatin, candicidin, and the like); antihypertensive agents (e.g., propanolol, propafenone, oxyprenolol, Nifedipine, reserpine, trimetafan camsylate, phenoxybenzamine hydrochloride, pargyline hydrochloride, deserpidine, diazoxide, guanethidine monosulphate, monoxidil, rescinamine, sodium nitroprusside, rauwolfia serpentine, alseroxyl, fentolamino mesylate, reserpine, and the like, anti-inflammatory (e.g., (non-spheroidal) ndometacin, naproxen, ibuprofen, ramifenazone, piroxicam cortisone (steroidal), dexamethasone, fluazacort, hydrocortisone, prednisolone, prednisone, and the like); antineoplastic; (for example, adriamycin, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and its derivatives, phenesterin, paclitaxel and its derivatives, taxotere and its derivatives, vinblastine, vincristine, tamoxifen, etoposide, piposulfan, and similar); antianxiety agents (eg, lorazepam, buspirone hydrochloride, prazepam, chlordiazepoxide hydrochloride, oxazepam, clorazepate dipotassium, diazepam, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, chlormezanone, dantrolen and the like); immunosuppressive agents (e.g., cyclosporin, azatrioprine, mizoribin, FK506 (tacrolimus), and the like); antimigraine agents (for example, ergotamine tartrate, propanolol hydrochloride, isometehptene mucate, dicloralphenazone, and the like); hypnotic sedatives (e.g., barbiturates (e.g., pentobarbital, sodium pentobarbital, secobarbital sodium), benzodiazapine (e.g., flurazepam hydrochloride, triazolam, tomazeparm, midazolam hydrochloride, and the like), antianginal agents (e.g., beta-blockers) adrenergic, calcium channel blockers (e.g., nifedipine, diltiazem hydrochloride, and the like) nitrates (e.g., nitroglycerin, isosorbide dinitrate, pentaerythritol tetranitrate, erythrityl tetranitrate), and the like); antipsychotic agents (e.g., haloperidol) , loxapine succinate, loxapine hydrochloride, thioridazine, thioridazine hydrochloride, thiothixene, fluphenazine hydrochloride, fluphenazine decanoate, fluphenazine enanthate, trifluoperazine hydrochloride, chlorpromazine hydrochloride, perphenazine, lithium citrate, prochlorperazine, and the like; antimalarial agents; (for example, lithium carbonate); antiarrhythmics (for example, broth tosylate) ethyl, esmolol hydrochloride verapamil hydrochloride, amiodarone, encainide hydrochloride, digoxin, digitoxin, mexiletine hydrochloride, disopyramide phosphate, procainamide hydrochloride, quinidine sulfate, quinidine gluconate, quinidine polygalacturonate, flecainide acetate, tocainide hydrochloride , lidocaine hydrochloride and similar antiarthritic agents; (e.g., phenylbutazone, sulindac, penicillamine, salsalate, piroxicam, azathioprine, indomethacin, sodium meclofenamate, gold sodium thiomalate, ketoprofen, auranofin, aurothioglucose, sodium tolmetin, and the like) antigout agents (e.g., colchicine, allopurinol , and the like), anticoagulants (e.g., heparin, sodium heparin, sodium warfarin, and the like), thrombolytic agents (e.g., urokinase, streptokinase, altoplases, and the like; antifibrinolytic agents (e.g., aminocaproic acid); hemoreológicos (for example, pentoxifilina), antiplatelet agents for example, aspirin, empirina, ascriptina, and similars); anticonvulsants (eg, valproic acid, sodium divalproex, phenytoin, sodium phenytoin, clonazepam, primidone, phenobarbitol, sodium phenobarbitol, carbamazepine, sodium amobarbital, methsuximide, metarbital, mephobarbital, mephenytoin, phensuximide, parametadione, ethotoin, phenacemide, sodium secobarbitol, clorazepate dipotassium, trimethadione, and the like); antiparkinson agents (e.g., ethosuximide and the like); antihestamines / antipruritics, (for example, hydroxyzine hydrochloride, diphenhydramine hydrochloride, chlorpheniramine maleate, brompheniramine maleate, ciproheptadine hydrochloride, terfenadine, clemastine fumarate, triprolidine hydrochloride, carbinoxamine maleate, dihydrate hydrochloride ina, phenindamine tartrate, azatadine maleate, tripelennamine hydrochloride, dexchlorpheniramine maleate, methylazine hydrochloride, trimprazine tartrate, and the like), useful agents in the regulation of calcium (eg, calcitonin, parathyroid hormone, and the like); antibacterial agents (e.g., amikacin sulfate, aztreonam, chloramphenicol, chloramphenicol palmitate, sodium chloramphenicol succinate, ciprofloxacin hydrochloride, clindamycin hydrochloride, clindamycin palmitate, clindamycin phosphate, metronidazole, metronidazole hydrochloride, gentamicin sulfate, hydrochloride lincomycin, sulfate tobramycin, vancomycin hydrochloride, polymyxin B sulfate, colistimethate sodium, colistin sulfate and the like): antiviral agents; (for example, gamma interferon, zidovudine, amantadine hydrochloride, ribavirin, acyclovir, and the like) r,, - antimicrobial (for example, cephalosporins (eg cefazolin sodium, cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium, cefoperazone sodium, cefotetan disodium, cefotoxime azothil, cefotaxime sodium, cefadroxil monohydrate, ceftazidime, cephalexin, cephalothin sodium, hydrochloride of cephalexin monohydrate, cefamandole naphtha, sodium cefoxitin, sodium cefonicide, ceforanide, sodium ceftriaxone, ceftazidime, cefadroxil, cephradine, sodium cefuroxime, and the like), penicillins (for example, ampicillin, amoxicillin, benzathine penicillin G, cyclacillin, sodium ampicillin, penicillin G potassium, penicillin V potassium, sodium piperacillin, sodium oxacillin, bacampicillin hydrochloride, sodium cloxacillin, disodium ticarcilin, sodium azlocillin, sodium indanyl carbenicilin, penicillin G potassium, procaine penicillin G, sodium methicillin, sodium nafcillin, and the like), erythromycins (e.g., erythromycin ethyl succinate, erythromycin, erythromylalate) icine, erythromycin lactobionate, erythromycin sesate, erythromycin ethyleuccinate, and the like, tetracyclines (e.g., tetracycline hydrochloride, doxycycline hyclate, minocycline hydrochloride, and the like), and the like; anti-infectives; (e.g., GM-CSF) bronchodilators (e.g., sympathomimetics (e.g., epinephrine hydrochloride, metaproterenol sulfate, terbutaline sulfate, isoetharine, isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate, albuterol, bitolterol, hydrochloride ~ ¿ k *? & mesylate isoproterenol, terbutaline sulfate, epinephrine bitartrate, metaproterenol sulfate, epinephrine, epinephrine bitartrate), anticholinergic agents (eg, pratropium bromide), xanthines (eg, aminophylline, difillin, metaproterenol sulfate, aminophylline), mast cell stabilizers; (for example sodium cromolyn, inalation corticosteroids (eg, flurisolidebeclomethasone dipropionate, beclomethasone dipropionate monohydrate), salbutamol, beclomethasone dipropionate (BDP), ipratropium bromide, budesonide, ketotifen, salmeterol, xinafoate, terbutaline sulfate, triamcinolone, theophylline, nedocromil sodium, metaproterenol sulfate, albuterol, solid silicon, and the like); hormones (eg, androgens (eg, danazol, testosterone cypionate, fluoxymesterone, -ethyl-testosterone, testosterone-enrichate, methyltestosterone, fluoxymesterone, testosterone cypionate), estrogens (eg, estradiol, estropipate, conjugated estrogens), progestins (eg, methoxyprogesterone acetate, norethindrone acetate), corticosteroids (eg, triamcinolone, betamethasone, sodium betamethasone phosphate, dexamethasone, dexamethasone sodium phosphate, dexamethasone acetate, prednisone, methylprednisolone acetate suspension, triamcinolone acetonide, methylprednisolone, sodium prednisolone phosphate, sodium methylprednisolone succinate, succinate sodium hydrocortisone, sodium methylprednisolone succinate, triamcinolone hexacatonide, hydrocortisone, hydrocortisone cipionate, prednisolone, fluorocortisone acetate, parametasone acetate, prednisolone tebulate, prednisolone acetate, sodium prednisolone phosphate, sodium hydrocortisone succinate, and the like); thyroid hormones (eg, sodium levothyroxine) and the like), and the like, hypoglycemic agents, eg, human insulin, purified insulin, purified pork insulin, glyburide, chlorpropamide, glipizide, tolbutamide, tolazamide, and the like) hypolipidemic agents , (for example, clofibrate, sodium dextrothyroxine, probucol, lovastatin, niacin, and the like) proteins (e.g., DNase, alginase, superoxide dismutase, lipase, and the like); nucleic acid (eg, sense or anti-sense nucleic acids that modify any therapeutically useful protein, including any of the proteins described herein, and the like; agents useful for the stimulation of erythropoiesis (e.g., erythropoietin); antiulcer agents; / anti-reflux. (eg, famotidine, cimetidine, ranitidine hydrochloride, and the like); anti-nauseating / antivominating (eg, meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate, promethazine hydrochloride, thiethylperazine, scopolamine, and the like); ., ^ J-liposoluble vitamins (e.g., vitamins A, D, E, K, and the like); and other drugs such as mitotane, visadin, halonitrosoureas, antrocyclines, elipcin, and the like. Examples of diagnostic agents contemplated for use in the practice of the present invention include ultrasound contrast agents, radiocontrast agents (e.g., iodo-octanes, halocarbons, renografin, and the like), magnetic contrast agents (e.g., fluorocarbons) , lipid-soluble paramagnetic compounds, and the like) as well as other diagnostic agents which can not readily be delivered without some physical and / or chemical modification to accommodate the substantially water-insoluble nature thereof. Examples of agents of nutritional value contemplated by the use in practice of the present invention include amino acids, sugars, proteins, carbohydrates, fat-soluble vitamins (eg, vitamins A, D, E, K and the like), fats, or combinations of any of two or more of them.

A. FORMATION OF NANOPARTICLES USING HIGH-SPEED HOMOGENIZATION The key differences between the pharmacologically active agents contained in a polymeric shell BL. £. according to the invention and the protein microspheres of the prior art are in the nature of the formation and the final state of the protein after the formation of the particle, and its ability to poorly transport agent substantially insoluble in water or soluble in water In accordance with the present invention, the polymer (eg, a protein) can be crossed as a result of exposure to high-speed cutting conditions in a high-pressure homogenizer. The high cutting speed is used to disperse a dispersing agent containing a pharmacologically active agent suspended or dissolved within an aqueous solution of a biocompatible polymer, optionally cutting sulfhydpide or disulfide groups (e.g., albumin) wherein a cross-linked polymer coating is formed around fine drops of a medium not watery. The high-speed cutting conditions produce cavitation in the liquid that causes tremendous local heating and results in the formation of superoxide ions that are capable of cross-linking the polymer, for example, by oxidizing the sulfhydryl residues (and / or by interrupting the disulfide bonds existing) to form new crossed disulfide bonds. In comparison with the process of the invention, the prior art method of crossing glutaraldehyde is non-specific and essentially reactive with any nucleophilic group present in the protein structure (e.g., amines and hydroxyl). The denaturation by heat as taught by the prior art significantly and irreversibly alter the structure of the protein. In comparison, a disulfide formation contemplated by the present invention does not substantially denature the protein. In addition, the particles of pharmacologically active agents substantially insoluble in water, contained within a shell differ from the heat denatured or crosslinked protein microspheres of the prior art because the polymeric shell produced by the process of the invention is relatively thin compared to the diameter of the coated particle. It has been determined (by a transmission electron microscopy) the "cover thickness" of the polymeric coating is approximately nanometers for a coated particle having a diameter of 1 miera (1000 nanometers). In contrast, the microspheres of the prior art do not have protein coatings, but have protein dispersed throughout the volume of the microsphere. Thus, according to the present invention, a pharmacologically active agent is dissolved in a solvent Suitable (for example, chloroform, methylene chloride, ethyl acetate, ethanol, tetrahydrofuran, dioxane, butanol, butyl acetate, acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide, methylpyrrolidinone, or the like, as mixtures of any of two or more of them). Additional solvents contemplated for use in the practice of the present invention include soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, sesame oil, orange oil, limonene oil, C1-C20 alcohols, C2-C20 esters, C3-C20 ketones, polyethylene glycols, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons and combinations thereof. Unlike conventional methods for the formation of nanoparticles, a polymer (for example, a polylactic acid) does not dissolve in the solvent. The oily part used in the preparation of the compositions of the invention typically contains only the pharmacologically active agent dissolved in solvent. Then, a protein (e.g., human serum albumin) is added (within the aqueous phase) to act as a stabilizing agent for the formation of stable nanobeams. The protein is added at a concentration in the range of approximately 0.05 to 25% (w / v), more preferably in the range of about 0.5% -5% (w / v). Unlike conventional methods for the formation of nanoparticles, no surfactant (eg, sodium lauryl sulfate, lecithin, Tween 80, pluronic F-68, or the like) is added to the mixture. Next, an emulsion is formed by homogenization at high pressure and cutting forces at high speed. Such homogenization is conveniently carried out in a high pressure homogenizer, typically operated at pressures in the range of 3,000 to 60,000 psi. Preferably, such processes are carried out at pressures in the range of about 6,000 to 40,000 psi. The resulting emulsion comprises very small nanogotes of the non-aqueous solvent (containing the dissolved pharmacologically active agent) and very small nanogotes of the protein stabilizing agent. Acceptable methods of homogenization include processes that transmit high speed cutting and cavitation such as high pressure homogenization, high speed shear mixers, sound formation, high speed shear boosters, and the like. Finally, the solvent is evaporated under reduced pressure to produce a colloidal system composed of nanoparticles coated with protein of the pharmacologically active agent and protein. Acceptable methods for evaporation include the use of rotary evaporators, falling film evaporators, spray dryers, freeze dryers and the like. Ultrafiltration can also be used for solvent removal. After evaporation of the solvent, the liquid suspension can be dried to obtain a powder containing the pharmacologically active agent and the protein. The resulting powder can be redispersed at any convenient time within a suitable aqueous medium such as a saline solution, buffered saline, water, a buffered aqueous medium, > - * - amino acid solutions, vitamin solutions, carbohydrate solutions, or the like, as well as combinations of any two or more thereof, to obtain a suspension that can be administered to mammals. The methods contemplated for obtaining this powder include freeze drying, spray drying, and the like. According to another embodiment of the present invention, an alternative method is provided for the formation of unusually small submicron particles (nanoparticles), ie, particles that are less than 200 nanometers in diameter. Such particles are capable of being sterile filtered before being used in the form of a liquid suspension. The ability to sterilely filter the final product of the formulation processes of the invention, (i.e., drug particles) is of great importance since it is impossible to sterilize dispersions containing high concentrations of protein (e.g., serum albumin) by means of conventional means such as autoclave. In order to obtain sterile filterable particles (i.e. particles <200nm), the pharmacologically active agent is initially dissolved in an organic solvent substantially invisible in water (eg, a solvent having less than about 5% water solubility, as for example, chloroform) at a high concentration, thereby forming an oil phase containing the pharmacologically active agent. Suitable solvents are mentioned above. Unlike conventional methods for the formation of nanoparticles, a polymer (for example polylactic acid) does not dissolve in the solvent. The oil phase used in the process of the present invention contains only the pharmacologically active agent dissolved in solvent. Then, a water-miscible organic solvent (eg, a solvent that is greater than about 10% in water solubility, such as for example, ethanol) is added in the oil phase to a final concentration in the range of about 1% - 99% v / v, more preferably in the range of about 5% - 25% v / v, of the total organic phase. The water-miscible organic solvent can be selected from such solvents as ethyl, acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, butanol, acetone, propylene glycol, glycerol, dimethyl sulfoxide, dimethyl formamide, methyl pyrrolidinone and the like. Alternatively, the water-immiscible solvent mixture with the water-miscible solvent is prepared first, after dissolution of the pharmaceutically active agent in the mixture. Next, human serum albumin or any other suitable stabilizing agent as described above is dissolved in an aqueous medium. This component acts as a stabilizing agent for the formation of stable nanogotes. Optionally, a sufficient quantity of the first solvent Organic (for example, chloroform) dissolves in the aqueous phase to bring it closer to the saturation concentration. A separate measured amount of the organic phase (which now contains the pharmacologically active agent, the first solvent and the second organic solvent) is added to the saturated aqueous phase, so that the phase fraction of the organic phase is between about 0.5% - 15% v / v, and more preferably between 1% and 8% v / v. Then, a mixture composed of micro and nanogota is formed by homogenization at low speed cutting forces. This can be achieved in a variety of ways, as can be easily identified by those skilled in the art, using, for example, a conventional laboratory homogenizer operated in the range of about 2,000 to about 15,000 rpm. It is then homogenized under high pressure (i.e. in the range of about 3,000 to 60,000 psi). The resulting mixture comprises an aqueous protein solution (e.g., human serum albumin), the pharmacologically active agent and soluble in water, the first solvent and the second solvent. Finally, the solvent is rapidly evaporated under vacuum to produce a colloidal dispersion system (pharmacologically active agent and proteins) in the form of extremely small nanoparticles (ie particles in the range of about 10nm - 200nm in diameters) can be filtered and sterilized . The preferred size range of the particles are between approximately 50 nm - xjue? A *. -,, * ** -. * ** 170 nm, depending on the formulation and operating parameters. The colloidal systems prepared according to the present invention can be additionally converted into powder by removal of water therefrom, for example, by lyophilization or spray drying to a suitable time-temperature profile. The protein itself (e.g., human serum albumin) acts as a cryoprotectant or lyoprotectant, and the powder is easily reconstituted by adding water, saline or a buffer, without the need to use conventional cryoprotectants such as mannitol, sucrose, glycine, and similar, while they are not required, of course it is understood that conventional cryoprotectants can be added to the formulations of the invention if so desired. The colloidal system of the pharmacologically active agent allows the delivery of high doses of pharmacologically active agent in relatively small volumes. This minimizes the patient's discomfort in receiving large volumes of fluid and minimizes hospital stay. In addition, the walls of the cover or polymeric coating are generally and completely degradable in vivo by proteolytic enzymes (for example, when the polymer is a protein), resulting in substantially no side effects of the delivery system, which is in direct contact with the significant side effects caused by the above formulations. A number of biocompatible polymers can be employed in the practice of the present invention for the formation of the polymeric shell which surrounds the pharmacologically active agent substantially insoluble in water. Essentially any polymer, natural or synthetic, optionally carrying sulfhydryl groups or disulfide bonds within its structure, can be used for the preparation of a cross-linked disulfide coating around particles of pharmacologically active agents substantially soluble in water. Sulfhydryl groups or sulfide bonds may be pre-existing within the polymer structure or may be introduced by suitable chemical modification. For example, natural polymers, proteins, peptides, polynucleic acids, polysaccharides (eg, starch, cellulose, dextrans, alginates, chitosan, peptide, hyaluronic acid and the like), proteoglycans, lipoproteins, etc., are candidates for modification. The proteins contemplated for use as stabilizing agents according to the present invention include albumins (which contain cysteine residues), and immunoglobulins, caseins, insulins (which contain 6 cysteines), hemoglobins (which contain 6 cysteine residues). per unit a2ß2) Msosomes (which contain 8 cysteine residues), immunoglobulins, alpha-2-macroglobulin, flbronectins, vitronectins, fibrinogens, lipases, and the like. The proteins, peptides, enzymes, antibodies, and any combination thereof, are general classes of stabilizers contemplated for use in the present invention. A currently preferred protein for use as a stabilizing agent is albumin. Optionally, proteins such as alpha-2-macroglubulin, a known opsonin, can also be used to improve the absorption of particles surrounded by the coating of pharmacologically active agents substantially insoluble in water by macrophage-like cells, or to improve the absorption of the particles surrounded by the cover inside the liver or spleen. Antibodies Specific ones can also be used to direct the nanoparticles to specific locations. Other functional proteins, such as enzyme antibodies, which can facilitate the direction of a biological to a desired location, can also be used as components of the stabilizing protein. Likewise, synthetic polymers are also good candidates for the formation of particles having a polymeric shell. In addition, polyalk or ileoglycol (ie, straight or branched chain), polyvinyl alcohol, polyacrylates, polyhydroxyethyl methacrylate, polyacrylate acid, polyethyloxazoline, polyacrylamides, polysopropol acrylamides, polyvinyl pyrrolidinone, -aaaafM »^^^ -_! --__________ polylactide / glycolide, and the like, and combinations thereof, are good candidates for the polymer / compatible in the formulation of the invention. Likewise, synthetic polypeptides are also good candidates as stabilizing agents for pharmacologically active agents substantially insoluble in water. In addition, contemplated for use in the practice of the present invention are those materials such as synthetic polyamino acids containing cysteine residues and / or disulfide groups; polyvinyl alcohol modified to contain its free Ifhydri groups and / or disulfide groups; polyalkylene glycol modified to contain free sulfhydryl groups and / or disulfide groups; polyacrylic acid modified to contain free sulfhydryl groups and / or disulfide groups; polyethyloxazoline modified to contain free sulfhydryl groups and / or disulfide groups: polyacrylamide modified to contain free sulfhydryl groups and / or disulfide group; polyvinyl pyrrolididone modified to contain free sulfhydryl groups and / or groups of Disulfide; polyalkylene glycols modified to contain free sulfhydryl groups and / or disulfide groups; polylactides, polysulfides, polycaprolactones, or copolymers thereof, modified to contain free sulfhydryl groups and / or disulfide groups; Like mixtures of any of two or more of the same.

In preparing the compositions of the invention, a wide variety of organic media can be employed to suspend or dissolve the pharmacologically active agent substantially insoluble in water. The organic medium contemplated for use in the practice of the present invention includes any non-aqueous liquid that is capable of suspending or dissolving the pharmacologically active agent, which does not chemically react either with the polymer used to produce the coating, or the agent pharmacologically active itself. Examples include vegetable oils (e.g., soybean oil, olive oil, and the like), coconut oil, safflower oil, cottonseed oil, sesame oil, orange oil. Limonene, aliphatic, cyclialiphatic or aromatic hydrocarbons oil having 4-30 carbon atoms (e.g., n-dodecane, n-decane, n-hexane, cyclohexane, toluene, benzene, and the like), aromatic aliphatic alcohols having 2-30 carbon atoms (e.g., octanol, and similar), aliphatic or aromatic esters having 2-30 carbon atoms (eg, ethyl caprylate (octanoate), and the like), alkyl, aryl, or cyclic ethers having 2-30 carbon atoms (eg, diethyl ether) , tetrahydrofuran, and the like), alkyl or aryl halides having 1-30 carbon atoms (and optionally more than one halogen substituent, eg, CH3Cl, CH2Cl2, CH2CI-CH2Cl, and the like), ketones having 3-3 pressure 30 carbon atoms (for example, * »To» acetone, methyl ethyl ketone, and the like), polyalkyl glycols, or combinations of any two or more thereof. * Especially preferred combinations of organic media contemplated for use in the practice of the present invention typically have a boiling point of no more than about 200 ° C, and include volatile liquids such as dichloromethane, chloroform, ethyl acetate, benzene, ethanol, butanol, butyl acetate, and the like (ie, solvents that have a degree of solubility for the pharmacologically active agent, and are soluble in other organic media used), together with an organic medium with a higher molecular weight (less volatile) . When added to another organic medium, these volatile additives help to activate the solubility of the pharmacologically active agent within the organic medium. This is desirable since this step usually takes time. After the dissolution, the volatile component can be removed (optionally under vacuum). The particles of the pharmacologically active agent associated with a polymeric coating, prepared as described above, are supplied as a suspension in a biocompatible aqueous liquid. This liquid can be selected from water, saline, a solution containing appropriate buffers, a solution containing nutritional agents such as amino acids, sugars, proteins, carbohydrates, vitamins or fats, and the like.

These biocompatible materials can also be employed in various physical forms such as gels, (reticulated or non-crosslinked) to provide matrices of which the pharmacologically active ingredient, for example paclitaxel, can be released by diffusion and / or degradation of the matrix. Temperature sensitive materials can also be used as the dispersion matrix for the formulation of the invention. Thus, for example, Capxol ™ can be injected into a liquid formulation of the temperature-sensitive material (for example, polyacrylamide copolymers or polyalkylene glycol copolymers and polylactide / glycolides) with gel at the tumor site and will provide a release Slow of Capxol ™. The Capxol ™ formulation can be dispersed to form a matrix of the aforementioned biocompatible polymers to provide a controlled release formulation of paclitaxel, which through the properties of the formulation of Capxol ™ (albumin associated with paclitaxel) results in lower toxicity to brain tissue as well as lower systemic toxicity as will be discussed later. This combination of Capxol ™ or other formulated chemotherapeutic agents similar to Capxol ™, together with a biocompatible polymer matrix, may be useful for controlled local delivery of chemotherapeutic agents to treat solid tumors in the brain and peritoneum (ovarian cancer), and applications local to other solid tumors. These combination formulations are not they limit the use of paclitaxel and can be used with a wide variety of pharmacologically active agents including anti-infectives, immunosuppressants, other chemotherapeutics, and the like. Colloidal particles substantially and completely contained within a polymeric stabilizer layer, or associated therewith, prepared as described above, are supplied hard, or optionally as a suspension in a biocompatible medium. This medium can be selected from water, buffered aqueous medium, saline solution, buffered saline solution, optionally buffered solutions of amino acids, optionally buffered solutions of proteins, optionally buffered solutions of sugars, optionally buffered solutions of carbohydrates, optionally buffered solutions of vitamins, solutions optionally cushioned synthetic polymers, emulsions containing lipids, and the like. In addition, the colloidal particles may be optionally modified by a suitable agent, wherein the agent is associated with the polymeric layer through an optional covalent bond. The covalent bonds contemplated for the linkages include ester, ether, urethane, diester, amide, secondary or tertiary amine, phosphate ester, sulfate ester, and the like. The appropriate agents contemplated for Optional modification of the polymeric shell include synthetic polymers (polyalkylene glycol (eg, straight or branched chain polyethylene glycol), polyvinyl alcohol, polyhydroxymethyl methacrylate, polyacrylic acid, polyethoxoxazoline, polyacrylamide, polyvinyl pyrillidone. , and the like), phospholipids (such as phosphatidyl choline PC), phosphatidyl ethyleneamine (PE), phosphatidyl nositol (Pl), sphlngomyelin, and the like), proteins (such as enzymes, antibodies, and the like), polysaccharides (such as starch, cellulose, dextrans, alginates, cytosal, pectin, hyaluronic acid, and the like), chemical modifying agents (such as pyridoxal 5'-phosphate, pyridoxal derivatives, dialdehydes, diaspirin esters, and the like), or combinations of any two or more of the same. Variations on the general theme of stabilized colloidal particles are possible. A suspension of fine particles of pharmacological agent in a biocompatible dispersing agent can also be used (instead of a biocompatible dispersing agent containing a dissolved biological) to produce a polymeric shell containing particles of suspended agents dispersed from the biological. In other words, the polymeric shell may contain a saturated solution of biological in a dispersing agent. Another variation is a polymeric shell containing a solid core of biological produced initially by dissolving in biological in a volatile organic solvent (for example benzene), forming the polymeric shell and Yes - evaporating the volatile solvent under vacuum, for example, in an evaporator, spray drying, or freeze drying the entire suspension. This results in a structure having a solid core of biological surrounded by a polymer coating. This latter method is particularly advantageous for delivering high doses of biologics to a relatively small volume. In some cases, the biocompatible material that forms the shell around the core can itself be a therapeutic or diagnostic agent, for example, in the case of insulin, which can be supplied as part of a polymeric shell formed in the process described. previously. In other cases, the polymer that forms the shell can participate in the biological supply, for example, in the case of antibodies used to direct, or in the case of hemoglobin, which can be supplied as part of a polymeric shell formed in the ultrasonic irradiation process described above, thereby providing a blood substitute having a high binding capacity for oxygen. Those skilled in the art will recognize that various variations are possible within the scope and spirit of this aspect of the invention. The organic medium within the polymeric coating can vary, a wide variety of pharmacologically active agents can be used, and a wide range of proteins just as other natural and synthetic polymers can be used in the formation of polymeric shell walls. The applications also vary widely. Other than biomedical applications such as drug delivery, diagnostic agents (in imaging applications), artificial blood, and parenteral nutrition agents, the polymeric shell structures of the invention can be incorporated into cosmetic applications such as skin creams , or hair care products, in perfumery applications, in pressure sensitive inks and the like. This aspect of the invention will now be described in more detail with reference to the following non-limiting examples.

Example 1 Preparation of Nanoparticles by Homogenization at 15 High Pressure. 30 mg of paclltaxel were dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human albumin solution (1% w / v). In the mixture, it was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transfer it into a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while the recycling of the emulsion was for at least 5 cycles. The resulting system was transferred into a rotary evaporator, 25 and the methylene chloride was rapidly removed at 40 ° C, to a reduced pressure (30 mm Hg), for 20-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting paclltaxel particles was 160-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours without adding any cryoprotectant. The resulting paste can be easily reconstituted to the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

Example 2 The Use of Surfactants and Conventional Proteins Result in the Formation of Large Crystals The following example demonstrates the effect of the vision of surfactants which are used in the conventional solvent evaporation method. A series of experiments were conducted using a procedure similar to that described in Example 1, but a surfactant such as Tween 80 (1% to 10%) is added to the organic solvent. It was found that after the removal of methylene chloride, large numbers of paclitaxel crystals were obtained having an average size of 1-2 microns, as seen by light microscopy and under polarized light. The crystals grow in a few hours to form crystals in the form of very large needles, with a size in the range of about5-15 mieras. A similar phenomenon is observed with other commonly used surfactants, such as Pluronic F-68, Pluronici F-127, Cremofor EL and Brij 58. From these results it can be concluded that the conventional solvent evaporation method using surfactants conventional in combination with a protein such as albumin is not suitable for the formation of drug particles with their microns (for example, paclitaxel) if a polymeric core, while a polar solvent (for example methylene chloride) is used.

EXAMPLE 3 The Use of Conventional Surfactants As a Result Only the Formation of Large Crystals This example demonstrates that it is not possible to form nanoparticles while using conventional surfactants, without a polymeric core material, with pharmacologically active agents which are soluble in solvents invisible in polar water (eg, chloroform). 30 mg of paclitaxel were dissolved in O.add ml of chloroform and 0.05 ml of ethanol. The solution was added to 29. 4 ml of Twee 80 solution (1% w / v), which was presaturated with 1% chloroform. The mixture was homogenized for 5 minutes at low RPM (Vitris model homogenizer: Tempest I.Q.) for to form a crude emulsion, and then it was transferred into a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while the recycling of the emulsion was for at least 6 cycles. The resulting system was transferred to a rotary evaporator, and the chloroform was rapidly stirred at 40 ° C, and a reduced pressure (30 mm Hg), for 15-30 minutes. The resulting dispersion was opaque, and contained large crystals in the form of drug needles. The initial size of the crystals (also observed by polarized light), can be 0. 7-5 mieras. Storage of the dispersion for several hours at room temperature led to an additional increase in the size of crystals, and finally to precipitation.

Example 4 Preparation of Sterile Filterable Nanoparticles of Less than 200 nm This example describes a process by which sterile filterable drug particles can be obtained. In this way, 30 mg of paclitaxel was dissolved in 0.55 ml of chloroform and 0.05 ml of ethanol. The solution is added to 29. 4 ml of human serum albumin solution (1% w / v), which was presaturated with 1% chloroform. The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transfer it into a high pressure homogenizer (Avestln). The emulsification was performed at 9000-40,000 psi while the recycling of the emulsion was for at least 6 cycles. The resulting system was transferred to a rotary evaporator, and the chloroform was rapidly stirred at 40 ° C, at a reduced pressure (30 mm Hg), for 15-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting particles was 140-160 nm (Z-average, Malvern Zetasizer). The dispersion was filtered through a 0.22 micron filter. (Millipore), without any significant change in turbulence, or particle size. HPLC analysis of the paclitaxel content revealed that more than 97% of the paclitaxel was recovered after filtration, thereby providing a sterile paclitaxel dispersion. The sterile dispersion was further lyophilized for 48 hours without adding any cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

Example 5 Preparation of Sterile Filterable Nanoparticles Less than 200 nm This example describes a process by which sterile filterable drug particles can be obtained. Of this BáritUtÉHÉdMita £ ^ & ^^^ ^ '^ Yes _ ^^ Iaa iai_Bß_H way 225 mg of paclitaxel were dissolved in 2.7 ml chloroform and 0.3 ml of ethanol?. The solution was added to 97 ml of solution of human serum albumin (3% w / v). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transferred inside to a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psl while the recycling of the emulsion was for at least 6 cycles. The resulting system was transferred to a rotary evaporator, and the chloroform was rapidly stirred at 40 ° C, at a reduced pressure (30 mm Hg), for 15-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting particles was 140-160 nm (Z-average, Malvern Zetasizer). The dispersion was filtered through a 0.22 micron filter. (Sartorius, sartobran 300), without any significant change in turbidity, or particle size. HPLC analysis of the paclitaxel content typically revealed that 70-100% of paclitaxel could be recovered after filtration, thereby providing a sterile paclitaxel dispersion. The sterile dispersion was filled aseptically inside sterile glasses, used without adding a cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline. The particle size after reconstitution is the same as before lyophilization.

Example 6 Effect of Phase Fraction of Organic Solvent on Particle Size The following example demonstrates the importance of having an unusually low phase fraction of the organic solvents in the system. In this way, a series of experiments were conducted following a procedure similar to that described for Example 4, except that the phase fraction of the organic solvent was altered, and the ethanol content was maintained at 10% v / v in the organic phase It was found that the increase of the phase fraction reached a significant particle increase: at a fraction of 4% v / v (above the saturation concentration, or 5% v / v total chloroform concentration) the particles resulting have a diameter of 250 nm; at 3% v / v phase fraction, the particles had a diameter of 200 nm, and at 2% v / v phase fraction, the particles had a diameter of 150 nm. Clearly, only the particles prepared at a very low phase fraction could be filtered and sterilized.

Example 7 Effect of Drug Concentration on the Particle Size The role of the drug concentration in the organic phase was demonstrated in the following example. Two experiments were performed in which the concentration of paclltaxel in the organic phase was 50 mg / ml or 75 mg / ml, while all other parameters were the same as described in Example 2. It was found that the low concentration of drug produces particles that have a diameter of approximately 150 nm, while those prepared at a higher drug loading were smaller, ie 130-138 nm. When a similar experiment was carried out, but with an ethanol concentration in the organic phase of approximately 50%, a similar tendency was observed, that is, the particles were 210 nm and 156 nm in diameter, for the concentration of 25 mg / ml and 50 mg / ml, respectively. These results directly contradict those reported by Sjostrom et al., Supra for the formation of nanoparticles in the presence of surfactants.

Example 8 Nanoparticle Formation of a Model Drug 30 mg of Isoreserpine (a model drug) was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human serum albumin solution (1% p / v). The mixture was homogenized for 5 minutes at low RPM (Vitris homogenizer, model: Tempest I.Q.) to be able to form a crude emulsion, and then transferred to a high pressure homogenizer (Avestln). The emulsification was performed at 9000-18,000 psi by recycling the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and the methylene chloride was rapidly removed at 40CC under a reduced pressure of (30 mm Hg), for 20-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting particles was 120-140 nm (Z-averaged, Malvern Zetaslzer). The dispersion was filtered through a 0.22 micron filter (Millipore). The sterile dispersion was lyophilized further for 48 hours without adding any cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

Example 9 Formation of Extremely Small Particles with a Drug Model The effect of ethanol in addition to reducing particle size is demonstrated for Isoreserplna. In this way, 30 mg of Isoreserplna were dissolved in 2.7 ml of methylene chloride and 0.3 ml. The solution was added to 27.0 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transfer it into a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40, 000 psi while recycling the emulsion was for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and the chloroform was rapidly stirred at 40 ° C, at a reduced pressure (30 mm Hg), for 20-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting particles was 90-110 nm (Z-averaged, Malvern Zetasizer). The dispersion was filtered through a 0.22 micron filter. (Millipore). The sterile dispersion was lyophilized further for 48 hours without adding any cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

Example 10 Use of a Solvent Visible in Water Only. Super-charged with a Drug Not Suitable for the Process of the Invention 30 mg of paclitaxel were dispersed in 0.6 ml of ethanol. At this concentration (50 mg / ml), paclitaxel is not completely soluble and forms a supersaturated dispersion. The dispersion is added to 29.4 ml of serum albumin solution human (1% p / v). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transfer it into a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while the recycling of the emulsion was for at least 6 cycles. The resulting system was transferred to a rotary evaporator, and the ethanol was rapidly stirred at 40 ° C, at a reduced pressure (30 mm Hg), for 15-30 minutes. The resulting dispersion particle size is extremely broad, varying from about 250 nm to several microns. Observation under the microscope revealed the presence of large particles and needle-like crystals typical of paclitaxel. These particles were too large for intravenous injection. This experiment demonstrates that the use of solvents such as ethanol that are freely visible in water in the process of the invention result in the formation of large particles with a very large particle size distribution and as such can not be used alone for the Process of the Invention. Thus, the process of the invention specifically excludes the use of miscible solvents in water when used alone for the dissolution or dispersion of the drug component. The process of the invention requires that the solvents, when used, must be mixed with solvents Z? Fev essentially immiscible in water to allow the production of the nanoparticles of the invention.

Example 11 The use of a Water Miscible Solvent Only Containing Drug Dissolved is Not Suitable for the Process of the Invention 30 mg of paclitaxel was dispersed in 1.3 ml of ethanol. At this concentration (approximately 24.5 mg / ml), paclitaxel is completely soluble in ethanol. The solution was added to 28.7 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transferred inside to a high pressure homogenizer (Avestin). The emulsification was performed at 9000-18,000 psi while the recycling of the emulsion was for at least 6 cycles. The resulting system was transferred to a rotary evaporator, and the ethanol was rapidly stirred at 40 ° C, under reduced pressure (30 mm Hg), for 15-30 minutes. The resulting dispersion particle size was extremely broad, varying from about 250 nm to several microns. Observation under the microscope revealed the presence of large particles and needle-like crystals typical of paclitaxel. These particles were too large for intravenous injection.

This example, in addition to Example 10 above, demonstrates that the use in the process of the invention of solvents such as ethanol are freely miscible in water results in the formation of large particles with a very large particle size distribution and as such is not they can use alone for the process of the invention. Thus, the process of the invention specifically excludes the use of miscible solvents in water when used alone for the dissolution or dispersion of the drug component. The process of the invention requires that the solvents, when used, be mixed with solvents essentially immiscible in water to allow the formation of the invention.

EXAMPLE 12 Determination of the Physical State of Paclitaxel in a Nanoparticle Form by X-Ray Powder Diffraction The crude material of paclitaxel is usually present as needle-shaped crystals of varying sizes, typically between 5-500 microns. The presence of crystals in a drug formulation for intravenous injection is obviously detrimental if the crystals are present in a size above a few microns due to the potential capillaries block. In addition, the solubility of the drug crystals in general will be lower than for the amorphous drug, thereby decreasing the bioavailability of the drug after i-! ^^^^^^^^ gjfe a-ZZ? -S?? intravenous administration. It is also known that as the loading of the drug in a formulation is increased, the tendency for its crystallization also increases. Thus, it is advantageous if the formulation contains the drug in an essentially amorphous form. X-ray powder diffraction was used to determine the crystalline or noncrystalline nature of paclitaxel in the lyophilized powder formulation. The following examples were analyzed: Sample 1 - paclitaxel powder; Sample 2 - Lyophilized serum albumin; Sample 3 - Physical mixture of paclitaxel and albumin; and Sample 4 - paclltaxel formulated. Each sample was tested with X-rays from 2 ° to 70 ° angles 2- teta using CuKa radiation, an accelerated voltage of 40KeV / 30mA, a step size of 0.05 ° 2-teta, and a data acquisition time of 2.0 seconds per He passed. Sample 1 showed strong typical peaks of a crystalline sample. The most intense paclltaxel peak was localized in 5.1 ° 2-theta. Sample 2 showed broad ridges typical of an amorphous material. Sample 3 broadly showed the broad crests of Sample 2 but in addition, the peak at 5.1 ° 2-theta paclitaxel was visible. Sample 4, paclitaxel formulated showed no evidence of crystallinity characteristics of paclltaxel, and appeared as identical to Sample 2, indicating the presence of a substantially amorphous pharmacologically active agent in the formulated sample.

AND? ace. t J & SM & * The amorphous nature of the nanoparticles produced according to the invention are in direct contrast to the products produced by other methods described in the art for producing nanoparticles. For example, the use of milling techniques, as described in U.S. Patent 5,145,684 (Liversidge et al.), And as described by Liversidge-Merisko et al., Pharmaceutical Research, 13 (2): 272-278 (1996 ), produces a substantially crystalline product.

Example 13 Preparation of Cyclosferine Nanoparticles (Capsorin I.V.) by High Pressure Homogenization 30 mg of cycoferin were dissolved in 3.0 ml of methylene chloride. The solution was then added to 27.0 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then transferred inside to a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while the recycling of the emulsion was for at least 5 cycles. The resulting system was transferred to a Rotavap, and the methylene chloride was rapidly stirred at 40 ° C, at a reduced pressure (30 mm Hg), for 20-30 minutes. The resulting dispersion was translucent, and the typical diameter of the resulting particles was 160-220 nm (Z-average, Malvern Zetasizer). jHlgiM Mjal MI¡¡ll The dispersion was lyophilized additionally for 48 hours, without adding any cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline solution. The particle size after reconstitution was the same as before lyophilization.

Example 14 Preparation of Cyclosporine Nanogotas (Oral Capsorin) by High Pressure Homogenization 30 mg of cyclosporin was dissolved in 3.0 ml of a suitable oil (sesame oil containing 10% orange oil). The solution was then added to 27.0 ml of human serum albumin solution (1% v / w). The mixture was homogenized for 5 minutes at RPM (under Vitris model homogenizer: Tempest I.Q.) to be able to form a crude emulsion, and then it was transferred to a high pressure homogenizer (Avestin). The emulsification was performed at 9000-40,000 psi while the emulsion was recycled for at least 5 cycles. The resulting dispersion had a typical diameter of 160-220 nm (Z-average, Malvern Zetasizer). The dispersion could be used directly or it could be lyophilized for 48 hours optionally by adding a suitable cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding sterile water or saline.

Example 15 Formulation for Inhalation of Anti-Asthmatic Drug Anti-asthmatic drugs have been prepared using microparticle techniques to produce effective formulations for dry powder inhalers (DPI). Beginning with a steroid drug (e.g., beclomethasone, decimethasone beclodipropionate, budesonide, dexamethasone, flunisollda, triamcinolone acetonide, and the like), a dry formulation of an appropriate particle size and release characteristics was prepared to ensure efficient delivery to the patient. respiratory system. The formulation was prepared using sound-forming techniques, or homogenization in which the active drug, dissolved in solvent, is dispersed to form an aqueous solution of protein to form a nanoparticle emulsion. This emulsion is then evaporated to remove the solvents, leaving the active drug coated with protein in solution. This liquid sample containing colloidal drug particles is measured by a Malvern Zetasizer and provides a Z-average size of 260 nm. In a preferred embodiment, the range of sizes of these colloidal particles is about 50-1,000 nm, and more preferably about 70-400 nm. In this liquid form, other excipients can be dissolved. Excipients include (but are not limited to) 0.5-15% mannitol, 0.1-5% lactose, and maltodextrin. In this step, the resulting solution of active drug, protein, and excipient can be either spray-dried or lyophilized and sprayed to form a dry powder. After spray drying, the dry particle size is determined by the Malvern Masterziser as D (v 0.5) of about 1-10 μm. The preferred size range for these particles is 0.5-15 μm, with a more preferred range of 0.7-8 μm. The spray-dried powder is then mixed with an excipient carrier powder. Again, several carriers are available, including lactose, trehalose, Pharmatosa 325 M, sucrose, mannitol, and the like. The size of the carrier powder is significantly larger than that of the formulated drug particles (-63-90 μm for lactose, 40-100 μm for the pharmatosa). The effectiveness of the dry powder formulation is demonstrated by testing it with an Andersen eight stage cascade impactor. The results of the impactor tests show a fine particle fraction (FPF) of -60%. This indicates a highly effective release of particles, appropriately sized for respiratory deposition. This FPF is surprisingly high and is a result of the composition of the formulation containing colloidal nanoparticles of the drug in larger formulation particles.

This formulation shows the application of spray and microparticle drying techniques in the processing and composition of dry powder formulations for aerosols with delivery via DPI. The high FPF results shown indicate an effective and promising approach to IPD formulations. Example 16 Summary of the Currently Preferred Manufacturing Process: Beginning with 1 Gram of Paclitaxel as BDS A solution of 3% HSA was prepared. To 51.7 ml of 25% Albutein add 379.3 ml of water for Injection. Mix evenly and filter the solution through a sterile 0.22 μm Nalgene disposable filter. Keep at 4 ° C until use. Weigh 1.0g of paclitaxel into a glass bottle. Combine CHCI3 and ethyl alcohol in appropriate proportions in a container. Mix well. To paclitaxel, add 13.33 ml of the chloroform / ethyl alcohol mixture. Shake to ensure that all paclitaxel dissolves in the solution. Filter the solution through a sterile 0.22 micron Teflon filter and collect in a sterile glass bottle. To the solution of paclltaxel dissolved in the glass bottle, add the HSA solution. Use the Sentry Microprocessor mixer to mix the paclitaxel / HSA solution. When the solution is mixed, pour the contents into the homogenizer. Cycle the ^ Gg mmám mix through the homogenizer at a pressure until the desired particle size is obtained. Collect the homogenized sample in a Kontes sterile round bottom flask. Fix the flask with the final sample to the rotary evaporator. Turn the vacuum and the rotation to a maximum on the rotary evaporator, and evaporate the organic solvent. This results in a colloidal solution of paclitaxel in human albumin. Save ~ 3ml of this rotoevaporated sample for particle size analysis. Under a sterile hood, filter the colloidal solution using a sterile 0.45 / 0.2μm filter and collect in a sterile receiving container. Save ~ 3ml of the filtered sample for HPLC analysis for paclitaxel concentration. Determine the fill volume to obtain 30 mg (or other derivative amount) of paclitaxel per container. Fill the sterile filtered sample in 30 ml containers placed in a Wheaton autoclave at approximately 17 ml each (based on the evaluation). Closing containers with Wheaton autoclave serum swabs. Each container should contain approximately 30 mg of paclltaxel. Lyophilize the samples in the FTS System Stoppering tray liner using a predetermined lyophilization cycle. After the samples have been freeze-dried, stop the containers and seal the containers Covering them with 20mm Wheaton aluminum disposable lids. Label the samples appropriately. The entire process is carried out in a clean room environment under aseptic conditions. The lyophilized samples contain residual solvent at levels of < 1000 ppm, and more preferably < 500 ppm, or up to < 100 ppm. Sterile Final Product Filtration: After removal of the solvent by evaporation, the colloidal solution of paclltaxel in the flask is sterile filtered through a combination of 0.45 / 0.2 micron sterilizing filters. The filtered solution is collected in a sterile beaker and filled sterilely into 30 ml containers. The containers are then placed on a lifting device. After completing the lyophilization cycle, the containers are covered with dry sterile nitrogen gas and capped under the nitrogen blanket. It should be noted that high pressure homogenization processes are used to break and kill bacteria and other cells to extract their contents.

EXAMPLE 17 Nanoparticle Formation Using a Preparation of Sound Formation of the Protein Cover with Oil Content Similar to the use of high speed cutting homogenization, the use of sound formation to form protein-coated nanoparticles of pharmacologically active agents and Water soluble are thought to cooperate by crosslinking the proteins through the formation of their intermolecular disulfide bonds. Many of the advantages over the prior art that are enjoyed by the high speed cutting homogenization technique described above apply equally to the sound forming methods described below. With respect to the organic solvents, proteins, and non-proteinaceous polymers that can be used in the sound forming method, reference is made to those components described above with respect to the high speed cutting homogenization method. It is expected that all the same components work equally well in both methods. This aspect of the invention will now be described in more detail with reference to the following non-limiting examples. Three ml of a USP (United States Pharmacopy) of 5% human serum albumin (Alpha Therapeutlca Corporation) were taken in a cylindrical vessel that could be attached to a sound-forming probe (Heat Systems, Model XL2020). The albumin solution was covered with a 6.5 ml layer of oil ^ ffi | soybean meal (soybean oil) grade USP. The tip of the sound-forming probe was placed to interfere between the two solutions and the assembly was kept in a cooling bath at 20 ° C. The system was allowed to equilibrate and the sound former was on for 30 seconds. A vigorous mixing occurred and a milky white suspension was obtained. The suspension was diluted 1: 5 with normal saline. A particle counter (Particle Data Systems, Elzone, Model 280 PC) was used to determine the size distribution and concentration of protein coatings with oil content. It was determined that the resulting protein coatings had a maximum cross-sectional dimension of approximately 1.35 ± 0.73 microns, and it was determined that the total concentration was -109 cells / ml in the original suspension. As a control, the above components, minus the protein, did not form a stable myocroemulsion when subjected to ultrasonic irradiation. This result suggests that the protein is essential for the formation of microspheres. This was confirmed by scanning electron micrograph and transmission electron micrograph studies as will be described below.

Example 18 Preparation of Polymer Covers Containing Dissolved Paclitaxel Paclitaxel was dissolved in USP grade soybean oil at a concentration of 2 mg / ml. 3 ml of a solution of 5% human serum albumin in USP was placed in a cylindrical container that could be attached to a sounding probe. The albumin solution was covered with a 6.5 ml layer of a soybean / paclitaxel oil solution. The tip of the sound-forming probe was made interfering between the two solutions of the assembly and kept in balance and the sound former was turned on for 30 seconds. A vigorous state was obtained until obtaining a stable milky white suspension containing the polymeric cover with protein walls covering the. solution of oil / paclitaxel. In order to obtain a higher charge of the drug within the reticulated protein coat, a mutual solvent for the oil and the drug (in which the drug has a considerably higher solubility) can be mixed in the oil. Taking into account that this solvent is relatively non-toxic (for example, ethyl acetate), it can be injected together with the original carrier. In other cases, it can be removed by vacuum evaporation of liquid after the preparation of the polymeric covers. It is recognized that several different methods can be employed to achieve the physical characteristics of the formulation.

The biological properties associated with this formulation of higher local concentrations at specific organ sites (prostate, lung, pancreas, bone, kidney, heart). , as the lower toxicities (LD50 increased, decreased myelosuppression, decreased cerebral toxicity), associated with higher efficacies is independent of the manufacturing method.

Example 19 Preparation of Nanoparticles by Sound Formation 20 mg of paclltaxel was dissolved in 1.0 ml of methylene chloride. The solution was added to 4.0 ml of human serum albumin solution (5% w / v). The mixture was homogenized for 5 minutes at low RPM (Vitris homogenizer, model: Tempest I.Q.) to be able to form a crude emulsion, and then transferred to a 40 kHz sound-forming cell. Sound formation was performed at 60-90% energy at 0 degrees for 1 minute (Sound Demerger 550). The mixture was transferred to a rotary evaporator, and methylene chloride was rapidly removed at 40 ° C, under reduced pressure (30 mm Hg), for 20-30 minutes. The typical diameter of the resulting particles was 350-420 nm (Z-average, Malvern Zetasizer). The dispersion was lyophilized additionally during 48 hours without adding a cryoprotectant. The resulting paste could be easily reconstituted to the original dispersion by adding water J Á ^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^^^^^^^ S? S ÁÁ or sterile saline. The particle size after reconstitution was the same as before lyophilization.

Example 20 In Vivo Biodistribution of Reticulated Protein Coats Containing a Fluorophore To determine the absorption and biodistribution of the trapped core within the polymeric coating of the protein after the intravenous invention, a fluorescent dye (rubrene, available from Aldrich) was retained within a polymeric cover of human serum albumin protein (HSA) and used as a marker. Thus, rubrene was dissolved in toluene, and the albumin-containing toluene / rubrene covers were prepared as described above by ultrasonic irradiation. The resulting milky suspension was diluted five times in a normal saline solution. Then 2 ml of diluted suspension was injected into the tail vein of a rat for 10 minutes. One animal was sacrificed one hour after Injection, and another 24 hours after Injection.

The frozen sections of 100 microns of lung, liver, kidney, spleen, and bone marrow were examined under a fluorescent microscope for the presence of fluorescent dye retained in the polymeric coating or dye released. At one hour, most of the polymeric covers appeared intact (it is say, they appeared as brightly fluorescent particles of approximately 1 miera of diameter) and they were located in the lungs and the liver. At 24 hours, the dye was observed in the liver, lungs, spleen and bone marrow. A general staining of the tissue was also observed, indicating that the cover wall of the polymeric covers had been digested, and the dye released from within. This result was consistent with what is expected and demonstrates the potential use of the compositions of the invention for the controlled or delayed release of a retained pharmaceutical people such as paclitaxel.

Example 21 Toxicity of Polymer Covers Containing Soybean Oil (SBO) Polymer Covers Containing Soybean Oil were prepared as described in Example 15. The resulting suspension was diluted in normal saline to produce two different solutions, one containing 20% SBO and the other containing 30% SBO. Intralipid, a commercially available agent of 20 TPN, containing 20% SBO. The LD50 for the intralipid in mice is 120 ml / kg, or approximately 4 ml for a 30 g mouse, when injected at 1 cc / mln. Two groups of mice (three mice in each group, each mouse weighing approximately 30 g) was treated with the The composition of the invention containing SBO as follows. Each mouse was injected with 4 ml of suspension prepared from polymeric covers containing SBO. Each member of a group received the suspension containing 20% of SBO, while each member of the other group received the suspension containing 30% of SBO. All three mice in the group that received the suspension containing 20% SBO survived such treatment, and did not show high toxicity in any of the tissues or organs when observed one week after treatment with SBO. Only one of the three mice in the group that received the suspension contained in 30% of SBO died after the injection. These results clearly demonstrate that the oil contained within the polymeric covers according to the present invention is not toxic in its LD50 doses, in comparison with the commercially available formulation of SBO (I ntralí pid). This effect can be attributed to the slow release (ie, controlled rate of becoming bioavailable) of the oil inside the polymeric shell. Such slow release prevents obtaining a lethal dose of oil, in contrast to the doses of high oil obtained with commercially available emulsions.

Example 22 In vivo Bioavailability of Soybean Oil Released from Covers Polymers A test was performed to determine the sustained or slow release of the material retained in the polymeric shell after the injection of a suspension of polymeric shells into the bloodstream of rats. Polymeric shells with a reticulated protein (albumin) wall containing soybean oil (SBO) were prepared by means of sound formation as described above. The resulting suspension of polymeric covers with oil content was diluted in a saline solution to obtain a final suspension containing 20% oil. 5 ml of this suspension was injected into the canola external jugular vein of the rat for a period of 10 minutes. The blood of these rats was collected at various time points after injection and the level of triglycerides (soybean oil is predominantly triglyceride) in the blood was determined by routine analysis. Five ml of a commercially available fat emulsion (I ntra lipid, an aqueous parenteral nutrition agent, containing 20% soybean oil, 1.2% egg yolk phospholipids, and 2.25% glycerin) were used as control. The control uses egg phosphatide as an emulsifier to stabilize the emulsion. A comparison of serum levels of the triglycerides in both cases would give a direct comparison of the bioavailability of the oil as a function of time. In addition to the suspension of the polymeric covers containing 20% of In addition, 5 ml of a sample of polymeric covers with oil content in saline in a final concentration in 30% oil was also injected. Two rats were used in each of the three groups. The blood levels of triglycerides in each case were tabulated in Table 1, given in units of mg / dL. Table 1 Blood levels before injection are shown in the column marked "Pre". Clearly, for optimal IP control, very high triglyceride levels are seen after the injection. The suspension of polymeric covers with oil content containing the same amount of total oil as the Intralipid (20%) shows dramatically availability different triglyceride detectable in the serum. The level increases to approximately twice its normal value and remains at this level for many hours, indicating a release ^^^^ j ^^ ^ tgÉÉ ^ jMjgH sustained or slow triglyceride within the blood at levels close to normal. The group that received the polymeric covers with oil content having 30% oil shows a higher level of triglycerides (concomitant with the highest dose administered) that falls from normal in 48 hours. Once again, blood levels of triglycerides do not increase astronomically in this group, compared with the control group that receives intrathecally. This once indicates the sustained and slow availability of the oil of the composition of the invention, which has the advantage of avoiding dangerously high blood levels of material held within the polymeric covers and availability over an extended period to acceptable levels. Clearly, the drugs provided within the polymeric covers of the present invention can achieve these same advantages. Such a polymeric shell system containing soybean oil can be suspended in an aqueous solution of amino acids, essential electrolytes, vitamins and sugars to form a total parenteral nutrient agent (TPN). Such TPN can not be formulated from currently available fat emulsions (for example, Ipid) because of the instability of the emulsion in the presence of electrolytes.

Example 23 Preparation of Polymer Covers with Protein Walls Containing a Solid Nucleus of Pharmaceutically Active Agent Another method of delivering a poorly water soluble drug such as paclitaxel into a polymeric shell is to prepare a polymeric material shell around a drug core. solid. Such a drug particle "coated with protein" can be obtained as follows. The procedure described in Example 16 is repeated using an organic solvent to dissolve paclitaxel at a relatively high concentration. The solvents generally used are organic such as benzene, toluene, hexane, ethyl ether, chloroform, alcohol and the like. The polymeric covers are produced as described in Example 15. 5 ml of the milky suspension of polymeric covers containing paclitaxel dissolved in 10 ml of normal saline was diluted. This suspension was placed on a rotary evaporator and the volatile organic was removed in vacuo. The resulting suspension is examined under a microscope to reveal opaque cores, indicating the removal of substantially all organic solvents, and the presence of solid paclitaxel. The suspension can be frozen and stored indefinitely and can be used directly or lyophilized later. Alternatively, the polymer coatings with dissolved drug core containing organic solvent are freeze-dried to obtain a dry powder that can be granulated. ^^^ g ^^^^^^^^^^^ g ^^^^ resuspend in saline solution (or other suitable liquid) at the time of use. Although the currently preferred protein for use in the formation of the polymeric shell is albumin, other proteins such as α-2-macroglobulin, a known opsonin, can be used to improve the absorption of the polymer coatings by macrophage-like cells. Alternatively, molecules such as PEG can be incorporated into particles to produce a polymeric shell with increased circulation time in vivo.

Example 24 Formation of Nanoparticles Mediant Spontaneous Microemulsion It is also possible to form nanoparticles without the use of sound formation, high speed cutting homogenization, or other high energy technique. In this way, it is possible to form a suspension (or dry powder) of an essentially pure drug, if desired. A microemulsion is a thermodynamically stable emulsion system that spontaneously forms when all its components come into contact, in the absence of the use of high speed cutting equipment or other substantial agitation. The microemulsions are substantially non-opaque, that is, they are transparent or translucent. The microemulslons comprise a dispersed phase, in which the typical drop size is below 1000 Angstrom (Á), hence its optical transparency. Drops in the microemulsion are typically spherical, although other structures such as elongated cylinders are also possible. (For further discussion see for example, Rosof, Progress in Surface and Membrane Science, 12: 405, Academic Press (1975), Friberg, Dispersion Science and Technology 6: 317 (1985).) As will be shown below, the present invention uses the unique characteristics of microemulsion as a first step towards obtaining extremely small nanoparticles, after the removal of the oil phase. • As described above, microparticles and nanoparticles can be formed by several processes, among them, the method of solvent evaporation. This method is based, in principle, on forming a simple oil in an emulsion of Water, in the presence of an active surface agent, while cutting forces are applied at high speed by means of various equipment such as rotor-stator mixers, sound formers, high pressure homogenizers, colloid mills etc. After forming such an emulsion, which contains a polymer and a drug dissolved in the dispersed oil droplets, the oil phase is removed by evaporation typically under reduced pressure and elevated temperature, and microparticles or nanoparticles of the dissolved drug and polymer are formed. Obviously, the size of the particles depends on the size of the emulsion drops; While the smaller the drops, the smaller the particles i? nd tmiijSí ^! jesns ... m¿. ... * r * 2? > * .. '. .-ay fya.- resulting. Small emulsion droplets can be achieved only by applying very high energy, and even then, using more advanced high-pressure homogenizers like the Microfluidizer, it is not practical to achieve emulsion droplets below 75 nm. Since emulsions are inherently unavoidable systems, and undergo processes such as droplet aggregation and coalescence, solvent evaporation processes for such emulsions can result in larger particles. The new method, which solves the problems associated with the application of the solvent evaporation method in conventional emulsions, consists of the following stages: a. Dissolve the drug insoluble in water in a solvent that has low solubility in water, and that has vapor pressure higher than water. The drug dissolves without any additional polymeric binder, although such a binder may be present, in principle. b. Mix the solvent with one or more suitable surfactants and one or more co-surfactant agents soluble in water. 0 c. Add an adequate amount of water or aqueous solution to this mixture, thereby spontaneously forming an oil-in-water microemulsion, without the use of any high-speed cutting equipment. The aqueous solution may contain electrolytes, amino acids, or any other additive that ¿G j & ^. MtSmm. ^ | fe¡ te ^ may affect the formation of the microemulsion during the first stage of preparation. d. Optionally, add a protein solution to the microemulsion. and. Remove the solvent by evaporation at reduced pressure, thereby causing drug precipitation in the form of extremely small amorphous nanoparticles, having a typical size below 1000 Angstroms. The particles in this step are dispersed and stabilized in an aqueous medium containing surfactants, co-surfactants and optionally protective agents such as proteins, sugars, etc. Acceptable methods of evaporation include the use of rotary evaporators, falling film evaporators, spray dryers, dryers freezing, and other standard evaporation equipment typically used in the industry. F. Optionally, the surfactant and co-surfactant agent can be removed by dialysis, ultrafiltration, adsorption, etc., thereby obtaining nanoparticles that are stabilized by the protein (if used). g. After evaporation of the solvent, the liquid dispersion of the nanoparticles can be dried to obtain a powder containing the pharmacological agent and optionally the protein, which can be redispersed to form a medium Aqueous solution suitable as saline, buffer, water and »- ¿-i»! sBvZ = a% Z4 «« ia > ¡¡¡AZ &i-¡, ...., ^^^^^ HÜ ^ B ^^ üteM! similar, to obtain a suspension that can be administered to an animal, having a particle size below 1000 Angstroms. Acceptable methods for obtaining this powder are by freeze drying, spray drying and the like. If the conversion to the solid form is carried out by lyophilization, several cryoprotectants can be added, such as mannitol, lactose, albumin, carboxymethylcellulose, polyvinylpyrrolldone, maltodextrins and / or polyethylene glycol. These nanoparticles can also be mixed with additional excipients or matrix-forming materials, in order to obtain a drug delivery system with high bloavailability, controlled release characteristics, and protection in gastric juice. The final product can be introduced to the subject as a tablet, capsule, reconstituted liquid, or the like. The formulation of the present invention has significant advantages over the methods previously used for the preparation of nanoparticles and microparticles, and the use of microcroussels or "concentrators of pre-mlcroemulsions". Many advantages are achieved using the process of the invention. The microemulsion is formed spontaneously, and the appropriate components are selected, and if there is no need for highly expensive equipment and high energy input. The droplet size is smaller by approximately one order of magnitude than the smaller emulsion droplets obtained by the high speed cutting equipment, and therefore extremely small nanoparticles can be obtained. The microemulsion is thermodynamically stable, and therefore, the usual problems associated with the instability of the emulsion (and thus the dependence of time on the size of the resulting particles) can be avoided. The whole process is much simpler than the conventional solvent-emulsion evaporation method, and less sensitive to various parameters. Since only simple mixing is involved in the process, increasing to large production volumes is very simple, compared to emulsification with equipment such as a high speed cutting homogenizer. Since the particle size obtained by the new process is so small, an order of magnitude less than the pore size of the membranes used for the Sterile filtration, the sterilization process is very effective, without problems associated with membrane blocking, such as increased filtration pressure, and high drug loss during the filtration process. Since there are no cutting forces at high speed in the emulsification process, there is no Increased temperature during emulsification, and therefore still temperature-sensitive drugs can be processed by the new method of the invention. The drug in the liquid formulation of the present invention has increased its chemical stability because it contains nanoparticles dispersed compared to conventional microemulsions ^ AM & amp; L. ' .... xáuvsa a ^. *; They contain dispersed nanogotas, that is, more chemical reactions carried out in a liquid state (microdroplets) against the solid state (nanoparticle). The present invention has increased chemical stability as a dry formulation as compared to conventional microemulsions which are liquid as the continuous microemulsion phase. The solid formulation allows the inclusion of the drug in several forms of solid doses, such as tablets, granules and capsules, in comparison with conventional microemulslons or "pre-microemulsion concentrates", which are present in the liquid form. The very narrow size distribution, in combination with the very low average particle size, ensures the increased adsorption of the drug in a more uniform manner than the microparticles and nanoparticles prepared by methods In this way, an increased bioavailability is expected. Although the examples presented in the next section make reference to water-insoluble molecules, the pharmacological agents contemplated as useful in the Preparation of nanoparticles They include, but are not limited to drugs, diagnostic agents, agents of therapeutic value, nutritional agents, and the like which are either soluble or insoluble in water. A non-limiting list of drug and compound categories includes but is not limited to all < ***** ** < - • - MÜÉÉSlfeMIrii MSl - "'* > *** - ~ * *«' - «- compounds listed above for use in a high speed cutting homogenization aspect of the invention The solvents described in the following examples are toluene and butyl acetate, however, any solvent or solvent mixture that is capable of dissolving the required drug will be suitable for use in the process of the invention, taking into account that a suitable microemulsion can be formed before removing the solvent. Such solvents may be chloroform, methylene chloride, ethyl acetate, butyl acetate, isobutylacetate, propyl acetate, tert-butyl methyl ether, butanol, propylene glycol, heptane, anisole, eumeno, ethyl formate ethanol, propanol, tetrahydrofuran, dioxane, acetonitrile, acetone. , dimethyl sulfoxide, dimethyl formamide, methylpyrrolidinone, soybean oil, coconut oil, castor oil, olive oil, safflower oil, cottonseed oil, alcohol C1-C20 esters, C2-C20 esters, C3-C20 ketones, polyethylene glycols, aliphatic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, d-limonene, combinations of these and the like. The protein (or a mixture of several proteins) used in this process should be such that it does not precipitate during the initial mixing or during the evaporation step. Many such proteins exist, including albumins (e.g., BSA, HSA, egg), gelatin, collagen, IgG, various enzymes, lactoglobulin, casein, soy proteins, and the like.

The surfactants used in this invention should be able to spontaneously form oil-in-water microemulslons, in the presence of a suitable co-surfactant agent and a solvent, without causing the drug to precipitate the protein (if present). The surfactants may be nonionic (Tween, Span, Triton, Pluronic, polyglycerol esters, and the like), anionic (SDS, cholates and deoxycholates, fatty acid soaps, and the like) cationic (ammonium chloride cetyltri methylo, and similar ones) or zwiteriónicos (lecithin, amino acids and the like). e ° The co-surfactant agent must have the ability to spontaneously form microemulsions with the selected surfactants, without causing the precipitation of the dissolved drug molecules (or protein if present) and without induce the formation of material with large crystals. Co-surfactant agents can be either water-soluble or oil-soluble, such as butanol, propylene glycol, benzyl alcohol, propanol, and the like. The conversion of the liquid dispersion of the nanoparticles by means of lyophilization may require the addition of cryoprotective agents, such as mannitol, lactose, amino acids, proteins, polysaccharides and the like. It is clear that the principles described in this invention can be applied in different variations of the process, by example: 1. The formation of drug particles that can be induced by diluting the microemulsion in a suitable solvent, in which the solvent is miscible. For example, the solvent has a low solubility in water, it might be possible to dilute the microemulsion to such an extent that the solvent will be below its water solubility limit. 2. The solvent and optionally the surfactant and co-surfactant can be removed using a selective extractor that does not dissolve the drug. 3. The surfactant and co-surfactant can be removed by ultrafiltration, while filters that have a cut below MW of the protein are used. Simple dialysis is also an option. 4. The formulation may contain only components that are acceptable for the intended use of the final formulation (oral, IV, topical, etc.), so there is no need for its removal. 5. Similarly, co-surfactants that can remain in the final product, such as glycerol, benzyl alcohol, etc., can be used. 6. The addition of several water-soluble molecules that can affect the microemulsion phase diagram (electrolytes, ethanol, etc.) is possible in this way, by controlling the ratio between the various components to provide the optimal drug loading. 7. The spontaneous emulsification stage can be carried out at a temperature different from the ambient temperature, in order to be able to affect the phase diagram (and the proportions of the component that lead to the formation of a microemulsion). In particular, it may be possible to use the temperature effect (in ethoxylated surfactants) to change the system from an oil-in-water microemulsion to a water-in-oil microemulsion. 8. It is possible to add other components to the solvent phase, in order to affect the bioavailability of the drug. In particular, the addition of an oil such as soybean oil is preferred, to improve oral absorption, and to protect the drug from chemical and enzymatic degradation. 9. Equally, the addition of a forming polymer matrix (such as PVP) to the solvent, along with the drug, can be done. 10. Stabilization and solid-form properties can be altered by the addition of a water-soluble polymer other than the protein (carboxymethylcellulose, gums and similar) to the external aqueous phase of the microemulsion. 11. The flow properties of the resultant solid form powder can be altered by adding colloidal particles (eg, silica) as filler, or the addition of reconstitution / anti-agglomeration aids. ^ '^^' - - - ^ - ^ - »- & ü ^ m máÉl ^ i 12. The same principles described in this invention can be applied to form water-soluble particles, while the stage is carried out of emulsification in the composition range in which a water-in-oil microemulsion is formed. The processes can be used, for example, to form extremely small protein nanoparticles.

Example 25 Preparation of Cyclosporin A Nanoparticles 115 mg of cyclosporin A was dissolved in 1 ml of butyl acetate, and mixed with 2 grams of 4: 1 of Triton X-100: n: Butanol solution. A clear system is obtained. 10 g of water were added in drops, while stirring slightly. A microemulsion of water in clear oil was obtained. 10 g of 1% casein solution was added, while stirring slightly. The system became slightly cloudy. The butyl acetate was removed in a Rotovap at 40 ° C, 80 mm Hg. The system became completely transparent. The particle size was measured by photon correlation spectroscopy. It was found that the Z-average size is 25-33 nm, while the size by number or volume distribution was only 9 nm. No particles were observed under the optical microscope, nor under polarized light. This result implies the absence of crystalline particles.

The liquid dispersion of these nanoparticles was lyophilized, after adding lactose (2% w / w). A white solid material was obtained, which, upon reconstitution in water, produced a transparent system, similar to that prior to lyophilization. The particle size in this reconstituted sample was very similar to that of the original formulation, Z-average of approximately 40 nm, and a diameter by number and volume distribution of between 10-12 nm.

Example 26 Preparation of Cyclosporin A Nanoparticles 119 mg of Clclosporin A were dissolved in butyl acetate, and mixed with 2 grams of 4: 1 Triton solution.

X-1 OO.propylene glycol. A transparent system was obtained. 7 g of water were added in the form of drops, while stirring slightly. A clear microemulsion of oil in water was obtained. 7g of 1% casein solution was added, while stirring slightly. The system became slightly cloudy. The sample was diluted 1: 1 with water, before evaporation of the solvent. The butyl acetate was removed in a Rotovap, at 40 ° C, 80 mm Hg. The system became completely transparent. This process also produced extremely small nanoparticles: Z-averaged 45 nm, and a diameter or distribution of number and volume 11 nm.

The liquid dispersion of these nanoparticles was boiled after adding lactose (2% w / w). A white solid material was obtained, which, upon reconstitution in water, produced a transparent system, similar to that prior to lyophilization. The particle size in this reconstituted sample was close to that of the original formulation, Z-average of approximately 25 nm and a diameter by number and volume distributions between 9-11 nm.

Example 27 Cyclosporin Nanoparticles Microemulsions were made with the following compositions: 50 mg of cyclosporin, 0.5 g of butyl acetate, 3.04 g of Tween 80: propylene glycol (1: 1) and 6.8 g of water. The microemulsion was evaporated to provide a clear liquid containing 5 mg / ml cyclosporin. In a control experiment, performed with the above components, simply mixing, but without butyl acetate, even after 17 hours, the clclosporin did not dissolve. There are several possibilities for surfactants, including polysorbates (Tween), sorbitan esters (span), sucrose esters, lecithin, monodiglycerides, polyethylene-polypropylene block copolymers (pluromics) soaps (sodium stearate, etc.), salts Sodium Glucose Glycolate, Ethoxylated Castor Oil, Sodium Stearoll Lactylate, Ethoxylated Fatty Acids (Myrj), Ethoxylated Fatty Alcohols (Brij), Sodium Dodecyl Sulfate (SDS), and the like. Also, in general, biopolymers such as starch, gelatin, cellulose derivatives, etc., can be used. Also for oral applications, all acceptable food grade surfactants can be used as surfactants presented in the McCutcheon Handbook of Surfactants of the CTFA Index. Possible co-solvents or co-surfactants for the microemulsion include propylene glycol, ethanol, glycerol, butanol, oleic acid, and the like.

Example 28 Preparation of BHT Nanoparticles 110 mg of butylated hydroxy toluene (BHT) was dissolved in 1 ml of toluene, and mixed with 2 ml of a 4: 1 solution of Triton X-100: n-butanol. 32 g of a 1% casein solution were added, and a microemulsion was spontaneously formed. The microemulsion was evaporated under reduced pressure, 80 mm Hg, at 40 ° C, until it became transparent. The size of the resulting particles was: Z-average 30 n, diameter by number distribution and volume is 16 and 15 nm, respectively.

Example 29 Preparation of BHT Nanoparticles A process similar to that described in Example 27 was carried out using water instead of casein solution.

After evaporation at 40 ° C, 80 mm Hg, the system became transparent, having an average Z size of approximately 10 nm.

Example 30 Preparation of Paclitaxel Nanoparticles 30 mg of paclltaxel was dissolved in 2 ml of butyl acetate, and 4 grams of 4: 1 Triton x-100: propylene glycol were added. 40 ml of water was added, and the system became slightly cloudy. After evaporation, the system became completely transparent. The Z-average size was 6 nm, the size by number and volume distribution was 7-9 nm. The same size was measured after one day at 4 ° C.

EXAMPLE 31 Identification of Microemulsion Phase Diagrams. [0102] Compositions were identified which produced microcrouslons, and which can be used to obtain nanoparticles by the solvent evaporation method. These compositions should contain a water-miscible solvent capable of dissolving hydrophobic molecules, an aqueous solution such as the continuous medium, surfactants, and possibly co-surfactants. The butyl acetate microemulsions in water can be formed into various compositions which are described by the phase diagrams (butyl acetate is classified as a solvent with a high acceptable residual concentration in the final product). In addition, both the surfactant and the co-surfactant agent are used in pharmaceutical and food applications: Tween 80 (ethoxylated sorbitan monooleate) and propylene glycol. Preliminary experiments were conducted using BHT as a model hydrophobic molecule, producing dispersions of particles in the range of 20-50 nm. After filtration, by 0.2 μm filters, approximately 100% of the BHT passed the membrane. Phase diagrams of various combinations of surfactant / co-surfactant agent were obtained by swirling the solvent with a mixture of surfactant-co-surfactant agent (prepared before mixing with the solvent, at various proportions), after addition in shape of drops in water. The turbidity of various compositions along with the "water line" was observed and the compositions that produced translucent systems were further analyzed by a scattering of light. When using various proportions of solvent / surfactant / co-surfactant, the areas in the phase diagrams that produced microemulsions were identified (only a small number of selected components of microemulsions). The same procedure was used for systems in which BHT was dissolved in butylacetate before conducting phase diagram experiments. The "filterability" of the microemulsion and the nanoparticles containing the BHT was evaluated by comparing the UV absorption spectrum before and after 0.2 μm filtration. The nanoparticles were obtained by vacuum evaporation of butylacetate (60 mm Hg, 40 ° C). It should be emphasized that no high-speed cutting equipment was used throughout the process. Microemulsion systems that could be useful for oral delivery were identified. The n-butylacetate was chosen as solvent. The following surfactants and cosurfactants were evaluated at various proportions: Tween 80: Glycerol 5 Tween 80: Glycerol 4 Tween 80: Glycerol 3 Tween 80: Glycerol 2 Tween 80: Glycerol 1 Span 80: Glycerol 4: 1 Span 80: Glycerol 3: 1 Tween 80: Propylene Glycol 4: 1 Tween 80: Propylene Glycol 3: 1 Tween 80: Propylene Glycol 2.5: 1 »* -f l ^ m mm Tween 80: Propylene Glycol 1.5: 1 Tween 80: Propllenglycol 1: 1 Tween 80: Propllenglycol 1: 2 ((Tween 80 + Span 80) 7: 1): Propylene glycol 3.5: 1 ((Tween 80 + Span 80) 7: 1): Propllenglycol 1: 1 ((Tween 80 + Span 80) 8: 1): Propylene glycol 4: 1 ((Tween 80 + Span 80) 5: 1): Propylene glycol 1: 1 Tween 80: ((Propllenglycol + Glycerol) 1: 1.2 2: 1 A suitable composition was found to be as follows: Tween 80 as a surfactant and propylene glycol as a cosurfactant at a ratio of 1: 1. The total phase diagram was evaluated For a system of n-butyl acetate, Tween 80: propllenglicol 1: 1, water Two additional solvents were tested: acetate sec-butyl and acetate ter-butyl The phase diagrams for these systems were the same as for those with acetate n-butyl The n-butyl acetate system, Tween 80: propylene glycol 1: 1, water was evaluated additionally The measurement of particle size for the sample 7% butyl acetate, 30% surfactant / PG, 63% water was performed.A Z average of about 20 nm was found.The nanoparticle formation process was conducted for an insoluble water dye, Sudan III, at a concentration of approximately 10 mg in 1 g of butyl acetate (5% acetate). Butyl, 23% agent ten surfactant / PG, 72% water). A particle size of approximately 17 nm was found. The nanoparticle formation process was also conducted for BHT at a concentration of 100 mg in 1 g of butyl acetate. The phase diagram for this system was determined. The particle size of about 20-50 nm was found depending on the composition. The control experiments with Sudan III and BHT were conducted. 14.4 g of water were added to 10 mg of Sudan III and 4.6 g of surfactant / PG was added to the mixture. The mixture was stirred for 24 hr with a magnetic stirrer. The dissolution of Sudan III was observed. However, when the same experiment was performed with BHT (100 mg of CBHT in 9 g of water and 4.3 g of surfactant / PG) the BHT solution was not observed. In this stage the evaporation was carried out (temperature 40 ° C, pressure of approximately 60 mm Hg). The measurement of the particle size for the samples was made before and after evaporation. The Z promised was approximately 20-50 nm and 30 nm was found for the samples before evaporation and after evaporation, respectively. The samples after evaporation were filtered through 0.2 μm filters, and the BHT concentration before and after after filtration was measured by UV absorption. HE < -%; | jf ^^^^ - H ^ j ^^^^^^^ ß found that there was no difference between the two samples. This result is obviously an indication of the very small size of the BHT nanoparticles. Two samples were prepared (the composition of these samples: sample No. 1: 4% butyl acetate, 14% surfactant / PG, 80% water, sample no 2: BHT 123 mg / g butyl acetate; 5% butyl acetate, 18% surfactant / PG, 77% water).

Example 32 Alternatives in the Choice of Process Equipment The process equipment used to produce the current batches will be modified for high-scale clinical production. There are several alternatives available in the choice of equipment to large scale for the production of Capxol ™. Some of these alternatives are shown in the following: Equipment Category Equipment Options Pre-mixer Blade Mixer, Rotameter Mixer High Pressure Equipment High Pressure Homogenizers (Avestin, Microfluidics, Stansted), Sound Formers (Head System) Rotary Evaporator Removal Equipment, - £ a £ & Example 33 Intravenous Delivery Systems Formulated from a Variety of Materials The materials used for the preparation of intravenous delivery systems can be polymeric (e.g., polyethylene, polyvinyl, polypropylene tubes, and the like), or glass. It is known that standard medical grade tubes contain hydrophobic portions on the internal surfaces thereof. These portions of this mode are available to come into contact with the injection solution. In fact, said tubes are specially manufactured, such as catheters, to present hydrophobic portions in contact with the treatment solution to reduce the absorption of aqueous material in the tube. However, any hydrophobic portion in the treatment solution will surely be attached to both the catheter tube and other components of the delivery system. As a result, a substantial portion of a pharmacologically active agent The hydrophobic can be sequestered in the inner walls of the tube catheter and the supply container. Consequently, the dosage of hydrophobic pharmacologically active agents can be erratic, since a substantial portion of the active agent can be absorbed into the walls of the tube. Critical therapeutic treatment, where the pharmacologically active hydrophobic agent is used to treat a disease, a significant reduction in the effective dose of the active agent to bring about a therapeutic failure. The failure is particularly surprising when the therapeutic portions are employed which require that the active agent be present above a certain level, but even the therapeutic window is narrow. A novel method for the intravenous introduction of a pharmacologically active hydrophobic agent has now been developed. By protecting the hydrophobic portions of the active agent, through association with the hydrophobic portions of a biocompatible coating (e.g., albumin), the propensity of the active agent to bind to the tube is dramatically reduced. Thus, the present invention allows the use of highly hydrophobic drug, in combination with standard medical grade polymers and hydrophobic glasses, in which the drug is protected and therefore not absorbed on the surface. The method of the invention comprises placing a protective coating of a biocompatible polymer (e.g. albumin) around a hydrophobic drug and placing the resulting composition in a hldrophobic polymeric delivery system. The methods of the invention are therefore capable of improving the delivery of a variety of hydrophobic therapeutics.

Example 34 HPLC Analysis of Paclitaxel Chromatography System: HPLC: Shimadzu LC-10AS Shimadzu SIL-10A Solvent Delivery System Shimadzu SCL-10A Auto Injector Shimadzu System Controller SPD-M10AV Shimadzu Diodoridation Detector CTO-10A Column Oven 5 Column: Curosil-PFP, 5μm, 4.6mm x 25cm, Fenomenex; or C-18 Mobile Phase: water / acetonitrile 65:45 Flow Rate: Socratic, 1.0 ml / min Detection: 228 nm 0 Identity of the Drug Substance Volume of paclltaxel (BDS) Paclitaxel BDS and standard paclitaxel (99.9 %, Hauser Chemical Research, Inc. Lot 1782-105-5) were quantitatively dissolved in acetonitrile and injected into HPLC 5 separately. 10 μl of 1.00 mg / ml of Paclltaxel was injected «Tí? ßf., TJ ^ _ j ~~~ -» ^ - ^ - ^^^ _ ^ t ^^ BDS and 10 μl of 2.07 mg / ml standard paclitaxel. The retention time of the dominant peak of Paclitaxel BDS is consistent with the retention time of the standard Paclitaxel Hauser.

Power of Paclitaxel BDS Paclitaxel BDS and standard paclltaxel were injected into the HPLC as described above. The potency of paclltaxel was derived based on the peak area ratio of Paclitaxel BDS over standard paclitaxel and the known potency of standard paclltaxel. * Impurity profile of Paclitaxel BDS. The chromatography system described above is capable of providing a high resolution of taxanes. 10-20 μl of 1.0 mg / ml of Paclitaxel BDS in acetonitrile which falls within the linear response range of the HPLC system was injected into the HPLC. The impurity profile was determined by the relative peak area.

Power Test of Paclitaxel in Capxol ™ Standard solutions (60, 100, 120, 140 and 160 μg / ml) were prepared quantitatively by dissolving Paclitaxel BDS in 3% HSA. The Capxol ™ samples were diluted in a saline solution at -100 μg / ml in a concentration of paclitaxel. The standard solutions and Capxol ™ samples were seeded with cephalomannin as the internal standard followed by the extraction of solid phase liquid phase extraction (see below). Equal volumes (20-30 μl) of the standard preparations and sample preparations of Capxol ™ were injected into the HPLC to measure the peak response ratios between paclltaxel and the internal standard cephalomannine. A calibration curve was generated by the ordinary minimum square regression in the results of the standard injections. The potency of paclitaxel in Capxol ™ was determined by comparing the peak response rate of the sample injections with the standard injections.

Paclitaxel Impurity Profile in Capxol ™ Capxol ™ was subjected to solid phase extraction or liquid phase extraction (see below) before being injected into HPLC. 30 μl of -1 mg / ml of paclltaxel extracted from Capxol ™ were injected to investigate the impurity profile as previously done.

Solid Phase Extraction A sample of Capxol ™ was reconstituted at approximately 100 μg / ml in saline. A solid phase extraction column, Bond-Elut (C-18) was conditioned with water. The column was loaded with the sample which was pulled through the column using a vacuum. The column then washed ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ The eluate containing the paclitaxel extracted in acetonitrile was injected into the HPLC.

Liquid Phase Extraction A sample of Capxol ™ is reconstituted at approximately 100 μg / ml in saline. To approximately 200 μl of this sample, 800 μl of acetonitrile was added. The mixture was swirled for 30 seconds and then centrifuged at 3,000 g for 5 minutes. The supernatant was removed and collected. The granule was resuspended in 200 μl of saline and the extraction step was repeated. The second supernatant was combined with the first. The supernatant extract was concentrated by evaporation after HPLC injection.

Example 35 Particle Size Distribution Using Photon Correlation Spectroscopy (PCS) The particle size distribution of the reconstituted Capxol ™ was analyzed by photon correlation spectroscopy (PCS) on the Malvern Zetasizer, Malvern Instruments Ltd. The Zetasizer was calibrated by NIST traceable Nanosphere ™ standards, Duke Scientific Corporation. The procedure of measuring the particle size of Capxol ™ in the Malvern Zetalzer included adjusting the following parameters: Temperature: 20.70 ° C Spreading angle: 90 ° Index disperser Refractor: 1.33 Honda length: 633 nm Viscosity: (Auto): 0.99 real refractor index: 1.59 imaginary refractor index: 0 After preparing the Zetasizer, the Sample dilution needed for a good size measurement of the kcts / sec readings (to start, aliquot 200 μl of sample into a cuvette then dilute it with approximately 2 ml of distilled water filtered on a 0.22 μm filter). Place the cuvette inside the cuvette holder inside the Zetasizer and start the measurement. Once the measurement begins, the Correlator Control display will appear. From the menu, choose the meter of deployment ratio. The deployment should be in an average range of 100-250 kcts / sec. If the ratio is either too high or too low, prepare another sample at a higher or lower dilution respectively. The size of the reconstituted Capxol ™ was analyzed, averaged, and recorded by a multimode analysis after three runs in Auto. The average particle size was 155 nm ± 23 nm for 25 batches of Capxol ™.

Example 36 Polymer Covers as Bearers for Polynucleotide Constructions. Enzymes and Vaccines While gene therapy is more widely accepted as a viable therapeutic option (currently, more than 40 human gene transfer proposals have been approved by the MIH associations and / or the FDA), one of the barriers to Moving on to implement this therapeutic approach is the refusal to use viral vectors for the incorporation of genetic material into the genome of a human cell. Viruses are inherently toxic. In this way, the risks involved in the use of viral vectors in gene therapy, especially for the treatment of non-genetic, non-lethal diseases, are unacceptable. Unfortunately, plasmids transferred without the use of a viral vector are usually not incorporated into the genome of the target cell. Also, as with conventional drugs, said plasmids have a finite half-life in the body. Thus, a general limitation in the implementation of gene therapy (as well as antisense therapy, which is a reversible form of gene therapy, where a nucleic acid or an oligonucleotide is introduced to inhibit gene expression) It has been the inability to effectively deliver nucleic acids or ollgonucleotides that are too large to penetrate the cell membrane. ? sa & * < * ~ * * - ^? = IÉjla ^ The encapsulation of DNA, RNA, plasmids, oligonucleotides, enzymes, and the like within coatings of protein microcapsules as described herein can facilitate their delivery to the liver, lung, spleen, lymph , and bone marrow. Thus, according to the present invention, said biologics can be delivered to intracellular sites without the risk associated with the use of viral vectors. This type of formulation facilitates the non-specific absorption or endocytosis of the polymer coatings directly from the bloodstream to the RES cells, inside the muscle cells by injection. • intramuscular, or by direct injection into tumors. In addition, monoclonal antibodies against nuclear receptors can be used to direct the encapsulated product to the nuclei of certain cell types. Diseases that may be targeted for such builders include diabetes, hepatitis, hemophilia, cystic fibrosis, multiple sclerosis, cancers in general, influenza, AIDS, and the like. For example, the gene for Insulin-like growth factor (IGF-1) can be encapsulated within envelope protein to deliver the treatment of diabetic peripheral neuropathy and cachexia. The genes encoding factor IX and factor VIII (useful for the treatment of hemophilia can be directed to the liver by encapsulating the present invention in protein microcapsule coatings. for the low density lipoprotein (LDL) receptor can be > ~ - *** d¡HtfeMfej ||| ? xaá .. ~ - directing the liver for the treatment of atherosclerosis encapsulating the present invention in protein microcapsule coatings. Other genes useful in the practice of the present invention are the genes which re-stimulate the body's immune response against cancer cells. For example, antigens such as HLA-B7, encoded by the DNA contained in a plasmid, can be incorporated into a protein coat of the present invention to be injected directly into a tumor (such as skin cancer). Once in the tumor, the antigen will bind to the tumor-specific cells that raise the level of cltokines (for example IL-2) that provide the tumor with a target for the immune system attack. As another example, plasmids containing portions of adeno-associated virus genomes are contemplated for encapsulation within protein microcapsule coatings of the present invention. In addition, the protein microcapsule coatings of the present invention can be used to deliver therapeutic genes to CD8 + T cells for adoptive immunotherapy against a variety of tumors and infectious diseases. The protein coatings of the present invention can also be used as a delivery system for fighting infectious diseases by means of targeted delivery of an antisense nucleotide, for example, against the virus Hepatitis B. An example of such antisense oligonucleotide is the 23-mer phosphorotloate against the polyadenylation signal of the hepatitis B virus. The protein coatings of the present invention can also be used for the delivery of the transmembrane regulatory gene of flbrosls. cystic (CFTR). Humans lacking this gene develop cystic fibrosis, which can be treated by the capsules of nebulized protein microcapsules of the present invention containing the CFTR gene, and by inhaling directly into the lungs. Enzymes can also be delivered using the protein coatings of the present invention. For example, the DNA enzyme can be encapsulated and delivered to the lung. Likewise, ribosomes can be encapsulated and directed to enveloping proteins of viruses or cells infected by viruses by attaching suitable antibodies to the outside of the polymeric shell. The vaccines can also be encapsulated within polymeric microcapsules of the present invention and used for intravenous, intramuscular or subcutaneous delivery.

Example 37 Localized Treatment of Tumors of the Brain and Tumors within the Peritoneum Providing chemotherapeutic agents locally to a tumor is an effective method for long-term exposure of the tumor. * gtóg ^ & drug while minimizing dose-limiting side effects. The aforementioned biocompatible materials can also be used in various physical forms as gels (cross-linked or non-crosslinked) to provide matrices of which the pharmacologically active ingredient, for example paclltaxel, can be released by diffusion and / or degradation of the matrix. Capxol ™ can be dispersed within a matrix of a biocompatible material to provide a sustained release formulation of paclitaxel for the treatment of brain tumors and tumors within the peritoneal cavity (ovarian cancer and metastatic diseases). Temperature sensitive materials can also be used as the dispersing matrix for the formulation of the invention. Thus, for example, Capxolt can be injected into a liquid formulation of the temperature sensitive materials (eg, copolymers of polyacrylamides or copolymers of polyalkylene glycols and polylactide / glycols and the like) with gel at the tumor site and provide a slow release t < to Capxol ™. The Capxol ™ formulation can be dispersed within a matrix of the bio c < ompatibles mentioned above to provide a controlled release formulation of paclitaxel, which through the properties of the Capxol ™ formulation (albumin associated with paclitaxel) results in lower toxicity to brain tissue as well as more systemic toxicity low as will be discussed later. This combination of Capxol, or other formulated chemotherapeutic agents similar to Capxol ™, together with a biocompatible polymer matrix, may be useful for the controlled local delivery of chemotherapeutic agents to treat solid tumors in the brain and peritoneum (ovarian cancer) and in local applications to other solid tumors. These combined formulations are not limited to the use of paclltaxel and can be used with a wide variety of pharmacologically active ingredients including anti-infectives, immune suppressants, and other chemotherapeutics and the like.

Example 38 Stability of Capxol ™ After Reconstitution The lyophilized Capxol ™ in glass containers was reconstituted with sterile normal saline at concentrations of 1, 5, 10, and 15 mg / ml and stored at room temperature and refrigerated conditions. It was found that the suspensions remained homogeneous for at least three days under these conditions. The measurements of particle size carried out at various time points did not indicate any change in the size distribution. No precipitation was seen under these conditions. This stability is unexpected and solves the problems associated with Taxol®, which precipitates in approximately 24 hours after reconstitution at the recommended concentrations of 0.6-1.2 mg / ml.

In addition, the reconstituted Capxol ™ remained stable in the presence of different polymeric tube materials such as teflon, silastic, polyethylene, tigon, and other standard infusion pipe materials. This is a great advantage over Taxol® which is limited to polyethylene infusion sets and glass infusion bottles.

Example 39 Unit Dose Forms for Capxol ™ Capxol ™ was prepared as a freeze-dried powder in containers of an appropriate size. In this way a desired dose can be filled into a suitable container and lyophilized to obtain a powder essentially containing albumin and paclitaxel in the desired amount. These containers then reconstitute with normal sterile saline or other aqueous diluents to the appropriate volume at the point of use to obtain a homogeneous suspension of paclitaxel in the diluent. This reconstituted solution can be administered directly to the patient either by injection or infusion with standard infusion sets intravenously. In addition, Capxol ™ can be prepared as a frozen solution ready for use in bottles or bags that can be thawed at the time of use and simply administered to the patient. This avoids the step of lyophilization in the process of manufacture.

-J **** ^ - ^ - 'tmá t má ^^ á h á M? I It is very surprising that when Capxol ™ and Taxol® are administered to rats in equivalent doses of paclitaxel, a much higher degree of Myelosuppression results for the group with Taxol® compared to the group with Capxol ™. This may result in lower incidences of infection and episodes of fever (eg, febrile neutropenia). You can also reduce the cycle time between treatments which is currently 21 days. With the use of pharmaceutical compositions prepared according to the present invention, this cycle time can be reduced to two weeks or less allowing a more effective treatment for cancer. Thus, the use of pharmaceutical compositions prepared according to the present invention can provide a substantial advantage over Taxol®.

Example 40 Oral Drug Delivery Taxol® is very poorly absorbed by the oral route. Particle formulations such as Capxol ™ can greatly improve the absorption of drugs such as paclltaxel. In addition, the formulations of the invention of paclitaxel prepared through a microcrouslon / evaporation process are useful for the oral absorption of drugs. The use of surfactants in combination with these formulations surprisingly improves the oral bioavailability of these drugs. The use of lipids, surfactants, enzyme inhibitors, permeation enhancers, ion pair-forming agents, metabolism inhibitors and the like was found to surprisingly increase the oral absorption of the paclitaxel formulations of the invention. Examples of ion pair-forming agents include but are not limited to trichloroacetate, trichloroacetate sallcylate, naphthalenesulfonic acid, glycine, bis-N, N-dibutylaminoethylene carbonate. Examples of membrane permeation enhancers include, but are not limited to sodium caprate, acyl glycerides, chickenxietllenalqulletersacil-carnltines, sodium cholate, sodium taurocholate, sodium taurodihydrofusidate, EDTA, sodium salicylate, and sodium methoxlsallcylate. A list without limitation of surfactants and lipids that can be used for the formulations of the invention have been described herein.

EXAMPLE 41 Mode of Administration of Capxol ™ and Formulation of the Invention of Other Drugs The formulations of the invention can be administered by intravenous infusion, intravenous bolus, intraperitoneal injection, intra-arterial injection, intra-portal injection, epithelial embolization, intratumoral injection or implantation, injection Ntraurethral or ontophoresis, intramuscular injection, subcutaneous injection, intrathecal injection, inhalation of dry powder or blistered fluid, and the like.

Example 42 Use of Capxol ™ to Direct it to Angiogenic Vasculature Angiogenesis has been implicated as a causative and / or exacerbating factor in the progression of diseases such as cancer, rheumatoid arthritis, and retinopathy. It has been surprisingly found that Capxol ™ can reverse or reduce the severity of rheumatoid arthritis as well as cure tumors in animal models. Therefore it is possible that Capxol ™ has antianglogenic activity. To make Capxol ™ even more effective, it is possible to direct it to the angiogenic vasculature by binding appropriate peptides to Capxol ™. An example of said peptide is RGD (arginine-glycine-aspartic acid). Many other peptides with similar activity can be bound to Capxol ™ or other drugs prepared by the processes of the invention, for targeted therapies. The peptide / Capxol ™ can be administered by conventional means to patients who need it.

Example 43 Use of Capxol ™ for Treatment of Liver Disease End-stage hepatocellular carcinoma and other liver cancers can be treated by administering Capxol ™ intraportally. Embolization directly into the liver greatly improves the range of the dose to the liver. In addition, much higher doses than conventional Taxol® can be used to treat the disease more efficiently. Also, suitable targeting agents such as proteins or peptides that are localized in liver tissue can be combined with Capxol ™ for better therapeutic efficiency.

Example 44 Toxicity / Myelosuppression Study of Paclitaxel - Comparison of Taxol® and Capxol ™ for a Study of Administration of a • Single dose in rats A summary of a preclinical study is presented below: Schedule: 1X, intravenous infusion of a single dose (day 1) 15 Animals: Sprague Dawley rats, 40 males, 40 females 5 rats / of each sex per group Weight: 300 ± 50 g Study duration: 15 days Treatment Groups: Taxol® (1 carrier + 3 treated groups 20 Capxol ™ (1 carrier * + 3 treated groups) Dose: Taxol® (0, 3, 6, and 9 mg / kg) Capxol ™ (0, 6, 9 and 12 mg / kg) Dose Concentration: 0.6 mg / ml (all rats) Volume of Dosage: Taxol® (15, 5, 10, 15 ml / kg ) 25 Capxol ™ (20, 10, 15, and 20 ml / kg) ^ | J ^^ É ^^ g &^ ^ ^^^ Infusion Rate: Approximately 0.75 ml / hr (all rats) Dosage Route: Infusion I.V., tail vein Obs. Clinics: 1X / day Clinical Trajectory: days 0 (before treatment), 1, 3, 7, 11, . Make standard list for rat Tox NCI Branched Body Weights: Days -1, 1, 3, 8 and 15 (* The carrier is prepared by a process identical to that described in the manufacturing section, with the exception that paclitaxel is omitted ).

Example 45 Study of Pilot Myelosuppression (Haematological Toxicity) Before starting the formal study, a pilot study with 3 rats in the Capxol ™ group and three rats in the Taxol® group were performed to determine the results. The doses used were 5 mg / kg with a dose volume of 7 ml / kg. The dose was given as an intravenous bolus through the vein of the tail. The results of this study are summarized in the graph (see Figure 3) which shows the percentage change in WBC accounts (an indicator of myelosuppression) for each formulation as a function of time.

Conclusions of the Pilot Myelosuppression Stage: ^^^^^^^^^^^^^ ugly ^^^^ U The data show significantly lower WBC counts (average + SD) in the Taxol® group compared to the Capxol ™ group, indicating a higher degree of myelosuppression for Taxol® (maximum WBC suppression of> 70% for Taxol®, maximum WBC suppression of <30% for Capxol ™). The analysis of the data shows a statistically significant difference (p <0.05) between the two groups for all the data points except for day 0, 13 and 14. In addition, the normal levels of WBC were recovered in 6 days in the group that received Capxol ™, while 14 days were required for recovery of normal WBC levels in the Taxol® group. This indicates a significantly reduced hematological toxicity for Capxol ™. If similar results are seen in human clinical trials, these data may suggest that the cycle time (currently 3 weeks for Taxol® between subsequent cycles of treatment can be significantly reduced (possibly to 2 weeks, or up to 1 week or less when Capxol ™ is used).

Example 46 Pilot Study of Antitumor Efficacy Before starting the previous study, a pilot study with Capxol ™ was conducted to determine the ranges and efficacy of the targeted dose. Mice (n = 10) were implanted subcutaneously with a breast tumor MX-1 and treatment was initiated when the tumor reached approximately 150-300 mg in size. This occurred on day 12 and treatment started on day 13 after the initial seeding. Capxol ™ was reconstituted in saline to obtain a colloidal solution of paclltaxel nanoparticles. Tumor-bearing mice (n = 5) were treated with reconstituted Capxol ™ at a dose of 20 mg / kg (denoted by VIV-1), administered by a bolus injection into the vein of the tail every day for 5 consecutive days. The control tumor-bearing group (n = 5) received only saline at the same time. The size of the tumors was verified as a function of time. The control group showed a tremendous increase in tumor weight up to an average of more than 4500 mg and all the animals in these groups were sacrificed between day 28 and day 39. The treatment group on the other hand showed a surprising efficacy and all the animals did not have measurable tumors at day 25. The animals in this group were all sacrificed at day 39 in which they showed no evidence of recurrence and no evidence of the tumor. The results are shown in Figure 4. 20 Conclusion: This study showed surprising antitumor activity for Capxol ™. Thus, the anti-tumor activity for paclitaxel is retained in the Capxol ™ formulation. This studio Indicates that intravenous administration of nanoparticles of ^ gg || jg | j | j iMMHtMiiiikMriiMI paclitaxel can be effective in administering the drug in the soluble form. In this way, Capxol ™ shows efficacy and potent antitumor activity without the toxic effects seen in the formulation of Taxol® with commercialized and approved creamur content. Note: Based on data from the literature, and in the experience of SRI (Southern Research Instltute) scientists, it has been established that the maximum tolerated dose (MTD) of paclitaxel dissolved in a diluent 12 (creamfor / ethanol, which is the same diluent used in Taxol® is 22.5 mg / kg for this particular genus of athymic mice.This result was obtained by dissolving paclltaxel at a much higher concentration in diluent 12 compared to Taxol® (6 mg / ml in creamfor / ethanol) This is done to minimize the amount of cream / ethanol administered to mice to avoid vehicular toxicity At a dose of 22.5 mg / kg, paclitaxel in diluent 12 has an efficiency similar to that of Capxol ™ previous.

Example 47 Treatment of Rheumatoid Arthritis in an Animal Model with Paclitaxel Nanoparticles The model of collagen-induced arthritis in the rat Louvaln was used to test the therapeutic effect of paclltaxel nanoparticles on arthritis. The sizes of the legs of the experimental animals were monitored to evaluate the seriousness of the arthritis. -s £ aiZZ "'.aal? Sc" After the arthritis developed completely (usually -9-10 days after the collagen injection), the experimental animals were divided into different groups to receive either the nanoparticles with paclitaxel 1 mg / kg qod, or nanoparticles with paclitaxel 0.5 mg / kg + prednisone 0.2 mg / kg qod (combination treatment) intraperitoneally for 6 doses, then one dose per week for 3 weeks. The sizes of the legs were measured at the beginning of the treatment (day 0) and each time the drug was injected. One group received only normal saline as control. At the end of the experiment, the group that received nanoparticles with paclitaxel achieved a 42% reduction in paw size, the combination treatment group showed a 33% reduction in paw size, while the control group had an increase about 20% of the size of the leg. The original size of the paw before the arthritis was induced was 50%. These results are shown in Figure 2. In conclusion, nanoparticles containing paclitaxel demonstrated therapeutic effect on arthritis. To avoid the side effects of long-term use of both paclitaxel and steroids, it is probably best to choose a combination treatment to obtain a similar effect but only half the dose of each drug.

Example 48 Effect of Capxol ™ on Arterial Restenosis The proliferation of abnormal vascular smooth muscle (VSMP) is associated with cardiovascular disorders such as atherosclerosis, hypertension and most endovascular procedures. Abnormal VSMP is a common complication of percutaneous transluminal coronary angioplasty (PTCA). The incidence of chronic restenosis resulting from VSMP after PTCA has been reported as high as 40-50% in 3-6 months. The high incidence of vascular reocclusion associated with PTCA has led to the development of an in vivo animal model of restenosis and the search for agents to prevent it. The following study describes the use of Capxol ™ to inhibit restenosis after intimate artery trauma. 15 Male Sprague-Dawley rats (Charles Rlver) weighing 350-400 gm were anesthetized with Ketamin and Rompun and the right common carotid artery exposed by a distance of 3.0 cm. Adherent tissue was removed from the path to allow two DIETRICH bulldog micro clips to be placed about 2 cm apart around the carotid without causing any injury by tightening the vagus or the associated upper cervical ganglion and the sympathetic cord. There are no branches along this segment of the vessel. A 30 gauge needle attached to a 3-way valve is inserted first and then pulled out from the lower end of the isolated segment ^ 9 & ^ á? - »ñv to make a hole in the wall of the vessel, and then Inserted in the upper end for Injection. 2-3 ml of phosphate-buffered saline were injected to rinse all the blood within the isolated segment after the 3-way valve is turned towards the other connection towards a regulated source of compressed air. A light stream of air (25 ml per minute) is passed along the lumen of the vessel for 3 minutes to cause drying of the endothelial lesion. The segment is then filled with saline solution before removing the needle from the vessel. The needle holes in the vessel wall are carefully cauterized to prevent bleeding. A swab moistened with saline can also be used to press on the needle holes to stop bleeding. The skin closes with 7.5 mm metal clips and washed with Betadine. All animals received the surgery described above and then sacrificed at day 14 after surgery. The carotid artery on each side was removed for pathological examination. The non-operated side will serve as self-control The experimental groups received different treatments as follows: Group 1: Treatment with high dose of Capxol ™: Paclltaxel 5 mg (p / 100 mg Human Albumin) / kg / week, IV 25 Group 2: Treatment with low dose of Capxol ™: Paclitaxel 1 mg (p / 20 mg Human Albumin) / kg / week, IV. Group 3: Control drug carrier. Human Albumin 100 mg / kg / week IV.

Carotid artery biopsy samples were preserved in Formalin and then cross sections (8 μm) are cut from paraffin blocks and stained with hematoxylin and eosin. The cross-sectional areas of the blood vessel layers (intima, media, and adventisia) were quantified. tf The injured carotid arteries in the control group showed a surprising accumulation of smooth muscle intimal cells and a VSMC invasion of basement membrane. The general thickness of the wall of the carotid artery doubled. The treatment groups showed a statistically significant decrease in intimal wall thickness compared to the control.

Example 49 20 In vivo Addressing of Nanoparticles By Incorporating certain targeting moieties such as proteins, antibodies, enzymes, peptides, oligonucleotides, sugars, polysaccharides, and the like, within the nanoparticle protein coat, it is possible to direct them to specific places in the body. This ability to - ~ * L ~ * - - ^ 2 ^ _. - ^ - - ^ * afa - ** ^ ... .., ** ** * * * address can be used for therapeutic or diagnostic purposes.

Example 50 Addressing Polymer Covers Antibodies The nature of the polymer coatings of certain aspects of the invention permits the binding of polyclonal or monoclonal antibodies to the polymeric shell, or the incorporation of antibodies within the polymer shell. The antibodies can be incorporated into the polymeric shell as the polymeric microcapsule shell is being formed, or the antibodies can be attached to the polymeric shell after preparation of the polymeric shell. Standard protein immobilization techniques can be used for this purpose.

For example, with protein microcapsules prepared from a protein such as albumin, a large number of amino groups in the albumin lysine residues are available for the binding of suitably modified antibodies. As an example, antitumor agents can be delivered to a tumor By incorporating antibodies against the tumor into the polymeric shell as it is being formed, or antibodies against the tumor can be attached to the polymeric shell after the preparation thereof. As another example, the gene products can be delivered to specific cells (e.g. epatosltos or certain cells of origin in the bone marrow) incorporating Antibodies against receptors on the cells directed into the polymeric shell as it is being formed or antibodies against receptors on the targeted cells can be attached to the polymeric shell after preparation thereof. In addition, monoclonal antibodies against nuclear receptors can be used to direct the encapsulated product to the nucleus of certain cell types.

Example 51 Addressing an Immunosuppressive Agent to Transplanted Organs Using Intravenous Supply of Polymeric Covers Containing Such Agents Immunosuppressive agents are used extensively after organ transplantation for the prevention of rejection episodes. In particular, cyclosporine, a potent immunosuppressive agent, prolongs the survival of allogeneic transplants involving skin, heart, kidney, pancreas, bone marrow, small intestine, and lung in animals. It has been shown that clclosporin suppresses some humoral immunity and to a large extent, cell-mediated reactions such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis, and graft-versus-recipient disease in many animal species. a variety of organs. Successful heart, liver, and kidney allogeneic transplants have been performed in humans using cyclosporine. Cyclosporin is currently administered orally, either as capsules containing a solution of chlorosporine in alcohol, and oils such as corn oil, polyethoxylated glycerides and the like, or as a solution in olive oil, chicken-oxygenated glycerides and the like. It is also administered by intravenous injection, in which case it is dissolved in a solution of ethanol (approximately 30%) and cremafor (polyoxyethylene castor oil) which must be diluted 1:20 to 1: 100 in a normal saline solution. or 5% dextrose before injection. In comparison with an intravenous infusion (i.v.), the absolute bioavailability of the oral solution is approximately 30% (Sandoz Pharmaceutical Corporation, Publication SDI-Z10 (A4), 1990). In general, the i.v. of ciclosporin suffers from similar problems that the i.v. currently practiced Taxol®, ie anaphylactic and allergic reactions that are believed to be due to cremafor. The supply carrier used for the formulation i.v .. In addition, the supply Intravenous drugs (eg, clclosporin) encapsulated as described herein avoid dangerous peak blood levels immediately after drug administration. For example, a comparison of the formulations currently available for cyclosporin with the encapsulated form previously described of clclosporin showed a decrease Fivefold peak blood levels of clclosporin immediately after injection. In order to avoid problems associated with cremafor, the clclosporin contained within the polymeric casings as described above can be delivered by iv injection.It can be dissolved in a biocompatible oil or a number of other solvent after which it can be dispersed within polymeric covers by means of sound formation as described above. In addition, an important advantage for supplying cyclosporin (or other immunosuppressant agent) in polymeric casings has a local targeting advantage due to the absorption of the material injected by the RES system in the liver. This can, to some extent, prevent slstic toxicity and reduce doses effective due to local management.

Example 52 Use of Capxol ™ for Antibody Targeting Monoclonal antibodies against various tumors or tissues can be linked to Capxol ™ to allow direct targeting of Capxol ™ or other drugs prepared by the process of the invention to sites of the disease. For example, antibodies against ovarian cancer linked to Capxol ™ and administered intraperitoneally will have a great benefit to the 25 patients with ovarian cancer.

Example 53 Intravenous Administration of Therapeutics Intravenous administration of therapeutics, for example, drugs, imaging agents, and the like, predisposes the therapeutic to at least one pass through the liver. As the therapeutic is filtered through the liver, a significant portion of that therapeutic is absorbed sequestered by the liver, and after that, it is not available for systemic distribution. On the other hand, once absorbed by the liver, it is very likely to be metabolized, and the resulting metabolic by-products very often have general systemic toxicities. By encapsulating the drug or other therapeutic agent in a coating according to the Invention (for example using a protein such as albumin), the sequestration by the liver when administered intravenously is relieved. Albumin, for example, is known to pass through the liver and is usually distributed through the patient. In this way, the sequestration of albumin by the liver does not occur to the same degree as toxic compounds or drugs that have hepatic receptors (or other mechanisms) that initiate processes that result in their removal from the bloodstream. By protecting the therapeutic with a coating of a biocompatible polymer (for example, a coating of human albumin), in the drug is then diverted without going through the liver and is generally distributed throughout all organ systems. In accordance with one aspect of the present invention, a novel method for evading the liver is provided, which comprises encapsulating a drug in a human liver albumin (essentially a physiological component) in this manner, plus the drug becomes available for systemic therapy In addition to the increased availability of the drug, there is a decrease in the production of metabolic byproducts of hepatocellular drug degradation. As much as the increase in the evasion of the liver and the decrease in the byproducts of drug metabolism provides a synergistic improvement in the efficacy of the drug in general. This improved efficacy extends to all drugs and materials that are encapsulated in albumin or human.

Example 54 Reduction of Myelosuppressive Effects (Haematological Toxicity) and General Drug Toxicity Several chemotherapeutic drugs have dose-limiting toxicity due to their myelosuppressive effects. Taxol® (paclitaxel) is a classic example of this drug. When administered in its currently approved formulation of cremafor / ethanol, Taxol® produces myelosuppressive effects that limit the repeated administration of the drug and prevent » ^ -jks & ^ .s. retreatment of a patient for at least 3 weeks in order to allow the patient's blood counts to return to normal. It has been postulated that due to the biocompatible, non-toxic nature of the drug carrier of certain aspects of the present invention, viz. human albumin, the toxic side-effect of myelosuppression can be greatly reduced. Sprague Dawley rats were supplied with paclitaxel in a commercial formulation (Taxol®) or were prepared by a method of the invention as nanoparticles with albumin, both formulations were administered by tail vein injection. mg / kg was administered for Taxol®, while two dose levels of 5 mg / kg and 12 mg / kg were administered for the formulation of the invention. The white blood cell counts of the rats were monitored daily after administration as an index of myelosuppression. For Taxol® (5 mg / kg) it was found that the WBC accounts fell by 47.6% and 63.5% on day 1 and day 2 after administration, respectively, while for the formulation of the invention at 5 mg / kg. kg, WBC accounts increased by 14.7% and 2.4% on day 1 and day 2, respectively For a higher dose of the formulation of the invention at 12 mg / kg, the WBC accounts increased 6.5% and 3.6% on day 1 and day 2, respectively. .- --g-; - * "- - ^^^^^^ - * ~ - ... j ek ?. ... > ... >. ^. ^ > -at__ > ^ .., These results indicate that short-term myelosuppression is greatly reduced by administering the drug in the formulation of the present invention.Another indicator of general toxicity is the body weight of the animal.The body weights of the rats were also monitored after administration of paclitaxel. at a dose of 5 mg / kg, Taxol® resulted in a reduction of body weight by 10.4% in 3 days after administration, while the same dose of paclitaxel administered in the formulation of the Invention resulted in only one 3.9% decrease in body weight, indicating the widely reduced toxicity of the formulation of the invention It is very surprising that when the formulation of the invention and Taxol® are administered to rats at doses equivalents of paclitaxel, a much higher degree of myelosuppression results for the Taxol® group compared to the group with the formulation of the invention. This may result in lower incidences of infectious episodes and fever (eg, febrile neutropenia). It can also reduce the cycle time between treatments which is currently 21 days for Taxol® with the use of pharmaceutical compositions prepared according to the present invention, this cycle time can be reduced to 2 weeks, 1 week, or less allowing a treatment more effective for cancers. Of this Thus, the use of pharmaceutical compositions prepared from according to the present invention can provide a substantial advantage over Taxol®.

Example 55 Administration of Bolus Dose of Nanoparticle Formulation The anti-cancer drug, paclitaxel, in its commercial formulation BMS with cremafor / ethanol, can not be administered as an intravenous bolus. This due to the high toxicity of the carrier which results in severe anaphylactic reactions and requires that patients receive the drug that is premedicated with steroids, antlestamines, and the like. The Taxol® formulation is administered as an intravenous infusion lasting from 1 hour to 24 hours. In comparison, formulations according to the present invention, due to the use of a non-toxic carrier, can be administered to a patient easily as an intravenous bolus (i.e., in a period less than 1 hour) without the toxicity problems seen in the Taxol® that is used clinically today. The effective dose of paclitaxel for a patient is typically between 200-500 mg, depending on the body weight of the patient or the body surface. Taxol® has to be delivered at a final dose concentration of 0.6 mg / ml, requiring large volumes of infusion (typically in the range of approximately 300-1000 ml). In comparison, ^^^^^^^^ J ^^^^^^^^^^^^^^^^^^^^^ gi ^^^^^^^^^^^^^^ The formulations of the invention do not have these limitations and can be administered at a desired concentration. This allows clinicians to treat patients through a rapid intravenous bolus that can be administered in as little as a few minutes. For example, if the formulation of the invention is reconstituted at a dose concentration of 20 mg / ml, the infusion volume for a total dose of 200-500 mg is only 10-25 ml, respectively. This is a great advantage in clinical practice.

Example 56 Reduction in Paclitaxel Toxicity in the Nanoparticle Formulation in Comparison with Taxol® It is well known that the anti-cancer drug paclitaxel in its commercial formulation (ie Taxol®) has a high toxicity resulting in severe anaphylactic reactions and it requires that patients receiving the drug be premedicated with steroids, antihistamines and the like. The toxicity of Taxol® was compared with the nanoparticle formulation of the present invention. Thus, the formulations were injected intravenously through the tail vein of C57BL mice at different dose levels and the toxic effects were monitored by a general observation of mice after injection. • - w v For Taxol®, a dose of 30 mg / kg was uniformly lethal within 5 minutes of intravenous administration. For the same dose, the formation of • nanoparticles according to the invention showed no apparent toxic effect. The formulation of nanoparticles at a dose of 103 mg / kg showed some reduction in the body weight of the mice, but even this high dose was not lethal. The doses of approximately 1000 mg / kg, 800 mg / kg and 550 mg / kg were all lethal but there was a difference in the time of the lethality, which varied between a few hours to 24 hours. Thus, the lethal dose of the formulation of the invention is greater than 103 mg / kg but less than 550 mg / kg. It is therefore clear that the lethal dose of the paclitaxel formulation is substantially higher than that of Taxol®. This has great significance in clinical practice where higher doses of chemotherapeutic drugs can be administered for more effective oncolytic activity with greatly reduced toxicity.

EXAMPLE 57 Determination of LD ^ in Mice for Taxol® Produced by Means of Methods of the Invention and Taxol After Single Intravenous Administration The LD50 of Capxol ™, Taxol®, and its carriers was compared, after only one intravenous administration. HE | j ßSj ^^^ á used a total of 48 CD1 mice. The Paclitaxel doses of 30, 103, 367, 548, and 822 mg / kg were tested for Capxol ™, and the doses of 4, 6, 9, 13.4 and 20.1 mg / kg of paclitaxel for Taxol®. Doses for human albumin, the carrier for Capxol ™, was only tested at 4.94 mg / kg (corresponding to a dose of 548 mg / ml of Capxol ™) human albumin is not considered toxic to humans. The doses tested for the Taxol® carrier (Cremophor EL®) were 1.5, 1.9, 2.8 and 3.4 ml / kg which corresponds to doses of 9, 11.3, 16.6 and 20.1 mg / kg of paclltaxel, respectively. Three to four mice were tf dosed with each concentration. The results indicated that paclltaxel administered in Capxol ™ is less toxic than Taxol® or Taxol® administered alone. LD50 and LD10 for Capxol ™ were 447.4 and 371.5 mg / kg paclitaxel, 7.53 and 5.13 mg / kg of paclltaxel in Taxol®, and 1325 and 794 mg / kg of Taxol® carrier, (corresponding to a dose of 15.06 and 9.06 mg / kg of paclltaxel). In this study, the LD50 for Capxol ™ was 59 times greater than Taxol® and 29 times greater than the Taxol® carrier alone. The LD10 for paclitaxel in Capxol ™ was 72 times greater than paclitaxel in Taxol® Summary of all these data in this study suggests that the Taxol® carrier is responsible for much of the toxicity of Taxol®. It has been shown that mice receiving Taxol® and the Taxol® carrier showed classic signs of severe hypersensitivity. Indicated by a bright pink skin coloration just after administration. No such reaction was seen for the Capxol ™ and Capxol ™ carrier groups. The results are presented in Table 2.

Table 2 These high doses of Capxol ™ were administered as bolus injections and represented the equivalent of approximately 80 - 2000 mg / m2 dose in humans. The LD10 or the maximum tolerated dose of Capxol ™ in this study is ^^^ gggfes ^^ ij ^ equivalent to approximately 1000 mg / m2 in humans. This is significantly higher than the approved human dose of 175 mg / m2 for Taxol®. To our surprise, it was found that the carrier, Cremophor / Ethanol, alone caused severe hypersensitivity reactions and death in several dose groups of mice. The LD50 data for the Taxol® carrier alone shows that it is considerably more toxic than Capxol ™, and significantly contributes to the toxicity of Taxol®. It is not clear in the literature, the cause of hypersensitivity, however, based on these data, it is believed that HSR's can be attributed to the Taxol® carrier. Example 58 Determination of LD ^ in Capxol ™ Taxol® Mice After Multiple Intravenous Administration The LD50 of Capxol ™ and Taxol® and their carriers were compared after multiple intravenous administrations. A total of 32 CD1 mice were used. Capxol ™ with 20 paclltaxel doses of 30, 69 and 103 mg / kg were administered daily for five consecutive days. Taxol® with paclltaxel doses of 4, 6, 9, 13.4 and 20.1 mg / kg were administered daily for 5 consecutive days. Four mice were dosed with each concentration. The results are presented in Table 3.

Table 3 Multiple Intravenous Administration The results indicated that Capxol ™ is less toxic than Taxol®. The LD50 and LD10 of Capxol ™ were 76.2 and 64.5 mg / kg of paclitaxel, respectively, compared to 8.07 and 4.3 mg / kg of paclitaxel in Taxol®, respectively. In this study, the LD50 for Capxol ™ was 9.4 times higher than for Taxol®. The LD10 for Capxol ™ was 15 times higher for Capxol ™ than for Taxol®. The results of this study suggest that Capxol ™ is less toxic than Taxol® when administered in multiple doses at daily intervals. ....? ^ i **? i ^ tU mt Example 59 Toxicity and Efficacy of Two Formulations of Capxol ™ and Taxolc A study was conducted to determine the efficacy of Capxol ™, Taxol® and the Capxol ™ carrier in afflicted NCr-nu female mice implanted with MX-1 human breast tumor fragments. 10 Intravenous injections were given to 5 groups W of mice with formulations of Capxol ™ VR-3 or VR-4 at doses of 13.4, 20, 30, 45 mg / kg / day for 5 days. Groups of 5 mice were also each supplied with intravenous injections of Taxol® at doses of 13.4, 20 and 30 mg / kg / day for five days. A control group of ten mice was treated with an intravenous injection of Capxol ™ control carrier (Human Albumin, 600 mg / kg / day) for 5 days. The evaluation parameters were the number of complete tumor regressions, the average duration of the complete regression, the survivors tumor-free, and tumor recurrenclas. Treatment with the Capxol ™ VR-3 formulation resulted in complete tumor regressions at all dose levels. The two highest doses resulted in 100% of survivors after 103 days. The formulation with Capxol ™ VR-4 resulted in a complete tumor regression in the three highest dose groups, and 60% regressions in 13.4 mg / kg / day. Survival portions after 103 days were somewhat less than with the VR-4 formulation. Treatment with Taxol® at 30, 20 and 13.4 mg / kg / day resulted in survival rates of 103 days of 40%, 20% and 20% respectively. The treatment with the control carrier had no effect on tumor growth and the animals were sacrificed after 33 to 47 days. The results are presented in Table 4. Table 4 tf CR = Complete tumor regression; TFS = Tumor-free survivor; TR = Recurrence of tumor; DCR = days of complete regression These unexpected and surprising results show an increased efficacy for the two formulations of Capxol ™ compared to Taxol®. In addition, higher doses of paclitaxel are achieved in the Capxol ™ groups due to lower toxicity of the formulation. These high doses were administered as bolus injections.

Example 60 Blood Kinetics and Tissue Distribution in 3H-Taxol® and Capxol ™ After a Single Intravenous Dose in Rat Two studies were conducted to compare the pharmacokinetics and tissue distribution of 3H-paclitaxel formulated in Capxol ™ and Taxol® Injection Concentrate. Fourteen male rats were Injected intravenously with 10 mg / kg of 3H-Taxol® and 10 rats with 4.9 mg / kg. Ten male rats were injected intravenously with 5.1 mg / kg of 3H-Capxol ™ in the previous study. The levels of both total radioactivity and paclitaxel decrease biphasically in the blood of rats after _- i ..? ~ je? ja * of 5 mg / kg of bolus IV dose of either 3H-Taxol® or 3H-Capxol ™. However, the levels of both total radioactivity and paclitaxel are significantly lower after administration of 3 H-Capxol ™ after a similar dose of 3 H-Taxol®. This lower level is distributed more quickly out of the blood. The HPLC profile of the blood shows a similar pattern of metabolism for highly polar metabolisms for both 3H-Capxol ™ and 3H-Taxol®. However, the rate of metabolism appears significantly lower for 3H-Capxol ™ as 44.2% of the blood radioactivity remains as paclltaxel 24 hours after the dose versus 27.7% of 3H-Taxol®. The excretion of radioactivity occurs only minimally in the urine and predominantly in the faeces for 3H-Capxol ™ which is similar to the reported excretion patterns for 3H-Taxol®. Blood kinetics for total radioactivity and after IV administration of 3H-Capxol ™ or 3H-Taxol® at 5 mg / kg are presented in Table 5.

Table 5 Treatment AU Q_24 Co c T1 / 2ß (mg Extrapolation observed observed (hr) eq.hr/ (mg eq / (ml) (mg eq / (ml) (hr) ml) Tissue radioactivity levels are higher after administration of 3H-Capxol ™ than in the administration of 3H-Taxol® for 12 of 14 tissues. The ppm tissue / blood ratios are highest in all tissues for animals dosed with 3H-Capxol ™ since blood levels are lower. This supports the rapid distribution of 3H-Capxol ™ from the blood to the tissue suggested by the blood kinetics data. The 3H-paclltaxel formulated in Capxol ™ shows a pharmacokinetic profile similar to 3H-paclitaxel formulated in Taxol® for the injection concentrate, but the ppm tissue / blood concentrations and the metabolism rates differ significantly. A significantly lower level of total radioactivity for animals treated with Capxol ™ than for animals treated with Taxol® in the blood sample 2 minutes after administration indicates that 3H-Capxol ™ is distributed more rapidly out of the blood. However, the rate of metabolism seems significantly slower for 3H-Capxol ™ since 44% of the blood reactivity remains as paclltaxel 24 hours after administration versus 28% for 3H-Taxol®. This result for Capxol ™ is surprising and provides a novel formulation to achieve sustained activity of paclltaxel compared to Taxol®. Taken together with the high local concentrations, this enhanced activity should result in increased efficacy for the treatment of primary tumors of metastases in organs with high local concentrations. The tissue distributions are presented in Table 6 below. The data represent the standard deviations on average of 10 rats in each group (Capxol ™ and Taxol®).

Table 6 Radioactive Wastes in Male Rat Tissues. Expressed as ppm after a single intravenous dose of 3H-Capxol ™ and 3H-Taxol® at 5 mg / kg Capxol TM Taxol® Sample Values ± SD Values ± SD Average Average Brain 0.106 0.008 0.145 0.020 Heart 0.368 0.063 0.262 0.037 Lung 1.006 0.140 0.694 0.057 Liver 1.192 0.128 1.37 0.204 Kidney 0.670 0.110 0.473 0.068 Muscle 0.422 0.120 0.386 0.035 Tract Gl 0.802 0.274 0.898 0.243 Testicles 0.265 0.023 0.326 0.047 Pancreas 0.963 0.357 0.468 0.070 Corpse 0.596 0.070 0.441 0.065 Bone 0.531 0.108 0.297 0.051 Spleen 0.912 0.131 0.493 0.070 Prostate 1.728 0.356 1.10 0.161 Vesicle Seminal 1.142 0.253 1.20 0.237 Blood 0.131 0.010 0.181 0.020 Plasma 0.131 0.012 0.196 0.026 The data show significantly higher levels of Capxol ™ accumulation in various organs when compared to Taxol®. These organs include prostate, pancreas, kidney, lung, heart, bone and vessel. In this way Capxol ™ may be more effective in Taxol® in the treatment of cancers of these organs at equivalent levels in paclltaxel.

The levels in the prostate tissue are of particular interest in the treatment of prostate cancer. This surprising and unexpected result has implications for the treatment of prostate cancer. Table 7 below shows the data for the individual rats (10 in each group) showing increased accumulation of paclitaxel in the prostate for Capxol ™ compared to Taxol®. The basis for localization within the prostate could be a result of the particle size of the formulation (20-400 nm), or the presence of albumin protein in the formulation which can cause localization within the tissue through the prosthetic tissue. of specific membrane receptors (gp 60, gp 18, gp 13, and the like). It is also likely to be biocompatible with others, biodegradable polymers other than albumin may show specificity to certain tissues such as The prostate results in a high local concentration of paclitaxel in these tissues as a result of the properties described above. Such compatible materials are contemplated for the paclltaxel of this invention. A preferred embodiment of the composition to achieve local concentrations paclltaxel high of the prostate is a formulation containing paclitaxel albumin with a size of paclitaxel in the range of 20-400 nm, and free of cremophor. This modality has also shown that it results in concentrations of higher levels of paclitaxel in the pancreas, kidney, lung, heart, bone and spleen compared to Taxol® in equivalent doses.

Table 7 Data for 10 Rats in Each Group Dose 5 mg / kg paclitaxel Capxol ™ Taxol® 1.228 1.13 2.463 1.04 1.904 0.952 1.850 1.42 1.660 1.31 1.246 1.08 1.895 1.03 1.563 0.95 1.798 0.94 1.676 1.18 Average 1.728 Average 1.103 SD 0.36 SD 0.16 These data show that the location of Capxol ™ in the prostate is approximately 150% compared to with Taxol®. This Unexpected location of paclltaxel in the prostate in the Capxol ™ formulation can be exploited for the delivery of other pharmacologically active agents to the prostate for the treatment of other disease states affecting that organ, for example, antibiotics in a similar formulation in the treatment of prostatitis (inflammation and infection of the prostate), effective therapeutic agents for the treatment of benign prostatic hypertrophy can be formulated in a similar manner to achieve high local delivery. Similarly, the surprising results that Capxol ™ provides high local concentrations in the heart can be exploited for the treatment of restenoses as well as atherosclerotic disease in coronary vessels. It has been shown that paclitaxel has therapeutic effect in preventing stenosis and atherosclerosis, and thus Capxol ™ is an ideal form of paclitaxel for these conditions. It has also been shown that polymerized albumin binds preferentially to inflamed endothelial vessels possibly through gp60, gp18 and gp13 receptors. Example 61 Blood Kinetics and Paclitaxel Tissue Distribution After Multiple Intravenous Dosage Levels of Capxol ™ in Rat 20 The study using 3H-Capxol ™ was supplemented by treating four additional groups of rats with a single bolus dose of 9.1 mg / kg, 26.4 mg / kg, 116.7 mg / kg, and 148.1 mg / kg of paclitaxel in Capxol ™. Blood was collected from the tail vein and the AUC0.24 was calculated. At 24 hours, the samples were collected ^^^^^? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ JMB H ^^^^ ^^ ^^ i ^ g ^^^^^^^^^^ ^^^^^^^ M l of blood, extrayeron, and the extract was injected into HPLC to determine the level of blood generating compound. The blood kinetics for total radioactivity and paclltaxel after IV administration of 3H-Capxol ™ are presented in Table 8.

Table 8 As the dose of paclitaxel increased, the area under the curve increased proportionally. The level of the generating compound after 24 hours was increased by a factor of 8.5 (0.04 ppm - 0.34 ppm), going from the dose of 9 mg / kg to the dose of 148 mg / kg.

Example 62 Determination of Rat Toxicity of Capxol ™ and Taxol® After Single Intravenous Administration The objective of the study was to determine the toxicity of Capxol ™ after a single IV administration in female and male rats. Capxol ™ was administered to 6 male rats and 6 female rats at doses of 5, 9, 30, 90 and 120 mg / kg. The portion of animals from each dose group was euthanized and necropsied on Day 8. The remaining animals were necropsied on Day 31. The results of the animals treated with Capxol ™ were compared with the results of the normal saline solution and of the carrier control groups as well as with the results of the animals treated with Taxol® 5, 9 and 30 mg / kg. The animals were examined immediately after dosing, 1 hour and 4 hours after administration, and once daily thereafter. Blood was collected from each animal for serum and blood determination before euthanasia. Thirteen deaths occurred during the 30-day observation period. All 12 animals treated with Taxol® at a dose of 30 mg / kg of paclltaxel died on day 4. Only the animal treated with Capxol ™ died. The animal treated with Capxol received 90 mg / kg of paclitaxel and was found dead on day 15. No other animal treated with Capxol ™ died at the dose of 90 kg to 120 mg / kg, therefore it is believed that death does not occur. It is related to the treatment.

During the first four observation periods, piloerection and wobble movement were observed in most of the animals treated with Taxol®, possibly due to the alcohol content of the drug. Piloerection was noted in a few animals treated with Capxol ™. Taxol®-treated animals were observed at a dose of 30 mg / kg of paclitaxel with piloerection and lethargy and were found dead by day 4. No visible signs of toxicity were observed in the animals treated with Capxol ™, except for a few incidences of piloerection at dose levels of 90 mg / ml and 120 mg / ml. No abnormalities were reported in the animals treated with Capxol ™. The results of the general necropsy for day 8 and day 31 were normal. Changes related to the significant dose occurred in the organs male reproducers in animals treated with Capxol ™. A degeneration and vacuolation of the epithelial cells of the epididymal ducts, very often accompanied by multifocal interstitial lymphocytic infiltrate, were observed. There was an increased severe atrophy of seminiferous tubes seen in all 20 testicles as the dose of Capxol ™ increased. In the opinion of the pathologist, there were significant lesions observed in the male reproductive organs of the animals treated with 9, 30, 90 and 120 mg / kg of Capxol ™. These changes involved diffuse degeneration and testicular necrosis. 25 These changes were the most prevalent in animals that • "H - * - ^ - - -.-Z-.za." ....., .. ^ - Jt »'- XX-i ^ s ^^^^^^^^ iX- received higher doses of Capxol ™ No changes were seen in the testes of control animals without treatment, control animals with carriers, or those treated with Taxol® These results are unexpected and have significant therapeutic implications for the treatment of cancer dependent of hormone such as prostate cancer The removal of the testicles (orchiotomy) is a therapeutic test for the treatment of prostate cancer Capxol ™ represents a novel formulation in the treatment of this disease achieving high local concentration of paclitaxel in this place , through the sustained activity of the active ingredient, by reducing testicular function and without the toxic carrier cremophor.Treatment with Capxol ™ in this way allows the reduction of levels of testosterone and other androgenic hormones. Cerebral cortical necrosis occurred at the average dose level of the animals treated with Taxol®. This may explain the deaths of animals treated with even higher doses of Taxol®. No brain lesions were seen in the animals treated with Capxol ™. This lack of brain neurological toxicity is surprising and has significant implications both in the treatment of brain tumors and the ability to achieve high systemic doses ranging from 5 - 120 mg / kg in rats (equivalent to 30 - 700 mg / m2 of dose in humans).

P ** ^^ "J- *» -. .- -a * - To summarize, Capxol ™ was considerably less toxic than Taxol®, none of the animals with Taxol® survived at higher doses of 9 mg / kg With the exception of an incidental death at 90 mg / kg Capxol ™, all animals that received Capxol ™ survived doses up to and including 120 mg / kg There was an effect related to high dose Capxol ™ in the reproductive organs The male rats showed no toxic effect from the administration of Capxol ™ in doses up to and including 120 mg / kg.These high doses were administered as bolus injections and represent the equivalent of -. 30 - 700 mg / m2 dose in humans.

Example 63 Pharmacokinetic Data (PK) for the Nanoparticles of Cyclosporine (Capsorine IV) After Intravenous Administration (Comparison with the Sandimmune IV Formulation Currently Commercialized by Sandoz The cyclosporine nanoparticles (Capsorine IV) prepared as described above (Examples 13 and 14) were reconstituted in saline and administered to a first group of 3 Sprague Dawley rats per intravenous bolus A second group of 3 rats was supplied with Sandimmune IV, which contains cremaphor / ethanol, after dilution in saline Each group received the same dose of 2.5 mg / kg cyclosporine Blood samples were taken at times of 0, 5, 15, 30 (minutes), and 1, 2, 4, 8, 24, 36, and 48 (hours) Cyclosporine levels in blood were evaluated by HPLC and the Typical PK parameters were determined.PK curves showed a typical deterioration with time as follows: Deterioration with AUC Time, mg-hr / ml Cmax, ng / ml Capsorine IV 12,228 2,853 Sandimmune I. V. 7,791 2,606 In addition, due to the toxicity of the Sandimmune I.V. formulation, 2 out of 3 rats in that group died within 4 hours after dosing. Thus the formulation of nanoparticles (Capsorine I.V.) according to the present invention shows an AUC and non-toxicity compared to the commercially available formulation (Sandimmune I.V.). Example 64 Pharmacokinetic (PK) Data for Ciclosporine Nanoglots (Capsorine Oral) After Oral Administration in Comparison with Neoral (Formulation Currently Commercialized by Sandoz) The cyclosporine nanogotes prepared above were administered in orange juice, to a first group of 3 Sprague Dawley rats by oral gavage. A The second group of 3 rats was supplied with Neoral, a commercially available microemulsion formulation containing emulsifiers, after dilution in orange juice, also by oral gavage. Each group received the same dose of 12 mg / kg of ciclosporine in an identical volume of orange juice. Blood samples were taken at times 0, 5, 15, 30 (minutes), and 1, 2, 4, 8, 24, 36 and 48 (hours). Cyclosporine levels in blood were evaluated by HPLC and typical PK parameters were determined. The PK curves showed a typical deterioration with time as follows: tf Deterioration with AUC Time, mg-hr / ml Cmax, ng / ml Capsorine I.V. 3,195 887 Sandimmune I.V. 3,213 690 15 In this way, the nanoglot formulation (Capsorine Oral) of the present invention exhibits PK behavior similar to that of commercially available formulation (Neoral).

Example 65 20 Clinical Investigation with Capxol ™ Objectives and Advantages The reason for selecting the initial dose for Phase I / II tests will be based on the dramatically lower preclinical toxicity data for the formulation with Capxol ™ compared to Taxol®. Preclinical data * The previous months indicate that the initial Capxol ™ toe-tes for the Phase I / II studies will use the established BAT (maximum tolerated dose) for the pacltaxel in the Taxol®. Based on current preclinical data, it is anticipated in these times that the clinical objectives for market approval will be to eliminate the need for pre-medication prior to the administration of paclitaxel.; determine the equivalent dose of Capxol ™ in Taxol®, that is, determine the doses at which the equivalent antitumor response is obtained; and eliminate the need for an i.v. continue (3 to 24 hours) for the administration of paclitaxel and replace administration for much shorter periods (<1 hour or bolus). There are many potential advantages of the Capxol ™ formulation for paclltaxel. Capxol ™ is a lyophilized powder that contains only paclitaxel and human serum albumin. Due to the nature of the colloidal solution formed in reconstituting the lyophilized powder, the toxic emulsifiers, such as cremophor (in the BMS formulation of paclitaxel) or polysorbate 80 (in the Rhone Poulenc formulation of docetaxel), and solvents such as ethanol to solubilize the drug, they are not required. The removal of toxic emulsifiers will reduce the incidence of anaphylactic reactions and hypersensitivity as is known to occur with products such as Taxol®. In addition, premedication with steroids and antihistamines is not anticipated before drug administration jgaáj ñiZZ ^ á- Due to the reduced toxicities, as evidenced by the LD10 / LD50 studies, higher doses can be used which results in greater efficacy. It is expected that the reduction of myelosuppression (compared to Taxol®) reduces the treatment cycle period (currently 3 weeks) and improves the therapeutic results. Capxol ™ can be administered at much higher concentrations (up to 20 mg / ml) compared to Taxol® (0.6 mg / ml), allowing much lower volume infusions, and possible administration as an intravenous bolus. The problem recognized with Taxol® is the precipitation of paclitaxel in internal catheters. This results in an erratic and deficient controlled dosage. Due to the inherent stability of the colloidal solution of the new formulation, Capxol ™, the problem of precipitation is solved. The literature suggests that particles in the size range of nanometers below one hundred are preferentially divided within tumors through permeable blood vessels of the tumor. It is expected that the particle cololities of paclitaxel in the Capxol ™ formulation show preferential targeting effect, extensively reducing the side effects of paclltaxel administered in the BMS formulation.

Example 66 Outline of the Clinical Trial Design of Capxol ™ Indication: Breast Cancer Metática Dosage Plan: The reason for selecting the initial dose for the Phase I / II trials will be based on the significantly lower pre-clinical toxicity data ( single dose LD10 data in mice) for the Capxol ™ formulation compared to Taxol®. The single dose in LD10 in mice is determined as 398.1 mg / kg. The conversion of this dose to a surface area base (3 times the mg / kg value) gives an estimate of 1194.3 or approximately 1200 mg / m2. A conservative initial dose of one-tenth of this value for humans results in a doses of 120 mg / m2. However, it is already well established that paclitaxel is safe at a dose of 175 mg / m2 and on the basis of a pile study with Capxol ™ that shows poorer myelosuppression in rats, a dose of 175 mg / m2 should be safe for the formulation of Capxol ™. The Capxol ™ solution will be supplied in about 15-30 minutes or less, if possible.

Example 67 Outline of the Capxol ™ Clinical Development Program Results of Phase I / II Combination Dosage 25 Study / Efficacy Test Liporcióna *** l ^ ^? ttai ^ ttut ^^ mt Patients / Purpose: Patients who have advanced breast metastatic disease refractory to standard therapies. The objective of this test will be to establish the speed of response of Capxol ™ as a single agent in patients with metastatic breast cancer. Dosage-Phase I Component: The initial dose to be used in the Phase I component of the test will be the known maximum tolerated dose (MTD) for paclitaxel (135 mg / m2). Subsequent doses will be increased in steps of 25% until the MTS is reached. There will be 3 patients in each of the initial dose levels of Capxol ™, expanding up to 6 patients in the BAT. The ability to move to the next dose level will be based on the adverse event pattern. That is, the study will be discontinued when two or more patients of 6 at a particular dose level exhibit non-myelosuppressive Grade 3 toxicity or Grade 4 myelosuppressive toxicity (on the WHO toxicity scale). The dose for Capxol ™ will be designated as the dose immediately preceding the dose at which the test was discontinued. Alternative schedules of drug administration, such as daily for 5 or 24-hour infusions can also be screened if necessary, based on the results of the initial single bolus dose schedule. Pharmacokinetics: For selected patients, a total pharmacokinetic study will be performed using serum extracted at appropriately designated time points. The TfüJJLarffl'T r -? FTr? IripTMr go mp? I r'Ttr? Wiil? T ?? r ?? mgmmam ^ z parameters such as t1 2 (phase a and ß, AUC, Cmax, clearance and volume of distribution will be determined.) Patients - Phase II components: Having established BAT, patients with breast cancer similar to those used in tests with The original paclitaxel will be selected for the Phase II component.The number will be based on the desire to establish a tumor response rate with acceptable accuracy at a confidence level of 95% as such.The study will be simply armed with the objective of establish equivalence with standard paclitaxel showing that the confidence interval contains the expected response rates for Capxol ™ The sample size of patients will be 30 patients, which is common for the Phase II component of a Phase I study 11. Measurement: The primary outcome will be the tumor response rate (CR / PR) for the recruited patients, and the response time will be longer. Response time and survival time will be monitored. The safety of the treatment will also be evaluated for proportions of adverse events and changes in the standard laboratory parameters.

Claims (65)

1. A method to reduce the toxicity of paclitaxel in a subject undergoing treatment with paclltaxel, such method is characterized in that it comprises systematically administering paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 175 mg / m2 for a period of administration of no more than 2 hours.

2. The method according to claim 1 characterized in that the dose is at least 250mg / m23.

The method according to claim 1 characterized in that the dose is at least 325mg / m2.

4. The method according to claim 1, characterized in that the administration period is not greater than 1 hour.

5. The method according to claim 1, characterized in that the administration period is not greater than 30 minutes.

6. The method according to claim 1, characterized in that paclitaxel is administered orally, intramuscularly, intravenously, intraperitonially, or by inhalation.

7. The method according to claim 1, characterized in that the treatment is for prostate cancer, orchidectomy, pancreatic cancer, or brain tumor.

8. The method according to claim 1, wherein the haematological or neurological toxicity of paclitaxel is reduced.

9. A method for administering paclitaxel to a subject in need, without the need for drugs prior to the administration of paclitaxel, the method is characterized in that it systematically comprises administering paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 135mg / m2 during a period of administration of not less than 2 hours.

10. The method according to claim 9, characterized in that the dose is at least 250 mg / m2.

11. The method according to claim 9, characterized in that the dose is at least 325 mg / m2.

12. The method according to claim 9, characterized in that the administration period is not greater than 1 hour.

13. The method according to claim 9, characterized in that the period of administration is not greater than 5 minutes.

14. The method according to claim 9, characterized in that paclitaxel is administered orally, intramuscularly, intravenously, or intraperitoneally, intrarterially, intraurethrally, intrathecally.

15. The method according to claim 9, characterized in that where the treatment is for prostate cancer, orchidectomy, pancreatic cancer, or brain tumor.

16. A method for administering paclitaxel to a subject in need thereof, the method is characterized in that it comprises systematically administering paclltaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 135 mg / m2 for a period of administration of no more than 2 hours, with a treatment cycle of less than 3 weeks.

17. The method according to claim 16, characterized in that the dose is at least 250 mg / m2.

18. The method according to claim 16, characterized in that the dose is at least 325 mg / m2.

19. The method according to claim 16, characterized in that the treatment cycle is less than 2 weeks.

20. The method according to claim 16, characterized in that the treatment cycle is less than 1 week.

21. The method according to claim 16, characterized in that paclitaxel is administered orally, intramuscularly, intravenously or intraperitoneally.

22. The method according to claim 16, characterized in that the treatment for prostate cancer, orchiectomy, pancreatic cancer or brain tumor.

23. A method for the administration of paclitaxel to a subject in need, the method is characterized in that 25 comprises systematically administering paclitaxel to the subject in a pharmaceutically acceptable formulation at a dose of at least 250 mg / m2.

24. The method according to claim 23, characterized in that the dose is at least 325 mg / m2.

25. The method according to claim 23, characterized in that the treatment cycle is less than 2 weeks.

26. The method according to claim 23, characterized in that the treatment cycle is less than 1 week.

27. The method according to claim 23, wherein the paclitaxel is administered orally ntramuscularmente, intravenously, or ntraperitonealmente

28. The method according to claim 23, wherein the treatment for prostate cancer, orchidectomy, pancreatic cancer or brain tumor.

29. A method for managing paclltaxel a subject in need thereof, the method is characterized by comprising paclitaxel administered systemically to the subject in a formulation that can be safely administered using medical tools made of materials containing extractable components.

30. The method according to claim 29, wherein the medical instruments are selected from the group consisting of tubes, catheters, infusion bags, bottles and syringes

31. A method for managing paclltaxel a subject in need thereof, the method is characterized by comprising paclitaxel administered systemically to the subject in a formulation that can be safely administered without using an inline filter.

32. A method for managing paclltaxel a subject in need thereof, the method is characterized by administering systematically paclltaxel a full dose of the subject in a volume of less than 250ml.

33. The method according to claim 32, characterized in that the volume is less than 150ml.

34. The method according to claim 32, characterized in that the volume is less than 60 ml. ^^ ££ & ^ i üfaß AJU CGU

35. A method for administering paclltaxel a subject in need thereof, the method is characterized by systematically managing the subject to paclitaxel • a speed of at least 50 mg / m2 / hour.

36. A paclitaxel formulation that has reduced to a subject undergoing treatment with paclitaxel hematological toxicity, the formulation is characterized by comprising paclltaxel a pharmaceutically acceptable formulation suitable for systemic administration at a dose of at least 175 mg / m2 for a period of administration of no more than 2 hours.

37. The formulation according to claim 15 36, characterized in that the dose is at least 250 mg / m2.

38. The formulation according to claim 36, characterized in that the dose is at least 325 mg / m2. 20

39. A formulation suitable for administering paclltaxel paclltaxel to a subject in need thereof, without the need for premedication before administration of paclitaxel, the formulation is characterized by comprising paclltaxel in a pharmaceutically acceptable formulation 25 suitable for systemic administration at a dose of It? mjgtíb Haa ^ tAÜMiMlHif least 135 mg / m2 over a period of administration of no more than 2 hours.

40. The formulation according to claim 5 39, characterized in that the dose is at least 250 mg / m2.

41. The formulation according to claim 39, characterized in that the dose is at least 325mg / m2.

42. A formulation suitable for administering paclitaxel paclltaxel to a subject in need thereof, a treatment cycle of less than 3 weeks, the formulation is characterized by comprising paclitaxel in a suitable pharmaceutically acceptable formulation for administration 15 systemic at a dose of at least 135 mg / m2 for a period of time of no more than 2 hours.

43. The formulation according to claim 42, characterized in that the dose is at least 250 mg / m2.

44. The formulation according to claim 42, characterized in that the dose is at least 325 mg / m2.

45. A formulation of paclitaxel suitable for the administration of paclltaxel to a subject in need thereof, The formulation is characterized in that it comprises paclltaxel in a pharmaceutically acceptable, creamfree free formulation.

46. A lyophilized formulation of paclitaxel suitable for the administration of paclltaxel to a subject in need thereof upon reconstitution.

47. A reconstituted formulation of paclitaxel suitable for the administration of paclitaxel to a subject in need thereof, the formulation is characterized in that it comprises the freeze-dried formulation of claim 41 and water or an aqueous solution.

48. A frozen formulation of paclitaxel suitable for the administration of paclitaxel to a subject who needs it when thawing

49. A liquid formulation of paclltaxel characterized in that it comprises paclitaxel water at a concentration of at least 2.0 mg / ml.

50. The liquid formulation of paclltaxel according to claim 49, wherein the concentration of paclitaxel is at least 5.0 mg / ml.

51. A liquid formulation of paclltaxel according to claim 49, wherein the concentration of paclltaxel is 10.0 mg / ml.

52. A drug formulation suitable for the administration of a drug to a subject in need thereof by inhalation, such a formulation is characterized in that it comprises microparticles of proteins having a size of about 1-10 μm, wherein the protein microparticles comprise drug nanoparticles which it has a size of approximately 50-1000 nm, more optionally an excipient.

53. A method to make nanoparticles that contain As an active agent, the method is characterized in that it comprises: a) combining a non-volatile phase, a volatile phase, and a surfactant that spontaneously forms a microemulsion, wherein the volatile phase contains the active agent; 20 and b) removing the volatile phase and thereby obtaining a suspension of solid nanoparticles in the non-volatile phase, wherein the nanoparticles contain the active agent and have an average diameter of less than 100 nm. 25

54. The method according to claim 53, characterized in that the nanoparticles have an average diameter of less than 50 nm.

55. The method according to claim 53, characterized in that the microemulsion comprises a co-surfactant agent.

56. The method according to claim 53, further characterized by comprising: tf c) removing the surfactant and / or the co-surfactant by dialysis, ultrafiltration, or adsorption.

57. The method according to claim 53, which is further characterized by comprising: c) essentially removing all remaining non-volatile phase by freeze drying, spray drying, or lyophilization, to obtain a dry powder of nanoparticles 20

58. The The method according to claim 57, further characterized in that it comprises: d) resuspending the dry powder of nanoparticles in a pharmaceutically acceptable carrier. 25

59. The method according to claim 58, which is further characterized by comprising: e) administering the resuspended nanoparticles to a patient.

60. The method according to claim 53, further characterized in that it comprises: c) filtering the suspension of solid nanoparticles through a filter of a pore size small enough to sterilize the suspension.

61. A method for making nanoparticles containing an active agent, the method is characterized in that it comprises: a) combining a non-volatile phase and a volatile phase that spontaneously form a microemulsion, wherein the non-volatile phase contains the active agent; and b) promoting the non-volatile phase and thereby obtaining solid nanoparticles in the volatile phase, wherein the nanoparticles contain the active agent and have an average diameter of less than 100nm.

62. A suspension of nanoparticles made by the method of claim 53.

63. Dry nanoparticles made by the method of claim 57

64. Suspension of nanoparticles made by the method of claim 58.

65. Suspension of nanoparticles made by the method of claim 61. ? - .. < * M®, z ^ -g ^ r ?. SUMMARY In accordance with the present invention, useful compositions and methods are provided for the in vivo release of substantially water insoluble pharmacologically active agents (such as the anti-cancer drug paclitaxel) in which the pharmacologically active agent is released in the form of suspended particles coated with protein (the one that acts as a stabilizing agent). In particular, the protein and the pharmacologically active agent in a biocompatible dispersant medium; they are subject to a high shear stress in the absence of any conventional surfactant and also in the absence of any polymeric core material in their particles. The process of the present invention produces particles with a diameter of less than about 1 micron. The use of the specific composition and preparation conditions (for example, the addition of a polar solvent to the organic phase) and the careful selection of the appropriate organic phase and the partial phase, result in the production of unusually small nanoparticles of less 200 nm in diameter that can be filtered sterile. The particle system produced in accordance with the invention can be converted to a redispersible dry powder comprising nanoparticles of a drug insoluble in water coated with a protein and free of protein to which molecules of the pharmacological agent are bound. This results in a single release system, in which a part of the pharmacologically active agent is rapidly bioavailable (in the form of molecules bound to the protein) and another part of it is present in the form of particles without any polymeric matrix therein. .