MXPA00002561A - Pharmaceutical compositions containing plasma protein - Google Patents

Abstract

The invention is related to water-soluble products and pharmaceutical formulations in solid or liquid form mainly for parenteral use. They consist of or comprise a therapeutically active substance (having low aqueous solubility and a substantial binding affinity to plasma proteins) and a plasma protein fraction in controlled aggregation state, whereby the said active substance and the said protein fraction are bound to each other by way of noncovalent bonds. It also covers processes for the preparation of the product and pharmaceutical formulation.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING PLASMA PROTEINS - DESCRIPTION OF THE INVENTION The present invention relates to a therapeutic method for releasing therapeutically active compounds with low solubility in water that substantially bind to plasma proteins. In particular, it relates to solid or liquid pharmaceutical formulations intended primarily for parenteral administration which contain or are composed of a therapeutically active compound with low water solubility and a substantial affinity for binding to plasma proteins (hereinafter referred to as ""). the active substance ") and a fraction of plasma protein in a state of controlled aggregation, said active substance being bound to the protein fraction through non-covalent bonds, and eventually other additives that are pharmaceutically acceptable and mainly acceptable for the parenteral administration such as water, stabilizers, controllers of protein aggregation.

The solid and homogeneous compounds of the invention, which are composed of the protein and the substance mentioned, are soluble in water and can be used for parenteral administration or to prepare pharmaceutical products intended for parenteral administration. It is known from the prior art that some biologically active compounds have a potent therapeutic activity, but they can not exert their beneficial effect due to their low solubility in aqueous medium. Some of them have never been formulated, while a few others have only reached the "phase I" of clinical development. Some of them appear in "poorly biocompatible" formulations with a relatively high toxicity due to the materials used in the formulation. A typical example is that of groups of taxa, more specifically paclitaxel, which is a potent cytostatic and whose application is reduced due to the toxicity of its formulation in Klucel: tween 80 or Klucel and diluent 12, a 1: 1 mixture of EL creamfor: ethanol [Cancer Chemotherapy and Pharmacology (1994) 34: 465-471; Journal of the National Cancer Institute (1990), 1247-1259]. El cremafor (castor oil pol i oxy i ted) has an inherent toxicity and produces vasodilation, lethargy, hypotension, etc. ? In order to reduce the toxic side effects of the solvent and the auxiliary agent, a number of special methods have been proposed: the application of very small doses over a long period of time, the pre-treatment before treatment, etc. (US Pat. 5665761, 5621001, 5670537, etc.). Another proposal is to combine the active substance with a dispersing agent contained within a shell with protein walls (U.S. Patent No. 5,560,933) which is formed by reaction of the protein with an oil such as soybean oil. -those formulations have been proposed for paclitaxel and phototherapy. However, even in the most recent literature there are warnings regarding the application treatment with, for example, paclitaxel (see, eg, Guidance for Industry issued by the US Department of Health and Human Service CDER September 1997, OGD-L -8) where - due to the reactions of ipersensibi 1 i da d all patients treated with paclitaxel should be premedicated with cor 11 co steroi, di fe nh i dr ami na and H2 - antagonists. It was further proposed to prepare the parenteral fo mulations of certain water-insoluble dihydrins or dihydrins by dissolving them in an organic solvent, or in a mixture of an organic solvent with water and adding an aqueous HSP solution to minimize the crystallization of water. the insoluble active substance (Hungarian patent No. 198381, German patent application No. 37 02105). However, the resulting liquid was not clear and contained an organic solvent . According to JP publication document No. 58216126 (See Japanese Patent Excerpts EPO) some carboxylic acid derivatives produce a highly inhibited aromatic functional group and approximately an aqueous solubility of 0.1 mg / ml were so ubiquized. by adding them to a dilute aqueous solution of human serum albumin. It is also known that oral pharmaceutical compositions were prepared by stirring an active ingredient with an egg albumin at a high speed of 5000 to 40,000 rpm in an aqueous solvent and then distilled the solvent (EPA 326618). As a result, an aqueous solution for oral use was obtained with the effect of relieving certain side effects of the active ingredient. It is also known that some of the active substances insoluble in water have a considerable affinity for the whey protein or protein. On this subject there is literature for paclitaxel [Cancer Chem. And Pharm. (1994) 34: 465-471]; miconazole, fluconazole, amphotericin B [Infection, 23 (5): 292-297 (1995) sept.]; c a r b ama z ep i n a [J. Chromatogr. B Biomed. Appl. 669 (2): 281-288 (July 21, 1995)]; azathioprine [Ann. N.Y. Acad. Sci., 685 (1993): 175-192]; propofol [J. Chromatogr. Sci (1992): 164-166]. According to the most recent literature [The Lancet vol. 352 (1998): 540-542], the Taxol0 drug produced the red kidney 11 ami nt of the red cells and the same caused the castor oil p o 11 e t o x 11 a do used as the solvent of this drug. For some water-insoluble drugs, toxic cream was used (c i c 1 or s p o r i na, teniposide, paclitaxel, amphoteric B). To the best of our knowledge is not available a series of highly active drugs but insoluble in water? whose forms for parenteral and intravenous administration have never been commercialized, for example, ritonavit, c a r b ama z e p a n a, canfotetina, azatiopina, miconazol, fluconazole, etc. Therefore, there is a great need to solve the problem so that products that are valuable from a therapeutic point of view but insoluble in water can be administered in a water-soluble form, preferably via a parenteral route, to patients who need to be treated with these ingredients. assets. The object of the present invention is to satisfy these requirements with respect to the active ingredients which are practically insoluble in water and which can substantially bind to plasma proteins. The present invention is based on the discovery that by binding the active substances to suitable proteins by non-covalent bonds before administration a new highly potent release system is obtained for the administration of the active ingredients with low water solubility. According to the invention homogeneous solid products are obtained which are then dissolved in water to obtain clear aqueous solutions and "*" And "biocompatibles" are suitable for parenteral administration, therefore, the invention offers a way to administer the desired and insoluble active ingredients in water without using toxic elements and, in some cases, in a a dose considerably more effective than the one used above The definitions used in this application and which are not repeated hereinafter are the following: R 1 represents an amide of terbutyloxycarboxylic acid or a benzoylamide, R 2 represents hydrogen or any acyl group , preferably acetyl, to solubility in water means that the solubility in water at room temperature is less than 1.10"4 M; a substantial affinity for binding to plasma proteins means that more than 90% of the ingredient binds to proteins in aqueous medium and in spontaneous equilibrium at ambient temperature, HSA means human serum albumin, API means water for injection.

An object of the invention is constituted by a human pharmaceutical formulation which is soluble in water and is intended primarily for parenteral administration, a formulation containing a therapeutically active compound with low solubility in water and with a substantial affinity for binding to plasma proteins and a Protein fraction of human plasma in a state of controlled aggregation. Another object of the invention is constituted by a veterinary pharmaceutical formulation soluble in water and intended mainly for parenteral administration, formulation containing a therapeutically active compound with low solubility in water and a substantial affinity to bind animal plasma proteins in a state of aggregation checked. The human or animal plasma which can include the products and the pharmaceutical formulations of the invention and which can be used in the methods for preparing the products and the compositions can be any of the protein fractions present in nature such as being or 1 buffy , an i nmuno g 1 obu 1 i na, a g 1 ic op rotein, an interferon and / or an interleukin and also the analogues recombine is of the same. Human and animal proteins can be used. In the compounds and compositions intended for the treatment of human beings, natural human serum and ecombinant proteins of human serum are preferred. Among the active ingredients of the invention which are practically insoluble in water, a large variety of compounds is included whose only limitation is that of exhibiting a substantial affinity for the selected plasma protein. As examples of the active ingredients, the following therapeutic agent groups can be named: a cytostatic such as a taxonoid, an antibiotic, a vitamin, an anti-inflammatory drug, an analgesic, an anticonvulsant, an immunosuppressant, an epileptic, an anxiolytic, a hypnotic, an antifungal, an anti-inflammatory agent, an inhibitor of lipid peroxidase, a coronary vasodilator, an anallergic agent, a cardioton, an urocosuric, an anabolic anteroid, a steroid hormone (progesterone, androgen, testosterone) and / or a photon ensib 11 izado r. Several active ingredients can be used at the same time as long as therapeutic doses and their adaptation are taken into account and the binding affinities to the selected protein that should be able to satisfy the different requirements are considered. According to one embodiment of the invention, pharmaceutical formulations as described above containing at least one of the following active substances are provided: amphotericin B, an analogue of adriamycin, apazone, azathioprine, bromazopam, camptothecin, carb ama z ep i na, clonazepam, cyclosporine, diazepam, dicumarol, digitoxin, dipyridamole, dis op amir ami da, f 1 a 11 raz ep am, gemfibrozil, ketochlorin, ketoconazole, miconazole, niflumic acid, oxazepam, fen ob orbit 1, phenytoin, progesterone, propofol, ritonavir, its 1 fi np iraz ona, suprofen, tacrolimus, tamoxifen, a taxonoid, testosterone, tirilazad, trioxsalen, valproic acid and / or warfarin. A preferred embodiment of the invention consists of a formulation as described above containing a taxanoid of general formula I. Another preferred embodiment of the invention contains or consists of paclitaxel and serum 1 bimin, immunoglobulin, g 1 ic or rotein , - human interferon and / or interleukin or another protein fraction of human plasma. Other special and important representatives of the invention are the solid and homogeneous products that are composed of and the pharmaceutical formulations containing the following components: paclitaxel, azathioprine, camptothecin, gemfibrozil, miconazole and propofol as active substances, and serum 1 b umina, i nmun og 1 obu 11 na, g 1 i co rote, human interferon and / or interleukin or the recombinant protein fraction of human plasma corresponding as proteins, in a molar ratio of 1: 0.05 to 1: 100, preferably from 1: 0.1 to 1:50. It is noted that in the present description it is recorded that the invention encompasses the pharmaceutical formulations described above both in the solid state and also in the form of an aqueous solution. In regard to their natural structures - more specifically to their chemical compositions - protein molecules tend to aggregate through their specific binding sites. The degree of aggregation depends on the parameters (temperature, composition, relative and absolute concentration of the components, consequently the pH, the ionic strength) of the solution in which the protein is found. The plasma proteins used in the invention are preferably in a stabilized or controlled state of aggregation. The objective is to avoid such an aggregation of proteins that can inhibit the optimal binding of the active ingredient used. The undesired aggregation of the proteins can be controlled by the presence of other molecules capable of occupying some or all of the binding sites on the macromolecules that participate in the aggregation in order to avoid multiple association of p r o t and n a - p r o t e í na. Some proteins can be acquired in stores in a state of controlled aggregation: containing stabilizers to prevent aggregation. However, this state is not always the most appropriate to promote the union with the active substance that is tried emp 1 e a r. According to the invention, the term "controlled aggregation state" represents the most favorable state for binding that occurs when the protein is capable of binding the active substance in exactly the manner desired for the purpose intended. It is not necessarily the state in which the maximum number of molecules of active substance is bound to the protein, but there are cases in which the highest proportion of union is preferred. This means that in some cases it is necessary to remove other excipients, for example, from a commercial serum albumin fraction, excipients such as stabilizers, ionic components, etc. This may be the necessary initial stage of the process when carrying out the method of the invention. The conditions required to establish the appropriate aggregation stage depend strictly on the active substance in question and on the relevant protein fraction. The examples illustrated below demonstrate, for example, that paclitaxel and cyclosporin A exhibit a greater binding to the plasma protein fraction in the absence of other excipients (such as stabilizers, ionic components, salts, etc.). However, there are other active substances (for example, amphotericin B and propofol) that do not show any interference with the binding of, for example, protein stabilizers. Therefore, for each of the pairs of substance a ct i va / p r o t e i n to employees according to the present invention it is necessary to establish the optimal state of aggregation of the protein used. When the pair composed of paclitaxel and HSA is used, it is important to eliminate all the agents that accompany the commercial HSA: stabilizers such as N - to ce ti 1 - D, L - tryptophan, the alkaline caprylates that are used to stabilize the protein during pasteurization at 60 ° C. Amphotericin B or propofol can be bound to the HSA in the presence of these stabilizers. In a certain way, when the desired state of aggregation can be reached with water, the other components must be removed and continue, for example, with the procedure described below in one of the examples. In order to achieve an optimal combination between specific substances and plasma-specific protein fractions, the following aspects must be taken into account: a) the characteristics of the binding sites occupied by the substance on the protein; ^ »*« ^ ¡! Ní ** "" «ja & samé? m? t .. b) other components that may be present in the solution and that may occupy the same binding sites or compete for them; c) the conditions f i s i c o - qui m a s for the conformation of the actual union site and the consequences for the union; d) the therapeutic aspects known as, for e j emp 1 o, i) paclitaxel on SA has unique transport characteristics; ii) paclitaxel on inte leuins with a proven therapeutic activity of the vehicle; iii) cyclosporin A on gamma-immunoglobulin with a proven therapeutic activity of the vehicle; iv) ritonavir on g amma - i nmu n g 1 or b u 1 n a with a proven therapeutic activity of the vehicle; e) the stability of the formulation. Water is one of the simplest agents that are used to control aggregation. The undesired aggregation can be inhibited by using an appropriate amount of water and the protein is ready to be used according to the invention - it is found in a "controlled form of aggregation". In an embodiment of the invention, the compounds and compositions can contain as an additive a controlling or stabilizing agent of the protein aggregation and / or an auxiliary stabilizer of the solution. As examples of these additives there may be named water, sodium chloride, a buffer, anhydrous alcohol such as glycerin, a sugar derivative which is soluble in water, preferably mannitol, sorbitol and / or dulcitol and other similar compounds. Another embodiment of the present invention includes the process for preparing the new products and the pharmaceutical formulations of the invention. The method comprises the following steps: a) the therapeutically active compound with low solubility in water and a substantial affinity for binding to plasma proteins ("the active substance") in an organic solvent miscible with water and acceptable from the point of contact are dissolved. of pharmaceutical sight, 1? b) said solution is combined with the aqueous solution of a plasma protein fraction in a state of controlled aggregation optionally containing c) another auxiliary additive which is pharmaceutically acceptable such as a protein aggregation controlling agent and / or a stabilizer, thereby obtaining a real solution in which said active substance is bound to said protein fraction through non-covalent bonds; d) the organic solvent is removed preferably by ultrafiltration, dialysis, digestion and / or lyophilization of the solution or its concentrate or by combination of these treatments, thereby obtaining a liquid or solid pharmaceutical product that is homogeneous and soluble in water and containing the active substance and the plasma protein fraction; e) the solid product is dissolved in water or the liquid product is diluted with water to obtain a liquid composition, clear and suitable for therapeutic administration and f) this product is eventually taken to a parenteral formulation (dosage form) for direct use . To prepare the new solid and homogeneous products composed of the active substances and the proteins bound through non-covalent bonds as described by the invention, it is preferred to use the process according to the invention comprising the following steps: a) the compound is dissolved Therapeutically active in a water-miscible and pharmaceutically acceptable organic solvent, b) combine said solution with the aqueous solution of the selected plasma protein fraction which is in a state of controlled aggregation, a solution that eventually contains c) another auxiliary additive that is pharmaceutically acceptable such as a protein aggregation controlling agent and / or a stabilizer, itffc? iaia? fy ^ 8 ^^^ d) obtaining in this way a real solution in which said active substance is bound to said protein fraction through non-covalent bonds; e) the organic solvent is removed and the solution or its concentrate is lyophilized. The most appropriate way to remove the solvent depends on the active substance and the protein used. From the nature of the active product (the pair that includes the active substance and the protein) it follows that the applied methods must guarantee mild treatment conditions. The 11 of 111 allows to obtain solid, homogeneous and water-soluble products that when redissolved in water can be administered through intr ap er 11 one a 1. It may be convenient to combine the steps described above to achieve, for example , that the process be more economical by first preparing a concentrate of the pair "active substance / protein" and then subjecting said concentrate to an 11. Some of the "active substance / protein" pairs (for example, the amphoteric pair B / s e r or lbúmi na) can be successfully concentrated by u 1 t r a f i 1 t r a c t ion or dialysis. Other pairs (for example, pa c 1 i t a xe 1 / H SA) are preferably treated by 11 or f 111 z a c on. Some pairs must first undergo a process of u 1 t r a f i 1 t r a c i on and the resulting concentrates must be 1 i or f i 1 i z a s. The person skilled in the art knows that when the preparation of pharmaceutical products intended for parenteral administration refers to the dilution with water, dilution with acid solutions containing other additives acceptable for parenteral administration, such as, for example, is also included. , sodium chloride. The appropriate solvent for dissolving the active ingredient according to step a) mentioned above must have the following properties: it must be capable of completely dissolving the active ingredient in its mixture with water and its mixture with an amount of less than 50% water It should not denature the protein given. Before carrying out the process of the invention with the active ingredient and the selected protein, it is necessary to choose the suitable solvent for which it is necessary to carry out the routine steps based on what has been stated above. It is preferable to use solvents whose mixtures containing an amount greater than 50% water are still capable of dissolving the active ingredient. Among the preferred solvents for step a) of the process described is, for example, any of the compounds of the group of the monoalcohols or polyalcohols aliphatic of 2 to 4 C atoms, preferably 70-100% ethanol, dimethylformamide, I gave you 1 fo rmami da. An agent controlling the aggregation and / or a stabilizing agent of the solution may be present during the preparation of the solution containing the protein. Such additives also contain an additional or optimum amount of water. They also include agents capable of partially occupying some of the binding sites of the protein to prevent aggregation. Some of these agents are sodium chloride, a buffer, a polyhydric alcohol such as glycerin and / or a derivative of a sugar which is soluble in water, preferably mannitol, sorbitol, dulcitol. To choose the optimal conditions of any active ingredient is necessary ~ 5 & fffe. ^ - "_ z - ¿yy7zi" ¿, *? & determine the optimal bonding properties and the corresponding aggregation properties by routine measurements.The complete method of such determinations is illustrated in the examples described below. Another preferred embodiment of the invention consists in the preparation of a solid, homogeneous and water-soluble product, which is composed of a taxanoid of general formula I and a plasma protein fraction and in which the active ingredient is bound to the plasma protein fraction by non-covalent linkages A preferred embodiment of the invention is that of a homogeneous, solid and water-insoluble product which is composed of paclitaxel and human serum albumin and wherein the active ingredient and the plasma protein fraction can be non-covalently bound According to a preferred embodiment of the invention, the compounds used in step a) are the paclit axel and a component of the natural plasma such as serum albumin, an i nmu no g 1 obu 11 n a, a g 11 cop r o t e i na, an interferon and / or an interleukin or the recombinants of these compounds. In other embodiments of the iMt? fe ^ B? a ^ J? MHMMBÍ stMa- --5á? ^ a ^ H invention is used as an active substance cytostatic insoluble in water such as a taxonoid, an antibiotic, a vitamin, an an 11 inf 1 amatorium, an analgesic, an anticon vu 1 sio, an immunosuppressant, an antipyretic, an anxiolytic, a hypnotic, an antifungal, a anticoagulant 1, an inhibitor of lipid peroxidase, a coronary vasodilator, an antiarrhythmic agent, a carotenoid agent, an urocosuric agent, an antithrombotic agent, a steroid hormone (progesterone, androgen, testosterone) and / or a fetosensibi 1 iza do r. Preferred active substances which can be used for the process according to the invention include the following: amphotericin B, an analogue of adriamycin, apazone, azathioprine, bromazopam, camptothecin, carb ama zepine, clonazepam, cyclosporin, diazepam, dicumarol, digitoxin , dipyridamole, di s op ir ami da, f 1 a 11 raz ep am, gemfibrozil, ketochlorin, ketoconazole, miconazole, niflumic acid, oxazepam, fen oba orbit 1, phenytoin, progesterone, propofol, ritonavir, its 1 fi np iraz ona , suprofen, tacrolimus, tamoxifen, a taxonoid, testosterone, tirilazad, trioxsalen, valproic acid and / or warfarin.

From what is described above it is understood that the present invention is not limited to any of the active substances or proteins listed above. Another preferred embodiment of the invention comprises the method of using the compounds and compositions of the invention for the treatment of human or animal patients. The method consists of administering an effective dose of the composition of the invention or prepared according to the invention to a patient who must be treated with the active ingredient. The dose to be administered depends on the active ingredient and the protein emp 1 e a da. Therefore, a method for treating humans or animals parenterally with a water-insoluble, therapeutically active substance having a substantial affinity for binding to a plasma protein is provided, which method is to parenterally administer an effective dose of the following products to a patient who needs to be treated with said active substance, preferably using the respective dosing ranges (calculated based on the active substance) indicated below: pa c 1 it ax 1 / a 1 buúna na 70- 280 mg / treatment; propofol / albumin 6-10 mg / kg / hour; ca ptotecin / albumin, gemfibrozil / albumin, cyclosporin A / albumin 3-5 mg / kg / day; amphotericin B / albumin up to 1.5 mg / kg / day, using the same dosing ranges for the compounds containing the respective protein proteins. The compounds, the compositions and methods of the present invention have the following advantages: they make it possible to avoid the use of incompatible vehicles from the biological point of view, by diminishing or eliminating totally the collateral effects that limit the dose and that are related to these components such as solvents toxic substances, surfactants, emu 1 scripts and other similar compounds the use of plasma protein fractions as vehicles for the drug does not present additional toxic effects, on the contrary, said fractions can increase the tolerance of patients, for example, in the In case of chemotherapy, in some desired cases the applied dose can be increased in comparison with the drugs that are currently in the stores that contain components that are incompatible from the biological point of view and whose toxicity is limiting for the dose, this being a possibility for improve the overall effect of the therapy . The examples described below illustrate the invention in more detail. EXAMPLES I. Methods of preparation, testing The methods described below were applied to determine the binding of a particular active ingredient (substance) to a protein: a) Ultrafiltration A sample of 1 ml of a clear solution, obtained by mixing the aqueous solution containing the protein in a state of controlled aggregation with the solution of the active ingredient in an appropriate solvent, through a membrane for ultrafiltration (limit of the passage closure >30000 Da) and the active ingredient was determined in the fraction of u 11 r a f i 11 r a do. By measuring the concentration of the active ingredient in the unfiltered solution the total amount (> 90%) was recovered in unaltered form. b) Freeze drying 1 ml of the solution described above was lyophilized. After lyophilization, the solid residue was dissolved in 1.00 ml of distilled water, obtaining a clear solution. When measuring the concentration of the active ingredient of this solution, no active ingredient was found in the aqueous phase, but 100% of the latter could be recovered in the protein fraction, c) Analysis of the active ingredient The tests for the determination of the active ingredient were carried out by HPLC with detection by UV spectroscopy. The HPLC analysis can be carried out, for example, with a Waters Millennium system (Waters, MA, USA) for HPLC which is composed of: a Waters 616 pump; a Waters 600S controller; a Waters 717 plus sample automatic injector, fitted with a thermostat set at + 5 ° C; a Waters 996 diode array UV / VIS detector. The system is driven and the data processing is carried out by a Waters Millenium v.2.02.0 device coupled to a P486 / 166 digital personal computer (Digital Equipments, Irvin, UK). The conditions must be optimized individually for each compound as illustrated below for a series of products. d) Evaluation of the chemical structure The LC / MS method is used to verify that the chemical structure of the substance recovered from the bound fraction remains unchanged. The LC / MS tests are carried out on a LC / MS mass spectrometer Finnigan Navigator (F nnigan, Manchester, UK) using the ES or APCI + ionization mode, with a MassLab data acquisition system v.2.0 registered by a Digital Computer Venturis FX / 166 (Digital Equipments, Irvm, UK). The conditions applied must be opti- mized individually for each specific substance and based on the references, as illustrated in several of the examples indicated below. e) Sample preparation The following describes a typical method of sample preparation that is used to determine by HPLC and / or LC / MS the total concentration of a substance present in a sample. The remaining solid of the lyophilization is reconstituted with water, then the solution is mixed with absolute ethanol 1: 1 producing the precipitation of the plasma proteins and keeping the substances dissolved. After rapid centrifugation a solution suitable for analysis by HPLC or LC / MS is obtained. The LC / MS analysis is carried out by directly introducing the sample, while the HPLC analysis proceeds separating the components from each other. Both methods provide valuable information on the chemical structure of the main compound and / or the possible degradation product, as exemplified below in detail for various products. The chromatography and mass spectroscopy data obtained from the HPLC and LC / MS analyzes can confirm the chemical equivalence between the known iologically active substance that is used as the starting material and the compound that is recovered after having been bound to the protein fraction according to the invention. f) Materials used All the active substances used have a quality USP XXIII. The following fractions of plasma protein were used in the experiments: 20% solution of human albumin HUMAN Serum Production and Manufacturing Co.Ltd. Ph. Eur. Godollo, Hungary Recombumm © 25% DELTA B i o t chno 1 ogy Ltd, Nottmgham, UK human albumin 20% Biotest Pharma GmbH, Dreieich, Germany Ph. Eur. Albumeon Centeon B i o - S e r v i c e s, Little Rock, AR, USA USP human albumin 20% Behring Centeon Pharma GmbH Vi e n a, Au s t r i a Ph. Eur. gammaglobul ina human 16% HUMAN Serum Production and Manufacturing Co.Ltd. Ph. Eur. Godollo, Hungary II. Preparation and chemical or physical tests In the following examples, the binding relationships between protein p 1 to smá t i c a: s u s t r a t o are within the average range between 1: 0, 1-100. The substrate binding ratios: HSA were calculated based on the assumption of a MW = 66500 for HSA and a MW = 150000 for g amma g 1 obu 1 i n a human [see 11 Science, vol. 244, pgs. 1195-1198, 1989; Vox Sang, 70: p. 203-209, 1996]. Example II .1 A 20% solution (3.08 x 10"3M) of human serum albumin in a controlled state of aggregation was mixed with a solution of 1 mg / ml (1.17 x 10 ~ 3M) of paclitaxel. in absolute ethanol in a ratio of 4: 1, then stirred until a clear solution was obtained, the solution was lyophilized, the solid residue was redissolved in a sufficient amount of water in order to obtain a clear solution with a 20% concentration of serum albumin The binding was determined from filtering the UF and retained fractions, showing a 99% binding of paclitaxel to human serum albumin, which represents a ratio of 1: 0.1 of serum albumin to urine: pac 1 itaxe 1.

E emplo II .2 A 4.44% solution (6.67 x 10"4M) of human seroalbumin in a controlled state of aggregation was mixed with a 2.0 mg / ml solution (2.34 x 10" 3M). ) of paclitaxel (MW = 853.92) in absolute ethanol in a ratio of 9: 1, then stirred until a clear solution was obtained. The solution was subsequently treated in the same manner as that described in Example II.1. The binding was determined from filtering the UF and retained fractions, observing a 99% binding of paclitaxel to human serum albumin. This represents a ratio of 1: 0.39 for human serum albumin: pa c 111 ax 1. E emp lo II .3 A 4.44% solution (6.67 x 10 ~ 4M) of recombinant human seroalbumin in a controlled state of aggregation was mixed with a solution of 2.0 mg / ml (1.40 x 10 ~ 3M) of paclitaxel in absolute ethanol in a ratio of 9: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in a sufficient quantity of water in order to obtain a clear solution with a concentration of 20% of human serum albumin ,. recombinant. The binding was determined from the filtrate of the UF and the retained fractions, observing a 99% binding of paclitaxel to the recombinant human serum albumin. This represents a ratio of 1: 0.24 of recombinant human serum albumin: paclitaxel. Use II .4 A 2.25% solution (1.5 x 10 ~ 4M) of human gamma g 1 obu 1 in a controlled state of aggregation was mixed with a 0.1 mg / ml solution (1.111 x 10 ~ 4M) of paclitaxel in absolute ethanol in a ratio of 9: 1, then stirred to obtain an & clear solution. The solution was lyophilized; The solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a concentration of 16% human gamma g 1 obu 1 i. The binding was determined from filtering the UF and the retained fractions, observing a 98% binding of paclitaxel to the human ammo g 1 obu 1 i na. This represents a ratio of 1: 0.71 of human gaimmaglobulin paclitaxel. The amount of paclitaxel in the examples I I1.1 to II.3 described above was measured by HPLC and with the following method: Column MN Nuclessil C18 5 um 250x2 mm mobile phase at c e t on i t r i 1: a gua = 73: 27 flow rate 0.30 ml / min ambient temperature detection at 273 nm characteristic retention time 5.9 min. k '= 2, 93 The substance was determined by LC / MS and found unchanged [see Rapid Communications in Mass Spectrometry vol. 11: p. 1025-1032, 1997, and Rapid Communications in Mass Spectrometry vol. 9: pp. 495-502, 1995]. In figs. 6 the comparative results are shown: Figure 6A shows the curve of the standard compound, Figure 6B shows the curve of the redissolved sample. Figure 6C shows the fragments of paclitaxel. Parameters of LC / M S: i or n i z a c i n n: i n t e r f a ce APCI +; flow rate of nitrogen gas: 300 l / h; solvent acetonitrile: buffer = 60: 40, 10 M ammonium formate buffer being at pH 5.0 adjusted with 10% formic acid; Flow rate: 0.300 ml / min. Assay for the determination of paclitaxel: ^ - ^^ i * For the quantitative determination of paclitaxel from different solutions of Examples II.1 to 11.27, the reverse phase HPLC method C-18 was used. The mixtures were injected into the HPLC system in solution e t a n o 11 c a = 50%, avoiding the precipitation of the substance. Union The binding of the substance to the plasma proteins was determined after 15 min. in equilibrium conditions at 8 ± 2 ° C. The distribution of the substance is measured after traction through an appropriate membrane (the passage closure must be> the PM of the protein), determining the concentration of the substance in the fraction of the 11 raf 111 ra do (represents the unbound fraction) and in the prefiltered solution, releasing the bound part by denaturing the protein (represents the total). For denaturation of the protein and the release of the bound fraction absolute ethanol was used previously cooled to 8 + 2 ° C in a ratio of 1: 1. The exact concentration values and the exact quantities were calculated taking into account the dilution factor.

Examples II.5 to 11.21 The human serum albumin solution in the concentration range between 20% (3.08x10"3M) and 0.02% (3.08x10" 6M) was combined with the solution of paclitaxel in absolute ethanol in a concentration range between 20 mg / ml (2.34 x 10 ~ 2M) and 0.01 mg / ml (1.17 x 10 ~ 5M), always obtaining clear solutions. The details are indicated in Table I. All measurements were carried out in triplicate and the calculated results were averaged. Table I Example [T] t (mM) [serum albumin n (TB) / nfseroal n (T, 3) / n (TT) Caption: [T] t total concentration of paclitaxel after addition to human serum albumin [HSA] concentration of human serum albumin n (TB) / n (HSA) number of moles of paclitaxel bound per mole of human serum albumin n (TB) / n (Tt) x 100% percentage of paclitaxel bound Figure 7 shows the variation in paclitaxel concentration (with 0.08% HSA, 10% ethanol, 0.002 mg / ml paclitaxel); Figure 8 shows the variation of the concentration of albumin (with 0.004-16.0% HSA, 20% ethanol, 0.2 mg / ml paclitaxel); Figure 9 shows the variation in the binding of paclitaxel to HSA (with 0.8% HSA, 10% ethanol, 0.1 to 2.0 mg / ml paclitaxel) as a function of pH and at pH values between 4 , 0 and 8.5.

The signs shown in the graph correspond to the following examples: Example 11.18 - - - Example 11.19 -O-O- Example 11.20 -x-x-x- Example 11.15 - < -0- Example 11.21 -A-A- T h e 11.22 The same methods were used as those described for examples II.2 to 11.21, but with serum albumin, immunoglobulin, g 1 i c op r or t e i n s, interferons or interleukins of animals. E j e lo 11.23 In the treatment of commercial human serum albumin or recombinant human serum albumin (hereinafter called albumin), which is carried out in order to reach the state of controlled aggregation that allows the best binding conditions of the molecule to be achieved, it includes the removal of stabilizers such as sodium caprylate, N-acetyl D, L-tryptophan and other ionic components and salts. a) Ultrafiltration method The pH of the solution containing 10% albumin is adjusted to 3.0 with hydrochloric acid and then diluted with bidistilled water to a content of 5% protein. Using the method of u 11 r a f i 11 r a c ón (limit of membran passage close to 3000 kD), the solution is concentrated to a content of 10% protein. The solution is diluted again to a content of 5% protein with 1.0 mM hydrochloric acid. Using the ultrafiltration method (limit of membran passage close to = 3000 kD), the solution is concentrated to a content of 10% protein. The procedure is repeated 12 times, then the pH is adjusted to 6.9 with aqueous solution of sodium hydroxide 2.0 M and the solution is diluted with bidistilled water to a concentration of 5% for protein. Using the ultrafiltration method (limit of membran passage closure = 3000 kD), the solution is concentrated again to a content of 10% protein. The solution is diluted again with bidistilled water to a content of 5% protein. Using the ultrafiltration method (limit of membran passage closure = 3000 kD), the solution is concentrated to a content of 10% protein. This procedure is repeated 10 times until obtaining a pure protein fraction that is sufficiently free of other excipients. In this state, the conductivity of the u 11 r a f i 11 r a do is equal to a of the bidistilled water used for the dilution. This protein is suitable to be linked, for example, to paclitaxel or cyclosporin. b) Using dialysis in place of u 11 r a f i 11 a tion, similar results can be obtained. The treatment lasts approximately 48 hours. E xemployment 11.24 a) A 0.8% solution (1,203 x 10 ~ 4M) of HSA was mixed with a 4.0 mg / ml solution (4.33x10"3M) of amphotecin B (MW = 924). , 09) in DMF in a relationship of 9: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a concentration of 20% for HSA. The binding was determined by filtering the UF and the retained fractions, observing 99.7% binding of amphotericin B to the HSA. This represents a ratio of 1: 4 for HSA: amphotericin B. b) The 1:40 ratio for HSA: an photoerici B was achieved by treating a 40.0 mg / ml solution of amphotericin B in the same way as described higher. E xemple 11.25 A 0.8% solution (1,203 x 10 ~ 4M) of recombinant human serum albumin was mixed with a 40.0 mg / ml (4.33xlO_2M) solution of amphotericin B in DMF + HC1 in a ratio of 9: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in sufficient amount of water so as to obtain a clear solution with a final concentration of 20% recombinant HSA. The binding was determined from filtering the UF and retained fractions, observing a 99.5% binding of amphotericin B to the HSA. This represents a ratio of 1:40 for HSA r e c omb i n a n a n t e r a n t e r i c e n a n a n c e n a n e n a n e n e n a n d e n e n e n a n d e n a n e n e n e n e n e n a n d e n e n Phase mó vi 1 acetonitrile: sc. t ampon = 1: 1 (sc buffer: 0.2% formic acid, pH adjusted to 4.0 with triethylamine) flow rate 0.30 ml / min ambient temperature detection at 365 nm characteristic retention time 5.3 min . k '= l, 41 The substance was determined by LC / MS and found unchanged. In figures 2 the comparative results are shown: figure 2A shows the curve of the standard compound, figure 2B shows the curve of the redissolved sample. Figure 2C shows the fragmentation of amphotericin B. LC / MS parameters: i on i z a c i on: i n t e r f a ce ESI +; flow rate of nitrogen gas: 300 l / h; solvent; 20 mM ammonium formate pH 4.0 adjusted with 10% formic acid; flow rate: 0, 300 ml / min. E xect 11.26 A 0.4% solution (6.015 x 10 ~ 5M) of HSA in a controlled aggregation state was mixed with a 0.14 mg / ml (4.02 x 0.00 M) solution of camptothecin. (MW = 348.36) in absolute ethanol in a ratio of 4: 1, then stirred until a clear solution was obtained, the solution was lyophilized, the solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a concentration of 20% for HSA The binding was determined from filtering the UF and retained fractions, observing a 98% union of camptothecin to HSA, this represents a ratio of 1: 5,34 for HS A: c amp toteci na 11.27 A 0.4% solution (6.015 x 10 ~ 5M) of recombinant human serum albumin in a state of controlled aggregation was mixed with a solution of 0.14 mg / ml (4.02xlO-M) of camptothecin in absolute ethanol in a ratio of 4: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in sufficient amount of water so as to obtain a clear solution with a final concentration of 20% recombinant HSA. The binding was determined from filtering the UF and the retained fractions, observing a 98% binding of camptothecin to the HSA. This represents a ratio of 1: 5.34 for recombinant HSA: camptothecin. Camptothecin was measured by HPLC with the following method: .Column MN Nucleosil Cie 5 um 250x2 mm. Mobile phase to ce t on i t r i 1: s c. buffer = 33:67 flow rate 0.30 ml / min t amb e e n t e d eetinction at 365 nm characteristic retention time 6.9 min. k '= 2.45 The substance was determined by LC / MS and found unchanged [Cancer Research, vol. 56: pgs. 3689-3694, 1996]. E xect 11.29 A 4.0% solution (6.015 x 10"4M) of HSA in a controlled aggregation state was mixed with a solution of 8.0 mg / ml (3.39x10" 2M) of carbamate. z ep i na (MW = 236.27) in absolute ethanol in a ratio of 19: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a final concentration of 20% HSA. The binding was determined from filtering the UF and the retained fractions, observing a 98% binding of the epidermal cell to the HSA. This represents a ratio of 1: 2.8 for HS A: ca r bama z op i na. The epizole was measured by HPLC with the following method: MN Nucleosil C column 8 8 5 um 250x2 mm Mobile phase a c e t o n i t r i 1: s c. buffer = 1: 1 (sc buffer: 0.2 formic acid, pH adjusted to 7.0 with trieti 1 amine) flow rate 0.25 ml / min ambient temperature detection at 285 nm characteristic retention time 5.3 min. k '= l, 12 The substance was determined by LC / MS and found unchanged [Eur. J. Clin. Chem. Clin. Biochem., Vol. 35 (10): p. 755-759]. In figs. 3 the comparative results are shown: Figure 3A shows the curve of the standard compound, Figure 3B shows the curve of the redissolved sample. Fig.3C shows the fragmentation of the c a r b ama z e p a n. Parameters of LC / M S: i on i z a c i on: i n t e r f a ce ESI +; flow rate of nitrogen gas: 300 l / h; solvent: 2 mM ammonium formate; Flow rate: 0.250 ml / min. E xample 11.30 A 4.0% solution (6.015 x 10"4M) of HSA in a controlled aggregation state was mixed with a 1.0 mg / ml solution (8.33 x 10" 4M) of cyclosporin A (MW = 1202.63) in absolute ethanol in a ratio of 9: 1, then stirred until a clear solution was obtained.

The solution was lyophilized; the solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a final concentration of 20% HSA. The binding was determined from filtering the UF and the retained fractions, observing a 97% binding of cyclosporin A to the HSA. This represents a ratio of 1: 0.14 for HSA: cyc 1 os po ri na A. E j emp lo 11.31 A 2.0% solution (3,008 x 10"4M) of recombinant HSA in a state of aggregation Controlled was mixed with a solution of 1.0 mg / ml (8.33 × 10 ~ 4M) of cyclosporin A in absolute ethanol in a ratio of 9: 1, then stirred until a clear solution was obtained, the solution was lyophilized, redissolved The solid residue in sufficient quantity of water to obtain a clear solution with a final concentration of 20% of recombinant HSA The binding was determined from the filtrate of the UF and the retained fractions, observing a 98% binding of cyclosporin A to ecombinant HSA This represents a ratio of 1: 0.29 for HSA rec omomb a nd: po ries A nn e r E xemployment 11.32 A solution at 2.25 1, 50x10 M g amma g 1 obu 1 i na human was mixed with a solution of 1.0 mg / ml (833xlO "4M) of cyclosporin A in absolute ethanol in a ratio of 9: 1, then stirred until a clear solution was obtained. The solution was lyophilized; The solid residue was redissolved in a sufficient amount of water so as to obtain a clear solution with a concentration of 16% for human ammonia. The binding was determined by filtering the UF and the retained fractions, observing a 98% binding of cyclosporin A to gamma g 1 obu 1 i na human. This represents a ratio of 1: 0.56 for g amma g 1 obu 1 i n a human: c i cl ospo ry A. Cyclosporin A was measured by HPLC with the following method: MN Nucleosil Cia column 5 um 250x2 mm Acetonitrile mobile phase: water: methanol: phosphoric acid = 700: 260: 40: 0.05. flow rate 0.350 ml / min temperature thermostat at 80 ° C detection at 205 nm characteristic retention time 7.5 min. k '= 2.95 The substance was determined by LC / MS [1] and found to be unchanged. LC / MS parameters: ionization: ESI interface + flow velocity of nitrogen gas: 300 l / h solvent: acetonitrile: water = 60: 40 flow rate: 0.350 ml / mint E j emp lo 11.33 0.4 solution % (6.015 × 10 ~ 5M) of HSA was mixed with a solution of 2.0 mg / ml (1.12 x 10"2M) of propofol (MW = 178.27) in absolute ethanol in a ratio of 9: 1. , then stirred until a clear solution was obtained, the solution was lyophilized, the solid residue redissolved in sufficient quantity of water to obtain a clear solution with a final concentration of 20% HSA. The binding was determined from the filtrate. of the UF and of the retained fractions, showing a 99% binding of propofol to the HSA This represents a ratio of 1: 18.3 for HSA: pr opo fo 1. E xemployment 11.34 A solution at 0.4% (6.015 × 10 ~ 5M) of recombinant HSA was mixed with a solution of 2.0 mg / ml (1.12 x 10 ~ 2 M) of propofol in absolute ethanol in a ratio of n of 9: 1, then stirred until a clear solution was obtained. The solution was lyophilized; the solid residue was redissolved in sufficient amount of water so as to obtain a clear solution with a final concentration of 20% recombinant HSA. The binding was determined from filtering the UF and the retained fractions, observing a 99% binding of propofol to the recombinant HSA. This represents a ratio of 1: 18.3 for recombinant HSA: propofol. Propofol was measured by HPLC with the following method: Column to MN Nucleosil C18 5 um 250x2 mm FFaassee mmóóvviil1 acet on itri 1 or: water = 73: 27 flow rate 0.30 ml / min t ambient ambient detection 273 nm characteristic retention time 6.1 min. k '= 1.77 The substance was determined by LC / MS and found unchanged [J. of Chromatography B, 669: p. 358-365, 1995]. In Figure 3 the comparative results are shown: Figure 3A shows the curve of the standard compound, Figure 3B shows the curve of the redissolved sample. Figure 3C shows the fragmentation of propofol. LC / MS Parameters: i on i z a c i on: i n t e r f a ce APCI +; flow rate of nitrogen gas: 300 l / h; solvent: acetonitrile / water = 73/23; speed of f lu j o: 0, 300 ml / min. T h e 11.35 9.0 ml of a 0.8% solution (1, 213X10"4M) of HSA were mixed with 1.0 ml of a 4.0 mg / ml solution (4.33X10" 3M) of amphotericin B in dimethylformamide to obtain a clear solution. This solution was dialysed against 2.0 liters of water (WFI) at 4 ° C, for a period of 20 hours and protected from light. Using the method of determination of example II.24. a 99.6% binding was found, representing a ratio of 1: 3.5 for HSA: annealing B. After repeating the dialysis process five times, the concentration of DMF in the solution was reduced to values below detection limit (2xl0 ~ 9M). III. Dosage Forms E j e plos III.1 to III.6 Using the lyophilization preparation procedure described above, an appropriate pharmaceutical formulation is obtained. If the solid is redissolved in the appropriate volume of water for injectable compositions so as to reach a concentration of 20% for HSA, a solution with an appropriate concentration for the therapeutic application is obtained, as summarized below for some active ingredients: Example Name Conc. mg / ml III.1 amphotericin B 11.09 III.2 camptothecin 6.8 III.3 carbamazepine 1.98 III.4 ciclosporin A 0.50 III.5 paclitaxel 1.0 III.6 propofol 10.0 The aforementioned dosage forms can be completed later to obtain bottles for injections and infusions. IV. Biological examples Studies on biological equivalence The biological equivalence was determined by comparing the new formulations of the invention with known formulations that are used in therapy and that contain the same active substance that is poorly soluble in water. Such known formulations were prepared in castor oil p or 11 e t ox 11 a do (Cremofor EL) and absolute ethanol. Materials used: Paclitaxel dissolved in a mixture of castor oil p o 1 i e t oxi 1 ado (Cremofor EL): absolute ethanol = 1: 1 with the aqueous solution of 1 to 5% of the invention, prepared according to the invention. Example IV.1 In vitro studies Comparative studies were carried out m vitro to determine the activity at n t i p r o 1 i f e r a n t e and cytotoxic in lines of human tumor cells. The formulations of paclitaxel in Cremophor EL / absolute ethanol and in HSA were compared on human myeloid leukemia cell lines K562, of mammary carcinoma MCF-7 and MDA-231 and of ovarian carcinoma OVCAR-5 [Anticancer Re s e a r ch, vo 1. 16: pages. 2469-2478, 1996]. Method: Colony growth inhibition assay: the monolayer cultures of the cell lines were treated with eight different concentrations of the drug contained in the two formulations described above, and also with a DMSO / s reference. on saline Cultures were incubated for 24, 48, 72, 96 and 120 hours, respectively. The colonies were stained with crystal violet and the percentage of colonies formed by untreated cells was calculated from the cells that survived the treatment. The results obtained with different cell lines are shown in Tables IIA to IV B. Each study shows the cells that survived the treatment and represent the percentage of colonies formed by untreated cells. All values indicate the averages of three experiments. TABLE II A Cell line: mammary carcinoma MCF7 Formulation: p a c 1 i t ax 1 / C r emo f o r EL and absolute ethanol TABLE II B Cell line: mammary carcinoma MCF7 Fo rmu 1 aci on: aclitaxel / HSA Ptx cc [uM / ti .empo (h) 24h 4 488hh 72h 96h 120h 0.005 91 8 844 75 41 27 0.01 88 8 811 69 35 23 0.02 84 7 766 64 31 20 0.025 80 7 700 59 28 16 0.05 77 6 666 53 25 12 0.1 75 5 599 46 20 9. 1.0 67 4 422 30 17 6. 10.0 58 3 311 21 9. (3.

TABLE III A Cell line: mammary carcinoma MDA-231 Formulation: p a c 1 i t a x e 1 / C r emo f o r EL and absolute ethanol 0. 005 97 89 80 47 34 0.01 94 87 75 41 30 0.02 89 82 69 37 28 0.025 86 76 65 34 23 O 05 84 72 59 29 21 O 1 83 66 53 26 18 1 or 73 49 34 21 9.5 10 or 65 37 24 14 8.2 TABLE III Cell line: mammary carcinoma MDA-231 Formulation: pa c 1 it ax 1 / HSA Ptx cc [uM / time (h) 24h 48h 72h 96h 120h 0.005 92 78 51 30 10 0.01 86 65 38 24 8.3 0.02 75 51 33 22 7.0 0.025 64 47 28 19 6.4 0.05 60 42 26 18 5.3 0.1 55 36 24 16 4.0 1.0 49 33 22 15 0.6 10.0 45 26 20 10 2.6 TABLE IV A Human myeloid leukemia cell line K562 Ptx cc [u / time (h) 24h 48h 72h 96h 120h 0.005 88 59 30 21 10 0.01 79 40 21 15 8.7 0.02 66 31 19 12 7.2 0.025 62 29 17 10 6.0 0.05 56 25 14 9.4 5.4 0.1 51 23 12 7.7 4.6 1.0 47 20 10.5 6.0 3.0 10.0 39 16 9.5 4.2 2.0 Formulation: pac 1 it ax 1 / C r emo for EL and absolute ethanol TABLE IV B Human myeloid leukemia cell line K562 Formulation: pa c 1 itaxe 1 / HSA x cc [uM / ti. Time (h) 24h 48h 72h 96h 120h 0.005 89 53 31 18 5.4 0.01 75 40 22 11 4.7 0.02 69 32 18 9.0 4.0 0.025 65 30 14 7.6 3.5 0.05 58 25 11 7.0 3.0 0.1 53 21 9.5 5.6 2.4 1.0 47 18 8.0 5.0 1.7 10.0 41 16 7.1 4.7 1.0 E xect IV .2 In vivo pharmacokinetic assay From the therapeutic point of view, one can consider the bi oe qu i va 1 ene as the demonstration of similar co-identical characteristics such as the ABC (area under curve), elimination constants, mean plasma life after administration of the same dose to the same species. This experiment was carried out with rats for both formulations as in example IV.1 [Semm. Oncol., Vol. 21 (5 Suppl 8): pp. 53-62, 1994].

ABC represents the area under the curve corresponding to the graph of the plasma concentration as a function of time. It can be obtained by measuring the plasma concentration of the compound administered at different times. Pharmaco-ethical studies in rats: Method: a dose of 2.5 mg / kg of paclitaxel was administered i.v. in a volume of 1.0 ml to CR (Wi) BR rats (body weight between 380 and 420 grams), and at each time value a 1.0 ml sample of blood was placed into a heparinized test tube. three animals, as indicated by the following graph: # 0 '10 * 20' 30 '45"60' 90 '2 3h 4h 5h 6h 1 + + + 2 + + + 3 + + + 4 + + + 5 + + + 6 + + + 7 + + + 8 + + 9 + + + 10 + + + 11 + + + 12 + + The plasma fraction was separated by high speed centrifugation at + 5 ° C and maintained > z- '; J £ ÉB¡2 & £ i Tíi- - .: frozen at -70 ° C until processed for analytical measurements. Sample preparation: the frozen plasma samples were heated to + 8 ° C, centrifuged for 5 min. at 5000 RPM. 0.300-0.500 ml of the clear plasma solution was taken and placed into an Oasis HLB 1 cc SPE (solid phase) extraction cartridge. Before introducing the plasma sample, the cartridge was rinsed with 1 ml of methanol and then with 1 ml of water for preconditioning. The content of paclitaxel was absorbed on the SPE cartridge while the rest of the components of the sample were removed by rinsing with 1 ml of water and 1 ml of acetonitrile / water at 30%. The cartridge was dried with air. The paclitaxel of the SPE cartridge was eluted with 1 ml of absolute ethanol. The sample was evaporated to dryness with nitrogen and stored at -20 ° C for analysis. The residue was dissolved in 0.200 ml of absolute ethanol and injected for analysis by HPLC. The HPLC conditions were the same as those used for the identification of the substances. Results: The obtained values represent the average of three measurements made with the individual samples of 3 animals. As a result, it can be said that the difference between the two curves obtained from the physical studies for equal doses of the two different formulations is within the deviation of the individual samples. If all the individual data are plotted, indicating the differences small or nonexistent in the physical characteristics of both formulations, a curve is obtained in a similar way. E v e v .3 The n vivo evaluation of the 15 investigations on the activity n 11 p r o 11 f e r a n t e and cytotoxic shows that the new formulations exert a positive effect against xenogeneic CH1 and Cltax human tumor grafts in nude mice. 20 E i x I V .4 Human Hyperactivity Tests Approximately 45% of patients treated with paclitaxel develop reactions of h i pe r s in s ib 111 da d. It was found that these effects collaterals are related to an excipient of *** & ** - * ~ t and% the formulation, the Cremofor EL, as they are also observed with other pharmaceutical products that contain the same component. This reaction of h i pe r s ib i i d d was determined as anaphylactic toxicity and manifested through the induction of histamine released by the Cremofor EL. The test was carried out with male CRL (Wl) BR rats weighing between 130 and 150 g [14]. The dose administered was approximately 7.0 mg / kg of paclitaxel and was applied i.v. in a total volume of 1.0 ml. The group for each dose and time contained 5 animals. Blood samples were collected after 2.5 and 10 minutes of treatment in tubes containing heparin. The plasma was separated by high speed centrifugation. The samples were stored at 70 ° C. The histamine content of the samples was C14 ~ methylated by a specific histamine-N -me t i 11 r a ss. The level of histamma in the plasma samples was determined by measuring the amount of C14 in the samples. The data obtained indicate that both Cremofor EL and the formulation containing it substantially induce the release of histamine, while both the HSA or the formulation containing it and paclitaxel have no such effect. E amp IV IV. The same effect as in Example IV.4 was found using in vitro human experiments from human blood samples and based on the quantitative assay of chromatin activation in blood lymphocytes [method: Analytical and Quantitative Cytology and Histology, vol. 8: p. 1, 1986]. E j amp lo I V .6 Comparison of toxic effects The evaluation of toxic effects was carried out in acute and subacute studies in mice with i.p. administration. and i.v. The experiments were carried out in CD2F1 mice weighing between 20 and 25 g. Because there are no consistent data on toxicity in mice with i.v. and i.p. doses of 40, 20 and 10 mg / kg were selected [method: J. Tox. Sol. Vol. Suppl. 1, pgs. 1 to 9, 1994].

Each of these doses was administered to 16 mice (8 males and 8 females) in an experiment as a single dose, and once a day for 5 days as a multiple dose. The symptoms of toxicity in the animals were tested, their body weight was measured daily for 10 days and after the last administration. Results: no specific toxicity signal was found, except fatigue and diarrhea. A loss of body weight was observed, with a minimum around the fourth day after the administration of the last dose. With the formulation containing the Cremofor EL a decrease of 15-20% was observed with a small recovery that lasted until the tenth day. With the formulation containing HSA a lower weight loss (5-10%) was observed with a rapid and complete recovery. All toxicity indications depended on the dose.

Claims (40)

1. Free product of organic solvent, soluble in water or the pharmaceutical formulation in 5 solid or liquid form and its true aqueous solutions free of organic solvent mainly for parenteral use containing a) a therapeutically active compound having low aqueous solubility (< 1.10 ~ 4 M / Lit) and a Substantial binding affinity for binding to plasma proteins (in the following "active substance"), b) a plasma protein fraction in a controlled aggregation state 15 whereby the active substance and the protein fraction bind each other by means of non-covalent bonds and optionally also contains c) pharmaceutically acceptable and parenterally acceptable additive formulations - such as water, stabilizer or stabilizers, controller or protein aggregation controllers. "^ y ^ - ^ z ^ *.

2. Water-soluble product or human pharmaceutical formulation according to claim 1, which contains a fraction of human plasma protein in a controlled aggregation state.

3. Water-soluble product or veterinary pharmaceutical formulation according to claim 1, which contains a protein fraction of animal plasma in the state of controlled aggregation.

4. A pharmaceutical product or formulation according to any of claims 1 to 3 containing as the plasma protein fraction a natural plasma component such as serum albumin or a serum albumin recombinant.

5. Product or pharmaceutical formulation according to any of claims 1 to 4, which contains as the plasma protein fraction an i nmonog 1 ob 1 u 1 i na natural, 1 ic op rote í na, interferol and / or interleukin or a recombinant of i nmu n og 1 obu 11 na, g 1 i cop r ot e i n, interferon and / or interleukin.

6. Product or pharmaceutical formulation according to any of the rei indications 1 to 5 containing as the water-insoluble active substance a cytostatics such as a taxonoid, an antibiotic, vitamin, antiinf 1 amatorium, analgesic, antiviral, anti-convulsive 11 e , immunosuppressant, anti epiphysis, epidural, anxiolytic, hypnotic, antifungal agent, anti-coagulant, lipid peroxidase inhibitor, coronary vasodilator, antiarrhythmic agent, urinary cation, uricosuric, antituberculosis agent , steroid hormone (progesterone, androgen, testosterone) and / or ophthalmosensitivity 1 iza do r.

7. Product or pharmaceutical formulation according to any of claims 1 to 6, which contains at least one of the following active substances: amphotericin B, an analogue to nd re aminacin, apazone, azathioprine, bromazepam, camptothecin, ca bb z ep ina, clonazepam, cyclosporin, diazepam, dicumarol, digitoxin, dipiridamol, di s op i rami da, f 1 an itraze pam, gemfibrozil, ketochlorin, ketoconazole, miconazole, niflumic acid, oxazepam, f enob to rb ita 1, phenytoin, progesterone, propofol, ritonavir, its 1 fi np iraz ona, suprofen, tacrolimus, tamoxifen, taxonoid, testosterone, tirilazad, trioxsalen, valproic acid, warfarin and wherein the molar ratio of the active ingredient: proteins is within the range of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1: fifty.

8. Product or pharmaceutical formulation according to any of claims 1 to 7, which contains a taxonoid of the general formula 10 I. - in the f or rmu 1 to R 1 represents tert.butyl-oxycarboxylic acid amide or benzoyl amide, R 2 represents hydrogen or an acyl group Preferably acetyl. -,,; * 6?

9. A pharmaceutical product or formulation according to any one of claims 1 to 8, which contains as a taxonoid paclitaxel and a human serum albumin, i nmonog 1 ub ina, g 1 ic or rotate, interferon and / or interleukin or some other fraction of natural recombinant human plasma protein in the molar ratio of 1: 0.5 to 1: 100, preferably in the molar ratio of 1: 0.1 to 1:50.

10. A pharmaceutical product or formulation according to any one of claims 1 to 9 containing azathioprine and human serum albumin, immunoglobulin, g 1 ic opr otei na, interferon and / or interleukin or some other recombinant or natural human plasma protein reaction in the ratio molar from 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

11. Formulation according to any one of claims 1 to 8, which contains camptothecin and human serum albumin, i nmonog 1 obub ina, g 11 c op rotein, interferon and / or natural recombinant human plasma protein in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

12. Formulation according to any one of claims 1 to 8, which contains gemfibrozil and human serum albumin, immunoglobulin, g 11 c op rotein, interferole and / or methyl ester or some other fraction of recombinant or natural human plasma protein in the molar ratio from 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

13. A pharmaceutical product or formulation according to any one of claims 1 to 8, containing miconazole and human serum albumin, immunoglobulin, g 1 ic or rotein, interferole and / or interleukin or some other protein fraction of recombinant or natural human plasma in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

14. A pharmaceutical product or formulation according to any one of claims 1 to 8, containing propofol and human serum albumin, immunoglobulin, g 1 ic or rotein, interleukin and / or interleukin or some other recombinant or natural human plasma protein fraction in the ratio molar from 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

15. Product or pharmaceutical formulation according to any of claims 1 to 8, which contains tamoxifen and human serum albumin, i nmono g 1 obu 1 i na, g 1 i coprotein, interferol and / or interleukin or some other protein fraction of Recombinant or natural human plasma in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

16. Product or pharmaceutical formulation according to any one of claims 1 to 8, which contains ptonavir and human seroalbumin, i nmon og 1 obu 1 ina, g 1 i coprotein, interferol and / or interleukin or some other fraction of plasma protein recombinant or natural human in the molar ratio of from 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

17. Pharmaceutical formulation according to any one of claims 1 to 8, containing tacrolimus and human serum albumin, i nmon og 1 obu 11 na, g 1 ic op rotein, interferol and / or interleukin or some other fraction of recombinant human plasma protein or natural in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

18. Pharmaceutical formulation according to any of claims 1 to 8, which contains tirilazad and human serum albumin, immunoglobulin, g 1 i c op r or t a n, interferol and / or 10 interleukin or some other protein fraction of recombinant or natural human plasma in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

19. A pharmaceutical formulation according to any one of claims 1 to 8, which contains trioxsalen and human serum albumin, immunoglobulin, g 1 i c opr or t and na, interferol and / or interleukin or some other protein fraction of 20 recombinant or natural human plasma in the molar ratio of 1: 0.05 to 1: 100, preferably 1: 0.1 to 1:50.

20. Pharmaceutical formulation according to any of the rei indications 1 to 20 which has a solid state that has the shape of an aqueous solution

21. Pharmaceutical formulation according to any of claims 1 to 20, which contains as an additive an agent that stabilizes the solution and / or the protein.

22. Pharmaceutical formulation according to claim 22, which contains as a solution and / or protein stabilizing agent any of the following: sodium chloride, a buffer, an alcohol such as glycerol and / or water-soluble sugar derivative, preferably mannitol, sorbitol, dulcitol.

23. Process for the preparation of a water soluble product or pharmaceutical formulation in solid or liquid form according to any of claims 1 to 22, or a product according to claims 29 to 36 and its true aqueous solution free of organic solvent characterized by preparing an aqueous solution by means of a) dissolving the therapeutically active compound having low aqueous solubility (< 1.10 ~ M / Lit) and a substantial binding affinity to the plasma proteins ("active substance") in a pharmaceutically acceptable, water-miscible organic solvent, b) combining the solution with the aqueous solution of the plasma protein fraction in a controlled aggregation state, c) and optionally with an additional pharmaceutically acceptable water-soluble auxiliary additive-such as a protein of controlled aggregation and / or a stabilizer - so a real solution is obtained containing the active substance and a protein fraction bound together by means of non-covalent bonds; d) removing the organic solvent and optionally the water preferably by ultrafiltration, dialysis, diafiltration and / or lyophilizing the solution or its concentrate or by combining these treatments so that a solid or liquid pharmaceutical product soluble in water, homogeneous, is obtained by containing the active substance interconnected with the plasma protein fraction e) optionally dissolving the solid product in water or diluting the liquid product with water so that a true, clear aqueous solution free of any organic solvent is obtains, which is suitable for therapeutic administration; and f) optionally terminating this product in a parenteral formulation (dosage form) for direct use.

24. Process for the preparation of a water soluble product or pharmaceutical formulation in its solid or liquid form according to any of claims 1 to 22, or a product according to claims 29 to 36, and its true aqueous solutions free of solvent organic characterized by a) dissolving the active compound therapeutically in a water-miscible, pharmaceutically acceptable organic solvent b) combining the solution with the aqueous solution of the selected plasma protein fraction in a controlled aggregation state. c) the solution optionally containing an additional pharmaceutically acceptable auxiliary additive - such as a protein aggregation controller and / or a stabilizer - whereby a real solution is obtained containing the active substance and the protein fraction bound together by of non-covalent bonds. d) remove the organic solvent and lyophilize the solution of your concentrate.

25. Process according to any of claims 23 and 24, wherein step a) is characterized by the use to dissolve the active substance of a solvent having the following properties: a) it is capable of completely dissolving the active substance in its mix with water and b) its mixture with < 50% water does not denature the protein used.

26. Process according to claim 25, characterized in that it uses as the solvent any of the group consisting of a monoalcohol or polyalcohol aliphatic C (2-4), ethanol 70-100%, tell me 1 fo rmami da, me ti 1 fo rmami da

27. Process according to claim 23 to 26 in which step a) is characterized in that it uses as the protein aggregate or stabilizer controller and / or the additive auxiliary solution is any of the following agents: water , sodium chloride, a buffer, a po-1 to 1 coh or 1, such as glycerol and / or a water-soluble sugar derivative preferably mannitol, sorbitol and / or dulcitol.

28. Process according to any one of claims 23 to 27 in which step a) is characterized by the use of paclitaxel and a component of the natural plasma such as serum albumin, an immunoglo b or 11 na, g 11 c op rotein , inferred and / or interleukin or a recombinant of seroa 1 bumi na, an i nmunog 1 ubu 11 na, g 11 copoteote, inferred and / or interleukin.

29. Product soluble in water, solid, homogeneous for pharmaceutical use consisting of at least one active substance that has low iS (?), áfeasüí aqueous solubility (<1.10 ~ M / Lit) of the amphotericin B group, an analogue of adriamycin, apazone, azathioprine, bromazopam, camptothecin, car bama z ep i na, clonazepam , cyclosporin A, diazepam, dicumarol, digitoxin, dipyridamole, dis op amiram, f 1 unitrazep am, gemfibrozil, ketochlorin, ketoconazole, miconazole, niflumic acid, oxazepam, faith nob arbita 1, phenytoin, progesterone, propofol, ritonavir, 1 fi np iraz ona, suprofeno, tacrolimus, tamoxifen, a taxonoid, testosterone, tirilazad, trioxsalen, valproic acid and / or warfarin and also consists of at least one protein from the serum albumin group, i nmu n og 1 obu 1 ina , g 1 ic op rotein inferred and / or interleukin or some other protein fraction of recombinant or natural human plasma where the active substance and the protein fraction are bound to each other by means of non-covalent bonds and where the molar ratio of the active substance and the protein fraction are within the range of from 1: 0.05 to 1: 100, preferably from 1: 0.1 to 1:50.

30. Water soluble, solid, homogenous product according to claim 30 which consists of a taxonoid of the general formula I in the f or rmu 1 to R 1 represents tert.butyl-oxycarboxylic acid amide or benzoyl amide, R 2 represents hydrogen or any acyl group preferably acetyl and a fraction of plasma protein.

31. Solid, homogeneous water-soluble product according to claim 29, consisting of paclitaxel and human seroalbumin, recombinant human plasma albumin and / or β-globulin.

The product is soluble in water, solid, homogeneous according to claim 29, which consists of amphotericin B and human serum albumin, recombinant human plasma albumin and / or β-globulin.

33. Water-soluble, solid, homogenous product according to claim 29, consisting of camptothecin and human serum albumin, plasma, recombinant human albumin and / or γ-globulin.

34. Water-soluble, solid, homogenous product according to claim 29, which consists of carbamazepine and human serum albumin, plasma, recombinant human albumin and / or β-globulin.

35. Water soluble product, solid, homogeneous according to rei indication 29, consisting of cyclosporin A and human serum albumin, recombinant human plasma albumin and / or β-globulin.

36. Water-soluble, solid, homogenous product according to claim 29, which consists of propofol and human serum albumin, plasma, recombinant human albumin and / or γ-globulin.

37. Method of treating human or veterinary patients with a terapeutically active substance having low aqueous solubility (<1.10 ~ 4 M / Lit) and having substantially plasma protein affinity characterized by administration to a patient in need of a treatment with the active substance and an effective dose of the product or pharmaceutical formulation according to or prepared according to any of claims 1 to 36.

38. Method of treating human or veterinary patients according to claim 37, with a therapeutically active substance having low aqueous solubility (< 1.10 ~ 4 M / Lit) and having substantial plasma protein affinity characterized by parenteral administration to a patient in need of a treatment with the active substance an effective dose of the following products preferably using the following dose averaged respectively (calculated on the active substance): pac 1 itaxe 1 / a 1 b umi na 70-280 mg / treatment; propof ol / albumin 6-10 mg / kg / hour; c amp t o c i n a / a 1 b umi na, g em f i b r o z i 1 / a 1 b um a, cyclosporin A / albumin 3-5 mg / kg / day; amphotericin B / albumin up to 1.5 mg / kg / day, whereby the same averaged doses are used for the compounds containing the respective recombinant proteins.

39. Method for parenteral delivery in the therapeutic use of pharmaceutically active ingredients with low solubility (<1.10 ~ 4 M / Lit) and substantial affinity for binding to plasma proteins characterized by administration to a patient in need of a treatment with the active substance an effective dose of the composition according to or prepared according to any of claims 1 to 28.

40. Product or method substantially as described in any of the examples.