MXPA96002007A - Liposomas with increased capacity of atrapment, method for its obtaining and its - Google Patents

Abstract

The present invention relates to a method for manufacturing a suspension of liposome vesicles with an improved high capacity. The method is characterized in that it comprises the steps of: a) dissolving one or more lipids forming a film in at least one organic solvent to form a solution in a reaction vessel, b) evaporating the solvent to form a thick viscous solution, c) subjecting the reaction vessel to reduced pressure and expanding the thickened solution into a foam, thereby producing a three-lipid structure dimensions, dry expanded with a volume density below 0.1g / cm3, d) contacting the three-dimensional lipid structure with a carrier phase in aqueous solution, thereby producing a suspension of liposome vesicles carrying the ported solution

Description

LIPOSOMES WITH INCREASED CAPACITY OF ATTRAPTION. METHOD FOR YOUR OBTAINMENT AND USE TECHNICAL FIELD The invention relates to a liposome vesicle precursor in the form of a dry lipid deposit and a method for manufacturing the liposome vesicles, with improved trapping ability by dissolving one or more lipids that form a film in at least one organic solvent in a reaction vessel, depositing the lipids by evaporation of the solvent, contacting the lipid deposit with a carrier phase in aqueous solution and producing the solution that traps the liposome vesicles. The invention also relates to an apparatus for carrying out the method, contrast agents comprising the liposome vesicle precursor and a method for making the contrast agents using the precursor.

ANTECEDENTS OF THE TECHNIQUE Liposome vesicles whose binding envelope consists of bi- or multilayers of lipid molecules have long been recognized as drug delivery systems, which can improve the therapeutic and diagnostic effectiveness of many drugs and contrast agents. Experiments with many different antibiotics and X-ray contrast agents have shown that better therapeutic activity or better contrast can be achieved with a higher level of safety by the encapsulation of drugs and contrast agents with liposomes. The great interest in liposomes as encapsulation systems for drugs has revealed that successful development and commercialization of such products requires reproducible methods of large-scale production of lipid vesicles with suitable characteristics. Therefore, an investigation has been initiated for the methods which will consistently produce liposome vesicles of the required size and concentration, size distribution and entrapment capacity without considering the nature of the lipid mixture. Such methods should provide liposomes with a consistent ratio of active substance to lipid, while adhering to currently accepted good manufacturing practices. As a result of the research and due to the fact that the behavior of the liposome can vary substantially with various production parameters, many different methods of manufacture have been proposed up to now.

Conventional liposome preparation methods include many steps in which the multilayer or bilayer forming components (phospholipids or mixtures of phospholipids with other lipids, eg cholesterol) are dissolved in a volatile organic solvent or solvent mixture in a flask of Round bottom, followed by evaporation of the solvent under conditions (temperature and pressure) which will prevent phase separation. By removing the solvent, a mixture of dry lipid, usually in the form of a film deposit on the walls of the reactor, is hydrated with an aqueous medium, which may contain dissolved buffers, salts, conditioning agents and an active substance which is going to be caught. The liposomes will be formed in the hydration stage, whereby a proportion of the aqueous medium becomes encapsulated in the liposomes. Hydration can be performed with or without energizing the solution by means of agitation, sound treatment or microfluidization with subsequent extrusion through one or more polycarbonate filters. The unencapsulated, free active substance can be separated for recovery and the product is filtered, sterilized, optional lyophilized and packaged.

Hydration, more than any other stage, influences the type of liposomes formed (size, number of lipid layers, trapped volume). The nature of dried lipid, its surface area and its porosity are of particular importance. In this way it has been established that the hydration and entrapment process are more efficient when the dried lipid film remains thin. This means that the greater the amount of lipid, greater surface area is required for lipid deposition, it also means that even when beads of glass and other inert insoluble particles are used to increase the surface area available for depositing the film, the thin film method remains largely measure a laboratory method. Other methods for manufacturing liposomes involve the injection of organic lipid solutions in an aqueous medium with continuous solvent removal, use of spray drying, lyophilization, microemulsification and microfluidization, etc., many publications or patents have been proposed such as for example US -A-4,529,561, US-A-4, 572,425, etc. An attempt to solve the problems of scaling up liposome production has been described in US-A-4, 935, 171 (Vestar). A method for preparing liposomes in commercial quantities is described by forming a homogenous and uniform lipid film in a thin film evaporator by evaporation of the organic solvent. After drying the thin lipid film, which is formed on the inner wall of the evaporator, the reservoir is hydrated in itself with an aqueous phase under stirring provided by the rotor. Although the solution proposed in that document seems to be a step in the right direction, the ratio of the lipid film surface to the reactor volume is only slightly, if not marginal, better than that of the round bottom flasks used at scale from laboratory. The yield space of the reactor or productivity is still too low for the process to be economically reasonable and competitive. Different aspects of liposome manufacturing have been considered and many improvements and different solutions to the scale enlargement problem have been proposed. Documents such as for example WO-A-86/00238, WO-A-87/00043, US-A-4,737,323, US-A-4, 753, 788 and US-A-4, 781, 871 have suggested the use of rapid freezing of multiple lamellar vesicles prepared previously, with the subsequent freezing and thawing treatment to improve their trapping capacity, the use of the technique of extrusion of multilamellar liposomes to improve their size distribution, etc.

Until now there has been no suggestion towards a large-scale industrial method, whose control of the production parameters will allow a reproducible process in which large volumes of liquid will be processed within a relatively small reactor space. All processes known at pilot or industrial scale would normally be linked to small batches in which the processing of small volumes of diluted liposome solutions would require a lot of floor and reactor space as well as the handling of large volumes of solutions and solvents. . In fact, due to relatively low space-time yields or reactor productivity, these methods are bulky and too expensive for large-scale commercial production.

BRIEF DESCRIPTION OF THE INVENTION Briefly summarized, the invention relates to a liposome vesicle precursor supported or unsupported, in the form of a three-dimensional structure of expanded lipids, with bulk density below 0.1 g / cm 3, preferably below 0.08, more preferably between 0.05 and 0.001 and even more preferred between 0.02 and 0.01. By supported structure, it is understood that the porous lipid deposit is formed in an arrangement or network of inert support material. The lipids forming the deposit are selected from synthetic or natural, saturated and unsaturated phospholipids including phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and mixtures thereof. The lipids may also contain substances selected from dicetyl phosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, α-tocopherol, stearic acid, stearylamine and mixtures thereof. The invention also relates to a method for making liposome vesicles with increased entrapment capacity, by dissolving one or more lipids that form a film in at least one organic solvent to form a solution. The lipid solution is introduced into a suitable reaction vessel and is subjected to evaporation, whereby drying the lipids form a three-dimensional, expanded porous structure whose bulk density is below 0.1 g / cm3, preferably below 0.08, more preferred between 0.05 and 0.001 and even more preferred between 0.02 and 0.01. Then, the porous structure is contacted with an aqueous carrier phase to produce liposome vesicles that trap a portion of the carrier phase.

The invention further comprises an apparatus for the manufacture of liposomes with high entrapment capacity according to the above method, comprising a reaction vessel with an inlet and outlet, a connection to a vacuum, means for cooling or heating, a medium of control and a package, comprising an arrangement of a pipe intimately packed with an inert material. Preferably, the pipe is stainless steel pipe with the inner diameter between 0.5 mm and 5 mm and the wall thickness between 0.5 mm and 2 mm. Alternatively, packaging, which may be stationary or moving, eg fluidized, may consist of Raschig rings, hollow glass spheres, crosslinked carbon, crosslinked vitreous carbon, crosslinked metal, glass wool or metal wool and glass fiber or metal fiber. The three-dimensional lipid structures of the invention are well suited for the large-scale manufacture of liposomes with high entrapment capacity. When incubated with an aqueous carrier phase containing a contrast medium, the three-dimensional lipid structures of the invention are particularly suitable for the manufacture of contrast agents for diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the reactor, with a separate section showing the expanded three-dimensional lipid structure of the invention. Figure 2 is a schematic diagram of the reactor with an inert pipe arrangement. Figure 3 is a graph of the lipid deposit versus concentration. Figure 4 is a graph of lipid deposit versus linear velocity. Figure 5 is a flow diagram of the production of a contrast medium using the expanded lipid structures of the invention.

DETAILED DESCRIPTION OF THE INVENTION This invention is based on the unexpected discovery that the formation of optimal liposome and increased reactor capacity is obtained if during the production of the vesicles, the deposition of the lipid obtained by the evaporation of the solvent from an organic solution of one or more film forming lipids in at least one organic solvent, before contact with an aqueous carrier phase, is expanded in a three dimensional structure whose bulk density is below 0.1 g / cm3, preferably below 0.08, greater preference between 0.05 and 0.001 and even more preferred between 0.02 and 0.01. Although the exact reasons for such unexpected results have not been fully established, it is assumed that the method provides an exceptionally large surface area to tank volume, whose subsequent hydration is therefore more efficient. In this way high yields of liposomes of the desired size and distribution are produced by the method, which is particularly easy to scale and control. Having a large surface to volume ratio, the expanded lipid structures improve the space-time performance of the reactors, so the technique becomes industrially very attractive. In addition, to facilitate scaling up and promote high productivity, the method provides other advantages, which include a reactor that rotates with the fastest time, ease of control of the hydration stage, reduced processing times, use of materials and, finally, use of the same reactor for the deposition, evaporation of the solvent, hydration of the expanded lipid structure and sterilization of the formed liposome vesicles. It has been established that the pure lipid porous structures of the invention have a very large surface to volume ratio. Unfortunately, due to the great fragility of the expanded structure, the exact surface area of the volume or weight unit of the expanded lipid structure may not have been established with great accuracy. However, a conservative estimate of the total surface area of 1 g of the expanded structure of the invention suggests that the total surface area may vary between 0.1 and 50 m2, which implies surface to volume ratios of between 10 to 0.5 x 10. ^. The expanded three-dimensional lipid structure can be obtained by evaporating the organic solvent from a reaction vessel, which contains a porous, inert network or a support which serves as a support material or a matrix surface for the deposit of lipids. The inert network can be any convenient material with a relatively large surface to volume ratio and can include a pipe arrangement or an inert packing arrangement such as hollow glass spheres, crosslinked carbon, crosslinked vitreous carbon, crosslinked metal, glass, ceramics or metal wool and fiberglass, ceramic or metal. When a pipe arrangement is used, the dimensions of the pipeline should be chosen in such a way that the maximum surface to volume ratio is achieved. The experiments carried out in the course of the development and characterization of the reactor according to the invention, have shown that in a given configuration the pipe with an inner diameter between 0.5 mm and 5 mm and a wall thickness between 0.5 and 2 mm has provided favorable results, however, another configuration of the reactor can favor other pipe dimensions. It has been established that since the lipid solution extends on the inner and outer surface of the pipe by gravity, a vertically disposed pipe arrangement is preferred although a helical arrangement is also possible. To facilitate the uniform deposition of the lipid films, the inert packing can be fluidized smoothly or the reactor packed with Raschig rings or any other inert material such as that mentioned in the above, it can be fed with the lipid solution from the top and left to gently percolate down the packaging. It is believed that the excellent results obtained in the percolation tower arrangement come from the fact that an efficient control of the thickness of the deposit is achieved by the contact drip shape of the lipid solution and the support. The excess liquid is constantly removed so that the uniform liquid thickness on the entire surface of the support is ensured. To further assist in uniform coating formation of the lipid solution on packaged air or an inert gas such as nitrogen, they can be introduced in a countercurrent fashion over a period of time. The gas is usually cold, however under certain conditions, it may be advantageous if the temperature of the gas is chosen in such a way that the drying and expansion of the lipid film is aided or carried out using a hot gas. After drying and expansion of the reservoir consisting of pure lipids or lipids with the usual degree of purity in the three dimensional structure, the reservoir is contacted with an aqueous carrier phase. Depending on the configuration of the reactor, the carrier phase can be introduced into the lower end of the reactor, for example in the case of the percolation tower or the fluidized bed configuration or in the upper part of the reactor column (in the case of a fixed pipe arrangement). The aqueous carrier phase used may be pure or may contain biologically active substances, contrast agents or both. Virtually any biologically active substance can be trapped in the liposomes produced according to the invention. Such substances include, but are not limited to, antibacterial compounds such as gentamicin, antiviral compounds such as rifamycins, antifungal compounds such as amphotericin B, antiparasitic compounds such as antimony derivatives, antineoplastic compounds such as mitomycin C, doxorubicin and cisplatinum, such proteins. such as albumin and lipoproteins, immunoglobulins, toxins such as diphtheria toxin, enzymes such as catalase, hormones, neurotransmitters, radiopaque compounds such as 99Tc, fluorescent compounds such as carboxyfluoroscein, anti-inflammatories such as salicylic acid and ibuprofen, anesthetics such as dibucaine or lidocaine , etc. Very good results and high entrapment loads are achieved with iodinated x-ray contrast agents such as yopamidol, yomeprol, yohexol, yopentol, yotrolan, iodixanol, yoglucol, etc. The iodine to lipid ratio of the liposome vesicles according to the invention is at least 2.75. The evaporation of the organic solvent or the mixture of solvents is carried out at above room temperature or reduced pressure or both. Experiments have shown that the rate of evaporation has a strong influence on the degree of expansion of the lipid structure. Therefore, for optimal expansion, the amount of heat and pressure inside the reactor will be properly controlled. The control becomes particularly important near the end of the evaporation of the solvent, ie when the solution thickens and becomes viscous. At this point, a slight reduction in pressure will result in a relatively rapid expansion (foaming). It has been established that by balancing the temperature and the pressure for a given solvent or a solvent mixture different degrees of expansion of the lipid deposit can be achieved. Best results are obtained when the organic solvent is selected from petroleum ether, chloroform, methanol, ethanol, propanol, isopropanol, n-butanol, tert-butanol, pentanol, hexanol, pentane, hexane, heptane, cyclohexane and mixtures thereof. Preferably, the solvent is an azeotropic mixture of two solvents. Good results have been obtained with azeotropic mixtures of ethanol with cyclohexane, chloroform with methanol and isopropanol with hexane. The lipids used for the production of liposomes vesicles are conventional and are selected from synthetic or natural, saturated and / or unsaturated phospholipids, including, phosphatidylcholine, phosphatidylethanol-a-ina, phosphatidylserine, phosphatidylglycerol, phosphatidyl-inositol and phosphatidic acid. The following phospholipids are particularly useful, dipalmitoylphosphatidylcholine, di-palmitoylphosphatidylglycerol, dipalmitoylphosphatidyl acid, dipalmitoylphosphatidylethanolamine and the corresponding counterparts distearoyl and dimyristyl and mixtures thereof. These lipids or their mixtures may also contain substances selected from dicetyl phosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, α-tocopherol, stearic acid, stearylamine and mixtures thereof. The invention also includes a supported or unsupported three-dimensional structure of expanded dry lipids with density below 0.1 g / cm 3, preferably below 0.08 g / cm 3 and more preferably with a density between 0.05 and 0.01 g / cm3. By supported structure it is meant that the porous lipid deposit is formed in an inert support material arrangement. Expanded three-dimensional lipid structures are extremely useful for the manufacture of liposomes with high entrapment capacity, particularly when these liposomes are used to transport drugs or diagnostic contrast agents. In such a case, the porous, three-dimensional lipid structure is contacted with an aqueous solution containing the drug or diagnostic agent as an active ingredient, whereby the liposomes will be formed and the ingredient encapsulated. The liposomes carrying the active substance are then processed as appropriate in a conventional manner. Alternatively, a suspension of "empty" liposome vesicles, ie, liposome vesicles containing only the aqueous liquid carrier can be formed first. In the subsequent step, these "empty" liposomes are contacted with a solution containing an active ingredient and charged vesicles using, for example, the transmembrane loading technique. The invention further comprises an apparatus for the manufacture of liposomes with high entrapment capacity, comprising a reaction vessel with an inlet and outlet, a connection to a vacuum, means for cooling or heating, a control means and a packaging, characterized in that the packaging is an intimately packaged pipe arrangement of an inert material. The pipe has an inner diameter of between 0.5 mm and 5 mm and the thickness of the wall of between 0.5 and 2 mm is preferably arranged in a vertical form although an arrangement similar to a coil is also possible. The following examples further illustrate the invention: Example 1 Reactor characterization A 316L stainless steel column, 1 meter high, thermoregulated, vertical with an internal diameter of 50 mm adapted at its lower end with a metal grid, it is filled with 12 stainless steel tubes. The inner diameter of the pipe was 4 mm and the wall thickness was 1 mm. Prior to insertion into the reactor, the pipe is welded per point to form a parallel pipe arrangement. The same configuration of the reactor, but with the pipe of 2 mm and 3 mm 'diameter have also been prepared and tested. Before the tests are directed to the expansion of the lipid deposits, the characterization of the reactor is carried out by depositing unexpanded lipid films using the following lipid composition: hydrogenated soy lecithin / dicetyl phosphate in a ratio molar of 9: 1. The experiments are carried out to determine the best conditions for the deposition of the lipids in the tubes and to establish the impact of the concentration of lipids, internal diameter and nature of the tubes and speed of drainage of the lipid solution. In all cases, lipid solutions in chloroform were introduced into the tubes at room temperature and after filling the tubes from the bottom the lipid solutions are drained at a controlled rate. The deposit is dried at 80 ° C under nitrogen, by evaporation of the solvent and the dry deposit is rinsed 3 times with a small amount of chloroform.

TABLE 1 Concentration Lipids deposited in lipid mg of lipid / 100 cm2 g / 1 | 3 mm tube | 4 mm tube I I 180 33.1 23-.3 220 43.7 29.7 260 62.5 40.0 300 81.5 56.5 320 94.5 80.4 340 111.3 84.2 As shown in Table 1 and Figure 3, the amounts of lipids deposited at various concentrations of lipid and two different diameters of stainless steel tube, the lipid coating increases with an increase in lipid concentration. In addition, the thickest deposits per unit area are obtained in the 3 mm tube than in the 4 mm tube. In both cases, the amounts of deposited lipids appear to be proportional to the square of the lipid concentration. As can be seen from Table 2, the amount of lipids deposited increases with the rate of drainage. However, if these results are expressed with good drainage velocity (in cm per minute) instead of drainage flow rate (in ml per minute) the amounts of deposited lipids appear to be almost proportional to the velocity linear. Regardless of the size of the actual tube. See Figure 4.

TABLE 2 Speed of lipids deposited in mg of lipid / 100 cm2 drainage in a tube of ml / minute 2 mm I 3 mm I 4mm 1 55.2 55.3 37.4 2 76.5 63.0 47.3 3.75 114.9 83.7 65.4 5 121.0 94.5 80.4 Further experiments are carried out on identical scale but using glass tubes instead of stainless steel tubes which have shown that there are no major differences between the two supports with respect to lipid deposition. The homogeneity of the lipid coating was determined by cutting the coated stainless steel tubes from the top at intervals of 10 cm and measuring the thickness of the film. The results obtained are given in Table 3.

TABLE 3 Fraction Lipids deposited in mg 3 mm tube | 4 mm tube 1 5.15 5.13 2 6.63 3.77 3 7.22 4.52 4 8.20 3.50 5 7.59 5.58 6 6.55 6.16 7 5.59 6.38 5.63 6.21 9 5.22 6.97 10 9.06 6.86 Mean ± S.D. 6.68 ± 1. 33 5.51 ± 1.24 The calculation of the apparent densities (volume) of the lipid deposits obtained in three different reactor configurations has shown that for the apparent densities of the 2 mm pipe they were between 0.04 and 0.06 g / cm3, for the 3 mm pipe between 0.02 and 0.04 g / cm3 and for the 4 mm pipe the apparent densities were between 0.01 and 0.03 g / cm3.

Production of Liposomes After characterization, a new reactor is prepared with 250 stainless steel tubes and connected in the circuit shown in Figure 5. 26 g of hydrogenated soy phosphatidylcholine (Nattermann) with 2 g of dicetyl phosphate and 106 g of chloroform, they are placed in a 1-liter glass reactor equipped with a stirrer, a heating jacket and the condenser (1) and heated to 60 ° C under agitation until complete dissolution. The filtered lipid solution through the sterile filter (3) is loaded onto the 316 L stainless steel column with a heating jacket filled with 250 tubes (4) of 304 stainless steel, 1 m long in parallel, by middle of the peristaltic pump (2). The excess solution is removed, the solvent evaporates and the lipids are deposited at 80 ° C by air circulation from the bottom of the column. The 2-liter glass reactor with stirrer, heating jacket, condenser (5) is filled with 849 g of yopamidol, 1196 g of water, 0.54 g of EDTA and 1.60 g of Tris and heated to 90 ° C under stirring until that complete solubilization is obtained. Then the solution of yopamidol is filtered, transferred to the glass reactor (6) and from there the column (4). The solution is circulated at 75 ° C between the reactor (6) and the column (4) by means of the gear pump (7). The liposome suspension formed is extruded through the filter (8), recovered in the reactor (9) and then concentrated using the microfiltration system (10) by means of the pump (11). The concentrated solution is washed with saline (12) to eliminate free yopamidol (diafiltration). Typical iodine to lipid relationships (I / L) for many experiments were run under different experimental condition, were in the range of 2.5-3.5 with lipid concentrations between 25 and 35 mg / ml with the average liposome size of 570 nm. The production unit can be sterilized (for example with steam) and is considered as a large-scale, aseptic, closed-circuit production unit.

Example 2 Example 1 is repeated in the experimental scale enlargement shown in Figure 5 but the size was amplified by a factor of four. 518.6 g of hydrogenated soy phosphatidylcholine (Nattermann) with 41.4 g of dicetyl phosphate and chloroform 2130.0 g, are placed in a 3 liter glass reactor equipped with a stirrer, a heating head and condenser (1) and heated to a 60 ° C under stirring until complete dissolution. The lipid solution is filtered on a sterile 0.22 μ filter (3) using the peristaltic pump (2). The lipid solution is then transferred to the 316 L stainless steel column with a heating jacket filled with 1000 tubes (4) of 304 stainless steel, 1 m long, in parallel and the excess of the lipid solution is removed . The chloroform evaporates and the dry lipids are deposited at 80 ° C by circulating air from the bottom of the column. The stainless steel reactor (316L) of 7 liters with stirrer, heating jacket, condenser (5) is charged with 2920 g of yopamidol, 4110 g of water, 1.87 g of EDTA and 5.50 g of Tris (HCl cs for pH 7.2 ) and heated to 90 ° C under agitation until complete stabilization is obtained. The solution of yopamidol is then passed through the sterile filter (not shown), transferred to column (4) using the gear pump (7) and circulated at 75 ° C between the reactor (6) and the column ( 4) for some time. The liposome suspension formed is recovered in the reactor (6), extruded through the filter (8) at 75 ° C and the liposomes are recovered in the reactor (9). The liposome solution is then concentrated using the microfiltration system (10). The iodine to lipid (I / L) ratios typical for many runs run under different experimental conditions were in the range of 2.5-3.5 with lipid concentrations between 25 and 35 mg / ml with the average liposome size of 570 nm. The apparent density of the lipid deposit varies as a function of the experimental conditions and is estimated to be between 0.08 and 0.05 g / cm3. However, the best liposomes were prepared with the apparent densities between 0.01 and 0.02 g / cm3. 3 Example 2 is repeated using the azeotropic mixture of chloroform and methanol (87/13 = v / v) as a solvent.

After evaporation of the solvent under reduced pressure, hot distilled water at 60 ° C is added to the reactor. The temperature of the added water was above the transition temperature (54 ° C) of the lipids used. The expanded three-dimensional lipid deposit obtained is allowed to hydrate and the liposomes formed are distributed homogeneously through the liquid. The MLV type liposomes are formed in high yield. After approximately 1 hour, the suspension of liposomes containing 5 mg / ml of lipids is extruded at 60 ° C through a 2 μm polycarbonate membrane (Nuclepore) and after cooling to room temperature, it is concentrated to 30 mg / ml by microfiltration using a 0.22 μm microfilter system Prostak (Millipore). To the suspension of concentrated liposomes, 1 liter of an aqueous solution containing 1040 g of (S) -N, N'-bis [2-hydroxy-1- (hydroxymethyl) -ethyl] -2,4,6- is added. triiodo-5-lactamido-isophthalamide (yopamidol) that is, 520 g / 1 of covalent iodine at 60 ° C. The resulting mixture (2 1) with iodine concentration of 260 g / 1 is incubated for about 30 minutes at 60 ° C, after which time the concentration of iodine outside and within the liposome nucleus have equalized. The resulting preparation is concentrated to 30 g of lipids / 1. The ratio of iodine to trapped lipid (I / L) was approximately 4.0.

Example 4 A glass column (500 mm high and 50 mm in diameter) is filled with Raschig rings and operated in a percolation tower reactor. A lipid solution containing 50 g / 1 of a mixture of distearoylphosphatidylcholine (DSPC), cholesterol and dicetyl phosphate with a molar ratio of 5: 4: 1 in chloroform was percolated down the column adapted with 35 layers of Raschig rings. spread over a nickel mesh as a support, until the last layer at the bottom is completely soaked with the solution. The excess solution is removed and a stream of hot nitrogen (80 ° C) is blown from the bottom up through the reactor. The lipid deposit is dried for 2 hours. The flow of nitrogen is stopped and the reactor is connected to a vacuum (1-2 Torr) and the tank is allowed to dry until all of the chloroform is removed. After evaporation of the solvent, the yomeprol solution with iodine concentration 260 g / 1 is added at 60 ° C to the reactor. The expanded, three-dimensional lipid deposit is allowed to hydrate for 30 minutes. The liposome suspension is extruded at 60 ° C through a 2 μm polycarbonate membrane (Nuclepore) and after cooling to room temperature, it is concentrated to 30 g of lipids / 1. The ratio of iodine trapped to lipid (I / L) was above 4.0. The same experiment was repeated with cross-linked carbon, crosslinked nickel and reticulated vitreous carbon as the packing of the column. The apparent densities of the three-dimensional lipid structure obtained in these experiments were between 0.05 and 0.005 g / cm3. Liposomes were obtained with a ratio of lipid to iodine of 3.5-4.5.

Example 5 A glass column (500 mm high and 50 mm diameter) filled with hollow glass beads as an inert packaging arrangement and operated as a fluidized bed reactor. A lipid solution containing 50 g / 1 of a mixture of dipalmitoylphosphatidylcholine (DPPC), cholesterol and dipalmitoylphosphatidic acid (DPPA) with a molar ratio of 5: 4: 1 in an azeotropic mixture of cyclohexane / ethanol (69.5 / 30.5 v / v) ) is introduced into the column containing a 100 mm high bed of hollow glass spheres, supported by a porous glass frit. The solution is allowed to wet the glass beads completely and the excess is removed. A stream of hot air (80 ° C) is blown from the bottom through the reactor and the spheres are fluidized until the lipid deposit was almost dry. Then the air flow stops, the reactor is connected to a vacuum (1-2 Torr) and the tank is allowed to dry until complete removal of the solvents. After evaporation of the solvent, 4% by weight of a solution of HCl lidocaine in water (pH 7.2) at 60 ° C is added to the reactor. The liposome solution formed is extruded at 80 ° C through a 2 μm polycarbonate membrane (Nuclepore) and then cooled to room temperature, concentrated to 35 mg lipid / ml. The lidocaine trapped in the liposomes was 0.35 mmoles of lidocaine / g of lipid. Several expansions (20-80%) of the bed during fluidization showed little influence on the quality of the deposit.

Example 6 Example 4 is repeated using a 500 mm high glass column without the inert packing. The column is filled with 100 ml of lipid solution (80 g / 1) prepared from azeotropic mixtures of ethanol / cyclohexane, chloroform / methanol and isopropanol / hexane. The organic solvent is first evaporated at 55 ° C and 300 mmHg pressure and then at 70 ° C and 10 mmHg until the formation of a dry foam deposit. In all cases, the three-dimensional expanded lipid structure obtained is hydrated at 70 ° C with an aqueous solution of yomeprol to produce suspensions of yomeprol encapsulated in the liposome. After extrusion at 70 ° C on a 0.6 μm polycarbonate membrane (Nuclepore) the liposome suspension is concentrated. Typical iodine to lipid (I / L) ratios for many runs run under different experimental conditions were in the range of 1.9-2.5 with lipid concentrations between 25 and 35 mg / ml. The apparent densities of the expanded lipid structure are calculated to be between 0.05 and 0.001 g / cm3. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (30)

1. A liposome vesicle precursor in the form of a dry lipid deposit, characterized in that the lipid deposit is an expanded, three-dimensional structure with bulk density of less than 0.1 g / cm 3 and preferably less than 0.08 g / cm 3.

2. The vesicle precursor according to claim 1, characterized in that the bulk density of the expanded three-dimensional lipid deposit is between 0.05 and 0.001.

3. The vesicle precursor according to claim 2, characterized in that the bulk density of the expanded three-dimensional lipid deposit is between 0.02 and 0.01.

4. The precursor of the vesicle according to claim 1, characterized in that the expanded three-dimensional structure is supported by a network of inert porous material.

5. The vesicle precursor according to claim 4, characterized in that the inert porous material comprises a pipe arrangement or an inert packing arrangement.

6. The vesicle precursor according to claim 5, characterized in that the pipe has an inner diameter between 0.5 mm and 5 mm and a wall thickness between 0.5 and 2 mm.

7. The vesicle precursor according to claim 5, characterized in that the inert packing is selected from hollow glass spheres, crosslinked carbon, crosslinked vitreous carbon, crosslinked metal, glass wool or metal wool and glass fiber or metal fiber .

8. The vesicle precursor according to any of the preceding claims, characterized in that the lipids are selected from synthetic or natural, saturated and unsaturated phospholipids including phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and mixtures thereof.

9. The vesicle precursor according to claim 8, characterized in that the lipids also contain substances selected from dicetyl phosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, α-tocopherol, stearic acid, stearylamine and mixtures thereof.

10. A method for preparing liposome vesicles with enhanced entrapment capacity by dissolving one or more film forming lipids in at least one organic solvent to form a solution in a reaction vessel, depositing the lipids by evaporation of the solvent, contacting the reservoir of lipid with a carrier phase in aqueous solution and produce liposome vesicles that trap the carrier solution, the method is characterized in that the deposit of lipid obtained by evaporation of the solvent is a three-dimensional, expanded structure whose apparent density is below of 0.1 g / cm3 and preferably below 0.08 g / cm3.

11. The method in accordance with the claim 10, characterized in that the bulk density is between 0.05 and 0.001 g / cm3.

12. The method according to claim 10, characterized in that the reaction vessel contains a network of porous, inert or packaged material which serves as a support or matrix for the deposition of lipids.

13. The method according to claim 12, characterized in that the packaging material is selected from cross-linked carbon, cross-linked vitreous carbon, cross-linked metal, glass or metal wool and glass or metal fiber.

14. The method in accordance with the claim 12, characterized in that the porous, inert packing is hollow glass spheres which are fluidized.

15. The method according to claim 12, characterized in that the inert packing comprises rings Raschig packed in a column as the reaction vessel and the lipid solution is introduced from the top in a percolation form.

16. The method in accordance with the claim 10, characterized in that after the evaporation of the organic solvent, the lipid deposit is in contact with the aqueous carrier phase and which is introduced at the lower end of the column.

17. The method according to claim 10, characterized in that the evaporation is carried out above room temperature or at reduced pressure.

18. The method in accordance with the claim 10 or 12, characterized in that the evaporation is carried out by means of blowing air or an inert gas, preferably nitrogen, through the reaction vessel.

19. The method in accordance with the claim 10 or 12, characterized in that the organic solvent is selected from chloroform, petroleum ether, methanol, ethanol, propanol, isopropanol, n-butanol, tert-butanol, pentanol, hexanol, pentane, hexane, heptane, cyclohexane and mixtures thereof.

20. The method in accordance with the claim 19, characterized in that the organic solvent is an azeotropic mixture of two solvents.

21. The method in accordance with the claim 20, characterized in that the solvent is a mixture of ethanol and cyclohexane, chloroform and methanol or isopropanol and hexane.

22. The method according to claim 10, characterized in that the aqueous carrier phase contains a biologically active substance.

23. The method in accordance with the claim 22, characterized in that the active substance is a contrast agent.

24. The method according to claim 23, characterized in that the contrast agent is an iodinated X-ray contrast agent.

25. The method according to claim 24, characterized in that the ratio of iodine to lipid of the liposome vesicles is at least 2.75.

26. A suspension of liposome vesicles comprising a dispersion of lipid particles hydrated in a carrier liquid, characterized in that the vesicles are obtained by hydration and dispersion of the liposome vesicle precursor according to claim 1-9 in the carrier.

27. A contrast agent, characterized in that it comprises a liposome vesicle suspension according to claim 26.

28. The use of the three-dimensional lipid structures according to claims 1-9, for the manufacture of liposomes with high entrapment capacity.

29. The use of the three-dimensional lipid structures according to claims 1-9, for the manufacture of diagnostic contrast agents.

30. An apparatus for the manufacture of liposomes with high entrapment capacity according to the method according to claims 10-25, comprising a reaction vessel with an inlet and outlet, a connection to a vacuum, a means for cooling or heating, a control means and a packaging, characterized in that the packaging is an arrangement of tubes intimately packed with an inert material, the tubes have an internal diameter between 0.5 mm and 5 mm and the wall thickness between 0.5 and 2 mm.