URIC ACID LIPOSOMES

Abstract

The present invention relates to liposomes that encapsulate uric acid, methods for its preparation and uses of said liposomes.

Claims

1-14. (canceled)

15. Liposome that encapsulates uric acid, uric acid salts, or combinations thereof, wherein said liposome has a lipid bilayer comprising double-chain phospholipids and positively charged cholesterol.

16. The liposome according to claim 15, wherein the encapsulated amount of uric acid is between 6*10.sup.20 and 6*10.sup.18 moles per liposome.

17. The liposome of claim 15, wherein said liposome is unilamellar.

18. The liposome according to claim 15, wherein said liposome has a hydrodynamic diameter measured by Dynamic Light Scattering (DLS) in water at a temperature of 25 C. of between 80 and 140 nm.

19. The liposome according to claim 15, wherein said liposome has a positively charge surface and a Z potential measured by Dynamic Light Scattering (DLS) at a temperature of 25 C. greater than 0 and lower than 15 mV.

20. The liposome according to claim 15, wherein between 2.5% and 10% molar of the double-chain phospholipids are phospholipids with one or more chains of a long-chain polymer that hinder the opsonization of blood liposomes attached to a polar head of said phospholipids; and between 90 and 97.5% molar of the double chain phospholipids are phospholipids with no polyethylene glycol attached to a polar head of said phospholipids.

21. The liposome of claim 20, wherein: said double chain phospholipids with one or more polyethylene glycol chains attached to a polar head are selected from 16:0 PEG2000 PE (or 1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]), 18:0 PEG2000 PE (or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]), 18:0 PEG5000 PE (or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000]) or combinations thereof; and said double chain phospholipids without polyethylene glycol attached to a polar head are selected from DSPC (18:0 PC or 1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (16:0 PC or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine)), 17:0 PC (or 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine) or combinations thereof.

22. The liposome according to claim 15, wherein said positively charged cholesterol is dimethylaminoethane-carbamoyl-cholesterol (DC-cholesterol) hydrochloride.

23. The liposome according to claim 22, comprising a molar fraction ratio between double chain phospholipids and DC-cholesterol between 0.6:0.4 and 0.75:0.25.

24. A method for the manufacture of liposomes that encapsulate uric acid, uric acid salts, or combinations thereof, said method comprising the steps of: a) preparing a solution comprising lithium and/or potassium ions in water; b) adjusting said solution to a pH value between 10 and 11; c) adding uric acid, uric acid salts, or combinations thereof, in which the lithium and/or potassium ions are present in an molar amount that is at least twice than of uric acid, keeping the pH in the range of 10-11 to obtain a complete dissolution; d) lowering the pH to a value between 7.2 and 7.4 to obtain a uric acid solution; e) forming a lipid film; f) rehydrating the lipid film formed in step e) with the uric acid solution of step d) to obtain liposomes; g) extruding the obtained liposomes in step f) using the lithium and/or potassium ions solution of step a) as solvent; and h) filtering the obtained liposomes in step g) using the lithium and/or potassium ions solution of step a) as solvent, wherein steps e) to h) are carried out free of calcium ions.

25. A pharmaceutical composition comprising a liposome according to claim 15 and at least one pharmaceutically acceptable excipient.

26. A medicine comprising the pharmaceutical composition according to claim 25.

27. A method for improving and/or treating cerebrovascular disease comprising administering the pharmaceutical composition according to claim 25 to a patient in need thereof.

29. A medicine comprising the liposome according to claim 15.

30. A method for improving and/or treating cerebrovascular disease comprising administering the liposome according to claim 15 to a patient in need thereof.

Description

[0158] FIG. 1 shows the liposomes stability of the present invention over time when stored at room temperature, as indicated in Example 2 included below. FIG. 1 shows the uric acid percentage that remains encapsulated in the liposomes of the present invention at different time intervals, as a measure of stability of said liposomes. In this sense, a 100% value is established for encapsulated uric acid in liposomes on day 1 and the result for the rest of days is indicated based on or compared to the value on day 1. On y-axis, encapsulated uric acid in liposomes percentage is indicated (considering as 100% the amount of encapsulated uric acid in the liposomes on day 1). x-axis shows the time (storage) in days since the start of the experiment.

[0159] FIG. 2 shows the experimental scheme of the first experiment included in Example 4 below.

[0160] FIG. 3 shows the volume of cerebral infarction (in percentage with respect to volume of the corresponding hemisphere (corrected for edema)) obtained in the first experiment mentioned in example 4. y-axis reflects the volume of cerebral infarction (in percentage with respect to volume of the corresponding hemisphere (corrected for edema)) and x-axis shows the different experimental groups, from left to right, as indicated in example 4: Lipo (empty liposomes diluted in saline), Lipo UA (Liposomes according to the present invention, i.e., that encapsulate uric acid), V (vehicle) and UA (free uric acid solution).

[0161] FIG. 4 shows the result of the neurological tests (neuroscore) obtained in the first experiment mentioned in example 4. y-axis reflects the neuroscore and x-axis shows the different experimental groups, from left to right, as indicated in example 4: Lipo (empty liposomes diluted in saline), Lipo UA (Liposomes according to the present invention, i.e., that encapsulate uric acid), V (vehicle) and UA (free uric acid solution).

[0162] FIG. 5 shows fluorescence confocal microscopy images obtained in Example 5 of mouse ischemic brains. In FIGS. 5A and 5B images, cell nuclei are shown in blue, cerebral blood vessels in red and liposomes of the present invention in green. Is observed that the liposomes of the present invention co-localize or localize in the cerebral vessels of mice. In both FIG. 5A and FIG. 5B, the dates show the co-localization areas of liposomes and cerebral blood vessels. The corresponding images on the right in both FIGS. 5A and 5B show in gray the cerebral liposomes and blood vessels co-localization zone as calculated by image analysis with the ImageJ Fiji program. FIG. 5C is the same as FIG. 5A but in black and white. FIG. 5D is the same as FIG. 5B but in black and white.

EXAMPLES

Example 1. Preparation Liposomes Method by Encapsulating Uric Acid, Comparison of the State of the Art Method for the Preparation of Liposomes with the Method of the Present Invention

a) State of the Art Method for Liposomes Preparation (See, for Example, Mulder W J, Striikers G J, Van Tilborg G a et al. Lipid-Based Nanoparticles for Contrast-Enhanced MRI and Molecular Imaging. NMR Biomed. 2006; 19 (1): 142-64; Needle J, Brea D, Arqibay B, et al. Quick Adjustment of Imaging Tracer Payload, for In Vivo Applications of Theranostic Nanostructures in the Brain. Nanomedicine. 2014; 10(4):851-8). Stealth or Silent or Long Circulating Blood Time Liposomes Preparation Based on DSPC and Cholesterol

[0163] For the preparation of DSPC liposomes (18:0 PC, or 1,2-distearoyl-sn-glycero-3-phosphocholine), PEG2000-PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000) and cholesterol are used in the amounts indicated in table 1, aiming at the preparation of liposomes containing a lipids total amount of 25 mol (normally between 25-100 mol are prepared).

TABLE-US-00001 TABLE 1 Molecular weight, molar fraction and total amount of each of the liposome components to obtain 25 mol of liposomes in the liposome manufacturing process of the state of the art. Molecular weight components (g/mol) Amount (mg) Molar fraction DSPC (18:0 PC) 790.1 12.18 0.617 18:0 PEG2000 PE 2805.5 3.51 0.050 Cholesterol 386.6 3.22 0.333

[0164] All these components are commercially available (for example, from Avanti Polar Lipids with references 850365P, 880120P and 700001P).

[0165] The method carried out, briefly, was:

Phase 1: Lipid Film Formation

[0166] 1) Remove the reagents from the freezer (stored at 20 C.) and allow them to reach room temperature before opening the containers that contain them. [0167] 2) Clean a reaction flask (preferably pear-shaped) with milliQ water or similar (3), followed by ethanol (3) and acetone (3). Clean in the same way a rotary evaporator and any other non-disposable glassware that needs to be used. [0168] 3) Dry the glassware to be used under nitrogen or argon gas stream. [0169] 4) Weigh the lipids and cholesterol required amount (see table 1), transfer them to the reaction flask and add 7 mL of CH.sub.3Cl:MeOH (chloroform-methanol, 6:1) mixture. Mix until the components are completely dissolved. [0170] 5) Place the reaction flask on a rotary evaporator and completely evaporate the organic solvent (typical working conditions are: bath at 30 C., rotation speed of about 140 rpm (revolutions per minute) and controlled pressure drops up to 200 mbar (20 kPa), and from this point on, reduce the pressure very slowly (or increase the vacuum very slowly) to the minimum possible over a period of 10 minutes). Is very important that once the solvent evaporates, a homogeneous lipid film remains at the bottom of the flask. If not, the reagents must be redissolved in 7 mL of the chloroform-methanol mixture and evaporated again. [0171] 6) Keep the flask with the film at maximum vacuum for at least 15 minutes. [0172] 7) Break the vacuum slowly and place the flask for a minimum of 1 hour under nitrogen or argon gas stream, or keep it in a high vacuum desiccator until the next day, to favor the total evaporation of organic solvents

Phase 2: Lipid Film Rehydration

[0173] 8) Heat the aqueous solution with the active principle to be encapsulated (uric acid) above the phase transition temperature (Tm) of the lipids used (65 C. in the present case), maintaining this temperature throughout the process. [0174] 9) Heat an extrusion system to the same temperature. In the present case, a 10 mL LIPEX Thermobarrel extruder from Evonik Industries was used, connected to a thermostatic water recirculation bath and a nitrogen gas stream, as the driving gas. [0175] 10) Place the reaction flask with the lipid film in water bath until reaching working temperature and then add 7 mL of the aqueous solvent prepared in step 8 and stir until the film is completely dissolved (the process can be facilitated adding a few units of glass beads of about 3 mm in diameter). Maintain stirring for about 10 min, with the flask always in the bath to avoid the temperature to drop below Tm.

Phase 3: Liposome Extrusion

[0176] 11) With the extruder balanced at a temperature above Tm (65 C. in the present case), wash it 3 times with a generous amount of aqueous solvent (prepared in step 8 explained above). [0177] 12) Mount a 0.4-micron pore size polycarbonate membrane on the extruder (Millipore) and wet it with solvent (lithium chloride solution). [0178] 13) Fill the extruder with the aqueous lipid mixture and allow the temperature to equilibrate (approximately 10 min). [0179] 14) Use a nitrogen or argon gas stream to force extrusion of the mixture through the membrane. Sample must be collected in a hot container, at a temperature above Tm (working temperature must never be lost). [0180] 15) Repeat the extrusion process on the 0.4-micron membrane one more time. [0181] 16) Repeat the process (4 times) using now a membrane, in this case 0.2 microns and, finally, repeat the extrusion process (8 times) using a 0.1-micron pore size membrane (in total extrusion process involves 20.4 microns+40.2 microns and 80.1 microns), to obtain nominal size liposomes (hydrodynamic diameter) around 100 nanometers (0.1 microns).

Phase 4: Liposome Filtration

[0182] The last step in liposome preparation consists of filtering the extruded solution through molecular mass cut filter systems (Amicon systems, for example) or subjecting them to dialysis processes. In this way, individual components that have not been integrated into liposomes are eliminated and their final concentration is adjusted (adjustment of the final volume of solution) and it is possible, if desired, to change aqueous solvent used for preparation of liposomes by another solvent of a hydrophilic nature (for example, by Serum or saline solution) [0183] 17) Fill a centrifugal filter unit (Amicon tube (or similar)) with 30 kDa cut-off membrane with solvent and filter it in a centrifuge (2 hours at 6000 g and at room temperature). Perform this filtering 3 times. [0184] 18) Pass the extruded liposome solution through the previously washed centrifugal filter unit (step 17) and centrifuge (2 hours at 6000 g). [0185] 19) Adjust the final volume of the liposome solution to the desired value (for example, 10 mL) and adjust the pH to the desired value (typically 7.2-7.4) with a few drops of HCl (1N) or NaOH (1N).

[0186] The liposomes thus prepared, according to the state of the art, can normally be stored at room temperature (or at 4 C. if preferred or if encapsulating agent so requires) for a prolonged period of time from days to months, depending on the composition thereof. In the specific case of uric acid liposomes, this state-of-the-art methodology was not adequate for two main reasons: [0187] 1) Low solubility of uric acid in organic solvents useful for the preparation of lipid films in the formation of liposomes (which prevented its incorporation into liposomes as part of the film, i.e., in step 4 described above) and the great instability which presents this compound in aqueous solutions (tendency to precipitate rapidly) under normal working conditions, made its incorporation into liposomes in stages 8-10 inefficient. [0188] 2) The fact that, after preparation, the liposomes encapsulating uric acid were not very stable in solution: In a period ranging from minutes to a few days, the precipitation of encapsulated uric acid was observed.

b) Method of the Present Invention

[0189] The process objective of the present invention as indicated above was to solve the problems observed with the state of the art process and to be able to obtain uric acid liposomes with two main features: [0190] 1) That they were stable in solution at 4 C. or higher (room temperature) temperatures for a period of at least 21 days. [0191] 2) That said liposomes present a minimum amount of uric acid of 1.6 mg/ml (or 9.5 mM) in solution.

[0192] With respect to what was indicated above in relation to the state of the art liposome manufacturing process, the manufacturing process of the liposomes that encapsulate uric acid of the present invention presented the following modifications: [0193] 1) For the lipid film formation, the steps explained above in the state of the art method were followed, but with the following composition for the lipid bilayer or liposome membrane:DSPC:DC-Cholesterol: 18:0 PEG2000-PE (0.617:0.333:0.050).

TABLE-US-00002 TABLE 2 Molecular weight, molar fraction and total amount of each of the liposome components to obtain 100 mol of liposomes in the method of the present invention. Molecular weight components (g/mol) Amount (mg) Molar fraction DSPC (18:0 PC) 790.1 48.72 0.617 18:0 PEG2000 PE 2805.5 14.04 0.050 DC-Cholesterol 537.26 17.89 0.333 [0194] 2) Preparation of the uric acid solution in aqueous medium and Phase 2:

[0195] To rehydrate the lipid film (Stage 2, steps 8-10 of the state of the art process) it was necessary to use an uric acid aqueous solution. This stage was essential in the preparation of uric acid liposomes and was key in obtaining liposome compositions of adequate concentration and stable over time. The process for preparing these solutions is described below and was:

a) Prepare a 40 mM Lithium Chloride Solution (1.696 g/l).

[0196] It was essential to use water that did not contain even traces of calcium ions (and preferably avoid the presence of sodium and carbonate ions). Distilled water, MilliQ or the like, as well as saline solutions, phosphate buffered saline or others, which are commonly used in laboratories for liposome synthesis were not suitable. With traces of calcium ions, uric acid solutions were unstable and tended to precipitate over time. Therefore, HPLC (High Performance Liquid Chromatography) grade water (Water, HPLC for Gradient Analysis, Fisher Chemical Ref 10449380) was used.

[0197] Since uric acid is divalent, the concentration of lithium chloride (or lithium and/or potassium salt) was at least twice that of uric acid since there must be at least twice as much lithium as uric acid (e.g., for an uric acid solution 20 mM, at least lithium chloride 40 mM was used). If a lower concentration is used, uric acid will end up precipitating over time, faster and in greater quantity the lower the concentration of lithium chloride. [0198] Once the lithium chloride solution was prepared, was essential to adjust the pH to a value between 10 and 11 using, for example, KOH 1M on demand. A pH meter electrode was introduced into the LiCl solution, and the pH was adjusted until it was in the desired range. The used KOH solution (or any other solution used in this step) did not contain calcium ions traces.

[0199] Next, uric acid was added:

b) Prepare a 20 mM Uric Acid Solution (3.362 g/l). [0200] At this point, uric acid was added little by little (adequate mass so that the final concentration was over 20 mM). It was essential to continuously monitor the solution pH as the solid uric acid was added to the LiCl, and that the pH never fell below 10 or rise above 11. In any case, the pH had to be corrected by adding a few drops of KOH 1M or HCl 1M so that the pH remained in 10-11 range. [0201] The HCl and KOH 1M solutions did not contain traces of calcium ions. [0202] At the end of the dissolution of all the uric acid in the lithium chloride solution, a clear solution was obtained, with no turbidity; the pH was lowered very slowly to 7.2-7.4 using a few drops of HCl 1M (solution without calcium traces).

[0203] The final result of this process was a uric acid solution 20 mM in LiCl 40 mM with pH 7.2-7.4, totally transparent, and which was stable at room T (and at 4 C.) for days. With this solution, the lipid film formed in phase 1 was rehydrated, following steps 8-10 as previously described (Working temperature T=65 C.) (i.e., as in the method for manufacturing or preparation liposome of the state of the art). [0204] In the process of the present invention, a lithium chloride solution was required (calcium ions free, for example using HPLC water, as already described) with a 0.65 g/l (0.65% weight/volume (w/v)-15 mM) concentration at pH 7.2-7.4 which was used as an aqueous solvent (even for cleaning material) in all steps of the liposome preparation process of the present invention.

C) Liposomes Extrusion and Filtration (Phases 3 and 4)

[0205] Extrusion followed the same steps as those indicated in the state of the art method, but in any case, the 0.65% (w/v) LiCl solution (15 mM) was used as solvent.

[0206] Filtration followed the same steps as those indicated in the state of the art method using as solvent the 0.65% (w/v) LiCl solution (15 mM) that was adjusted to 10 mL and to pH 7.2-7.3 with KOH 1M, or HCl 1M, as required.

[0207] Following all these modified steps, a clear solution (with no turbidity) of uric acid liposomes (which encapsulate said uric acid) was obtained, which had about a 11 mM concentration of said uric acid (the exact amount will depend on the performance of the synthesis process).

Results Obtained:

[0208] 26 batches of uric acid liposomes were prepared. [0209] 1liposomes preparation according to the conventional method of the state of the art explained above in this example, including the use of phosphate buffer saline as a vehicle
Batches: 3 Batchess with Problems: 3 Failed Batches: 100% [0210] 2liposomes preparation according to the conventional method of the state of the art explained above in this example, but with pH control by dissolving uric acid and the use of Saline Serum as a vehicle
Batches: 10 Batches with Problems: 4 Failed Batches: 40% [0211] 3Preparation of liposomes according to the conventional method of the state of the art explained above in this example, with pH control when dissolving uric acid and of the final solution of liposomes, use of HPLC water in synthesis and use of LiCl 0.65% w/v as a vehicle (material washes made with MiliQ water)
Batches: 7 Batches with Problems: 1 Failed Batches: 14% [0212] 4Process of the present invention (the entire process done with HPLC water, including material washing)
Batches: 6 Batches with Problems: 0 Failed Batches: 0%

[0213] Observed problems in the cases of batches 1 to 3 were basically formation of uric acid crystals in suspension and precipitation, at different times, after preparation. Surprisingly, with the process of the present invention, all these problems were solved, and all the manufactured batches were successful.

Example 2. Characterization of the Liposomes Obtained by the Method of the Present Invention

[0214] Following the method of the present invention described in Example 1, 22 batches of uric acid liposomes (AU liposomes) and 22 batches of liposomes of identical composition, but with no uric acid inside (control liposomes) were prepared. Each of the preparations was characterized, as indicated below.

1) Size and z-Potential of Liposomes.

[0215] Liposomes size (in the form of hydrodynamic diameter) and Z potential determination was done by means of DLS, for which a Malvern z-sizer equipment was used, operated according to the manufacturer's instructions, at a temperature of 25 C. Briefly, for this, a 100-microliter sample was extracted from the final liposome solution, bringing it to 1 mL (dilution 1 to 10) in HPLC grade water that was introduced into a cuvette. The equipment was turned on and the laser was allowed to stabilize for at least 30 minutes. The sample was then thermostated in the cuvette inside the equipment for at least 3 minutes, to proceed later with the measurement. In no case the sample concentration was greater than 1 mg/mL.

[0216] The size (in the form of hydrodynamic diameter), polydispersity and potential Z obtained was indicated in Table 3 included below:

TABLE-US-00003 TABLE 3 Liposomes that encapsulate uric acid obtained according to the method of the present invention and control liposomes size and Z potential. Hydrodynamic Sample diameter (in nm) Polydispersity Z Potential (mV) AU liposomes 113.3 13.7 0.008 0.003 10.42 2.1 Control liposomes 104.1 9.4 0.010 0.008 10.43 1.0

2) Production Performance

[0217] Is normal that in the multiple stages involved in the synthesis, extrusion and filtering of liposomes, a certain amount of material is lost. To determine the performance of the process of the present invention, the amount of lipids in the final liposome solutions was determined, using the well-known Rouser colorimetric method (Rouser G, Fkeischer S, Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May; 5(5):494-6. doi: 10.1007/BF02531316. PMID: 5483450).

TABLE-US-00004 TABLE 4 Yield results in production obtained in Example 2. Sample Heavy lipids (mol) Final lipids (mol) Yield (%) AU liposomes 66.7 0.02 44.4 2.5 66.6 3.7 Control liposomes 66.7 0.02 44.9 0.9 67.3 1.4

3) Encapsulation Efficiency

[0218] In each uric acid liposomes preparation, the exact amount of therapeutic agent encapsulated in the liposomes was calculated using a colorimetric technique based on the uricase digestion method described by Hamzah H H et al. (Hamzah H H, Zain Z M, Musa N L W, Lin Y C, Trimbee E (2013) Spectrophotometric Determination of Uric Acid in Urine Based-Enzymatic Method Uricase with 4-Aminodiphenylamine Diazonium Sulfate (Variamine Blue RT Salt). J Anal Bioanal Tech S7: 011. doi:10.4172/2155-9872.S7-011). In summary, 25 L of non-encapsulated uric acid solution were taken (after encapsulation, the non-encapsulated uric acid sample is obtained in the filtration phase by centrifugation), 25 L of Variamin 0.1 mM and 50 L of uricase (50 g/ml), bringing the final volume to 1 ml. The mixture was kept at 37 C. for 30 minutes and the amount of uric acid in solution was determined by measuring the absorbance at 261 nm, determining the concentration by means of a calibration line obtained from standard uric acid solutions measured in the same way.

[0219] To calculate encapsulation efficiency, the exact uric acid amount added in each liposome preparation was recorded in phase 2 of the liposome preparation process of the present invention (hydration of the lipid film) and the non-encapsulated uric acid amount was determined by colorimetry, from the obtained filtrate in phase 4 of the liposome preparation process of the present invention (filtrate in an Amicon tube with a cut-off point of 30 kDa). Obtained results are summarized in Table 5 included below:

TABLE-US-00005 TABLE 5 Yield results in encapsulation obtained in Example 2 for the liposome preparation process of the present invention. Initial uric acid Encapsulated uric Encapsulation Sample (mol) acid (mol) Efficiency (%) AU liposomes 140.58 0.26 112.01 8.68 79.7 6.2

4) Liposomes Stability

[0220] Finally, a study was carried out in which stability of the liposomes in solution at room temperature was observed for a period of 21 days. In this period, no turbidity or precipitate formation was observed. On the other hand, the liposomes were filtered every 3-7 days and the uric acid amount that remained encapsulated and which part had been released in solution were determined, using the colorimetric method described in the previous section. Obtained results showed that 7 days after the liposomes synthesis or elaboration, more than 90% of uric acid remained encapsulated while less than 10% had been released, the liposomes remaining, therefore, stable in solution. At 15 and 21 days from the liposomes synthesis or elaboration, the initial uric acid that remained encapsulated amounts were greater than 80%, demonstrating the great stability of the liposomes (see FIG. 1). It is important to note that despite the fact that at long storage times a small portion of uric acid is released in solution (released from liposomes), the solutions retained their transparency and uric acid crystallization was not observed.

Example 3. Analysis of Different Compositions of the Lipid Bilayer in Liposomes that Encapsulate Uric Acid

[0221] Four different lipid compositions used in the process of the present invention were analyzed for the preparation of liposomes that encapsulate uric acid: [0222] 1DOPE:cholesterol:18:0 PEG2000-PE Molar fractions (0.583:0.333:0.083) [0223] 2DSPC:Cholesterol:18:0 PEG2000-PE Molar fractions (0.583:0.3333:0.083) [0224] 3DOTAP:DSPC:Cholesterol:18:0 PEG2000-PE Molar fractions (0.3:0.283:0.33:0.083) [0225] 4DSPC:DC-Cholesterol:18:0 PEG2000-PE Molar fractions (0.617:0.333:0.050) [0226] DOPE: 18:1 (9-Cis) PC (DOPC) 1,2-dioleol-sn-glycero-3-phosphocholine [0227] DOTAP: N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium

[0228] The lipid bilayer compositions 1 to 3 showed non-optimal results of encapsulation and uric acid release and lower than those of 4, which did show optimal results of encapsulation and uric acid release. Specifically, it was observed that the kinetics of uric acid release from the liposomes to dissolution were faster in compositions 1 to 3, compared to 4, in such a way that 7 days after preparation less than 65% of the UA remained. encapsulated in the liposomes of formulations 1 to 3 (compared to 90% of formulation 4) and after 15 days less than 45% of the UA remained encapsulated in the liposomes of formulations 1 to 3 (compared to 80% of formulation 4).

Example 4. Efficacy Analysis of Uric Acid Liposomes of the Present Invention in a Mouse Model of Cerebral Ischemia/Reperfusion

[0229] In this example, the liposomes of the present invention efficacy was analyzed in a mouse model of brain ischemia/reperfusion. The experimental details were as follows: [0230] Species: Mouse C57BL/6 from the supplier Laboratorios Janvier; sex: males; age: 10 to 14 weeks. [0231] Dose: 16 mg uric acid/kg mouse weight. [0232] Ischemia/reperfusion: 30 min middle cerebral artery occlusion (MCAO) (monitored with laser Doppler) followed by 24 h of reperfusion. [0233] Treatment: a) Intravenous infusion (20 minutes duration) of the different treatments. Treatment began 30 min after reperfusion. Treatments were administered blind. [0234] Neurological test: At 24 h the neuroscore was performed. [0235] Euthanasia: After neurological test, the animals were sacrificed. Blood samples were taken, and the brain was extracted which was cut out for staining with TTC (tetrazolium chloride) to measure the cerebral infarct volume. [0236] Inclusion/exclusion criteria: All mice that had a drop in blood flow greater than 65% and a reperfusion greater than 70% received the treatment. Of these, animals were not included if the injection was not correct. Of the correctly administered animals, mice that did not develop an infarct or with a very small infarct (<10%), and mice that had an infarct outside the territory of the middle cerebral artery were excluded. Animals that died were counted but were not included in the study due to the lack of volume/neuroscore data at end point. [0237] See summary of the experimental protocol included in FIG. 2.

[0238] The treatment was randomized, and the administration of the drugs was performed blind. The treatment groups were: [0239] a) Liposomes that encapsulate uric acid diluted in saline solution (Lipo-UA) (Liposomes according to the present invention). [0240] b) Empty liposomes diluted in saline solution (Lipo) (i.e., liposomes prepared according to the method of the present invention but with no uric acid). [0241] c) Uric acid solution (dissolved in mannitol and lithium) (UA). [0242] d) Corresponding vehicle (V): is the same solution as that used in group c) but with no uric acid (that is, a mannitol and lithium solution with no uric acid).

Results:

[0243] Table 6 includes a summary of the animals included in each of the experimental groups:

TABLE-US-00006 TABLE 6 Summary of the mice included in each of the experimental groups of Example 4. The numbers indicated in the table refer in all cases to the number of mice. Lipo-UA Lipo UA V Total 18 20 16 18 Excluded 1 5 4 3 Included 16 15 12 13 Mortality 1 0 0 2 Mortality % 5.6% 0% 0% 11%

[0244] Analysis of the results was done in a blinded mode.

[0245] The obtained results for the cerebral infarction volume and neuroscore in the different experimental groups are summarized in FIGS. 3 and 4.

[0246] Administration of Lipo-UA produced a significant decrease (31.79%) in the volume of the cerebral infarct compared to the control group (Lipo) (see FIG. 3).

[0247] UA administration produced an also significant decrease (28.61%) in the cerebral infarct volume compared to the control group (vehicle; V) (see FIG. 3).

[0248] Treatment with Lipo-UA and treatment with UA caused a similar reduction in cerebral infarct volume, however, a tendency was observed for the effect to be greater with Lipo-UA (see FIG. 3).

[0249] Additionally, and surprisingly, treatment with Lipo-UA produced an improvement in neurological function as deduced from the significant reduction (25.6%) in the neuroscore test score. This effect was not observed in UA treatment (see FIG. 4) Additionally, it was possible to corroborate that the liposomes of the present invention reached the blood capillaries of the brain effectively (see example 5 and FIG. 5).

[0250] Finally, the liposome manufacturing method effect on their effectiveness was also studied. For this, the same protocol indicated above was followed but with uric acid liposomes prepared according to the state of the art method and uric acid liposomes prepared according to the method of the present invention. The results obtained were those shown in Tables 7 and 8 included below:

TABLE-US-00007 TABLE 7 Results obtained with uric acid liposomes prepared according to the state of the art method. Lipo- Reduction Lipo UA % p No. of mice 7 7 Cerebral infarct volume 42.57 40.03 5.97 p = 0.6983 (mm.sup.3) Cerebral infarction volume 26.24 23.99 8.57 p = 0.5590 (% with respect to the volume of the corresponding hemisphere (corrected for edema)) Neuroscore 11.29 9.71 13.96 p = 0.4202

TABLE-US-00008 TABLE 8 Obtained results with uric acid liposomes prepared according to the method of the present invention. Lipo- Reduction Lipo UA % p No. of mice 10 9 Cerebral infarct volume 45.53 31.86 30.02 p = 0.0215 (mm.sup.3) Cerebral infarction volume 27.26 19.83 27.26 p = 0.0222 (% with respect to the volume of the corresponding hemisphere (corrected for edema)) Neuroscore 9.60 7.89 17.82 p = 0.1216

[0251] As derived from Tables 7 and 8, the method of the present invention makes it possible to obtain liposomes that encapsulate uric acid that show a greater therapeutic effect (in the form of a smaller volume of cerebral infarction and a better neuroscore) compared to uric acid liposomes obtained with the process of the state of the art.

Example 5. Study of the Localization of Liposomes of the Present Invention in the Mouse Brain

[0252] In this case, the ischemic mice (obtained as indicated in Example 4) were administered liposomes of the present invention (with uric acid) or control liposomes (with saline solution), all of them with a green fluorescent protein (Dioc18), according to the liposome administration method set forth above. Two hours after reperfusion, euthanasia was carried out, the brain was fixed with 4% paraformaldehyde, and the tissue was processed for immunofluorescence and confocal microscopy. For this, coronal sections of the brain were made with vibratome (50 m thick) that were cryoprotected in glycerol and stored at 20 C. Blood vessels were stained with the anti-Glut1 antibody followed by a secondary antibody AlexaFluor-556 (red). Nuclei are visualized with DAPI staining (blue). A confocal microscopy study (DragonFly) was carried out, making 1 m planes to carry out a co-localization study with the ImageJ software (Colocalization threshold). FIG. 5 shows a representative image that illustrates the presence of the green fluorescent protein Dioc18 in the capillary wall (red) after the liposomes that encapsulate uric acid and Dioc18 administration.

[0253] Therefore, Examples 1 to 5 demonstrate that the inventors of the present invention have been able to effectively obtain liposomes that encapsulate uric acid, that said liposomes are stable over time and are superior (superior therapeutic effect) for the treatment of stroke.

[0254] Additionally, the results collected in examples 1 to 5 demonstrate the surprising results obtained with the process of the present invention for the preparation of liposomes that encapsulate uric acid, both in terms of performance and stability, as well as a surprising superior therapeutic effect.