Co-crystals of uric acid for the treatment of neurological diseases

Abstract

The present invention are co-crystals of Uric Acid with increased solubility with respect to crystals of Uric Acid, suitable for the pharmaceutical use. The invention comprises co-crystals with L-lysine, L-arginine, ethylene diamine, and tromethamine as co-formers, pharmaceutical compositions thereof and their therapeutical use in the prevention or treatment of cerebrovascular diseases.

Description

[0001] CO-CRYSTALS OF URIC ACID

[0002] Field of the invention

[0003] The present invention belongs to the fields of chemistry and medicine, in particular to the crystallographic chemistry of uric acid in the form of co-crystals, and to the pharmaceutical use thereof.

[0004] Background

[0005] The potential therapeutical medical effect of uric acid (UA) as active agent against neurological diseases has been extensively disclosed in the art, in particular for cerebrovascular diseases.

[0006] UA is a powerful antioxidant agent that blocks the reaction between superoxide anion and nitric oxide, which would damage cells by nitrosylating tyrosine residues of proteins. The plasma concentration of UA is almost 10 times higher than other antioxidant substances, such as vitamins C or E, and its antioxidant capacity is also higher. Beyond these antioxidant effects, UA also acts on transcription factors as therapeutic targets, among others having a positive regulation in the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF).

[0007] It is known that the administration of UA before an occlusion of the middle cerebral artery or after reperfusion significantly reduces resulting cerebral infarction, suppresses accumulation of Reactive Oxygen Species (ROS) and decreases lipid peroxidation. Further, the administration of UA has been shown neuroprotective in a thromboembolic model of focal cerebral ischemia. The relationship between higher UA levels in the blood at the time of a cerebral infarction and a lower neurological severity is also well known in the art. Among others, the international application WO 2010112113 A1 discloses the combined use of uric acid and citicoline for the treatment of stroke.

[0008] However, the use of UA as a therapeutical agent involves a serial of preventions that need to be watched out. The main one is that UA has limited solubility in organic fluids, with tendency to crystallize, which complicates its incorporation in pharmaceutical formulations for a medical use.

[0009] Unwanted formation of crystals of UA in organic fluids has been reported in a serial of publications, always referring to crystallization at an excess of the compound. These crystals are prejudicial because they harm the organs where they show up and have no therapeutical effect by themselves. For example, RU 2260801 C1 reports the formation of atypical crystals of LIA of different forms in combination with cholesterol crystals at purine exchange disorders.

[0010] To overcome these difficulties, transport systems or platforms by nanomaterials and controlled release scaffolds have been developed as an alternative to the use of free LIA as a therapeutical agent. These delivery agents facilitate stability, allowing better logistics in the clinical setting and a suitable administration into the body in therapeutically relevant doses.

[0011] WO2025172340 describes the obtention of crystals of LIA in a synthetic pathway capable for therapeutical use. The obtention and characterization of crystals offer improved pharmaceutical features that enable effective dosages of the compound. In fact, the crystals described in WO2025172340 are the starting material for the obtention of the cocrystals of the present invention; the aim is that these co-crystal forms of LIA help solving the problems that still present at the pharmaceutical delivery of the active agent.

[0012] Hall V. M. discloses five co-crystals of LIA 1:1 with several pyridines and pyrimidines in the form of hydrates (Hall V. M. etal. «llric Acid Crystallization Interrupted with Competing Binding Agents» Crystal Growth & Design, Vol. 19, 12, 2019,

[0013]

[0014] Some of the obtained co-crystals present a solubility in simulated urine six times higher than sodium urate and two times higher than anhydrous LIA. The document also refers to a new co-crystal of llric-trimethoprim with intermediate solubility between anhydrous LIA and sodium urate that serves as an antibiotic. These results aimed to study the inhibition of crystallization of LIA in vivo related to kidney diseases and gout. However, pyridine and some derivatives of pyrimidine are toxic, and their use in pharmaceuticals is therefore restricted.

[0015] Sours R. E. incorporates small concentrations of methylene blue molecules in the crystalline matrices of anhydrous LIA during the grow of crystals, with great specificity, in the form of hexahydrate (Sours R. E. et al. «Dyeing uric acid crystals with methylene blue», Journal of the American Chemical Society, Vol 124, 29, 2002 (https: / / pubs.acs.Org / doi / 10.1021 / ja026083w). Methylene blue is anyway toxic for the human body, and at least for this reason these crystals have no application in the pharmaceutical industry.

[0016] Vickovic I. discloses the crystalline structure of urate of guanidium monohydrate (Vickovic I. et al. «Crystal structure of guanidinium urate monohydrate, CsHal^ Oa CHeNa) ■ H2O», Z. Krystallogr. NCS 215, 4, 583-584, 2000, https: / / www.degruyterbrill.eom / document / doi / 10.1515 / ncrs-2000-0455 / html). Crystals of UA obtained from biological sources do not comply with the pharmaceutical standards because of the presence of eventual impurities and, therefore, may not be suitable for GMP-compliant products intended for animal or human administration. Eventual co-crystals of UA of increased solubility but obtained from these crystals would suffer the same drawback.

[0017] The problem of the art is to increase the pharmaceutical delivery of uric acid with respect to the crystals of the active agent obtained by the synthetic pathway described in WO2025172340. The solution of the present invention is the development of new cocrystal forms of improved solubility.

[0018] Description of the Invention

[0019] The present invention is directed to novel crystalline forms of uric acid (UA) co-crystallized with different co-formers.

[0020] Accordingly, a first aspect is a co-crystal comprising UA and L-lysine. Another aspect is a co-crystal comprising UA and L-arginine. Another aspect is a co-crystal comprising UA and ethylenediamine. Still another aspect is a co-crystal comprising UA and tromethamine.

[0021] A preferred aspect of the invention refers to a co-crystal consisting of UA and L-lysine. Another preferred aspect is a co-crystal consisting of UA and L-arginine. Another preferred aspect is a co-crystal consisting of UA and ethylenediamine. Still another preferred aspect is a co-crystal consisting of UA and tromethamine.

[0022] The UA used as starting material in the synthesis of the co-crystals referred herein is obtained by a synthetic process according to WO2025172340 and is termed in the present application as sample SP308-FD-P1.

[0023] I. P IL- H. id

[0024]

[0025] Another preferred aspect of the invention is the co-crystal of UA and L-lysine with a stoichiometric ratio of UA to L-lysine of 1:1. A more preferable aspect is a first polymorph A of the co-crystal of LIA and L-lysine containing 0.8 equivalents of water. This is shown by the Thermogravimetry Coupled to Fourier Transform Infrared Spectroscopy (TG-FTIR) thermogram (Figure 6), revealing a water loss of 4.23 wt.% from 22°C to 200°C. It is also seen that decomposition at higher temperatures starts at about 200°C.

[0026] In a preferable aspect of the invention, polymorph A has a powder X-ray diffraction pattern (PXRD) comprising a 20 angle at value of 14.80 and at least three 20 angle values selected from the group consisting of 11.88, 16.44, 17.61, 20.04, 20.80, and 23.93. In another preferable aspect, the PXRD comprises a 20 angle at value of 14.80 and at least six 20 angle values selected from the group consisting of 11.38, 11.88, 16.44, 16.68, 17.61, 20.04, 20.63, 20.80, 21.30, 23.93, 25.56, 29.21, 34.81, and 36.25. In another aspect, the PXRD comprises all the values referred to. The obtained diffractogram accepts a ±0.1 tolerance in the 20 angle values, preferably a ±0.2 tolerance.

[0027] The obtained PXRD pattern of the L-lysine co-crystal Polymorph A (Figure 5) is different than the pattern of crystals of uric acid (Figure 1) and also than those of anhydrous and hydrated forms of L-lysine of the art.

[0028] The Differential Scanning Calorimetry (DSC) curve of said polymorph A (Figure 7) shows a first large endothermal event at 86.8°C that likely corresponds to the release of water. Then a series of different thermal events can be observed at higher temperatures. Melting occurs likely under decomposition, although the meting point of the L-lysine co-crystal has not been exactly determined.

[0029] Another preferable aspect of the invention is a second polymorph B of the co-crystal of UA and L-lysine containing 0.85 eq. of water. This is shown by TG-FTIR thermogram (Figure 11) revealing a water loss of 4.65 wt.% from 22°C to 220°C. Decomposition is observed at higher temperatures.

[0030] Preferably, this polymorph B has a PXRD (Figure 9) comprising a 2© angle at value of 17.88 and at least three 2© angle values selected from the group consisting of 11.90, 16.65, 20.03, 20.56, 23.95, and 24.36. In another preferable aspect, the PXRD comprises a 2© angle at value of 17.88 and at least six 20 angle values selected from the group consisting of 11.90, 16.65, 17.64, 20.03, 20.56, 21.54, 23.20, 23.95, 24.36, 25.06, 27.65, 29.61, and 35.39. In another aspect, the PXRD comprises all the values referred to. The obtained diffractogram accepts a ±0.1 tolerance in these 20 angle values, preferably a ±0.2 tolerance. When overlapping the PXRD patterns of the co-crystal uric acid and L-lysine Polymorph A and Polymorph B (Figure 10), it can be observed that the two PXRD patterns show some significant deviations. As said, the relevant peak at 14.80 of polymorph A is absent in the diffractogram of polymorph B; in turn, polymorph B shows a peak at 17.88 that is absent in polymorph A. Likewise, a double peak at 20 angle of 17.64 / 17.88 is shown for Polymorph B whereas only one peak appears at 17.61 for Polymorph A.

[0031] These results confirm the presence of two different crystalline forms of the co-crystal of LIA with L-lysine.

[0032] In the present application, a relevant peak in a PXRD spectrum is considered a specific diffraction peak that has a peak intensity typically at least three times higher than the background noise, significant enough to be distinguished, with the most intense peak in a phase normalized to 100%, and that contains crucial information about a material's crystalline structure.

[0033] The DSC curve of said Polymorph B (Figure 12) shows a first large endothermal event at 54.8°C that likely corresponds to the release of water. Then, the melting peak is observed at 271.6°C with an onset at 265°C. Therefore, a further aspect of the invention is the polymorph B of the co-crystal with L-lysine with a melting point onset at 271.6°C.

[0034] The Dynamic Vapor Sorption (DVS) isotherms of samples of both Polymorph A (Figure 8) and Polymorph B (Figure 13) indicate hygroscopic samples, based on a water uptake at 85% relative humidity (r.h.) compared to the mass of the sample at the start of the DVS measurement.

[0035] The co-crystals of uric acid and L-lysine of the present invention shows increased solubility with respect to UA, increasing the bioavailability of UA as active agent and thereby achieving an advantage for the pharmaceutical delivery of the active agent.

[0036] According to the art, pure UA has a solubility of about <1 g / L, whilst polymorph A referred herein has a solubility of 13.5 g / L and polymorph B of 29.5 g / L of uric acid (Example 4). Polymorph B has a higher solubility than Polymorph A, showing that the latter may be more stable as both crystalline forms contain water.

[0037] In another aspect of the present invention, the co-crystals of UA contain L-arginine, ethylene diamine, or tromethamine as co-formers. These co-formers were selected based on their structure attending to functional groups and interactions, an aqueous solubility of higher than 2 mg / mL, a molecular weight < 200 g / mol, and their pharmaceutical acceptance. The obtained co-crystals were characterized by PXRD, TG-FTIR, and elemental analysis to confirm crystallinity, the solvent content, and their composition.

[0038] For the co-crystal of LIA and L-arginine, the elemental analysis (Example 6) match with the theoretical content of monohydrate of a 1:1 co-crystal. Therefore, another preferable aspect of the invention is the co-crystal of LIA and L-arginine in the form of a monohydrate, even more preferably having a stoichiometric ratio of LIA to L-arginine of 1 :1.

[0039] Consistently, the TG-FTIR thermogram of co-crystal of UA and L-arginine (Figure 15) reveals that the sample contains water. A first water loss of 0.4% is observed from 25°C to 110°C. Then, 6.01% of loss was observed from 110°C to 235°C and corresponds likely to the release of water and carbon dioxide. Decomposition is observed at higher temperatures.

[0040] More preferably, the co-crystal of UA and L-arginine has a PXRD (Figure 14) comprising at least three 2G angle values selected from the group consisting of 6.65, 17.04, 19.47, 20.14, 25.85, and 28.32. In another preferable aspect, the PXRD comprises at least six 2G angle values selected from the group consisting of 3.31, 6.65, 17.04, 18.01, 18.69, 19.11, 19.47, 20.14, 21.44, 23.68, 25.85, 26.24, 28.32, and 31.00. In another aspect, the PXRD comprises all the values referred to. The obtained diffractogram accepts a ±0.1 tolerance in these 2G angle values, preferably a ±0.2 tolerance.

[0041] For the co-crystal of UA and ethylene diamine, the results of the elemental analysis (Example 7) fit well with a theoretical content of a 2:1 co-crystal rather than a 1:1 species. Therefore, another preferable aspect of the invention is the co-crystal of UA and ethylene diamine with a stoichiometric ratio of UA to ethylene diamine of 2:1.

[0042] The TG-FTIR thermogram of the co-crystal of UA and ethylene diamine (Figure 17) reveals a first loss of 2.42% from 25°C to 185°C that corresponds likely to the release of water and carbon dioxide. Then, 0.31% of water loss was observed from 185°C to 240°C. Based on these results, the observed water content seems to be surface adsorbed water. Decomposition is observed at higher temperatures.

[0043] More preferably, the co-crystal of UA and ethylene diamine has a PXRD (Figure 16) comprising at least three 2G angle values selected from the group consisting of 11.27, 16.93, 23.35, 26.45, and 28.85. In another preferable aspect, the PXRD comprises at least six 2G angle values selected from the group consisting of 11.27, 12.57, 16.93, 23.35, 26.45, 27.81, and 28.85. In another aspect, the PXRD comprises all the values referred to. The obtained diffractogram accepts a ±0.1 tolerance in the 20 angle values, preferably a ±0.2 tolerance.

[0044] For the co-crystal of LIA and tromethamine, the results of the elemental analysis (Example 8) fit relatively well with the theoretical content of hemihydrate of a 1:1 co-crystal. Therefore, another preferable aspect of the invention is the co-crystal of LIA and tromethamine in the form of a hemihydrate, even more preferably having a stoichiometric ratio of LIA to tromethamine of 1:1.

[0045] Consistently with this, the TG-FTIR thermogram of co-crystal of LIA and tromethamine (Figure 19) revealed 3.5% of water, wherein the theoretical water content for a hemihydrate of the co-crystal of uric acid and tromethamine is 3.0%. The TG-FTIR reveals a water loss of 3.5% from 25°C to 220°C. Decomposition is observed at higher temperatures.

[0046] More preferably, the co-crystal of LIA and tromethamine has a PXRD (Figure 18) comprising at least three 20 angle values selected from the group consisting of 6.41, 13.60, 16.81, and 27.24. In another preferable aspect, the PXRD comprises at least six 20 angle values selected from the group consisting of 6.41, 13.60, 14.22, 15.78, 16.81, 17.67, 19.17, 21.75, and 27.24. In another aspect, the PXRD comprises all the values referred to. The obtained diffractogram accepts a ±0.1 tolerance, preferably a ±0.2 tolerance, in the 20 angle values.

[0047] Another aspect of the present invention is a pharmaceutical composition of any of the cocrystals of the present invention, or a combination thereof, and a pharmaceutically acceptable excipient.

[0048] In a preferred aspect, the pharmaceutical composition is in the form of a tablet, a pill, or a gel, and even more preferably is to be administered orally or mucosally.

[0049] In a more preferred aspect, the pharmaceutical composition is a liquid composition of the co-crystal of LIA and L-lysine, where the liquid composition has a molar ratio of UA:L-lysine of 1:1. This molar content comes out directly from the dilution of the co-crystal of 1:1 stochiometric ratio of the present invention. In fact, eventual formulations out of the scope of the present invention are feasible by the dilution of LIA with L-lysine adding each component separately, at least to form meta-stable solutions, but would need a molar ratio LIA: L-lysine of 1 :1.5 or higher of L-lysine to be able to dissolve the active agent (Example 9). In a preferable aspect, this liquid composition is administered orally intradermally, intravenously, subcutaneously, or intramuscularly, and most preferably it is in the form of an injection.

[0050] In another preferred aspect, the pharmaceutical composition is a liquid composition of the co-crystal of LIA and L-Arginine, where the liquid composition has a molar content of LIA: L-Arginine of 1:1.

[0051] In another preferred aspect, the pharmaceutical composition is a liquid composition of the co-crystal of LIA and ethylene diamine, where the liquid composition has a molar content of LIA: ethylene diamine of 2:1.

[0052] In still another preferred aspect, the pharmaceutical composition is a liquid composition of the co-crystal of LIA and tromethamine, where the liquid composition has a molar content of LIA: tromethamine of 1 : 1.

[0053] The crystallographic forms of LIA of the present invention are capable for releasing the active agent in a therapeutical use. Therefore, a further aspect of the invention is the cocrystals or the pharmaceutical compositions as referred to for use in the prevention or treatment of a cerebrovascular disease, preferably stroke.

[0054] Another aspect is the use of the co-crystals or the pharmaceutical compositions of the present invention in the preparation of a medicament for the prevention or treatment of a cerebrovascular disease, preferably stroke.

[0055] And still another aspect is a method of preventing or treating a cerebrovascular disease, preferably stroke, the method comprising administering a therapeutically effective amount of the co-crystals or the pharmaceutical compositions of the present invention to a subject in need thereof.

[0056] Description of the Figures

[0057] Figure 1 shows the Powder X-ray diffraction (PXRD) pattern of uric acid free drug as starting material, sample SP308-FD-P1.

[0058] Figure 2 shows a Raman spectrum recorded for the crystal of uric acid sample SP308-FD-P1 , from 2000 to 200 cm-1.

[0059] Figure 3 shows an1H-NMR spectrum of the crystal of uric acid sample SP308-FD-P1 in DMSO. 600 MHz, number of scans: 8, temperature: 298 K, 10.6 ppm (s, 1H), 10.7 ppm (s, 1H), 11.4 ppm (s brought, 1 H), 11.8 ppm (s brought, 1 H), 3.34 (H2O), 2.51 (DMSO), 0.00 (TMS). Figure 4 shows a TG-FTIR performed on the crystal of uric acid sample SP308-FD-P1 at a heating rate of 10°C / min up to 350°C. 0.18% residual water was released from 23°C to 170°C. Decomposition is observed at higher temperature, starting from 170°C.

[0060] Figure 5 shows a PXRD pattern conducted on the co-crystal of LIA and L-lysine, sample SP308-LYS-P3 (Polymorph A), revealing a crystalline material.

[0061] Figure 6 shows the TG-FTIR thermogram of sample SP308-LYS-P3d, obtained after 1 day drying of the co-crystal of LIA and L-lysine sample SP308-LYS-P3 (< 20 mbar, 40°C). A heating rate of 10°C / min was used and the measurement was conducted up to 350°C.

[0062] 4.23% of residual water was released from 23°C to 200°C. 4.2% of water corresponds to approximately 0.8 equivalents.

[0063] Figure 7 shows the DSC curve of sample SP308-LYS-P3d. Exothermic events are going down and endothermic events up. DSC measurement was conducted with a heating rate of 10°C / min up to 100°C using a sample pan with pinhole in the lid in order to remove the majority of the water. Then, the temperature was decreased to 25°C and a second heating rate was conducted with 10°C / min up to 300°C.

[0064] Figure 8 shows the DVS isotherm of sample SP308-LYS-P3d, with the change of the estimated water content (thin line) and relative humidity (thick line) as a function of time.

[0065] Figure 9 shows the PXRD pattern of the co-crystal of uric acid and L-lysine, sample SP308-LYS-P5 (Polymorph B), revealing a crystalline material.

[0066] Figure 10 overlaps the PXRD patterns of the co-crystal of uric acid and L-lysine Polymorph A sample SP308-LYS-P4 (top trace) and Polymorph B sample SP308-LYS-P5 (bottom trace).

[0067] Figure 11 shows the TG-FTIR thermogram of sample SP308-LYS-P5. A heating rate of 10°C / min was used and the measurement was conducted up to 350°C.

[0068] Figure 12 shows the DSC curve of sample SP308-LYS-P5 with a heating rate of 10°C / min using a sample pan with pinhole in the lid.

[0069] Figure 13 shows the DVS isotherm of sample SP308-LYS-P5: the change of the estimated water content (thin line) and relative humidity (thick line) as a function of time.

[0070] Figure 14 shows the PXRD pattern of the co-crystal of uric acid and L-arginine, sample SP308-ARG-P1d.

[0071] Figure 15 shows the TG-FTIR thermogram of sample SP308-ARG-P1d with a heating rate of 10°C / min up to 300°C. Figure 16 shows the PXRD pattern of the co-crystal / solvate of uric acid and ethylene diamine, sample SP308-EDA-P1d.

[0072] Figure 17 shows the TG-FTIR thermogram of sample SP308-EDA-P1d with a heating rate of 10°C / min up to 300°C.

[0073] Figure 18 shows the PXRD pattern of the co-crystal of uric acid and tromethamine, sample SP308-TRO-P1.

[0074] Figure 19 shows the TG-FTIR thermogram of sample SP308-TRO-P1d with a heating rate of 10°C / min up to 300°C.

[0075] EXAMPLES

[0076] Example 1: Obtention of the co-crystal of UA and L-lysine, and characterization thereof.

[0077] Obtention process

[0078] 100 mg of crystals of uric acid free drug, sample SP308-FD-P1 , were suspended in 2 mL of a concentrated solution of L-lysine (200 mg / mL) in water at room temperature (r.t. 20-25°C) in a closed 4 mL glass reactor (Supelco). After overnight stirring, a solution was obtained. The reactor was then opened to let the solvent evaporate and, after 4 days of further stirring, a suspension was obtained. The solid was isolated by filtration obtaining sample SP308-LYS-P3. The sample was dried under vacuum (< 20 mbar) at 40°C for 1 day to obtain SP308-LYS-P3d, and was characterized with PXRD (Figure 5), TG-FTIR (Figure 6), DSC (Figure 7), confirming a 100% obtention of only one polymorph named “polymorph A”. Its chemical identity was confirmed by elemental composition analysis using C, H and N content determinations.

[0079] Purity determinations were conducted on two L-lysine co-crystal samples using HPLC. The produced co-crystals show purities of 100 area %.

[0080] Elemental Analysis, sample SP308-LYS-P3d

[0081] The chemical identity of dry SP308-LYS-P3 sample (SP308-LYS-P3d) was further confirmed by elemental composition analysis using C, H and N content determinations. The obtained results are summarized in Table 1. The obtained values are closed to the theoretical values of a 1:1 species with a molecular mass of 328.71 g / mol and the formula CnHisNeO. 0.8 H2O. However, a difference of 0.6-1.5% compared to the theoretical values is observed for the H and N contents. Since the experiment was conducted with an excess of L-lysine, it can be assumed that the sample can contain residual L-lysine. Table 1: Result from C, H, N, and O content analysis for sample SP308-LYS-P3d compared with the theoretical composition of a 1 :1 species with L-lysine, a 1 :2 uric acid / L-lysine species, a 2:1 uric acid / L-lysine species, all containing 4.2% of water (0.8 eq), as well as pure uric acid and pure L-lysine.

[0082]

[0083] Dynamic Vapor Sorption (DVS) of sample SP308-LYS-P3d

[0084] The behavior of the L-lysine co-crystal sample SP308-LYS-P3d was investigated in the presence of variable water vapor pressure at 25°C using DVS (Figure 8). At the start of the measurement, the sample contained 4.6% of water; this value is consistent with the 4.2 % of water found by TG-FTIR (Figure 6). By decreasing the relative humidity to 0%, the sample slowly lost about 1.5% of weight until 6% r.h. Then a rapid decrease in the water content was observed by decreasing the relative humidity further to 0% r.h. When the relative humidity was again increased, a rapid mass update was first observed (3.5% until 6% r.h.). The water content stayed between 3.5% and 4.9% from 10% r.h. to 62% r.h. Finally, a rapid increase in the water content was observed from 62% r.h. with a final water content of about 39%, reached upon storage at 95%. The second DVS cycle was almost identical to the first one.

[0085] Example 2: Obtention of polymorph B of the co-crystal of UA and L-lysine, and characterization thereof.

[0086] In a first stage, 5.0 g of crystals of uric acid free drug, sample SP308-FD-P1, were suspended in 50 mL of water containing lysine (20.44 g) at r.t. (20-25°C). After 5 min stirring a yellow solution was obtained and after 2 hours it could be observed a first precipitation. The stirring continued at room temperature in the closed reactor for 66 h and, thereafter, a white suspension was obtained. The product was filtered off and dried on the filter nutsch for 30 min to obtain 8.71 g of product. A PXRD revealed that this product was already a co-crystal of UA and L-lysine.

[0087] Besides, 2.5 g of crystals of uric acid free drug, sample SP308-FD-P1, were sequentially added to 10 ml water containing lysine (1.74 g) at r.t. with stirring. After addition of 20% of the uric acid a clear solution was obtained. Then, 10 mg of the product obtained at the first stage were added to form a turbid mixture. After 20 min the remaining 80% of the uric acid free drug was added. The resulting suspension was stirred overnight at r.t. The product was filtered off, washed with 2.5 ml ethanol / water (4:1) and 2.5 I ethanol / water (9:1) and dried under vacuum at 60°C. 1.87 g of product was obtained and was characterized with PXRD (Figure 9), confirming a 100% obtention of only one polymorph of co-crystals of LIA and L-lysine named “polymorph B”, sample SP308-LYS-P5.

[0088] Elemental Analysis, sample SP308-LYS-P5

[0089] The chemical identity of the Polymorph B sample was confirmed by elemental composition analysis using C, H, N and O content determinations. The obtained results are summarized in Table 2. These results fit very well with the theoretical content of a 1:1 cocrystal with L-lysine containing 4.65% of water (0.85 equivalents).

[0090] Table 2. Result from C, H, N, and O content analysis of sample SP308-LYS-P5 compared with the theoretical composition of a 1 :1 species with L-lysine.

[0091] Expected content for a 1 :1 Experimental

[0092] Element co-crystal with 4.65% of water SP308-LYS-P5

[0093] (0.85 eq)

[0094] C 40.1 % 40.1 % ± 0.3 %m / m

[0095] H 6.1% 6.0% ± 0.3 %m / m

[0096] N 25.6% 25.5% ± 0.3 %m / m

[0097] O 28.3% 28.4% ± 0.3 %m / m

[0098] Water content 4.63% 4.65%

[0099] Dynamic Vapor Sorption (DVS), sample SP308-LYS-P5

[0100] The behavior of the L-lysine co-crystal Polymorph B sample was investigated in the presence of variable water vapor pressure at 25°C using DVS (Figure 13). At the start of the measurement, the sample contained 4.63% of water; this value is consistent with the 4.65 % of water found by TG-FTIR (Figure 11). By decreasing the relative humidity to 0%, the sample slowly lost about 0.2% of weight until 28.6% r.h. Then, a rapid decrease in the water content was observed by decreasing the relative humidity further to 0% r.h. When the relative humidity was again increased, a rapid mass update was first observed (4.5% until 40% r.h.) The water content stayed between 4.5% and 5% from 40% r.h. to 80% r.h. Finally, a rapid increase in the water content was observed from 80% r.h. with a final water content of about 6.4%, reached upon storage at 95%. The second DVS cycle was similar to the first one. Relevant to note is that the sample SP308-LYS-P5 was recovered after this test and submitted to PXRD analysis. The obtained PXRD pattern corresponded well to Polymorph A, meaning that during the DVS measurement happened a form conversion from B to A.

[0101] Example 3: Purity of the co-crystals of UA and L-lysine.

[0102] The purity of the free uric acid starting material SP308-FD-P1 as well as the purity of Polymorph A sample SP308-LYS-P4 of the L-lysine co-crystal were tested by HPLC. Sample SP308-LYS-P4 was obtained by the procedure of Example 1. Sample SP308-FD-P1 showed a purity of 99.98% and no impurity were observed for the salts. The purity of the L-lysine co-crystal Polymorph B sample SP308-LYS-P5 was also tested by HPLC, with no observed impurities.

[0103] Table 3: Purity data of the co-crystals of uric acid and L-lysine.

[0104]

[0105] Example 4: Solubility in Water of the co-crystals of UA and L-lysine.

[0106] Precise solubility determinations were conducted in water at 25°C for the L-lysine cocrystal sample SP308-LYS-P4. 12.8 mg of the co-crystal was suspended in 0.5 mL of water at 25°C. After 15 minutes, a solution was obtained, and the corresponding pH was 8.27. Additional 21.2 mg of SP308-LYS-P4 material was added and the obtained suspension was equilibrated at 25°C. After 15 minutes, a pH of 8.64 was recorded. The suspension was equilibrated for 24 hours at 25°C with a shaking rate of 700 rpm. After the equilibration, the pH of the suspension was 8.6. The suspension was then filtered using a centrifugal unit filter (0.2 pm, PVDF, 1 minute, 3000 relative centrifugal force (ref)). The pH of the recovered solution was measured, and the concentrations of uric acid in the recovered filtrate was assessed using HPLC method with two injections per sample. The solubility value obtained after 24 hours of equilibration in water is 13.49 mg / mL of uric acid (free drug equivalent); the obtained value corresponds to 24.3 mg / mL of the L-lysine co-crystal dissolved in water.

[0107] No form conversion was observed after 24 hours of equilibration in water at 25°C. Likewise, a precise solubility determination was conducted in water at 25°C for the L-lysine co-crystal Polymorph B, sample SP308-LYS-P5. 50.1 mg of the co-crystal was suspended in 0.5 mL of water at 25°C. After 15 minutes, a pH of 7.8 was recorded. The suspension was equilibrated for 24 hours at 25°C with a shaking rate of 700 rpm. After the equilibration, the suspension was then filtered using a centrifugal unit filter (0.2 pm, PVDF, 1 minute, 4000 ref). The pH of the recovered solution was measured, and the concentration of uric acid in the recovered filtrate was assessed using HPLC with two injections per sample.

[0108] Furthermore, it was observed that Polymorph B converted to Polymorph A after 24 hours of equilibration in water at 25°C or after storage at 40°C / 75% r.h. Therefore, Polymorph A seems to be a stable hydrated form.

[0109] Table 4. Summary of the solubility data in water.

[0110] <

[0111]

[0112] Example 5: Obtention Process of co-crystals of UA and other coformers.

[0113] For each experiment, 75 mg ± 2 mg of UA starting material, sample SP308-FD-P1, was weighed into a 2.5 mL Eppendorf tube. The desired amount of co-former (1:1 mol ratio) was weighed (± 2 mg) and then added to the tube containing UA. Three glass balls of 3 mm diameter were added to the tube. The tube was closed and shaken vigorously 3-5 times by hand in order to homogenize the powder mixture. Then, 30 pL (2-3 drops) of the selected solvent (distilled water of ethanol (Merck) were added in the tube, and the mixture was milled for 5 minutes with 30 Hz using a Retsch Mixer Mill MM400. The obtained product was analyzed by PXRD without pretreatment.

[0114] Table 5 summarizes the conducted experiments and the PXRD results. New hits for possible co-crystal formation were obtained for L-Arginine as co-former in water, sample SP308-ARG-P1 , for tromethamine as co-former in water, sample SP308-TRO-P1 , and for ethylenediamine as co-former in both solvents, sample SP308-EDA-P1. The hits were observed as unknown PXRD reflections in a mixture with the uric acid starting material.

[0115] Table 5. Results of the co-crystal screening using SP308-FD-P1 as starting material.

[0116]

[0117] In order to confirm co-crystal formation, the hits were reproduced in “solution” experiments (suspension experiment and cooling crystallization), where part of the mixture is in solution. The produced co-crystals were then filtered, and the obtained solids been characterized in order to confirm the stoichiometry and the nature of the obtained solid forms.

[0118] Example 6: Obtention of the co-crystal of UA and L-Arginine, and characterization thereof.

[0119] For sample SP308-ARG-P1, Hit 1 was reproduced in a solution-based co-crystal formation experiment. The uric acid free drug was suspended in an aqueous solution of L-arginine (1.1 equivalents). After 2 hours, a solution was observed but crystallization was observed after overnight stirring at r.t. Further equilibration was conducted for 4 days and the solid was then isolated by filtration. Drying was conducted for 2 days under vacuum (<5 mbar, r.t.) The dried sample SP308-ARG-P1d was then characterized by PXRD (Figure 14), TG-FTIR (Figure 15), and elemental analysis to confirm crystallinity, solvent content, and composition.

[0120] Elemental Analysis, sample SP308-ARG-P1d

[0121] The chemical identity of the SP308-ARG-P1d sample was confirmed by elemental composition analysis using C, H, N, and O content determinations. These results fit well with the theoretical content of monohydrate of a 1:1 co-crystal with L-arginine.

[0122] Table 6. Result from C, H, N, and O content analysis for sample SP308-ARG-P1d compared with the theoretical composition of a solvent-free 1 :1 co-crystal with L-arginine (342.31 g / mol, CnHiaNaOb) and a 1 :1 co-crystal containing 1 equivalent of water (360.33 g / mol, CnHisNsOs H2O). Experimental Expected content for a 1 :1 coElement

[0123] SP308-ARG-P1d crystal containing 1 eq of water

[0124] C 36.4% 36.7% ± 0.3 %m / m

[0125] H 5.7% 5.6% ± 0.3 %m / m

[0126] N 30.9% 31.1% ± 0.3 %m / m

[0127] O 27.0% 26.6% ± 0.3 %m / m

[0128] Example 7: Obtention of the co-crystal of UA and ethylenediamine, and characterization thereof.

[0129] For sample SP308-EDA-P1 (Hit 5), a new PXRD pattern was found using water or ethanol as the solvent system in a liquid-assisted grinding experiment. This hit was reproduced in a solution-based co-crystal formation experiment. The uric acid free drug was suspended in an aqueous solution of containing 1.1 equivalent of ethylene diamine. Gel formation was first observed but rapidly converted to a suspension. Further equilibration was conducted for 5 days and the solid was then isolated by filtration. Drying was conducted for 2 days under vacuum (<5 mbar, r.t.) The dried sample SP308-EDA-P1d was then characterized by PXRD (Figure 16), TG-FTIR (Figure 17), and elemental analysis to confirm crystallinity, solvent content, and composition.

[0130] Elemental Analysis, sample SP308-EDA-P1d

[0131] The chemical identity of the SP308-EDA-P1d sample was further confirmed by elemental composition analysis using C, H, N, and O content determinations. The results fit better with the theoretical content of a 2:1 co-crystal (free drug to ethylene diamine) rather than a 1:1 species.

[0132] Table 7. Result from C, H, N, and O content analysis for sample SP308-EDA-P1d compared with the theoretical composition of a solvent-free 2:1 co-crystal with ethylene diamine (198.16 g / mol, CeHaNsCh) and a 1 :1 co-crystal containing (228.21 g / mol, C7H12N6O3).

[0133] Experimental Expected content for a Expected content for a Element

[0134] SP308-EDA-P1d 2:1 co-crystal 1:1 co-crystal C 35.8% 36.4% ± 0.3 %m / m 36.8% ± 0.3 %m / m H 4.2% 4.1% ± 0.3 %m / m 5.3% ± 0.3 %m / m N 34.7% 35.3% ± 0.3 %m / m 36.8% ± 0.3 %m / m O 24.8% 24.2% ± 0.3 %m / m 21.0% ± 0.3 %m / m

[0135] Example 8: Obtention of the co-crystal of UA and tromethamine, and characterization thereof. For sample SP308-TRO-P1, Hit 4 was reproduced in a solution-based co-crystal formation experiment. The uric acid free drug and 1.1 equivalent of tromethamine were suspended in water. The obtained suspension was equilibrated for 3 days and the solid was then isolated by filtration. Drying was conducted for 2 days under vacuum (<5 mbar, r.t.) The dried sample SP308-TRO-P1d was then characterized by PXRD (Figure 18), TG-FTIR (Figure 19), and elemental analysis to confirm crystallinity, solvent content, and composition.

[0136] Elemental Analysis, sample SP308-TRQ-P1d

[0137] The chemical identity of the SP308-TRO-P1d sample was further confirmed by elemental composition analysis using C, H, N, and O content determinations. The results fit well with the theoretical content of hemihydrate of a 1 :1 co-crystal with tromethamine.

[0138] Table 8. Result from C, H, N, and O content analysis for sample SP308-TRO-P1d compared with the theoretical composition of a solvent-free 1 :1 co-crystal with tromethamine (289.25 g / mol, C9H15N5O6) and a 1 :1 co-crystal containing 0.5 equivalent of water (298.26 g / mol, C9H15N5O6 0.5H2O).

[0139] Expected content for a Expected content for a 1 :1 co- Experimental

[0140] Element 1:1 co-crystal crystal containing 0.5 eq. of SP308-TRO-P1d

[0141] water

[0142] C 36.7% 37.4% ± 0.3 %m / m 36.2% ± 0.3 %m / m

[0143] H 5.3% 5.2% ± 0.3 %m / m 5.4% ± 0.3 %m / m

[0144] N 24.0% 24.2% ± 0.3 %m / m 23.5% ± 0.3 %m / m

[0145] O 34.1% 33.2% ± 0.3 %m / m 34.9% ± 0.3 %m / m

[0146] Example 9: Dilution of crystals of UA with L-lysine

[0147] Example 9a: 0.50 g UA crystals (SP308-FD-P1) was added to a solution of 435 mg Lysine (1.0 eq) in 5 ml water (10 vol) at r.t. A suspension was obtained which did not clarified, showing that the original product could not be dissolved. After stirring overnight, the suspension was tried to be filtered, but the solid run through the filter and could not be isolated.

[0148] Example 9b: 0.50 g UA (SP308-FD-P1) was added to a solution of 652 mg Lysine (1.5 eq) in 5 ml water (10 vol) at r.t. After 30 min a clear meta-stable solution was obtained, in which a solid started to precipitate after 60 minutes. After stirring overnight, the product was filtered off. After drying 800 mg product were obtained that ultimately corresponded to the co-crystal of UA and L-lysine polymorph B. Example 9c: 0.50 g UA (SP308-FD-P1) was added to a solution of 1.74 g Lysine (4.0 eq) in 5 ml water (10 vol) at r.t. After 5 min a clear meta-stable solution was obtained, in which a solid started to precipitate after 100 minutes. After stirring overnight, the product was filtered off. After drying 780 mg product were obtained.

[0149] Instrumental - Typical Measurement Conditions

[0150] DSC: Differential scanning calorimetry was carried out with a TA Instruments Q2000 instrument. An aluminum pan, with or without a pinhole in the lid, was used for each measurement. The aluminium sample pan was filled with sample under ambient conditions. The measurement was conducted with a heating rate of 10°C / min. The melting point is understood as the peak maximum.

[0151] 1H-NMR. Bruker DPX300 spectrometer;1H-NMR spectra were recorded using a proton frequency of 300.13 MHz; 30° excitation pulse; recycle delay of 1 s; accumulation of 16 scans, and deuterated DMSO-de as the solvent. The chemical shifts were referenced relative to TMS at 0 ppm.

[0152] DVS: Dynamic vapor sorption was carried out with a Prollmid SPS23-100n instrument.

[0153] The following program was applied: 50% relative humidity (r.h.) - 0% r.h. - 95% r.h. - 0% r.h. - 95% r.h. - 50% r.h., scan rate of 5% r.h. per hour, T = 25°C.

[0154] Hygroscopicity is classified based on criteria inspired by the European Pharmacopoeia1:

[0155] - very hygroscopic (vh): increase of the mass > 15 %;

[0156] - hygroscopic (h):increase of the mass is less than 15 % and greater or equal to 2 %;

[0157] - slightly hygroscopic (sh): increase of the mass is less than 2 % and greater or equal to 0.2 %;

[0158] - not hygroscopic (nh): increase of the mass is less than 0.2 %;

[0159] - deliquescent (d): sufficient water is absorbed to Polymorph A liquid.

[0160] The classification is based on comparison to the mass of the sample at the start of the DVS measurement.

[0161] Powder X-Ray Diffraction: Stoe Stadi P equipped with a MythenIK Detector; Cu-Ka1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; curved Ge monochromator; 0.02°29 detector step size, 48 s. step time, 1.5-5O.5°20

[0162] 1The European Pharmacopoeia classifies hygroscopicity after 24 hours storage at 80% r.h. The inventors perform DVS instead of storage at constant r.h.; the mass change at 85% r.h. is used for classification to compensate for the shorter exposure time in DVS. scanning range; detector mode: step scan; 1°20 detector step. The samples (10-20 mg of powder) were measured between two acetate foils or Kapton foils. No special treatment was used in preparing the samples other than the application of slight pressure to distribute the powder over the irradiated surface area. An ambient air atmosphere was used for all measurements, and each sample was rotated during the measurement. RAMAN: The Raman spectrum was recorded with a Bruker Multi RAM FT-Raman spectrometer, which is operated with a Nd:YAG laser (1064 nm wavelength) and a liquid-nitrogen cooled germanium detector. A nominal laser power of 100 mW was used to accumulate 64 scans with a resolution of 2 cm-1. Peak positions are generally accurate to within + / - 2 cm-1.

[0163] Solubility: solubilities were determined by incremental addition of solvent to 10 mg of the compound. If the substance was not dissolved by addition of a total of at least 10 mL solvent, the solubility is indicated as <1 mg / mL.

[0164] Solvents: Fluka, Sigma-Aldrich, Merck, or ABCR analytical grade solvents were used. TG-FTIR: Thermogravimetric measurements were carried out with a Netzsch ThermoMicrobalance TG 209 F1 Nevio coupled to a Bruker FT-IR Spectrometer Tensor II. Aluminium sample pans with a pinhole, N2 atmosphere, heating rate 10°C / min.

[0165] HPLC: The HPLC device is an Agilent 1260 Infinity Series 2 HPLC system.

[0166] Table 9. HPLC method used in this study

[0167] Parameter Description

[0168] column YMC-Pack ODS-AQ, 250 x 4.6 mm, 3 pm sample dilution solvent 0.05 M phosphate buffer

[0169] mobile phase A acetate buffer pH 5

[0170] mobile phase B acetonitrile

[0171] gradient 0.0 min - 100% A

[0172] 15.0 min - 100% A

[0173] 25.0 min - 80% A

[0174] 35.0 min - 60% A

[0175] 45.0 min - 60% A

[0176] 45.1 min - 100% A

[0177] 60.0 min - 100% A

[0178] flow 0.5 mL min-1

[0179] injection volume 10 pL

[0180] detection 285 nm

[0181] column temperature 25°C

[0182] run time 60 min

[0183] maximum pressure 140 bar

Claims

CLAIMS1. A co-crystal comprising uric acid (UA) and L-lysine.

2. The co-crystal according to claim 1, characterized in that said co-crystal is of UA and L-lysine.

3. The co-crystal according to claim 1 or 2, characterized by a stoichiometric ratio of UA to L-lysine of 1:1.

4. The co-crystal according to any one of claims 1-3 as polymorph A, characterized in that said polymorph A contains 0.8 eq. of water.

5. The co-crystal according to claim 4, characterized by a powder X-ray diffraction pattern comprising a 20 angle at value of 14.80 and least three 20 angle values selected from the group consisting of: 11.88, 16.44, 17.61, 20.04, 20.80, and 23.93.

6. The co-crystal according to claim 4, characterized by a powder X-ray diffraction pattern comprising a 20 angle at value of 14.80 and at least six 20 angle values selected from the group consisting of: 11.88, 16.44, 16.68, 17.61, 20.04, 20.63, 20.80, 21.30, 23.93, 25.56, 29.21, 34.81, and 36.25.

7. The co-crystal according to any one of claims 1-3 as polymorph B, characterized in that characterized in that said polymorph B contains 0.85 eq. of water.

8. The co-crystal according to claim 7, characterized by a powder X-ray diffraction pattern comprising a 20 angle at value of 17.88 and at least three 20 angle values selected from the group consisting of 11.90, 16.65, 20.03, 20.56, 23.95, and 24.36.

9. The co-crystal according to claim 7, characterized by a powder X-ray diffraction pattern comprising a 20 angle at value of 17.88 and at least six 2© angle values selected from the group consisting of: 11.90, 16.65 17.64, 20.03, 20.56, 21.54, 23.20, 23.95, 24.6, 25.06, 27.65, 29.61 and 29.61.

10. A co-crystal comprising UA and L-Arginine.

11. The co-crystal according to claim 10, characterized in that said co-crystal is of UA and L-Arginine.

12. The co-crystal according to claim 10 or 11 , characterized in that said co-crystal is a monohydrate.

13. The co-crystal according to any one of claims 10-12, characterized by a stoichiometric ratio of LIA to L-Arginine of 1:1.

14. The co-crystal according to any one of claims 10-13, characterized by a powder X-ray diffraction pattern comprising at least three 20 angle values selected from the group consisting of: 6.65, 17.04, 19.47, 20.14, 25.85, and 28.32.

15. The co-crystal according to any one of claims 10-13, characterized by a powder X-ray diffraction pattern comprising at least six 20 angle values selected from the group consisting of: 3.31, 6.65, 17.04, 18.01, 18.69, 19.11, 19.47, 20.14, 21.44, 23.68, 25.85, 26.24, 28.32, and 31.00.

16. A co-crystal comprising LIA and ethylene diamine.

17. A co-crystal according to claim 16, characterized in that said co-crystal is of LIA and ethylene diamine.

18. The co-crystal according to claim 16 or 17, characterized by a stoichiometric ratio of LIA to ethylene diamine of 2: 1.

19. The co-crystal according to any one of claims 16-18, characterized by a powder X-ray diffraction pattern comprising at least three 20 angle values selected from the group consisting of: 11.27, 16.93, 23.35, 26.45, and 28.85.

20. The co-crystal according to any one of claims 16-18, characterized by a powder X-ray diffraction pattern comprising at least six 20 angle values selected from the group consisting of: 11.27, 12.57, 16.93, 23.35, 26.45, 27.81, and 28.85.

21. A co-crystal comprising LIA and tromethamine.

22. A co-crystal according to claim 21, characterized in that said co-crystal is of LIA and tromethamine.

23. The co-crystal according to claim 21 or 22, characterized in that the co-crystal is a hemihydrate.

24. The co-crystal according to any one of claims 21-23, characterized by a stoichiometric ratio of LIA to tromethamine of 1 :1.

25. The co-crystal according to any one of claims 21-24, characterized by a powder X-ray diffraction pattern comprising at least three 20 angle values selected from the group consisting of: 6.41, 13.60, 16.81, and 27.24.

26. The co-crystal according to any one of claims 21-24, characterized by a powder X-ray diffraction pattern comprising at least six 20 angle values selected from the group consisting of: 6.41, 13.60, 14.22, 15.78, 16.81, 17.67, 19.17, 21.75, and 27.24.

27. A pharmaceutical composition, comprising at least one co-crystal as defined in any one of claims 1 to 26, and a pharmaceutically acceptable excipient.

28. The pharmaceutical composition according to claim 27, characterized in that said composition is in the form of a tablet, a pill, or a gel.

29. The pharmaceutical composition according to claim 28, characterized in that said composition is to be administered orally or mucosally.

30. The pharmaceutical composition according to claim 27, characterized in that said composition is a liquid composition of the co-crystal of LIA and L-lysine, wherein said liquid composition has a molar content of II A: L-lysine of 1 : 1.

31. The pharmaceutical composition according to claim 30, wherein said composition is administered orally intradermally, intravenously, subcutaneously, or intramuscularly.

32. The pharmaceutical composition according to claim 30 or 31, wherein said pharmaceutical composition is in the form of an injection.

33. The co-crystal of any one of claims 1-26, or the pharmaceutical composition of any one of claims 27-32, for use in the prevention or treatment of a cerebrovascular disease.

34. The co-crystal or the pharmaceutical composition for use according to claim 33, characterized in that said cerebrovascular disease is stroke.

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