Pharmaceutical compositions of nanocrystallized xanthatin and use thereof as an Anti-tumor agent

Abstract

The present invention describes the procedure for obtaining nanocrystals of isolated xanthatin from a natural source. It further describes pharmaceutical compositions comprising such nanocrystals, as well as the uses and methods of treatment in which they may be used.

Description

[0001]PHARMACEUTICAL COMPOSITIONS OF NANOCRYSTALLIZED XANTHATHINE AND ITS USES AS AN ANTITUMORAL. DESCRIPTION Technical field The present invention belongs to the technical field of pharmaceutical products, and in particular relates to new pharmaceutical compositions containing xanthine as an active pharmaceutical ingredient, its preparation methods and medical use. Background of the invention Xanthine is a natural sesquiterpene lactone with molecular formula C15H18O3, isolated mainly from the aerial parts of species belonging to the genus Xanthium (fam. Asteraceae). The members of this genus are perennial plants that are distributed in America, Pakistan, India, China and Eurasia. From a structural point of view, said compound exhibits the presence of an α-methylene-γ-butyrolactone ring fused in a trans arrangement with a carbocycle of 7 carbon atoms and an unsaturated ketone side chain.Several studies with xanthanolides have shown that this structural arrangement is an essential feature in the different types of biological activities exhibited by xanthan, including the remarkable antiproliferative activity against different tumor cell lines. Several reports demonstrate the effectiveness of xanthanide in significantly reducing cell proliferation and inducing apoptosis in a wide variety of tumor cell lines including: ovarian carcinoma (SK-OV-3 and ES-2), melanoma cells (SK-MEL-2), central nervous system (XF498), colon carcinoma (HCT-15 and CT26WT), human gastric carcinoma (MKN-45), non-small lung cancer cells (A549 and H1299), breast cancer (MDA-MB-231) and cervical cancer (HeLa). Several mechanisms for the antitumor action of xanthanines have been proposed.Takeda and colleagues demonstrated that xanthines act as a catalytic inhibitor of topoisomerase IIα, promoting DNA damage in breast cancer cells. In addition, it acts on the STAT3 signaling pathway and the Wnt / β-catenin pathway, which are closely related to antiproliferative effects. It has also been reported that xanthines can downregulate the expression of Chk1 and Chk2 and alter the phosphorylation of CDC2, which are key regulators of the G2 / M transition. Likewise, its antimitotic potential is reported from the interruption of the metaphase-to-anaphase transition by acting on the formation of mitotic spindle microtubules, leading to apoptotic cell death. It has also been reported that xanthine effectively inhibits the proliferation and tumorigenicity of malignant glioma cells mainly by suppressing autophagy through the activation of the PI3K / Akt / mTOR pathway.Studies of the underlying mechanisms have shown that xanthine has antiproliferative, antiangiogenesis, and proapoptotic effects on various types of cancer, both in vitro and in vivo. All these studies suggest that xanthines, isolated from X. strumarium, have potential in cancer prevention and therapy. Xanthine is a lipophilic drug that is poorly soluble in water. It can be rapidly metabolized and eliminated from the body according to a pharmacokinetic study performed in rats after intravenous administration. Patent JPH09188671A of 1996 refers to pharmaceutical compositions prepared by conventional methods containing xanthine as an antitumor for oral use in the form of tablets and syrup, and injectable as an intravenous infusion.Conventional methods for preparing injectables with poorly water-soluble active ingredients involve high amounts of cosolvents and / or surfactants, which can cause anaphylactic reactions in patients. Patent CA2688486A1 of 2008 refers to the formulation of sesquiterpenes as water-insoluble antitumor agents together with one or more antioxidants and one or more solubilizers selected from the group consisting of PEG400, an animal or vegetable oil (e.g., olive oil), a castor oil derivative and ethylene oxide (Cremophor RH 40), and polysorbate 80. It is known that high amounts of both organic solvents and surfactants are required to achieve the solubilization of water-insoluble active ingredients such as xanthine, which can cause adverse reactions.Patent CN110123754A of 2019 refers to the production of a polymeric nanomicelle of xanthine functionalized with the cyclic peptide (NGR) for the active targeting of dendritic cells in the treatment of allergic rhinitis as an anti-inflammatory. The aqueous pharmaceutical formulation without any solubilizer or organic solvent is presented as a strategy to overcome the limitation of xanthine's hydrophobicity, allowing targeted therapy, sustained release, greater stability, increased cellular internalization, effectiveness and reduction of side effects. In 2020, Zhou and collaborators synthesized polymeric polyadopamine nanoparticles containing xanthine, where they improved gastric adhesion and bioavailability of xanthine. They demonstrated that the nanoparticles may be potentially effective in inhibiting gastric cancer through oral administration.Zheng X et al. in 2020 obtained xanthine polymeric micelles functionalized with the CD13-specific cyclic peptide (NGR) targeting dendritic cells as a potential drug for the treatment of refractory allergic rhinitis through nonclinical evaluation of intranasal delivery in a murine (mouse) model of allergic rhinitis. Nanocrystal technology is one of the most widely used strategies to improve the solubility and bioavailability of poorly water-soluble drugs. They are nanometric-sized particles of pure drug stabilized by a small amount of suitable surfactants / polymers. To avoid particle aggregation in the liquid state, lyophilization is considered one of the quintessential techniques along with spray drying, supercritical fluids, granulation, and pelletization to improve the long-term stability of colloidal nanoparticles.Drug nanocrystals are a versatile formulation approach for improving the pharmacokinetic and pharmacodynamic properties of poorly water-soluble drugs. They offer the opportunity to modify the composition of matter and its physical and / or chemical properties without altering existing covalent bonds. The decrease in size results in an increase in surface area, which increases the dissolution rate, and improves drug solubility, penetrability, and adhesion. All of these properties contribute to improved drug bioavailability. Compared with other drug delivery systems, nanocrystals are formed directly from drugs and offer the advantages of high drug loading, easy industrial production, and relatively low preparation costs.They can also be administered via various drug delivery routes, such as subcutaneous injection, intravenous injection, oral administration, vaginal administration, transdermal administration, and the like. The absence of carrier substances offers a theoretical drug loading of up to 100%, compared to a typical 50–90% (w / w), resulting in satisfactory therapeutic concentrations at lower doses. Toxic side effects resulting from encapsulating / solubilizing excipients, as is the case with Cremophor RH 40, are also eliminated. Preparation techniques can be divided into top-down, bottom-up, and their combination. Top-down approaches, which primarily include media milling and high-pressure homogenization, employ high mechanical force to convert large drug powders into nanosized particles.These approaches are simple and rapid, do not require organic solvents, and are highly reproducible. However, the process is typically energy- and time-consuming, and the high shear and temperature can cause crystal instability and subsequent aggregation. There are also concerns about the likelihood of product contamination by grinding media. Bottom-up approaches, primarily involving solvent-antisolvent precipitation, precisely control drug precipitation and crystallization to achieve the desired nanosized particles. These approaches are favorable in terms of small and narrow particle size distribution. In situ gelation has been developed over the years for contraception or for the treatment of bacterial, fungal, and sexually transmitted infections.It allows for controlled and sustained drug release, reduces the frequency of administration, requires lower doses, increases drug bioavailability, and reduces side effects. In particular, in situ thermogelation uses systems presented as liquids that can be introduced or deposited on the body's surface using a minimally invasive technique before solidifying or gelling at body temperature in the target tissue, organ, or body cavity. In situ thermogelation polymer matrices offer advantages over other systems that require surgical implantation procedures and sometimes must even be removed at the end of treatment. The in situ thermogelation system formulation (thermosensitive hydrogel) is used, among other routes, for vaginal drug delivery.Compared with other types of cancer, therapeutic agents can be administered locally to the vaginal mucosa. Furthermore, cervical cancer is an excellent option for localized drug delivery, as is the case with systems such as in situ gelation. Sustained and localized delivery of anticancer drugs to the female reproductive tract can avoid the adverse effects associated with systemic administration, improve efficacy by ensuring adequate drug concentration at the site of action, and allow for convenient self-administration. Currently, there are no commercial formulations containing xanthine approved by any regulatory body.Despite the existence of the above solutions, there are no injectable formulations of xanthine as an antitumor agent in the state of the art that contain xanthine nanocrystals without the use of organic solvents that give it high solubility, bioavailability, and effectiveness that is easily scalable. Likewise, there is no xanthine formulation in the form of an in situ gellable thermosensitive hydrogel containing xanthine nanocrystals for localized therapy and its application as an antitumor agent. Therefore, the design of new effective and easy-to-use xanthine formulations has important clinical value. BRIEF DESCRIPTION OF THE INVENTION The present disclosure relates to pharmaceutical compositions containing xanthine as the active pharmaceutical ingredient. In particular, one aspect of the present invention relates to a new process for the preparation of xanthine nanocrystals.Another aspect of the present invention is a composition of nanocrystals prepared according to the described process, comprising xanthine, a surfactant and a cryoprotectant. Another aspect described is a pharmaceutical composition comprising the composition of xanthine nanocrystals, together with one or more pharmaceutically acceptable excipients. A particular aspect of the present invention is a pharmaceutical composition formulated for injectable administration. Additionally, another aspect of the present invention is the use of the injectable formulation in the treatment of colon, lung, breast, cervical, or stomach cancer. In another aspect, a method is described for treating a patient suffering from colon, lung, breast, cervical, or stomach cancer with the injectable formulation. Another particular aspect of the present invention is a pharmaceutical composition formulated as a thermosensitive hydrogel that can be gelled in situ.Additionally, another aspect of the present invention is the use of the thermosensitive hydrogel formulation in the treatment of cervical, colorectal, or ocular cancer. In another aspect, a method is described for treating a patient suffering from cervical, colorectal, or ocular cancer with the in situ gellable thermosensitive hydrogel. BRIEF DESCRIPTION OF THE FIGURES The figures concern the lyophilized xanthine nanocrystals of example 2 according to the present invention. Figure 1. Images of crystals of: a) isolated xanthine and b) nanocrystallized xanthine. Figure 2. UV spectra, recorded in the range of 200-400 nm of the nanocrystallized xanthine. Figure 3. Chromatograms of the xanthine nanocrystals determined by HPLC, recorded at 210 (A) and 280 (B) nm. Figure 4. Determination of the melting point. Figure 5. Particle diameter distribution. Figure 6. X-ray diffraction spectra. Figure 7.Fourier transform infrared spectroscopy. Figure 8. Heteronuclear single quantum correlation (HSQC) spectrum of xanthine nanocrystals. A magnification of the aliphatic region of the spectrum is shown. Figure 9. Scanning electron microscopy. Figure 10. Effect of commercial xanthine (XCtrol), isolated xanthine (XAisl), and nanoxanthine (XNano) on cell viability in HT-29 (A) and Hela (B) cell lines at 24 h. Medium supplemented with 1% FBS was used as a viability control (Ctrol). Figure 11. Effect of nanocrystallized xanthine on cell cycle progression in HT-29 colon cancer cells by flow cytometry. 1% FBS medium was used as an untreated control (Ctrol), and docetaxel was used as a positive control (DTX). Cells were treated with 10 and 20 μM of nanocrystallized xanthine, isolated xanthine, and 10 μM of commercial xanthine for 48 h. DMSO was used as a control vehicle.The data represent the percentage distribution of cells in each phase of the cycle. XCtrol: Commercial Xanthine, XA: Isolated Xanthine, XNano: Nanocrystallized Xanthine. Figure 12. In vivo antitumor effectiveness of the nanocrystal injectable: a) Evaluation of tumor volume and b) Evaluation of tumor weight. Figure 13. The figure concerns the in vivo antitumor effectiveness of embodiment number 6 of the in situ gelation formulation containing xanthine nanocrystals of embodiment number 2: a) Evaluation of tumor weight and b) Evaluation of the tumor inhibition rate (%). DETAILED DESCRIPTION OF THE INVENTION The objective of the present invention is to obtain xanthine nanocrystals by the antisolvent precipitation method, drying the nanocrystal suspension, with the lyophilized nanocrystals, and the formulation of an injectable and an in situ gelation comprising the lyophilized nanocrystals.The preparation of the xanthine nanocrystals of the present invention is carried out by the following technical scheme: (1) a 0.3% - 10% (w / v, g / ml) portion of xanthine is dissolved in organic phase such as ethanol, solution A; The aforementioned organic phase is selected from one or more of dimethyl sulfoxide, propylene carbonate, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, methylpyrrolidone, hexamethylphosphoramide, methanol, ethanol or their combination. (2) performing sterilization treatment of solution A.(3) 0.01%-10% (w / v, g / ml) of the stabilizing agent or surfactant is dissolved in water as a dispersed medium, solution B; The aforementioned surfactant is selected from one or more of Tween 80, Tween 20, Tween 40, Tween 60 and Tween 50, poloxamer, phosphatidylcholine or lecithin, acacia, tragacanth, sodium lauryl sulfate, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose or carboxymethylcellulose; PVP polyvinylpyrrolidone, for example a PVP with a molecular weight in the range of 10,000 to 100,000 Daltons, such as 50,000 to 60,000; Bile acid salts, such as salts of deoxycholic acid, taurocholic acid or glycocholic acid, for example a sodium salt of a bile acid, such as sodium taurocholate, sodium deoxycholate or sodium glycocholate; 12-OH ethoxylated stearic acid (e.g. Solutol HS15), Pluronic, Tetronic or Lutrol surfactants such as Lutrol F68 or Lutrol F127.The aqueous phase may contain a single stabilizer or a mixture of two or more stabilizers. (4) 0.01%-10% (w / w, g / g) of freeze-drying protectant relative to the amount of xanthine used (Solution C) is added to the aforementioned solution. One of the following is the freeze-drying protectant: glucose, lactose, mannitol, sucrose, trehalose. (5) Perform sterilization treatment on Solution C. (6) Obtain suspension D by adding A to C under stirring for 5-30 min. (7) Remove organic solvent and obtain aqueous suspension of nanocrystals. (8) Freeze suspension D between -40. o C and -80 o C, and subsequent sublimation by heating between +5 o C and +50 oC, lyophilized formulations of xanthine nanocrystals are obtained. The drying of the nanocrystals can be carried out in addition to lyophilization, by spray drying, fluidized bed, pelletization, granulation, supercritical fluids. The preparation of injectable formulations follows the following steps. (9) The lyophilized formulations of xanthine nanocrystals are added with saline or glucose solution to redissolve for intravenous injection by infusion to be administered at doses of 0.5 -2 mg / Kg. The preparation of formulations in in situ gelation follows the following steps (10) slowly add under stirring the mucoadhesive polymer at a concentration between 0.1 and 5% (w / v, g / v) in cold deionized water (between 4 and 15 oC) under stirring, solution E. The aforementioned mucoadhesive polymer is selected from one or more of carbopol 940, HPMC (hydroxypropyl methylcellulose), NaCMC (sodium carboxymethylcellulose), Polycarbophil AA1, HPC (hydroxypropyl cellulose), sodium alginate, guar gum. (11) then add the thermosensitive polymer at a concentration between 15 and 25 % (w / v, g / v) in solution D under stirring, and allow to dissolve at 4 oC between 8 and 16 h. solution E The aforementioned thermosensitive polymer is selected from one or more of poloxamer 407 (Pluronic 127), poloxamer 188 (Pluronic F68), methylcellulose, PLA-PEG, PLGA-PEG, polycaprolactone-polyethylene glycol block copolymer. (12) A portion of 0.2-10% of lyophilized xanthine nanocrystals (w / v, g / mL) is dispersed in solution E with gentle mixing. Administer intravaginally between 1-3 mL. EMBODIMENTS The present invention will now be illustrated, but not limited, by the following examples. Example 1.Preparation of xanthine nanocrystals 0.5 g of xanthine is dissolved in 100 mL of ethanol and sterilized by filtration through a 0.2 μm filter. This solution is then added under stirring to 400 mL of an aqueous solution previously sterilized through a 0.2 μm filter containing 1.2% Tween 80 with respect to the volume of water used and 1% sucrose with respect to the amount of xanthine added. Stirring is continued for 20 min at 75 rpm. Concentration is carried out under vacuum until the ethanol is eliminated. The resulting aqueous suspension is frozen at -80. o C for 3 hours and lyophilized for 48 h 30 oC. The particle diameter can reach 505 nm. A mass of 0.355 g is obtained. Example 2. Preparation of xanthine nanocrystals 0.5 g of xanthine is dissolved in 100 mL of ethanol and sterilized filtration is performed using a 0.2 μm filter. This solution is then added under stirring to 400 mL of an aqueous solution previously sterilized by a 0.2 μm filter of Tween 80 at 1% with respect to the volume of water used and mannitol at 1% with respect to the mass of xanthine added. Stirring is continued for 20 min at 100 rpm. It is concentrated under vacuum until the ethanol is eliminated. The resulting aqueous suspension is frozen at -80 o C for two hours and freeze-dried for 24 h 40 oC. The particle diameter can reach 669 nm. A mass of 0.386 g is obtained. Example 3. Preparation of xanthine nanocrystals 0.5 g of xanthine is dissolved in 50 mL of dimethyl sulfoxide (DMSO) and sterilized by filtration through a 0.2 μm filter. Subsequently, under stirring, 400 mL of an aqueous solution previously sterilized through a 0.2 μm filter of Tween 80 at 0.8% with respect to the volume of water used and mannitol at 1% with respect to the mass of xanthine added are added. Stirring is continued for 20 min at 100 rpm. It is concentrated under vacuum until the dimethyl sulfoxide is eliminated. The resulting aqueous suspension is frozen at -80 o C for three hours and freeze-dried for 24 hours. oC. The particle diameter can reach 650 nm. A mass of 0.372 g is obtained. Example 4. Preparation of xanthine nanocrystals 0.5 g of xanthine is dissolved in 50 mL of dimethyl sulfoxide (DMSO) and sterilized by filtration through a 0.2 μm filter. Subsequently, 400 mL of an aqueous solution of Tween 20 at 1% with respect to the volume of water used and mannitol at 1% with respect to the mass of xanthine added are added under stirring. Stirring is continued for 15 min at 100 rpm. Concentration is carried out under vacuum until the ethanol is eliminated. The resulting aqueous suspension is frozen at -80 o C for two hours and freeze-dried for 24 h 40 o C. The particle diameter can reach 685 nm. A mass of 0.368 g is obtained. Example 5. Preparation of the in situ gellable thermosensitive hydrogel containing xanthine nanocrystals Dissolve 1 g of HPMC in 50 mL of cold deionized water at 4 ° C. oC, then add 20 g of Pluronic 127 under stirring, keep for 10 hours until completely dissolved. Then add 5 g of xanthine nanocrystals under stirring until completely dispersed. Add deionized water in sufficient quantity to complete 100 mL. Example 6. Preparation of the in situ gellable thermosensitive hydrogel containing xanthine nanocrystals Dissolve 2 g of Carbopol in 50 mL of cold deionized water at 4 o C. Subsequently, add 15 g of Pluronic 127 and 10 g of Pluronic F68 under stirring and keep for 12 hours until completely dissolved. Add 10 g of xanthine nanocrystals under stirring until completely dispersed. Add deionized water in sufficient quantity to complete 100 mL. Example 7. Preparation of the in situ gellable thermosensitive hydrogel containing xanthine nanocrystals Dissolve 2 g of HPMC in 50 mL of cold deionized water at 4 oC. Subsequently, add 15 g of Pluronic 127 and 10 g of Pluronic F68 under stirring and keep for 12 hours until completely dissolved. Add 10 g of xanthine nanocrystals under stirring until completely dispersed. Add deionized water in sufficient quantity to complete 100 mL. Example 8. Preparation of the in situ gellable thermosensitive hydrogel containing xanthine nanocrystals Dissolve 1 g of Carbopol in 50 mL of cold deionized water at 4 oC, then add 20 g of Pluronic 127 under stirring and keep for 12 hours until completely dissolved. Then add 5 g of xanthine nanocrystals under stirring until completely dispersed. Add deionized water in sufficient quantity to complete 100 mL. Example 9. Physicochemical characterization of xanthine nanocrystals 9.1.Determination of the organoleptic characteristics in terms of appearance and color. The visual determination of appearance and color was carried out. Figure 1 shows the image of the isolated xanthine (A) and the lyophilized nanocrystallized xanthine (B). There are marked differences in the organoleptic characteristics in terms of appearance and color since in the naked eye the crystals in sample a) can be seen with the slight yellow color in contrast to sample b where fine white particles are observed. 9.2.Ultraviolet (UV) spectroscopy: Recorded on a Thermo Scientific SPECTRONIC GENESYS spectrophotometer in the 200-400 nm range, using “chromatographic analysis grade” methanol as a blank. The nanocrystallized xanthine sample was prepared at a concentration of 0.25 mg / mL. Absorption maxima are observed at 207.2 and 277.1 nm, corresponding to the maxima described for this compound. 9.3. Analysis by High Performance Liquid Chromatography (HPLC) with a diode array detector: An HPLC system was used using Shimadzu equipment with a Supelco reversed-phase column (12.5x4.6mmx5μm) with a DAD detector. The samples were dissolved in methanol at a concentration of 1 mg / mL and eluted using an acetonitrile / water polarity gradient, as shown in Table 1. The injection volume of the samples was 20 μL and the absorbance was read at 210 and 280 nm, methanol was used as a blank.Chromatographic analysis determined a peak purity of 99.99%. 9.4. Melting point determination: The purity was determined using a Büchi Melting Point M-565 automatic melting and boiling point determination system. Büchi Labortechnik AG capillaries were used to introduce the xanthine sample into the column until it formed a compact column 4-6 mm high. This column was compressed using an M569 sample loader prior to introduction into the system. The melting point of the nanocrystallized xanthine was 109.3 ± 0.4 °C. 9.5. Particle size determination: The particle size distribution of the isolated and nanocrystallized xanthine samples was determined using a Shimadzu (Kyoto, Japan) SALD 7101 particle analyzer coupled with WingSALD II 3.0.4 software. Ultrasonic bath was applied to the diluted samples for 10 minutes before measurement.The particle size distribution of nanocrystallized xanthine with an average particle size of 669 ± 43 nm. 9.6. X-ray diffractometry (XRD): The X-ray diffraction spectra of isolated xanthine and nanocrystallized xanthine were obtained on a MAXIMA-X, XRD-7000 X-ray diffractometer from the manufacturer Shimadzu (Kyoto, Japan) with CuKα radiation at a voltage of 30.0 kV and a current of 30.0 mA. The records were made at an angular interval 2θ of (5-80) ° and a scanning speed of 2 ° / min with continuous scanning. OriginPro 2018 software was used to obtain the spectra. The characteristic peaks of the crystalline state of xanthine are observed. 9.7. Fourier transform infrared spectroscopy (FTIR): Isolated xanthine and the nanocrystals obtained from it were analyzed by Fourier transform infrared spectroscopy using an IR Prestige 21 system from Shimadzu (Kyoto, Japan).The Attenuated Total Reflectance method was used for the test, using a SPECAC Golden Gate model with a diamond optical window (n=2.4), with minimal contact between the sample and the window. The scanning range was 4000–600 cm. -1 , with a mirror speed of 2.0 scans / min and a resolution of 4 cm -1 . Prior to sample analysis, a background scan was performed by covering the diamond with the fine powder tip and then with the accessory (Volatile Cover) for the analysis of liquid samples. The spectrum was obtained from a total of 30 scans. The characteristic functional groups for xanthine are observed in the FTIR spectrum, which shows that there was no degradation during the nanocrystallization process. The peak near and below 3000 cm -1 corresponds to the CH bond, around 1750 cm -1A typical lactone absorption band is observed. The sharp peaks at approximately 1670 cm -1 and 1600 cm -1 represent the unsaturated carbonyl system, while the 1150 cm -1 represents the methyl group and the peak around 960 cm -1corresponds to vinyl. 9.8. Nuclear magnetic resonance (NMR): The 1H and 13C NMR spectra were recorded on a Varian VXR-Unity NMR spectrometer at 400 and 100 MHz, as well as the multipulse experiments (HMBC, HSQC, NOESY and COSY). The solvent used to dissolve the samples was DMSO-d6. The chemical shifts (δ) were expressed in ppm and the coupling constants (J) in Hz. The δ values ​​are referred to tetramethylsilane as an internal reference (TMS). The correspondence with the signals reported for this compound is shown. 9.9.Scanning electron microscopy (SEM): For the measurement of xanthine and nanocrystals, a small portion of each sample was deposited on the sample holders with carbon tape, then they were coated with a 15 nm thick layer of gold in order to make the study samples conductive. Once this process was completed, the analysis was carried out on the TESCAN Scanning Electron Microscope, model FE-SEM MIRA3 from Micra Nanotecnologia (Federal District of Mexico, Mexico) with an Energy Dispersive X-ray detector (EDS). The nanocrystals show particles with small needle-like morphology, without agglomerations and with uniform size. Example 10. Inhibition of cell growth of nanocrystallized xanthine by MTT assay. The viability of the cultured cells was evaluated by the MTT reduction assay. The assay was performed using a variation of the method described by Mosmann (Mosmann, 1983).HT-29 and HeLa cells were seeded in 96-well plates at 1x104 cells / ml and 5x103 cells / ml respectively. They were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, triple antibiotic (3A) and glucose (25 mM), and maintained at 37°C in a 5% CO2 incubator until 80–90% confluent. After 24 h of incubation at 37°C, to ensure cell adherence, the medium was removed and different concentrations (10 and 20 μM) of the isolated commercial xanthine and xanthine nanocrystals were added as a negative control medium supplemented with 1% DMSO. Commercial and laboratory-isolated xanthine samples were dissolved in DMSO prior to treatment, unlike nanocrystallized xanthine, which was directly added to the cell culture medium.The prepared plates were incubated for 24 h, and 50 µL of MTT (5 mg / ml) was added per well, for a final concentration of 1 mg / ml. Cells were incubated for 4 h at 37 °C, and then the medium was discarded. 100 µL of DMSO was added per well and resuspended to dissolve the formazan crystals. Relative cell viability was obtained by measuring the absorbance in an ELISA plate reader (Tecan Sunrise MR20-301, TECAN Austria) at 540 nm. Absorbance is directly proportional to the level of cell proliferation or viability. ^^^^^^^^^^^^^^^^^^^^ (%) =^^^^^^^^^^^^^ ^^^^ ^^^^ ^^^^ ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^ ^^^^^^ ^^^^^^^^^^^^^^ ^^^^ ^^^^ ^^^^ ^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^^^^. × 100Figure 10 shows the graphs of the results of the effect of xanthine nanocrystals, commercial xanthine and xanthine isolated in the laboratory on the cell viability of human colorectal adenocarcinoma (HT-29) (Fig A) and human cervical carcinoma (HeLa) tumor lines (Fig B). The MTT assay was used as described by Mosmann in 1983, using DMSO as a negative control. It can be seen that the inhibition of viability exerted by xanthine isolated in the laboratory and xanthine nanocrystals on both cell lines was significantly greater than that of commercial xanthine.There are no statistically significant differences in the effect between xanthine isolated in the laboratory (dissolved in DMSO prior to treatment) and nanocrystallized xanthine obtained from it but added directly to the cell culture medium, which is indicative of the effectiveness of xanthine nanocrystals by increasing the solubility of xanthine through nanocrystallization since the use of organic solvents such as DMSO, which is necessary to dilute drugs that are poorly soluble in water, as is the case with xanthine, prior to treatment in cell cultures, was not required. Example 11. Assay to evaluate the in vitro antiproliferative activity of nanocrystallized xanthine by flow cytometry. Antiproliferative activity on cell cycle arrest was evaluated in the HT-29 cell line. Cells were grown in 6-well plates (2×10. 5 cells / ml and 1x10 5, respectively) until reaching 90% confluence. Three xanthine samples were evaluated: commercial, laboratory-isolated, and nanocrystallized xanthine, each at concentrations of 10 and 20 μM. Prior to treatment, both the commercial and isolated xanthine samples were dissolved in DMSO, unlike nanocrystallized xanthine, which was directly added to the cell culture medium. Medium supplemented with 1% fetal bovine serum (FBS) was used as a control. 2 μM docetaxel was used as a positive control for cell cycle arrest in G2 / M. Treatments were added to medium supplemented with 1% FBS the day after cell seeding on the plates. Cells were incubated with the treatments for 48 h, then harvested, fixed in 100% EtOH, and stored at -20 °C. Cell cycle distribution was assessed by flow cytometry.On the day of the assay, the samples were incubated with a PI / RNase mixture at room temperature and in the dark for 20 minutes prior to analysis. Figure 11 shows the results associated with the effect on cell cycle progression for cells treated with commercial xanthine, isolated xanthine, and xanthine nanocrystals in the human colorectal adenocarcinoma tumor line HT-29 by flow cytometry analysis. There are no statistically significant differences between the effect of commercial xanthine and the untreated control group in the G2 / M phase. There are no statistically significant differences between xanthine isolated in the laboratory (dissolved in DMSO prior to treatment) with respect to nanocrystallized xanthine obtained from it, but added directly to the cell culture medium.The graph shows that both isolated xanthine (dissolved in DMSO prior to treatment) and nanocrystallized xanthine (added directly to the cell culture medium) induce cell cycle arrest in the G2 / M phase without any statistically significant differences with respect to the positive control group (Docetaxel). The result is indicative of the effectiveness of xanthine nanocrystals in increasing its solubility since an organic solvent such as DMSO, which is necessary for substances that are poorly soluble in water (as is the case with xanthine), was not used prior to treatment. Example 12. Evaluation of the in vivo antitumor effect of injectable xanthine nanocrystals. The in vivo antitumor effect of injectable xanthine nanocrystals was evaluated. For this purpose, colon carcinoma cells (CT26WT) were inoculated into male Balb / c mice with average weights of 22-24 g.Tumor implantation was performed by inoculating 0.2 ml of cell suspension with a total of 5x10 subcutaneously (sc) into the right flank of each animal. 6cells / mouse. Four groups of seven animals each were randomly assigned. The xanthine nanocrystal injectable was prepared by diluting the nanocrystals at a concentration of 4 mg / ml in water for injection. The treatment regimen consisted of 10 days of daily intravenous administration of 20 mg / kg of nanocrystalized xanthine injectable, placebo injectable (without xanthine nanocrystals), and every two days a daily administration of 6 mg / kg of oxaliplatin (4 administrations) intraperitoneal. Tumor weight and volume were assessed. The tumor volume was calculated using the following formula: ^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ = 0.5 × ^^^^^^^^^^ × ^^^^^^ℎ^^2The length and width of the tumors were measured every 3 days using a caliper. The in vivo antitumor effect of the nanocrystal injectable in male Balb / c mice inoculated with colon carcinoma cells (CT26WT) is shown in Figure 12.It is observed in the graphs that the injectable nanocrystallized xanthine inhibits tumor growth by decreasing the volume (a) and the tumor weight (b) in Balb / c mice intravenously in CT26WT colorectal cancer tumor cells without any statistically significant differences with the Oxaliplatin injectable. Example 13. Evaluation of the in vivo antitumor effect of in situ gelation containing xanthine nanocrystals. The evaluation of the in vivo antitumor effect of in situ gelation containing xanthine nanocrystals was carried out using murine cervical carcinoma cells (U14) inoculated into the cervix of female Balb / c mice with average weights of 22-24 g. Tumor implantation was carried out by inoculating 25 µL of cell suspension in PBS with a total of 4 x 10 into the submucosa near the cervix of each animal. 5cells / mouse. Four groups of 6 animals per group were randomly assigned. The treatment regimen was carried out for 5 days with a daily intravaginal infusion of the thermosensitive hydrogel containing xanthine nanocrystals (8.5 µL, 0.850 mg of nanocrystallized xanthine per mouse) and placebo, and for three days (once daily on alternate days) of the positive control of Carboplatin by intravenous injection (0.675 mg of Carboplatin per mouse). Tumor weight and tumor inhibition rate were evaluated. The tumor inhibition rate was calculated with the following formula (1): ^^^^^^ = ^^^^ − ^^^^^^ × 100 ^^English: Where: TIT means tumor inhibition rate in % PC means the average weight of the tumors in the control group Px means the average weight of the tumors in the treated group Figure 13 shows the in vivo antitumor effect of embodiment number 6 of the in situ gelation formulation containing xanthine nanocrystals of embodiment number 2, in murine cervical carcinoma cells (U14) inoculated into the cervix of Balb / c mice. A significant decrease in tumor weight is observed in the groups treated with the nanoxanthine and carboplatin injectable with respect to the placebo and the control group, with no statistically significant differences between the in situ gelation of the xanthine nanocrystals and the Carboplatin injectable. The tumor inhibition rate of the xanthine nanocrystal hydrogel and the Carboplatin injectable was 48.2 and 54.4% respectively.References Patent documents JPH09188671A CA2688486A1 CN110123754A Other publications 1. Favier, L.S., et al., Anti-ulcerogenic activity of xanthanolide sesquiterpenes from Xanthium cavanillesii in rats. Journal of Ethnopharmacology 2005.100: p.260-267. 2. Ferrer, J.P. and A.S. Lamar, Mitotic targets of natural drugs and new strategies for anti-cancer therapy. Revista Cubana de Ciencias Biológicas, 2016.4(3): p.3-15 3. Geng, Y.-d., et al., Xanthatin mediates G2 / M cell cycle arrest, autophagy and apoptosis via ROS / XIAP signaling in human colon cancer cells. Natural product research, 2020.34(18): p. 2616-2620 4. Jean Legault, Andre Pichette, Serge Lavoie, owner. Sesquiterpene formulations, kits and methods of use thereof. Canadian patent CA2688486A1. June 2, 2008. [accessed April 6, 2023] Available at: https: / / patents.google.com / patent / CA2688486A1 / en 5. J. Xanthanolides with antitumor. activity from Xanthium italicum. Z Naturforsch C J Biosci.2009 May-Jun;64(5-6):343-9 6. Li, W. D., Wu, Y., Zhang, L., Yan, L. G., Yin, F. Z. et al. Characterization of xanthatin: anticancer properties and mechanisms of inhibited murine melanoma in vitro and in vivo. Phytomedicine.2012., 20(10): 865-873. 7. Liu, M., Xiao, C. Q., Sun, M. W., Tan, M. J., Hu, L. H., & Yu, Q. Xanthatin inhibits STAT3 and NF ‐ κ B s i g n a l l i n g b y c o v a l e n t l y b i n d i n g t o J A K a n d I K K k i n a s e s . J o u r n a l o f Cellular and Molecular Medicine.2019; 23(6), 4301-4312. 8. Lu, L., Xu, Q., Wang, J., Wu, S., Luo, Z., & Lu, W. Drug nanocrystals for active tumor- targeted drug delivery. Pharmaceutics.2022; 14(4), 797. 9. Lu, Y., Li, Y., & Wu, W. Injected nanocrystals for targeted drug delivery. Acta Pharmaceutica Sinica B.2016; 6(2), 106-113. 10. Nibret, E., Youns, M., Krauth-Siegel, R. L. and Wink, M. Biological activities of xanthatin from Xanthium strumarium leaves. Phytother Res.201125(12): 1883-1890. 11. Ramirez-Erosa, I., Huang, Y., Hickie, R. A., Sutherland, R. G. and Barl, B. Xanthatin and xanthinosin from the burs of Xanthium strumarium L. as potential anticancer agents. Can J Physiol Pharmacol.2007; 85(11): 1160-1172. 12. Ran, Q., Wang, M., Kuang, W., Ouyang, J., Han, D., Gao, Z., & Gong, J. Advances of Combinative Nanocrystal Preparation Technology for Improving the Insoluble Drug Solubility and Bioavailability. Crystals.2022; 12(9), 1200. 13. Shen, M., Zhou, X. Z., Ye, L., Yuan, Q., Shi, C., Zhu, P. W., ... & Shao, Y. (2018). Xanthatin inhibits corneal neovascularization by inhibiting the VEGFR2 mediated STAT3 / PI3K / Akt signaling pathway. International journal of molecular medicine.2018; 42(2), 769-778. 14. Takeda, S., et al., (-)-Xanthatin induces the prolonged expression of c-Fos through an N- acetyl-L-cysteine (NAC)-sensitive mechanism in human breast cancer MDA-MB-231 cells. Journal of Toxicological Sciences 2013.38(4): p.547-557 15. Tao, L., et al., Xanthatin triggers Chk1-mediated DNA damage response and destabilizes Cdc25C via lysosomal degradation in lung cancer cells. Toxicology and Applied Pharmacology, 2017.337: p.85-94. 16. Tao, L., Fan, F., Liu, Y., Li, W., Zhang, L. et al. (2013). Concerted suppression of STAT3 and GSK3beta is involved in growth inhibition of non-small cell lung cancer by Xanthatin. PLoS One.2013; 8(11): e81945. 17. Vasas, A. and J. Hohmann, Xanthane sesquiterpenoids: structure, synthesis and biological activity. Natural Products Reports 2011.28: p.824-842. 18. Xia Ming, Wang Cheng, Zhao Miaoqing, Liu Chengcheng, Sol Chao, Zheng Xue, , titular. A kind of targeting is in the Xanthatin nano-micelle and preparation method and application of dendritic cells. Patente china CN110123754A. 24 de junio de 2019. [consultado el 6 de abril de 2023] Disponible en: https: / / patents.google.com / patent / CN110123754A / en?q=(xanthatin)&oq=xanthatin 19. Yan C, Li H, Wu Y, Xie D, Weng Z, Cai B, Liu X, Li W, Chen Z.Determination of xanthatin by ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry: application to pharmacokinetic study of xanthatin in rat plasma. J Chromatogr B Analyt Technol Biomed Life Sci.2014 Feb 1;947-948:57-61. 20. Yoichi Sato, Tetsuya Otsubo, Tomihiko Higuchi, titular. Xanthatin-containing antitumor medicinal composition. Patente japonesa JPH09188671A.9 de enero de 1996. [consultado el 6 de abril de 2023]. Disponible en: https: / / patents.google.com / patent / JPH09188671A / en 21. Yu, Y., Yu, J., Pei, C. G., Li, Y. Y., Tu, P. et al. Xanthatin, a novel potent inhibitor of VEGFR2 signaling, inhibits angiogenesis and tumor growth in breast cancer cells. Int J Clin Exp Pathol.2015; 8(9): 10355-10364. 22. Zhang, L., Ruan, J., Yan, L., Li, W., Wu, Y. et al. Xanthatin induces cell cycle arrest at G2 / M checkpoint and apoptosis via disrupting NF-kappaB pathway in A549 non-small-cell lung cancer cells. Molecules.2012a.17(4): 3736-3750. 23. Zhang, L., et al., Xanthatin Induces G2 / M Cell Cycle Arrest and Apoptosis in Human Gastric Carcinoma MKN-45 Cells. Planta Medica, 2012b.78: p.890-895 24. Zheng X, Sun C, Yu R, Chu X, Xu J, Liu C, Zhao M, Xu X, Xia M, Wang C. CD13-specific ligand facilitates Xanthatin nanomedicine targeting dendritic cells for therapy of refractory allergic rhinitis. Int J Pharm.2020 Mar 15;577:119034. doi: 10.1016 / j.ijpharm.2020.119034. Epub 2020 Jan 25. PMID: 31991183. 25. Zhou Y, Zhu X, Lin S, Zhu C, Wu L, Chen R, Chen Z, Li W. A Novel Nanoparticle Preparation to Enhance the Gastric Adhesion and Bioavailability of Xanthatin. Int J Nanomedicine.2020 Jul 14;15:5073-5082.

Claims

CLAIMS 1. A method for preparing nanocrystals with improved solubility, characterized in that the preparation process in powder form comprises the following steps: a) dissolving xanthine in an organic solvent. b) dissolving surfactant in water together with the cryoprotectant. c) transferring the mixture obtained in a) to the aqueous solution obtained in b), stirring and mixing continuously for 5 to 30 minutes. d) removing the organic solvent to obtain an aqueous suspension of nanocrystals. e) drying the product until a powder is obtained.

102. The method for preparing nanocrystals according to claim 1, characterized in that the organic solvent used in step a) is dimethyl sulfoxide, propylene carbonate, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, methylpyrrolidone, hexamethylphosphoramide, methanol, ethanol or a combination thereof. 3.

4. The method for preparing nanocrystals according to claim 1, characterized in that proportions of 0.3% - 10% (w / v) of xanthine are used.

4. The method for preparing nanocrystals according to claim 1, characterized in that the surfactant used in step b) is added in concentrations between 0.01-10% and is selected from the group comprising Tween 80, Tween 20, Tween 40, Tween 60 and Tween 50, poloxamer, hydroxypropyl methylcellulose, hydroxyethylcellulose, 30 ethylhydroxyethylcellulose or 20 carboxymethylcellulose; PVP polyvinylpyrrolidone; bile acid salts; 12 OH ethoxylated stearic acid, Pluronic, Tetronic or a combination thereof.

5. The method of preparing nanocrystals according to claim 1, characterized in that the cryoprotectant used in step b) is added at 0.5%-10% (w / w, g / g) and is selected from the group consisting of lactose, mannitol, sucrose, trehalose, fructose, glucose, sodium alginate, gelatin or a combination thereof.

6. The method of preparing nanocrystals according to claim 1, characterized in that the drying step in step e) is carried out by lyophilization, spray drying, fluidized bed, pelletization, granulation, supercritical fluids.

7. A lyophilized nanocrystal composition with improved solubility prepared by the method according to claim 1, characterized in that it comprises xanthine, surfactant and a cryoprotectant.

8. The lyophilized nanocrystal composition according to claim 7, characterized in that the average size of the nanocrystals is between 100 - 700 nm.

9. The lyophilized nanocrystal composition according to claim 7, characterized in that xanthine is present between 85 - 99.5 %.

10. The lyophilized nanocrystal composition according to claim 7, characterized in that the surfactant is present in concentrations between 0.01 -10% and is selected from the group comprising Tween 80, Tween 20, Tween 40, Tween 60 and Tween 50, poloxamer, phosphatidylcholine or lecithin, gum arabic, gum tragacanth, sodium lauryl sulfate, 5-hydroxypropylmethylcellulose, hydroxyethylcellulose, 30-ethylhydroxyethylcellulose or carboxymethylcellulose; PVP, polyvinylpyrrolidone; bile acid salts; 12-OH ethoxylated stearic acid, Pluronic, Tetronic, or a combination thereof.

11. The lyophilized nanocrystal composition according to claim 7, characterized in that the cryoprotectant is present between 0.4 and 5%, and is selected from the group comprising lactose, mannitol, sucrose, trehalose, fructose, glucose, sodium alginate, gelatin, or a combination thereof. 12.A pharmaceutical composition comprising the lyophilized nanocrystal composition according to claims 7 to 11 and pharmaceutically acceptable excipients.

13. The pharmaceutical composition according to claim 12, wherein the composition is formulated for intravenous administration and is formulated in powder form for reconstitution with saline or glucose solution for infusion.

14. The pharmaceutical composition according to claim 13, wherein the composition is formulated for direct injection or infusion into the tumor or infusion.

15. The lyophilized nanocrystal composition according to claim 11, wherein the composition is formulated for vaginal, rectal or ocular administration and comprises a thermosensitive hydrogel.

16. The thermosensitive hydrogel according to claim 15 comprising: - a 0.2 - 10% portion of lyophilized nanocrystals - a thermosensitive polymer - a mucoadhesive excipient 2517.The thermosensitive hydrogel according to claim 15, characterized in that the thermosensitive agent is used in concentrations between 15 and 25% (w / v, g / v) and is selected from the group consisting of poloxamers; PLA-PEG copolymers; PLGA-PEG copolymers; methylcellulose; polycaprolactone block copolymer; preferably poloxamers, and more preferably it is poloxamer 407 and / or poloxamer 188 or a combination thereof. 3018. The thermosensitive hydrogel according to claim 15, characterized in that the mucoadhesive excipient is used in a concentration between 0.1 and 5% (w / v, g / v) and is selected from the group consisting of carbopol 940, HPMC (hydroxypropylcellulose), NaCMC (sodium carboxymethylcellulose), Polycarbophil AA1, HPC (hydroxypropylcellulose), sodium alginate, guar gum.

19. A pharmaceutical composition as defined in any one of claims 12 to 18, for use in the treatment of cancer.

20. Use of a pharmaceutical composition according to claims 13 and 14 for the treatment of colon, lung, breast, cervical, or stomach cancer.

21. A method of treating a disease in a patient, the method comprising administering to said patient a therapeutically effective amount of the pharmaceutical composition according to claims 13 and 14, wherein the disease is selected from: colon, lung, breast, cervical, or stomach cancer.

22. Use of a pharmaceutical composition according to claims 15 and 18 for the treatment of cervical, colorectal, or ocular cancer.

23. A method of treating a disease in a patient, the method comprising administering to said patient a therapeutically effective amount of the pharmaceutical composition according to claims 15 and 18, wherein the disease is selected from: cervical, colorectal, or ocular cancer.