Pharmaceutical compositions of nanocrystallized xanthotoxin and their use as antitumor agents

Abstract

Methods of obtaining isolated xanthotoxin nanocrystals from natural sources are described. Pharmaceutical compositions comprising such nanocrystals are also described, as are uses and methods of treatment in which they can be used.

Description

Technical Field

[0001] Xanthin is a natural sesquiterpene lactone with the molecular formula C15H18O3, mainly isolated from the aerial parts of species belonging to the genus Xanthium (Asteraceae). Members of this genus are perennial plants distributed in the Americas and Eurasia.

[0002] From a structural perspective, the compound exhibits an α-methylene-γ-butyrolactone ring fused in trans configuration to a 7-carbon ring, and contains an unsaturated ketone side chain. Multiple studies on xanthogen sulfadiazine compounds have shown that this structural arrangement is a fundamental characteristic of the different types of biological activities exhibited by xanthogen sulfadiazine, particularly its significant antiproliferative activity against various tumor cell lines. Background of the Invention

[0004] Multiple reports have demonstrated the effectiveness of xanthin in significantly reducing cell proliferation and inducing apoptosis in various tumor cell lines, including: ovarian cancer (SK-OV-3 and ES-2), melanoma (SK-MEL-2), central nervous system cancer (XF498), colon cancer (HCT-15 and CT26WT), human gastric cancer (MKN-45), non-small cell lung cancer (A549 and H1299), breast cancer (MDA-MB-231), and cervical cancer (HeLa) cells.

[0005] Several mechanisms for the antitumor effects of xanthin have been proposed. Takeda and colleagues demonstrated that xanthin, as a catalytic inhibitor of topoisomerase IIα, promotes DNA damage in breast cancer cells.

[0006] Furthermore, xanthogenetic acid 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 xanthogenetic acid can negatively regulate the expression of Chk1 and Chk2 and alter the phosphorylation status of CDC2, both key regulators of the G2 / M phase transition. Xanthogenetic acid has also been reported to have antimitotic potential, disrupting the metaphase-anaphase transition by acting on the formation of microtubules in the mitotic spindle, thereby leading to apoptosis. It has also been reported that xanthogenetic acid primarily inhibits autophagy by activating the PI3K / Akt / mTOR pathway, thereby effectively inhibiting the proliferation and tumorigenicity of malignant glioma cells. Studies of potential mechanisms have shown that xanthogenetic acid has antiproliferative, anti-angiogenic, and pro-apoptotic effects in various types of cancer, both in vitro and in vivo. All these studies indicate that xanthogenetic acid (from Xanthium sibiricum)... X. strumarium Xanthin, isolated from [a specific source], has potential in cancer prevention and treatment.

[0007] Xanthin is a lipophilic molecule with poor solubility in water. Pharmacokinetic studies in rats after intravenous administration show that it is rapidly metabolized and eliminated from the body.

[0008] Patent JPH09188671A (1996) relates to a pharmaceutical composition containing xanthin as an antitumor agent, prepared by conventional methods, for oral administration in tablet and syrup form, and for injection as an intravenous infusion. Conventional methods for preparing injectables containing poorly water-soluble active ingredients involve large amounts of co-solvents and / or surfactants that may trigger allergic reactions in patients.

[0009] Patent CA2688486A1 (2008) relates to a formulation of a sesquiterpene compound, acting as a water-insoluble antitumor agent, with one or more antioxidants and one or more solubilizers, said solubilizers being selected from the group consisting of PEG400, animal or vegetable oils (e.g., olive oil), castor oil derivatives, ethylene oxide (Cremophor RH 40), and polysorbate 80. It is known that large amounts of organic solvents and surfactants are required to dissolve water-insoluble active ingredients such as xanthin, which may cause adverse reactions.

[0010] Patent CN110123754A (2019) relates to obtaining a cyclic peptide (NGR)-functionalized xanthin polymeric nanomicelles for active targeting of dendritic cells as an anti-inflammatory agent in the treatment of allergic rhinitis. An aqueous pharmaceutical formulation free of any solubilizers or organic solvents is proposed as a strategy to overcome the hydrophobicity limitation of xanthin, achieving targeted therapy, sustained release, increased stability, enhanced cellular internalization, efficacy, and reduced side effects.

[0011] In 2020, Zhou et al. synthesized polydopamine polymer nanoparticles containing xanthogen sulfadiazine, improving the gastric adhesion and bioavailability of xanthogen sulfadiazine. They demonstrated that the nanoparticles may have potential effectiveness in oral inhibition of gastric cancer.

[0012] In 2020, Zheng X et al. obtained xanthin micelles functionalized with CD13-specific cyclic peptide (NGR) polymers targeting dendritic cells through non-clinical evaluation of intranasal administration in a mouse model of allergic rhinitis, as a potential drug for the treatment of refractory allergic rhinitis.

[0013] Nanocrystal technology is one of the most widely used strategies for improving the solubility and bioavailability of poorly soluble drugs. These are nanoscale particles of pure drug stabilized by small amounts of suitable surfactants / polymers. To avoid particle aggregation in the liquid state, freeze-drying is considered one of the superior techniques for improving the long-term stability of colloidal nanoparticles, alongside spray drying, supercritical fluids, granulation, and pelleting. Drug nanocrystals are a versatile formulation method for improving the pharmacokinetic and pharmacodynamic properties of poorly soluble drugs, offering the opportunity to alter the composition and physical and / or chemical properties of a substance without changing existing covalent bonds. The reduction in size leads to an increase in surface area, thereby increasing the dissolution rate and improving drug solubility, permeability, and adhesion. All these properties contribute to improved drug bioavailability. Compared to other drug delivery systems, nanocrystals are formed directly from drugs, offering advantages such as high active ingredient loading, ease of 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, and transdermal administration. Because it contains no carrier material, the theoretical drug loading capacity is as high as 100%, typically between 50-90% (w / w), thus achieving satisfactory therapeutic concentrations at lower doses. Toxic side effects caused by encapsulating / solubilizing excipients (such as Cremophor RH40) are also eliminated.

[0014] Preparation techniques can be categorized into "top-down," "bottom-up," and combinations thereof. Top-down methods primarily involve media milling and high-pressure homogenization, utilizing high mechanical forces to transform bulk drug powders into nanoscale particles. These methods are simple, rapid, solvent-free, and highly reproducible. However, the process is typically energy-intensive and time-consuming; high shear and temperature can lead to crystal instability and subsequent aggregation. There are also concerns about potential product contamination by the milling media. Bottom-up methods primarily involve solvent-antisolvent precipitation, precisely controlling drug precipitation and crystallization to obtain the desired nanoparticles. These methods are advantageous for small and narrow particle size distributions.

[0015] In-situ gelation technology has been developed for many years for contraception or treatment of bacterial, fungal, and sexually transmitted infections. It allows for controlled and sustained release of drugs, reducing dosing frequency and requiring lower doses, while improving drug bioavailability and reducing side effects. In particular, in-situ thermogelation utilizes a liquid system that can be introduced or deposited onto the body surface using minimally invasive techniques before solidifying or gelling at body temperature within the target tissue, organ, or body cavity. The in-situ formed polymer matrix offers advantages over other systems that require surgical implantation procedures and sometimes even removal at the end of treatment. In-situ gel system formulations (thermosensitive hydrogels) are used for various routes of administration, including vaginal administration.

[0016] Compared to other cancers, therapeutic agents can be administered locally to the vaginal mucosa, making cervical cancer an excellent option for local drug delivery, as is the case with systems such as in situ gelation. Continuous, local application of antitumor drugs to the female reproductive tract avoids the adverse effects associated with systemic administration, enhances efficacy by ensuring adequate drug concentrations at the site of action, and allows for convenient self-administration.

[0017] Currently, no commercially available formulations containing xanthin have received approval from any regulatory agency. Despite the existence of such formulations, there is currently no state-of-the-art injectable xanthin antitumor formulation containing xanthin nanocrystals that does not use organic solvents, exhibits high solubility, bioavailability, efficacy, and ease of large-scale production. Similarly, there are currently no xanthin formulations in the form of in-situ gelled thermosensitive hydrogels containing xanthin nanocrystals for local treatment and antitumor applications. Therefore, designing novel, effective, and easy-to-use xanthin formulations has significant clinical value.

[0018] Invention Summary

[0019] This disclosure relates to pharmaceutical compositions containing xanthin as an active pharmaceutical ingredient. In particular, one aspect of the invention relates to a novel method for preparing xanthin nanocrystals. Another aspect of the invention is a nanocrystal composition prepared according to the method, comprising xanthin, a surfactant, and a cryoprotectant.

[0020] Another aspect described is a pharmaceutical composition comprising a composition of xanthin nanocrystals and one or more pharmaceutically acceptable excipients.

[0021] One specific aspect of the invention is the formulation of a pharmaceutical composition for injection administration. Furthermore, another aspect of the invention is the use of said injectable formulation in the treatment of colon cancer, lung cancer, breast cancer, cervical cancer, and gastric cancer. In yet another aspect, a method of treating a patient suffering from colon cancer, lung cancer, breast cancer, cervical cancer, uterine cancer, or gastric cancer with said injectable formulation is described.

[0022] Another specific aspect of the invention is a pharmaceutical composition formulated into an in-situ gelled thermosensitive hydrogel.

[0023] Furthermore, another aspect of the invention is the use of the said thermosensitive hydrogel formulation in the treatment of cervical cancer, uterine cancer, colorectal cancer, and eye cancer. In another aspect, a method for treating patients with cervical cancer, uterine cancer, colorectal cancer, or eye cancer using the said in-situ gelled thermosensitive hydrogel is described. Brief description of the attached diagram

[0025] The accompanying drawings relate to freeze-dried xanthin nanocrystals according to Embodiment 2 of the present invention.

[0026] Figure 1Crystal images: A) isolated xanthin and B) nanocrystallized xanthin.

[0027] Figure 2 The ultraviolet spectrum of nanocrystalline xanthin recorded in the 200-400 nm range.

[0028] Figure 3 Chromatograms of xanthin nanocrystals determined by HPLC are recorded at A) 210 nm and B) 280 nm.

[0029] Figure 4 Melting point determination.

[0030] Figure 5 Particle size distribution.

[0031] Figure 6 X-ray diffraction spectrum.

[0032] Figure 7 Fourier transform infrared spectroscopy (FTIS).

[0033] Figure 8 Heteronuclear single quantum correlation (HSQC) spectrum of xanthin nanocrystals. A magnified view of the aliphatic region of the spectrum is shown.

[0034] Figure 9 Scanning electron microscope (SEM).

[0035] Figure 10 The effects of commercially available xanthin (XCtrol), isolated xanthin (XAisl), and nanocrystalline xanthin (XNano) on cell viability at 24 hours in HT-29 and HeLa cell lines A) and B) were investigated. Medium supplemented with 1% fetal bovine serum (FBS) was used as a viability control (Ctrol).

[0036] Figure 11 The effect of nanocrystalline xanthin on cell cycle progression of HT-29 colon cancer cells was evaluated by flow cytometry. Medium supplemented with 1% FBS served as the untreated control (Ctrol), and docetaxel (DTX) was used as the positive control. Cells were treated for 48 hours with 10 and 20 μM nanocrystalline xanthin, isolated xanthin, and 10 μM commercially available xanthin. DMSO was used as the control vector. Data represent the percentage distribution of cells at each stage of the cell cycle. XCtrol: commercially available xanthin; XAisl: isolated xanthin; XNano: nanocrystalline xanthin.

[0037] Figure 12In vivo antitumor effects of nanocrystal injections: A) tumor volume assessment, and B) tumor weight assessment.

[0038] Figure 13 In vivo antitumor effects of the in-situ gelled formulation of Example 6 containing the xanthin nanocrystals of Example 2: A) tumor weight assessment and B) tumor inhibition rate (%) assessment. Invention Details

[0040] The purpose of this invention is to obtain xanthin nanocrystals by antisolvent precipitation, dry the nanocrystal suspension to obtain freeze-dried nanocrystals, and formulate injections and in-situ gels containing freeze-dried nanocrystals.

[0041] The preparation of xanthin nanocrystals in this invention is carried out through the following technical solution:

[0042] (1) Dissolve 0.3%-10% (w / v, g / ml) of xanthin in an organic phase such as ethanol to form solution A;

[0043] The organic phase mentioned above is selected from one or more of dimethyl sulfoxide, propylene carbonate, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, methylpyrrolidone, hexamethylphosphoramide, methanol, ethanol, or combinations thereof.

[0044] (2) Sterilize solution A.

[0045] (3) Dissolve 0.01%-10% (w / v, g / ml) of stabilizer or surfactant in water as a dispersion medium, which is solution B;

[0046] The surfactants mentioned above are selected from Tween 20, Tween 40, Tween 50, Tween 60 and Tween 80, poloxamer, phosphatidylcholine or lecithin, gum arabic, tragacanth gum, sodium lauryl sulfate, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethyl hydroxyethylcellulose or carboxymethylcellulose; polyvinylpyrrolidone (PVP), for example, PVP with a molecular weight in the range of 10,000 to 100,000 Daltons, such as 50,000 to 60,000; bile salts, such as deoxycholate, taurocholate or glycocholate, such as sodium salts of bile acids, such as sodium taurocholate, sodium deoxycholate or sodium glycocholate; 12-hydroxyethoxylated stearic acid (e.g. Solutol HS15), Pluronic, Tetronic or Lutrol surfactants, such as Lutrol F68 or Lutrol F127.

[0047] The aqueous phase may contain a single stabilizer or a mixture of two or more stabilizers. (4) Add 0.01%-10% (w / w, g / g) of lyophilization protectant to the above solution (solution C) relative to the amount of xanthin used.

[0048] The freeze-drying protectant is one of the following: glucose, lactose, mannitol, sucrose, or trehalose.

[0049] (5) Sterilize solution C.

[0050] (6) A suspension D is obtained by adding A to C with stirring for 5-30 minutes.

[0051] (7) Remove the organic solvent to obtain an aqueous suspension of nanocrystals.

[0052] (8) The suspension D was frozen between -40°C and -80°C, and then sublimated by heating between 5°C and 50°C to obtain a lyophilized formulation of xanthin nanocrystals. In addition to lyophilization, the nanocrystals can be dried by spray drying, fluidized bed drying, pelleting, granulation, or supercritical fluid drying.

[0053] The preparation of injectable formulations follows these steps.

[0054] (9) The lyophilized preparation of xanthogenan nanocrystals is added to saline or glucose and redissolved for intravenous infusion. The dosage is 0.5-2 mg / kg.

[0055] The preparation of in-situ gelling formulations follows these steps.

[0056] (10) The mucosal adhesion polymer, solution E, is slowly added to cold deionized water (between 4 and 15°C) at a concentration between 0.1 and 5% (w / v, g / v) with slow stirring.

[0057] The aforementioned mucosal adhesive polymers are selected from one or more of the following: carbomer 940, HPMC (hydroxypropyl methylcellulose), NaCMC (sodium carboxymethyl cellulose), Polycarbophil AA1, HPC (hydroxypropyl cellulose), sodium alginate, and guar gum.

[0058] (11) Then, with stirring, a 15-25% (w / v, g / v) concentration of the thermosensitive polymer is added to solution E and dissolved at 4°C for 8 to 16 hours. Solution F.

[0059] The aforementioned thermosensitive polymers are selected from one or more of poloxamer 407 (Planic 127), poloxamer 188 (Planic F68), methylcellulose, PLA-PEG, PLGA-PEG, and polycaprolactone-polyethylene glycol block copolymers.

[0060] (12) Disperse 0.2-10% of freeze-dried xanthin nanocrystals (w / v, g / mL) in solution F by gentle mixing. Administer 1-3 mL vaginally.

[0061] Specific implementation form

[0062] The present invention will be described through the following embodiments, but is not limited thereto.

[0063] Example 1.

[0064] Preparation of Xanthin Nanocrystals

[0065] 0.5 g of xanthin was dissolved in 100 mL of ethanol and sterilized through a 0.2 μm filter. This solution was then added, with stirring, to 400 mL of a pre-sterilized aqueous solution containing 1.2% Tween 80 (by volume of water) and 1% sucrose (by the amount of xanthin). The mixture was shaken at 75 rpm for 20 minutes. The solution was concentrated under vacuum until the ethanol was removed. The resulting aqueous suspension was frozen at -80°C for 3 hours and freeze-dried at 30°C for 48 hours. The particle diameter reached 505 nm. A yield of 0.355 g was obtained.

[0066] Example 2.

[0067] Preparation of Xanthin Nanocrystals

[0068] 0.5 g of xanthin was dissolved in 100 mL of ethanol and aseptically filtered through a 0.2 μm filter. The solution was then added, with stirring, to 400 mL of a pre-sterilized aqueous solution containing 1% Tween 80 (by volume of water) and 1% mannitol (by mass of the added xanthin). The mixture was shaken at 100 rpm for 20 minutes. The solution was concentrated under vacuum until the ethanol was removed. The resulting aqueous suspension was frozen at -80°C for 2 hours and then freeze-dried at -40°C for 24 hours. The particle diameter reached 669 nm. A yield of 0.386 g was obtained.

[0069] Example 3.

[0070] Preparation of Xanthin Nanocrystals

[0071] 0.5 g of xanthin was dissolved in 50 mL of dimethyl sulfoxide (DMSO) and sterilized through a 0.2 μm filter. Then, 400 mL of a pre-sterilized aqueous solution containing 0.8% Tween 80 (by volume of water) and 1% mannitol (by mass of the added xanthin) was added with stirring. The mixture was shaken at 100 rpm for 20 minutes. The solution was concentrated under vacuum until the DMSO was removed. The resulting aqueous suspension was frozen at -80°C for 3 hours and freeze-dried at -20°C for 24 hours. The particle diameter reached 650 nm. A yield of 0.372 g was obtained.

[0072] Example 4.

[0073] Preparation of Xanthin Nanocrystals

[0074] 0.5 g of xanthin was dissolved in 50 mL of dimethyl sulfoxide (DMSO) and aseptically filtered through a 0.2 μm filter. Then, 400 mL of an aqueous solution containing 1% Tween 20 (by volume of water) and 1% mannitol (by mass of the added xanthin) was added with stirring. The mixture was shaken at 100 rpm for 15 minutes. The solution was concentrated under vacuum until the DMSO was removed. The resulting aqueous suspension was frozen at -80°C for 2 hours and then freeze-dried at -40°C for 24 hours. The particle diameter reached 685 nm. A yield of 0.368 g was obtained.

[0075] Example 5.

[0076] Preparation of thermosensitive in-situ gelled hydrogels containing xanthin nanocrystals

[0077] Dissolve 1 g of HPMC in 50 mL of cold deionized water at 4°C, then add 20 g of Prönkel 127 while stirring, and continue for 10 hours until completely dissolved. Next, add 5 g of xanthin nanocrystals while stirring until completely dispersed. Add sufficient deionized water to bring the volume to 100 mL.

[0078] Example 6.

[0079] Preparation of in-situ gelled thermosensitive hydrogels containing xanthin nanocrystals

[0080] Dissolve 2 g of carbomer in 50 mL of cold deionized water at 4°C. Then, while stirring, add 15 g of Pranic 127 and 10 g of Pranic F68, and continue for 12 hours until completely dissolved. Add 10 g of xanthin nanocrystals while stirring until completely dispersed. Add sufficient deionized water to bring the volume to 100 mL.

[0081] Example 7.

[0082] Preparation of in-situ gelled thermosensitive hydrogels containing xanthin nanocrystals

[0083] Dissolve 2 g of HPMC in 50 mL of cold deionized water at 4°C. Then, while stirring, add 15 g of Pranic 127 and 10 g of Pranic F68, and continue for 12 hours until completely dissolved. Add 10 g of xanthin nanocrystals while stirring until completely dispersed. Add sufficient deionized water to bring the volume to 100 mL.

[0084] Example 8.

[0085] Preparation of thermosensitive in-situ gelled hydrogels containing xanthin nanocrystals

[0086] Dissolve 1 g of carbomer in 50 mL of cold deionized water at 4°C, then add 20 g of Pranic 127 while stirring, and continue for 12 hours until completely dissolved. Next, add 5 g of xanthin nanocrystals while stirring until completely dispersed. Add sufficient deionized water to bring the volume to 100 mL.

[0087] Example 9.

[0088] Physicochemical characterization of xanthin nanocrystals

[0089] 9.1. Sensory characteristics of appearance and color were measured. Visual measurements of appearance and color were performed. Figure 1 Images of A) isolated xanthin and B) freeze-dried nanocrystalline xanthin are shown.

[0090] Significant differences exist in sensory characteristics (appearance and color), as the crystals in sample A) are pale yellow, while fine white particles are observed in sample B).

[0091] 9.2. Ultraviolet (UV) Spectroscopy: Recorded on a Thermo Scientific SPECTRONIC GENESYS spectrophotometer, in the range of 200–400 nm, using analytical grade methanol as a blank. Nanocrystalline xanthogenetic samples were prepared at a concentration of 0.25 mg / mL.

[0092] Maximum absorption values ​​were observed at 207.2 and 277.1 nm, corresponding to the maximum values ​​described for this compound.

[0093] 9.3. High-performance liquid chromatography (HPLC) analysis with diode array detector: A Shimadzu HPLC system equipped with a Supelco reversed-phase column (12.5 x 4.6 mm x 5 μm) and a DAD detector was used. Samples were dissolved in methanol at a concentration of 1 mg / mL and eluted using an acetonitrile / water polar gradient, as shown in Table 1. The sample injection volume was 20 μL, and absorbance was read at 210 and 280 nm. Methanol was used as a blank. Peak purity was determined to be 99.99% by chromatographic analysis.

[0094] 9.4. Melting Point Determination: The melting point was determined in an automated melting and boiling point measuring apparatus, Melting Point M-565. Using a Buchi Labortechnik AG capillary tube, the xanthin sample was introduced to form a dense column with a height of 4-6 mm, compacted using an M569 sample loader before introduction into the apparatus.

[0095] The melting point of nanocrystalline xanthin is 109.3±0.4℃.

[0096] 9.5. Particle Size Determination: The particle size distribution of the separated xanthin and nanocrystalline xanthin samples was determined using a Shimadzu SALD 7101 particle analyzer (Kyoto, Japan) and WingSALD II 3.0.4 software. The average particle size of the nanocrystalline xanthin was 669 ± 43 nm.

[0097] 9.6. X-ray Diffraction (XRD): X-ray diffraction patterns of the separated xanthin and nanocrystalline xanthin were obtained using a MAXIMA-X XRD-7000 X-ray diffractometer at Shimadzu (Kyoto, Japan), using CuKα radiation at a voltage of 30.0 kV and a current of 30.0 mA. Recording was performed in the 2θ angle range (5–80)° at a scan rate of 2° / min, with continuous scanning. Spectra were obtained using OriginPro 2018 software. Characteristic peaks of the crystalline form of xanthin were observed.

[0098] 9.7. Fourier Transform Infrared Spectroscopy (FTIR): Xanthin and the nanocrystals obtained from it were analyzed using the attenuated total reflectance method on a Prestige 21 IR instrument from Shimadzu (Kyoto, Japan). Tests were performed using an accessory with a diamond optical window (n=2.4), brand SPECAC, model Golden Gate, with minimal contact between the sample and the window. The scanning range was 4000–600 cm⁻¹. -1 Mirror speed 2.0 scans / minute, resolution 4 cm. -1Prior to sample analysis, background scanning was performed by covering the diamond with a tip used for fine powders and then with a volatile cover for liquid sample analysis. Spectra were obtained from a total of 30 scans.

[0099] FTIR spectroscopy revealed the characteristic functional groups of xanthogen sulfadiazine, clearly indicating that no degradation occurred during nanocrystallization. (Approximately below 3000 cm⁻¹) -1 The peak corresponds to the CH bond, at approximately 1750 cm⁻¹. -1 A typical lactone absorption band was observed at approximately 1670 cm⁻¹. -1 and 1600 cm -1 The sharp peak at 1150 cm⁻¹ represents an unsaturated carbonyl system, while the peak at 1150 cm⁻¹ represents an unsaturated carbonyl system. -1 The peak at approximately 960 cm⁻¹ represents methyl groups. -1 The peak at that location corresponds to vinyl groups.

[0100] 9.8. Nuclear Magnetic Resonance (NMR): 1H and 13C NMR spectra were recorded on a Varian VXR-Unity NMR spectrometer at frequencies of 400 and 100 MHz, respectively, using multi-pulse experiments (HMBC, HSQC, NOESY, and COSY). DMSO-d6 was used as the solvent for sample dissolution. Chemical shifts (δ) are expressed in ppm, and coupling constants (J) are expressed in Hz. δ values ​​were referenced to tetramethylsilane as an internal standard (TMS).

[0101] This shows consistency with the reported signal of this compound.

[0102] 9.9. Scanning Electron Microscopy (SEM): For measurements of xanthin and nanocrystals, a small portion of each sample was deposited onto a sample stage with carbon tape, and then coated with a 15 nm thick layer of gold to make the sample under study conductive. After this process, analysis was performed using a TESCAN scanning electron microscope (model FE-SEM MIRA3, from MicraNanotechnology (Federal District of Mexico)) equipped with an energy-dispersive X-ray detector (EDS).

[0103] The nanocrystals exhibit particles with a fine needle-like morphology, without agglomeration and with uniform size.

[0104] Example 10.

[0105] The cell growth inhibition of nanocrystalline xanthin was assessed using the MTT assay.

[0106] Cell viability was assessed using the MTT reduction assay. This assay was performed using the method described by Mosmann (1983). HT-29 and HeLa cells were respectively injected with 10...4 cells / ml and 5x10 3 Cells were seeded at 100 cells / ml in 96-well plates. Cells were cultured in Duchenne Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamine, triple antibiotic (3A), and 25 mM glucose at 37°C in a 5% CO2 incubator until 80–90% cell confluence. After 24 hours of incubation, to ensure cell adhesion, the medium was removed, and different concentrations (10 and 20 μM) of commercially available xanthin, isolated xanthin, and xanthin nanocrystals were added, or, as a negative control, medium supplemented with 1% DMSO was added. Commercial and isolated xanthin samples were dissolved in DMSO prior to treatment, unlike nanocrystalized xanthin added directly to the cell culture medium. The prepared plates were incubated for 24 hours, and 50 μL of MTT (5 mg / ml) was added to each well to a final concentration of 1 mg / ml. Cells were incubated at 37°C for 4 hours, then the medium was discarded. Add DMSO (100 μl per well) to each well and resuspend to dissolve the formazan crystals. Relative cell viability was determined by measuring absorbance at 540 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Austria). Absorbance is directly proportional to cell proliferation or viability levels.

[0107]

[0108] Figure 10 The results show the effects of xanthin nanocrystals, commercially available xanthin, and laboratory-isolated xanthin on the cell viability of human HT-29 colorectal adenocarcinoma (Figure A) and human HeLa cervical cancer cell lines (Figure B). MTT assays were performed according to the method described by Mosmann in 1983, using DMSO as a negative control. It can be seen that laboratory-isolated xanthin and xanthin nanocrystals had significantly higher inhibitory effects on the viability of both cell lines than commercially available xanthin. There was no statistically significant difference between the effects of laboratory-isolated xanthin (dissolved in DMSO before treatment) and nanocrystalline xanthin obtained from it but added directly to the cell culture medium. This indicates the effectiveness of xanthin nanocrystals in improving xanthin solubility through nanocrystalization, as it eliminates the need for dilution with organic solvents such as DMSO before cell culture treatment, as is typically required when treating poorly water-soluble drugs like xanthin.

[0109] Example 11.

[0110] An experiment to evaluate the in vitro antiproliferative activity of nanocrystalline xanthogen sulfadiazine using flow cytometry.

[0111] Antiproliferative activity via cell cycle arrest was evaluated in HT-29 cells. Cells were cultured in 6-well plates (2 x 10⁶ cells per well). 5 and 1x10 5 Cells / ml were used until 90% cell confluence was achieved. Three xanthin samples were evaluated: commercial, laboratory-isolated, and nanocrystalline xanthin at concentrations of 10 and 20 μM. Commercial and isolated xanthin were dissolved in DMSO prior to treatment, unlike nanocrystalline xanthin which was added directly to the cell culture medium. Medium supplemented with 1% FBS was used as a control. 2 μM docetaxel was used as a positive control for G2 / M phase cell cycle arrest. Treatment was added to medium supplemented with 1% FBS the day after cell seeding. Cells were incubated for 48 hours post-treatment, collected, fixed in 100% ethanol, and stored at -20°C. Cell cycle distribution was then assessed using flow cytometry. On the day of assay, samples were incubated in the dark with a Pl / Rnase mixture at room temperature for 20 minutes before analysis.

[0112] Figure 11 Results from flow cytometry analysis of the effects of commercially available xanthin, isolated xanthin, and xanthin nanocrystals on cell cycle progression in the human HT-29 colorectal adenocarcinoma cell line are presented. At the G2 / M phase, there was no statistically significant difference in the effect of commercially available xanthin compared to the untreated control group. There was also no statistically significant difference between laboratory-isolated xanthin (dissolved in DMSO before treatment) and nanocrystalized xanthin obtained from it but added directly to the cell culture medium. The graphs show that both isolated xanthin (dissolved in DMSO before treatment) and nanocrystalized xanthin (added directly to the cell culture medium) induced cell cycle arrest at the G2 / M phase, with no statistically significant difference compared to the positive control group (docetaxel). This result demonstrates the effectiveness of xanthin nanocrystals in increasing xanthin solubility, as no organic solvent like DMSO, which is necessary for poorly water-soluble substances like xanthin, was used prior to treatment.

[0113] Example 12.

[0114] Evaluation of the in vivo antitumor effect of injectable xanthin nanocrystals

[0115] The in vivo antitumor effect of injectable xanthin nanocrystals was evaluated. For this purpose, CT26WT colon cancer cells were inoculated into male BALB / c mice with an average weight of 22–24 g. 0.2 ml of cell suspension (total 5 x 10⁻⁶ ml) was injected subcutaneously (sc) on the right side of each animal. 6Tumor implantation was performed using cells / mouse. Animals were randomly assigned to four groups of seven each. Injectable xanthogenan nanocrystals were prepared by diluting the nanocrystals in water for injection to a concentration of 4 mg / ml. The treatment regimen lasted 10 days, with daily intravenous administration of the nanocrystalized xanthogenan injection at a dose of 20 mg / kg, an injectable placebo (without xanthogenan nanocrystals), and intraperitoneal administration of oxaliplatin at a dose of 6 mg / kg every two days (for a total of four doses). Tumor weight and volume were assessed. Tumor volume was calculated using the following formula:

[0116] Tumor volume = 0.5 × length × width 2

[0117] The length and width of the tumor were measured every three days using calipers.

[0118] Figure 12 This study demonstrates the in vivo antitumor effect of injectable nanocrystals in male BALB / c mice inoculated with CT26WT colon cancer cells. The graphs show that intravenously administered nanocrystalized xanthogenan injection inhibited tumor growth by reducing A) tumor volume and B) tumor weight in BALB / c mice, without statistically significant differences compared to oxaliplatin injection.

[0119] Example 13.

[0120] Evaluation of the in vivo antitumor effect of in situ gel containing xanthin nanocrystals.

[0121] The in vivo antitumor effect of an in situ gel containing xanthin nanocrystals was evaluated using mouse U14 cervical cancer cells inoculated into the cervix of female BALB / c mice with an average weight of 22–24 g. Cell suspension in PBS was submucosally injected into the cervix of each animal (total 4 x 10⁻⁶ cells / mL). 5 Tumor implantation was performed using cells / mouse. Four randomly distributed groups of six animals each were formed. The treatment regimen consisted of daily intravaginal infusion of a thermosensitive hydrogel containing xanthogen sulfadiazine nanocrystals (8.5 μL, 0.850 mg of nanocrystalline xanthogen sulfadiazine per mouse) and placebo for five days, and daily intravaginal infusion of xanthogen sulfadiazine nanocrystals (8.5 μL, 0.850 mg of nanocrystalline xanthogen sulfadiazine per mouse) and placebo for three days. The positive control was administered intravenously (0.675 mg of carboplatin per mouse) for three days (every other day). Tumor weight and tumor inhibition rate were assessed. The tumor inhibition rate was calculated using the following formula:

[0122]

[0123] in:

[0124] TIR represents tumor inhibition rate, expressed as a percentage (%).

[0125] AW represents the average tumor weight in the control group.

[0126] Ax represents the average tumor weight in the treatment group.

[0127] Figure 13 The in situ gel formulation of Example 6, containing xanthin nanocrystals from Example 2, demonstrates its in vivo antitumor effect against mouse U14 cervical cancer cells inoculated into the cervix of female BALB / c mice. Tumor weight was significantly reduced in the groups treated with nano-xanthin and carboplatin injection compared to the placebo and control groups; there was no statistically significant difference between the in situ gel of xanthin nanocrystals and carboplatin injection. The tumor inhibition rates of the xanthin nanocrystal hydrogel and injectable carboplatin were 48.2% and 54.4%, respectively.

[0128] References

[0129] Patent documents

[0130] JPH09188671A

[0131] CA2688486A1

[0132] CN110123754A

[0133] Other publications

[0134]

[0135]

[0136]

Claims

1. A method for preparing nanocrystals with improved solubility, characterized in that, The preparation process of powder form includes the following steps: a) Dissolve xanthin in an organic solvent. b) Dissolve the surfactant together with the cryoprotectant in water. c) Transfer the mixture obtained in a) to the aqueous solution obtained in b), stir and mix continuously for 5 to 30 minutes. d) Remove the organic solvent to obtain an aqueous suspension of nanocrystals. e) Dry the product until a powder is obtained.

2. 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. The method for preparing nanocrystals according to claim 1, characterized in that, Use 0.3%-10% (w / v) of xanthin.

4. The method for preparing nanocrystals according to claim 1, characterized in that, The surfactant used in step b) is added at a concentration between 0.01% and 10%, and is selected from the group consisting of: Tween 80, Tween 20, Tween 40, Tween 60 and Tween 50, poloxamer, hydroxypropyl methylcellulose, hydroxyethylcellulose, 30-ethyl hydroxyethylcellulose or carboxymethylcellulose; polyvinylpyrrolidone (PVP); bile salts; 12-hydroxyethoxylated stearic acid, Pranic, Tetronic or combinations thereof.

5. The method for 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 for preparing nanocrystals according to claim 1, characterized in that, The drying step in step e) is carried out by freeze drying, spray drying, fluidized bed, pelletizing, granulation, or supercritical fluid.

7. A composition of freeze-dried nanocrystals with improved solubility prepared by the method according to claim 1, characterized in that, It contains xanthogen sulfadiazine, surfactants, and cryoprotectants.

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

9. The composition of freeze-dried nanocrystals according to claim 7, characterized in that, Xanthin exists in concentrations ranging from 85% to 99.5%.

10. The composition of freeze-dried nanocrystals according to claim 7, characterized in that, The surfactant is present at a concentration between 0.01% and 10%, and is selected from the group consisting of: Tween 80, Tween 20, Tween 40, Tween 60 and Tween 50, poloxamer, phosphatidylcholine or lecithin, gum arabic, tragacanth, sodium lauryl sulfate, hydroxypropyl methylcellulose, hydroxyethylcellulose, 30-ethyl hydroxyethylcellulose or carboxymethylcellulose; polyvinylpyrrolidone (PVP); bile salts; 12-hydroxyethoxylated stearic acid, Pluronic, Tetronic or combinations thereof.

11. The composition of freeze-dried nanocrystals according to claim 7, characterized in that, The cryoprotectant is present in the range of 0.4-5% and is selected from the group consisting of lactose, mannitol, sucrose, trehalose, fructose, glucose, sodium alginate, gelatin, or combinations thereof.

12. A pharmaceutical composition comprising the composition of freeze-dried nanocrystals according to claims 7 to 11 and a pharmaceutically acceptable excipient.

13. The pharmaceutical composition of 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, characterized in that, The composition is formulated for direct intratumoral injection, perfusion, or infusion.

15. The composition of freeze-dried nanocrystals according to claim 11, wherein the composition is formulated for vaginal, rectal or ocular administration and comprises a thermosensitive hydrogel.

16. The thermosensitive hydrogel of claim 15, comprising: -0.2-10% share of freeze-dried nanocrystals. - A thermosensitive polymer - A mucosal adhesive excipient.

17. The thermosensitive hydrogel according to claim 15, characterized in that, The heat sensitizer is used at a concentration between 15 and 25% (w / v, g / v) and is selected from the group consisting of: poloxamer; PLA-PEG copolymer; PLGA-PEG copolymer; methylcellulose; polycaprolactone block copolymer; preferably poloxamer, more preferably poloxamer 407 and / or poloxamer 188, or combinations thereof.

18. The thermosensitive hydrogel according to claim 15, characterized in that, The mucosal adhesive excipient is used at a concentration between 0.1% and 5% (w / v, g / v) and is selected from the group consisting of: carbomer 940, HPMC (hydroxypropyl cellulose), NaCMC (sodium carboxymethyl cellulose), Polycarbophil AA1, HPC (hydroxypropyl cellulose), sodium alginate, and guar gum.

19. The pharmaceutical composition as defined in any one of claims 12 to 18, for treating cancer.

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

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

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

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

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