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    • 专利摘要:
    Procedure for obtaining a pharmaceutical composition using acetogenins with supramolecular polymeric micelles for the treatment of skin cancer. The present invention describes a process for obtaining a pharmaceutical composition using acetogenins with supramolecular polymeric micelles for the treatment of skin cancer. The procedure includes the biodirected isolation of acetogenins from, for the first time, the leaves of Annona cherimola and the encapsulation of acetogenins generating supramolecular polymeric micelles, mixing an aqueous solution of α-cyclodextrin and urea with a solution of acetogenin in an organic solvent., and subjecting the final solution to dialysis in water to purify it. The encapsulated compound obtained by this procedure has a higher potency than that shown by the unencapsulated compound, which also favors the administration of the drug as it can be carried out in an aqueous medium and does not require any organic solvent or formulation. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2854523A1
    申请号:ES202031011
    申请日:2020-10-06
    公开日:2021-09-21
    发明作者:Mejías Francisco Javier Rodríguez；Valdivia Manuel Jesús Martínez；Domínguez Francisco Antonio Macías；Vázquez María Teresa Gutiérrez；Molinillo José María González；Durán Alexandra García
    申请人:Universidad de Cadiz；
    IPC主号:
    专利说明:

    [0002] PROCEDURE FOR OBTAINING A PHARMACEUTICAL COMPOSITION USING ACETOGENINS WITH SUPRAMOLECULAR POLYMERIC MICELLAS FOR THE TREATMENT OF SKIN CANCER
    [0004] TECHNICAL SECTOR
    [0006] The invention belongs to the field of chemical compounds encapsulated in Core / Shell systems, applicable to in vitro tumor cell systems. The isolation of acetogenins is carried out by biodirected isolation using as starting material, the leaves of Annona cherimola.
    [0008] BACKGROUND OF THE INVENTION
    [0010] Within the isolation and chemical synthesis of compounds destined for the pharmacological field, a great deal of progress has been made with respect to the activity of the compounds. Modifications of functional groups or additions of different fragments have turned out to be key to obtain very specific molecules towards molecular targets. Although the isolation of natural products already has recognized activity [1].
    [0012] One of the main problems faced by the pharmacological industry is that of increasing bioavailability, ensuring that the bioactive molecule performs the desired activity when exposed to a complex living system and that the solubility in the medium is adequate. Side reactions of oxidation or structural modification can occur in the process of molecular transport to the target. Even a low solubility of the compound in the cell or application environment can limit its use. As a solution to these problems presented, compound encapsulation systems outperform others due to their ability to fully preserve the properties of the encapsulated compound. Secondary forces keep the molecule of interest completely trapped within others, masking the physicochemical properties of the host.
    [0013] One of the most notable compounds within the field of encapsulation are cyclodextrins, of which there are a great variety of derivatives. These have been extensively studied for encapsulation in the last 25 years [2-6]. However, delving into the use of encapsulation systems, the generation of core / shell systems are being used with better results due to the ability to encapsulate a greater number of bioactive molecules [7-9]. In addition, the generation of different types of rotaxane substructures using cyclodextrins has made it possible to use these toroidal molecules as generators of new organic nanoparticles, known as supramolecular polymeric micelles (SMPMs) [10-12].
    [0015] Acetogenins are a specific case of bioactive compounds with refuted anti-tumor activity against HeLa, HepG2 and human mammary adenocarcinoma (MCF-7 / Adr) among others. Its chemical structure comprises one or two aliphatic chains, with 35-37 carbon atoms, a lactonic ring a, p unsaturated, between 1 and 3 tetrahydrofuran rings and several hydroxyl groups (see example Figure 2). These compounds are only soluble in organic media, and therefore are of difficult real application to living systems, whose main medium is aqueous; which limits its potential application in future clinical cases.
    [0017] REFERENCE LIST 1234567
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    [0023] Rubiales, FA Macías, P. Delavault, Org. Biomol. Chem. 2017, 34, 28-35.
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    [0026] [6] C. Lucas-Abellán, I. Fortea, JA Gabaldón, E. Núñez-Delicado, J. Agric. Food Chem. 2008, 56, 255-9.
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    [0028] Biomed. Nanotechnol. 2015, 11, 1093-1105.
    [0029] [8] W. H. Lee, CY Loo, CR Leong, PM Young, D. Traini, R. Rohanizadeh, Expert Opin. Drug Deliv. 2017, 14, 937-957.
    [0030] [9] FJR Mejías, M. López-Haro, LC Gontard, A. Cala, M. Femández-Aparicio, JMG Molinillo, JJ Calvino, FA Macías, ACS Appl. Mater. Interfaces 2018, 10, 2354-2359.
    [0031] [10] F. J. R. Mejías, M. T. Gutiérrez, A. G. Durán, J. M. G. Molinillo, M. M. Valdivia, F.
    [0032] A. Macías, Colloids Surfaces B Biointerfaces 2019, 173, 85-93.
    [0033] [11] H. Dong, Y. Li, S. Cai, R. Zhuo, X. Zhang, L. Liu, Angew. Chemie 2008, 47, 5573-5576.
    [0034] [12] J. Hong, Y. Li, Y. Xiao, Y. Li, Y. Guo, H. Kuang, X. Wang, Colloids Surfaces B Biointerfaces 2016, 145, 319-327.
    [0036] Description of the invention
    [0038] The present invention relates, among other aspects, to a bio-targeted isolation process using a cell viability bioassay on tumor cells. Thus, in the present invention, the obtaining of acetogenins from Annona cherimola leaves is described for the first time. For this purpose, the Annona cherimola leaves necessary for the extraction were dried and crushed in an industrial mill to achieve a larger extraction surface. Extracts of plant material usually have a large amount of fatty acids and chlorophylls. For their elimination and to avoid their interference in the bio-directed isolation, different extraction and separation methods were used for their elimination. The plant material was defatted by the Soxhlet extraction method using a low polarity organic solvent in order to remove the fatty acids present in the sample. Then, by means of a vacuum chromatography column using silica as adsorbent and gradient elution, the elimination of chlorophylls was carried out. Subsequently, by means of different chromatographic techniques, such as column chromatography or high efficiency liquid chromatography, the acetogenins were obtained. In each purification stage, a cell viability bioassay was performed on skin cancer cells, to select those fractions with the highest activity. Likewise, by means of mono- and two-dimensional spectroscopic and spectrometric techniques, the structural elucidation of acetogenins was carried out.
    [0039] Therefore, in a first aspect of the invention, bio-directed isolation comprises the following steps:
    [0041] I) Drying and crushing the plant material, preferably the leaves of Annona cherimola ;
    [0042] II) Degreasing by extraction with an organic solvent of low polarity; III) Elimination of chlorophylls by gradient elution with binary mixtures of water and organic solvents of higher polarity;
    [0043] IV) Biodirected fractionation of acetogenins; and
    [0044] V) Structural characterization of acetogenins.
    [0046] The invention also includes the process for encapsulating acetogenins using cyclodextrin and urea. A supramolecular polymeric micelle is generated around acetogenin (Figure 4) that allows the improvement of the solubility of acetogenin in aqueous medium, improves its bioavailability and maintains or improves cytotoxic activity. Hence, a second aspect of the invention refers to a process for obtaining supramolecular polymeric micelles of cyclodextrin and urea comprising a drug with an encapsulated acetogenin structure, where said encapsulated drug is characterized by comprising the following structure:
    [0051] Where:
    [0053] - "n" is an integer between 1 and 3;
    [0054] - "m" is an integer between 8 and 12;
    [0055] - "q" is an integer between 6 and 12;
    [0056] - "R i" can be a hydroxyl (OH) or a hydrogen atom (H);
    [0057] - "R 2 " can be a hydroxyl (OH) or a hydrogen atom (H);
    [0058] - "R 3 " can be a hydroxyl (OH), a hydrogen atom (H) or a ketone group (C = O);
    [0059] - "R 4 " can be any substituent, preferably C 1 -C 4 alkyl groups, a hydrogen atom (H), hydroxyl group (OH) or halogen group from the whole: fluorine (F), bromine (Br), chlorine ( Cl) or iodine (I);
    [0060] - "R 5 " represents one or more substituents of the hydroxyl type that may or may not be present in the alkyl chain whose length is determined by the parameter "q". Preferably the number of substituents of the hydroxyl type "R5" is one or two ;
    [0061] - "R6" can be any substituent, preferably C 1 -C 4 alkyl groups, a hydrogen atom (H), hydroxyl group (OH) or halogen group of the bromine (Br), chlorine (Cl) or iodine (I) group ;
    [0062] - "R 7 " can be a hydroxyl (OH), a hydrogen atom (H), or a combination of both for the values of m; and between positions 1 and 2 there may or may not be a double bond.
    [0064] and where said process is characterized in that it comprises the following steps: I) dissolving the drug in an organic solvent; II) Dissolution of the encapsulating agents, urea and cyclodextrin, in an aqueous solution (polar); III) Mixing the solutions (I II) and preferably continuous stirring of the mixture, and IV) obtaining the encapsulated drug; and where the cyclodextrin that is selected is preferably alpha-cyclodextrin. In the context of the present invention, the alkyl chain "R5" is composed of methylenes, except in those positions where there is a hydroxyl substituent.
    [0066] In a preferred embodiment of the second aspect of the invention, the process is characterized in that it comprises the following steps: I) Dissolution of acetogenins in an organic solvent; II) Dissolution of the encapsulating agents, urea and cyclodextrin, in an aqueous solution; III) Mixing the solutions (I II), preferably by continuous stirring of the mixture; IV) Evaporation of the organic solvent in the mixture to obtain the drug in an aqueous solvent and optionally V) Purification of the encapsulated drug and evaporation of the solvent where the purified drug is found. Preferably the encapsulated drug is purified by dialysis, preferably using a dialysis membrane of 1000 Daltons or higher.
    [0068] In another preferred embodiment of the second aspect of the invention, the method further comprises evaporating the solvent where the drug is found. purified and optionally preservation of supramolecular polymeric micelles (SMPMs) at low temperature.
    [0070] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the isolated drug is obtained from the leaves of Annona cherimola.
    [0072] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the drug is annonazine with the following chemical structure:
    [0077] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the drug is molvizarin with the following chemical structure: (2S) -4- (2R, 11 R) -2,11-dihydroxy-11 - [(2R , 5R) -5 - [(2R, 5R) -5 - [(1 S) -1-hydroxyundecyl] oxolan-2-yl] undecyl] -2-methyl-2H-furan-5-one.
    [0082] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the drug is cherimoline with the following chemical structure: 4- [9- [5- [1,4-dihydroxy-4- [5- (1-hydroxyundecyl ) oxolan-2-yl] butyl] oxolan-2-yl] -2-hydroxynonyl] -2-methyl-2H-furan-5-one.
    [0085] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the drug is bullatacin with the following chemical structure: (S) -3 - ((2R, 13S) -2,13-dihydroxy-13 - ((2S , 2'S, 5S, 5'S) -5 '- ((R) -1-hydroxyundecyl) octahydro- [2,2'-bifuran] -5-yl) tridecyl) -5-methylfuran-2 (5H) -one.
    [0092] In another preferred embodiment of the second aspect of the invention, the method is characterized in that the drug is motrillin with the following chemical structure: (2S) -4 - [(13R) -13 - [(2R, 5R) -5 - [( 2R, 5R) -5 - [(1S, 6S) -1,6-dihydroxyundecyl] oxolan-2-yl] oxolan-2-yl] -13-hydroxytridecyl] -2-methyl-2H-furan-5-one.
    [0097] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the drug is annonisine with the following chemical structure: 2-methyl-4- [2,6,11-trihydroxy-11- [5- [5- ( 1-hydroxyundecyl) oxolan-2-yl] oxolan-2-yl] undecyl] -2H-furan-5-one.
    [0100] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the organic solvent in which the acetogenins are dissolved (the drug) is selected from the list consisting of: Dimethylsulfoxide (DMSO), Chloroform (CHCl3), Dichloromethane (CH2Cl2), Methanol (CH3OH), Ethanol (CH3CH2OH) or Tetrahydrofuran (CH2CH2CH2CH2O). Preferably, the organic solvent in which the acetogenins (the drug) are dissolved is selected from the list consisting of: Chloroform, Dichloromethane and Tetrahydrofuran.
    [0102] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the polar solvent is selected from the list consisting of: Water (H2O), Glycerol (HOCH2CHOHCH2OH), Ethanol (CH3CH2OH) and Acetonitrile (CH3CN). Preferably, the polar solvent is water, and this is preferably buffered using buffers from 6.5 to 7.8, more preferably the water comprises sodium chloride in order to slightly increase the ionic strength of the medium.
    [0104] On the other hand, in the case of using tetrahydrofuran (THF) and water, the optimal range for each of the solvents is between 70% and 30%. These percentages make it possible to obtain the highest percentage of encapsulation of acetogenins, 35% of encapsulation being the optimum when it is used at 50/50 THF / Water. Thus, in another preferred embodiment of the second aspect of the invention, the process is characterized in that the polar solvent is water and tetrahydrofuran, preferably in a ratio between 70:30 and 30:70. Preferably, the polar solvent is water and tetrahydrofuran, preferably in a 70:30 ratio.
    [0106] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the polar solvent is water and tetrahydrofuran, preferably in a 50:50 ratio.
    [0108] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the synthesis of the supramolecular polymeric micelles with encapsulated acetogenins is carried out at temperatures between 0 and 60 ° C, preferably between 30 ° C-55 ° C. The optimum working temperature is between 30 ° C-55 ° C, thus achieving encapsulation percentages between 30-35%. The decomposition temperature of acetogenins is close to 60 ° C, therefore the synthesis is carried out below this temperature.
    [0110] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the stirring takes place at speeds between 1 and 3000 rpm.
    [0112] In another preferred embodiment of the second aspect of the invention, the process is characterized in that the elimination of the organic solvent in the immiscible mixture is carried out by streams of gases such as nitrogen or oxygen, heat, under vacuum, lyophilization or any combination of these.
    [0114] In another preferred embodiment of the second aspect of the invention, the method is characterized in that the concentration range of urea is between 35% and 50% w / volume of the micelle shell, and that of cyclodextrin, preferably alpha-cyclodextrin , is between 65% and 50% w / volume of the micelle shell. Although it is true that in the present invention we have used alpha-cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin and the polymeric agent Pluronic F-127, of these compounds, the desired micelles were only obtained with alpha cyclodextrin. Optimal concentration ranges for urea are 35% to 50%, while for cyclodextrin they are 65% to 50%. These ranges also provide the highest encapsulation percentage of acetogenins between 30 and 35%.
    [0116]
    [0117] In the table above we show the encapsulation percentages measured for each of the encapsulation systems that we have tested. Considering these percentages, it is clear that the best percentage was obtained with alpha-cyclodextrin.
    [0119] A third aspect of the invention relates to supramolecular polymeric cyclodextrin micelles obtainable or obtained according to the process according to any of the embodiments of the first aspect of the invention.
    [0121] A fourth aspect of the invention refers to supramolecular polymeric cyclodextrin micelles characterized in that they comprise a core and a shell, where said shell comprises cyclodextrin and urea, and the core comprises the drug as defined in any of the embodiments of the first aspect of the invention. Preferably, the cyclodextrin is selected from the list consisting of alpha cyclodextrin, gamma-cyclodextrin, or 2-hydroxypropyl-beta-cyclodextrin. Preferably, said structures have a mean size between 60 nm and 140 nm, preferably 96 nm. More preferably, the concentration range of urea is between 35% and 50% w / volume of the shell, and that of cyclodextrin is between 65% and 50% w / volume of the shell. More preferably, the encapsulated drugs have a spherical geometry.
    [0123] A fifth aspect of the invention relates to polymeric micelles according to any of the third or fourth aspects of the invention, for use in therapy.
    [0125] A sixth aspect of the invention refers to a pharmaceutical composition comprising the supramolecular polymeric cyclodextrin micelles according to any of the third or fourth aspects of the invention.
    [0127] A seventh aspect of the invention relates to the composition according to the sixth aspect of the invention or to the supramolecular polymeric cyclodextrin micelles according to any of the third or fourth aspects of the invention, for the treatment or prevention of cancer, in particular for the following . The acetogenin micelles of the invention have been tested in cell lines of cervical cancer (HeLa), ovarian cancer (IGROV-1), skin cancer (SK-MEL-28) and non-tumor cells (HEK-293 ). The results of the acetogenins encapsulated with micelles correspond to annonacin, which is the acetogenin with which the study was carried out, because it was the majority that we isolated from the plant.
    [0129] The results for three lines are shown in Figure 9. However, the IC50 values for each cell line were as follows: HeLa: 19.32 | j, M, IGROV-1: 46.54 | j, M and HEK-293: 68.76 ^ M.
    [0131] Therefore, it is shown that micelles with encapsulated acetogenins do not attack non-tumor cells, by showing such a high IC50 value. The IC50 value of the skin cancer line could not be measured, but at the concentration of 100. ^ M it kills 85% of the tumor cells.
    [0133] In addition to these in vitro studies, in vivo studies were also performed. Three mice were injected with the micelles dispersed in a phosphate buffer (PBS) for two weeks, making 7 injections in total. Blood samples were taken every two weeks and after 14 weeks the mice did not show any adverse effects. At 16 weeks the mice were sacrificed. The concentrations tested in the mice were 20 mM and 5 mM micelles concentration.
    [0135] BRIEF DESCRIPTION OF THE DRAWINGS
    [0137] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description, in which, with an illustrative and non-limiting nature, the following has been represented :
    [0139] Figure 1.- Biodirected isolation of annonacin from deciduous leaves of Annona cherimola "Fino de Jete".
    [0141] Figure 2.- Spectroscopic data of annonazine.
    [0143] Figure 3.- Example of acetogenin with cytotoxic activity, annonacin.
    [0144] Figure 4.- Diagram of the structure of supramolecular polymeric micelles with annonacin.
    [0146] Figure 5.- Transmission microscopy images in which the morphology of the supramolecular polymeric micelles is observed.
    [0148] Figure 6.- NMR proton spectrum in which different chemical shifts are observed for α-cyclodextrin. All are made in D2O A) α-cyclodextrin. B) Urea encapsulated in α-cyclodextrin. C) Annonacin encapsulated in the supramolecular polymeric micelle generated by α-cyclodextrin and urea. D) Sample C treated with phenol.
    [0150] Figure 7.- Release of annonacin in a physiological medium.
    [0152] Figure 8.- (Left) Viability assay in different cell lines. (Right) Light microscope images of the appearance of cells after 24 hours of treatment with: 1) DMSO. 2) α-cyclodextrin. 3) Annonacin dissolved in DMSO. 4) Annonacin encapsulated in the supramolecular polymeric micelle.
    [0154] Figure 9.- This figure shows the results for three cell lines using annonacin encapsulated in the micelles of the invention. The IC50 values for each cell line were as follows: HeLa: 19.32 ^ .M, IGROV-1: 46.54. ^ M and HEK-293: 68.76 ^ M.
    [0156] EXAMPLES
    [0158] Example 1: Bio-rigid isolation of annonacin from the deciduous leaves of Annona cherimola variety Fino de Jete
    [0160] The leaves of Annona cherimola variety "Fino de Jete" necessary for the initial extraction were collected in October 2014 in Almuñecar (36 ° 44'2.1''N 3 ° 41'26.6''W). Once dried at room temperature, 2.2 kg of the resulting leaves were mechanically crushed and defatted by Soxhlet using 4 L. of hexane, obtaining a mass of dry extract equal to 159 g (7.4% Rto). resulting was extracted using the same procedure, with 4 L of dichloromethane. Removal of the solvent under reduced pressure resulted in obtaining the initial extract E1, (140 g, 6.5% Rto.), Which was separated by reverse phase silica gel vacuum column chromatography (RP-18). Seven new fractions (E1.1 - E1.7) were obtained, after using different proportions of the MeOH-H 2 O and dichloromethane mixture: E1.1 (MeOH-H 2 O; 0:10), E1.2 (MeOH -H 2 O 2: 8), E1.3 (MeOH-H 2 O 4: 6), E1.4 (MeOH-H2O 6: 4), E1.5 (MeOH-H2O 8: 2), E1.6 (MeOH-H2O 10: 0), E1.7 (dichloromethane). Fraction E 1.6 , (28 g, 1.3% Rto), was again fractionated after showing the lowest value of cell viability. Separation was carried out by column chromatography on silica gel using a gradient of hexane: ethyl acetate (from 5 to 100% ethyl acetate in 10% increments). Ten fractions were obtained (E 1. 6.1 -E 1. 6.10 ) and in this case, the most active (E 1. 6.5 , 2.57 g, 0.12% Rto.) Was subjected to a new purification following the same procedure previously described with ethyl acetate and hexane. In this new separation, 13 fractions were obtained, attending to similarity patterns in thin layer chromatography: E 1 . 6 . 5.1 - E 1 . 6 . 5.13 . At this time, the presence of acetogenins is confirmed, by 1H-NMR experiments, in fractions that show lower cell viability. Starting from the fraction E 1 . 6 . 5.9 , by column chromatography on silica gel using hexane: ethyl acetate (7: 3) to 100% ethyl acetate as gradient elution system. A total of 60 mg was obtained. Annonacin, with 0.003% Rto (see figure 1).
    [0162] The presence of annonacin was confirmed in the 1H-NMR and 13C-NMR spectrum (see figure 2) after observing the characteristic signals of the lactonic group a, p-unsaturated substituted in position y and a hydroxyl group in position 4. Del Similarly, the signals corresponding to a single tetrahydrofuran ring flanked by two hydroxyl groups and a fourth in position 10 were appreciated. On the other hand, the mass spectrum confirmed a molecular formula calculated for C 35 H 64 O 7 [M + H] + of 597.4736, found at 597.4730.
    [0164] Example 2: Preparation of the supramolecular polymeric micelle with annonacin and characterization
    [0166] 40 mg of annonacin is dissolved in 4 mL of dry THF. In a different flask, 160 mg of alpha-cyclodextrin and urea are weighed together, which are dissolved in 4 mL of H 2 O type I with the help of an ultrasound probe to facilitate the process of dissolution. The annonacin solution is added dropwise to the cyclodextrin-urea solution, and the new mixture is stirred for 24 hours at 60 ° C. The final solution is dialyzed for 30 minutes in type I water to purify it. For this, a dialysis membrane is used that restricts the passage of molecules with a molecular weight greater than 6000-8000 AMU. The sample remaining inside the membrane is frozen and lyophilized to obtain a solid state sample.
    [0168] The structure of the complexes formed has a spherical or pseudospherical morphology, in which an external halo (corresponding to the membrane of the polymeric micelle composed of cyclodextrins and urea) and an internal cavity where annonacin is housed (Figure 5).
    [0170] The determination by nuclear magnetic resonance spectroscopy of the compound object of the invention was carried out by comparing the chemical shift of the cyclodextrin signals when it is free in solution, when it is forming a complex with urea, when it forms supramolecular micelles with acetogenin in its interior and when an organic medium is applied to break the micellar structure (Figure 6).
    [0172] The release of acetogenins to the physiological environment (37 ° C and pH = 7.2) begins after 20 hours of exposure. Acetogenins are completely bioavailable in the medium at 39 hours, and remain stable in said medium for more than 6 days (Figure 7).
    [0174] The bioavailability of acetogenins when encapsulated in SMPMs is studied using the simulation method of the human gastrointestinal system. The method is divided into five phases:
    [0176] 1. The oral phase comprises the simulation of salivary fluid, which is prepared by dissolving a concentration of 30 g / L of mucin in PBS and shaking it vigorously. The pharmaceutical formulation is dissolved in this mixture in a 1: 1 ratio and the pH is adjusted to 6.8. Subsequently, it is stirred for 10 minutes at 100 rpm and 37 ° C.
    [0177] 2. The gastric phase comprises the formation of a solution of NaCl 2 g / L, HCl 7 mL / L and pepsin 3.2 g / L, all of them dissolved in pure type I water with pH 2. This new solution is mixed with solution 1 of the oral phase in a 1: 1 ratio. This new mixture is stirred at 100 rpm, for 2 hours and 37 ° C.
    [0178] 3. The small intestine phase comprises the formation of five solutions, namely: CaCh 36.7 mg / mL in water, NaCl 218.7 mg / mL in water, cholic acid 187.5 mg / mL in PBS, pancreatin 60 mg / mL in PBS and 60 mg / mL lipase in PBS. These solutions are added to the gastric phase 2 solution in the following proportions: 20: 1 of CaCl 2 and NaCl, 8.5: 1 of cholic acid and 12: 1 of pancreatin and lipase. The resulting final solution is stirred at 100 rpm for 2 hours at 37 ° C.
    [0179] 4. The sample obtained from the previous phase is cooled in an ice bath and centrifuged at 12000 rpm and 4 ° C for 30 minutes. The supernatant is analyzed in UV-Vis to calculate the dissolved annonacin concentration.
    [0180] 5. The percentage of bioavailability is determined by following the following formula:
    [0182] Anonazine concentration in the supernatant Bioavailability = --------------------------; - -------------- ------------ ----------- 100
    [0183] Initial anonazine concentration
    [0185] The bioavailability achieved by annonacin encapsulated in SMPMs was thirteen times higher compared to free annonacin.
    [0187] Example 3: Biological Activities of the Supramolecular Polymeric Micelle with Annonacin
    [0189] The cytotoxicity bioassays for the compound object of the invention are performed using the trypan blue exclusion protocol and subsequent counting of the stained and unstained cells using an automatic cell counter (Automated Cell Counter T20 Bio-Rad)
    [0191] The result of the cell viability test is measured, after 24 hours of treatment of human origin tumor cells SK-Mel-28, for the purpose of the invention. This treatment is applied under controlled incubation conditions: CO 2 pressure of 5%, humidity of 95% and temperature of 37 ° C.
    [0192] Viable cells, and therefore alive, will be considered those that keep their cell membrane intact. Non-viable cells, stained by the dye in question, are also detected by the measurement equipment.
    [0194] The tests are carried out in triplicate with dilution of 100 µM. The sample of cells treated with organic solvent (dimethyl sulfoxide: DMSO) is established as a negative control, as well as the components of the supramolecular polymeric micelle. Acetogenin in DMSO is considered as a positive control.
    [0196] The effect of the compound encapsulated and suspended in water is of greater potency than that shown by the unencapsulated compound, applied to the cells dissolved in organic solvent (Figure 7). This fact offers great interest from the point of view of the applicability of acetogenins as cytotoxic agents.
    [0198] Example 4: Structure of the supramolecular polymeric micelle of the invention
    [0200] The structures of the micelles correspond to those of the image shown in Figure 5. These "Shell / Core" structures (Core / Shell) comprise an outer membrane composed of cyclodextrin and urea, while the core is composed of acetogenins.
    [0202] The scheme in Figure 4 explains in more detail the composition of the micelles. Said structures have an average size of 96 nm, but two groups of micelles are observed, one with an average size of about 60 nm and another distribution with an average size of 140 nm.
    [0204] The outer membranes have a diameter of 8 nm, and depending on their size, they are comprised of two molecules of cyclodextrin and 32 molecules of urea.
    [0206] Example 5. Geometry of the micelles of the invention.
    [0208] Micelles geometry
    [0209] The pseudospherical geometry has special relevance in the study of the bioavailability of acetogenins when they are administered encapsulated within the micelle and reach the digestive system.
    [0210] In the proposed example, it is shown how we performed an oral and gastrointestinal simulation study, and it is shown that micelles allow acetogenins to remain stable for much longer until they reach the small intestine and can be absorbed. The bioavailability value of free acetogenin is 3.68%, while the bioavailability of acetogenin encapsulated in the micelle is 47.01%.
    [0212] Apart from said stability, a study of the release under physiological conditions was carried out, where the sphericity of the micelles shows a gradual release in which the micelles reduce their volume while they release the acetogenins (see Figure 7).
    权利要求:
    Claims (29)
    [1]
    1. Procedure for obtaining supramolecular polymeric micelles of cyclodextrin and urea comprising a drug with an encapsulated acetogenin structure, where said drug is characterized by comprising the following structure:

    [2]
    2. Procedure for obtaining supramolecular polymeric micelles of cyclodextrin and urea according to the preceding claim, characterized in that it comprises the following steps: I) Dissolution of acetogenins in an organic solvent; II) Dissolution of the encapsulating agents, urea and cyclodextrin, in an aqueous solution; III) Mixing the solutions (I II), preferably by continuous stirring of the mixture; IV) Evaporation of the organic solvent in the mixture to obtain the drug in an aqueous solvent and optionally V) Purification of the encapsulated drug and evaporation of the solvent where the purified drug is found.
    [3]
    3. Procedure for obtaining supramolecular polymeric micelles of cyclodextrin and urea according to the preceding claim where the encapsulated drug is purified by dialysis, preferably using a dialysis membrane of 1000 Daltons or higher.
    [4]
    4. Procedure for obtaining supramolecular polymeric micelles of cyclodextrin and urea according to any of claims 2 or 3, where the procedure additionally comprises evaporation of the solvent where the purified drug is found and optionally the preservation of the supramolecular polymeric micelles (SMPMs ) at low temperature.
    [5]
    5. Process according to any of claims 1 to 4, characterized in that the isolated acetogenin is obtained from the leaves of Annona cherimola.
    [6]
    6. Process according to any of claims 1 to 5, characterized in that the drug is annonacin with the following chemical structure:

    [7]
    7. Method according to any of claims 1 to 5, characterized
    because the drug is molvizarin with the following chemical structure: (2S) -4- (2R, 11R) -2,11-dihydroxy-11 - [(2R, 5R) -5 - [(2R, 5R) -5- [ (1S) -1-hydroxyundecyl] oxolan-2-yl] undecyl] -2-methyl-2H-furan-5-one.

    [8]
    8. Method according to any of claims 1 to 5, characterized
    because the drug is bullatacin with the following chemical structure: (S) -3 - ((2R, 13S) -2,13-dihydroxy-13 - ((2S, 2'S, 5S, 5'S) -5 '- ((R) -1-hydroxyundecyl) octahydro- [2,2'-bifuran] -5-yl) tridecyl) -5-methylfuran-2 (5H) -one.

    [9]
    9. Process according to any of claims 1 to 5, characterized in that the drug is motrillin with the following chemical structure: (2S) -4 - [(13R) -13 - [(2R, 5R) -5 - [(2R, 5R) -5 - [(1S, 6S) -1,6-dihydroxyundecyl] oxolan-2-yl] oxolan-2-yl] -13-hydroxytridecyl] -2-methyl-2H-furan-5-one.

    [10]
    10. Process according to any of claims 1 to 5, characterized in that the drug is annonisine with the following chemical structure: 2-methyl-4- [2,6,11-trihydroxy-11- [5- [5- (1- hydroxyundecyl) oxolan-2-yl] oxolan-2-yl] undecyl] -2H-furan-5-one.

    [11]
    11. Process according to any of the preceding claims, characterized in that the organic solvent in which the acetogenins (the drug) are dissolved is selected from the list consisting of: Dimethylsulfoxide (DMSO), Chloroform (CHCl3), Dichloromethane (CH2Cl2), Methanol (CH3OH), Ethanol (CH3CH2OH) or Tetrahydrofuran (CH2CH2CH2CH2O).
    [12]
    12. Process according to any of claims 1 to 11, characterized in that the organic solvent in which the acetogenins are dissolved (the drug) is selected from the list consisting of: Chloroform, Dichloromethane and Tetrahydrofuran.
    [13]
    13. Process according to any of the preceding claims, characterized in that the polar solvent is selected from the list consisting of: Water (H2O), Glycerol (HOCH2CHOHCH2OH), Ethanol (CH3CH2OH) and Acetonitrile (CH3CN).
    [14]
    14. Process according to any of the preceding claims, characterized in that the polar solvent is water, and this is preferably buffered using buffers from pH 6.5 to 7.8, more preferably the water comprises sodium chloride in order to slightly increase the ionic strength of the medium.
    [15]
    15. Process according to any of the preceding claims, characterized in that the polar solvent is water and the organic solvent is tetrahydrofuran, preferably in a ratio between 70:30 and 30:70.
    [16]
    16. Process according to any of the preceding claims, characterized in that the polar solvent is water and the organic solvent is tetrahydrofuran, preferably in a 70:30 ratio.
    [17]
    17. Process according to any of the preceding claims, characterized in that the polar solvent is water and the organic solvent is tetrahydrofuran, preferably in a 50:50 ratio.
    [18]
    18. Process according to any of the preceding claims, characterized in that the synthesis of the supramolecular polymeric micelles with encapsulated acetogenins is carried out at temperatures between 0 and 60 ° C, preferably between 30 ° C-55 ° C.
    [19]
    19. Process according to any of the preceding claims, characterized in that the stirring takes place at speeds between 1 and 3000 rpm.
    [20]
    20. Process according to any of the preceding claims, characterized in that the elimination of the organic solvent in the immiscible mixture is carried out by gas currents such as nitrogen or oxygen, heat, under vacuum, lyophilization or any combination of them.
    [21]
    21. Process according to any of the preceding claims, where the concentration range of urea is between 35% and 50% w / volume of the micelle shell, and that of cyclodextrin is between 65% and 50% % w / volume of micelle shell.
    [22]
    22. Supramolecular polymeric micelles of alpha-cyclodextrin obtainable or obtained according to the process according to any of claims 1 to 21.
    [23]
    23. Supramolecular polymeric alpha-cyclodextrin micelles characterized in that they comprise a core and a shell, wherein said shell comprises cyclodextrin and urea, and the core comprises the drug as defined in any of claims 1 to 21.
    [24]
    24. The supramolecular cyclodextrin polymeric micelles according to claim 24, wherein said structures have a mean size between 60 nm and 140 nm, preferably 96 nm.
    [25]
    25. The supramolecular polymeric micelles of cyclodextrin according to any of claims 23 or 24, where the concentration range of urea is between 35% and 50% w / volume of the shell, and that of cyclodextrin is between 65 % and 50% w / volume of the cover.
    [26]
    26. Supramolecular polymeric cyclodextrin micelles according to any of claims 23 to 25, characterized in that the encapsulated drugs have a spherical geometry.
    [27]
    27. Polymeric micelles according to any of claims 23 to 26, for use in therapy.
    [28]
    28. Pharmaceutical composition comprising the supramolecular polymeric cyclodextrin micelles according to any of claims 23 to 27.
    [29]
    29. The pharmaceutical composition according to claim 28 or the micelles according to any of claims 23 to 27, for the treatment or prevention of skin cancer.
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