WO2013167782A1

WO2013167782A1 - Amphiphilic copolymers carrying alpha-tocopherol having antitumoural properties

Info

Publication number: WO2013167782A1
Application number: PCT/ES2013/070287
Authority: WO
Inventors: Raquel Palao Suay; Francisco Jesús PARRA RUIZ; María Rosa Aguilar de Armas; Mar Fernández Gutiérrez; Julio SAN ROMÁN DEL BARRIO; Juan Parra Cáceres
Original assignee: Consejo Superior De Investigaciones Cientificas; Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina (Ciber-Bbn)
Priority date: 2012-05-07
Filing date: 2013-05-07
Publication date: 2013-11-14
Also published as: ES2433248B1; ES2433248A1

Abstract

The invention relates to the use of a family of amphiphilic copolymers that form polymeric micelles of a micrometric or nanometric size and consist of acrylic monomers derived from the alpha-tocopherol molecule and highly hydrophilic monomers. This family of copolymers exhibits an antitumoural activity per se, but they can also act as vehicles for other active principles having an antitumoural effect.

Description

ALFA-TOCOFEROL CARRYING AMPHIFOLIC COPOLYMERS WITH

ANTITUMOR PROPERTIES

The present invention relates to the use of a family of amphiphilic copolymers that form polymeric micelles of nanometric or micrometric size and that are constituted by acrylic monomers derived from the alpha-tocopherol molecule and highly hydrophilic monomers. This family of copolymers have "per se" antitumor activity, but they can also serve as vehicles for other active ingredients with antitumor effect.

Therefore, the invention could be framed in the field of pharmaceutical chemistry and pharmacology.

STATE OF THE TECHNIQUE

The anticancer treatments that currently exist in the market are characterized by their high toxicity due to their low specificity since they affect both tumor cells and healthy cells, especially if they are in active division. Hence, at present, research and development of new drugs has been directed towards drugs capable of selectively killing cancer cells (anticancer activity) and / or drugs capable of inhibiting tumor growth by preventing the development of the vasculature that irrigate (antiangiogenic activity) using as a strategy the search for differences between tumor cells and healthy cells.

An example of a recently discovered drug is alpha-tocopherol succinate, known to be a "mitochondrially targeted anti-cancer drugs" therapeutic agent that induces selective apoptosis of tumor cells and not their healthy counterparts. In addition, it also induces apoptosis of growing endothelial cells and not of those quiescent. Therefore it can be said that it presents dual anti-cancer activity and antiangiogenic, which positions him as a potent chemotherapeutic agent. It has been shown that the biological activity of alpha-tocopherol succinate is enhanced when it is conjugated with a hydrophilic polymer such as polyethylene glycol (TPGS) ("Vitamin E Analogs, a Novel Group of" Mitocans, "as Anticancer Agents: The Importance of Being Redox-Silent "Jiri Neuzil et al. Molecular Pharmacology 2007 vol. 71 no. 5 pp 1 185-1 199 DOI: 10.1 124 / mol.106.030122). This compound has been widely used as a surfactant in the stabilization of nanoparticles, but there are no formulations for proper medical administration. In recent years there have been some publications stably incorporating it into liposomes (Koudelka S, Masek J, Neuzil J, Turanek J (2010) Lyophilised liposome-based formulations of alpha-tocopheryl succinate: Preparation and physico-chemical characterization J Pharm Sci 99, 2434-2443), but its biological activity has not been demonstrated. Also, it has been incorporated into the formulation of nanoparticles based on degradable polyesters. However, all of them incorporated a single molecule of D-tocopherol per macromolecule.

Alpha-tocopherol succinate is highly hydrophobic, has low solubility in physiological media and is therefore difficult to administer. Therefore, the research interest in this field of application has focused on improving the solubility of hydrophobic drugs in physiological medium, by incorporating them into amphiphilic macromolecules that are capable of self-organization resulting in stable particles with a hydrophobic core and a cover hydrophilic The nature of the particles will also allow incorporating alpha-tocopherol into the particle, encapsulating other hydrophobic drugs.

Patent application US 201 1/0129540 A1 describes the modification of acrylic polymers to anchor alpha-tocopherol molecules by means of a spacer based on amino acids or peptides. It also describes the use of these systems as carriers of other hydrophobic drugs. But nevertheless, On the one hand, it does not detract from the possible anti-tumor activity of the "per se" systems and on the other, the grafting of alpha-tocopherol molecules in the system does not exceed 30% of the total monomers. In addition, the modification of the polymers is carried out in a random and uncontrolled manner, without taking into account the microstructure of the macromolecules formed.

The following scientific articles 1) "Hydrophilic polymers dehved from vitamin E" B. Vázquez et al. J Biomater Appl (2000) 15 (2) 1 18-139, M.A. 2) "Resorbable polyacrylic hydrogels derived from vitamin E and their application in the healing of tendons" Plasencia et al. J Mater Sci: Mater Med (1999) 10 (10- 1 1) 641-648 and 3) "Hydrophilic acrylic biomaterials derived from vitamin E with antioxidant properties "C. Ortiz et al J Biomed Mater Res (1999) 45 (3) 184-191 describe acrylic polymeric derivatives carrying alpha-tocopherol of up to 20% of the total monomers, where alpha-tocopherol It is directly linked to the acrylic group of the polymer derivative. The application of these systems is oriented in relation to the antioxidant capacity associated with the incorporated alpha-tocopherol.

Therefore, it is of interest to find antitumor agents of high activity and specificity that are "per se" soluble in physiological media.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of a family of amphiphilic copolymers that form polymer micelles of micrometer or nanometer size and that are constituted by acrylic monomers derived from the alpha-tocopherol molecule and highly hydrophilic monomers. This family of copolymers have a dual activity (anticancer and antiangiogenic) "per se", but they can also serve as vehicles for other active ingredients with antitumor effect. "Copolymer" in the present invention is understood as a macromolecule composed of two or more different repetitive units, called monomers, which can be joined in different ways by means of chemical bonds. The monomers that form the copolymer can be distributed randomly or periodically.

The term "amphiphilic copolymer" refers to copolymers formed by lipophilic blocks linked with hydrophilic blocks.

In the present invention the term vitamin E refers to a family comprising alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol and delta-tocotrienol. Of these, alpha-tocopherol is the form that remains active in the body, making it the preferred form of vitamin E. It acts as a fat-soluble antioxidant that prevents lipid peroxidation of polyunsaturated fatty acids in cell membranes. Its formula is as follows:

α-TOCOPHEROL (VITAMIN E) The compounds of the present invention have a dual proapoptotic activity of growing (antiangiogenic) and anticancer endothelial cells (decreases the cell viability of cancer cells without affecting their healthy homologs) provided by the alpha molecule. tocopherol, as shown in example 1 and 2 below. The synergy between both anti-tumor capabilities is an additional advantage at the time of using them in anticancer treatments compared to the compounds currently used.

In the present invention the alpha-tocopherol molecule is linked to an acrylic moiety by an enzymatically hydrolysable separator segment, R ₂ , which prevents its accumulation in the organism, since it gives rise to products totally soluble in the physiological environment. The advantage of these alpha-tocopherol-carrying acrylic monomers is, therefore, their solubility in physiological medium, which allows them to be eliminated from the organism using the most normal metabolic pathway, being commonly eliminated by filtration through the kidney.

The compounds of the present invention have the ability to form micelles of micrometer and micrometer size, with hydrophobic core and hydrophilic cortex. They have an advantage to highlight, the size and morphology of the micelles can be modulated by controlling the molar composition and the concentration of these copolymers. Micelles of sizes between 5 and 300 nm are easily administered by injection, in the area where they are required.

The compounds of the present invention can be prepared in a wide range of molar compositions, all of which induce the death of growing endothelial cells (antiangiogenic capacity) at high concentrations and, some, also do so at relatively low concentrations. This last fact gives it an advantage over the compounds currently used since pharmaceutically the use of any medication at low concentrations is always beneficial.

In a first aspect, the present invention relates to the use of a polymeric compound of formula (I):

(i) where Ri is selected from hydrogen and linear or branched d-Cs alkyl,

R ₂ is vitamin E or -X-vitamin E, where X is the group - [0- (CH ₂ ) _to -R3-

where a and b have a value independently selected from 1 to 6,

c has a value selected from 1 to 6,

R ₃ and R ₄ are independently selected from C = 0, 0-C = 0, NH-C = 0, NH, -S-, SC = 0 and Si (R ₂ ) 0,

G is a hydrophilic monomer; Y

(m + n) is equal to 1,

where ^* is understood as the repetition of the myn monomers, or their pharmaceutically acceptable isomers or salts, for the manufacture of a pharmaceutical composition for the treatment of lung, breast, liver, colon, skin and other cancers in which The mitochondrial activity of cancer cells is altered.

Preferably, a and b have a value independently selected from 2 to 6.

The term "alkyl" refers in the present invention to aliphatic, linear or branched chains, having 1 to 8 carbon atoms, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, tert- butyl, sec-butyl, n-pentyl, etc. Preferably the alkyl group has between 1 and 4 carbon atoms. The alkyl radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido, alkoxide, thiol , amino, acylamino, cyano, carboxylate, carboxamide, carboxy ester, aryl or heteroaryl or combinations of these groups. When the alkyl group is substituted, it is preferably substituted by one or more amine, amide or ether groups, which in turn may or may not be substituted by alkyl, amide, cycloalkyl or ethers groups and these, in turn, may also be substituted or no.

The compounds of the present invention represented by formula (I) may include isomers, depending on the presence of multiple bonds (eg, Z, E), including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention. The individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques.

The term "pharmaceutically acceptable salts" indicates a formulation of a compound that does not cause significant irritation in an organism and does not impair the biological activity or properties of the compound. Pharmaceutical salts can be obtained by reacting a compound of the invention with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-acid. -toluenesulfonic acid, salicylic acid and the like.

Unless otherwise indicated, the compounds of the invention also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having said structure, except for the substitution of a hydrogen for a deuterium or for Tritium, or the substitution of a carbon for a carbon enriched in ¹³ C or ¹ C or a nitrogen enriched in ¹⁵ N, is within the scope of this invention.

In a preferred embodiment, vitamin E is alpha-tocopherol.

In one preferred embodiment G is selected from the following group N- vinylpyrrolidone, 1-vinylimidazole and / V, / Vd¡met¡lacr¡lam¡da, / V-isopropylacrylamide, 2- hydroxyethylmethacrylate, 2-hydroxyethyl acrylate, polyethylene glycol methacrylate), polyethylene glycol acrylate), 2-hydroxypropyl methacrylate, N-ethylmorpholine methacrylate, acrylate / V-ethylmorpholine, methacrylate / V-hydroxyethyl pyrrolidone, / V-hydroxyethyl pyrrolidone acrylate and 2-vinylpyridine.

In one preferred embodiment a has a value of 2. In a preferred embodiment b has a value of 2.

In a preferred embodiment R ₃ is 0-C = 0.

In a preferred embodiment R ₄ is C = 0.

In a preferred embodiment, c is 1.

For their application in therapy, the compounds of formula (I), will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels. The purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and still more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I) or its pharmaceutically acceptable isomers or salts. In a more preferred embodiment, the present invention relates to the polymeric compound of formula (

a-tocopherol

(II) where (m + n) is equal to 1 and ^* is understood as the repetition of monomers m and n.

In the present invention the compound of formula (II) is also called poly (VP-co-MTOS). The alpha-tocopherol molecule binds through the hydroxyl group of the bicycles, so, in the polymeric compound of formula (II), the hydrogen of this hydroxyl group has disappeared and an ester group has formed.

In another preferred embodiment, the present invention relates to the polymeric compound of formula (III),

where (m + n) is equal to 1 and ^* is understood as the repetition of monomers m and n.

In the present invention the compound of formula (III) is also called poly (VP-co-MVE). The alpha-tocopherol molecule binds through the hydroxyl group of the bicycles, so, in the polymeric compound of formula (III), the hydrogen of this hydroxyl group has disappeared and an ester group has formed.

In a preferred embodiment, m has a value between 0.05 to 0.80.

In a more preferred embodiment, m has a value between 0.1 to 0.2.

In a preferred embodiment, n has a value between 0.20 to 0.95.

In a more preferred embodiment, n has a value between 0.8 to 0.9.

In a second aspect, the present invention relates to the polymeric compound of formula (IV)

or its pharmaceutically acceptable isomers or salts,. where G is described above and (m + n) is equal to 1 and ^* is understood as the repetition of monomers m and n.

In a preferred embodiment, the polymeric compound of formula (IV) is the polymeric compound of formula (II).

(II) In a preferred embodiment, m has a value between 0.05 to 0.80. In a more preferred embodiment, m has a value between 0.1 to 0.2.

In a preferred embodiment, n has a value between 0.20 to 0.95.

In a more preferred embodiment, n has a value between 0.8 to 0.9. In a third aspect, the present invention relates to the pharmaceutical composition comprising the compound of formula (IV) defined above and more preferably the compound of formula (II).

In a preferred embodiment, this composition further comprises a pharmaceutical vehicle.

The pharmaceutical vehicles that can be used in said compositions are those known to those skilled in matena and commonly used in the elaboration of therapeutic compositions.

In another preferred embodiment, the pharmaceutical composition comprises another active ingredient and more preferably an active ingredient with antitumor effect. By "active ingredient" is meant in the present invention a compound that contains at least one high purity chemical substance used in the prevention of a disease, or to prevent the occurrence of an unwanted physiological process. In a more preferred embodiment, the pharmaceutical composition is in an amount between 0.010 mg / ml and 1.25 mg / ml. In a fourth aspect, the invention relates to the use of the polymeric compound of formula (IV), more preferably the compound of formula (II) for the manufacture of a pharmaceutical composition. The fifth aspect of the present invention relates to the process for obtaining a polymeric compound of formula (IV) comprising the following steps: a) Mixture of alpha-tocopherol with mono-2- (methacryloxy) ethyl succinate in the presence of a catalyst and a reaction activator. b) Mixture of the monomer obtained in step (a) with a hydrophilic monomer, in the presence of a reaction initiator. In a preferred embodiment, the catalyst of step (a) is an amine.

In a preferred embodiment, the activator of step (a) is a measure.

In a preferred embodiment, the reaction time of step (a) has a duration between 1 and 72 hours.

In a preferred embodiment, step (a) is performed at a temperature of 15 ° C to 60 ° C. In a preferred embodiment, the hydrophilic monomer of step (b) is selected from the following group: / V-vinyl pyrrolidone, 1-vinylimidazole and N, N-dimethylacrylamide,

/ V-isopropylacrylamide, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, polyethylene glycol methacrylate), polyethylene glycol acrylate), 2-hydroxypropyl methacrylate, / V-ethylmorpholine methacrylate, acrylate / V-ethyl acrylate, v-ethyl acrylate of / V-hydroxyethyl pyrrolidone and 2-vinylpyridine. In a more preferred embodiment, the hydrophilic monomer of step (b) is N-vinyl pyrrolidone, the compound of formula (II) being obtained.

In a preferred embodiment, the initiator of step (b) is a radical initiator.

In another preferred embodiment, the concentration of mono-2- (methacryloxy) ethyl succinate employed in (a) is between 0.01 and 20 equivalents.

In another preferred embodiment, the catalyst concentration employed in (a) is between 0.01 and 1 equivalent.

In another preferred embodiment, the concentration of reaction activator employed in (a) is between 0.9 and 1.6 equivalents. In another preferred embodiment, the concentration of monomer used in step (b) is between 0.01 and 10 M.

In another preferred embodiment, the concentration of initiator employed in step (b) is between 0.001 and 0.1 M.

The sixth aspect of the invention relates to a micellar particle comprising the polymeric compound of formula (I).

The term "micellar particle" in the present invention refers to that polymeric micelle whose size can be micrometric or nanometric.

In a preferred embodiment the micellar particle is formed by the polymeric compound of formula (III). In a preferred embodiment the micellar particle is formed by the polymeric compound of formula (IV). In a preferred embodiment the micellar particle is formed by the polymeric compound of formula (II).

In another preferred embodiment, the micellar particle formed by the polymeric compound of formula (I) comprises an active ingredient with antitumor effect.

These micellar particles incorporate the active ingredient by encapsulation in the metric size micelles.

In a more preferred embodiment, the active ingredient encapsulated in the micellar io particle is selected from the group comprising cyclosporine, colchicine, mitomycin C, mycophenolic acid, rapamycin, everolimus, tacrolimus, paclitaxel, QP-2, actinomycin, estradiols, dexamethasone, metharexate , cilostazol, prednisone, doxorubicin, ranpirnas, troglitazone, valsartan, pemirolast, C-MYC antisense, angiopeptin, vincristine, PCNA ribozyme, 2-15 chloro-deoxyadenosine, mTOR-directed compounds and fludarabine.

In a preferred embodiment, the micellar particle described above also comprises a biomolecule.

In a seventh aspect, the invention relates to the use of the micellar particle described above for the manufacture of a pharmaceutical composition.

In a more preferred embodiment, the use of the micellar particle refers to a pharmaceutical composition for the treatment of lung, breast, liver, colon, skin and other types of cancer in which the mitochondrial activity of the cancer cells is altered

Another aspect of the present invention relates to the use of the micellar particle as a pharmaceutical vehicle.

30

The eighth aspect of the present invention relates to the method of obtaining the micellar particle where any of the compounds Polymers of formula (I) described above are dissolved in an organic solvent and mixed with aqueous medium.

In a preferred embodiment, the organic solvent is miscible in water.

In a more preferred embodiment the water miscible organic solvent is selected from the group comprising dioxane, tetrahydrofuran and dimethylformamide. In a preferred embodiment, the organic solvent is in a concentration of between 2 and 20 mg / ml.

In another preferred embodiment, the aqueous medium is in a concentration of between 0.02 and 3 mg / ml.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Shows the synthesis scheme of methacrylic monomer derived from the alpha-tocopherol molecule (MTOS).

FIG. 2. Shows the ¹ H-NMR spectrum (400 MHz, CDCI ₃ ) of the methacrylic monomer derived from the alpha-tocopherol molecule (MTOS) FIG. 3. Shows the ¹ H-NMR spectrum (400 MHz, CDCI ₃ ) of the poly (VP-co-MTOS) copolymer (95: 5)

FIG. 4. Shows the ¹ H-NMR spectra (400 MHz, 1,4-dioxane-d ₈ ) of the evolution of the vinyl protons during the copolymerization reaction of the poly (VP-co-MTOS) (70:30). The reaction was carried out in the resonance tube under the same reaction conditions as described in example 1, but using deuterated dioxane as solvent.

FIG. 5. It shows the variation of the particle size according to the composition of the copolymer (poly (VP-co-MTOS) 95: 5, 70:30 and 50:50) and its concentration during the nanoprecipitation process.

FIG. 6. Shows the scanning electron microscopy (SEM) (a) and atomic force microscopy (AFM) (b) images of nanoparticles obtained with methacrylic copolymers derived from the alpha-tocopherol and N-vinylpyrrolidone (VP) molecule called poly (VP-co-MTOS).

FIG. 7. Shows the confocal microscopy images of HPMEC-ST1 human microvascular endothelial cells exposed for 5 hours and a half to poly (VP-co-MVE) particles (80:20) that encapsulated coumarin-6 inside.

FIG. 8. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 80% confluence exposed to nanoparticles obtained with methacrylic copolymers derived from the alpha-tocopherol molecule (poly (VP-co-MTOS)) of Molar compositions 95: 5, 90: 10, 85:15, 80:20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 9. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 80% confluence exposed to nanoparticles obtained with methacrylic copolymers derived from the alpha-tocopherol molecule (poly (VP-co-MVE)) of molar compositions 95: 5, 90: 10 and 85: 15. MTT assay, the mean of cell viability is represented relative ± standard deviation (n = 8; ^* : p <0.05).

FIG. 10. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 50% confluence exposed to poly (VP-co-MTOS) nanoparticles with 95: 5, 90: 10, 85:15 molar compositions , 80:20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. eleven . It shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 100% confluence exposed to poly (VP-co-MTOS) nanoparticles with 95: 5, 90: 10, 85:15, 80 molar compositions : 20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 12. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 50% confluence exposed to poly (VP-co-MVE) nanoparticles with 95: 5, 90: 10 and 85: 15 molar compositions MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 13. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 100% confluence exposed to poly (VP-co-MVE) nanoparticles with 95: 5, 90: 10 and 85: 15 molar compositions MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented. FIG. 14. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 100% and 50% confluence exposed to polyparticles nanoparticles (VP-co-MTOS) 90: 10 and 80:20. Test MTT, at each point the mean is represented, the 95% confidence interval for the mean (n = 8), and the results obtained in the Tukey HSD test ( ^* : p <0.05) performed to compare the relative cell viability values measured between the cultures of the two cell lines maintained with the same concentration of particles from each of the systems included in the figure.

FIG. 15. Shows the results of the viability test of HPMEC-ST1 human microvascular endothelial cells at 100% and 50% confluence exposed to poly (VP-co-MVE) nanoparticles 90: 10 and 85: 15. MTT test, in each point represents the mean, the 95% confidence interval for the mean (n = 8), and the results obtained in the Tukey HSD test ( ^* : p <0.05) performed to compare the feasibility values relative cell measured between the cultures of the two cell lines maintained with the same concentration of particles of each of the systems included in the figure.

FIG. 16. Shows the results of the viability test of human breast adenocarcinoma cells MCF-7 exposed to poly (VP-co-MTOS) nanoparticles 95: 5, 90:10, 85:15, 80:20 and 70:30 . MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 17. Shows the results of the viability test of human HMEpC breast epithelial cells exposed to poly (VP-co-MTOS) nanoparticles 95: 5, 90:10, 85:15, 80:20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 18. Shows the results of the viability test of human breast adenocarcinoma MCF-7 exposed to nanoparticles (poly (VP-co-MVE)) of molar compositions 95: 5, 90:10 and 85: 15. MTT assay, se represents the mean relative cell viability ± standard deviation (n = 8; ^* : p <0.05). FIG. 19. Shows the results of the viability test of human HMEpC breast epithelial cells exposed to nanoparticles (poly (VP-co-MVE)) of molar compositions 95: 5, 90:10 and 85:15. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 20. Shows the results of the viability test of human breast adenocarcinoma MCF-7 exposed to freshly prepared nanoparticles (poly (VP-co-MVE)) of 95: 5, 90: 10 and 85:15 molar compositions. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 21. It shows the results of the viability test of human breast adenocarcinoma cells MCF-7 exposed to poly (VP-co-MVE) nanoparticles 95: 5 and 90: 10 with different storage periods prior to use. MTT test, at each point the mean and 95% confidence interval for the mean are represented (n = 8).

FIG. 22 a and b. It shows the results of the viability test of human breast epithelial cells (HMEpC) and in breast adenocarcinoma cultures (MCF-7) exposed to poly (VP-co-MTOS) 90:10 and 70:30 nanoparticles, and poly (VP-co-MVE) 90: 10 and 85: 15. MTT test, at each point the mean is represented, the 95% confidence interval for the mean (n = 8), and the results obtained in the test Tukey HSD ( ^* : p <0.05) performed to compare the relative cell viability values measured between the cultures of the two cell lines maintained with the same particle concentration of each of the systems included in the figure.

FIG. 23. Shows the results of the viability test of human adenocarcinoma cells of the WiDr colon exposed to poly (VP-co-MTOS) nanoparticles 95: 5, 90:10, 85:15, 80:20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented. FIG. 24. Shows the results of the viability test of human adenocarcinoma of the WiDr colon exposed to the nanoparticles (poly (VP-co-MVE)) of molar compositions 95: 5, 90:10 and 85: 15. MTT assay, the mean relative cell viability ± standard deviation (n = 8; ^* : p <0.05).

FIG. 25. Shows the results of the viability test of human adenocarcinoma cells of the WiDr colon exposed to 90: 10 poly (VP-co-MTOS) nanoparticles loaded with -tocopherol succinate (10, 5 and 1%). MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 26. It shows the results of the viability test of human adenocarcinoma cells of the WiDr colon exposed to 85: 15 poly (VP-co-MVE) nanoparticles loaded with -tocopherol succinate (10, 5 and 1%). MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 27. It shows the results of the feasibility test of FaDu human larynx adenocarcinoma cells exposed to poly (VP-co-MTOS) nanoparticles 95: 5, 90:10, 85:15, 80:20 and 70:30. MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented. FIG. 28. Shows the results of the FeDu human larynx adenocarcinoma viability test exposed to nanoparticles (poly (VP-co-MVE)) of 95: 5, 90:10 and 85: 15 molar compositions. MTT Assay, the mean relative cell viability ± standard deviation (n = 8; ^* : p <0.05). FIG. 29. It shows the results of the viability test of FaDu human larynx adenocarcinoma cells exposed to 90: 10 poly (VP-co-MTOS) nanoparticles loaded with -tocopherol succinate (10, 5 and 1%). MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

FIG. 30. It shows the results of the feasibility test of FaDu human larynx adenocarcinoma cells exposed to 85: 15 poly (VP-co-MVE) nanoparticles loaded with -tocopherol succinate (10, 5 and 1%). MTT assay, the mean of the relative cell viability ± standard deviation (n = 8; ^* : p <0.05) is represented.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the use as an anticancer drug of the poly (VP-co-MTOS) copolymer belonging to the family referred to in the present invention.

First, the synthesis of the monomer derived from the alpha-tocopherol molecule through which the alpha-tocopherol molecule is attached to an acrylic moiety of the monomer by an enzymatically hydrolyzable separator element is described.

The synthesis of poly (VP-co-MTOS) copolymers from the acrylic monomer carrying the alpha-tocopherol and vinyl pyrrolidone molecule as a highly hydrophilic monomer is described below. The molar composition of the copolymers formed and their reactivity are checked, to confirm their ability to produce micelle-like polymeric self-assembled structures. The method of preparing micella particles is described below and its size and morphology are determined, which vary according to the composition. molar copolymers of poly (VP-co-MTOS) and their concentration during the nanoprecipitation process.

Finally, feasibility tests are carried out, where the low toxicity of poly (VP-co-MTOS) nanoparticles is demonstrated throughout the compositional gradient of the prepared copolymers. In addition, all the compositions tested induce the death of growing endothelial cells at high concentrations and, some, also do so at relatively low concentrations. In addition, the feasibility results obtained with MCF-7 breast adenocarcinoma cultures and human HMEpC breast epithelial cells are compared. This test demonstrates that the compounds described here significantly decrease the cell viability of cancer cells without practically affecting healthy ones in the concentrations tested.

Example 1

Synthesis of a methacrylic monomer derived from the alpha-tocopherol molecule (MTOS)

The monomer was prepared by reacting the alpha-tocopherol molecule with mono-2- (methacryloxy) ethyl succinate (MES) in the presence of dimethylaminopyridine (DMAP) as a catalyst and dicyclohexyl carbodiimide (DCC) as a reaction activator. Dichloromethane was used as solvent. The reaction scheme is shown in Figure 1.

In a round bottom flask, the alpha-tocopherol molecule (1 equivalent), the MES (1.3 equivalents) and the DMAP (0.1 equivalent) were introduced in 100 ml of dichloromethane. DCC (1.4 equivalents) was added slowly, drop by drop, with constant stirring at room temperature. The reaction mixture was maintained for 24 hours under the same conditions. After 24 hours, the reaction mixture was filtered to remove solid by-products. The DMAP and the unreacted alpha-tocopherol molecule were removed by successive extractions with solutions of NaOH and 1 N HCI and subsequently the solvent was removed under reduced pressure. The resulting medium was redissolved in hexane and washed 3 times with the same NaOH and HCI solutions used previously in order to improve the purification efficiency. The overall reaction yield was between 85 and 90%. Figure 2 shows the MTOS spectrum with the assignment of the resonance signals that verify the correct synthesis of the monomer. The signals corresponding to the vinyl protons and the protons of the group (CH ₃ ) of the acrylic group can be clearly observed in the spectrum. Synthesis of the copolymer carrying the alpha-tocopherol poly molecule (VP-co-MTOS)

Copolymers were prepared by reacting the obtained MTOS monomer and N-vinyl-2-pyrrolidone (VP) as hydrophilic monomer from compositions in the VP: MTOS feed (% -molar) of 95: 5, 90:10, 85: 15, 80:20, 70:30, 50:50, 30:70 and 20:80.

All polymers were prepared by radical polymerization at high conversion. The reaction was carried out by dissolving the monomers in dioxane (0.25 M) using 2,2'-Azobisisobutyronitrile (AIBN) as initiator (1.5x10 ^"2 M). The prepared solution was deoxygenated by a stream of N ₂ (g) for 30 minutes at room temperature The reaction mixture was kept at 60 ° C inside an oven for 24 hours Finally, the product obtained was purified by dialysis and lyophilized so that an amorphous powder was obtained white with yields shown in Table 1. The molar composition of the copolymers prepared was calculated from their corresponding ¹ H-NMR spectra. Figure 3 shows as an example the spectrum of the copolymer prepared from molar fractions in the VP: MTOS feed of 95: 5. The disappearance of the 5 characteristic signals of the vinyl protons is observed. In addition, the widening of the signals as a consequence of the polymerization and with it, of the macromolecular nature of the synthesized polymers is appreciated.

For the calculation of the molar compositions of the copolymers, the values of the normalized integrals of the characteristic signals of each monomer are considered. Specifically, the signals that appear in the range of 3-5 ppm corresponding to 3 protons of the VP and 4 protons of the MTOS (CH ₂ -13 and CH ₂ -14) and the signal at 0.86 ppm are taken into account corresponding to 12 protons of MTOS (CH ₃ -4a ', CH ₃ -8a', CH ₃ -12a 'and 15 CH ₃ -13'). Table 1 summarizes the MTOS composition values of the copolymers prepared together with the yields obtained in each case.

Table 1 . Molar composition of MTOS in the feed (FMTOS) and in the different copolymers prepared (IT), yield of the copolymerization reactions, number average molecular weight (M _n ), weight (M _w ) and polydispersity index { Pdl).

FMTOS f TOS Rto (%) M _w -10 ³ M „-10 ³ Pdl

0 0 85 35.0 14.2 2.2

5 7 69 55.6 30.5 1, 8

10 15 79 82, 1 46.8 1, 8

15 28 79 89.3 46.9 1, 9

20 34 79 1 12.8 58.8 1, 9

30 46 83 106.0 58.2 1, 8

50 63 73 124.2 64, 1 1, 9 70 82 75 123.8 48.0 2.5

80 90 63 164.3 58.8 2.8

100 100 60 262, 1 81, 5 3.2

Table 1 also includes the molecular weights average in weight (M _w ), in number (M _n ) and the polydispersity index (Pdl) of the prepared copolymers. These results are obtained by size exclusion chromatography (SEC). For this, a Perkin-Elmer chromatograph equipped with a Socratic LC-250 series pump was used, connected to a Series 200 refractive index detector. The samples were eluted using three columns connected in series of polystyrene-divinylbenzene series (Polymer Laboratories) pore size of 10 ³ , 10 ⁴ and 10 ⁵ A at 30 ° C. As eluent io tetrahydrofuran (THF) was used at a flow rate of 1 ml / min. For calibration, methyl polymethacrylate standards of molecular weight between 10,300 and 1,400,000 Da were used.

To know the microstructural organization of the polymer chains and the reactivity of each of the monomers, the reactivity ratios between the monomers were calculated by quantitative analysis of ¹ H-NMR in situ.

Copolymerizations of the different products were carried out in an NMR system using deuterated dioxane as a solvent at 60 ° C. The 20 reactions were carried out within the previous resonance tube deoxygenation with nitrogen and placing a capillary tube with dichlorobenzene inside which will serve as a reference signal. Different concentrations of comonomers were studied in order to cover the entire range of concentrations, the total concentration of monomers being 0.25 M.

25

A sequence of spectra at different times of the polymerization reaction obtained for the copolymer prepared with a VP: MTOS feed of 70:30 is shown in Figure 4. To calculate the instantaneous concentrations of the monomers, the signals Hi (6, 1 ppm) and Ai have been integrated (7, 1 ppm) assigned to the acrylic and vinyl protons of the VP and MTOS monomers respectively. The values of the reactivity ratios obtained are r _T os = 1, 2 and r _VP = 0.12. With this procedure, it is verified that the MTOS is almost 10 times more reactive than the VP and, therefore, the large difference in reactivity of the monomers used. This results in macromolecular chains with long sequences of both monomers and few intermediate composition chains. This compositional gradient along the macromolecular chains together with the different hydrophilicity of the monomers gives rise to polymeric self-assembled structures of the micellar type.

Preparation of nanoparticles from poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers

Nanoparticles were prepared from the poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers synthesized as described above by the nanoprecipitation method. For this, the copolymers (10mg / ml) were dissolved in a water-miscible organic solvent, in particular, dioxane. Then, this solution was added dropwise and with vigorous stirring, over the necessary amount of water to obtain a concentration of nanoparticles between 0.05 and 2.5 mg / ml.

For cell assays, nanoparticle solutions were prepared at a concentration of 2.5 mg / ml in PBS and the dioxane was removed by dialysis for 3 days. In addition, for fluorescence assays, the nanoparticles were loaded with coumarin-6 which was added in a concentration of 100 pg / ml in the dioxane solution. The nanoparticle size distribution was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS equipment (Malvern Instruments) equipped with a He-Ne laser at 633 nm and with an angle of 173 °. The measurements were carried out in polystyrene cuvettes at room temperature, after sonication and filtration through a 0.45 pm membrane.

Table 2 summarizes the sizes and polydispersities obtained for the nanoparticles prepared at a concentration of 0.5 mg / ml. In addition, Figure 5 compares the size of the particles as a function of the concentration of nanoparticles for three copolymers of different molar composition in the feed (VP: MTOS 95: 5, 70:30 and 50:50). The results obtained demonstrate that the size and morphology of the micelles can be modulated by controlling the molar composition of these copolymers and the concentration of nanoparticles in the medium.

Table 2. Hydrodynamic diameter and polydispersity (Pdl) of nanoparticles prepared at a concentration of 0.5 mg / ml from the family of

copolymers poly (VP-co-MTOS)

Hydrodynamic diameter

FMTOS ÍTOS Pdl

(nm)

0, 136 ±

5 7 109.5 ± 6.5

0.028

0.088 ±

10 15 1 13.4 ± 1.5

0.01 1

0.090 ±

15 28 124.2 ± 5.0

0.006

0.088 ±

20 34 137.3 ± 6.7

0.006

0.076 ±

30 46 142.0 ± 3.3

0.01 1

0.022 ±

50 63 201, 9 ± 1, 2

0.013 The morphology of the nanoparticles was observed by Scanning Electron Microscopy (SEM, Philips XL 30 ESEM) and Atomic Force Microscopy (AFM) using the intermittent contact mode (tapping). For this, a drop of the nanoparticle suspension was deposited at a 1: 50 dilution, which was allowed to dry for 24 hours on a 14 mm glass disk. Figure 6 includes the images obtained in such a way that it is verified that the particles are spherical and do not tend to aggregate.

Table 3 includes the results obtained from particle sizes and i or polydispersities for the nanoparticles prepared from the family of poly (VP-co-MVE) copolymers. It is observed that poly (VP-co-MVE) particles are comparatively larger and with values that are not significantly affected by changing the content of MVE in the copolymer. On the contrary, the particles formed from the poly (VP-co-MTOS) system 15 have a size that increases slightly with the increase in the amount of MTOS in the polymers.

Table 3. Hydrodynamic diameter and polydispersity (Pdl) of nanoparticles prepared at a concentration of 0.5 mg / ml from the family of

20 copolymers poly (VP-co-MVE)

Hydrodynamic diameter

FMVE flVlVE Pdl

(nm)

0.091 ±

5 8 142.5 ± 1, 1

0.009

0.068 ±

10 22 160.7 ± 1.5

0.003

0, 197 ±

15 39 142.9 ± 4.3

0.008

0.203 ±

20 65 148.6 ± 2.9

0.019 In vitro biocompatibility test of poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers. Study of its antiangiogenic activity in growing endothelial cells.

For the viability and induction assays of cell death of growing endothelial cells, human lung microvascular endothelial cells (HPMEC-ST1) were used. For this, cell cultures were prepared and maintained and multiplied at 37 ° C in an atmosphere with 5% CO2, using HEPES-modified M-199 culture medium, supplemented with 10% bovine fetal serum, 1% of a penicillin-streptomycin solution, 0.1% endothelial growth factor and 0.1% sodium heparin. First, it was found that the poly (VP-co-MTOS) and poly (VP-co-MVE) (1.25 mg / ml) particles are endocyted by the cells. For this, the particles of an HPMEC-ST1 culture with coumarin-6 are loaded in a solution of 100 mg / ml to be visualized by confocal fluorescence micrography. After 5 and a half hours of incubation it is observed that the particles accumulate around the nucleus, as shown in Figure 7. In addition, these results show that the nanoparticles are capable of effectively encapsulating biomolecules, in this case taking as a model molecule Coumarin-6. Therefore, the particles may serve as vehicles for other chemotherapeutic drugs of high toxicity and difficult administration due to their hydrophobic structure, such as paclitaxel, simvastatin or doxorubicin.

The feasibility test was carried out by an MTT test. This assay is based on the metabolic reduction of 3- (4,5-dimethylthiozol-2-lo) -2,5-diphenyltetrazole (MTT) bromide performed by the mitochondrial enzyme succinate dehydrogenase in a colored compound blue (formazan), allowing to determine the mitochondrial functionality of the treated cells. After a 24 h growth period in an atmosphere with 5% CO2, the culture medium was exchanged for 50 μΙ of the nanoparticle solutions that were kept in contact with the cells for 24 hours under the same conditions. After this period of time, the contents of the wells were removed and a solution of the MTT reagent was added which was allowed to act for 4 hours. Finally, the contents of the wells were emptied to add in each of them 100 μΙ of a DMSO solution and proceed to perform the optical density readings at 570 nm. The viability tests were carried out with 80% confluence cultures (growing cells) in order to determine if the compositions were toxic to this type of cells and affected mitochondrial activity in vitro. Poly (VP-co-MTOS) nanoparticles were prepared from poly (VP-co-MTOS) copolymers for compositions in the VP: MTOS (% -molar) feed of 95: 5, 90: 10, 85: 15, 80:20 and 70:30. On the other hand, poly (VP-co-MVE) nanoparticles were prepared from poly (VP-co-MVE) copolymers for compositions in the VP: MVE (% -molar) 95: 5 feed. 90:10 and 85: 15. To that end, in all cases, solutions of the nanoaggregates in PBS at a concentration of 2.5 mg / ml were used, from which dilutions of a gradually lower concentration were prepared.

The results compiled in Figures 8 and 9 show how the cell viability of endothelial cells at 80% confluence decreases at high concentrations of the nanoparticles tested. Only concentrations of poly (VP-co-MTOS) and poly (VP-co-MVE) nanoparticles greater than 0.078 mg / ml significantly affect cell viability. Since the MTT assay measures the activity of the mitochondrial enzyme succinate dehydrogenase, we can state that the particles significantly affect the mitochondrial activity of growing endothelial cells and induce their death at high concentrations, demonstrating their antiangiogenic action. In order to study the selectivity of the nanoparticles towards the growing endothelial cells, feasibility tests were also carried out with cultures of the same cells but at 50% (highly proliferative cells) and 100% (quiescent cells, at rest ) of confluence.

Figures 10, 1 1, 12 and 13 show the results of MTT tests obtained with HPMEC-ST1 cells at 50 and 100% confluence, for the polyparticles of poly (VP-co-MTOS) and poly (VP-co -MVE). For both polymer systems it is observed how the cell viability of highly proliferative endothelial cells (50% confluence, Figures 10 and 12) decreases significantly. Specifically, a 70% and 60% decrease in cell viability is obtained with the most active nanoparticles of both polymer systems at a concentration of 1, 25 and 0.625 mg / ml, respectively. However, the cell viability of the same resting endothelial cells (100% confluence, Figures 1 1 and 13) is only significantly affected for higher nanoparticle concentrations.

Figures 14 and 15 compare the results obtained with cultures of endothelial cells at 100 and 50% confluence for the most active nanoparticles (poly (VP-co-MTOS) 90: 10 and 80:20 and poly (VP-co- MVE) 90: 10 and 85: 15). It is observed how the decrease in cell viability for highly proliferative cultures is significant for concentrations of nanoparticles equal to or greater than 0.078 mg / ml. In addition, the selectivity towards growing cells is greater in the case of nanoparticles based on poly (VP-co-MVE) copolymers. These results allow us to conclude that nanoparticles selectively affect the viability of highly proliferative endothelial cells giving these systems antiangiogenic capacity.

In vitro biocompatibility test of poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers. Study of its anticancer activity in human breast adenocarcinoma cells MCF7 Trials were carried out with human breast adenocarcinoma tumor cells (MCF7 cell line; Health Protection Agency Culture Collections - HPAC-Cat. No. 86012803; Lot No. 10K025, pass 9+) in order to study the anti-cancer activity of the copolymers carrying alpha-tocopherol. The culture medium used in the maintenance of this cell line was DMEM (Dulbeco's modified Eagle's Medium) with glucose, sodium pyruvate and L-glutamine (SIGMA D6429), supplemented with penicillin / streptomycin (SIGMA P0781), 1% of a non-essential amino acid solution (SIGMA M7145) and 10% fetal bovine serum (SBF; GIBCO 10270-106). An MTT test was performed in order to determine the activity of the particles on these cancer cells, following the same working protocol as described above.

The results for poly (VP-co-MTOS) copolymers demonstrate that some compositions (eg 90: 10, 85: 15 and 80:20) significantly affect cell viability at relatively low concentrations (greater than 0.010 mg / ml), as seen in Figure 16. All the compositions tested significantly affect the cell viability of MCF-7 at high concentrations. Since the MTT assay measures the activity of the mitochondrial enzyme succinate dehydrogenase, we can state that poly (VP-co-MTOS) particles significantly affect the mitochondrial activity of these cells and induce their death at high concentrations, which demonstrates their action against this type of tumor cells. In addition, trials with human breast epithelial cells (HMEpC) were carried out. The culture medium used in the maintenance of this line was HuMEC Basal Serum Free Medium (GIBCO) supplemented with HuMEC supplemented kit (GIBCO), and 10% fetal bovine serum (SBF; GIBCO 10270-106). An MTT assay was performed in order to determine the activity of the particles on these healthy cells, following the same working protocol as described above. The results show that poly (VP-co-MTOS) copolymers (90: 10, 85: 15, 80:20 and 70:30) do not significantly affect the cell viability of HMEpC cells, as observed in the Figure 17. Since the MTT assay measures the activity of the mitochondrial enzyme succinate dehydrogenase, we can affirm that these particles do not significantly affect the mitochondrial activity of this type of healthy cells.

Figures 18 and 19 show the MTT test results obtained with MCF7 and HMEpC cells respectively, for poly (VP-co-MVE) copolymers, under the conditions described above. Figure 18 shows a dose-dependent decrease in cell viability of MCF-7 cancer cells (the higher the concentration of particles, the lower the cell viability). The activity of the copolymers is greater, the higher the MVE content. Finally, Figure 19 shows that the MVE carrier copolymers tested do not significantly affect the cellular viability of HMEpC in the range of compositions tested, except for the 90: 10 poly (VP-co-MVE) copolymer in concentration 1, 25 mg / ml whose viability is still greater than 80%. The antitumor activity of these systems is affected both by the mode and by the storage time prior to their use in biocompatibility tests. It is important to keep the nanoparticle solutions in PBS at a temperature between 4-8 ° C and sterilize them previously by filtration. To demonstrate the influence of the particle preservation process, the same biocompatibility tests were performed with MCF-7 cells using poly (VP-co-MVE) nanoparticles but, in this case, with a storage period short (less than a week). It should be noted that for the previous experiments the period of storage of the particles was greater (8 weeks). The results obtained in the MTT test for nanoparticles tested after a short storage period of poly (VP-co-MVE) are shown in Figure 20. In addition, a comparison of the cell viability obtained in MCF-7 cultures using nanoparticles with different storage periods is included in Figure 21. It is observed how the anticancer activity of the particles is significantly reduced when the particles are stored for long periods of time.

Figure 22 summarizes the results obtained for poly (VP-co-MTOS) and poly (VP-co-MVE) using cancer cell cultures (MCF-7) and their healthy homologs (HMEpC). It is observed how the particles affect the cell viability of breast adenocarcinoma cultures, while they do not do so in the case of human breast epithelial cells. Example 2

Preparation of nanoparticles loaded with -tocopherol succinate from poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers One of the major attractions of prepared nanoparticles is the possibility of combining and improving their antitumor activity " per se "with the encapsulation of active ingredients that are highly hydrophobic and therefore difficult to administer. In this way, polymeric nanoparticles act as a vehicle for these drugs with antitumor effect, allowing controlled dosing and improving their solubility in physiological medium.

Nanoparticles loaded with alpha-tocopherol succinate (10, 5 and 1% by weight) were prepared from the copolymers poly (VP-co-MTOS) (90: 10) and poly (VP-co-MVE) (85: 15) that have demonstrated greater anticancer activity in the different cell lines used. Again, the nanoprecipitation method was used so that the copolymers and succinate of alpha-tocopherol were dissolved in dioxane (10mg / ml). Then this solution is added dropwise and with vigorous stirring, on the necessary amount of PBS to obtain a final nanoparticle concentration of 2.5 mg / ml. Finally, dioxane was removed by dialysis for 3 days.

The size distribution of the nanoparticles was determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS equipment (Malvern Instruments) equipped with a He-Ne laser at 633 nm and at an angle of 173 °. The measurements were carried out in polystyrene cuvettes at room temperature. Table 4 summarizes the sizes and polydispersities obtained in each case.

Table 4. Hydrodynamic diameter and polydispersity. (Pdl) of the nanoparticles loaded with alpha-tocopherol succinate from the copolymers poly (VP-co-MTOS) 90: 10 and poly (VP-co-MVE) 85: 15.

In vitro biocompatibility test of poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers. Study of its anticancer activity in human colon adenocarcinoma cells.

Once the anticancer properties of nanoparticles based on alpha-tocopherol derivatives in breast adenocarcinoma cells were confirmed, in vitro biocompatibility tests were performed with these particles using other cell lines to determine if they are also active so that they could be used to treat different types of cancers. Particularly, viability tests with human colon adenocarcinoma tumor cells were carried out following the same working protocol described above (WiDr cell line; Health Protection Agency Culture Collections -HPAC-Cat. No.851 1 1501; pass 9+) to determine if the alpha-bearing copolymers also have an optimal anticancer activity towards this type of cells. The culture medium used in the maintenance of this cell line was DMEM (Dulbeco's modified Eagle's Medium) with a high glucose content, sodium pyruvate and supplemented 2 mM L-glutamine (SIGMA D6429), 1% penicillin / streptomycin (SIGMA P0781) , 1% of a solution of nonessential amino acids (SIGMA M7145) and 10% fetal bovine serum (SBF; GIBCO 10270-106). The results obtained from the MTT assay with WiDr cells exposed to poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers are summarized in Figures 23 and 24, respectively. Cell viability decreases significantly below 70% for higher nanoparticle concentrations (0.625 and 1.25 mg / ml).

In the case of poly (VP-co-MTOS) copolymers, the viability reduction reaches values up to 40% for the most active nanoparticles ((poly (VP-co-MTOS) 95: 5 and 85: 15). In contrast, a dose-dependent decrease in cell viability is observed for poly (VP-co-MVE) copolymers, specifically, poly (VP-co-MVE) nanoparticles (85: 15) with a higher concentration of Vitamin E methacrylate reduces cell viability by 50% with the highest concentration of nanoparticles of 1.25 mg / ml Therefore, it can be concluded that these polymeric nanoparticles induce the death of colon adenocarcinoma cells at high concentrations, significantly affecting their mitochondrial activity. In vitro biocompatibility test and study of the anticancer activity of the most active nanoparticles (poly (VP-co-MTOS) 90:10 and poly (VP-co-MVE) 85:15) loaded with alpha-tocopherol succinate in cells of human colon adenocarcinoma.

The "per se" anti-cancer activity of the poly (VP-co-MTOS) and poly (VP-co-MVE) polymer systems studied can be enhanced by encapsulating different chemotherapeutic drugs inside the polymeric nanoparticles that act as vehicles that release the drug in the localized area of the tumor. In this case, poly (VP-co-MTOS) (90: 10) and poly (VP-co-MVE) (85: 15) particles loaded with alpha-tocopherol succinate were prepared, as described in the example 2. To study the effect of these particles with alpha-tocopherol succinate, in vitro biocompatibility MTT assays were performed using WiDr colon adenocarcinoma cell cultures, which were maintained under the same conditions described above.

The results obtained from the MTT assay with WiDr cells exposed to nanoparticles loaded with alpha-tocopherol succinate are summarized in Figures 25 and 26. For both polymer systems, a dose-dependent decrease in cell viability is observed so that the higher the alpha-tocopherol succinate load in the particles, the lower the cell viability. In the case of poly (VP-co-MTOS) nanoparticles (Figure 25), cell viability is significantly reduced below 70% at high concentrations for particles with a 5% charge. This effect is observed even at lower concentrations (up to 0.078 mg / ml) for particles with a 10% charge. In this case, a viability reduction of up to 75% is observed at the highest particle concentration (1.25 mg / ml). These results are significantly better than those obtained with the same empty particles. Therefore, we can conclude that these particles They affect the cell viability of this cell line so that the loading of the particles with a bioactive drug such as alpha-tocopherol succinate enhances the anti-cancer activity of these systems, probably due to their controlled dosage to the environment where they exert their therapeutic activity.

The results of cell viability for poly (VP-co-MVE) charged particles (Figure 26) demonstrate that their anti-cancer activity towards WiDr cells is significant for high concentrations of the particles (0.625 and 1.25 mg / ml) with the highest succinate content of alpha-tocopherol. In this case, the charged particles also significantly affect the mitochondrial activity of the WiDr cells but no differences are observed in the behavior of the same particles without charge.

In vitro biocompatibility test of poly (VP-co-MTOS) and poly (VP-co-MVE) copolymers. Study of its anticancer activity in human larynx adenocarcinoma cells

In order to deepen the anti-cancer activity of alpha-tocopherol-bearing copolymers, tests were carried out with squamous cell carcinoma larynx cells (FaDu cell line; American Type Culture Collection-Cat. No. HTB-43; step 2 +) to determine whether nanoparticles affect the cell viability of FaDu cultures. The culture medium used in the maintenance of this cell line was DMEM (Dulbeco's modified Eagle's Medium) with high glucose content, pyridoxine chloride and modified with 25 mM HEPES buffer. Supplemented with 2 mM L-glutamine (SIGMA D6429), 1% penicillin / streptomycin (SIGMA P0781) and 10% fetal bovine serum (SBF; GIBCO 10270-106). Again, in vitro biocompatibility was quantified by an MTT assay, following the same working protocol described above.

The in vitro biocompatibility results for poly nanoparticles (VP-co-MTOS) demonstrate their low toxicity for this type of tumor cells, such and as shown in Figure 27. For none of the compositions and concentrations tested, a viability reduction is achieved below 70% so that, in this case, the particles do not significantly affect the mitochondrial activity of tumor cells of larynx.

Figure 28 shows the results obtained from the MTT assay with FaDu cell cultures exposed to poly (VP-co-MVE) nanoparticles. In this case, the copolymers are more active and a 40% reduction in cell viability is achieved for the concentration of nanoparticles (poly (VP-co-MVE) 90:10 and 85: 15) tested higher (1, 25 mg / ml)

In vitro biocompatibility test and anti-antigenic activity of the most active nanoparticles (poly (VP-co-MTOS) 90:10 and poly (VP-co-MVE) 85:15) loaded with alpha-tocopherol succinate in human adenocarcinoma cells of larynx

In vitro biocompatibility assays were performed on FaDu human larynx adenocarcinoma cells exposed to poly (VP-co-MTOS) (90: 10) and poly (VP-co-MVE) (85: 15) particles loaded with succinate of alpha-tocopherol. The objective is to determine if they have an optimal anticancer activity, improving the results obtained with the same particles without charge. The MTT assay and the maintenance of FaDu cultures were performed following the same protocols described above. The results compiled in Figures 29 and 30 show that the poly (VP-co-MTOS) (90: 10) and poly (VP-co-MVE) (85: 15) particles significantly reduce cell viability in a dose- dependent (the greater the load of the bioactive drug in the particles, the lower the viability) with the highest concentrations studied (0.625 and 1.25 mg / ml).

Poly (VP-co-MTOS) nanoparticles (90: 10) reduce cell viability up to 60 and 75% with an alpha-tocopherol succinate load of 5 and 10%, respectively. On the other hand, poly (VP-co-MVE) particles (85: 15) decrease cell viability by 50% with the maximum bioactive drug load at a concentration of 1.25 mg / ml. These results show that these particles significantly affect the mitochondrial activity of FaDu cells. In addition, the particle charge enhances its anticancer activity. Specifically, the same empty poly (VP-co-MTOS) (90: 10) particles did not significantly affect the viability of these cells.

Claims

1 .- Use of a polymeric compound of formula (I),

or its pharmaceutically acceptable isomers or salts, where Ri is selected from hydrogen and linear or branched d-Cs alkyl;

where a and b have a value independently selected from 1 to 6,

c has a value selected from 1 to 6,

R ₃ and R ₄ are independently selected from C = 0, 0-C = 0, NH-

C = 0, NH, -S-, SC = 0 and Si (R ₂ ) 0,

G is a hydrophilic monomer; Y

(m + n) is equal to 1,

where ^{* it} is understood as the repetition of myn monomers, for the manufacture of a pharmaceutical composition for the treatment of lung, breast, liver, colon, skin and other cancers in which the mitochondrial activity of cancer cells is altered 2 - Use according to claim 1 wherein vitamin E is alpha-tocopherol.

3. - Use according to any of claims 1 or 2 wherein G is selected from the following group: / V-vinylpyrrolidone, 1-vinylimidazole and / V, / V-dimethylacrylamide, / V-isopropylacrylamide, 2-hydroxyethylmethacrylate, 2-hydroxyethyl acrylate, polyethylene glycol methaclate), polyethylene glycol acrylate), 2-hydroxypropyl methacrylate, / V-ethylmorpholine methacrylate, acrylate / V-ethylmorpholine, methacrylate / V-hydroxyethyl pyrrolidone, / V-hydroxyethyl pyrrolid and pyrrolid acrylate.

4. - Use according to any of claims 1 to 3, where a is 2.

5. - Use according to any of claims 1 to 4, wherein R ₃ is 0-C = 0.

6. - Use according to any of claims 1 to 5 wherein b is 2.

7. - Use according to any of claims 1 to 6 wherein R ₄ is C = 0.

8. - Use according to any of claims 1 to 7, wherein c is 1.

9. - Use according to any of claims 1 to 3, wherein R ₂ is vitamin E.

10. Use according to claim 1, wherein said compound is the compound of formula (II):

where (m + n) is equal to 1 and ^* is understood as the repetition of the monomers m and n.

1 .- Use according to claim 1, wherein said compound is the compound of formula (III):

(III)

12. - Use according to any of the preceding claims wherein m has a value between 0.05 to 0.80.

13. - Use according to the preceding claim wherein m has a value between 0.1 and 0.2.

14. - Use according to any of the preceding claims wherein n has a value between 0.20 to 0.95. 15. Use according to the preceding claim wherein n has a value between 0.8 to 0.9.

- Polymeric compound of formula (IV),

a-tocopherol

(IV) or its pharmaceutically acceptable isomers or salts, wherein G is described in claims 1 or 3 and (m + n) is equal to 1 and ^* is understood as the repetition of monomers m and n. - Polymeric compound, according to the preceding claim, of formula (II),

(II)

18. - Compound according to any of the preceding claims 16 or 17, wherein m has a value between 0.05 to 0.80.

19. - Compound according to the preceding claim, wherein m has a value between 0.1 to 0.2.

20. - Compound according to any of claims 16 to 19, wherein n has a value between 0.20 to 0.95.

twenty-one . - Compound according to the preceding claim, wherein n has a value between 0.8 to 0.9.

22. - Pharmaceutical composition comprising the compound of formula (IV) described according to any of claims 16 to 21.

23. - Pharmaceutical composition according to the preceding claim, further comprising a pharmaceutical vehicle.

24. - Pharmaceutical composition according to any of claims 22 or

23, which also includes another active substance with antitumor effect.

25. - Pharmaceutical composition according to any of claims 22 a

24, wherein the compound of formula (IV) is in an amount between 0.010 mg / ml and 1.25 mg / ml.

26. - Use of the polymeric compound of formula (IV), according to any of claims 16 to 21, for the manufacture of a pharmaceutical composition.

27. - Method of obtaining a compound of formula (IV) according to any one of claims 16 to 21 comprising the following steps: a) Mixing the alpha-tocopherol with mono-2- (methacryloxy) ethyl succinate in the presence of a catalyst and a reaction activator. b) Mixture of the monomer obtained in step a) with a hydrophilic monomer, in the presence of a reaction initiator.

28. - Method according to claim 27, wherein the catalyst of step a) is an amine.

29. - Method according to any of claims 27 or 28, wherein the activator of step a) is a measure.

30. Method according to any of claims 27 to 29, wherein the reaction time of step a) has a duration between 1 and 72 hours.

31. - Method according to any of claims 27 to 30, wherein step a) is carried out at a temperature of 15 ° C to 60 ° C.

32. - Method according to any of claims 27 to 31 wherein the hydrophilic monomer of step b) is selected from the following group: N- vinyl pyrrolidone, 1-vinylimidazole and / V, / Vd¡met¡lacr¡lam¡da, / V-isopropylacrylamide, 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, polyethylene glycol methaclate), polyethylene glycol acrylate), 2-hydroxypropyl methacrylate, / V-ethylmorpholine methacrylate, acrylate / V-ethyl acrylate, p-hydroxyl methacrylate / V-hydroxyethyl pyrrolidone and 2-vinylpyridine.

33. - Process according to any of claims 27 to 31 wherein the hydrophilic monomer of step b) is / V-vinyl pyrrolidone.

34. - Method according to any of claims 27 to 33, wherein the initiator of step b) is a radical initiator.

35. - Method according to any one of claims 27 to 34 wherein the concentration of mono-2- (metachloilox¡) ethyl succinate employed in (a) is between 0.01 and 20 equivalents.

36. - Method according to any of claims 27 to 35, wherein the concentration of catalyst employed in (a) is between 0.01 and 1 equivalent.

37. - Method according to any of claims 27 to 36, wherein the concentration of the activator of the reaction employed in (a) is between 0.9 and 1.6 equivalent.

38. - Method according to any of claims 27 to 37, wherein the concentration of monomer used in step (b) is between 0.01 and 10 M.

39.- Method according to any of claims 27 to 38, wherein the concentration of initiator used in step (b) is between 0.001 and 0.1 M. 40.- Micellar particle comprising the compound of formula (I) described according to any of claims 1 to 25.

41. - Particle according to claim 40, wherein said compound is the compound of formula (III).

42. - Particle according to claim 40, wherein said compound is the compound of formula (IV).

43. - Particle according to the preceding claim, wherein said compound is the compound of formula (II)

44. - Particle according to any of claims 40 to 43 which further comprises an active ingredient with antitumor effect. 45.- Particle according to claim 44, wherein the active ingredient is selected from the group comprising cyclosporine, colchicine, mitomycin C, mycophenolic acid, rapamycin, everolimus, tacrolimus, paclitaxel, QP-2, actinomycin, estradiols, dexamethasone, metharexate, cilostazol, prednisone, doxorubicin, ranpirnas, troglitazone, valsartan, pemirolast, C-MYC antisense, angiopeptin, vincristine, PCNA ribozyme, 2-chloro-deoxyadenosine, compounds targeting mTOR and fludarabine.

46. - Use of the particle according to any of claims 40 to 45 for the manufacture of a pharmaceutical composition.

47. - Use of the particle according to any of claims 40 to 45 for the manufacture of a pharmaceutical composition for the treatment of cancer. of lung, breast, liver, colon, skin and other types of cancer in which the mitochondrial activity of cancer cells is altered.

48. - Use of the particle, according to any of claims 40 to 45, as a pharmaceutical vehicle.

49. - Procedure for obtaining the particle according to any of claims 40 to 45 wherein the polymeric compound of formula (I) is dissolved in an organic solvent and mixed with aqueous medium.

50. - Process according to claim 49, wherein the organic solvent is miscible in water.

51. - Process according to any of claims 49 or 50, wherein the water miscible organic solvent is selected from the group comprising dioxane, tetrahydrofuran and dimethylformamide.

52. - Method according to any of claims 49 to 51, wherein the organic solvent is in a concentration between 2 and 20 mg / ml.

53. - Method according to any of claims 49 to 52 wherein the aqueous medium is in a concentration between 0.02 and 3 mg / ml.