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    • 专利摘要:
    The present invention relates to acrylic hydrogels comprising a nitrogen base selected from cytosine, adenine, guanine, uracil and thymine, to methods for obtaining said hydrogels and to contact lenses prepared from said hydrogels. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2755415A1
    申请号:ES201931063
    申请日:2019-11-29
    公开日:2020-04-22
    发明作者:Garcia Angela Varela；Nine Angel Concheiro；Lorenzo Carmen Alvarez
    申请人:Universidade de Santiago de Compostela；
    IPC主号:
    专利说明:

    [0001]
    [0002] Hydrogels comprising a nitrogenous base
    [0003]
    [0004] Field of the Invention
    [0005]
    [0006] The present invention relates to acrylic hydrogels comprising a nitrogenous base selected from cytosine, adenine, guanine, uracil and thymine, to methods for obtaining said hydrogels, as well as to the medical, especially ophthalmic, use of said hydrogels.
    [0007]
    [0008] Background of the Invention
    [0009]
    [0010] The hydrophilic nature of hydrogels hinders the efficient loading of low-polar molecules, as are the vast majority of drugs and active substances.
    [0011]
    [0012] To solve this problem, different solutions have been studied. One of them is the incorporation of the drug or the active substance in a nano or micrometer-sized vehicle that is then incorporated into the hydrogel (as described for example in Y. Kapoor, A. Chauhan, Drug and surfactant transport in Cyclosporine A and Brij 98 laden p-HEMA hydrogels, J. Coll. Int. Sci. 322 (2008) 624-633). However, in most of these studies the drug load on the hydrogel remains relatively low.
    [0013]
    [0014] Thus, it is still necessary to develop hydrogels that are capable of capturing low-polar substances and releasing them in a controlled manner over time.
    [0015]
    [0016] Brief description of the invention
    [0017]
    [0018] The authors of the present invention have designed a new hydrogel with the capacity to incorporate molecules, including low polar molecules, in particular drugs and low polar active substances.
    [0019]
    [0020] The hydrogels of the present invention are designed in such a way that they present micro domains in which molecules are incorporated, more specifically, drugs and active substances can be incorporated, which can also form complexes with nitrogenous bases that are incorporated into the hydrogel.
    [0021] The hydrogels of the present invention are useful in the treatment of pathological or physiological states, in the elaboration of topical, transdermal or transmucosal release systems for molecules and in the preparation of cosmetics.
    [0022]
    [0023] Thus, in a first aspect, the present invention is directed to a hydrogel in the form of a three-dimensional network characterized in that it comprises cross-linked methacrylic and / or acrylic chains, where the chains comprise alkyl groups to which a nitrogenous base selected from cytosine is attached. , adenine, guanine, uracil and thymine.
    [0024] A second aspect of the invention is directed to a method for preparing a hydrogel as described in the first aspect of the invention, comprising the steps of:
    [0025] to. polymerizing a mixture comprising i) monofunctional methacrylic or acrylic monomers, or combinations thereof, ii) bifunctionalized methacrylic or acrylic monomers, and iii) methacrylic or acrylic monomers comprising an electrophile group, to form a three-dimensional network base framework; and
    [0026] b. react a nitrogenous base selected from cytosine, adenine, guanine, uracil and thymine, with the base framework.
    [0027]
    [0028] In another aspect, the present invention is directed to a hydrogel obtainable by a method as described in the second aspect of the invention.
    [0029]
    [0030] In another aspect, the present invention relates to a contact lens comprising a hydrogel as described in the first aspect of the invention. In another aspect, the present invention relates to a method for preparing said contact lens.
    [0031]
    [0032] In a further aspect, the present invention is directed to a hydrogel and a contact lens, as described in the foregoing aspects, for use in medicine, preferably ophthalmology. More specifically, said hydrogel or contact lens is used to treat or prevent dry eyes, cataracts and corneal ulcers.
    [0033]
    [0034] In yet another aspect, the present invention is directed to the use of the hydrogel in the manufacture of topical, transdermal or transmucosal delivery systems, and in cosmetics.
    [0035]
    [0036] Description of the figures
    [0037] Figure 1. Schematic representation of the reaction between the nitrogenous base and the glycidyl methacrylate group in the three-dimensional network of intermediate hydrogel.
    [0038]
    [0039] Figure 2. Amount of TA incorporated into the hydrogels after 48 hours of immersion of the same in a TA solution (0.01 mg / mL) at 25 ° C.
    [0040]
    [0041] Figure 3. Profiles of TA transfer in simulated tear fluid at 35 ° C from non-functionalized nitrogenous hydrogels (solid lines) and from functionalized hydrogels (dotted lines).
    [0042]
    [0043] Figure 4. Amount of TA permeated through the cornea and sclera when TA is given from an aqueous solution (white bars), or from non-functionalized hydrogels (black bars) and functionalized (gray bars).
    [0044]
    [0045] Figure 5. Amount of TA accumulated in the cornea and in the sclera when TA is transferred from an aqueous solution (white bars), or from non-functionalized hydrogels (black bars) and functionalized (gray bars).
    [0046]
    [0047] Detailed description of the invention
    [0048]
    [0049] In one aspect, the present invention is directed to a hydrogel in the form of a three-dimensional network characterized in that it comprises cross-linked methacrylic and / or acrylic chains, where the chains comprise alkyl groups to which a nitrogenous base selected from cytosine, adenine, is attached. guanine, uracil and thymine.
    [0050] In the present invention, a three-dimensional network is understood to be a polymeric framework that extends in all three dimensions of space. This three-dimensional network is formed when the polymer chains that constitute it are crosslinked or crosslinked by monomers that have the function of crosslinking agents (A. Yamauchi. Gels: Introduction. In: Y. Osada, K. Kajiwara. Gels Handbook, Volume 1 The Fundamentals, Academic Press, London, 2001, pages 4-12).
    [0051]
    [0052] In the present invention, by methacrylic or acrylic chain is meant a polymeric chain that is the result of the polymerization of methacrylic or acrylic monomers. In the present invention, "methacrylic and acrylic chain" is understood to mean a polymeric chain that is the result of the polymerization of methacrylic and acrylic monomers.
    [0053] By methacrylic or acrylic unit is meant each monomer unit that constitutes the polymer chain after the polymerization of methacrylic or acrylic monomers.
    [0054]
    [0055] Monofunctionalized methacrylic or acrylic units are understood to be methacrylic or acrylic units that are the result of the polymerization of monomers containing a single methacrylic or acrylic group. By monofunctionalized acrylic or methacrylic unit combinations is meant that one methacrylic monomer has been mixed with a different methacrylic monomer and / or with one or more acrylic monomers.
    [0056]
    [0057] Bifunctionalized methacrylic or acrylic units are understood to be methacrylic or acrylic units that are the result of the polymerization of monomers containing two or more methacrylic or acrylic groups. In the present invention, the bifunctionalized acrylic or methacrylic units have the function of crosslinking agents.
    [0058]
    [0059] Methacrylic or acrylic units comprising an electrophilic group are understood to be methacrylic or acrylic units that are the result of the polymerization of methacrylic or acrylic monomers that comprise an electrophilic group.
    [0060]
    [0061] By electrophile group is meant a carbon atom to which a good leaving group is attached and has the ability to react in a nucleophilic, bimolecular, or unimolecular substitution reaction. Thus, by a methacrylic or acrylic monomer comprising an electrophilic group is meant one having a carbon to which a good leaving group is attached. There are commercial examples of such monomers that are useful for the present invention, such as, for example, glycidyl methacrylate, glycidyl acrylate, 3-chloro-2-hydroxypropyl methacrylate. But they can also be prepared by simple reactions known to the person skilled in the art, for example, the chlorine group of 3-chloro-2-hydroxypropyl methacrylate can be transformed into a better leaving group such as tosylate, triflate, mesylate, phosphate, etc, and there is also a literature describing the preparation of these methacrylic or acrylic monomers that comprise an electrophilic group and that is known to a person skilled in the art.
    [0062]
    [0063] In a preferred embodiment, the methacrylic and / or acrylic chains of the hydrogels of the invention comprise or consist of monofunctional acrylic or methacrylic units, or combinations thereof; methacrylic or acrylic units bifunctionalized; and methacrylic or acrylic units comprising an electrophilic group. In one embodiment the chains do not comprise another type of unit.
    [0064]
    [0065] In a preferred embodiment, the hydrogels of the invention are characterized in that the proportion by weight of the bifunctionalized acrylic or methacrylic units is preferably between 0.1% and 10% with respect to the weight of the hydrogel. In a preferred embodiment, the weight ratio of the methacrylic or acrylic units comprising an electrophile group is preferably between 0.1% and 10% with respect to the weight of the hydrogel.
    [0066]
    [0067] The hydrogels of the present invention possess the ability to incorporate water in high proportions without dissolving. Furthermore, the hydrogels of the invention have high optical clarity, excellent biocompatibility, and physical and mechanical properties that make the hydrogels of the present invention useful as components of contact lenses, and in particular soft contact lenses.
    [0068]
    [0069] The hydrogels of the invention further have the ability to incorporate molecules, preferably molecules that have an affinity for the nitrogenous base that is present in the hydrogel. Furthermore, the hydrogels of the invention have the ability to control the transfer of said molecules, preferably the transfer to physiological media.
    [0070]
    [0071] In a preferred embodiment, the molecule with an affinity for the nitrogen base is an antioxidant. In a more preferred embodiment, the molecule with an affinity for the nitrogenous base is selected from trans-ferulic acid, edaravone, idebenone, N-acetylcysteine, a-Lipoic acid, flavonoids, isoflavones, rutoxides, silibinin, baicalein, quercetin, catechins, polyphenols, resveratrol, curcumin, vitamin A, vitamin C, vitamin E and coenzyme Q. In an even more preferred embodiment the antioxidant is trans-ferulic acid.
    [0072]
    [0073] In a preferred embodiment, the invention is directed to a hydrogel in the form of a three-dimensional network characterized in that it comprises cross-linked methacrylic chains, where the chains comprise alkyl groups to which cytosines are attached.
    [0074]
    [0075] In another preferred embodiment, the invention is directed to a hydrogel in the form of a three-dimensional network characterized in that it comprises cross-linked methacrylic chains, in where the chains comprise alkyl groups to which cytosines are attached, and the hydrogel further comprises an antioxidant molecule, preferably transferulic acid. In a more preferred embodiment, the methacrylic chains are formed by: i) monofunctionalized methacrylic units derived from the monofunctionalized monomers hydroxyethyl methacrylate and ethylene glycolphenyl methacrylate; ii) bifunctionalized methacrylic units from the bifunctionalized monomer ethylene glycol dimethacrylate; iii) methacrylic units with an alkyl group derived from the monomer with a glycidyl methacrylate electrophile group, where the alkyl group is covalently linked to a cytosine via an amino bond.
    [0076]
    [0077] In another more preferred embodiment, the methacrylic chains are formed by: i) monofunctionalized methacrylic units derived from the monofunctionalized hydroxyethyl methacrylate monomers in a weight ratio of between 60% and 99% with respect to the weight of the hydrogel, preferably between 70% and 99% , more preferably between 80% and 98%, and ethylene glycolphenyl ether methacrylate in a proportion by weight of between 0.1% and 10% with respect to the weight of the hydrogel; ii) bifunctionalized methacrylic units derived from the bifunctionalized monomer ethylene glycol dimethacrylate in a proportion by weight of between 0.1% and 10% with respect to the weight of the hydrogel; iii) methacrylic units with an alkyl group from the monomer with a glycidyl methacrylate electrophile group in a weight ratio of between 0.1% and 10% with respect to the weight of the hydrogel, where the alkyl group is covalently linked to a cytosine by means of a bond Not me.
    [0078]
    [0079] In another preferred embodiment, the molecule with an affinity for the nitrogen base present in the hydrogel is a pharmaceutical active ingredient. In a more preferred embodiment, the pharmaceutical active ingredient is capable of forming hydrogen bonds or pi pi stacking interactions with the nitrogenous base present in the hydrogel.
    [0080]
    [0081] In a further aspect, the invention is directed to a contact lens comprising the hydrogel of the invention as described in any of its embodiments.
    [0082]
    [0083] In one embodiment, the contact lens is a soft contact lens. In the context of the present invention, a soft contact lens is a contact lens that has a modulus of elasticity (i.e., Young's modulus) of less than 2.5 MPa.
    [0084]
    [0085] In one embodiment, the hydrogel or contact lens has a water content by weight with respect to the weight of the hydrogel or lens of 20% to 80%.
    [0086] In one embodiment, the hydrogel or contact lens further comprises additional active agents that serve to treat dry eye or any derived symptoms such as inflammation.
    [0087]
    [0088] In one embodiment the additional active agent is an anti-inflammatory agent. More particularly, the anti-inflammatory agent is selected from the group consisting of ibuprofen, ketoprofen, flurbiprofen, fenoprofen, naproxen, piroxicam, tenoxicam, isoxicam, meloxicam, indomethacin, aceclofenac, diclofenac, and a combination thereof.
    [0089]
    [0090] In another aspect the invention is directed to a process for the preparation of the hydrogels of the invention, which comprises the steps of:
    [0091]
    [0092] to. polymerizing a mixture comprising i) monofunctional methacrylic or acrylic monomers, or combinations thereof, ii) bifunctionalized methacrylic or acrylic monomers, and iii) methacrylic or acrylic monomers comprising an electrophile group, to form a three-dimensional network base framework; and
    [0093] b. react a nitrogenous base selected from cytosine, adenine, guanine, uracil and thymine, with the base framework.
    [0094]
    [0095] Through the polymerization of step a) methacrylic or acrylic chains, or acrylic and methacrylic chains are formed. Crosslinking of these chains as they grow can be accomplished in a number of ways, for example through the use of monomers or crosslinking agents that comprise more than one reactive group that reacts with different growing chains without terminating the polymerization. In a preferred embodiment, crosslinking is achieved through the use of bifunctionalized acrylic or methacrylic monomers in step a) In the context of the present invention the meaning of crosslinking and crosslinking is the same.
    [0096]
    [0097] In a preferred embodiment, the base framework of the network that is prepared in step a) is prepared by polymerizing monofunctional methacrylic or acrylic monomers, or combinations thereof, bifunctional methacrylic or acrylic monomers, and methacrylic or acrylic monomers comprising an electrophilic group . In this way, a base framework is obtained, comprising methacrylic and / or acrylic chains that comprise or consist of monofunctionalized acrylic or methacrylic units, or their combinations, bifunctionalized methacrylic or acrylic units, and methacrylic or acrylic units comprising an electrophilic group.
    [0098]
    [0099] The base framework that is prepared in step a) is itself a three-dimensional hydrogel.
    [0100] Monomers giving rise to methacrylic or acrylic units comprising an electrophile group are preferably glycidyl methacrylate, glycidyl acrylate, 3-chloro-2-hydroxypropyl methacrylate.
    [0101]
    [0102] The monomers that give rise to methacrylic or acrylic units that have a methacrylic or acrylic group in their structure (monofunctionalized) are preferably hydroxyethyl methacrylate, ethylene glycolphenyl ether methacrylate, 1- (tristrimethylsiloxysilylpropyl) -methacrylate, methylmethacrylate, N, N-dimethyl, N-dimethyl -diethylacrylamide, methacrylic acid, acrylic acid, aminopropyl methacrylate, cyclohexyl methacrylate, or fluorosiloxane acrylate.
    [0103]
    [0104] The monomers that give rise to methacrylic or acrylic units that have two methacrylic or acrylic groups in their structure (bifunctionalized) are preferably ethylene glycol dimethacrylate, 1,3-Butanediol diacrylate, 1,4-Butanediol diacrylate, 1,6-Hexanediol diacrylate, Ethylene glycol diacrylate, Fluorescein O, O'-diacrylate, Glycerol 1,3-diglycerolate diacrylate, Pentaerythritol diacrylate monostearate, 1,6-Hexanediol ethoxylate diacrylate, 3-Hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2-dimethylpropionate diacrylate , Bisphenol A ethoxylate diacrylate, Di (ethylene glycol) diacrylate, Neopentyl glycol diacrylate, Poly (ethylene glycol) diacrylate, Poly (propylene glycol) diacrylate, Propylene glycol glycol diacrylate, Tetra (ethylene glycol-diacrylate) 1,4-Butanediol dimethacrylate, 1,6-Hexanediol dimethacrylate, Bisphenol A dimethacrylate, Diurethane dimethacrylate, Ethylene glycol dimethacrylate, Fluorescein O, O'-dimethacrylate, Glycerol dimethacrylate, Bisphenol Ethoxyl Ato dimethacrylate, Bisphenol A glycerolate dimethacrylate, Di (ethylene glycol) dimethacrylate, Poly (ethylene glycol) dimethacrylate, Poly (propylene glycol) dimethacrylate, Tetraethylene glycol dimethacrylate, Tri (ethylene glycol) dimethyl acrylate, Tri (ethylene glycol dimethyl acrylate) -ethylene glycol dimethacrylate), Poly (methyl methacrylate-co-ethylene glycol dimethacrylate).
    [0105]
    [0106] In a preferred embodiment, the methacrylic monomers from step a) are a combination of the hydroxyethyl methacrylate and ethylene glycolphenyl ether methacrylate monomers. In a preferred embodiment, the mixture from step a) presents Ethylene glycol polyphenyl ether methacrylate in a concentration of between 50 and 1000 mM, more preferably between 100 and 800 mM, even more preferably between 200 and 400 mM, and in particular 400 mM.
    [0107]
    [0108] In one embodiment, the mixture from step a) presents glycidyl methacrylate in a concentration of between 50 and 1000 mM, more preferably between 100 and 800 mM, even more preferably between 200 and 600 mM, and in particular 400 mM.
    [0109]
    [0110] In a preferred embodiment, the mixture from step a) comprises hydroxyethyl methacrylate, ethylene glycolphenyl ether methacrylate, glycidyl methacrylate and ethylene glycol dimethacrylate. In a more preferred embodiment, the mixture from step a) comprises hydroxyethyl methacrylate in a weight ratio of between 60% and 99% with respect to the weight of the hydrogel, preferably between 70% and 99%, more preferably between 80% and 98% ,, ethylene glycolphenyl ether methacrylate in a weight ratio of between 0.1% and 10% based on the weight of the hydrogel, glycidyl methacrylate in a weight ratio of between 0.1% and 10% based on the weight of the hydrogel and ethylene glycol dimethacrylate in a weight ratio of between 0.1% and 10% with respect to the weight of the hydrogel.
    [0111]
    [0112] In a preferred embodiment, the mixture of step a) consists of monofunctionalized acrylic or methacrylic monomers, or combinations thereof, bifunctionalized acrylic or methacrylic monomers, and methacrylic or acrylic monomers comprising an electrophile group.
    [0113]
    [0114] Preferably step a) is carried out in the presence of a polymerization initiator, for example azobisisobutyronitrile (AIBN). The initiation of the polymerization can be carried out by heating the monomer mixture or by exposing it to ultraviolet-visible radiation.
    [0115]
    [0116] The polymerization process can be carried out in molds of suitable dimensions to provide the hydrogels as required. Therefore, in one embodiment, the polymerization reaction is carried out in a mold that provides the hydrogel with a contact lens shape. In a preferred embodiment, the mold is a glass, polypropylene, polyethylene, or polytetrafluoroethylene mold.
    [0117]
    [0118] In step b), the amino groups of the nitrogenous bases, which are nucleophilic groups, react with the electrophilic groups present in the hydrogel base framework, giving rise to amino bonds, by means of a nucleophilic substitution reaction.
    [0119]
    [0120]
    [0121] The nitrogenous base of step b) is in solution, preferably in aqueous solution or in a mixture of aqueous solution and organic solvent.
    [0122]
    [0123] In a further embodiment, the procedure described above further comprises a step in which a molecule with affinity for the nitrogenous base present in the hydrogel is incorporated into it in a simple process in which said molecule is contacted with the hydrogel obtained in step b) previously described. This stage takes place in an aqueous medium.
    [0124]
    [0125] In the process of the present invention, a hydrogel is obtained in which the nitrogenous base has not interfered in the polymerization and crosslinking process, and therefore no base frameworks are obtained in which the nitrogenous base is a structural link of the chains that make up the hydrogel.
    [0126]
    [0127] In one embodiment, the resulting hydrogels from any of the foregoing embodiments are washed, and optionally dried.
    [0128]
    [0129] The polymerization reaction can take place in a mold with the appropriate design to obtain a contact lens. If the polymerization reaction has not been carried out in a mold that provides the hydrogel with a contact lens shape, the contact lens can be prepared by lathe cutting the hydrogel.
    [0130]
    [0131] In another aspect, the present invention is directed to hydrogels obtainable by a method according to any of the previously described embodiments.
    [0132]
    [0133] Through the hydrogel or contact lens of the present invention, molecules are administered to the eye, and more specifically to the surface of the front of the eye (or anterior segment of the eye), and even more particularly to the cornea. Furthermore, advantageously, the hydrogel or contact lens of the present invention allows said administration to be controlled / sustained and not immediate.
    [0134]
    [0135] In one embodiment, the invention is directed to a hydrogel of the present invention, and more specifically to a contact lens of the present invention, for use in medicine. More particularly, the use in medicine is a use in ophthalmology.
    [0136]
    [0137] In a more specific embodiment, the invention is directed to a hydrogel of the present invention, and more specifically to a contact lens of the present invention, for use in treating or preventing dry eye.
    [0138] In an alternative embodiment, the invention is directed to the use of a hydrogel of the present invention, and more specifically of a contact lens of the present invention, for the preparation of a medicament for the treatment or prevention of dry eye.
    [0139]
    [0140] In an alternative embodiment, the invention is directed to a method of preventing or treating dry eyes, which comprises administering to a patient suffering from dry eyes a hydrogel of the present invention, and more specifically a contact lens of the present invention.
    [0141]
    [0142] In a particular embodiment, the contact lens of the present invention is used for the purposes described above and in addition to correct the vision of the user / patient.
    [0143]
    [0144] The invention is described below by means of the following examples which are to be considered merely illustrative and in no way limiting the scope of the present invention.
    [0145]
    [0146] Examples
    [0147]
    [0148] The following materials were used in the examples that follow:
    [0149]
    [0150] Transferuric acid (TA) from AlfaAesar (Kandel, Germany). 2-Hydroxyethyl methacrylate (HEMA) from Merck (Darmstadt, Germany); ethylene glycol dimethacrylate 98% (EGDMA), glycidyl methacrylate (GMA), ethylene glycolphenyl ether methacrylate (EGPEM), 2,2'-azobis (2-methylpropionitrile) (AIBN), dichlorodimethylsilane, cytosine, 2,2'-azobis (2-amidinropane) dihydrochloride (AAPH) and Trolox were purchased from Sigma-Aldrich (Steinheim, Germany); 1,4-dioxane from Panreac (Barcelona, Spain); NaOH from VWR Chemicals (Leuven, Belgium); water purified by reverse osmosis (resistivity> 18M Qcm, MilliQ, Millipore® Spain). Simulated lacrimal fluid (FLS) prepared with the following composition: 6.78 g / L NaCl, 2.18 g / L NaHCO3 and 1.38 g / L KCl and 0.084 g / L CaCf2HaO with pH 7.5.
    [0151]
    [0152] Example 1. Synthesis of acrylic hydrogels
    [0153]
    [0154] Hydrogels were prepared from the monomer mixtures summarized in Table 1.
    [0155] Table 1
    [0156] Hydrogel HEMA EGDMA GMA EGPEM
    [0157] (mL) 0 ¿ L) 0 ¿ L) 0 ¿ L)
    [0158]
    [0159]
    [0160]
    [0161]
    [0162]
    [0163]
    [0164]
    [0165]
    [0166]
    [0167]
    [0168]
    [0169] The hydrogels were coded based on the concentration of GMA, indicated as G in the code, the concentration of EGPEM, indicated as E in the code, and in the case that they have been functionalized with cytosine (see example 2) the code ends in C, whereas if it is not functionalized the termination is zero.
    [0170]
    [0171] EGDMA (8mM), GMA (0, 100, 200, 400 or 600mM) and EGPEM (0, 100 or 200mM) were added to HEMA and the mixtures were kept under magnetic stirring (300rpm, at room temperature 20- 23 ° C) for 15 minutes. AIBN (10mM) was then added and stirred for an additional 15 minutes. Finally, each solution was injected into a mold and polymerization was carried out at 50 ° C for 12 hours and then at 70 ° C for an additional 24 hours.
    [0172]
    [0173] The intermediate hydrogels thus obtained were characterized by IR spectrometry (FTIR Varian 670-IR equipped with a PIKE GladiATR Diamond Crystal), the characteristic bands of the polyHEMA frameworks were observed and the presence of glycidyl groups at bands of between 900 cm was evidenced " 1 and 690 cm "1.
    [0174]
    [0175] Example 2. Functionalization with a nitrogenous base
    [0176]
    [0177] Each hydrogel obtained in Example 1 was immersed in 100 mL of a water: dioxane mixture (1: 1), 1.11 g of cytosine was added, and the mixture in a closed container was
    [0178]
    [0179]
    [0180] kept in oscillatory agitation (30 osc / min) at 80 ° C for 24 hours. This process is illustrated in Figure 1.
    [0181]
    [0182] In parallel, another hydrogel underwent the same treatment, but without the presence of cytosine. After functionalization, the hydrogels were cut into discs (10 mm in diameter) and washed with miliQ water (1L) under magnetic stirring (200 rpm), changing the medium every 24 hours until the monomers were absent. Finally, the discs were dried at 50 ° C for 24 hours.
    [0183]
    [0184] The hydrogels thus obtained were characterized by IR spectrometry (FTIR Varian 670-IR equipped with a PIKE GladiATR Diamond Crystal), the characteristic bands of the polyHEMA frameworks were observed, and the presence of cytosine was observed when observing the band of the amido group at approx. 1655 cm-1.
    [0185]
    [0186] Example 3. Properties of hydrogels
    [0187]
    [0188] The degree of swelling was tested in water and FLS for each hydrogel prepared in examples 1 and 2 (measurements made at room temperature, 20-23 ° C) and calculated as follows:
    [0189] Degree of performance (%) = Wt Wo ■ 100
    [0190] Where W 0 and Wt represent the weight of the dry and swollen disc, respectively.
    [0191] It was observed that both the hydrogels prepared in Example 1 and the cytosine functionalized hydrogels prepared in Example 2, had a similar degree of swelling, and also similar when comparing the results in water and in FLS. The degree of swelling in all cases is in a range of between 40% and 65%.
    [0192]
    [0193] All discs reached swelling equilibrium in approximately one hour.
    [0194] Light transmission was measured on the swollen disks in water and in FLS, on a UV-Vis spectrophotometer between 190 and 800 nm (Agilent 8453, Germany). All the discs showed excellent light transmission properties, with transmittance values in the visible in a range greater than 90%. It was further observed that hydrogels incorporating cytosine absorb more in the UV range.
    [0195]
    [0196] All of these properties make the hydrogels of the invention suitable for use as soft contact lenses, even those containing cytosines are also suitable for protecting the eye from UV radiation.
    [0197] Example 4. Charge of molecules with affinity for nitrogenous bases
    [0198] The dried hydrogel disks were weighed and placed in 5 mL of aqueous TA solutions (0.01 mg / mL) prepared with 0.05% EDTA as a stabilizer. They were kept at 25 ° C under orbital shaking (300 rpm) for 48 hours. The absorbance of the medium was monitored at 320 nm at certain time intervals (Agilent 8543 UV / Vis spectrophotometer, Germany). The amount of TA loaded was estimated by the difference between the initial and final amount of TA in the solution using a previously validated calibration curve. The results obtained are shown in Table 2 and Figure 2.
    [0199] Table 2
    [0200]
    [0201]
    [0202]
    [0203] The hydrogels prepared in Example 1 were observed to complete the charge in the first 8 hours, while the cytosine functionalized hydrogels obtained in Example 2 completed the charge after 24 hours. The discs that showed the highest load were those from hydrogels prepared with a concentration of GMA of 400 mM and with EGPEM at a concentration of 200 mM and functionalized with cytosine.
    [0204]
    [0205]
    [0206] Example 5. Assignment of molecules with affinity for nitrogenous bases
    [0207]
    [0208] The discs from Example 4 were rinsed with water, excess water was removed from the surface with filter paper, and each disc was placed in a vial with 5 mL of FLS and the vials were kept under oscillating shaking (300 rpm) at 35 ° C. At preset times (0.5, 1, 2, 4, 6, 8, and 24 h) 2.5 mL of the transfer medium was taken, the absorbance at 320 nm was measured (UV / VIS Agilent 8453, Germany) and The sample was returned to the source vial. To calculate the amount of TA transferred by each hydrogel, the concentration in the transfer medium was calculated from the absorbances using the corresponding TA calibration line in FLS, and then the concentration was multiplied by the volume, and the values were they referred to the initial weight of each hydrogel disk.
    [0209]
    [0210] The results are shown in Figure 3. It can be seen that the cytosine-functionalized hydrogels provided a more sustained release over time and a less intense burst in the first hour than in the case of the delivery with hydrogels that do not incorporate cytosine. Furthermore, cytosine-functionalized hydrogels yield a higher amount of TA than those that are not functionalized, which is an advantage from a therapeutic point of view. The best results were obtained with cytosine functionalized hydrogels that were prepared with a concentration of GMA and EGPEM of 400 mM and 200 mM, respectively, and those that were prepared with a concentration of GMA and EGPEM of 600 mM and 200 mM, respectively.
    [0211]
    [0212] Example 6. Study of antioxidant activity
    [0213]
    [0214] To verify that the antioxidant activity of TA was maintained after being incorporated into the hydrogels and yielding, an analysis of the antioxidant activity was performed. A biologically relevant source of radicals, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), was selected to carry out the standard ORAC test (C. Lucas-Abellan, MT Mercader-Ros, MP Zafrilla, JA Gabaldon and E. Nunez-Delicado, Food Chem. Toxicol., 2011, 49 , 1255-1260). This method quantifies the ability of a substance to protect fluorescein from degradation by free radicals. Thus, the greater the antioxidant capacity, the lower the decrease in fluorescein fluorescence.
    [0215]
    [0216]
    [0217] The FLS release medium was analyzed in which uncharged and TA-loaded hydrogels were soaked. The uncharged cytosine functionalized hydrogels were tested to verify that there were no leaching substances that could cause an artifact (false antioxidant activity) during the test. Results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC) for comparative purposes. As expected, uncharged hydrogels resulted in values close to zero (or even negative), indicating that they did not release any antioxidant substances. In contrast, TA loaded G400E200-C led to the highest antioxidant activity, equivalent to 53.28 ± 2.44 Trolox, compared to its TA loaded cytosine free counterpart G400E200-0 which had an activity of 37.48 ± 1.53 ^ M Trolox . This finding corroborates the usefulness of cytosine in providing hydrogels with an enhanced ability to harbor TA and, even more notably, that the TA-cytosine interaction has no detrimental effect on the antioxidant activity of TA. In fact, the antioxidant activity expressed as ^ molTolox / | imolTA gave values in the range of 4.8-5.0, which demonstrates the utility of hydrogels as vehicles for the transfer of substances, in this case of TA.
    [0218]
    [0219] Example 7. Cellular Compatibility Study
    [0220]
    [0221] Cellular compatibility was evaluated in a surrogate model using chorioallantoic membrane of fertilized chicken egg (HET-CAM) (F. Alvarez-Rivera, D. Fernández-Villanueva, A. Concheiro and C. Alvarez-Lorenzo, J. Pharm. Sci., 2016, 105, 2855-2863). Discs of each hydrogel loaded with TA as obtained in Example 4 were placed on the chorioallantoic membrane. Possible changes in membrane vasculature were observed for 5 min, recording lysis, bleeding and coagulation time. As a negative control, the 0.9% NaCl solution was used, and as a positive control, a 0.1 N NaOH solution. All the discs passed the compatibility test, causing no lysis, bleeding or coagulation.
    [0222]
    [0223] In addition, in vitro cytocompatibility of the G0E0-0, G400E200-0, and G400E200-C hydrogels in human corneal epithelial cells (HCEC; ATCC PCS-700-010) was evaluated. The cell line was grown in Keratinocyte-Serum Free medium (Gibco, Great Britain), supplemented with hydrocortisone (500 ng / mL), insulin (5 microg / mL), penicillin / streptomycin 1%, and antifungal 1%, prior to treatment with Fibronectin BSA-Bovin coligen I. HCEC (20,000 cells / well) were seeded in a 48-well plate in Keratinocyte-Serum Free medium and cultured for 24 h at 37 ° C (95% RH and 5% CO 2 ). The discs were loaded for 24 hours in a 10 µg / mL TA solution, cut into four pieces, and then autoclaved (steam heated) in the same solution, at 121 ° C for 15 minutes. Then, a disk piece was placed in each well for 24 h. Aqueous TA solution (10 µg / mL) was also added in triplicate (pre-sterilization) and negative controls included untreated cells. After 24 hr in cell culture, the disks and drug solution were removed from the wells, and the cell viability assay was performed following the manufacturer's instructions using WST-1 (Roche, Switzerland). Absorbance was read at 450 nm (Bio-Rad Model 680 UV Microplate Reader, USA). Cell viability (%) was calculated as follows:
    [0224] uhc
    [0225] V ia b ilid adce lu la r (%) = —----- t - r - —------ x 100
    [0226] A b S c o n o l n eg a tive
    [0227]
    [0228] This study showed that hydrogels at the used charge concentration (10 | ig / mL, approx. 51.5 | iM) are compatible, showing cell viability levels higher than 80% after 24 hours of direct contact.
    [0229]
    [0230] Example 8. Corneal permeability study
    [0231]
    [0232] Fresh cattle eyes were collected in the abattoir and transported submerged in PBS (phosphate buffered saline) with added antibiotics (penicillin at 100 IU / mL and streptomycin at 100 | ig / ml) in an ice bath. The cornea was isolated with 2 3 mm of surrounding sclera and the sclera and cleaned with PBS before mounting in a Franz-type vertical diffusion cell, separating the donor chamber from the recipient. The chambers were filled with bicarbonate buffer pH 7.2. The diffusion cells were placed in a thermostated bath at 37 ° C and kept under stirring for 30 min. Then, the buffer was removed from the donor chamber and replaced by the samples to be tested: 2 mL of aqueous TA solution (10 microL / mL), or G400E200-0 and G400E200-C disks in 2 mL of 0.9% NaCl. The area available for permeation was 0.785 cm2. The chambers were covered with parafilm. Samples were taken at 1 hr intervals
    [0233]
    [0234]
    [0235] 1 mL from the receiving chambers and the same volume was replaced with buffer, taking care to remove bubbles from the diffusion cells.
    [0236]
    [0237] Samples taken from the receiving chambers were filtered through nylon membranes (0.45 | im) and measured with an HPLC kit (Waters 717 Autosampler, Waters 600 Controller, 996 Photodiode Array Detector) conditioned with a C18 column (Waters Symmetry C18 5p, m; 4.6x250 mm) and Empower 2 as software. The mobile phase consisted of methanol: acetonitrile: phosphate buffer (20:15:65) at 1 mL / min and 35 ° C (KH 2 PO 4 buffer 0.68 g / L adjusted to pH 3-3.1 with phosphoric acid ). The injected volume was 50 ^ L, and the TA was quantified at 320 nm (retention time 5 min). The ALA calibration line in aqueous phase was prepared in the concentration range between 0.009 and 10.0 mg / mL in carbonate buffer. The TA content of the samples was calculated from the calibration curve.
    [0238]
    [0239] After 6 hr of testing, a sample was taken from the donor chamber for further analysis. Furthermore, the corneas and sclera were visually examined to ensure that they had not been damaged during the trial.
    [0240]
    [0241] The corneas and sclera were kept overnight in 3 mL of an ethanol: water solution (50:50 v / v), sonicated for 99 min at 37 ° C, centrifuged (1000 r.p.m.
    [0242] 5 min, 25 ° C), filtered and centrifuged again (14000 rpm, 20 min, 25 ° C). The coefficient of permeability of TA through the cornea and sclera was calculated as the ratio of the steady state flow and the concentration of TA in the donor chamber.
    [0243] Both in the cornea and in the sclera, the G400E200-C hydrogels loaded with transferulic acid gave similar permeability coefficients to those obtained as the free drug solution, indicating that the transfer from the cytosine functionalized hydrogel is not an obstacle to its penetration into the cornea and sclera, unlike what occurs for non-functionalized hydrogels (Figure 4). Similar values were also obtained for the amount of accumulated trans-ferulic acid in the cornea and sclera (Figure 5).
    [0244]
    [0245]
    one
    权利要求:
    Claims (22)
    [1]
    1. Hydrogel in the form of a three-dimensional network characterized in that it comprises cross-linked methacrylic and / or acrylic chains, where the chains comprise alkyl groups to which a nitrogenous base selected from cytosine, adenine, guanine, uracil and thymine are attached.
    [2]
    Hydrogel according to claim 1, characterized in that the methacrylic and / or acrylic chains comprise: monofunctional methacrylic or acrylic units, or combinations thereof; methacrylic or bifunctionalized acrylic units; and methacrylic or acrylic units comprising an electrophilic group.
    [3]
    3. Hydrogel according to any of the preceding claims, characterized in that the proportion by weight of the bifunctionalized acrylic or methacrylic units is preferably between 0.01% and 10% by weight with respect to the weight of the hydrogel.
    [4]
    Hydrogel according to any of the preceding claims, characterized in that the proportion by weight of the methacrylic or acrylic units comprising an electrophilic group is between 0.1% and 10% with respect to the weight of the hydrogel.
    [5]
    5. Hydrogel according to any of the preceding claims, wherein the hydrogel further comprises a molecule with an affinity for the nitrogenous base present in the hydrogel.
    [6]
    6. Hydrogel according to claim 5, wherein the molecule with affinity for a nitrogen base present in the hydrogel is an antioxidant molecule.
    [7]
    7. Hydrogel according to claim 5, wherein the molecule with affinity for a nitrogen base present in the hydrogel is a pharmaceutical active ingredient.
    [8]
    8. Method for preparing a hydrogel as defined in claim 1, comprising the steps of:
    to. polymerizing a mixture comprising i) monofunctional methacrylic or acrylic monomers, or combinations thereof, ii) bifunctionalized methacrylic or acrylic monomers, and iii) methacrylic or acrylic monomers comprising an electrophile group, to form a three-dimensional network base framework; and
    b. react a nitrogenous base selected from cytosine, adenine, guanine, uracil and thymine, with the base framework.
    2
    [9]
    9. Method according to claim 8, further comprising step c) contacting a molecule with affinity for the nitrogenous base with the hydrogel obtained in step b), in the presence of an aqueous medium.
    [10]
    10. The method according to claim 8 to 9, wherein the monofunctional methacrylic or acrylic monomers are selected from the group consisting of hydroxyethyl methacrylate, ethylene glycolphenyl ether methacrylate, 1- (tristrimethylsiloxysilylpropyl) -methacrylate, methylmethacrylate, N, N-dimethylacrylamide diethylacrylamide, methacrylic acid, acrylic acid, aminopropyl methacrylate, cyclohexyl methacrylate, and fluorosiloxane acrylate, or combinations thereof.
    [11]
    The method according to any of claims 8 to 9, wherein the bifunctionalized acrylic or methacrylic monomers are selected from the group consisting of ethylene glycol dimethacrylate, 1,3-Butanediol diacrylate, 1,4-Butanediol diacrylate, 1,6-Hexanediol diacrylate , Ethylene glycol diacrylate, Fluorescein O, O'-diacrylate, Glycerol 1,3-diglycerolate diacrylate, Pentaerythritol diacrylate monostearate, 1,6-Hexanediol ethoxylate diacrylate, 3-Hydroxy-2,2-dimethylpropyl 3-hydroxy-2,2- Dimethylpropionate diacrylate, Bisphenol A ethoxylate diacrylate, Di (ethylene glycol) diacrylate, Neopentyl glycol diacrylate, Poly (ethylene glycol) diacrylate, Poly (propylene glycol) diacrylate, Propylene glycol glycerol diacrylate, Tetra (glycollate). dimethacrylate, 1,4-Butanediol dimethacrylate, 1,6-Hexanediol dimethacrylate, Bisphenol A dimethacrylate, Diurethane dimethacrylate, Ethylene glycol dimethacrylate, Fluorescein O, O'-dimethacrylate, Glycerol dimethacrylate, Bisphenol A ethoxylate or dimethacrylate, Bisphenol A glycerolate dimethacrylate, Di (ethylene glycol) dimethacrylate, Poly (ethylene glycol) dimethacrylate, Poly (propylene glycol) dimethacrylate, Tetraethylene glycol dimethacrylate, Tri (ethylene glycol) dimethyl acrylate, Triet (ethylene glycol) -ethylene glycol dimethacrylate) and Poly (methyl methacrylate-co-ethylene glycol dimethacrylate).
    [12]
    12. Method according to any of claims 8 to 9, wherein the mixture of step a) further comprises a polymerization initiator.
    [13]
    13. Method according to any of claims 8 to 9, wherein the reaction of step b) takes place in the presence of an organic solvent.
    [14]
    14. Hydrogel obtainable by a process as defined in any one of claims 8 to 13.
    [15]
    15. Contact lens comprising a hydrogel as defined in any one of claims 1 to 7 or 14.
    [16]
    16. Method for preparing a contact lens as defined in claim 15, comprising forming the contact lens from the hydrogel as defined in any one of claims 1 to 7 or 14 by lathe cutting of the hydrogel or by molding the hydrogel, or by a combination of these techniques.
    [17]
    17. Hydrogel as defined in any one of claims 1 to 7 or 14, or contact lens as defined in claim 15, for use in medicine, preferably ophthalmology.
    [18]
    18. Hydrogel or contact lens for use according to claim 17, for use in the treatment or prevention of dry eye.
    [19]
    19. Hydrogel or contact lens for use according to claim 17, for use in the treatment or prevention of cataracts.
    [20]
    20. Hydrogel or contact lens for use according to claim 17, for use in the treatment or prevention of corneal ulcers.
    [21]
    21. Use of a hydrogel as defined in any one of claims 1 to 7 or 14, in the manufacture of topical, transdermal or transmucosal delivery systems.
    [22]
    22. Use of a hydrogel as defined in any one of claims 1 to 7 or 14, in cosmetics.
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    ES2755415B2|2021-03-05|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20080124378A1|2005-02-04|2008-05-29|Byrne Mark E|Therapeutic contact lenses with anti-fungal delivery|
    CN108102117A|2017-12-28|2018-06-01|长春工业大学|A kind of bionical preparation method for gluing tough aquogel system of covalently cross-linked base|
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