JP2007187897A - Hydrogel ophthalmic lens for gene therapy of eye - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a practical ophthalmic lens for gene therapy which is a hydrogel capable of exhibiting a satisfactory slow-releasing effect by holding genes that can appear within animal cells and is excellent in transparency without causing shape changes before and after the slow-release. <P>SOLUTION: The hydrogel ophthalmic lens is capable of including and slowly releasing the medicine used for the gene therapy of the eyes. Also, the hydrogel ophthalmic lens is obtained by treating a copolymer containing a nonionic hydrophilic monomer and (meth)acrylamide at a ratio of (40 to 95):(60 to 5) wt.% in an alkaline buffer solution. The ophthalmic lens is characterized by using a plasmid DNA as the medicine for the gene therapy of the eyes to be included and slowly released. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a hydrogel ophthalmic lens that can be used for gene therapy. More specifically, the present invention relates to ophthalmic lenses such as contact lenses and intraocular lenses that can effectively transfer plasmid DNA into ocular cells.

  Genetic eye diseases such as corneal dystrophy are known to be caused by mutations in genes expressed in eye cells. A method for transferring a normal gene into eye cells is promising for the treatment of such a genetic eye disease.

  Conventionally, transfer of genes into ocular cells has been performed by a surgical method of transplanting cultured cells or administration by eye drops. However, the surgical technique increases the burden on the patient and may cause sequelae such as decreased visual acuity. In addition, although administration with eye drops is relatively easy, there is a drawback in that the drug flows out with tears in a short time after instillation.

  In order to effectively transfer a gene into a cell, a carrier is required, and a method of using an improved virus, a method of encapsulating in a liposome, and a method of using plasmid DNA are known. When using viruses and liposomes, there are concerns about infectivity and cytotoxicity, and there are problems with safety. Further, when these are added to eye drops, they can be transferred into cells to some extent, but they are not effective for transfer into eye cells because they are diluted with tears.

  As an example using a liposome, there is an eye drop containing a liposome encapsulating an expression vector incorporating a gene DNA that can be expressed in mammalian cells (see Patent Document 1). This is a method of transferring a gene into an eye cell by eye drop for the purpose of gene therapy for an abnormal gene DNA involved in an eye disease caused by a gene abnormality. However, as described above, since the gene is diluted with tears in a short time after instillation, it is necessary to devise a method for making the contact time with the cornea as long as possible. When gene DNA is used in an eye ointment or the like, it is superior to eye drops in that the contact time with the cornea is longer, but has the disadvantage that the eye irritation and the field of view become cloudy.

  On the other hand, when plasmid DNA is used, it is safer than when viruses are used, and its structure and size can be freely selected. Therefore, gene therapy technology using this has recently attracted attention. ing. However, even if plasmid DNA is used by adding it to eye drops, it cannot exert a sufficient transfer effect to eye tissue.

By the way, it is generally known to utilize the properties of hydrogel as a means for releasing a drug continuously. For example, there is a composition using a concentration gradient distribution for drugs such as proteins, genes, and physiologically active substances in the hydrogel (see Patent Document 2). In this method, an acrylamide-based hydrogel is sandwiched between a phosphate buffer and an alkaline sodium hydroxide aqueous solution, and an alkali concentration gradient due to the aqueous sodium hydroxide solution is formed in the gel. In other words, the amide group is hydrolyzed by the strong alkalinity of the sodium hydroxide aqueous solution to form a gradient distribution of carboxyl groups inside the gel, and a cationic drug is carried on this carboxyl group to effectively release the drug. Technology. However, in this method, the concentration distribution of the carboxyl group in the hydrogel may be uneven, and when used as an ophthalmic lens, the gel shape is not stable and the visual acuity is reduced. When a molecular drug is loaded, the gel becomes cloudy due to the concentration distribution. In addition, an anionic drug cannot be supported in the gradient distribution of the carboxyl group concentration by hydrolysis.
Japanese Patent Laid-Open No. 10-67688 JP 2004-323774 A

  An object of the present invention is to provide a hydrogel that can easily perform gene therapy for an abnormal gene DNA involved in an eye disease caused by a gene abnormality, and particularly to provide a hydrogel ophthalmic lens. More specifically, it is a hydrogel that retains a gene that can be expressed in animal cells and can exhibit a good sustained release effect, and has no change in shape before and after the sustained release, and is a practical gene therapy with excellent transparency. An ophthalmic lens is provided.

  The present invention is a hydrogel ophthalmic lens that can contain and gradually release drugs used for gene therapy of the eye.

  Further, the present invention is obtained by treating a copolymer containing a nonionic hydrophilic monomer and (meth) acrylamide in a ratio of 40 to 95:60 to 5% by weight in an alkaline buffer solution. This is a hydrogel ophthalmic lens used for gene therapy of the eye.

  Furthermore, the present invention is a hydrogel ophthalmic lens for use in ocular gene therapy, characterized in that plasmid DNA is used as an ophthalmic gene therapy drug that is comprehensively and slowly released.

  The ophthalmic lens of the present invention includes genes that can be expressed in animal cells and can be released over 24 hours or more, and is excellent in practical transparency and shape stability as an ophthalmic lens.

  The present inventor, as a means for transferring a drug used for ocular gene therapy into an eye cell, a method of effectively releasing the drug with a hydrogel ophthalmic lens, in particular, the drug having an anionic functional group and This is based on knowledge obtained by earnestly examining a method using electrostatic interaction with an electrolytic group or a polar group in a gel.

  In the present invention, in order to effectively use a drug used for eye gene therapy, hydrogel having an amide group is alkali-treated under specific conditions to convert the amide group to a carboxyl group by hydrolysis. Without making it, the bias of the electric charge to the nitrogen atom of the amide group in the said hydrogel is formed. Specifically, a suitable interaction between the hydrogel and a gene therapy drug containing at least one anionic functional group in the molecular structure, particularly a carboxyl group, and the hydrogel is effectively included. And let it release slowly.

  Examples of the drug used for ocular gene therapy preferably used in the present invention include plasmid DNA containing at least one anionic functional group, particularly a carboxyl group in the molecular structure. Plasmid DNA is circular DNA and can be used as it is, but it can also contain desired DNA according to the intended treatment. For example, colloidal corneal dystrophy is an autosomal recessive genetic disease that causes severe amyloid deposition in the corneal epithelium. In this disease, amyloid is deposited on the cornea, so that transparency is lost and visual acuity is reduced. So far, it has been found that this causative gene is M1S1 (membrane component, chromosome 1, surface marker 1) gene. It is also possible to incorporate this M1S1 gene into plasmid DNA and use it as a carrier to transfer it to the target eye cell. In the present invention, the target DNA for the hydrogel ophthalmic lens, in particular, plasmid DNA is immersed in an aqueous solution so that the plasmid DNA is included in the lens.

  The gene concentration in the aqueous solution should be appropriately selected for each gene depending on its solubility and the minimum effective concentration and maximum safe concentration for transfer into animal cells. A concentration of 0.1 μg / μL is preferred.

  In the ophthalmic lens of the present invention, methacrylamide and acrylamide are used as monomers having an amide group. The present invention utilizes the interaction between the positive charge of the nitrogen atom in the molecule and the carboxyl group of the plasmid DNA. However, even if it has a nitrogen atom, the desired effect cannot be obtained with other compounds. I can't. For example, methacrylamidopropyltrimethylammonium chloride (MAPTAC), which similarly has a nitrogen atom at the molecular end, is too charged to provide a sustained release effect even if it can carry plasmid DNA. In addition, dimethylacrylamide cannot form a sufficient charge bias due to a methyl group bonded to a nitrogen atom, and cannot carry plasmid DNA.

  If the methacrylamide and acrylamide used in the present invention are appropriately selected, the physical properties of the resulting gel can be selected. For example, if acrylamide is used, an ophthalmic lens with high flexibility can be obtained, and the water content can be increased. If methacrylamide is used, a firm ophthalmic lens can be obtained, the strength can be improved, and the water content can be reduced. These can be used in combination as desired.

  Any nonionic hydrophilic monomer can be used as long as it has at least one hydrophilic group in the molecule and is nonionic. For example, 2-hydroxymethyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2,3-dihydroxypropyl (meth) acrylate and 2-polyethylene glycol mono (meth) acrylate, Examples thereof include 2-polypropylene glycol (meth) acrylate and N-vinylpyrrolidone, and two or more of these may be used in combination. If these hydrophilic monomers are also appropriately selected, the water content of the ophthalmic lens can be changed.

  The main component monomer is preferably used in an amount of 5 to 60% by weight of the amide monomer with respect to the hydrophilic monomer. If the amount is less than 5% by weight, the amount of genes that can be expressed in animal cells is extremely small in the ophthalmic lens, and the amount of genes transferred to the eye cells is extremely small. If it exceeds 60% by weight, the gene inclusion amount increases, and the shape of the ophthalmic lens is not stabilized along with the release thereof, and the mechanical strength is lowered, which is not preferable.

  In the present invention, in addition to the above components, any copolymerizable monomer can be used. For example, by using a crosslinkable monomer, it is possible to form a network structure of the ophthalmic lens and adjust the mechanical strength. Examples of the crosslinkable monomer include ethylene glycol dimethacrylate, methylene bisacrylamide, 2-hydroxy-1,3-dimethacryloxypropane, trimethylolpropane triacrylate, and the like. The amount of the crosslinkable monomer used is preferably 0.1 to 4.0 parts by weight with respect to 100 parts by weight of the main monomer. Particularly preferred is 0.1 to 1.0 part by weight. When the crosslinkable monomer is less than 0.1 parts by weight, a remarkable effect is not seen in the shape stability of the ophthalmic lens, and when it exceeds 4.0 parts by weight, the crosslinking effect becomes excessive and the polymer gel becomes brittle. Absent.

  In addition, if a hydrophobic monomer is used as an optional copolymerizable monomer, the water content and swelling rate of the resulting ophthalmic lens can be adjusted, and the target gene can be finely adjusted in the ophthalmic lens. Can be achieved. Any hydrophobic monomer may be used as long as it is compatible with the hydrophilic monomer or (meth) acrylamide, which is an essential component of the present invention. For example, methyl methacrylate, isobutyl methacrylate, 2,2,2-trimethyl may be used. Examples thereof include fluoromethacrylate and cyclohexyl methacrylate.

  One feature of the present invention is the treatment of the hydrogel synthesized for the present invention with an alkaline aqueous solution. As a result, a sufficient charge bias can be formed in the gel. In the case of alkaline aqueous solution treatment, the amide group inside the gel is hydrolyzed to a carboxyl group under strong alkalinity, and the ophthalmic lens itself is negatively charged. Similarly, the negatively charged plasmid DNA is repelled. An ophthalmic lens capable of sustained release cannot be obtained.

  The alkaline aqueous solution used in the present invention is preferably a buffer system having a value exceeding pH 7.0 and 11.0 or less. A particularly preferred range is pH 8-10. Treatment with an aqueous solution having a buffering action is preferable because alkali treatment can be performed in a state in which the shape of the ophthalmic lens is stabilized. That is, when a simple alkaline aqueous solution that is not a buffer system is used, the ophthalmic lens becomes large or deforms due to an electrical change that occurs as a result of the treatment, so that it does not return to its original shape, and the strength is also not preferred. .

  In addition, at pH 7 or lower, the amide group in the gel is neutral and cannot interact with the plasmid DNA. Therefore, it is difficult to maintain the state even when the target gene is incorporated, and it is released within a few hours. Resulting in. Moreover, when it becomes strong alkalinity exceeding pH 11.0, the side chain in a gel will be hydrolyzed and will deform | transform and cannot be used as an ophthalmic lens.

  What is necessary is just to adjust a process liquid according to a conventional method, and what is necessary is just to immerse in this for about 30 to 90 minutes under normal temperature.

  Examples of the buffer include phosphate buffer, maleic acid / Tris / NaOH buffer, triethanolamine HCl / NaOH buffer, 5,5-diethylbarbituric acid Na / HCl buffer, and pyrophosphate Na / HCl buffer. , Tris / HCl buffer, diethanolamine / HCl buffer, boric acid / NaOH buffer, sodium borate / HCl buffer, ammonia / ammonium chloride buffer, glycine / NaOH buffer, sodium carbonate / sodium bicarbonate buffer, Examples thereof include a sodium borate / NaOH buffer solution and the like, as long as it has a value exceeding pH 7.0 and can be adjusted to the buffering action range at 11.0 or less.

  In the production of the ophthalmic lens of the present invention, a polymerization initiator is first added to the mixture of monomers, and further stirred and dissolved. As the polymerization initiator, any radical polymerization initiator can be used, and the addition amount of the polymerization initiator is preferably about 10 to 3500 ppm external to the total amount of monomers. Polymerization is carried out by putting the monomer mixture into a mold for an ophthalmic lens such as metal, glass or plastic and sealing it. The polymerization can be carried out by any method as long as it is radical polymerization, and the polymerization can also be carried out by adding water or an organic solvent to the monomer mixture.

  After completion of the above polymerization, the mixture is cooled to room temperature, and the resulting polymer is taken out of the mold and cut and polished as necessary. The obtained ophthalmic lens is hydrated and swollen to obtain an ophthalmic lens. Examples of the liquid (swelling liquid) used for this hydration swelling include water, physiological saline, isotonic buffer and the like. The swelling liquid is heated to 60 to 100 ° C. and dipped for a certain period of time to quickly enter a hydrated and swollen state. Moreover, it becomes possible to remove the unreactive monomer contained in the polymer by the swelling treatment.

  Next, after being immersed in a buffer adjusted to pH 8 to 10 at room temperature for 1 hour and subjected to alkali treatment, the ophthalmic lens is sufficiently washed with pure water to dissolve the previously prepared plasmid DNA. The hydrogel ophthalmic lens of the present invention is obtained in the step of immersing in an aqueous solution.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

(Evaluation methods)
[Evaluation of shape stability]
The ophthalmic lens before and after plasmid DNA release was measured in saline using a contact lens projector. The ophthalmic lens before release of plasmid DNA was evaluated as ◯ when no change in shape was observed, and x when change in size or deformation was observed.

[Moisture content]
As an evaluation, the water content was measured based on the “water content measurement with hydrogel lens (ISO10339: 1997)”.

[Measurement of light transmittance]
The light transmittance of the lens after inclusion of the plasmid DNA was measured using an ultraviolet-visible spectrophotometer (UV-3150: manufactured by Shimadzu Corporation).

[Measurement of drug release]
The lens after inclusion of the plasmid DNA was immersed in 3 mL of physiological saline at 25 ° C., and the amount released was measured by electrophoresis using an agarose gel every 24 hours. 1 kbp was used as a standard marker for measurement. When the release of plasmid DNA was observed over 12 hours, it was marked as ◯, and when it was not confirmed, it was marked as x.

[Evaluation of release into tears]
The lens after inclusion of the plasmid DNA was attached to a rabbit, and 5 μL of tear fluid after a certain time was collected with a capillary tube. The plasmid DNA in the collected tears was amplified by PCR (Polymerase Chain Reaction) method, and the presence of plasmid DNA in the tears was confirmed by electrophoresis using an agarose gel. For electrophoresis, 1 kbp was used as a standard marker. When the presence of plasmid DNA was observed in the tears for 12 hours, it was marked as ◯, and when it was not recognized, it was marked as x.

[Evaluation of Rabbit Corneal Epithelial Cell Importability]
The lens after inclusion of the plasmid DNA was attached to a rabbit, and the rabbit corneal epithelial cells after attachment were collected, cultured for 48 hours, and observed with a fluorescence microscope. Cells transferred with the plasmid DNA were colored with fluorescence using GFP (Green Fluorescent Protein) to evaluate the transfer of the plasmid DNA into the corneal epithelial cells. When transfer to corneal epithelial cells was observed, it was marked with ◯.

Example 1
2-hydroxyethyl methacrylate (HEMA) 95% by weight, acrylamide (AAm) 5% by weight, ethylene glycol dimethacrylate (EDMA) 0.3 parts (external), azobisisobutyronitrile (AIBN) 0.3 Part (external) was added and stirred for about 1 hour with sufficient nitrogen substitution. After stirring, the monomer mixture was placed in a mold for an ophthalmic lens and polymerized by raising the temperature in the range of 50 to 100 ° C. over 25 hours. The polymer was taken out of the container, immersed in distilled water at about 80 ° C. for about 4 hours, and hydrated and swollen to obtain an ophthalmic lens of this example. This ophthalmic lens was immersed in a phosphate buffer solution (PBS) adjusted to pH 8 at room temperature for 1 hour for alkali treatment, and then thoroughly washed with pure water. The ophthalmic lens after washing was soaked in 3 mL of a 0.01 μg / μL aqueous solution of plasmid DNA (manufactured by CLONTEC) prepared in advance at 25 ° C. for 48 hours to encapsulate the plasmid DNA. The ophthalmic lens covered with the plasmid DNA was immersed in 3 mL of distilled water at 25 ° C. for 24 hours to remove the free plasmid DNA, and then subjected to the various evaluations described above.

(Examples 2 to 10)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with a phosphate buffer solution (PBS) adjusted to the pH shown in Table 1, and plasmid DNA was included in the same manner as in Example 1 to perform the above various evaluations.

(Comparative Examples 1-5)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with a phosphate buffer solution (PBS) adjusted to the pH shown in Table 1, and plasmid DNA was included in the same manner as in Example 1 to perform the above various evaluations. Since Comparative Example 2 had a large change in the lens shape before and after the drug release and its practicality was poor, evaluation of tear release and rabbit corneal epithelial cell transferability was not performed.

(Comparative Example 6)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with physiological saline adjusted to pH 7, and plasmid DNA was included in the same manner as in Example 1 to perform the above various evaluations.

(Comparative Example 7)
An ophthalmic lens having the composition shown in Table 1 and hydrated and swollen in the same manner as in Example 1 was obtained. This ophthalmic lens was treated with an aqueous sodium hydroxide solution adjusted to pH 12, and the plasmid DNA was included in the same manner as in Example 1 to perform the above various evaluations. Since the lens shape changes greatly before and after the drug release and its practicality is poor, the release into tears and the corneal epithelial cell transferability were not evaluated.

The symbols in [Table 1] are as follows.
HEMA 2-hydroxyethyl methacrylate
HPMA 2-hydroxypropyl methacrylate
NVP N-Vinylpyrrolidone
AAm Acrylamide
MAm methacrylamide
MAPTAC methacrylamidopropyltrimethylammonium chloride
DMAA dimethylacrylamide
EDMA ethylene glycol dimethacrylate

Claims (3)

  A hydrogel ophthalmic lens that contains drugs used in gene therapy for the eye and can be released slowly.   A copolymer obtained by treating a nonionic hydrophilic monomer and (meth) acrylamide in a ratio of 40 to 95:60 to 5% by weight in an alkaline buffer, and obtained by treatment. The hydrogel ophthalmic lens according to 1. The hydrogel ophthalmic lens according to claim 1 or 2, wherein plasmid DNA is used as a drug for gene therapy for ocular inclusion and sustained release.