WO2012111878A1 - Pharmaceutical composition containing the superoxide dismutase fusion protein for preventing or treating eye diseases - Google Patents

Abstract

The present invention relates to an agent for treating dry eyes, and more particularly, to a pharmaceutical composition for treating dry eyes, which contains, as an active ingredient, the superoxide dismutase fusion protein, which is capable of permeating into ocular tissue.

Description

Pharmaceutical composition for preventing or treating ophthalmic diseases containing superoxide dismutase fusion protein

The present invention relates to a dry eye treatment agent, and more particularly to a pharmaceutical composition for treating dry eye syndrome containing a superoxide dismutase fusion protein that can penetrate into the eye tissue as an active ingredient.

Recently, in the field of ophthalmology, the use of immunosuppressive agents as therapeutic agents is increasing due to the increase of corneal transplantation and autoimmune disease patients, and new drugs are actively developed to reduce side effects of such immunosuppressive agents. In the case of eye diseases, immunosuppressive agents are being studied as eye drop dosage forms, and the side effects are increasing because corneal permeability of drugs currently being used is low due to excessive use. The cornea is well permeable if the drug to be permeated has both hydrophilic and fatty properties.

Corneal transplantation is a surgical procedure that restores vision by replacing the cloudy cornea of a patient with vision loss due to corneal disease with a donated clean cornea. Corneal transplantation is the most frequently performed transplantation worldwide due to the development of corneal preservation methods, the development of surgical techniques and instruments, and the treatment of rejection reactions. The eyeball can be transplanted even after cardiopulmonary arrest. This is the only activated organ transplantation in Korea, where brain death is not legally recognized with a high success rate compared to other organs. According to the National Organ Transplantation Management Center in Korea, corneal transplantation is increasing every year, and the number of corneal transplants in 2007 was 405, and in the United States, the number exceeds 40,000 from 1990 to 2007.

As cornea transplantation increases, various problems appear, and efforts are being made to reduce them. Corneal graft rejection accounts for the majority of corneal graft failures. Although steroids have been used mainly to reduce rejection in corneal transplantation, complications such as increased intraocular pressure, delayed wound healing, and cataracts can occur if steroids are used for a long time, and high doses do not prevent rejection. Recently, attempts have been made to reduce rejection by using immunosuppressants rather than steroids. In the high-risk corneal graft group, the group treated with cyclosporine A, an immunosuppressive agent, showed higher rejection than the steroid-only group. Was reported. In experimental animal models, there was less postoperative invasion of inflammatory cells after surgery than in the FK506 (Tacrolimus) group. The use of FK506 (Tacrolimus) or cyclosporin A in organ transplant patients is excellent in immunosuppressive effects, but side effects of drugs such as nephrotoxicity, hypertension, metabolic disorders, and risk of diabetes have been pointed out as problems of use.

Autoimmune disease is an autoimmune disease that occurs when the body's defense mechanisms, ie when the immune system starts to damage it against its body tissues, and patients are still on the rise. Autoimmune disease is a disease caused by excessive or inadequate control of the autoimmune response by inducing an inappropriate immune response as the environment of the body's cytokine (signaling substance that controls and stimulates the body's defense system) against viruses or bacteria is induced. However, the exact cause of the disease is not known and may be caused by environmental, genetic, or immunological factors. There are more than 80 known autoimmune diseases, of which US research shows that 2.1 million people have rheumatoid arthritis, 3.7 million people have fibromyalgia, and 4-4 million people have psoriasis. 30% are also arthritis patients, and 4 million vitiligo patients, and between 2 and 3 million people with Sjogren syndrome. In addition, known autoimmune eye diseases include uveitis, Behcet's disease, and keratoconjunctivitis, and dry eye is also regarded as an inflammatory disease due to an abnormal immune response, and dryness is also regarded as an autoimmune disease. Most autoimmune diseases use steroids or immunosuppressants in recent years. One of the representative autoimmune eye diseases is uveitis. The uvea consists of the iris, ciliary and choroid. The uvea is rich in blood vessels and connective tissue. Inflammation of the uvea is called uveitis, and the cause of the disease is unclear until now, but the cause is considered syphilis and tuberculosis. The cause of uveitis is currently looking at the immune system abnormalities. Inflammation at the uveal may be caused by bacteria or viruses, but the immune response is a lot of inflammation is classified as an autoimmune disease. Uveitis has been treated with topical or systemic steroids. However, topical treatments such as eye drops or systemic steroid treatments require high doses when the drug does not reach the uveal tissue, which often results in serious side effects. In addition, immunosuppressive drugs are used in patients who do not respond to steroid treatment or patients who cannot tolerate the severe side effects of systemic steroids, which have been discontinued due to side effects such as bone marrow suppression, hemorrhagic cystitis and kidney damage. These patients often lead to blindness. Therefore, it is urgent to develop eye drops for eye diseases with good eye penetration and few side effects.

Dry eye syndrome is a syndrome in which tear film is insufficient or the tear film is excessively evaporated and the tear film becomes unstable, resulting in various symptoms such as foreign body or stinging. Dry eye syndrome is associated with decreased tear secretion, disease of the eyeballs and eye appendages, such as eyelid abnormalities or inflammation, and skin diseases (Stevenson-Johnson syndrome, pemphigoid), and systemic diseases ( Vitamin A deficiency, Sjogren's syndrome) (Ophthalmology, 7th edition, Sculpture, Yoon Dong-ho. According to a recent survey by Chung-Ang University Hospital, 75% of Korean adults suffer from dry eye, and one out of three is a serious patient with inflammation of the cornea.

Treatment for dry eye syndrome focused on maintaining a certain amount of tears by conservative methods such as supplementing artificial eye drops or temporarily or permanently preventing tears, but recently dry eye syndrome has been newly recognized as an inflammatory disease. Anti-inflammatory treatments for dry eye syndrome have been actively attempted, and the effects have been reported for severe dry eye patients.

Inflammation of the ocular surface became a concern in the treatment of dry eye syndrome, as evidenced by increased T-lymphocytes and high levels of cytokines and various inflammatory mediators. One such drug is cyclosporin A 0.05% eye drop (Restasis). However, side effects occur in dry eye patients, and the most common adverse reaction is burning sensation in the eye (a sensation of burning when the drug touches the skin or tongue) and conjunctival hyperemia in 1-5% of patients. , Secretions, seborrhea, eye pain, foreign body sensation, pruritus, pain, visual acuity (often blurred vision) have been reported. Therefore, there is a need for the development of new inflammatory and immunosuppressive agents with fewer side effects.

Although the use of immunosuppressants is gradually increasing in ophthalmic diseases, the side effects are severe and there is a limit to use. Immunosuppressants refer to a variety of substances used by the host to reduce or block the ability to make antibodies (humoral immunity) or cellular immune responses, and mainly include selective immunosuppression, such as autoimmune diseases and fetal fibrosis; Used for therapeutic purposes to prevent rejection after organ transplantation. In addition to immunosuppressive functions, immunosuppressants have side effects such as anemia, leukopenia, thrombocytopenia, and hair loss, and are therefore limited in use. Cytotoxic agents with low cytotoxicity have been developed from secondary metabolites of bacteria and fungi, and cyclosporin A and FK506 (Tacrolimus), which are currently the most widely used in organ transplant patients, are typically used, but side effects are not completely reduced.

Ophthalmic disease therapies need to be changed from systemic to topical use and new drug development to reduce side effects of existing drugs. Recently, the most commonly used immunosuppressive agents, cyclosporin and FK506, are used systemically. Both drugs can cause damage to the kidneys and nervous system, which makes it difficult to use them widely. Development studies into eye drop formulations are actively underway. There has been a published paper showing that the use of FK506 in eye drops to suppress rejection after corneal transplantation has been shown to have an excellent effect on delaying rejection. All have been reported to have had a good effect. In the Department of Ophthalmology, the Catholic University of Korea, eye drops containing cyclosporine in patients with dry eye syndrome were reported to have increased tear secretion, especially in dry eye associated with systemic diseases. However, the clinical period is so short that limitations exist. The Pennsylvania School of Medicine's research team, through the Journal of the American Medical Association, is very effective in treating dry eye with cyclosporine in patients with moderate to moderate dry eye syndrome who do not respond to existing treatment among 15 to 34% of elderly people. Appeared.

Although eye drop formulations are more effective than systemic administration and can minimize side effects, intraocular penetration of drugs is very difficult and requires the introduction of new technologies. In particular, high molecular weight protein preparations are less easily penetrated than chemicals. There are many disturbing factors because the intracorneal penetration of the drug depends on the chemical composition of the epithelium and the parenchyma of the cornea. The epithelium of the cornea has a higher lipid molecular weight than the parenchymal portion, but the dissociative drug permeates well, but the parenchyma penetrates only the dissociated form. In addition, the endothelial contains a lipoid (lipoid) is characterized by passing only fat-soluble substances. Therefore, corneas are well penetrated by drugs having amphoteric and hydrophilic amphoteric properties. Therefore, in order to increase the permeability of the drug that does not penetrate the cornea well, the amount to be used in excess of the amount originally administered may result in side effects.

Patent No. 472938 relates to a transport domain-target protein-transport domain fusion protein and its use for improved cell penetration efficiency, wherein protein transport domains such as HIV-1 Tat peptide, oligolysine, oligoarginine, Disclosed is a fusion protein covalently attached to the terminal and / or C-terminus to enhance cell permeability.

In addition, Patent No. 493662 discloses a technique in which oligolysine as a protein transport domain is covalently bonded to the N-terminus and / or C-terminus of a protein to improve cell permeability.

Many researchers' papers, including those patents, have shown that HIV-a Tat peptide, oligolysine, oligoarginine, oligos (lysine, arginine) and PEP-1 peptides enhance the cellular permeability of proteins.

Patent Publication No. 2002-10445 discloses a Tat-superoxide dismutase fusion protein and discloses the use of therapeutic agents such as glomerulonephritis and vasculitis.

However, to date, no superoxide dismutase fusion protein has been identified for use in treating eye diseases such as dry eye.

Accordingly, an object of the present invention is to provide an ocular disease protein therapeutic agent for eye drops.

In order to achieve the above object, the present inventors studied to develop a protein agent, which is an immune response suppressing drug, into a formulation for enhancing the ocular penetration ability, and completed the present invention by identifying an ophthalmic disease therapeutic effect of an ophthalmic protein preparation.

The inventors of the present invention found that one of the protein transport domains, preferably PEP-1 peptides, derivatives thereof, HIV-1 Tat peptides, derivatives thereof, oligolysine, oligoarginine, oligo (lysine + arginine), which carries proteins in the cell in nature. More than one species were fused to the N- and / or C-terminus of the external protein human superoxide dismutase (hereinafter referred to as "SOD"), which was overexpressed in E. coli and metal chelating affinity chromatography Purification was easy and convenient by chromatography. In addition, it was confirmed through experiments that the purified fusion protein effectively improves eye diseases including dry eye. The present invention has raised the possibility of applying superoxide dismutase fusion proteins as protein therapeutics for ophthalmic diseases.

According to an aspect of the present invention, the present invention provides a superoxide dismutase fusion in which protein transport domains such as PEP-1 and Tat are covalently bonded to at least one of the N-terminus and C-terminus of the superoxide dismutase. Provided is a pharmaceutical composition for the treatment of ophthalmic diseases containing a protein (combined with "SOD fusion protein" hereinafter).

According to another aspect of the invention, the invention contains a superoxide dismutase fusion protein, and provides a pharmaceutical composition for the treatment of eye diseases such as dry eye. The superoxide dismutase fusion proteins of the invention are useful for the treatment of eye diseases, typically for the treatment of dry eye syndrome.

As used herein, the term "ophthalmic disease" refers to Stevenson-Johnson syndrome, Sjogren's syndrome, dry eye syndrome, trauma, eye trauma by eye surgery (eye surgery means any surgery to incision the eye, typically cataract surgery, Glaucoma surgery, retinal surgery, LASIK surgery, Lasek surgery, etc.), infectious / non-infective uveitis, immunorejection after corneal transplantation, or corneal epithelial disorder due to exogenous disease caused by wearing hard contact lenses. The ophthalmic disease is not necessarily limited thereto, and preferably includes dry eye syndrome.

As used herein, the term “dry eye” refers to a syndrome in which tear generation is insufficient or tears of the tear film are excessively evaporated, resulting in unstable tear film and various symptoms such as foreign body or stinging. More specifically, "dry eye syndrome" refers to Stevenson-Johnson syndrome or pemphigoid, which is a condition that is accompanied by decreased tear secretion or disease of the eyeballs and eye appendages, such as eyelid abnormalities, inflammation, or skin diseases. Vitamin A deficiency and Sjogren's syndrome, which is associated with the disease, impairs the surface of the eyeballs in the gaps of the exposed eyelids and causes irritation such as discomfort, foreign bodies, and dryness, and inflammation of the eye surface when the corneal damage is severe. Say disease. As the lesion progresses, congestion may be seen. Complications may include mild visual impairment at first, followed by corneal ulcers, corneal perforation, and secondary bacterial infections, and severe corneal scarring and angiogenesis. .

In the present invention, the superoxide dismutase fusion protein is basically an immunoassay method (eg, a radioimmunoassay method, a radioimmunoprecipitation method, an enzyme-linked immunity) using genetic recombination of a protein, an antigen-antibody reaction. Adsorption method (ELISA), dot blot analysis, western blot, inhibition or competition assay and sandswitch analysis (see Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Quantitative or qualitative analysis was performed according to an enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984), and molecular testing using PCR.

Efficacy verification for the ophthalmic disease represented by the superoxide dismutase fusion protein in the present invention can be carried out using a number of related papers and known dry eye animal models.

For example, the inventors used a dry blowing induced lett eye dry model as a short-term dry eye syndrome model. Surgical epithelial damage of 0.4 mm2 was applied to the median portion of the cornea using a surgical knife, and then dry eye was caused by exposure to a dry blower with a humidity of 25-30% and 2.4 m / sec. The tear secretion was measured, and corneal damage rate was measured using a fluorescent dye. The conjunctival eye and bleeding conjunctiva were extracted and examined histopathologically. Apoptosis of corneal epithelial cells was evaluated immunohistochemically using PARP {poly (ADP-ribose) polymerase}. In addition, the efficacy of the superoxide dismutase fusion protein on dry eye syndrome was compared to the validated 0.1% sodium hyaluronate (see Johnson et al. 2006, 2008) eye groups.

As another dry eye model, a botulinum toxin-A induced mouse dry eye model was used. 20 mU of BTX-A is injected into the lacrimal gland into the transconjunctival root to inject BTX-A into the tear-secreting portion of the lacrimal gland orbital lobe, causing dry eye I was. The corneal damage rate was measured using fluorescent dyes for dry eye efficacy test, and the conjunctiva attached eye and bleeding conjunctiva were removed and histopathologically observed.

Superoxide dismutase fusion protein as a novel dry eye treatment substance was calculated as 0.01mg / kg corresponding to 6 times of the effective dose 0.1% solution for the single dose intravenous toxicity test, and 500mg corresponding to 500 times. Toxicity experiment was performed by setting / kg as high dose group and 2.5mg / kg as low dose group.

As a result, the superoxide dismutase fusion protein according to the present invention significantly increased tear secretion in the dry eye animal model, increased the thickness of conjunctival epithelium thinned due to inflammation, and increased the number of mucus producing cells. In addition, the superoxide dismutase fusion protein according to the present invention reduced apoptosis during eye injury and decreased corneal damage.

The present invention relates to an eye drop composition for preventing and treating ophthalmic diseases comprising a fusion protein having a protein transport domain covalently attached to at least one of the N-terminus and the C-terminus of a superoxide dismutase.

In the present invention, the protein transport domain

a) a hydrophobic domain consisting of 15-30 amino acids, comprising at least five tryptophan, a hydrophilic domain containing at least four lysines, and a spacer separating the two domains ( protein transport domain, consisting of

b) a protein transport domain consisting of 6-12 amino acid residues and comprising at least 3/4 of arginine or lysine residues,

c) oligolysine protein transport domain consisting of 6 to 12 lysines,

d) an oligoarginine protein transport domain consisting of 6 to 12 arginine and

e) one or more selected from oligo (lysine, arginine) protein transport domains consisting of 6 to 12 lysine or arginine and derivatives of a) to e).

In addition, the present invention is characterized in that the fusion protein is one selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14.

In addition, the ophthalmic disease is characterized by corneal conjunctival epithelial disorder caused by Stevenson-Johnson syndrome, Sjogren's syndrome, dry eye syndrome, or an exogenous disease caused by wearing trauma or hard contact lenses.

Pharmaceutical compositions containing a superoxide dismutase fusion protein as an active ingredient can be formulated into eye drops by conventional methods in combination with a carrier that is conventionally acceptable in the pharmaceutical art. The eye drop composition is preferably an isotonic aqueous solution or suspension, and the composition mentioned is sterile and / or contains an adjuvant such as a preservative, stabilizer, wetting agent or salt / or buffer for osmotic pressure control. In addition, they may contain other therapeutically valuable substances.

In general, eye drop compositions have been known to moisturize and lubricate anionic polymers such as hyaluronic acid and carboxymethylcellulose or their pharmaceutically acceptable salts in eye drops, and include, in addition to these components, pharmaceutically acceptable carriers. Can be. Examples of the pharmaceutically acceptable carrier include isotonic agents, buffers, stabilizers, pH adjusting agents and solvents. Isotonic agents play a role in controlling the isotonicity of eye drops, and typically, sodium chloride or potassium chloride may be selected. The buffer performs the function of adjusting the acidity or alkalinity of the eye drops. Buffers usually used in the preparation of eye drops include aminocapronic acid, sodium dihydrogen phosphate and sodium dihydrogen phosphate. Stabilizers play a role of stabilizing eye drops, and it is usually possible to use sodium edetate and / or sodium perborate as stabilizers. pH adjusters adjust the pH of the eye drop composition, such as hydrochloric acid and / or sodium hydroxide. It is preferable to use sterile purified water or distilled water for injection as a solvent. The eye drop according to the present invention is preferably a liquid formulation. A preservative, a preservative, etc. can be added to the said eye drop composition as needed.

The recommended amount of the eye drop composition and the number of times of use according to the present invention can be appropriately increased or decreased depending on the symptoms, once or three times a day, 5 to 6 times a day. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health status, time of administration, frequency of administration, severity of the disease, and the like.

Intracellular delivery of a superoxide dismutase protein molecule according to the present invention consists of 15-30 amino acids in superoxide dismutase, a non-hydrophobic domain comprising five or more tryptophans, four lysines The target protein is a cell permeable transport domain including Pep-1 or a HIV Tat cell permeable domain, in which a transport domain including a hydrophilic domain including a plurality of the above and a spacer separating the two domains is covalently bonded. This is accomplished by constructing a fusion protein in a covalently bonded form with the N-terminus and / or C-terminus of the superoxide dismutase. An example of the transport domain of the present invention includes a PEP-1 peptide consisting of 21 amino acids and an amino acid sequence such as SEQ ID NO: 1, and an HIV Tat 49 to 57 peptide consisting of an amino acid sequence such as SEQ ID NO: 2. However, the protein transport domain of the present invention is not limited only to the PEP-1 peptide of SEQ ID NO: 1, the HIV Tat 49-57 residue peptide of SEQ ID NO: 2, and lacks a partial substitution, addition or addition of the amino acid sequence of PEP-1 or HIV Tat. It is easy for a person skilled in the art to prepare a peptide having a function similar to a PEP-1 peptide or an HIV Tat peptide, and thus it is composed of 15 to 30 amino acids and includes 5 or more tryptophans. A protein transport domain consisting of a non-hydrophobic domain, a hydrophilic domain containing four or more lysines, and a spacer separating the two domains, and the same and similar amino acid substitutions. Consists of a protein transport domain, or 6-12 amino acid residues, that performs protein transport function A transport domain comprising at least 3/4 of an arginine or lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoarginine transport domain consisting of 6 to 12 arginines or 6 to 12 lysine or arginine It will be apparent that the oligo (lysine, arginine) transport domain also belongs to the scope of the invention.

Specifically, the present invention relates to an eye drop composition for treating ophthalmic diseases comprising a superoxide dismutase fusion protein covalently bonded with the above protein transport domain.

Definitions of main terms used in the detailed description of the present invention are as follows.

"Superoxide dismutase fusion protein" includes a protein transport domain and a superoxide dismutase, and is a genetic fusion of a protein transport domain with a cargo molecule (ie, superoxide dismutase in the present invention). Or covalent complexes formed by chemical bonds. In the specification and drawings, "SOD fusion protein" was used interchangeably. As a specific example, "Tat-SOD" refers to a Tat protein transport domain coupled to the N-terminus of SOD in a superoxide dismutase fusion protein.

In addition, the "genetic fusion" means a linear, covalent linkage formed through the genetic expression of the DNA sequence encoding the protein.

"Target cell" also means a cell to which a cargo molecule is delivered by a transport domain, i.e., a target cell is a cell in the body, i. It is meant to include the microorganisms found. In addition, target cells are meant to include extracellular cells, ie cultured animal cells, human cells or microorganisms. Specifically, in the present specification, the target cell refers to an eye cell.

As used herein, the term "protein transport domain" refers to a covalent bond with a cargo molecule (target protein) peptide or protein, so that the peptide or protein can be introduced into a cell without the need for a separate receptor, carrier, or energy. For example, PEP-1 peptide (SEQ ID NO: 1).

As used herein, “target protein” refers to a molecule that is covalently bound to a PEP-1 protein transport domain and introduced into a cell to exhibit activity. It is synonymous with "cargo molecule".

In addition, the present specification is interchangeable with the expressions "transduction", "transport", "penetration", "transport", "transfer", "transmission", "pass" for "introducing" a protein or peptide into a cell. It was.

In the present invention, the protein transport domain is composed of 15 to 30 amino acids, a non-hydrophilic domain including 5 or more tryptophan, a hydrophilic domain including a large number of lysine and a protein transport domain consisting of a spacer separating the two domains, A transport domain consisting of 6 to 12 amino acid residues and containing at least 3/4 of an arginine or lysine residue, an oligolysine transport domain consisting of 6 to 12 lysines, an oligoarginine transport domain consisting of 6 to 12 arginines, or 6 And oligo (lysine, arginine) transport domains composed of from twelve lysine or arginine. In addition, the target protein (cargo molecule) is superoxide dismutase. The protein transport domain and the target protein may be substituted with other amino acid (s) of similar polarity in which one or more amino acids in the sequence are functionally equivalent in response to a silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, hydrophobic amino acid classifications include alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, proline and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positive basic amino acids include arginine, lysine and histidine. Negatively charged acidic amino acids include aspartic acid and glutamic acid. In addition, fragments or derivatives thereof having the same or similar biological activity within a range of homology between the fusion protein and the amino acid sequence of the present invention, such as 85-100%, are also included in the scope of the present invention.

In addition, the present invention is characterized in that the cell introducing superoxide dismutase fusion protein has an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

In addition, the present invention is characterized in that the protein transducing domain (PTD) is covalently bonded to one or both sides of the carboxy terminus and amino terminus of the superoxide dismutase.

The present invention also relates to an eye drop composition for treating ophthalmic diseases comprising the cell-transducing superoxide dismutase fusion protein as an active ingredient.

The present invention also relates to an eye drop composition for treating ophthalmic diseases comprising the cell-introduced superoxide dismutase fusion protein as an active ingredient and a pharmaceutically acceptable carrier.

The superoxide dismutase fusion protein according to the present invention significantly increased tear secretion in dry eye animal models.

In addition, the superoxide dismutase fusion protein according to the present invention increased the thickness of conjunctival epithelium thinned due to inflammation and increased the number of mucus producing cells.

In addition, the superoxide dismutase fusion protein according to the present invention reduced apoptosis during ocular injury, decreased corneal damage, and did not find any abnormality as a result of conducting toxicological experiments in vivo.

FIG. 1 is a diagram showing a cumulative test using 1 × 15 mm cobalt chloride paper and showing changes in tear secretion before and after dry blowing. In FIG. 1, B represents before dry blowing, and A represents after dry blowing. The two controls were intact, that is, the group that did not perform dry blowing and the negative eye group that caused dry eye and did not receive the drug. The drug control group is a control group to which hyaluronic acid is added, and the test group is a group treated with the superoxide dismutase fusion protein of the present invention. Each drug was administered 5 μl per eye once every hour for 5 hours.

FIG. 2 is a histopathological picture of the effect of superoxide dismutase fusion protein drug on the conjunctival epithelial thickness and the number of mucus producing cells in a dry blowing-induced dry eye model using PAS staining. In Figure 2 A is a normal control group, B is a dry blowing induced negative control group, C is a drug control group administered hyaluronic acid, D is a test group administered a superoxide dismutase fusion protein. Each drug was administered 5 μl per eye for 1 hour for 5 hours. ThE in FIG. 2A shows the epithelial thickness measurement range.

Figure 3 is a photograph of the immunohistochemical observation of the degree of apoptosis of the cornea in a dry blowing induced eye dry model using PARP {Poly (ADP-ribose) polymerase}. A is a normal control group, B is a dry blowing induced negative control group, C is a drug control group, D is a test group administered the superoxide dismutase fusion protein of the present invention. Each drug was administered 5 μl per eye for 1 hour for 5 hours. The scale bar in the figure is 80 μm.

4 is a photograph taken with a slit lamp after illuminating a fluorescent light source after 1 μl of 1% fluorescein in a BTX-A-induced dry eye syndrome model. "Control" is normal control group, "Saline" is saline added control group, "BTX" is BTX-A added control group, "SOD" is a test group administered superoxide dismutase protein, not fusion protein, "Tat" -SOD "is a test group administered superoxide dismutase fusion protein. Each drug was administered 5 μl per eye for 1 hour for 5 hours.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention in more detail, and it is apparent to those skilled in the art that the scope of the present invention is not limited to the description of these examples. In particular, as a result of each experimental example, the data of the Tat-SOD fusion protein was mainly described among the various fusion proteins prepared in the specific examples, but other fusion proteins were also the result of the Tat-SOD fusion protein as a sample. The results showed similar results (70-110%).

<Material>

Restriction enzymes and T4 DNA ligase were purchased from Promega (USA) and Pfu polymerase was purchased from stratagene (USA). Tat oligonucleotides were synthesized in Gibco BRL custom primer (USA). IPTG was purchased from Duchefa (Netherland). pET-15b and BL21 (DE3) plasmids were purchased from Novagen (USA) and Ni-nitrilo-triacetic acid Sepharose superflow was purchased from Qiagen (Germany). Human superoxide dismutase (mixed herein with the same meaning as "superoxide dismutase", "SOD") cDNA was isolated from human placental cDNA library by polymerase chain reaction method [Kang, JH, Choi, BJ and Kim, S. M. (1997) J. Biochem. Mol. Biol. 30, 60-65]. All other reagents were used as express products. In addition, in the embodiment of the present invention, the fusion protein was prepared using human superoxide dismutase, but the scope of the present invention does not only affect the fusion protein using human superoxide dismutase, but it is a yeast-derived, bacterial-derived enzyme. It is also found that the effect of the present invention can be obtained even when using the above.

<Example 1: Preparation and transformation of expression vector of PEP-1-SOD fusion protein>

In order to develop a technique for infiltrating a protein with a function into a cell, a fusion protein expression vector capable of delivering a target protein into a cell was prepared, and a human super-penetration was performed to easily analyze the ability of the PEP-1 peptide to deliver the protein into the cell. Oxide dismutase was selected.

First, PEP-1-SOD To produce a fusion protein, a pET-PEP expression vector containing a PEP-1 peptide (KETWWE TWWTEW SQP KKKRKV) (SEQ ID NO: 1) was prepared. Two types of oligonucleotides corresponding to the PEP-1 peptide (top chain, 5'-TATGAAAGAAACCTGGTGGGAAACCTGGTGGACCGAATGGTCTCAGCCGAAAAAAAAACGTAAAGTGC-3 '(SEQ ID NO: 15);NdeⅠ-XhoⅠ It was inserted by ligation to pET-15b cut with restriction enzyme. Subsequently, two kinds of oligonucleotides were synthesized based on the cDNA sequence of human superoxide dismutase. Forward primer is 5'-CTCGAGGCGACGAAGGCCGTGTGCGTG-3 '(SEQ ID NO: 17)XhoⅠRestriction primer and reverse primer is 5'-GGATCCTTATTGGGCGATCCCAATTAC-3 '(SEQ ID NO: 18)BamHⅠ It has a restriction site.

Polymerase chain reaction (PCR) was carried out in a thermal cycle reactor (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl silicon tube and heated at 94 ° C. for 5 minutes. PCR reactions were performed. After PCR, the reactants were separated by agarose gel electrophoresis to isolate the reactants and ligation to the TA cloning vector (Invitrogen, Sandiego, USA), and then transformed into competent competent cells (competent cells). Plasmids were isolated from the transformed bacteria by alkaline lysis method. Cutting the TA vector containing the human SOD cDNA as XhoⅠ BamHⅠ and then was inserted into the PEP expression vector. E. coli BL21 (DE3) transformed with PEP-1-SOD was selected and overexpressed of the recombinant PEP-1-SOD fusion protein by inoculating colonies into 100 ml LB medium and adding IPTG (0.5 mM) into the medium. Induced. Overexpressed PEP-1-SOD fusion protein was confirmed by SDS-PAGE and Western blot analysis.

The method was modified to produce SOD-PEP-1 fusion protein and PEP-1-SOD-PEP-1 fusion protein.

Example 2: Expression and Purification of PEP-1-SOD Fusion Proteins

E. coli BL21 (DE3) cells containing human SOD cDNA prepared in Example 1 in PEP-1-SOD form were placed in LB medium containing ampicillin and cultured at 37 ° C. at 200 rpm. When the bacterial concentration in the culture medium was OD ₆₀₀ = 0.5 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 and 1 mM, followed by further incubation at 30 ° C for 12 hours. The cultured cells were collected by centrifugation, and then 5ml binding buffer (5mM imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) was added and pulverized by an ultrasonic grinder. The supernatant was immediately centrifuged Ni ²⁺ - acrylonitrile triazol setik acid Sepharose Super flow ^{(Ni 2+ -nitrilotriacetic acid sepharose super flow} ) load the column and binding buffer of 10 times by volume with 6-fold volume wash buffer (60mM of The fusion protein was washed with imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) followed by elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected and PD-10 column chromatography was performed to remove salts contained in the fractions.

Purified protein concentration was determined by Bradford method using bovine serum albumin as a standard.

In the same manner as above, the SOD-PEP-1 fusion protein and PEP-1-SOD-PEP-1 fusion protein were overexpressed and purified.

<Example 3: Preparation and transformation of Tat-SOD fusion protein expression vector>

To overexpress Tat-SOD fusion protein, pET-Tat-SOD expression vector containing superoxide dismutase, transduction site of HIV-1 Tat (Tat49-57) and cDNA for 6 histidines in sequence Prepared. First, a pET-Tat expression vector containing a basic domain of HIV-1 Tat (ie, amino acids 49-57) was prepared. Two kinds of oligonucleotides corresponding to the Tat base domain (top strand, 5'-TAGGAAGAAGCGGAGACAGCGACGAAGAC-3 '(SEQ ID NO: 19); bottom strand, 5'-TCGAGTCTTCGTCGCTGTCTCCGCTTCTTCC-3' (SEQ ID NO: 20) )) Was ligated into pET15b cut with NdeI-XhoI restriction enzyme. Then, two kinds of oligonucleotides were synthesized based on the sequence of cDNA of human SOD. The forward primer has a Xho I restriction site as SEQ ID NO: 17 of Example 1 above, and the reverse primer has a BamH I restriction site with SEQ ID NO: 18 of Example 1 above.

Polymerase chain reaction (PCR) was performed in a thermal cycler (Perkin-Elmer, model 9600). The reaction mixture was placed in a 50 μl silicon tube (siliconized reaction tube) and heated at 94 ° C. for 5 minutes. PCR reactions consisted of 30 extensions for 40 seconds at 94 ° C, denaturation for 1 minute at 54 ° C, annealing for 3 minutes at 70 ° C, and final extension for 5 minutes at 20 ° C for 10 minutes at 72 ° C. final extension). After PCR, the reactants were separated by agarose gel electrophoresis and ligated to TA cloning vectors (Invitrogen, Sandiego, USA). This vector was then transformed into transformant cells and the plasmids were isolated from the transformed bacteria by alkaline lysis method [Sambrook, J., Fritsch, F.E. and Maniatis, T (1989) Molecular cloning, Cold spring harbor laboratory press, Cold spring harbor]. TA vectors containing human superoxide dismutase cDNA were digested with Xho I and BamH I and inserted into pET-15b and pET-15b-Tat expression vectors. Expression of the vector is under the control of the T7 promoter and lacO-operator.

E. coli BL21 (DE3) transformed with pET-Tat-SOD was selected, then colonies were inoculated in 100 ml LB medium and IPTG (0.5 mM) was added into the medium to induce overexpression. E. coli cells, which induced overexpression of the fusion protein with IPTG, were disrupted by sonication at 4 ° C, and then centrifuged to separate proteins from the supernatant by 15% SDS-polyacrylamide gel electrophoresis. Overexpressed SOD and Tat-SOD were confirmed by SDS-polyacrylamide gel electrophoresis and Western blot analysis.

The method was modified to produce SOD-Tat fusion protein and Tat-SOD-Tat fusion protein.

Example 4 Tat-SOD Fusion Protein Overexpression and Purification

Transformed E. coli BL21 was put in LB medium containing ampicillin and incubated at 37 ° C. at 200 rpm. When the bacterial concentration in the culture medium showed O.D 600 = 0.5 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, followed by further incubation for 3 hours. The cultured cells were collected by centrifugation and sonicated with 5 ml binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9).

The supernatant was immediately loaded by centrifugation into a 2.5 ml Ni 2+ -nitrilotriacetic acid sepharose column and 10-fold volume of binding buffer and 6-volume washing buffer (60 mM imidazole). , 0.5M NaCl, 20mM Tris-HCl, pH 7.9), and then the fusion protein was eluted with elution buffer (1M imidazole, 0.5M NaCl, 20mM Tris-HCl, pH 7.9). Subsequently, the fractions containing the fusion protein were collected, and Sephadex G-15 column chromatography was performed to remove the salts contained in the fractions. Since the fusion protein contained six histidines at the N-terminus, the fusion protein was purified almost purely (purity> 90%) in a single step of immobilized metal-chelate affinity chromatography. Protein concentrations of the fractions were determined by Bradford method using bovine serum albumin as standard [Bradford, M.A. (1976) Anal. Biochem. 72, 248-254.

In the same manner as above, the SOD-Tat fusion protein and Tat-SOD-Tat fusion protein were overexpressed and purified.

Example 5: Sample Preparation

Various SOD fusion proteins purified in Examples 2 and 4 were dissolved in 5 μg / 5 μl in physiological saline to be used as samples in subsequent experiments.

Experimental Example 1: Changes in tear secretion by Schirmer method in dry blowing induced eye dry model

1-1. Dry Blowing Dry Eye Induced Animal Model

Surgical epithelial damage of 0.4 mm2 was applied to the median cornea with a surgical knife, and then dry eye was induced by exposure to dry air with a humidity of 25-30% and 2.4 m / sec.

1-2. Changes in tear secretion

Tear secretion changes were tested according to the Schirmer method (cobalt chloride paper; Toyo Roshi Kaisha, Japan). In general, the Schirmer test using a phenol red thread is known as the most common method of measuring changes in tear secretion and is the most basic method for evaluating corneal dryness. Fujihara et al. 2001, Invest.Ophthalmol.Vis.Sci. 2001; 42: 96-100, Nakamura et al., Cornea 2004; 23: 390-7). In this example, a change in tear secretion amount before and after dry blowing was observed using 1 × 15 mm cobalt chloride paper.

In the dry eye-induced control group, a significant decrease in tear secretion occurred compared to the normal control group, and the superoxide dismutase fusion protein of the present invention compared with the control drug hyaluronic acid (Hyaluronate; Samil Pharm. Co., Seoul, Korea) Reduction of tear secretion was suppressed in the test group. The results are summarized in FIG. 1 and Table 1. FIG.

Table 1

		Tear secretion (mm)
		Before the test	After the test	Change
Control	normal	8.86 ± 0.97	10.19 ± 0.70	1.33 ± 1.15
	Dry Eye Induction	9.02 ± 1.50	7.65 ± 0.72	-1.36 ± 1.43
Drug control	Hyaluronic Acid (Hyaluronate)	8.95 ± 0.84	9.43 ± 1.52	0.48 ± 1.16
Experimental group	Tat-SOD	9.05 ± 1.24	9.67 ± 1.30	0.62 ± 1.02

Experimental Example 2: Histopathological Changes of the Eye Conjunctiva in Dry Blow-Induced Dry Eye Model

The drug was administered using a dry blowing-induced dry eye model, and the conjunctiva-attached eye was extracted and histopathological observation was performed by PAS (Periodic acid-Schiff) staining.

Dry eye results in corneal and conjunctival damage, and such changes can be readily determined histopathologically (see Nakamura et al., Invest.Ophthalmol. Vis. Sci. 2003; 44: 4682-8, Higuchi et al. , Curr Eye Res 2007; 32: 83-8). Compared to the normal group, the dry eye-induced control group showed damage mainly due to local dropout of the conjunctival epithelium and reduction of the mucous producing cells. There was a significant decrease in the thickness of the conjunctival epithelium, the number and proportion of the mucous producing cells, and the conjunctival epithelium. There was a significant increase in the damage area, respectively. This conjunctival injury was significantly suppressed in the superoxide dismutase fusion protein treated group as compared to the dry eye induced control group. The results are summarized in Table 2 and FIG.

TABLE 2

		Corneal Epithelial Thickness (μm)	Mucous producing cells
			Number (cells / mm epithelium)	Range (% of mm epithelium)
Control	normal	113.21 ± 12.06	17.25 ± 3.15	40.03 ± 9.56
	Dry Eye Induction	38.13 ± 10.26	4.00 ± 1.93	10.02 ± 5.35
Drug control	Hyaluronic Acid (Hyaluronate)	63.22 ± 10.53	8.63 ± 2.26	22.19 ± 7.02
Test group	Tat-SOD	88.17 ± 14.83	10.88 ± 2.36	25.37 ± 7.33

Experimental Example 3: Changes in Apoptosis of the Eye Cornea in a Dry Blow-Induced Dry Eye Model

The corneal epithelial cell death was assessed immunohistochemically using PARP {poly (ADP-ribose) polymerase}.

PARP is a representative apoptosis marker (see Barrett et al., J Histochem Cytochem 2001; 49: 821-32). Increased PARP in corneal epithelium indicates increased corneal epithelial damage due to apoptosis and is caused by dry eye Eye injury is also known to be involved in some degree of cell death (see Yeh et al., Invest.Ophthalmol. Vis. Sci. 2003; 44: 124-9).

In the dry eye induced control group, a significant increase in the apoptosis marker PARP immunoreactivity was observed compared to the normal group. The control drug hyaluronic acid (Hyaluronate) and the test group superoxide dismutase fusion protein administration group similarly reduced apoptosis significantly compared to the dry eye induced control group. The results are summarized in FIG.

Experimental Example 4: Eye Corneal Damage in BTX-A-Induced Dry Eye Model

4-1. BTX-A Dry Eye Induced Animal Model

Dry-secreted portion of the botulinum toxin-A-induced mouse dry eye model was injected with 20 mU of BTX-A into the transconjunctival root in the lacrimal gland and the tear-secreting portion of the lacrimal gland orbital lobe. ) Was injected with BTX-A to cause dry eye syndrome.

4-2. Changes in Corneal Fluorescent Dye Penetration

In the BTX-A induced dry eye model, corneal damage was observed using a fluorescent dye (fluorescein sodium salt, Sigma Co., USA) for the dry eye efficacy test of superoxide dismutase fusion protein.

Corneal penetration rate of fluorescent dyes is the most common method of assessing corneal permeability. Increased permeability is known to mean increased corneal damage (see Yokoi & Kinoshita, Cornea 1995; 14: 485-9, Nakamura et. al., Invest.Ophthalmol.Vis.Sci. 2003; 44: 4682-8, Nakamura et al., Invest.Ophthalmol.Vis.Sci.2005; 46: 2379-87, Steinfeld et al., Br.J.Ophthalmol 2004; 88: 48-53), it is easy to identify invasive damaged areas by the deposition or transmission of fluorescent dyes on the cornea (Koh et al., Am J Ophthalmol 2003; 136: 513-9).

In the dry eye group, the corneal permeability was significantly increased compared to the normal group. The test group was divided into a superoxide dismutase protein and a superoxide dismutase fusion protein. The superoxide dismutase fusion protein group significantly decreased the fluorescence transmittance compared to the superoxide dismutase protein group. The results are summarized in FIG.

Experimental Example 5: Lett single intravenous toxicity test of superoxide dismutase fusion protein

For the single-dose intravenous toxicity test of the novel dry eye therapeutic substance superoxide dismutase fusion protein, 500 mg of 5 mg, calculated as 0.01 mg / kg corresponding to the total 6 eye drops of 0.1% solution Toxicity experiment was performed by setting / kg as high dose group and 2.5mg / kg as low dose group. Each experimental group was tested five dogs each.

The main toxicological observations were mortality, clinical symptoms, weight change, autopsy findings, organ weight, histopathological changes, and long-term observations were lung, heart, thymus, kidney, adrenal gland, spleen, testis / ovary, liver, pancreas, brain, 17 sites including the epididymis / uterus, the submandibular lymph nodes, the bladder, the prostate gland, and the dorsal vein.

As a result, the superoxide dismutase fusion protein showed no death in both low and high dose groups, and the clinical symptoms, weight, organ weight, gross and histological abnormalities due to the drug administration would be very safe. Judging.

Formulation Example 1

An eye drop composition comprising the SOD fusion protein prepared in the above Example was prepared as shown in Table 3.

TABLE 3

Ingredient		Content (mg)
chief ingredient	Tat-SOD	5
pH regulator	Hydrochloric acid	Quantity
	Sodium hydroxide	Quantity
Tonicity	Sodium chloride	700
	Potassium chloride	150
Buffer	Aminocaproic acid	200
stabilizator	Sodium Ethate	10
solvent	Sterilized Purified Water	Quantity
Total amount		100 ml

Formulation Example 2

An eye drop composition comprising the SOD fusion protein prepared in the above Example was prepared as shown in Table 4.

Table 4

Ingredient		Content (mg)
chief ingredient	Tat-SOD	5
pH regulator	Hydrochloric acid	Quantity
	Sodium hydroxide	Quantity
Tonicity	Sodium chloride	700
	Potassium chloride	150
Buffer	Aminocaproic acid	200
stabilizator	Sodium Ethate	10
Preservative	Benzalkonium chloride	30
solvent	Sterilized Purified Water	Quantity
Total amount		100 ml

The superoxide dismutase fusion proteins according to the present invention can be used as protein therapeutics for ophthalmic diseases.

Sequences attached to the present invention are primer sequences, fusion protein sequences associated with protein transport domain sequences and protein transport domains.

Claims (4)

1. An eye drop composition for preventing and treating ophthalmic diseases comprising a fusion protein having a protein transport domain covalently bonded to at least one of the N-terminus and C-terminus of superoxide dismutase.
2. The method according to claim 1,

   The protein transport domain is

   a) a hydrophobic domain consisting of 15-30 amino acids, comprising at least five tryptophan, a hydrophilic domain containing at least four lysines, and a spacer separating the two domains ( protein transport domain, consisting of

   b) a protein transport domain consisting of 6-12 amino acid residues and comprising at least 3/4 of arginine or lysine residues,

   c) oligolysine protein transport domain consisting of 6 to 12 lysines,

   d) an oligoarginine protein transport domain consisting of 6 to 12 arginine and

   e) at least one selected from oligo (lysine, arginine) protein transport domains consisting of 6 to 12 lysine or arginine.
3. The method according to claim 1,

   The fusion protein is a composition, characterized in that one selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.
4. The method according to claim 1,

   The ocular diseases include Stevenson-Johnson syndrome, Sjogren's syndrome, dry eye syndrome, trauma, ocular trauma due to eye surgery, uveitis (infectious or non-infectious), exogenous rejection after corneal graft surgery or wearing hard contact lenses. The composition characterized in that the corneal conjunctival epithelial disorder.

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