JP2010083829A - Cornea protectant and cornea trouble ameliorating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cornea protectant or a cornea trouble ameliorate agent, further an corneal epithelium trouble healing accelerator which are useful for cornea trouble developed according to various causes and especially for ameliorating corneal epithelium trouble, and in which safety is high. <P>SOLUTION: The cornea protectant or the cornea trouble ameliorating agent is characterized by comprising sericin as an active ingredient. Moreover, the corneal epithelium trouble healing accelerator is characterized by comprising the sericin as an active ingredient. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a corneal protective agent or corneal disorder ameliorating agent comprising sericin as an active ingredient. Specifically, the present invention relates to a corneal epithelial disorder healing promoter useful for improving corneal epithelial disorder, comprising sericin as an active ingredient.

Background Art The cornea is composed of five layers: a corneal epithelial layer, a Bowman membrane, a corneal stroma layer, a Descemet's membrane, and a corneal endothelium layer. Of these, the corneal epithelial layer is located on the outermost surface of the cornea and is liable to be damaged due to the influence from the outside. The corneal epithelial disorder literally means that the corneal epithelial cells that constitute the corneal epithelial layer are damaged. , Corneal erosion, persistent corneal epithelial defect or corneal ulcer.

  The causes of corneal epithelial disorders are diverse and can be broadly divided into those caused by external factors and those caused by internal factors. External factors include, for example, trauma, burns, chemical substances, drugs, ultraviolet rays, contact lens wear, foreign matter, eyelashes, dry air, infection, and the like. In addition, the corneal epithelium may be damaged by surgical invasion of the cornea such as corneal refractive surgery and cataract surgery. In addition, there are corneal epithelial disorders caused by intrinsic diseases (internal factors) such as Sjogren's syndrome, dry eye syndrome (dry eye), neuroparalytic keratitis, and diabetic keratopathy.

For example, dry eye refers to a disease in which the surface of the eye is dried and damaged due to a decrease in tears or qualitative abnormality. It is said that the onset increases due to overuse of the eyes by operating the personal computer, drying of the air by air conditioning, wearing of contact lenses, etc., and it has been increasing in recent years. In addition, dry eye may develop as a symptom of an endogenous disease (typically, Sjogren's syndrome). Since dry eye is a disease accompanied by indefinite complaints, appropriate treatment is important in terms of improving the quality of life (QOL) of patients.
With the modern visual information society and the widespread use of contact lenses, eye diseases are becoming increasingly diversified, and the protection of corneal epithelial cells that are constantly exposed to external risk factors and the improvement of the damage are important issues.

  Traditional treatments for corneal epithelial disorders have been inactive, primarily protecting the cornea from external factors and relying solely on natural healing. In such a treatment method, healing is often delayed, and infectious diseases caused by microorganisms are accompanied in the meantime, which may eventually lead to visual impairment or blindness, and is regarded as a problem.

  When the corneal epithelial layer is damaged and damaged, epithelial cells around the defect firstly expand, move, and adhere to the defect, and then cell proliferation proceeds, the cells differentiate, and the disorder is cured. It is supposed to be. With the development of cell biology, substances involved in these healing processes have been elucidated, for example, fibronectin, hyaluronic acid, EGF (epidermal growth factor), IL-6 (interleukin-6), FGF (fibroblast growth). factor), KGF (keratinocyte growth factor), and vitamin A have been reported as regulatory factors (Non-patent Document 1).

  However, these have only been confirmed to be involved in epithelial cell spreading, proliferation, or differentiation in in vitro culture tests. Actually, the improvement of corneal epithelial disorder by instillation or the like has been limited in vivo. This is because the situation and environment of cells, substances, temperature, pH conversion, metabolism, etc. involved are extremely complex in vivo (in vivo), and the results of in vitro culture tests are simply expressed as in vivo results. It is difficult to expect.

  For example, the use of growth factors such as EGF and FGF having a strong cell growth promoting action for the treatment of disorders of the corneal epithelial layer has been studied. This is because by applying these growth factors to damaged tissues, it is expected that the growth of corneal epithelial cells is promoted to promote cell extension and movement, and as a result, the repair of the corneal epithelial layer is promoted.

  However, in fact, it has been reported that growth factors such as EGF and FGF did not have a definitive effect in clinical trials for corneal ulcers (Patent Document 1, Non-Patent Document 2). . In addition, it has been pointed out that cell proliferation effects such as EGF can also promote proliferation of corneal parenchymal cells and endothelial cells other than corneal epithelial cells, thereby causing corneal opacity and reducing patient QOL (Patent Literature). 2). Furthermore, these growth factors have a carcinogenic problem and a risk of inducing angiogenesis, and are considered to be serious side effects when used as a medicine. These factors are difficult to manufacture and are barriers to pharmaceuticals, and there are many problems that should be cleared in terms of safety and manufacturing cost.

Thus, even if a factor having a cell growth promoting effect is used for the treatment of a disorder of the corneal epithelial layer, it is only related to the process of cell proliferation in the repair of the disorder of the corneal epithelial layer, It does not necessarily guarantee that the epithelial cells extend / move and adhere around the defect and differentiation of the cells into tissues, nor does it guarantee that they will not adversely affect them. In addition, having a cell proliferating action is considered to cause the proliferation of unexpected cells and tissues other than corneal epithelial cells in vivo, and also abnormal cell growth such as carcinogenic action. There is no denying the danger of incurring.
For this reason, it is totally unpredictable whether or not it is possible to improve the damage of the corneal epithelial layer normally and safely only by finding that the substance has a cell proliferation action.

  In addition to such factors that have a cell growth promoting action, those that have been clinically applied and actively confirmed for active treatment of corneal epithelial disorders include, for example, fibronectin and hyaluronic acid. It is done. However, since fibronectin is a blood-derived component, it is still not satisfactory in terms of safety and antigenicity. In addition, hyaluronic acid is limited to corneal epithelial disorders not accompanied by lacrimal secretion, and is not versatile.

  Therefore, it can be said that a highly safe substance that is useful for improving corneal epithelial disorder is still strongly desired.

  Sericin, which is a silk protein, is composed of 18 types of amino acids. Due to its excellent biocompatibility, its application to foods, cosmetics, pharmaceuticals, etc. has been studied, and some have been put into practical use. Japanese Patent Laid-Open No. 2006-115837 (Patent Document 3) discloses that sericin has a cell cryoprotective action, and International Publication WO2002 / 86133 (Patent Document 4) discloses sericin. It is disclosed that it is added to a culture medium for growing cultured cells.

  However, as far as the present inventors know, it has not been reported so far that sericin has an ameliorating effect on corneal epithelial disorder, particularly a healing promoting effect. In addition, as far as the present inventors know, the administration of sericin itself has not been reported.

JP 2001-64198 A JP 2004-59432 A JP 2006-115837 A International Publication WO2002 / 86133 Pamphlet Ophthalmology 39: 1387-1393,1997 N.Engl.J. Med 338 (17) 1222 (1998)

Summary of the Invention

  The present inventors have now discovered that the silk protein sericin has an excellent healing promoting effect on corneal epithelial disorders. This indicates that sericin is useful for protecting the cornea and improving corneal damage. The present invention is based on these findings.

  Therefore, the present invention provides a corneal protective agent or corneal disorder ameliorating agent, and further a corneal epithelial disorder healing accelerator, which is useful for improving corneal disorders caused by various causes, particularly corneal epithelial disorders, and is highly safe. For the purpose.

  The corneal protective agent or corneal disorder ameliorating agent according to the present invention comprises sericin as an active ingredient. Preferably, the agent is used for protecting the corneal epithelium or ameliorating corneal epithelial disorders.

  The corneal epithelial disorder healing promoter according to the present invention comprises sericin as an active ingredient.

  According to one preferred embodiment of the present invention, in the agent according to the present invention, that is, the corneal protective agent, the corneal disorder ameliorating agent, or the corneal epithelial disorder healing promoter, sericin is obtained by extraction from silk thread.

  According to one more preferred embodiment of the present invention, the average molecular weight of sericin in the medicament of the present invention is 3,000 to 300,000.

  According to another preferred embodiment of the present invention, sericin is a hydrolyzate in the medicament of the present invention.

  According to another more preferred aspect of the present invention, the agent of the present invention is provided as an eye drop.

  According to the corneal protective agent, corneal disorder ameliorating agent, or corneal epithelial disorder healing promoter of the present invention, it is possible to safely and effectively prevent, improve, and promote healing of corneal disorders caused by various causes, particularly corneal epithelial disorders. It can be carried out.

Detailed description of the invention

Active ingredient In the present invention, sericin used as an active ingredient may be derived from a natural product or artificially synthesized by a conventional chemical and / or genetic engineering technique. Are also included. Therefore, for example, sericin produced by a rabbit or a barb can be used as sericin. A natural product is advantageous because it is highly safe and relatively inexpensive. Preferably, sericin is highly purified and substantially free of impurities.

  The sericin in the present invention includes sericin and a hydrolyzate thereof. Here, the hydrolyzate of sericin can be obtained by subjecting a non-hydrolyzed product of sericin to hydrolysis using conventional means such as alkali or enzyme.

  In the present invention, sericin derived from a natural product can be obtained by extracting sericin present in silk thread by a conventional method. Specifically, for example, sericin can be obtained by using a silkworm, raw silk, silk fabric, or the like as a raw material, and hydrolyzing or extracting the raw material partially with hot water, acid, alkali, enzyme, or the like. . The obtained sericin is purified to a high purity (for example, a total nitrogen amount of 13% or more of sericin solids) by the method described in Japanese Patent No. 3011759 as necessary, and prepared in a solid form such as a powder. The Commercial products (for example, pure sericin (trade name), available from Wako Pure Chemical Industries, Ltd.) can also be used.

  In the present invention, the average molecular weight of sericin is not particularly limited, but is preferably 3,000 to 300,000, more preferably 5,000 to 100,000, and still more preferably 10,000 to 50,000. It is. When the average molecular weight of sericin is 300,000 or less, the aqueous solution can be prevented from gelation at a concentration of about 0.5% (w / v), and the use form can be prevented from being limited. it can. Moreover, it is advantageous when the average molecular weight of sericin is 3,000 or more when exhibiting the healing promotion effect with respect to a corneal epithelial disorder.

  Sericin (preferably hydrolyzed sericin) used in the present invention is easily water-soluble and can maintain stable properties as an aqueous solution. For this reason, it is excellent in miscibility with other components usually added to pharmaceuticals. In addition, sericin is non-toxic, tasteless and odorless and has excellent safety. For this reason, there are almost no worries about the influence on a sensory characteristic and a side effect.

  Sericin, which is an active ingredient in the present invention, is known to have a cell growth promoting action, but as shown in the examples such as EGF and FGF described above, even if the proliferation of corneal epithelial cells is merely promoted, As described above, it does not necessarily promote the healing of corneal epithelial disorder. Such a fact can be said to have been well known to those skilled in the art. Under such circumstances, it can be said that it was unexpected and surprising that sericin was able to exert a healing promoting effect on corneal epithelial disorder without causing side effects such as corneal opacity. This is thought to be due to the enhancement of cell adhesion in addition to cell proliferation by sericin, thereby suppressing cell shedding and further promoting the healing effect of corneal epithelial disorder. These are theories and do not constrain the present invention.

Drug As described above, the corneal protective agent or corneal disorder ameliorating agent according to the present invention is characterized by containing sericin as an active ingredient. Preferably, the agent is used for protecting the corneal epithelium or ameliorating corneal epithelial disorders. More preferably, the corneal epithelial disorder improving effect is a corneal disorder healing promoting effect, and more preferably a corneal epithelial healing promoting effect. That is, a preferred embodiment of the corneal protective agent or corneal disorder ameliorating agent includes a corneal epithelial disorder healing promoter.
Therefore, according to the present invention, as described above, a corneal epithelial disorder healing promoter comprising sericin as an active ingredient is also provided.

  In the present specification, the term “medicament” is used in a sense including the corneal protective agent or the corneal disorder ameliorating agent according to the present invention, and the corneal epithelial disorder healing promoter according to the present invention.

Here, “corneal protection” refers to protecting corneal tissue from corneal damage that may occur due to various factors. Specifically, “corneal protection” here can be broadly divided into protection from corneal damage due to external factors and protection from corneal damage due to internal factors. External factors include, for example, trauma, burns, chemical substances, drugs, ultraviolet rays, contact lens wear, foreign matter, eyelashes, dry air, infection, and the like. Further, external factors include those caused by surgical invasion of the cornea such as corneal refractive surgery and cataract surgery. On the other hand, internal factors include Sjogren's syndrome, dry eye syndrome (dry eye), diabetic keratopathy and the like.
In addition, the “cornea” is composed of the corneal epithelial layer, Bowman's membrane, corneal stroma layer, Descemet's membrane, and corneal endothelium layer as described above, and the corneal epithelial layer is located on the outermost surface of the cornea. However, it is easy to cause a failure due to the influence from the outside world. Thus, according to a preferred embodiment of the present invention, the cornea in “corneal protection” is the corneal epithelium.

  The term “amelioration of corneal disorders” means improvement of corneal disorders that can be caused by various factors as described above, symptom reduction, symptom reduction, symptom relief, and treatment, healing, and healing promotion. It is. According to a preferred embodiment of the present invention, the cornea in “amelioration of corneal injury” is corneal epithelium.

Therefore, as a use form of the corneal protective agent according to the present invention, when the occurrence or onset of corneal disorder is predicted due to various factors, the corneal protective agent is applied in advance to prevent the corneal disorder, etc. Is mentioned. As a specific example, the corneal protective agent according to the present invention can be used for the prevention of symptoms such as corneal disorders based on trauma due to dryness, for example, dry eye. In addition, the usage form of the corneal disorder ameliorating agent according to the present invention may be applied to the affected part of the disorder or its peripheral part in order to improve the corneal disorder caused by various factors. As a specific example, the corneal disorder ameliorating agent according to the present invention can be used for amelioration of symptoms such as dry eye.
Since sericin, which is an active ingredient, has moisturizing properties, it can be said that the drug of the present invention is effective for protecting from corneal damage due to trauma due to dryness or for improving the damage.

  In the present specification, “healing promotion” of “corneal epithelial disorder” means assisting or enhancing the healing ability of corneal epithelial tissue damaged by various factors, that is, self-repairing ability. Therefore, “healing promotion” of “corneal epithelial disorder” includes the promotion of corneal epithelial cell division, extension, or secretion of a corneal protective component contained in the tear film, etc. in the vicinity of the disorder. Used.

  As described above, the drug according to the present invention comprises sericin as an active ingredient. Here, “comprising as an active ingredient” means that in the case of the corneal protective agent of the present invention, it contains an amount sufficient to exert a corneal protective effect, that is, an effective amount of sericin. In the case of a disorder-improving agent, it means that it contains an amount of sericin sufficient to exert an ameliorating effect on corneal disorder, that is, an effective amount of sericin. It contains an amount of sericin that is sufficient to exert a healing promoting effect on the skin, that is, an effective amount.

  The drug according to the present invention is usually administered in the form of a composition as an ophthalmic pharmaceutical preparation by blending it with a solvent, a carrier or other additives that are acceptable for formulation. The preferred administration method is mainly local administration, but is not limited to this, and various administration methods can be adopted. As the dosage form, eye drops, eye ointments, intraocular perfusion / cleaning agents and the like are suitable for local administration, but are not limited thereto. In the present invention, the drug is preferably provided as an eye drop.

When the dosage form is an eye drop or an intraocular perfusion / cleaning agent, it may be prepared in the form of a liquid in advance, or it may be prepared as a solid and dissolved, suspended or emulsified in a solvent at the time of use. It may be. Examples of solid agents include tablets, granules, powders, capsules and the like.
These can be appropriately produced by known methods.

  Solvents or carriers that are acceptable for the formulation described above include, for example, water (sterilized purified water, purified water, etc.), physiological saline, buffer (citric acid and salts thereof (sodium citrate, etc.), boric acid, etc. And salts thereof (borax, etc.), phosphoric acid and salts thereof (sodium monohydrogen phosphate, etc.), acetic acid and salts thereof (sodium acetate, etc.), tartaric acid and salts thereof (sodium tartrate, etc.) Examples include vegetable oils (olive oil, soybean oil, sesame oil, cottonseed oil, etc.), mineral oils (liquid paraffin, petroleum jelly), alcohols (propylene glycol, p-octyldodecanol), and the like.

  The drug according to the present invention may further contain other pharmaceutically active ingredients as necessary, as long as the effects of the present invention are not hindered. Examples of other components that can be used in combination include, for example, a decongestant component, an eye regulator component, an anti-inflammatory component or an astringent component, an antihistamine component or an antiallergic component, vitamins, amino acids, and antibacterial agents. Ingredients, bactericidal components, saccharides, local anesthetic components, steroid components, glaucoma therapeutic components and the like. In addition, for example, in the drug of the present invention, it is conceivable to add a moisturizing component such as hyaluronic acid in addition to sericin to enhance water retention.

  The pH of the drug according to the present invention is not particularly limited as long as it is in an ophthalmically acceptable range, and is usually 4.0 to 10.0, preferably 4.5 to 9.5. More preferably, it is 5.0-9.0. The pH adjustment may be performed using a pH adjuster such as hydrochloric acid, citric acid, boric acid, phosphoric acid, acetic acid, tartaric acid or a salt thereof, sodium hydroxide, sodium carbonate, sodium hydrogen carbonate, or the above buffer. it can.

  The osmotic pressure of the drug according to the present invention is not particularly limited as long as the osmotic pressure is within an ophthalmically acceptable range, and is usually 100 to 1200 mOsm, preferably 100 to 600 mOsm, and more preferably 150 to 400 mOsm. Moreover, the osmotic pressure ratio with respect to physiological saline is 0.3-4.1 normally, Preferably it is 0.3-2.1, More preferably, it is 0.5-1.4. The osmotic pressure is adjusted by using an isotonic agent such as inorganic salts (sodium chloride, potassium chloride, calcium chloride, etc.), sugars (mannitol, glucose, etc.), polyhydric alcohols (glycerin, propylene glycol, etc.). it can.

  In addition to this, the agent according to the present invention may contain other additives usually used in preparing individual preparations as necessary, as long as the effects of the present invention are not hindered. Examples of such additives include solubilizers, stabilizers, preservatives, thickeners, chelating agents, and cooling agents.

  The dose of the drug according to the present invention varies depending on the type of sericin used, the type of disorder targeted, the age, weight, symptoms, desired effect, administration method, treatment period, etc. of the patient. As sericin which is an active ingredient, for example, it may be administered once to several times a day so as to be 1 μg to 100 mg / eye per day for an adult.

  Therefore, the medicament according to the present invention preferably comprises sericin as an active ingredient in an amount to be administered in a range of 1 μg to 100 mg per day. The drug according to the present invention preferably contains sericin so that the final concentration is 0.01 to 20% (w / v), more preferably 0.1 to 10% (w / v). . A drug containing sericin within this range is administered once to several times a day for adults at a dose of 10 to 100 μl.

  The present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

  Seven-week-old male Sprague-Dawley rats (available from Shimizu Experimental Materials Co., Ltd.) were divided into 4 groups, and Nembutal Injection (available from Dainippon Sumitomo Pharma Co., Ltd.) was injected intraperitoneally at 30 mg per kg body weight of the rat. General anesthesia was given by administration. Then, in order to peel the corneal epithelial layer of a certain area, it was cut out using a biopsy trepan having a diameter of 2.5 mm (available from KAI Industries Co., Ltd.), and a beaver with an edge of 3.5 mm and an angle of 30 ° across the center of the cornea Peeled with a blade (available from Nippon Becton Dickinson Co., Ltd.). Based on the time when the corneal epithelial layer was detached, the following experiment was further advanced.

After peeling off the corneal epithelial layer, sericin hydrolyzate containing sericin hydrolyzate at concentrations of 1% (w / v), 5% (w / v) and 10% (w / v), respectively, and physiological saline as a control Was instilled 50 μl at 3 hour intervals 5 times a day immediately after peeling (0 hour), 3 hours, 6 hours, 9 hours and 12 hours later.
The sericin solution was prepared by dissolving a predetermined amount of a commercially available powdered sericin hydrolyzate (pure sericin (trade name), available from Wako Pure Chemical Industries, Ltd.) in physiological saline, and then having a pore size of 0.2 μm. This was performed by filter sterilization with a filter (available from Minisart, Sartorius Co., Ltd.).

1% (w / v) fluorescein containing Benokiseal (available from Santen Pharmaceutical Co., Ltd.) after detachment, immediately after detachment (0 hour), 12 hours, 24 hours, and 36 hours after detachment The solution is instilled 50 μl at a time to stain the corneal wound site and photographed with a fundus camera connected to a digital camera (a new ophthalmology, “Attaching the latest digital camera to an old fundus camera”, 24: 1125-1127 (2007)) did.
The result was as shown in FIG.

The stained portion of the obtained image was quantified with image analysis software (Image J, National Institutes of Health (NIH)) to obtain the corneal epithelial layer defect area, and the cure rate was calculated according to the following formula: .
Healing rate (%) = (defect area immediately after debonding−defect area during measurement) / defect area immediately after delamination × 100

  The results were as shown in Table 1 and FIG.

From the results, in the group instilled with physiological saline, the disorder was observed up to 36 hours after the corneal epithelial layer was detached. On the other hand, in the group instilled with sericin solution, a significant healing promoting effect was observed, and the effect increased with increasing sericin concentration.
Moreover, from the visual judgment of the obtained image, it was confirmed that no corneal opacity occurred in the group using sericin according to the present invention.

It is a photograph which shows the healing promotion effect with respect to the corneal epithelial disorder of this invention. It is a graph which shows the healing promotion effect with respect to the corneal epithelial disorder of this invention.

Claims (7)

  A corneal protective agent or a corneal disorder ameliorating agent, comprising sericin as an active ingredient.   The agent according to claim 1, which is used for protecting a corneal epithelium or improving a corneal epithelial disorder.   A corneal epithelial disorder healing promoter comprising sericin as an active ingredient.   The medicine according to any one of claims 1 to 3, wherein sericin is obtained by extracting from silk thread.   The chemical | medical agent as described in any one of Claims 1-4 whose average molecular weights of sericin are 3,000-300,000.   The medicine according to any one of claims 1 to 5, wherein sericin is a hydrolyzate.   The medicine according to any one of claims 1 to 6, which is provided as an eye drop.