JP2005060360A - Ophthalmic medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new ophthalmic medicine specified for the subconjunctival administration effective for the suppression of a rejection in a local transplantation in eye, glaucoma and the suppression of eye allergy inflammation. <P>SOLUTION: This ophthalmic medicine consists of a compound containing at least 1 kind selected from the group consisting of N,N'-diacylcystine and its esters. The medicine suppresses the rejection of an allo transplanting piece considered as a Th1 response of a cellular immunity, and the eye allergy and eye inflammation considered to be a Th2 response. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a new ophthalmic drug, and more particularly to a drug comprising an inhibitor for rejection of ocular tissue transplantation by subconjunctival administration, an ocular allergy, and an ocular inflammation inhibitor.

  The immune system is a system for protecting the self from invasion from the outside such as viruses and bacteria, or biological invasion by cells (cancer cells and the like) generated by abnormalities in self-derived cells. However, this immune system becomes abnormal, and it works excessively, or the immune system works in the direction of eliminating self-components. Diseases that cause such a condition are collectively referred to as immune diseases. For example, acute and chronic inflammatory diseases such as atopic dermatitis, hay fever, asthma, sarcoidosis, allergic diseases, rheumatoid arthritis, diabetes (IDDM), SLE, chronic fatigue syndrome and other autoimmune diseases, hepatitis , Liver cirrhosis, ulcerative colitis, Crohn's disease and other diseases such as gastrointestinal inflammation (IBD) and cancer cachexia. There are various causes of these immune diseases, and it is impossible to create a drug that is truly useful for a disease by simply thinking of immunosuppression and immune enhancement. On the other hand, in various organ transplantation reactions such as kidney, liver, heart, lung, skin and cornea, it is well known that the transplant is rejected and dropped due to the immune response reaction of the living body. Although it is collectively called allograft rejection, its immunological mechanism has not been clarified at all. Although it has been found that non-selective suppression of the immune response suppresses rejection, most of them lack the selectivity for the target cell, and there are serious problems such as increased carcinogenesis and increased chances of infection. Often has serious side effects.

  T lymphocytes and B lymphocytes are well known as cells responsible for immunity, and each exerts various functions as a carrier of cellular immunity and humoral immunity. On the other hand, macrophages / monocytes (including dendritic cells and Langerhans cells) are cells that are deeply involved in cellular immunity and humoral immunity, such as allergies, immune diseases such as rheumatism, cancer, bacterial infections, transplanted tissues, etc. He is deeply involved in self-exclusion. T lymphocytes, B lymphocytes and macrophages / monocytes have an extremely important role in the development of inflammatory, allergic and immune diseases as described above and the pathological progress of rejection after organ transplantation. Nevertheless, the application to the treatment, improvement, prevention and transplantation field of human diseases assuming each cell-specific function is still in a trial and error state.

  In recent years, a shift in the type of helper T cell subpopulation in peripheral blood has been associated with disease, and helper T lymphocytes, which are subpopulations in T lymphocytes, are further classified into two subpopulations Th1 and Th2. These two abundance ratios are being proved to be important indicators of the immune function of living bodies. Attempts have been made to establish a more appropriate treatment method by diagnosing the disease state of the disease based on this index and improving its abundance ratio. That is, when Th2 causing IgE production from B cells is greater than Th1 (Th1 <Th2), allergic diseases are said to be worsened. By measuring Th1 / Th2, the state of immunity can be assayed or Th1> Th2 Attempts have been made to control allergies. On the other hand, the presence of diseases and biological reactions caused by the situation where Th1 is dominant has been raised such as rheumatoid arthritis and transplant rejection. However, in recent years it has become clear that this simple idea is a complete mistake.

It is disclosed that cystine derivatives are useful as immunomodulators (Patent Document 1) and inflammatory factor activation inhibitors (Patent Document 2). However. It is not known that administration under the conjunctiva suppresses rejection and ocular allergic inflammation in local ocular transplantation.
JP 11-246435 A JP 2002-226457 A

  Elucidate whether N, N'-diacylcystine or its ester suppresses the cellular immune response typified by the Th1 response, and at the same time aggravates the allergic inflammatory response as a Th2 response observed at the time of allotransplantation in the ocular region Examining whether or not there is a possibility, providing new inhibitors and methods of rejection in allotransplantation to the local area of the eye, and medical treatment called allotransplantation, which is indispensable for visual acuity recovery in patients with visual acuity, is widely safe It is an object of the present invention to provide a technique to be performed. That is, it is impossible to develop a new ophthalmic drug that can suppress allergic inflammatory response (considered to involve humoral immunity) simultaneously with immunological rejection (which may be thought to involve cellular immunity) It is an object to clarify whether or not a dosage form is desirable for the expression of its efficacy.

We thought that N, N′-diacylcystine or its ester may suppress the Th1 response, and the present inventors proceeded with intensive medical research and aimed at pharmaceutical preparation. It was newly found that by suppressing chemokine production from human corneal epithelial cells and conjunctival fibroblasts, the Th2 response was suppressed unexpectedly. Furthermore, it has been found that administration under the conjunctiva efficiently and locally suppresses ocular allergic inflammation considered to be a Th2 response and suppresses allograft rejection considered to be a Th1 response. In addition to suppressing the rejection associated with allotransplantation, it was found that the harmful inflammatory response observed during rejection can also be suppressed, and the present invention has been completed.
In other words, we succeeded in developing a new ophthalmic drug that can suppress allergic inflammatory responses (considered to involve humoral immunity) as well as immunological rejection (which may be thought to involve cellular immunity). The present invention has been completed by clarifying that its efficacy is exerted more strongly by a specific specific method called subconjunctival administration.

At present, the Th1 / Th2 balance is defined by IL-6, IL-10, or IL-4 and IL-12, IL-18, and the ratio at which they are produced in vivo. It is already known that Th2 involved in humoral immunity is induced by the former three, and Th1 involved in cellular immunity is induced by IL-12 and IL-18. However, although IL-6, IL-10, IL-12, and IL-18 have been found to be produced from macrophages, the same macrophage cells are IL-6, IL-10, IL-12, IL- If 18 is also produced, there will be one macrophage involved in both Th1 induction and Th2 induction, which is a major contradiction when considering the immune response of the living body. The present inventors produce IL-12 only by a reduced macrophage having a high GSH content, causing Th1 deviation, and by producing oxidized macrophages, production of IL-6 and IL-10 is enhanced, and Th2 deviation is induced. I found out that It was also found that IL-6 that induces Th2 is produced in large quantities by the action of IFNγ when macrophages are inclined to the oxidized form even if IFNγ, which is representative of Th1 cytokine, is produced. On the other hand, it was also found that when reduced macrophages are present, the reduced phenotype of macrophages is further enhanced by IFNγ, which is representative of Th1 cytokines. When oxidized macrophage is induced and IL-4, which is a representative of Th2 cytokine, acts, the trait of oxidized macrophage is further enhanced. These findings show that the immune response in the opposite direction of humoral immunity and cellular immunity is unambiguously defined by the redox state of macrophages, and is an important finding related to the fundamentals of immunology. The inventors have shown that N, N′-diacylcystine or its ester induces oxidized macrophages. That is, according to this idea, N, N′-diacylcystine or its ester is expected to enhance the Th2 response.
However, contrary to the expectation of the present inventors in the present invention, and completely different from the expectation of the trader at the time of completion of the present invention, the allergic inflammatory response considered to be a Th2 response was actually suppressed.

  Two types of costimulation, CD80 / 86-CD28 and CD40-CD40L, play a central role in allograft rejection. On the other hand, there are many questions about the immunosuppressive action of Th2 cytokines IL4 and IL10 in vivo based on the Th1 / Th2 paradigm. There is also a finding that skin grafts are rejected early by adoptive transfer of Th2-type CD4 + T lymphocytes to SCID mice, and a cellular immune response is also found in IL-4-/-and IL-10-/-mice On the contrary, there are many reports that it is suppressed. Recently, it is thought that a T lymphocyte subpopulation called Tr-1 that produces TGFβ rather than Th1 plays a central role in immune regulation. Typical Th1 cytokines IFN-γ and IL-2 enhance cellular immunity according to the above-mentioned idea. According to recent findings, there are many reports that cellular immunity is conversely enhanced in mice deficient in IFN-1γ or IL-2. Inhibition of T lymphocyte proliferation by CTLA-4Ig administration is observed in wild type mice but not in IFN-γ − / − mice. It shows that IFN-γ has an immunosuppressive effect in vivo. This situation is similar for IL-2. The immunosuppressive action of IL-2 is due to the induction of apoptosis through the activation of T lymphocytes. In mice lacking IL-2-/-or high-affinity IL-2R, autoimmune diseases such as hyperproliferation of T lymphocytes and IBD frequently occur. The fact that autoimmunity occurs frequently in mice lacking Th1 cytokines was revealed.

  In such mice, the induction of apoptosis called AICD (activation induced cell death) via Fas is severely impaired. IL-2 induces apoptosis in antigen-activated T lymphocytes by enhancing FasL expression on T lymphocytes and suppressing the expression of FLIP, a protein that suppresses Fas / FasL system This is because it is caused by repeated recovery. As a result, IL-2 works in an immunosuppressive manner. In addition, when the functions of IL-2, IL-2Rα, and IL-2Rβ are neutralized, AICD is suppressed, but when the function of IL-2Rγ is neutralized, the expression of Bcl-2 is suppressed and AICD is enhanced. IL-2 promotes AICD of CD8 + T lymphocytes through two mechanisms: enhancing FasL expression and down-modulating γ chain expression on CD8 + T lymphocytes. IL-4, IL-7, and IL-15 sharing the γ chain have no effect on FasL expression or γ chain expression, and have no effect of promoting AICD.

  It is known that N-acetylcysteine (NAC) that induces reduced macrophage suppresses the Th2 immune response and induces the allergic reaction by inducing the Th1 immune response. Therefore, it is assumed that N, N'-diacylcystine or an ester thereof according to the present invention, which reduces the amount of glutathione in cells and induces oxidized macrophages, enhances allergic reactions.

Like other sites of inflammation, local chemokine expression is deeply involved in the development and maintenance of allergic conjunctivitis, corneal transplant rejection, alkaline trauma, uveitis, and glaucoma.
It has been suggested that tissue damage by eosinophils is involved in the onset and progression of severe allergic conjunctivitis, and that eotaxin plays an important role as an eosinophil migration factor in this disease. I came. Therefore, eotaxin suppression can prevent allergic conjunctivitis from becoming severe.
It is clear that various cytokines are associated with the onset of corneal transplant rejection in relation to antigen presentation by Langerhans cell migration and establishment of delayed type hypersensitivity reaction by T cell, macrophage, and neutrophil migration. In particular, it has been found that IP-10 is involved in regional lymph nodes.
Alkaline trauma is an eye trauma that is the most serious and leads to blindness, but the onset is closely related to local production of IL-1, IL-6, IL-8, and RANTES. Experimentally, IL-8 and RANTES production can be suppressed by instillation of IL-1 receptor antagonist, and corneal turbidity and inflammation after alkaline trauma can be suppressed.
With regard to uveitis, Verma et al. Detect high concentrations of IL-8, IP-10, MCP-1, RANTES, MIP-1β in the anterior aqueous humor of patients with anterior uveitis, and their concentrations correlate with disease state Showed that. Furthermore, it is known that chemokines not only attract inflammatory cells locally but also participate in T lymphocyte differentiation. For example, RANTES and MIP-1α are involved in Th1 cell and MCP-1 is involved in Th2 cell differentiation. T lymphocytes that have infiltrated into the eyeball are subsequently stimulated by intraocular antigen-presenting cells under the influence of various chemokines to modify the disease state. In the study in rats, the administration of anti-MCP-1, RANTES, MIP-1α, and MIP-1β antibody had an inhibitory effect on experimental autoimmune uvitis. Production of IL-8, GRO, and MCP-1 in the anterior chamber has been confirmed in endotoxin-induced uvitis, which is an experimental uveitis by administration of Lipopolysaccharid (LPS). It has been found that administration of anti-IL-8 antibody suppresses infiltration of neutrophils and anti-MCP-1 antibody into monocytes in the eye.
In glaucoma, eyelid adhesion is a major cause of increased intraocular pressure, but abnormal production of extracellular matrix such as fibronectin and collagen due to local inflammation that occurs in conjunction with chemokine production such as IL-8 and MCP-1 The present inventors have revealed that this is caused by an abnormal decomposition.
In this way, it is becoming clear that chemokine expression in the local area of the eye is deeply involved in the onset and maintenance of allergic conjunctivitis, corneal transplant rejection, alkaline trauma, uveitis, glaucoma, etc. Drugs that suppress chemokine production have not yet been provided.

  Treatment of eye diseases by systemic administration of drugs requires a large amount of drugs, and side effects cannot be ignored. Compared to this, local administration by subconjunctival injection has the advantage that the local area of the eye can be efficiently treated with a small amount of high-concentration drug. Furthermore, since the barrier of the conjunctival epithelium can be ignored, it moves more effectively into the eye than eye drops. In addition, since the drug stored under the conjunctiva gradually flows out into the tear fluid, it also has the effect of continuous instillation. In the case of corneal transplantation, there is no blood flow to the donor cornea until the new blood vessel reaches the donor cornea, and therefore there is no effect on the donor cornea by systemic administration other than the route through the tear fluid. Therefore, it can be said to be the most effective administration method for suppressing locally dependent inflammation, rejection, antigen presentation, and the like.

  Against this background, the present inventors have conducted intensive research and found that N, N′-diacylcystine or its ester, which has been shown to induce oxidized macrophages, is expected to enhance the Th2 response. Nevertheless, it has been found to exhibit a completely different pharmacological effect. In the present invention, contrary to the expectation of the inventors of the present invention, and completely different from the expectation of the trader at the time of completion of the present invention, the allergic inflammatory response considered to be a Th2 response was actually suppressed.

The N, N′-diacylcystine of the present invention is N, N′-diacetylcystine, and the ester of N, N′-diacylcystine is N, N′-diacetylcystine dimethyl ester, N, N′-diacetyl. Examples include cystine diethyl ester, N, N′-diacetylcystine isopropyl ester, and N, N′-di-L-alanylcystine dimethyl ester. Preference is given to N, N′-diacetylcystine dimethyl ester.
N, N′-diacylcystine or an ester thereof may be administered alone, or a mixture thereof may be administered.

  The pharmaceutical agent for subconjunctival administration of the present invention is used in the form of injection, implantation, etc. Examples of the dosage form include solutions such as injections, gels, ointments, solids and the like.

That is, the present invention is as follows.
By administering N, N′-diacylcystine or its ester under the conjunctiva, it is possible to safely and efficiently suppress ocular allergy and ocular inflammation that are considered to be local Th2 responses, and to reject allografts that are considered to be Th1 responses. The provision of ophthalmic drugs that suppress the reaction. The present invention provides an ophthalmic pharmaceutical product that can suppress rejection associated with allotransplantation and also suppress harmful inflammatory responses observed during rejection.
In other words, we succeeded in developing a new ophthalmic drug that can suppress allergic inflammatory responses (considered to involve humoral immunity) as well as immunological rejection (which may be thought to involve cellular immunity). Providing N, N'-diacylcystine or an ester thereof as an ophthalmic pharmaceutical for subconjunctival administration by clarifying that its efficacy is exerted by a specific specific method called subconjunctival administration Providing Ophthalmic Drugs for Subconjunctival Administration as Rejection Inhibitors in Local Ocular Transplantation, and Ophthalmic Drugs for Subconjunctival Administration as Eye Allergies and Eye Inflammation Inhibitors for Local Ocular Transplantation in Which Organs Are Cornea, Corneal Endothelium, Corneal Stem Cells It is an invention whose main point is to do.

  The usefulness, inventive step and novelty of the present invention in suppressing organ transplant rejection will be described below. Unlike various immune diseases, in organ transplantation, the time of transplantation is the first exposure to donor antigen, and immunological manipulation can be performed easily. In organ transplantation, immunosuppressive drugs are currently used to suppress rejection, but this treatment suppresses all immune systems in a non-antigen-specific manner. There are side effects such as infectivity. Therefore, establishment of a method for selectively suppressing only an immune response to a donor antigen is important and is the most ideal treatment. The present inventors have intensively studied the development of a therapeutic method that aims to induce immune tolerance against a donor antigen, can obtain a permanent engraftment similar to autotransplantation, and requires an immunosuppressant. Was completed. I am convinced that the development of this method is worthy of the 21st century treatment. The merit of the treatment according to the present invention is that only the response to the transplanted organ antigen is suppressed (antigen specificity), and after the appearance of these cells, the suppression mechanism becomes stronger and the antigen presentation is performed promptly as the antigen is exposed. It can be applied to corneal transplantation to high-risk eyes that are prone to rejection.

  The present inventors administer N, N′-diacylcystine or an ester thereof subconjunctivally to suppress ocular allergic inflammation that is considered to be a local Th2 response safely and efficiently, and allovirus that is considered to be a Th1 response. An ophthalmic drug that simultaneously suppresses rejection of a graft has been found. It is an ophthalmic pharmaceutical that can suppress the rejection associated with allotransplantation and also suppress the harmful inflammatory response observed during rejection.

  There have been many attempts to administer large amounts of IL-4 and IL-10 and to introduce genes for the suppression of donor antigen-specific rejection by Th2 deviation to date. It is difficult to influence the response in local lymph nodes. This is because cytokines themselves have no endocrine action in the body and are mainly paracrine or autocrine actions in the vicinity of cells. In addition, long-term continuous administration requires a large amount of cost and labor. The present invention is a method of administering a drug locally to a recipient transplanted with a transplanted organ, and has few systemic side effects. Furthermore, since the reaction to the donor is biased to a reaction that suppresses rejection, suppression that always exceeds rejection is generated. At present, only the present inventors have established this method in organ transplantation. The greatest merit of this method is that it can be realized inexpensively, simply and safely without breaking the immune balance in the living body.

Corneal transplantation is a safe transplant with a higher engraftment rate than other organ transplants, and the current situation is that 50,000 operations are performed annually in the United States and 2000 in Japan. Furthermore, in recent years, the existence site of the corneal epithelial cell Stem cell has been clarified, and a new surgical technique has been established from the standpoint of cell biology. It has become possible. Among these, the biggest problem at present is rejection of transplants, and only a large amount of local and systemic steroids and cyclosporine has been administered clinically. These side effects are a problem.
In addition, establishment of transplant technology for high-risk eyes is a major issue as an expansion of indications. Transplantation into the cornea into which new blood vessels have invaded is accelerated by antigen sensitization, and the effector cell tends to reach the donor cornea. Transplantation into the cornea where Langerhans cells are migrating after inflammation is performed at an early stage of antigen sensitization.
Furthermore, there are corneal limbal transplantation and epithelial transplantation as problems in the expansion of the surgical procedure. The corneal limbal transplant brings in the donor Langerhans cells and is therefore presented with the antigen. Further, both the limbal transplantation and the seven-skinned transplantation are likely to be rejected because the transplanted site is the corneal limbus and is rich in vascular tissue. The corneal epithelium is highly antigenic. Conventional non-antigen-specific immunosuppression methods include systemic side effects such as susceptibility to infection, diabetes, gastrointestinal ulcers, neurological symptoms, obesity, and renal dysfunction as local side effects such as steroid glaucoma and steroid cataract Infectivity is pointed out, and this problem can be solved by carrying out the present invention.

  High-risk eyes with neovascularization in the recipient's cornea are prone to transplant rejection and steroids and immunosuppressants are used, but there are still many problems. In reports on corneal transplant rejection, it is known that MHC (major histocompatibility antigen) adaptation has no effect on the reduction of rejection, and that minor antigens other than MHC significantly reduce rejection. This is because the cornea has an immunologically special environment called the “immune private site”. (1) MHC expression is low in the corneal tissue, (2) there are no blood vessels and lymph vessels, (3) the corneal tissue Is involved in the absence of antigen-presenting cells. Due to these special circumstances, the donor antigen in corneal transplantation is thought to be presented to the recipient T cells mainly by the recipient antigen-presenting cells (Indirect path of allo- collocation). By this mechanism, the recipient is sensitized, and CD4 + T cells that have received the antigen cause a delayed type hypersensitivity reaction (DTH) to the donor antigen and cause rejection. In corneal transplantation, it is thought that these operations are made simpler in that the cornea is an immunoprivilege site. Corneal transplantation is a safe transplant with the best engraftment rate among organ transplants, and many have been performed in the world. However, in recent years, the concept of transplantation has been developed, and the indications and transplantation methods for carrying out transplantation have expanded, so that corneal transplants that are likely to cause rejection are increasing.

  The significance of the present invention will be described using corneal transplantation as an example. By subconjunctival administration, suppression of Th1 reaction and allergic inflammatory response are suppressed in the same way, so that (1) only response to corneal transplant antigen can be suppressed (antigen specificity), and (2) rejection against donor antigen. It is possible to produce an inhibitory reaction that always exceeds the reaction, and (3) it can be applied to a high-risk eye in which antigen presentation is rapid and rejection occurs at a high rate. In organ transplantation, the functional role of the organ must also be taken into account. Corneal transplantation is the most suitable transplant as a model for rejection of minor antigens because the shape and function of the graft are simple and there are no donor antigen-presenting cells. The novelty relating to suppression of corneal transplant rejection and graft colonization, which is one of the components of the present invention, is (1) a therapeutic method that has never been seen before, in which antigen-presenting cells are manipulated to change immune responses. (2) The fact that there was no other method than immunosuppression that was not antigen-specific as a conventional method was overcome, and (3) tolerance induction by antibodies was used at the research level. The point which can be suppressed, and (4) suppression of Th1 reaction and allergic inflammatory response can be suppressed equally by (4) subconjunctival administration. In terms of inventive step, (1) the possibility of antigen-specific immunosuppression from conventional non-specific immunosuppression methods, (2) the use of conventional immunosuppressive agents with strong side effects of systemic administration can be restricted A point is mentioned. As usefulness, (1) a point that can be specifically suppressed, (2) a point that can be achieved at low cost, (3) a point that the suppression mechanism becomes stronger as the antigen is exposed after the treatment according to the present invention, ( 4) The present invention can be applied to high-risk eyes that are promptly presented with antigens and easily cause rejection.

An embodiment of the present invention will be described.
The present invention suppresses ocular allergic inflammation considered to be a local Th2 response safely and efficiently by administering N, N′-diacylcystine or its ester under the conjunctiva in the local area of the eye, and considers it as a Th1 response. It is applied as an ophthalmic drug that simultaneously suppresses rejection of allografts. It is an ophthalmic pharmaceutical that can suppress the rejection associated with allotransplantation and also suppress the harmful inflammatory response observed during rejection. These agents can be used alone or as a mixture thereof.

The pathological conditions that can be used as a target include all ocular allergic diseases and ocular inflammation, but, among other things, allergic conjunctivitis, acute conjunctivitis, chronic conjunctivitis, corneal fructen, Includes keratoepitheliosis, scleritis, uveitis, choroiditis, iritis, alkaline trauma, glaucoma. In addition, representative examples of organs to be transplanted when used as an inhibitor of transplant rejection include transplantation of classic eye tissues such as cornea, corneal endothelium, retina, corneal stem cells, retinal stem cells, and cells as regenerative medicine Transplantation and artificial organ transplantation are also included.
It is clear that the present invention can be widely applied to inflammatory conditions associated with rejection.

  The ophthalmic drug handled in the present invention can be administered alone in an actual medical field. In addition to the ophthalmic drug included in the present invention, for example, steroidal compounds, IL-4, IL-10, TGF- It can also be mixed or used in combination with substances derived from in vitro and in vivo such as cytokines represented by β. In addition, the ophthalmic drug handled in the present invention can be used alone or in combination with other drugs handled in the ophthalmic field.

The outline of the embodiment of the present invention will be described for corneal transplantation.
To determine the induction of corneal engraftment in allogeneic corneal transplantation, 8-10 week old mice are used, BALB / c (H-2d) mice as recipients, and only minor antigens differ as donors. D2 (H-2d) mice are used. In order to induce the corneal neovascularization, which is a high-risk eye that is prone to rejection at a high rate and early, to the recipient, three 11-0 nylon threads were sewn into the corneal stroma and left for 14 days. This nylon thread is removed at the time of corneal transplantation.
Corneal transplantation is performed as follows. Recipient mice are anesthetized by intraperitoneal administration of 3 mg of ketamamine and 0.0075 mg of xylazine for transplantation. A central part of the cornea of the donor mouse is excised in a circular shape with a diameter of 2 mm to prepare a graft. The central part of the cornea of the host mouse is excised 2 mm in diameter, and the donor cornea is sutured end-to-end using 8-0 nylon. After the operation, an antibiotic ointment is applied, and eyelid suture is performed using 8-0 nylon. The eyelid suture is removed 72 hours after the operation, and the corneal suture is removed 7 days after the transplantation.

  A slit lamp microscope is used to determine corneal rejection, and the corneal rejection is observed twice a week. Rejection is determined by scoring corneal opacity in 6 grades. The determination possible period is 14 days after transplantation, and score 3 or higher, and score 21 or higher after 21 days are determined as rejection. (Score 0: transparent engraftment, 1: mild corneal surface turbidity and iris blood vessels / iris crest irrational see-through possible, 2: mild corneal parenchyma and iris vessels / iris crest permeation allowed, 3: moderate corneal opacity and iris irreversible Unable to see blood vessels and iris crests, 4: High corneal opacity and iris border see-through, 5: Complete corneal opacification and no anterior chamber see-through).

  Drug administration method: In a single administration method, the drug is dissolved or suspended in PBS to a predetermined concentration, and then injected into the mouse abdominal cavity or subconjunctiva. The injection time is 3 times 7 days before, 4 days before and 1 hour before corneal transplantation. Control mice are injected with PBS at the same time. Kaplan-Meier Survival curves were used for the significant difference test of the survival rate of corneal transplantation, and comparison was made by Breslow-Gehan-Wilcoxon test. A P value of less than 0.05 is considered significant.

Corneal epithelial transplantation is performed as follows. The cornea is composed of five layers, and from the outside are the corneal epithelium, Bowman's membrane, corneal stroma, Desmetmet membrane, and corneal endothelium. The corneal epithelium has a high proliferation ability, and cells are supplied from a stem cell in the corneal limbus. Therefore, the corneal epithelium can be replaced with the host corneal epithelium within a few months even when a full-thickness corneal transplantation or superficial corneal transplantation, which is a transplant in the central part of the cornea, is used. Of these five-layer structures, the corneal epithelium has the highest antigenicity and is likely to be a target for early rejection after transplantation. Finally, the corneal epithelium is a layer that is very easy to fall off due to inflammation and rejection, and it is very difficult to maintain the donor epithelium. Currently, corneal transplantation is used to replace healthy tissue or to compensate for missing tissue. The significance of corneal transplantation is as follows: (1) The vision is improved by making the turbid cornea transparent (mainly treated by corneal full-layer transplantation and corneal surface transplantation); (2) non-proliferating corneal endothelial cells are surgically invaded To improve the visual acuity and relieve pain for the purpose of supplementing the endothelial cells (mainly treated by full-thickness corneal transplantation), (3) corneal epithelium reduces stem cell In addition, the damaged cornea is prevented from entering the conjunctival epithelial cells and covered with transparent corneal epithelial cells. Corneal epithelial transplantation is mainly used for the purpose of (3), but there are many cases with corneal opacity and often has the meaning of (1).
Corneal epithelial transplantation is performed as a surgical treatment for ocular surface diseases in which the corneal epithelium is deficient, covered with the conjunctival epithelium, and becomes clouded. Corneal epithelial transplantation is a method in which only the corneal limbal epithelium and superficial parenchyma are excised from the donor cornea and transplanted to the host limbus. The corneal epithelium of the graft repeats constant growth and the host corneal epithelium is It is intended to maintain. However, among them, the prognosis is extremely poor in intractable keratoconjunctival diseases accompanied by strong inflammation such as Stevens-Johnson syndrome, ophthalmic pemphigus, and corneal corrosion. The most important reason is that the foreign (allo) corneal epithelium having strong antigenicity is recognized and rejected by the host immune system. In addition, since the transplanted site is a corneal limbus having a lot of vascular tissue or Langerhans cells, antigen sensitization or rejection reaction to a donor antigen is likely to occur. Furthermore, complications caused by systemic and local administration of a large amount of immunosuppressant after surgery for prevention of rejection are also a major factor in the poor prognosis. The side effects of steroids are systemic, easily infectious, diabetes, gastrointestinal ulcer, neurological symptoms, obesity, and steroid glaucoma, steroid cataract, and easily infectious in the eyes. Therefore, the method according to the present invention that selectively suppresses only the immune response to the donor antigen is considered ideal. This means that new safe treatments can be provided for intractable keratoconjunctival diseases that are not currently being actively treated, and the social contribution is considered to be extremely high.

  It describes about corneal stem cell transplantation (Stem cell). In diseases such as Stevens-Johnson syndrome, pemphigoid, and corneal corrosion, the corneal epithelium including the Stem cell is dropped, and an invasion of an opaque conjunctival epithelium accompanied by blood vessels is caused. In addition, in a disease in which the corneal epithelium is genetically abnormal, such as a keratoid corneal degeneration, the Stem cell itself is abnormal. As a method for treating these, there is no method other than transplanting healthy corneal epithelium Stem cells in the limbus of normal eyes and always supplying normal corneal cells. However, allogeneic corneal epithelial transplants are easily rejected and difficult to maintain. Therefore, suppression of rejection using the method according to the present invention creates a turning point in the treatment of these diseases.

  As described above, the useful efficacy in the medical field of the organ transplant rejection inhibitor according to the present invention is obvious from its novel mechanism of action, and is useful in both acute and chronic phases of disease rejection. It is. In particular, subconjunctival administration is effective for transplantation of ocular tissues. The present invention can be widely applied to pathological conditions that depend on Th2 deviation such as ocular allergic diseases and ocular inflammation.

  The present invention will be described more specifically with reference to the following examples, but these examples do not limit the scope of the present invention.

を作成した。
（２）結膜下投与方法
３０Ｇ注射針を用い、０時、３時、６時、９時方向のマウス結膜下に５μｌずつ、計２０μｌ（８μｇ）の薬剤Ｎ，Ｎ’−ジアセチルシスチンジメチルエステル溶液を注入した。注入頻度は計３回、０日、３日、７日に投与した。
（３）酸化型マクロファージ同定方法
角膜および結膜組織を凍結し、５μｍに薄切した。Ｐｏｌｙ−ｌｙｓｉｎｅでコーティングしたスライド上で空気乾燥し、アセトン固定を行った。５％ＢＳＡ／ＰＢＳで非特異的抗体結合部位を阻害したのち、ＣＤ１１ｂ−ＦＩＴＣで染色を行った。洗浄後、１０μＭのＭｏｎｏｃｈｌｏｒｏｂｉａｍｉｎｅ（以下、ＭＣＢ）を３０分作用させ、洗浄後蛍光顕微鏡で螢光強度を判定した。結果、Ｎ，Ｎ’−ジアセチルシスチンジメチルエステル結膜下投与群では、角膜および結膜におけるＣＤ１１ｂ陽性細胞だけでなく、その他の細胞もＭＣＢによる螢光減弱がみられ、細胞内グルタチオン濃度が低下していることが明らかとなった。Example 1. Induction of oxidized macrophages on the ocular surface by subconjunctival administration Method (1) Materials Mouse: BALB / c mice of 8 to 10 weeks of age were used.
Drug: Drug N, N′-diacetylcystine dimethyl ester adjusted to 4000 μg / ml

It was created.
(2) Subconjunctival administration method Using a 30G needle, 5 μl each of the mouse conjunctiva at 0 o'clock, 3 o'clock, 6 o'clock, and 9 o'clock, 20 μl (8 μg) of drug N, N′-diacetylcystine dimethyl ester solution Injected. The injection frequency was 3 times in total, administered on the 0th, 3rd, and 7th days.
(3) Oxidized macrophage identification method The cornea and conjunctival tissue were frozen and sliced to 5 μm. It was air-dried on a slide coated with Poly-lysine and fixed with acetone. After inhibiting non-specific antibody binding sites with 5% BSA / PBS, staining was performed with CD11b-FITC. After washing, 10 μM Monochlorobiamine (hereinafter referred to as MCB) was allowed to act for 30 minutes, and the fluorescence intensity was determined with a fluorescence microscope after washing. As a result, in the subconjunctival administration group of N, N′-diacetylcystine dimethyl ester, not only CD11b positive cells in the cornea and conjunctiva but also other cells showed fluorescence attenuation due to MCB, and the intracellular glutathione concentration was decreased. It became clear.

Example 2 Rejection suppression effect of corneal limbal transplantation by donor and recipient subconjunctival administration 8 to 10-week-old mice were used. BALB / c (H-2d) mice as transplant recipients, differing only minor antigens as donors B10. D2 (H-2d) mice were used. From the donor eyeball, the cornea piece including the sclera and cornea was excised. After scuffing and removing the corneal endothelium, the corneal limbus was excised into a donut shape having a width of 0.5 mm to prepare a donor graft. Recipient mice were anesthetized by intraperitoneal administration of 3 mg of ketamine and 0.0075 mg of xylazine for transplantation. After scraping and removing the corneal epithelium of the recipient mouse, the donor graft was transplanted to the corneal limbus with 11-0 nylon thread by end-to-end suture, and the eyelid was sutured to complete the surgical procedure. The eyelid suture was removed after 3 days, after which rejection was observed daily.
The drug N, N′-diacetylcystine dimethyl ester is 5 μl of a 400 μg / ml solution at a time, for a total of 20 μl, B10. D2 donor eyeballs and BALB / c recipient eyeballs were injected subconjunctivally at 0, 3, 6 and 9 o'clock. The donor was infused three times 7 days, 4 days, and 1 day before transplantation, and the recipient was infused three times, 7 days, 4 days, and 1 hour before transplantation. As a control, PBS base was injected under the conjunctiva. Kaplan-Meier Survival curves were used for the significant difference test of the engraftment rate of limbal transplantation, and comparison was made by Breslow-Gehan-Wilcoxon test. A P value of less than 0.05 was considered significant.
In the PBS administration group (N = 7), all donor corneas were rejected on day 5 after transplantation, and in the drug administration group (N = 14), donor corneas were born on 12 out of 14 eyes even on day 22 after transplantation. The survival rate increased significantly (P <0.0001).

Example 3 Rejection suppression effect of corneal limbal transplantation by subconjunctival administration of recipient alone. Corneal limbal transplantation was performed by the above method. The drug N, N′-diacetylcystine dimethyl ester was injected only under the conjunctiva of the BALB / c recipient eye using the above method, and the rejection suppression effect was determined by examining the engraftment rate.
In the drug administration group (N = 11), 5 eyes were engrafted even 14 days after transplantation, and the engraftment rate was significantly increased compared to the control group (P <0.05).

Example 4 Rejection suppression effect of corneal limbal transplantation by subconjunctival administration of donor alone Corneal limbal transplantation was performed by the above method. The drug N, N′-diacetylcystine dimethyl ester was obtained using the above method B10. Injection was performed only under the conjunctiva of the D2 donor eye, and the rejection suppression effect was determined by examining the engraftment rate.
In the drug administration group (N = 12), 4 eyes were engrafted even 14 days after transplantation, and the engraftment rate was significantly increased compared to the control group (P <0.05).

Embodiment 5 FIG. Rejection suppression effect of corneal limbal transplantation on donors with different MHC 8 to 10 week old mice were used. BALB / c (H-2d) mice were used as transplant recipients, and C57BL / 10 (H-2d) mice with different MHC and minor antigens were used as donors. The above was used for the transplantation method and the determination of rejection.
The drug N, N′-diacetylcystine dimethyl ester was injected into C57BL / 10 donor eyeballs and BALB / c recipient eyeballs by the above method, and the rejection inhibition effect was determined by examining the engraftment rate.
The drug administration group (N = 13, MST = 7.5 ± 0.8 days) has a significantly increased survival rate compared to the PBS administration control group (N = 7, MST = 4.1 ± 0.1 days). (P <0.002).

状態を検討した。ドナー移植片を凍結し、５μｍに薄切した。Ｐｏｌｙ−Ｌ−ｌｙｓｉｎｅでコーティングしたスライド上で空気乾燥し、アセトン固定を行った。５％ＢＳＡ／ＰＢＳで非特異的抗体結合部位を阻害したのち、ＣＤ１１ｂ−ＦＩＴＣで染色を行った。洗浄後、１０μＭのＭＣＢを３０分作用させ、洗浄後蛍光顕微鏡で螢光強度を判定した。結果、Ｎ，Ｎ’−ジアセチルシスチンジメチルエステルを添加保存液中で保存した移植片では、基剤であるオプチゾール保存液と比較して、ＣＤ１１ｂ陽性細胞だけでなく、その他の細胞もＭＣＢによる螢光減弱がみられ、細胞内グルタチオン濃度が低下していることが明らかとなった。
また、この保存角膜を上記方法によりレシピエントマウスに移植し、拒絶反応抑制効果を生着率を検討することにより判定した。薬剤投与群（Ｎ＝１３、ＭＳＴ＝６．５±０．８日）は上記コントロールと比較して有意な差が認められなかったが、拒絶反応は促進されなかった。Example 6 Donor cornea preservation method using a preservation solution to which the drug N, N′-diacetylcystine dimethyl ester was added Mice aged 8 to 10 weeks were used. BALB / c (H-2d) mice as transplant recipients, differing only minor antigens as donors B10. D2 (H-2d) mice were used. Donor grafts were made as described above. With drug N, N′-diacetylcystine dimethyl ester 400 μg / ml

The condition was examined. Donor grafts were frozen and sliced to 5 μm. It was air-dried on a slide coated with Poly-L-lysine and fixed with acetone. After inhibiting non-specific antibody binding sites with 5% BSA / PBS, staining was performed with CD11b-FITC. After washing, 10 μM MCB was allowed to act for 30 minutes, and the fluorescence intensity was determined with a fluorescence microscope after washing. As a result, in the grafts preserved in the preservation solution containing N, N′-diacetylcystine dimethyl ester, not only the CD11b-positive cells but also other cells were fluorescent by MCB as compared with the base optizole preservation solution. Attenuation was observed, and it was revealed that the intracellular glutathione concentration was decreased.
Further, this preserved cornea was transplanted into a recipient mouse by the above method, and the rejection suppression effect was determined by examining the engraftment rate. In the drug administration group (N = 13, MST = 6.5 ± 0.8 days), no significant difference was observed compared to the control, but rejection was not promoted.

Example 7 Suppression of rejection by full-thickness corneal transplantation by donor and recipient subconjunctival administration 8 to 10-week-old mice were used. BALB / c (H-2d) mice as transplant recipients, differing only minor antigens as donors B10. D2 (H-2d) mice were used. A full-thickness cornea with a diameter of 2 mm was excised from the center of the donor cornea to prepare a donor graft. As recipients, high-risk eyes in which 11-0 nylon was sewn to the corneal stroma 14 days before transplantation to induce corneal neovascularization were used. Recipient mice were anesthetized by intraperitoneal administration of 3 mg of ketamine and 0.0075 mg of xylazine for transplantation. A little less than 2 mm in the central part of the cornea of the recipient mouse was excised, the donor graft was transplanted with an end-to-end suture using 11-0 nylon thread, and the eyelid was sutured to complete the surgical procedure. The eyelid suture was removed after 3 days, after which rejection was observed twice a week.
The drug N, N′-diacetylcystine dimethyl ester is 5 μl of a 400 μg / ml solution at a time, for a total of 20 μl, B10. D2 donor eyes and BAL / c recipient eyes were injected into the subconjunctiva at 0, 3, 6, and 9 o'clock. The donor was infused three times 7 days, 4 days, and 1 day before transplantation, and the recipient was infused three times, 7 days, 4 days, and 1 hour before transplantation. As a control, PBS base was injected under the conjunctiva. Kaplan-Meier Survival curves were used for the significant difference test of the engraftment rate of the full-thickness corneal transplantation, and comparison was made by Breslow-Gehan-Wilcoxon test. A P value of less than 0.05 was considered significant.
In the PBS administration group (N = 15), all donor corneas were rejected on day 21 after transplantation, and in the drug administration group (N = 14), donor corneas were born in 12 out of 14 eyes even at 56 days after transplantation. The survival rate increased significantly (P <0.0001).

Example 8 FIG. Anti-rejection effect of full-thickness corneal transplantation on donors with different MHC 8 to 10 week old mice were used. BALB / c (H-2d) mice were used as transplant recipients, and C57BL / 10 (H-2b) mice with different MHC and minor antigens were used as donors. The above was used for the transplantation method and the determination of rejection.
The drug N, N′-diacetylcystine dimethyl ester was injected into C57BL / 10 donor eyeballs and BALB / c recipient eyeballs by the above method, and the rejection inhibition effect was determined by examining the engraftment rate.
The donor and recipient subconjunctival drug-administered group (N = 25, MST = 43.6 ± 1.5 days) was significantly more significant than the PBS-administered control group (N = 10, MST = 16.1 ± 1.1 days). The engraftment rate increased (P <0/001). Also, the engraftment rate significantly increased (P <0.001) in the drug administration group only under the recipient conjunctiva (N = 10, MST = 36.8 ± 4.5 days).

Example 9 Rejection suppression effect of full-thickness corneal transplantation using F1 of donor recipient as donor 8 to 10-week-old mice were used. BALB / c (H-2d) mice were used as transplant recipients, and BALF / c and C57BL / 6 F1 CBF1 (H-2b / d) mice were used as donors. The above was used for the transplantation method and the determination of rejection.
The drug N, N′-diacetylcystine dimethyl ester was injected into C57BL / 10 donor eyeballs and BALB / c recipient eyeballs by the above method, and the rejection inhibition effect was determined by examining the engraftment rate.
The donor and recipient subconjunctival drug-administered group (N = 15) significantly increased the engraftment rate compared to the PBS-administered control group (N = 10) (P <0.001).

Example 10 Examination of rejection in INF-γ knockout mice and IL-4 knockout mice 8-10 week old mice were used. INF-γ knockout mice (C57BL / 6 background (H-2b)), IL-4 knockout mice (C57BL / 6 background (H-2b)), and C57BL / 6 (H-2b) as transplant recipients Mice, BALB / c (H-2b) mice with different MHC and minor antigen as donors, BALB. B (H-2d) mice were used. A full-thickness cornea with a diameter of 2 mm was excised from the center of the donor cornea to prepare a donor graft. Transplantation was performed on untreated recipients. Recipient mice were anesthetized by intraperitoneal administration of 3 mg of ketamine and 0.0075 mg of xylazine for transplantation. A little less than 2 mm in the central part of the cornea of the recipient mouse was excised, the donor graft was transplanted with an end-to-end suture using 11-0 nylon thread, and the eyelid was sutured to complete the surgical procedure. The eyelid suture was removed after 3 days, after which rejection was observed twice a week.
When BALB / c mice were used as donors, INF-γ knockout mice (N = 12, MST = 34.9) compared to C57BL / 6 recipients (N = 30, MST = 30.1 ± 2.1) No significant corneal engraftment was observed in either ± 2.6) or IL-4 knockout mice (N = 10, MST = 28.1 ± 3.1). Also, BALB. Similarly, when B mice were used as donors, INF-γ knockout mice (N = 10, MST = 35.6) compared to C57BL / 6 recipients (N = 10, MST = 25.9 ± 3.2). No significant corneal engraftment was observed in neither ± 4.0) nor IL-4 knockout mice (N = 10, MST = 23.3 ± 1.3).

Example 11 Inhibition of chemokine production by modification of thiol redox state of cells in the local area of the eye The intracellular thiol redox state of macrophages and dendritic cells controls local tissue inflammation pathology. Using human tenon sac fibroblasts and cultured corneal epithelium (HCE), the drug N, N′-diacetylcystine dimethyl ester (400 μg / ml) that biases the redox state to the oxidized form and glutathione monoethylester (GSH−) that biases the reduced form to the oxidized form OEt) (10 mM) was allowed to act in vitro for 8 hours. Subsequently, IL-1α (1 ng / ml) was added and stimulated. IL-8 and MCP-1 in the culture supernatant after 16 hours were measured by ELISA. Nuclear protein was extracted from the cells 2 hours after addition of IL-1α, and NF-κB activation was measured by gel shift assay.
As a result, IL-8 and MCP-1 production was higher than that of controls (14106 ± 553 pg / ml, 10311 ± 834 pg / ml, hereinafter mean ± SD), respectively, with N, N′-diacetylcystine dimethyl ester (7028 ± 569 pg). / Ml, 1929 ± 41 pg / ml) was significantly suppressed (P <0.05). No significant inhibition was observed with GSH-Oet (16199 ± 650 pg / ml, 10082 ± 491 pg / ml), which is a reduced inducer. Moreover, the activation of transcription factor NF-κB was suppressed by N, N′-diacetylcystine dimethyl ester.
These results indicate that the modification of the intracellular redox state exerts an anti-inflammatory action through inhibition of neutrophil and macrophage migration, and N, N′-diacetylcystine dimethyl ester has an inhibitory effect on inflammatory diseases. It became clear that there was. That is, an effect can be expected for allergic conjunctivitis, acute conjunctivitis, chronic conjunctivitis, corneal frickten, punctate keratoepitheliopathy, scleritis, uveitis, choroiditis, and iritis.

Claims (7)

  An ophthalmic pharmaceutical comprising at least one selected from the group consisting of N, N'-diacylcystine and esters thereof.   An ophthalmic pharmaceutical agent for subconjunctival administration containing at least one selected from the group consisting of N, N'-diacylcystine and its ester.   An ophthalmic pharmaceutical product for subconjunctival administration, which is a rejection inhibitor for local ocular transplantation, containing at least one selected from the group consisting of N, N'-diacylcystine and esters thereof.   The transplant rejection inhibitor according to claim 3, wherein the organ to be transplanted containing at least one selected from the group consisting of N, N'-diacylcystine and an ester thereof is a cornea, a corneal endothelium, a corneal stem cell or the like.   The ophthalmic pharmaceutical according to claim 1, which is an inhibitor of glaucoma and ocular allergy inflammation, containing at least one selected from the group consisting of N, N'-diacylcystine and esters thereof.   The ophthalmic pharmaceutical product for subconjunctival administration according to claim 2, which is an inhibitor of glaucoma and ocular allergy inflammation, containing at least one selected from the group consisting of N, N'-diacylcystine and esters thereof.   Allergic conjunctivitis, acute conjunctivitis, chronic conjunctivitis, corneal fricken, punctate keratoepitheliosis, scleritis, uveitis, containing at least one selected from the group consisting of N, N′-diacylcystine and esters thereof, The ophthalmic pharmaceutical agent for subconjunctival administration according to claim 2, which is an ocular allergic inflammation inhibitor such as retina choroiditis or iritis.