JP7083479B2 - Preventive or therapeutic agent for type 1 diabetes - Google Patents

Description

The present invention relates to a prophylactic or therapeutic agent for type 1 diabetes. More specifically, the present invention relates to a preventive or therapeutic agent for type 1 diabetes, a pharmaceutical composition for the prevention or treatment of type 1 diabetes, and a kit for the prevention or treatment of type 1 diabetes.

Diabetes mellitus is roughly classified into type 1 diabetes and type 2 diabetes according to its onset factors. Type 1 diabetes is a disease mainly caused by insulin deficiency due to the destruction of pancreatic islet β cells by an autoimmune disease. Type 1 diabetes, also known as insulin-dependent diabetes mellitus, is fatal if insulin is not routinely administered. Until now, it has been said that the onset usually occurs in juveniles, but it has become clear that there are many cases of onset after the age of 30 due to type 2 diabetes.

Allergic diseases such as allergic rhinitis and atopic dermatitis caused by mites are known. As one of the therapeutic methods for these diseases, desensitization therapy using a mite-derived antigen has been put into practical use. For example, Patent Document 1 describes a rapidly dissolving sublingual tablet containing a tick-derived antigen.

Japanese Unexamined Patent Publication No. 2016-193929

Although symptomatic treatments exist for type 1 diabetes, no effective preventive or therapeutic method that can cure type 1 diabetes is known. Therefore, an object of the present invention is to provide a prophylactic or therapeutic agent for type 1 diabetes.

The present invention includes the following aspects.
[1] A prophylactic or therapeutic agent for type 1 diabetes containing a mite-derived antigen as an active ingredient.
[2] The prophylactic or therapeutic agent for type 1 diabetes according to [1], which is for subcutaneous administration, transdermal administration or intraperitoneal administration.
[3] A pharmaceutical composition for preventing or treating type 1 diabetes, which comprises the prophylactic or therapeutic agent for type 1 diabetes and an adjuvant according to [1] or [2].
[4] The pharmaceutical composition for preventing or treating type 1 diabetes according to [3], which is for subcutaneous administration, transdermal administration or intraperitoneal administration.
[5] The pharmaceutical composition for preventing or treating type 1 diabetes according to [3] or [4], which is administered once or twice.
[6] A kit for preventing or treating type 1 diabetes, which comprises the preventive or therapeutic agent and adjuvant for type 1 diabetes according to [1] or [2].

INDUSTRIAL APPLICABILITY According to the present invention, a prophylactic or therapeutic agent for type 1 diabetes can be provided.

It is a representative histological image of pancreatic islets of NOD mice in the control group in Experimental Example 1. It is a representative histological image of pancreatic islets of NOD mice in the mite-derived antigen-administered group in Experimental Example 1. It is a graph which shows the result of having calculated the islet inflammation score of the mouse of each group in Experimental Example 1. 3 is a graph showing the results of analysis of the Th1 / Th2 balance of mouse spleen cells in Experimental Example 3.

[Preventive or therapeutic agent for type 1 diabetes]
In one embodiment, the present invention provides a prophylactic or therapeutic agent for type 1 diabetes, which comprises a mite-derived antigen as an active ingredient.

The Non Obese Diabetes (NOD) mouse is a type 1 diabetes model mouse. In NOD mice, induction of cytotoxic T cells into β cells of islets begins at 2 weeks of age, and isletitis is observed from around 4 weeks of age. After that, females develop diabetes from about 14 weeks of age, while males develop diabetes from about 21 weeks of age. The proportion of NOD mice that develop diabetes is 90% or more in females and about 10% in males.

As will be described later in the Examples, the inventors have shown that administration of a tick-derived antigen to NOD mice can prevent or treat the onset of diabetes. Therefore, the preventive or therapeutic agent of the present embodiment can prevent or treat type 1 diabetes.

As the mites that cause allergic diseases, Dermatophagoides farinae (hereinafter, may be referred to as "Derf"), Dermatophagoides pteronysinus (hereinafter, may be referred to as "Derp") and the like are known. ..

Among the antigens (allergens) produced by these mites, the ones that are particularly problematic are Group I (DerI), which is abundant in its excrement and has a molecular weight of about 25,000, and Group I (DerI), which is abundant in the insect body and has a molecular weight of about 14. It is a protein called 000 Group II (DerII).

The Delphi-derived Group I and Group II proteins are referred to as Delphi I protein and Delphi II protein, respectively. Further, the Group I protein and the Group II protein derived from Derp are called Derp I protein and Derp II protein, respectively.

In the prophylactic or therapeutic agent of the present embodiment, the mite-derived antigen includes an extract of mite worm body, an extract of mite excreta, a recombinant DelfI protein or a fragment thereof, a recombinant DelfII protein or a fragment thereof, and a recombinant. A DerpI protein or a fragment thereof, a recombinant DerpII protein or a fragment thereof, a mixture thereof, or the like can be used. In addition, one type of mite may be used alone, or two or more types may be mixed and used.

In particular, the mite-derived antigen for desensitization therapy for mite allergic rhinitis has a well-established production technique and is easily available, so that it can be suitably used for the prophylactic or therapeutic agent of the present embodiment.

Type 1 diabetes is classified into three categories, acute onset, slow progression, and fulminant disease, depending on the progression of symptoms. In acute onset and slowly progressive type 1 diabetes, islet-related autoantibodies, anti-glutamate decarboxylase (GAD) antibodies, are detected in the patient's blood. Therefore, it is considered that pancreatic islets still remain while the autoimmune reaction progresses in type 1 diabetic patients with acute onset and slowly progressing. On the other hand, in fulminant type 1 diabetic patients, anti-GAD antibody was not detected in the blood of the patients, and it is considered that the pancreatic islets were completely destroyed.

The prophylactic or therapeutic agent of the present embodiment is particularly effective for administration to a patient with acute onset and slowly progressive type 1 diabetes in which an anti-GAD antibody is detected and an autoimmune reaction is in the process of progress.

The prophylactic or therapeutic agent of the present embodiment can be administered to a patient by, for example, subcutaneous administration, transdermal administration, intraperitoneal administration, or the like. The dose of the prophylactic or therapeutic agent varies depending on the patient's symptoms, age, gender, etc., and cannot be unconditionally determined, but is not particularly limited as long as it can induce an immune response in the living body to be administered. For example, an administration schedule in which the active ingredient (tick-derived antigen) of 10 to 100 μg / person is administered once a week can be mentioned, but is not limited to this.

[Pharmaceutical composition for prevention or treatment of type 1 diabetes]
In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of type 1 diabetes, which comprises the above-mentioned prophylactic or therapeutic agent and adjuvant.

As will be described later in the examples, the pharmaceutical composition of the present embodiment can improve the preventive or therapeutic effect of type 1 diabetes and suppress the onset of anaphylactic shock by containing an adjuvant.

As the adjuvant, the same adjuvant as that used for a usual vaccine preparation can be used. More specifically, for example, aluminum hydroxide gel, Freund's complete adjuvant, Freund's incomplete adjuvant, hemozoin, TitterMax Gold (Titamax) and the like can be used. TitterMax Gold is an adjuvant composed of a water-in-oil emulsion of a nonionic copolymer having a hydrophilic region made of polyoxyethylene and a hydrophobic region made of polyoxypropylene.

The pharmaceutical composition of the present embodiment may be formulated as a pharmaceutical composition containing the above-mentioned prophylactic or therapeutic agent, adjuvant and pharmaceutically acceptable carrier.

The pharmaceutical composition is preferably in a dosage form used parenterally, for example, an injection, an external skin preparation, or the like. Specific examples of the external skin preparation include dosage forms such as ointments and patches.

As the pharmaceutically acceptable carrier, those usually used for the preparation of a pharmaceutical composition can be used without particular limitation.

The pharmaceutical composition may contain additives. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and martitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffering agents such as salts and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like can be mentioned.

The pharmaceutical composition is formulated by appropriately combining the above-mentioned mite-derived antigen, adjuvant and the above-mentioned pharmaceutically acceptable carrier and additive, and mixing them in a generally accepted unit dose form required for pharmaceutical practice. can do.

The pharmaceutical composition can be administered to a patient by, for example, subcutaneous administration, transdermal administration, intraperitoneal administration, or the like. The dose of the pharmaceutical composition varies depending on the patient's symptoms, age, gender, etc., and cannot be unconditionally determined, but is not particularly limited as long as it can induce an immune response in the living body to be administered. For example, an administration schedule in which the active ingredient (tick-derived antigen) of about 100 μg / person is administered once or twice can be mentioned, but is not limited thereto.

For example, as will be described later in the examples, by administering the pharmaceutical composition of the present embodiment only once (single administration), pancreatic isletitis is improved without the development of a mite-derived antigen-specific IgE antibody. Conceivable.

[kit]
In one embodiment, the present invention provides a kit for preventing or treating type 1 diabetes, which comprises the above-mentioned preventive or therapeutic agent for type 1 diabetes and an adjuvant. It can be said that the kit of this embodiment is a kit containing a mite-derived antigen and an adjuvant.

In the kit of this embodiment, the tick-derived antigen and the adjuvant are the same as those described above. According to the kit of the present embodiment, the mite-derived antigen and the adjuvant can be mixed and administered to a patient or a patient at the time of use. Further, the kit of the present embodiment may further include equipment such as an injection tube.

[Other embodiments]
In one embodiment, the invention provides a method of preventing or treating type 1 diabetes, comprising administering an effective amount of a tick-derived antigen to a patient in need of treatment. As the mite-derived antigen, the same antigens as those described above can be used.

In one embodiment, the present invention provides a method for preventing or treating type 1 diabetes, which comprises administering an effective amount of a pharmaceutical composition containing a mite-derived antigen and an adjuvant to a patient in need of treatment. As the mite-derived antigen and the adjuvant, the same ones as those described above can be used.

In one embodiment, the invention provides a tick-derived antigen for the prevention or treatment of type 1 diabetes. As the mite-derived antigen, the same antigens as those described above can be used.

In one embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of type 1 diabetes, which comprises a tick-derived antigen and an adjuvant. As the mite-derived antigen and the adjuvant, the same ones as those described above can be used.

In one embodiment, the present invention provides the use of a tick-derived antigen for producing a prophylactic or therapeutic agent for type 1 diabetes. As the mite-derived antigen, the same antigens as those described above can be used.

In one embodiment, the invention provides the use of tick-derived antigens and adjuvants for the manufacture of prophylactic or therapeutic agents for type 1 diabetes. As the mite-derived antigen and the adjuvant, the same ones as those described above can be used.

Next, the present invention will be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental Example 1]
(Administration of mite-derived antigen to NOD mice)
A 2-week-old female NOD mouse was given 10 μg / mite-derived antigen (Df insect body extract, product number 10107, manufactured by ITEA Inc., Tokyo Environmental Allergy Research Institute) once a week from 2 to 8 weeks of age. It was administered intraperitoneally (tick-derived antigen-administered group, n = 7). No adjuvant was used. In addition, the mice in the control group were administered with only the medium instead of the mite-derived antigen (n = 7).

Subsequently, the pancreas was excised by euthanasia when either the condition of becoming positive for urinary sugar or reaching the age of 10 weeks was satisfied. Subsequently, the excised pancreas was fixed with paraformaldehyde, embedded in paraffin, and then tissue sections were prepared.

Subsequently, the obtained pancreatic tissue sections were stained with hematoxylin and eosin and observed under a microscope to evaluate pancreatic isletitis. Three or more islets were evaluated per individual, and the highest score was taken as the isletitis score of the individual. The evaluation criteria for pancreatic islet inflammation are as shown in Table 1 below.

1 (a) to 1 (g) are representative histological images of pancreatic islets of each mouse (No. 1 to No. 7) in the control group. In addition, FIGS. 2 (a) to 2 (g) are representative histological images of pancreatic islets of each mouse (No. 1 to No. 7) in the mite-derived antigen-administered group. Table 2 below shows the isletitis scores evaluated based on the photographs of FIGS. 1 and 2.

FIG. 3 is a graph showing the results of calculating the isletitis scores (mean ± standard deviation) of the mice of each group based on the results in Table 2. In FIG. 3, “**” indicates that there is a significant difference when the risk rate (p value) is less than 0.01.

As a result, it was confirmed that the infiltration of lymphocytes into the pancreatic islets was significantly suppressed in the mite-derived antigen-administered group as compared with the control group.

In this experiment, administration of the mite-derived antigen was started from 2 weeks before the onset of isletitis in NOD mice, and the administration was continued until 8 weeks. Since the onset of type 1 diabetes is caused by isletitis, it has been clarified that type 1 diabetes can be prevented or treated by administration of a mite-derived antigen.

[Experimental Example 2]
(Combined use of mite-derived antigen and adjuvant)
It is considered that 4-week-old NOD mice have already developed isletitis. In this experimental example, a 4-week-old NOD mouse was used for the study. First, 4-week-old female NOD mice were divided into 5 groups, 5 in each group. Subsequently, mice in each group were administered a mite-derived antigen (Df insect body extract, product No. 10107, manufactured by ITEA Inc., Tokyo Environmental Allergy Research Institute) and an adjuvant under the conditions shown in Table 3 below.

In Table 3, E-1 represents the adjuvant hemozoin (In vivo Gen), and E-2 represents the adjuvant TitterMax Gold (TiterMax).

Subsequently, each mouse was euthanized when it reached 11 weeks of age, and the pancreas was removed. Subsequently, the excised pancreas was fixed with paraformaldehyde, embedded in paraffin, and then tissue sections were prepared.

Subsequently, the obtained pancreatic tissue sections were stained with hematoxylin and eosin and observed under a microscope to evaluate pancreatic isletitis. The evaluation criteria for pancreatic isletitis were the same as in Experimental Example 1. Three or more islets were evaluated per individual, and the highest score was taken as the isletitis score of the individual. Table 3 shows the isletitis scores. In Table 3, "**" indicates that there was a significant difference at P <0.01 as a result of Wilcoxson's rank sum test.

As a result, it was found that the onset of pancreatic isletitis tended to be suppressed by a single intraperitoneal administration of a mixture of mite-derived antigen and hemozoin (group 2-2). In addition, it was clarified that the onset of pancreatic isletitis can be significantly suppressed by a single subcutaneous administration of a mixture of a mite-derived antigen and TitterMax Gold (Groups 2-4). In addition, the effect of suppressing the onset of pancreatitis when a single dose of mite-derived antigen is administered using an adjuvant (groups 2-2 and 2-4) is that the mite-derived antigen is intraperitoneally administered 5 times without using an adjuvant. It was higher than the effect of suppressing the onset of pancreatitis when administered (group 2-5).

That is, it was clarified that the isletitis can be improved by a single administration of the mite-derived antigen using an adjuvant even after the onset of isletitis.

[Experimental Example 3]
(Examination of Th1 / Th2 balance)
It is known that when a foreign substance invades a living body, it is taken up by dendritic cells or the like and presented as an antigen to naive CD4 positive T cells, and the activated T cells differentiate into Th1 cells or Th2 cells. Th1 cells produce interferon (IFN) -γ and the like, and are mainly involved in cell-mediated immunity and infection defense.

On the other hand, Th2 cells produce interleukin (IL) -4, IL-5, IL-9, IL-13 and the like. IL-4 and IL-13 promote immunoglobulin class switching in B cells to enhance IgE production. IL-5 also activates eosinophils. In addition, IL-4 and IL-9 act on mast cells to induce allergic inflammation.

A subset of Th1 cells and Th2 cells form a Th1 / Th2 balance by the inhibitory actions of the cytokines produced by each. Type 1 diabetes is an organ-specific autoimmune disease, in which case the Th1 / Th2 balance is thought to be Th1 predominant.

In this experimental example, the Th1 / Th2 balance in each group of mice was examined using the spleen cell suspension of each mouse collected in Experimental Example 2.

Specifically, the spleen cell suspension was stimulated with concanavalin A, which is a kind of lectin, to promote cytokine production of T cells. Subsequently, monensin was added to the cells to inhibit intracellular protein transport. Subsequently, CD4, which is a cell surface antigen, was stained with FITC-labeled anti-mouse CD4 antibody.

Subsequently, saponin was added to the cells to enhance cell membrane permeability, and the intracellular cytokines IFN-γ and IL-4 were used with APC-labeled anti-mouse IFN-γ antibody and PE-labeled anti-mouse IL-4 antibody, respectively. Stained.

Subsequently, cells were analyzed using a flow cytometer. CD4-positive IFN-γ-positive cells were designated as Th1 cells, and CD4-positive IL-4-positive cells were designated as Th2 cells.

FIG. 4 is a graph showing the results of cell analysis. In FIG. 4, "*" indicates that there was a significant difference at P <0.05 as a result of Student's t-test, and "**" indicates that there was a significant difference at P <0.01 as a result of Student's t-test. Indicates that there was a significant difference.

As a result, it was clarified that the Th1-dominant state could be improved by a single administration of the mite-derived antigen to the 4-week-old NOD mice using an adjuvant.

[Experimental Example 4]
(Examination of appearance of IgE class antibody by administration of mite-derived antigen)
4-week-old female NOD mice were divided into 4 groups, 5 in each group. Subsequently, mice in each group were administered a mite-derived antigen (Df insect body extract, product No. 10107, manufactured by ITEA Inc., Tokyo Environmental Allergy Research Institute) and an adjuvant under the conditions shown in Table 4 below.

In Table 4, E-1 represents the adjuvant hemozoin (In vivo Gen), and E-2 represents the adjuvant TitterMax Gold (TiterMax).

Subsequently, when each mouse reached the age of 11 weeks, blood was collected to detect a tick-derived antigen-specific IgE antibody. Detection of IgE antibody was evaluated by passive cutaneous anaphylaxis reaction (PCA reaction) using rats.

Specifically, sera collected from mice in each group were intradermally administered to rats, and 24 hours later, the same rats were administered with a dye (Evans blue) and a tick-derived antigen by tail vein. Here, when IgE is present in the serum derived from mice, the tick-derived antigen binds to the mouse IgE bound to the receptor of rat mast cells, and a type I allergic reaction is induced locally on the skin, resulting in histamine and the like. It is released and induces hyperpermeability of peripheral blood vessels. As a result, blood components containing pigment flow out of the blood vessels of the rat. Therefore, by observing the peeled rat skin, the distribution of the pigment flowing out from the blood vessels can be visually observed as spots.

In this experimental example, when the diameter of the spot was 5 mm or more, it was determined that the PCA reaction was positive, and when the diameter of the spot was less than 5 mm, it was determined that the PCA reaction was negative. When the PCA reaction was positive, the maximum dilution factor of the positive mouse serum was taken as the antibody titer. Table 4 shows the results of the PCA reaction. In Table 4, for example, "negative (5/5 cases)" indicates that the result of the PCA reaction of the mouse sera of 5 out of 5 cases was negative.

As a result, when a single dose of mite-derived antigen was administered to mice using an adjuvant, no generation of mite-derived antigen-specific IgE antibody was observed (groups 4-1 and 4-2). In addition, when the mite-derived antigen was administered to mice twice using an adjuvant, the development of mite-derived antigen-specific IgE antibody was observed in 1 of 5 cases (Group 4-3).

The antibody titer of the tick-derived antigen-specific IgE antibody in the mouse serum of the 4-4 group was 20 times or more in all 5 cases. In addition, the antibody titer of one case that was positive in the 4-3 group was 32 times.

From the above results, even after the onset of pancreatic isletitis, it is possible to improve pancreatic isletitis without the development of IgE antibody specific to the mite-derived antigen by administering a single dose of the mite-derived antigen using an adjuvant. It became clear.

INDUSTRIAL APPLICABILITY According to the present invention, a prophylactic or therapeutic agent for type 1 diabetes can be provided.

Claims (3)

A prophylactic or therapeutic agent for type 1 diabetes, which contains Dermatophagoides farinae extract as an active ingredient and is used for one or two subcutaneous administrations. A pharmaceutical composition for preventing or treating type 1 diabetes, which comprises the prophylactic or therapeutic agent for type 1 diabetes and an adjuvant according to claim 1 and is for one or two subcutaneous administrations. A preventive or therapeutic agent for type 1 diabetes and an adjuvant, which comprises the prophylactic or therapeutic agent and an adjuvant for type 1 diabetes according to claim 1, wherein the prophylactic or therapeutic agent and the adjuvant are mixed and used by subcutaneous administration once or twice. Treatment kit.

Images