JP4801868B2 - Systemic lupus erythematosus disease suppressant - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention may, for example pathogenesis related systemic lupus erythematosus disease drugs suppressing systemic Eritematode scan.
[0002]
[Prior art]
Recently, self systemic lupus erythematosus, known as a representative of immunological diseases (Systemic Lupus Eryhtrmatosus: SLE) is the background of some genetic predisposition since concordance rate in identical twins is about 63%, infection It is considered to be a multifactorial disease that develops due to environmental factors such as sex hormones, ultraviolet rays, and drugs.
[0003]
In this systemic lupus erythematosus, autoantibodies against various systemic organs are produced, causing lesions in the skin, joints, kidneys, nerves, heart, blood vessels, lungs, intestinal tract, or bladder. These autoantibodies mainly recognize nuclear and cellular component antigens, and directly invade each organ, or form immune complexes in blood and deposit in tissues. Furthermore, it is thought to promote the activation of the trapping system and promote inflammation and destruction of the tissue.
[0004]
The main autoantibody in the pathology of systemic lupus erythematosus is an anti-double-stranded DNA antibody (anti-dsDNA Ab), which is one of the antinuclear antibodies. Glomerulonephritis (lupus nephritis), which is very important as an indicator and induced by immune complexes, is a serious factor leading to death and is a central antibody that affects the progression of the disease state.
[0005]
On the other hand, the relationship of genetic predisposition to systemic lupus erythematosus is being elucidated by analysis of susceptibility genes of the human body and endogenous retroviruses (ERV) that have been incorporated into the body in advance. The relationship of environmental factors to systemic lupus erythematosus is being elucidated through the analysis of sex viruses, but the full picture of systemic lupus erythematosus has not yet been clarified.
[0006]
Then, the treatment of this systemic lupus erythematosus, but are common to essentially autoimmune disease, in a fundamental treatment except pair disease therapy is corticosteroids expect anti-inflammatory and immunosuppressive action Adrenocortical steroids such as predonisolone are frequently used, and in particular, they are forced to be used in large quantities in the early stage and severe cases. Furthermore, administration of an immunosuppressive drug such as cyclophosphamide or azathioprine is considered in cases of corticosteroid resistance or cases in which side effects appear.
[0007]
[Problems to be solved by the invention]
However, in the case of treating systemic lupus erythematosus that has already developed disease, it is necessary to systemically administer the above-mentioned corticosteroids, immunosuppressive drugs, etc. over a long period of time. As a result, intermittent high-dose therapy is forced along with relapse, and there is a risk of recurrence due to a decrease in the dose of the drug to be administered.
[0008]
Thus, with current therapies, patients with systemic lupus erythematosus are able to control bone marrow function with immunosuppressants, such as osteoporosis due to corticosteroids, boneless non-rotating necrosis, peptic ulcers, diabetes, infection or depression. In addition to being constantly suffering throughout the life of side effects such as interstitial pneumonia, liver dysfunction, kidney dysfunction or infection, there is a method to prevent and prevent systemic lupus erythematosus It has the problem that it is not yet known.
[0009]
The present invention has been made in view of the above problems, and an object thereof is to provide a systemic lupus erythematosus disease drugs that can suppress the onset of systemic lupus erythematosus.
[0010]
[Means for Solving the Problems]
The systemic lupus erythematosus disease suppressant of the present invention contains estrogen as an active ingredient and is administered during the neonatal period to suppress the onset of systemic lupus erythematosus .
[0011]
Then, as an active ingredient an estrogen, by being administered during the neonatal period, it is possible to perform the immune tolerance against pathogenic antigens produced by estrogen stimulation, systemic in descending Yo以 immune Hiroshi by the estrogen-stimulated Can control the onset of lupus erythematosus .
[0012]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the configuration of a systemic lupus erythematosus disease inhibitor of one embodiment of the present invention will be described with reference to the drawings.
[0013]
First, systemic lupus erythematosus is a representative disease in collagen diseases as autoimmune diseases (Systemic Lupus Eryhtrmatosus: SLE), together emit good young women with puberty, the concordance rate in identical twins It is considered to be a multifactorial disease caused by environmental factors such as infections, sex hormones, ultraviolet rays, drugs, etc. on the basis of some genetic predisposition since it is about 63%.
[0014]
In this systemic lupus erythematosus, autoantibodies against various systemic organs are produced, causing lesions in the skin, joints, kidneys, nerves, heart, blood vessels, lungs, intestinal tract, or bladder. These autoantibodies mainly recognize nuclear and cellular component antigens, and directly invade each organ, or form immune complexes in blood and deposit in tissues. Furthermore, it is thought to promote the activation of the trapping system and promote inflammation and destruction of the tissue.
[0015]
The main autoantibody in the pathology of systemic lupus erythematosus is an anti-double-stranded DNA antibody (anti-dsDNA Ab), which is one of the antinuclear antibodies. Glomerulonephritis (lupus nephritis), which is very important as an index and induced by immune complexes, is a serious factor leading to death and is a central antibody that affects the progression of the disease state.
[0016]
Then, estrogen formulation onset is systemic lupus erythematosus disease drugs you suppress the systemic lupus erythematosus, for example estradiol valerate is estrogen (Estrogen) is contained as an active ingredient is a generic name of estrogen. Moreover, this systemic lupus erythematosus disease inhibitor is administered only once in the early part of the birth, before the systemic lupus erythematosus develops.
[0017]
Here, estrogen is the most basic female sex steroid hormone, and has not only the development of internal and external genitals but also the extragenital effects on the central nervous system, metabolic system, breast and the like. Moreover, as this estrogen, there exist estrone (estrone), estradiol (estradiol), estriol (estriol), etc.
[0018]
Estradiol has the highest physiological activity, is produced mainly from the ovary, exhibits a characteristic secretion pattern with follicular development, and is secreted in large quantities as part of placental estrogen during pregnancy. In addition to the growth and proliferation of the gonadal system, this estradiol is involved in bone metabolism, has a skeletal growth effect, and as an index of placental function during pregnancy, it is an ovary in adolescence, infertility, menopause and menopausal women It has an important meaning as a function evaluation.
[0019]
Next, the operation of the above embodiment will be described with reference to experimental examples.
[0020]
(Experimental example)
Using an animal model, we investigated the disease suppression by estrogen administration for systemic lupus erythematosus.
[0021]
As animal models of systemic lupus erythematosus, NZB / NZW F1 mice derived from NZB (New Zealand black mice) / BINJ and NZW (New Zealand white mice) / LacJ are known.
[0022]
In this NZB / NZW F1 female mouse, the expression of anti-double-stranded DNA antibody (anti-dsDNA Ab) and hyperglobulinemia were observed from around 4 weeks of age. Increased strand DNA antibody titer, spontaneously induced immune complex-mediated glomerulonephritis, lupus nephritis, and death due to renal failure within about 10 months after birth Take. Thus, the clinical course of this NZB / NZW F1 female mouse is very similar to that of human systemic lupus erythematosus.
[0023]
Then, within 2 weeks after birth, more specifically within 3 days, female NZB / NZW F1 mice were treated with 17-β-estradiol (17E ₂ ) valerate peranine depot (Pelanin Depot: Mochida Pharmaceutical). Co., Ltd.) was administered only once, and the proteinuria, the expression level of anti-DNA antibody (anti-dsDNA Ab), and the survival rate over time were observed about 3 times over 18 months.
[0024]
In a detailed method, this peranin depot (estrogen) was diluted with sesame oil (sesame oil: manufactured by Sigma Chemical) for inoculation and adjusted to 2 ng / ul subcutaneously at the back at a ratio of 200 ng / g to the body weight of the mouse. Administration was performed. Sesame oil used for dilution as a control at the same time as estrogen inoculation was administered subcutaneously at the back in the same volume.
[0025]
The determination amount analysis of urine protein was performed using a tetrabromophenol blue coated urine analysis stick (manufactured by Terumo).
[0026]
Furthermore, the antibody titer of the anti-double-stranded DNA antibody (anti-dsDNA Ab) is measured by enzyme immunoassay (ELISA method), and the antigen is double-stranded calf thymus DNA (manufactured by Sigma Chemical), A peroxidase-labeled anti-mouse IgG Fab fragment (manufactured by Nordic Immunology Laboratories) was used as the secondary antibody, and a monoclonal mouse anti-DNA antibody (manufactured by CHEMICON International) was used as the standard for antibody titer measurement.
[0027]
In female NZB / NZW F1 mice administered with 8- to 10-week-old estrogen capable of reproduction, genital maturation was observed histopathologically, and estrogen receptor ER-α mRNA in each organ was observed. Expression was evaluated by a gene amplification method called RT-PCR (Riverse Transcription-Polymerase Chain Reaction) as a molecular biological technique to confirm the retention of functions related to estrogen.
[0028]
At this time, RNA was extracted by physically homogenizing tissues of each organ and then using TRIZOL LS Reagent (GIBCO BRL) according to the experiment method of the reagent company. The extracted RNA has 6 to 8 exon regions of estrogen receptor α as primer pairs (sense [AGG GTG AAG AGT TTG TGT GC; 1534-1543] and antisense [AGG AAT GTG CTG AAG TGG AG; 1918 to 1937]. ) Using a one-step RT-PCR kit (QIAGEN). At the same time, a mouse-glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) RNA that is continuously expressed in each organ is used as an internal control, and a primer kit manufactured by Maxim Biotec. Was amplified using.
[0029]
As a result, in many cases, anti-DNA antibodies become apparent after approximately 4 weeks of age in female NZB / NZW F1 mice, and each time after the aging, the antibody positivity rate and the positivity are increased. Increased amounts are allowed as they mature.
[0030]
At this time, a similar result was also obtained in the control group, which was a group of 20 female NZB / NZW F1 mice administered with control oil within 3 days of life, but 20 female estrogens administered with estradiol. In the administration group, as shown in FIG. 2, the antibody positive rate and the suppression of the enhancement of the amount after positivity were significantly confirmed.
[0031]
In addition, the female NZB / NZW F1 mice in the control group after 7 weeks of age died sequentially as the antibody amount increased, and those with low antibody titers became alive. Although not presented due to the reduced reliability of statistical treatment, the mean antibody titer of female NZB / NZW F1 mice was lower in the estrogen-treated group than in the female control group.
[0032]
Furthermore, as shown in FIG. 1, the survival rate of the female control group decreases after 7 months, and reaches 10% or less at 12 months. On the other hand, the survival rate of the estrogen administration group decreased after 11 months after birth, 4 months later than the female control group, and maintained a survival rate of 50% even after 18 months. In addition, the bar | burr shown in FIG. 1 shows the average of the number for every female estrogen administration group and a control group, and an error bar shows a standard deviation.
[0033]
As a result, the survival rate of female NZB / NZW F1 mice treated with estrogen is clearly higher than that of female control groups. Results were similar to or better than survival. In addition, regarding proteinuria, which is an index of renal dysfunction in all dead mice, all died within 3 months after the proteinuria increased to 250 mg / dl or more in common in the estrogen administration group and the control group. ing.
[0034]
Furthermore, in anatomy in 8-10 week old female NZB / NZW F1 mice, which are reproductive, there was no significant difference in uterine and ovarian weights between the estrogen-treated group and the control group. No abnormal morphology was observed, and the pathological findings were similar.
[0035]
In addition, estrogen receptor expression in each organ of NZB / NZW F1 mice was analyzed by RT-PCR, and as shown in FIG. 3, it was strong in the brain, liver and spleen, and in heart, lung and uterus. It was below the detection limit. GAPDH shown in FIG. 3 is glyceraldehyde-3-phosphate dehydrogenase.
[0036]
Therefore, the estrogen receptor mRNA expression in each organ of female NZB / NZW F1 mice to which estrogen was administered and the pathological findings in estrogen-related tissues were not significantly different between the estrogen administration group and the control group, This treatment was confirmed to be safe without causing hormonal dynamic changes in the body.
[0037]
As described above, according to the above-mentioned embodiment, systemic lupus erythematosus is most frequently caused by puberty in women (female), and estrogen is strongly considered as one of the triggers that cause this disease. ing. On the other hand, male NZB / NZW F1 mice, which are animal models of systemic lupus erythematosus, have been reported to exacerbate the disease caused by estrogen and to suppress the disease caused by castration.
[0038]
However, exacerbation of diseases caused by administration of these estrogens and suppression of onset by castration are both considered to be multifactorial diseases, and the etiology is not yet clear. Elucidation of a susceptibility gene is advancing as a genetic predisposition, but it is not clear whether this susceptibility gene is involved as an antigen, or whether it is involved in an immune response or tolerance tolerance. No strong suspicious substances or organisms have been identified.
[0039]
Therefore, as a second genetic predisposition, there is an endogenous retrovirus or other gene that has an estrogen-induced promoter in the somatic cell gene. Focusing on the possibility that the constituents are similar, when a substance that is recognized for the first time after birth is produced by increasing the amount of estrogen produced in puberty, antibodies against that substance are produced and crossover with self-constituents in the body It was assumed that one of the etiologies was to cause a reaction. In other words, the purpose is to induce pathogenic substances in vivo and tolerate immune tolerance by performing estrogen stimulation for a short period of time immediately after birth.
[0040]
Here, the dose of estrogen used this time was in line with the general amount used in humans, and useful findings were not obtained at 1/10 of 20 ng / g. However, the estrogen administration group was different from the control group. and we followed the same course, despite it was clear that can suppress the progress of the disease and clinical picture by administration of estrogen, prior to death in all lethal mice, growth and antibody production of anti-DNA antibodies, The increase in proteinuria suggests that it is necessary to administer a higher concentration of estrogen, and there is a possibility that pathogenesis is suppressed in a concentration-dependent manner.
[0041]
This is because estrogen is produced in patients with systemic lupus erythematosus before the onset of illness, and is produced at low concentrations continuously, and high concentrations of estrogen are produced periodically after adolescence leading to menstruation. If you think you have the disease, you agree theoretically.
[0042]
Similarly, men with low levels of estrogen have a lower incidence of systemic lupus erythematosus, and in animal studies, male NZB / NZW F1 mice can be affected by exposure to high levels of estrogen. Can be backed up. Probably, there is a possibility that the promoter is stimulated by abnormal estrogen at a certain concentration or more and production of an antigen as a pathogen is started.
[0043]
Therefore, in the above experiment, female NZB / NZW F1 mice, which are animal models of systemic lupus erythematosus, are given the same dose as male NZB / NZW F1 mice, which are basically unaffected by administration of estrogen once in the early stages of birth. Estrogen receptor essential for maintaining homeostasis by administration of a single estrogen to a newborn baby, because it did not cause abnormalities in the expression and reproductive function of estrogen receptor in each organ It is considered that the onset of systemic lupus erythematosus after maturation can be suppressed and prevented, the progression can be suppressed, and the survival rate can be increased without reducing the expression of genital organs and without causing genital abnormalities.
[0044]
In addition, it has been elucidated cause gene by gene analysis proceeds, if possible prenatal diagnosis, Ru more selective treatment can be permanently increase of systemic lupus erythematosus.
[0045]
【The invention's effect】
According to the present invention, the administration of a systemic lupus erythematosus disease-suppressing drug containing estrogen as an active ingredient in the neonatal period , where immunological tolerance is possible, can provide immune tolerance against pathogenic antigens produced by estrogen stimulation. since it can suppress the onset of systemic lupus erythematosus in descending immune Hiroshi Yo以 by the estrogen-stimulated.
[Brief description of the drawings]
FIG. 1 is a graph showing the survival rate when a systemic lupus erythematosus disease inhibitor of the present invention is administered.
FIG. 2 is a graph showing the antibody titer of an anti-DNA antibody when a systemic lupus erythematosus disease inhibitor is administered.
FIG. 3 is data showing the expression of mRNA in cells of each organ when a systemic lupus erythematosus disease inhibitor and control oil are administered.

Claims (1)

Contains estrogen as an active ingredient,
Given during the neonatal period ,
A systemic lupus erythematosus disease suppressant characterized by suppressing the onset of systemic lupus erythematosus .

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