JP5382683B2 - Preventive or therapeutic agent for chronic fetal lung disease - Google Patents

Description

  The present invention relates to a thioredoxin family polypeptide (hereinafter sometimes referred to as TRXs) and a drug for maternal administration containing at least one of its inducers as an active ingredient.

The premature birth rate in advanced Asian countries is said to be about 10%, which is the highest in the world after North America. In addition, the rate of premature birth is increasing in recent years.
Therefore, the maintenance of the uterine environment to prevent premature birth is an important issue.

One cause of preterm birth is ascending infection that causes inflammation in the womb.
Here, when signs of ascending infection in the uterus appear in the clinical field, inflammation in the womb due to cytokine overproduction (cytokine storm) or neutrophil infiltration has already progressed in many cases. Conventionally, antibiotics have been administered to suppress the growth of bacteria that cause cytokine storms, but there are many cases where the effect is low, and there are many cases where it is difficult to suppress preterm birth Met.
Furthermore, many recent studies have shown that if the time from birth of the uterus to the delivery of the baby increases, the effect of the uterine inflammation on the fetus increases, and complications include inflammation of the fetal lung (such as chronic lung disease). It has been found that this occurs (see Non-Patent Documents 1 to 4).
In order to reduce the effects of fetal inflammation on the fetus and to reduce fetal lung inflammation, it is better to give birth early. However, if childbirth was accelerated, there was a problem that a newborn infant (premature baby) was born.

In addition, chorioamnionitis is a characteristic pathological image in the placental placenta involved in infection and inflammation. The main cause of inflammation in the chorionic tissue and amniotic tissue is due to neutrophil infiltration triggered by the production of various cytokines.
In view of such a current situation, in order to prevent premature birth, it has been desired to effectively suppress in utero cytokine and neutrophil infiltration.

On the other hand, thioredoxin (TRX) has a 12 kDa redox activity by a disulfide / dithiol exchange reaction between two cysteine residues of its active site sequence: -Cys-Gly-Pro-Cys-: (See Non-Patent Document 5). Thioredoxin has been isolated and identified from many prokaryotes and eukaryotes since it was isolated from E. coli as a hydrogen ion donor to ribonucleotide reductase or as an enzyme important for the synthesis of deoxyribonucleotides.
In addition, adult T cell leukemia inducing factor (ADF), which is a kind of thioredoxin, was first identified as an IL-2 receptor inducing factor produced by T lymphocytes infected with HTLV-1. , Human thioredoxin.
Moreover, intracellular thioredoxin plays an important role in radical scavenging and control of transcription factors relating to redox such as activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) (Non-patent Document 6). reference).
In addition, human thioredoxin controls signal transduction of p38 mitogen activating protein kinase (MAPK) and apoptosis signal regulating kinase-1 (ASK-1).
Furthermore, the present inventors have also reported that thioredoxin is released extracellularly and exhibits a cytokine or chemokine action (see Non-Patent Document 7), and that extracellular thioredoxin moves into the cell (see Non-Patent Document 8). I came.

  However, the relationship between preterm birth and thioredoxin has not yet been reported.

Anti-Annexin A2 IgM Antibody in Preterm Infants: Its Association with Chorioamnionitis.PEDIATRIC RESEARCH Vol.60, No.6,2006; 699-704 Pathology of new bronchopulmonary dysplasia. Seminars in Neonatology 2003; 8, 73-81 Control Mechanisms of Lung Alveolar Development and Their Disorders in Bronchopulmonary Dysplasia.PEDIATRIC RESEARCH Vol.57, No.5, Pt2, 2005; 38R-46R The New BPD. NeoReviews 2006; 7; e531-e545 Redox regulation of cellular activation. Ann.Rev.Immunol.1997; 15: 351-369 AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. PNAS.1997; 94: 3633-3638 Circulating thioredoxin suppresses lipopolysaccharide-induced neutrophil chemotaxis. PNAS.2001; 98: 15143-15148 Redox-sensing release of thioredoxin from T lymphocytes with negative feedback loops. J. Immunol. 2004; 172: 442-448

  In the course of research on the relationship between preterm birth and thioredoxin family polypeptides (TRXs), the present inventors have unexpectedly confirmed that TRXs administered to the mother not only have the effect of preventing preterm birth, but also suppress the inflammation of the fetal lung. It was also found that the effect of can be expected. In other words, the present inventors have concluded that TRXs are effective as a maternally administered agent that can suppress not only premature birth prevention effects but also fetal lung inflammation that often occurs as a complication of premature birth, leading to the completion of the present invention. It was.

  Accordingly, it is an object of the present invention to provide a maternally-administered drug that suppresses not only premature birth prevention effects but also fetal lung inflammation, which frequently occurs as a complication of premature birth, and has few side effects.

The invention according to claim 1 includes -Cys-Gly-Pro-Cys-, -Cys-Pro-Tyr-Cys-, -Cys-Pro-His-Cys-, -Cys-Pro-Pro-Cys- the thioredoxin family of polypeptides and / or active ingredient sulforaphane its inducing agents having, administered to the mother, to preventive or therapeutic agent for chronic lung disease fetal.
Invention, prophylactic or therapeutic agent for chronic lung disease of claim 1 fetal of, wherein the thioredoxin family of polypeptides is a human thioredoxin consisting of the amino acid sequence shown in SEQ ID NO: 1 according to claim 2 About.
The invention according to claim 3 is that the polypeptide of the thioredoxin family comprises an amino acid sequence in which one or several amino acids other than positions 32 and 35 are substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 1. about preventive or therapeutic agent for chronic lung disease fetal according to claim 1, characterized in that it is a thioredoxin variants.
The invention according to claim 4 relates to the preventive or therapeutic agent for chronic lung disease fetal according to any of claims 1 to 3, characterized in that a liquid or gel.
The invention according to claim 5 relates to the preventive or therapeutic agent for chronic lung disease fetal according to claim 4, characterized in that it is for vaginal administration.

Preventive or therapeutic agent for chronic lung disease engagement Ru fetal the present invention (hereinafter, also referred to as a parent administering drugs), since the polypeptide (TRXs) and active ingredient one or more of its inducing agents thioredoxin family Inhibits inflammation in the womb, and further prevents the effects of fetal inflammation on the fetus. Thereby, premature birth can be prevented. In addition, when TRXs are administered to a mother, TRXs affect the lungs of the fetus. Therefore, inflammation of the fetal lung, which often occurs as a complication of preterm birth, can be suppressed.

Furthermore, since the drug for maternal administration according to the present invention contains TRXs that are endogenous thiol proteins expressed in the body or substances that induce the expression of TRXs in the body as an active ingredient, there is no worry about side effects, and the mother is highly safe. It becomes a drug for administration.
In addition, since the thioredoxin family polypeptide is human thioredoxin or a modified thioredoxin, the possibility of side effects can be reduced, and a safer drug for maternal administration can be obtained.
Moreover, since the drug for maternal administration is liquid or gel, parenteral administration such as application to a specific place is possible, and many administration routes can be realized.
For example, by using a liquid or gel, it can be used for vaginal administration. Specifically, the drug for maternal administration can be administered to the mother by washing it in the vagina as a liquid cleaning solution for vaginal washing or by applying it directly in the vagina as a gel. Inflammation in the womb, which is the cause of premature birth, is caused by ascending infection from within the vagina. By using a maternally administered drug for vaginal administration, inflammation in the womb can be suppressed even from the initial stage. Can be reliably prevented. In addition, vaginal administration is safer than other administration routes, and is optimal as an administration route to the mother.

As a result of intensive studies, the present inventors have discovered that a new function of thioredoxin family polypeptides (TRXs) suppresses the production of inflammatory cytokines TNFα, IFNγ, IL-6, and MCP1 in the maternal womb. did.
By suppressing the expression of inflammatory cytokines in the maternal womb, inflammation in the womb caused by cytokine storms in the womb can be suppressed.
Moreover, since TRXs can inhibit neutrophil migration, neutrophil infiltration in the womb can be suppressed, and inflammation in the womb resulting from neutrophil infiltration can also be suppressed.
Due to the above-described effects of TRXs, inflammation due to ascending infection, which is the cause of premature birth, can be suppressed, and premature birth can be prevented.
Furthermore, since the inflammation in the womb can be suppressed, the influence of the uterine inflammation on the fetus can be suppressed, and it is not necessary to accelerate the delivery.
In addition, the present inventors have also found that an unexpected effect that TRXs administered to a mother body also affects the fetal lung can be expected. When TRXs affect the lungs of the fetus, not only premature delivery but also fetal lung inflammation (such as chronic lung disease) that often occurs as a complication of premature birth can be effectively suppressed.

Based on the above, the present inventors have found that TRXs are optimal for maternally administered drugs (those that have not only premature birth prevention effects but also anti-inflammatory effects on fetal lungs) and complete the present invention. It came to.
Hereinafter, an embodiment of a drug for maternal administration containing as an active ingredient one or more of thioredoxin family polypeptides and derivatives thereof according to the present invention will be described in detail.

[TRXs]
TRXs used for maternally administered drugs are endogenous thiol proteins that are expressed in the body, so there is no worry about side effects and they are highly safe drugs for maternally administered.

Examples of TRXs include human thioredoxin (hTRX) and the like, as long as they belong to the “thioredoxin family”, and the active center thereof is -Cys-Gly-Pro-Cys-, -Cys-Pro-Tyr- Examples include those having polypeptides having Cys-, -Cys-Pro-His-Cys-, -Cys-Pro-Pro-Cys-.
Among these, thioredoxin or thioredoxin 2 (mitochondrial-specific thioredoxin) having the sequence -Cys-Gly-Pro-Cys- at the active center is preferable.

  The origin of thioredoxin is not particularly limited as long as it has the above-mentioned active center, and thioredoxin of animals including humans (ADF of animals including humans), thioredoxins of bacteria such as E. coli, thioredoxins of yeast, human ADF activity And a polypeptide (human ADFP), glutaredoxin such as human and Escherichia coli, and the like. Particularly preferred are human thioredoxin (hTRX) and yeast thioredoxin. Yeast thioredoxin may be used in a state isolated from yeast, but can also be used in the form of a yeast rich in thioredoxin.

  In this specification, human thioredoxin (hTRX) refers to a polypeptide consisting of 105 amino acids shown in SEQ ID NO: 1. The nucleotide sequence of hTRX is shown in SEQ ID NO: 2. Since hTRX has been identified from humans, it is preferable because it has fewer side effects than other TRXs.

Further, a TRX variant prepared by a known genetic engineering technique based on the human thioredoxin of SEQ ID NO: 1 may be used as long as it suppresses inflammation in the womb and fetus.
Examples of the TRX variant include those in which one or several amino acids other than positions 32 and 35, preferably positions 32 to 35 of SEQ ID NO: 1 are substituted, deleted, added, or inserted.

  In the present invention, polypeptides (TRXs) belonging to the above thioredoxin family can be used alone or in combination of two or more.

The drug for maternal administration according to the present invention includes both oral administration and parenteral administration, and may be appropriately selected depending on the administration route. However, in consideration of the effect on the fetus, parenteral administration is preferable. .
In addition, intravenous (IV) administration may be performed in the treatment of maternal infection, fetal inflammatory response syndrome, and the like.
In addition, various forms of administration such as painting can be realized by changing the form of the drug for maternal administration to liquid or gel, but the initial stage of inflammation in the womb can be used for vaginal administration. It is preferable that
Inflammation in the womb, which is the cause of premature birth, is caused by ascending infection from the vagina, so it is possible to suppress inflammation in the womb from the initial stage by administering the maternally administered drug for vaginal administration. This is because premature birth can be reliably prevented. In addition, vaginal administration is safer than other administration routes, and is optimal as an administration route to the mother.
Specifically, the drug for maternal administration can be administered to the mother by washing it in the vagina as a liquid cleaning solution for vaginal washing or by applying it directly in the vagina as a gel.

  When orally administering the drug for maternal administration according to the present invention, as the drug form, solid preparations (tablets, pills, powders, coated tablets, granules, capsules, etc.), liquid preparations (solutions, suspensions, Emulsions, syrups, etc.), inhalants (aerosols, atomizers, nebulizers, etc.) and liposome encapsulating agents.

  The drug for maternal administration according to the present invention is put into practical use as a pharmaceutical preparation by using a pharmaceutically acceptable pharmaceutical carrier together with the above active ingredients. Pharmaceutical carriers include binders, disintegrants, surfactants, absorption enhancers, humectants, adsorbents, lubricants, fillers, extenders, moisturizers, preservatives, stabilizers, emulsifiers, solubilizers Examples thereof include salts that adjust osmotic pressure, diluents such as buffers, excipients, and the like, and these are appropriately selected and used depending on the dosage unit form of the resulting preparation.

In the case of forming into a tablet form, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. Agent (water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, etc.), disintegrant (carboxymethylcellulose sodium, carboxymethylcellulose calcium, low substituted hydroxypropyl) Cellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, etc.), surfactant (polyoxyethylene sorbitan fatty acid esters, lauryl) Acid sodium, stearic acid monoglyceride, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption promoters (quaternary ammonium base, sodium lauryl sulfate, etc.), humectants (glycerin, starch, etc.) Adsorbents (starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.), lubricants (purified talc, stearate, boric acid powder, polyethylene glycol, etc.) and the like can be used.
Furthermore, the tablet can be made into a tablet coated with a normal coating as necessary, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, or a film-coated tablet. Moreover, it can also be set as a double tablet and a multilayer tablet by changing the number of the coating layers applied to a tablet.
In the case of forming into a pill form, for example, excipients (glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.), binders (gum arabic powder, tragacanth powder, gelatin, ethanol) Etc.), disintegrating agents (laminaran, agar etc.) and the like can be used.

  In the case of parenteral administration of the drug for maternal administration according to the present invention, an injection (solution, emulsion, suspension) used for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, etc. Agents), liquid agents (eg, eye drops, nasal drops, etc.), drops, inhalants (aerosols, powder inhalers, etc.) and the like.

When the drug for maternal administration according to the present invention is prepared as an injection such as solution, emulsion, suspension, etc., it is preferable that these are sterilized and isotonic with blood. In addition, when adjusting to an injection, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like are used as diluents. be able to.
In this case, a sufficient amount of sodium chloride, glucose or glycerin for preparing an isotonic solution may be contained in the injection. Ordinary solubilizing agents, buffering agents, soothing agents and the like may be added.

  When the drug for maternal administration according to the present invention is a liquid preparation, it may be stored after removing moisture by freeze storage or freeze drying. The lyophilized liquid preparation (lyophilized preparation) can be used by dissolving it again by adding distilled water for injection at the time of use.

  When the maternally administrable drug according to the present invention is used as an inhalant, it may be prepared using a known inhalant additive. Examples of the additive for the suction agent include propellant, solid excipient (sucrose, lactose, glucose, mannitol, sorbit, etc.), liquid excipient (propylene glycol, etc.), binder (methylcellulose, hydroxypropylcellulose, Polyvinylpyrrolidone, polyethylene glycol, sucrose, etc.), lubricant (magnesium stearate, light anhydrous silicic acid, talc, sodium lauryl sulfate, etc.), preservative (sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.), stable Agent (citric acid, sodium citrate, etc.), suspending agent (methyl cellulose, polyvinylpyrrolidone, lecithin, sorbitan trioleate, etc.), dispersant (surfactant, etc.), solvent (water, etc.), tonicity agent (Sodium chloride, etc.), pH adjusters (sulfuric acid, hydrochloric acid, etc.), solubilizing agents ( Ethanol, etc.), and the like.

  Furthermore, in the medicine for maternal administration according to the present invention, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other medicines may be contained as necessary.

  The dose of TRXs can be easily determined by those skilled in the art with reference to well-known techniques. For example, when administered to the mother, the dose is preferably about 6 to 24 mg / kg per person per day. In addition, the dose can be administered once or divided into several times per day. However, the dosage is preferably adjusted as appropriate according to the form of various preparations and the degree of disease.

[TRXs inducer]
The present invention also includes a case where TRXs are induced in the body using a TRXs inducer such as sulforaphane to suppress inflammation in the womb and fetal lung.
Sulforaphane, which is a TRXs inducer, is contained in, for example, cabbage, purple cabbage, broccoli, kale, rocket vegetable, cauliflower, radish, Chinese cabbage, turnip, komatsuna, chingensai, etc., and it is preferable to use shoots of these plants.
Among them, sulforaphane is abundant in broccoli sprouts (broccoli sprout), radish sprouts (kaiwared radish), murasaki cabbage sprouts, and the like.
The sulforaphane-containing extracts obtained from these plants contain sulforaphane of about 0.001 to 200 mg / g, preferably about 0.005 to 80 mg / g, more preferably about 0.01 to 50 mg / g. It is desirable that In addition, the sulforaphane-containing extract is subjected to treatment such as purification and freeze-drying as necessary.

  Further, the amount of sulforaphane to be ingested may be appropriately selected depending on its usage, body weight, health condition, other conditions, the degree of symptoms, and the like.

  Sulforaphane may be used in combination with geranylgeranylacetone (GGA), which is a TRXs inducer.

  The sulforaphane-containing extract may be used as it is, or may be used in the form of a pharmaceutical preparation containing a TRXs inducer as an active ingredient. When used in the form of a pharmaceutical preparation, it can be prepared together with a pharmaceutically acceptable pharmaceutical carrier.

  Moreover, when administering the formulation which uses the TRXs inducer of this invention to an active ingredient to a parent | base, it can also be used with the form of food-drinks. When used in the form of a food or drink, it may be prepared by adding a plant as a raw material to the sulforaphane-containing extract.

  Examples of the foods and drinks include health foods, nutritional supplements (balance nutritional foods, supplements, etc.), functional nutritional foods, foods for specified health use, foods for the sick, and the like. The method for producing these foods is not particularly limited as long as the effect of induction of thioredoxin expression is obtained.

  Moreover, the pharmaceutical for maternal administration according to the present invention may be prepared by combining thioredoxin family polypeptides (TRXs) and TRXs inducers.

  Hereinafter, the effects of the present invention will be made clearer by showing examples. However, the present invention is not limited to the following examples.

(Test Example 1)
In Test Example 1, LPS (Lipopolysaccharide: Ribopolysaccharide) is administered to a pregnant female mouse in a state in which premature birth is likely to occur and TRX is administered to a female mouse that is likely to have premature delivery. Verified.
Hereinafter, the experimental method of Test Example 1 will be described in detail.

First, LPS and TRX administered to female mice will be described.
LPS used in Test Example 1 was prepared through the following steps (i) to (iv).
(I) 10 mg of LPS (E. coli O55: B5 (SIGMA)) bulk powder is dissolved in 2 ml of physiological saline to a concentration of 5 mg / ml.
(Ii) LPS dissolved in physiological saline is vortexed for 10 seconds and sonicated for 15 minutes.
(Iii) Dispense 20 μl each into a polystyrene tube (volume: 0.5 ml) and store at −80 ° C.
(Iv) Dissolve on ice immediately before use and dilute to 20 μg / ml with physiological saline.
The TRX used in this example is a human recombinant TRX diluted to 0.2 mg / ml with PBS (Phosphate Buffered Saline).

Next, a method for administering LPS and TRX to female mice will be described.
The female mouse used in Test Example 1 is a C3H / HeN female mouse.
A 9-15 week-old C3H / HeN female mouse (hereinafter simply referred to as a female mouse in Test Examples 1 to 3) was mated with a B6D2F1 male mouse to confirm the plug. The day when the plug was confirmed was defined as the 0th day of pregnancy.
On the 15th day of pregnancy, LSP 10 ml / kg (200 μg / kg) was administered into the abdominal cavity of female mice twice every 3 hours (specifically at 14:00 and 17:00).
In addition, TRX is 10 ml / kg (2 mg / kg) by female injection by intravenous injection from the tail vein three times every 3 hours (specifically at 13:00, 16:00, 19:00) from 1 hour before LPS administration. To give female mice of Example 1.
And the time when the placenta or fetal delivery was confirmed was taken as the time of delivery, and the number of days from pregnancy to delivery was measured.
In addition, female mice not administered anything (no LPS administered) were Comparative Example 1, female mice administered only LPS were Comparative Example 2, PBS was administered instead of TRX (LPS and PBS were administered) The female mouse was Comparative Example 3, the female mouse administered with 0.2 mg / ml OVA (ovalbumin: ovalbumin) instead of TRX (LPS and OVA administered) was Comparative Example 4, and the female mice of Comparative Examples 1 to 4 were used. In contrast, the number of days until delivery was measured in the same manner as in Example 1.

  Table 1 shows the results of Test Example 1. The premature birth rate in Table 1 is a value obtained as premature birth when giving birth before 18th. The number of solids in Table 1 indicates the number of female mice tested.

*: p < 0.05， **: p < 0.01 versus Control，††： p < 0.01 versus LPS

*: p <0.05, **: p <0.01 versus Control, ††: p <0.01 versus LPS

  From Table 1, it can be seen that Example 1 has a lower premature birth rate than Comparative Examples 2 and 3. That is, it can be said that preterm birth can be prevented by administering TRX.

(Test Example 2)
The female mice of Example 1 were subjected to cardiac puncture under ether anesthesia 6 hours after the initial LPS administration, and blood was collected. The collected blood was centrifuged at 1000 g for 20 minutes at 4 ° C., and the serum was separated and stored at −80 ° C.
Then, the serum is lysed on ice, and six types of cytokines (IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10, IL-6 are used using a BD Cytometric Bead Array, Mouse Inflammation Kit (BD Biosciences). ) Was measured with an automated cell analyzer (FACScan). Specific measurement methods using an automatic cell analyzer are as follows (i) to (iii).
(I) Mix 50 μl of Capture beads, 50 μl of sample, and 50 μl of PE detection reagent, and vortex.
(Ii) After vortexing, incubate for 2 hours at room temperature in a light-shielded state, add 1 ml of Wash Buffer, vortex to wash the beads.
(Iii) After washing, it is centrifuged at 200 g for 5 minutes, and after removing the supernatant by suction, 0.3 ml of Wash Buffer is added, resuspended and measured with an automatic cell analyzer.

Moreover, about the female mouse | mouth of Comparative Examples 1-3, it measured about 6 types of cytokine similarly to the female mouse | mouth of Example 1. FIG.
FIG. 1 is a diagram showing the results of Test Example 2. Specifically, FIGS. 1A to 1F show IL-12p70, TNF-α, IFN-γ, MCP-1, IL-10, and IL-6 in this order. The vertical axis indicates the amount of each cytokine (pg / ml), and the horizontal axis indicates Comparative Example 1 (Control), Comparative Example 2 (LPS), Comparative Example 3 (LPS + PBS), and Example 1 (LPS + TRX) in order from the left. Show.

  As shown in FIG. 1, by administering TRX, five types of cytokines, TNF-α, IFN-γ, MCP-1, IL-10, and IL-6, are markedly greater than those in Comparative Examples 2 and 3. It turns out that it becomes low. These cytokines are substances that cause inflammation in the womb and cause preterm birth. That is, it can be said that TRX can suppress inflammation in the womb and prevent premature birth by suppressing these cytokines.

(Test Example 3)
FIG. 2 is a photograph showing a mouse placental labyrinth 6 hours after the initial administration of LPS in female mice. (A) is Comparative Example 1 (Control), (b) is Comparative Example 2 (LPS), (c) Comparative Example 3 (LPS + PBS), (d) shows Example 1 (LPS + TRX).
As shown in FIG. 2, it can be seen that the inflammation in Example 1 is suppressed as compared with Comparative Examples 2 and 3. That is, it can be seen that administration of TRX suppresses inflammation of blood vessels in the placental labyrinth.

(Test Example 4)
Next, as Test Example 4, the effect on the fetus when TRX is administered to the mother is verified.
In Test Example 4, C57BL / 6 female mice were used as female mice, and human recombinant TRX diluted to 4 mg / ml with PBS was used as TRX.
Seven to ten week old C57BL / 6 female mice (hereinafter simply referred to as female mice in Test Examples 4 and 5) were mated with C57BL / 6 male mice, and plugs were confirmed. The day when the plug was confirmed was defined as the 0th day of pregnancy.
One female mouse on day 18 of gestation was administered with 400 μg (100 μl) of TRX by intraperitoneal injection. And 1 hour after TRX administration, protein was collected from each tissue of fetal brain, lung, liver and kidney, and the presence of TRX was confirmed by Western blot analysis for each.
As a comparative example, 100 μl of PBS was administered by intraperitoneal injection to one female mouse on the 18th day of pregnancy. Then, Western blot analysis was similarly performed using proteins of fetal brain, lung, liver, and kidney tissues 1 hour after PBS administration.
The Western blot analysis was performed by adjusting the protein concentration to 1 μg / μl with RIPA buffer and electrophoresis of 10 μg / lain protein.
FIG. 3 is a diagram showing the results of Western blot analysis, where 1 is the fetal brain tissue at 18 days of gestation when PBS is administered to the mother and 2 is the fetus at 18 days of gestation when TRX is administered to the mother. Brain tissue of 3 days, fetal lung tissue of fetal age 18 when PBS is administered to mother, 4 fetal lung tissue of fetal age 18 when TRX is administered to maternal, 5 is PBS to mother Fetal liver tissue at 18 days of gestation when administered, 6 fetal liver tissue at 18 days of gestation when TRX is administered to the mother, and fetus at 18 days of gestation when PBS is administered to the mother. 8 shows a lane in which the protein of the kidney tissue of fetus on the 18th day of gestation when TRX was administered to the mother was transferred to 8.

  The band (A) in lane 4 (pulmonary tissue administered with TRX) in FIG. 3 is a band indicating TRX. As shown in FIG. 3, in the fetus, TRX was confirmed only in the lung. From this result, it can be seen that TRX administered to the mother affects the fetal lung. Therefore, it can be said that administration of TRX to the mother can suppress not only premature delivery but also fetal lung inflammation that often occurs as a complication of premature birth.

(Test Example 5)
FIG. 4 shows a photograph obtained by immunostaining the fetal lung tissue of a female mouse by the ABC method, and TRX is stained reddish brown. Specifically, (a) shows fetal lung tissue 1 hour after PBS administration used in Test Example 4, and (b) shows fetal lung tissue 1 hour after TRX administration used in Test Example 4. .
In addition, the ADF-21 antibody (100-fold dilution) that specifically recognizes hTRX is used as the primary antibody of the ABC method, and vectortain Elite ABC Kit (mouse IgG) (manufactured by Vector) is used after the secondary antibody. It was. For the color development, impact DAB (manufactured by Vector) was used.
FIG. 4 also shows that TRX is present in the fetal lung when TRX is administered to the mother. In other words, it can be said that by administering TRX to the mother, not only premature delivery but also inflammation of the fetal lung can be suppressed.

  The present invention can be suitably used for prevention of premature birth, prevention and treatment of fetal lung inflammation, and the like.

It is a figure which shows the result of Test Example 2. It is a photograph which shows the placenta maze part in the female mouse of Example 1 and Comparative Examples 1-3. It is a figure which shows the result of a Western blot analysis. It is the photograph at the time of performing immunostaining by ABC method about the lungs of the fetus of a female mouse.

Claims (5)

Thioredoxin family polypeptides having -Cys-Gly-Pro-Cys-, -Cys-Pro-Tyr-Cys-, -Cys-Pro-His-Cys-, -Cys-Pro-Pro-Cys- in the active center / or sulforaphane its inducer as an active ingredient, it is administered to the mother, preventive or therapeutic agent for chronic lung disease fetal. Preventive or therapeutic agent for chronic lung disease fetal according to claim 1, wherein the thioredoxin family of polypeptides is a human thioredoxin consisting of the amino acid sequence shown in SEQ ID NO: 1. The thioredoxin family polypeptide is a modified thioredoxin comprising an amino acid sequence in which one or several amino acids other than positions 32 and 35 are substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 1. preventive or therapeutic agent for chronic lung disease fetal according to claim 1,. Preventive or therapeutic agent for chronic lung disease fetal according to any of claims 1 to 3, characterized in that a liquid or gel. Preventive or therapeutic agent for chronic lung disease fetal according to claim 4, characterized in that it is for vaginal administration.

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