JP2007063219A - Agent for preventing neonatal necrotizing enterocolitis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent exhibiting excellent effect for preventing neonatal necrotizing enterocolitis. <P>SOLUTION: The neonatal necrotizing enterocolitis prevention agent contains an n-3 long-chain polyunsaturated fatty acid or its ester as an active component. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a medicament for preventing neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (hereinafter “NEC”) is an extremely serious acquired bowel disease that is characterized by ischemic necrosis of the intestine and is accompanied by intestinal perforation, peritonitis, sepsis, shock, and the like. NEC is known to occur frequently in preterm and very low birth weight infants. In recent years, although neonatal medical care has improved, the death rate from NEC is still high, and the cause and prevention method have not yet been clarified. Among them, immaturity of the gastrointestinal tract, hypoxia before and after delivery, damage in the intestinal lumen, bacterial infection of the intestinal wall, administration of artificial milk, etc. are considered as main factors.
Conventionally, neonatal infants who have developed NEC have been treated by cessation of oral administration, antibiotics, or surgical intervention. In addition, regarding prevention of NEC, birth prevention of low birth weight infants, infection protection by oral administration of immunoglobulin (see Non-Patent Document 1); method of promoting intestinal maturation by maternal administration of steroid (see Non-Patent Document 2) A method for promoting stabilization of intestinal flora by administration of bifidobacteria or lactobacilli (see Non-Patent Document 3); a method of administering polyunsaturated fatty acids to newborns (see Non-Patent Document 4), and the like have been reported.

In general, multicellular organisms drive the intracellular signal transduction system based on information transmission between cells via cytokines, etc., and control the activation of multiple transcription factors to induce the expression of genes related to cell functions. I have control. One of transcription factors, NF-κB, is a dimer identified as a factor that binds to an enhancer of immunoglobulin κ light chain (see Non-Patent Document 5), and its activity is tumor necrosis factor α (TNF−). α), activated by IL-1. In the cytoplasm, the inhibitor IκB binds to the NF-κB dimer to make it inactive, but the TNF-α or IL-1 signal ultimately leads to degradation of IκB and activates the dimer. To do. When IκB is degraded, NF-κB moves into the nucleus and induces expression of the target gene. As a group of genes considered to be involved in the expression control of NF-κB, genes mainly involved in inflammation / immune response and genes involved in processes such as apoptosis are known. Therefore, it is expected that various diseases can be prevented and treated by blocking the cascade leading to NF-κB activity.
Rubaltelli FF, et al. Dev Pharmacol Ther 17, 138-143 (1991) Nanthakumar NN, et al. Am J Physiol Gastrointest Liver Physiol 288, G85-92 (2005) Hoyos AB. Int J Infect Dis. 3, 197-202 (1999) Caplan MS, et al. Pediatric Research 49, 647-652 (2001) Sen R, Baltimore D, Cell 46, 705-716 (1986)

Neonatal necrotizing enterocolitis disease, which is considered a serious disease, has an extremely high mortality rate of 55 to 65% (Shoji Koji, Pediatrics, Vol. 11, pp. 1807-1811, 1999). Preventing at the stage has been a challenge.
An object of this invention is to provide the chemical | medical agent which exhibits the effect excellent in prevention of the neonatal necrotizing enterocolitis disease by administering to the mother in pregnancy.

  The present inventors created an NEC disease model using a rat fetus and examined NEC onset, and in the NEC onset group, the expression of IκB family IκB-α and IκB-β proteins was recognized, We found the involvement of NF-κB, an inflammatory signal molecule, in the developmental mechanism. In addition, in the onset of NEC, the expression of peroxisome proliferator-activated receptor family γ (PPARγ), which is an anti-inflammatory signal molecule, was also significantly high, suggesting that it may be anti-inflammatory in NEC pathology . In addition, focusing on n-3 long-chain polyunsaturated fatty acids (n-3PUFA), the anti-inflammatory effect in newborns was examined by administering n-3 long-chain polyunsaturated fatty acids to the mother before delivery. However, the present inventors completed the present invention by discovering that PPARγ expression is increased, suppression of inflammation via NF-κB occurs, and that NEC onset of neonates is suppressed.

  That is, this invention provides the neonatal necrotizing enterocolitis preventive agent which contains n-3 type | system | group long-chain polyunsaturated fatty acid or its ester as an active ingredient.

  Since the drug of the present invention can suppress the degradation of IκB, which is an inhibitor of NF-κB, and suppress inflammation through NF-κB, which is an inflammatory signal molecule, it is particularly relevant to diseases involving NF-κB, particularly neonatal necrosis. It is useful as an agent for preventing enterocolitis. Moreover, inflammation of the intestinal tract can be mildly suppressed by its anti-inflammatory action.

Examples of n-3 long-chain polyunsaturated fatty acids or esters thereof (n-3PUFA) used in the present invention include n-3 long-chain polyunsaturated fatty acids having 11 or more carbon atoms or esters thereof. Examples of the unsaturated fatty acid include α-linolenic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), and the like. Examples of unsaturated fatty acid esters include lower alkyl esters having 1 to 6 carbon atoms, and specific examples include methyl esters, ethyl esters, propyl esters, and isopropyl esters. Of these, methyl ester and ethyl ester are preferred. Of these, highly unsaturated fatty acids having 20 to 24 carbon atoms or esters thereof are preferred, and eicosapentaenoic acid, docosahexaenoic acid or esters thereof are particularly preferred. These compounds may be used alone or in combination of two or more.
n-3PUFA is not particularly limited, and for example, one isolated and purified from fish oil, sesame oil, algae, marine mammals and the like can be used.

  The drug of the present invention is for administering an n-3 long-chain polyunsaturated fatty acid or an ester thereof to a prenatal mother. As shown in the Examples described later, by administering the drug of the present invention to the mother body, the incidence of NEC can be remarkably reduced, and the effect of preventing neonatal necrotizing enterocolitis is excellent. Moreover, the severity of NEC is also low, and inflammation of the intestinal tract can be mildly suppressed.

  The drug of the present invention can be made into pharmaceutical preparations of various dosage forms according to usage, and examples of such dosage forms include oral administration such as powders, granules, fine granules, tablets, capsules, and dry syrups. Or parenteral administration, such as an injection and a suppository, is mentioned.

  These preparations are pharmaceutically acceptable excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents depending on the dosage form as long as they do not affect the matrix. , And can be appropriately mixed, diluted or dissolved with pharmaceutical additives such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers, solubilizing agents, etc., and produced according to a conventional method.

  The drug of the present invention preferably contains at least 100 mg, particularly 200 mg or more of n-3 long-chain polyunsaturated fatty acid or ester thereof per gram of drug.

  The drug of the present invention is administered to the mother as described above, and the administration time is considered to be late in pregnancy, particularly in pregnancy, considering that NEC frequently occurs in preterm infants or extremely low birth weight infants. It is preferred to administer from 10 to 10 months.

  The dosage of the drug of the present invention is appropriately selected in consideration of the body weight, age, essential fatty acid amount (nutrient requirement) required for one day, and the like. For example, for docosahexaenoic acid (DHA), 120 to 1500 mg per day, preferably 400 to 1200 mg, can be administered once a day or several times a day.

  Moreover, the neonatal necrotizing enterocolitis disease preventive agent of the present invention can be produced in the form of a nutritional composition contained in a general food and drink, and the food and drink are eaten as a dietary therapy. Useful for the prevention of neonatal necrotizing enterocolitis. In this case, the n-3 long-chain polyunsaturated fatty acid or ester thereof, which is an active ingredient of the neonatal necrotizing enterocolitis preventive agent of the present invention, is 0.1 to 10.0% by mass, particularly 0.5. It is preferable to make it contain in food-drinks in the ratio of -5.0 mass%.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Reference Example Examination of NEC Disease Model and Inflammatory Signal Substance Using Rat Fetus 1) A rat fetus (SD system) on the 20th day of fetal life was removed by abdominal incision. After birth, 0.05, 0.1, and 0.15 mL of artificial milk were administered once with an oral catheter, and the intestinal tract was excised at 24 hours after birth, and the incidence of NEC was evaluated (N = 18). As a result, as shown in Table 1, the occurrence of NEC was observed at a high rate by administration of 0.15 mL of artificial milk.
2) From the above, rats administered with 0.15 mL of artificial milk were divided into an NEC onset group (N = 8) and an NEC non-onset group (N = 10), and the expression intensity of inflammatory signal substances was examined.
Total RNA was extracted from the sample by a conventional method using an RNA extraction kit (RNA STAT 60 Reagent, Tel-Test). After DNaseI treatment, cDNA was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR), and the mRNA expression levels of IκB-α, IκB-β and PPARγ were semi-quantified. As primers for RT-PCR, PPAR-γ: sense primer 5′-GACACAGAGATGCCATTCTG-3 ′ (SEQ ID NO: 1) and antisense primer 5′-CACAAGCATGGACTCCATAG-3 ′ (SEQ ID NO: 2), IκB-α: sense primer 5 '-GACGAGGATTACGAGCAGAT-3' (SEQ ID NO: 3) and antisense primer 5'-CCTGGTAGGTTACTCTGTTG-3 '(SEQ ID NO: 4), IκB-β: sense primer 5'-CCCAAGAGATGCCTCAGATA-3' (SEQ ID NO: 5) and antisense primer 5′-GCCTTCATCTCTGTTGTCAC-3 ′ (SEQ ID NO: 6), GAPDH: sense primer 5′-GGGTGATGCTGGTGCTGAGTATGT-3 ′ (SEQ ID NO: 7) and antisense primer 5′-AAGAATGGGAGTTGCTGTTGAAGTC-3 ′ (SEQ ID NO: 8) were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and each mRNA expression was quantified by the ratio of the product obtained by RT-PCR to GAPDH.
3) The results are shown in FIG. As shown in FIG. 1, when each mRNA expression level of IκB-α, IκB-β, and PPARγ was compared as a ratio to the expression amount of GAPDH, it was significantly increased in the NEC onset group compared to the NEC non-onset group. This suggested the involvement of NF-κB in the mechanism of NEC development. In addition, the expression of PPARγ mRNA in the NEC onset group was also significantly high, suggesting that it is acting anti-inflammatory in NEC pathology.

Example 1 Examination of changes in immune response by maternal administration of n-3 long-chain polyunsaturated fatty acids 1) DHA, EPA or soybean oil shown in Table 2 (control group) for 12-week-old SD pregnant rats ) Each 2.5 g / kg was orally administered for 2 weeks from the 7th day of pregnancy to fetal excision (N = 2). The rat fetus was removed through an abdominal incision, and 0.15 mL of artificial milk was administered once by oral catheter after birth. The intestinal tract was removed at the time of breeding for 24 hours in an incubator, and the presence or absence of NEC was confirmed. The DHA or EPA administration group requires administration of 52% soybean oil as an essential fatty acid.

2) Examination of mRNA expression level of inflammatory signal substance
Rats that developed NEC were used as specimens. In the same manner as in the above Reference Example, total RNA was extracted from the digestive tract by a conventional method using an RNA extraction kit. After DNaseI treatment, cDNA was synthesized by reverse transcriptase polymerase chain reaction (RT-PCR), and the mRNA expression levels of IκB-α, IκB-β and PPARγ were semi-quantified. PCR products were stained and visualized by electrophoresis. The correction was made using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard.
3) Results
Figure 2 shows the NEC incidence. Rats born from DHA and EPA administration groups had a lower incidence of NEC compared to the control group. In addition, NEC cases in the DHA and EPA administration groups had milder inflammation (intestinal necrosis, hyperemia, edema, ascites) and histological findings than those in the control group.
FIG. 3 shows the mRNA expression levels of IκB-α, IκB-β and PPARγ in NEC cases. When each mRNA expression level was compared as a ratio with respect to the expression level of GAPDH, the expression of PPARγ mRNA was more enhanced in the DHA and EPA administration groups than in the control group in the cases with NEC. On the other hand, mRNA expression of IκB-α and IκB-β was attenuated.
These results indicate that maternal administration of n-3PUFA suppresses IκB degradation in the rat fetus, increases PPARγ expression, suppresses inflammation via NF-κB, and suppresses fetal NEC onset It was confirmed.

Example 2 Manufacture of a nutritional composition for prevention of NEC Using commercially available DHA oil, various ingredients were mixed based on the formulation shown in Table 3, a 20% milk preparation was prepared, homogenized, and then sterilized It concentrated and dried by the conventional method, and the NEC prevention nutrition composition (milk powder) was manufactured.

It is a figure which shows the mRNA expression level of IκB-α, IκB-β and PPARγ in the NEC onset group and the NEC non-onset group. It is a figure which shows the NEC incidence in a control group, a DHA administration group, and an EPA administration group. It is a figure which shows the mRNA expression of I (kappa) B- (alpha), I (kappa) B- (beta), and PPAR (gamma) by electrophoresis (NEC onset example).

Claims (5)

  A preventive agent for neonatal necrotizing enterocolitis comprising an n-3 long-chain polyunsaturated fatty acid or an ester thereof as an active ingredient.   The preventive agent according to claim 1, wherein the n-3 long-chain polyunsaturated fatty acid is docosahexaenoic acid or eicosapentaenoic acid.   The prophylactic agent according to claim 1 or 2, which is administered to a mother.   The preventive agent according to claim 1 or 2, which is based on the transcriptional activity inhibition of the transcription factor NF-κB.   Food-drinks containing the preventive agent of any one of Claims 1-4.