WO2011004843A1 - Pharmaceutical composition for treatment of sirs or highly pathogenic influenza infection - Google Patents

Pharmaceutical composition for treatment of sirs or highly pathogenic influenza infection Download PDF

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WO2011004843A1
WO2011004843A1 PCT/JP2010/061541 JP2010061541W WO2011004843A1 WO 2011004843 A1 WO2011004843 A1 WO 2011004843A1 JP 2010061541 W JP2010061541 W JP 2010061541W WO 2011004843 A1 WO2011004843 A1 WO 2011004843A1
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group
sirs
clp
endothelin
etr
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PCT/JP2010/061541
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French (fr)
Japanese (ja)
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秀親 岡田
有武 岡田
則子 岡田
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株式会社蛋白科学研究所
公立大学法人名古屋市立大学
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Priority to JP2011521944A priority Critical patent/JP5686734B2/en
Publication of WO2011004843A1 publication Critical patent/WO2011004843A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to a composition for SIRS treatment.
  • the invention also relates to a composition for the treatment of highly toxic influenza infections, in particular H5N1 avian influenza infections.
  • the invention further relates to a method for the treatment of SIRS as well as highly toxic influenza infections.
  • SIRS Systemic Inflammatory Response Syndrome
  • zepsis is a fatal disease that causes 300,000 patients to die each year in North America, and there is still no effective therapeutic agent.
  • APC activated protein C
  • Using activated protein C (APC) can only improve 30% mortality to 24%.
  • pathogenic bacteria have already increased in the body, and even if bacteria are suppressed with antibiotics, endotoxin (LPS) cannot be removed, and antibiotic treatment and treatment with symptomatic treatment such as heparin and steroids The current situation remains.
  • SIRS systemic inflammatory response syndrome
  • DIC disseminated intravascular coagulation
  • MOF multiple organ failure
  • amino acids 37 to 53 (RAARISLGPRCIKAFTE) (SEQ ID NO: 1) of C5a previously published by the present inventors are an anti-sense against the antisense homology box (AHB) peptide (Non-patent Document 3) of C5a receptor (C5aR). It is a sense peptide and is called PL37 (Non-Patent Document 4). This region of C5a is presumed to be a potential site for C5aR stimulation (Non-Patent Document 5).
  • HAB antisense homology box
  • the present inventors created a complementary peptide ASGAPAPGPAGPLRPMF (SEQ ID NO: 2) to PL37 using a computer program, MIMETIC (6-8), which binds to C5a anaphylatoxin and is mediated by complement. After confirming that lethal shock is prevented in rats, it was named PepA (Patent Document 1, Non-Patent Document 9).
  • PepA or AcPepA SEQ ID NO: 3
  • influenza viruses There are three types of influenza viruses: A, B and C. It is type A that causes a global epidemic and causes many deaths. Influenza A viruses are further classified into a number of subtypes based on the antigenicity of hemagglutinin and neuraminidase, which are viral surface proteins. So far, at least 16 mutations have been found in HA and 9 mutations in NA, and there can be as many subtypes as there are combinations thereof. Differences between these subtypes are expressed by abbreviations such as H1N1 to H16N9.
  • H5N1 is particularly called highly pathogenic avian influenza virus (in Japan, it is regulated by the Livestock Infectious Disease Prevention Law), and when infected, both humans and birds have extremely high mortality rates. .
  • chickens are said to die within 2 days if they become infected with H5N1 avian influenza and develop influenza.
  • Endothelin is a peptide that was first isolated from the culture supernatant of porcine aortic endothelial cells, and acquires a strong vasoconstrictive action when it is cleaved by an enzyme from a precursor and converted to a mature peptide.
  • This is known to be the most potent vascular smooth muscle contraction and blood pressure raising action among existing substances, and is one of the physiologically active substances currently in the limelight. It has been reported to have not only vasoconstrictive action but also various physiological actions such as cardiotonic action and renal mesangial contractile action, and at least three isoforms (endothelin-1, endothelin-2, endothelin-3) have been reported. Has been.
  • endothelin receptors type A receptor specific for endothelin-1 and type B receptor nonspecific for three isopeptides of endothelin.
  • the amino acid sequence has also been deduced.
  • the present inventors have previously identified an antisense homology box peptide of endothelin receptor A type (Non-patent Documents 3 and 2), and endotoxin shock and hemorrhagic shock caused by the peptide via endothelin receptor A type. Have been reported to attenuate inflammatory responses such as histamine and to suppress the action of histamine release. As a result of intensive studies, the present inventors have found that endothelin activity inhibitors are extremely effective in treating SIRS and highly toxic influenza infections, and have completed the present invention.
  • JP 2004-269520 A Japanese Patent No. 3923515
  • the object of the present invention is to provide a pharmaceutical composition for SIRS treatment.
  • the present invention also aims to provide a pharmaceutical composition for the treatment of highly virulent influenza virus infection.
  • the present invention further aims to provide a method for the treatment of highly virulent influenza virus infection as well as SIRS treatment.
  • the present invention provides a pharmaceutical composition for SIRS treatment containing an endothelin activity inhibitor.
  • the present invention also provides a pharmaceutical composition for the treatment of highly virulent influenza virus infection, for example, highly pathogenic avian influenza infection, containing an endothelin activity inhibitor.
  • the present invention provides a method for treating SIRS, comprising administering an effective amount of an endothelin activity inhibitor to a patient in need of SIRS treatment. Also provided is a method for treating a highly toxic influenza infection, comprising administering an effective amount of an endothelin activity inhibitor to a patient in need of treatment for a highly toxic influenza virus infection.
  • the present invention provides the use of an endothelin activity inhibitor in the manufacture of a pharmaceutical composition for SIRS treatment.
  • the present invention also provides the use of an endothelin activity inhibitor in the manufacture of a pharmaceutical composition for the treatment of highly toxic influenza infection.
  • the endothelin activity inhibitor is not particularly limited, but the peptides of SEQ ID NOs: 4 to 18 in the sequence listing, particularly the peptide of SEQ ID NO: 11 is preferably used.
  • the SIRS and highly toxic influenza virus infection can be effectively treated by the pharmaceutical composition of the present invention.
  • Example 3 is a graph showing the results of Example 1.
  • 3 is a graph showing the survival time of Example 2.
  • the survival periods of the ETR-P1 / fl group ( ⁇ ), CLP group ( ⁇ ), and Siam group (dotted line) are shown.
  • Mean survival was significantly longer in the ETR-P1 / fl group than in the CLP group: 18.9 ⁇ 2.3 (ETR-P1 / fl) vs. 9.0 ⁇ 0.8 (CLP) p ⁇ 0.005 Mann-Whitney test.
  • Two animals (33.3%) in the ETR-P1 / fl group were alive at 24 hours. All sham groups survived for 24 hours.
  • the change of the mPAP ratio of the ETR-P1 / fl group ( ⁇ ), the CLP group ( ⁇ ), and the sham group ( ⁇ ) is shown. Changes in heart rate (HR) of ETR-P1 / fl group ( ⁇ ), CLP group ( ⁇ ), and Sham group ( ⁇ ) are shown. At 6 hours and 9 hours, a significantly higher heart rate was observed in the CLP group than in the other groups (p ⁇ 0.05). The change of the mPAP / MAP ratio of the ETR-P1 / fl group ( ⁇ ), the CLP group ( ⁇ ), and the sham group ( ⁇ ) is shown. mPAP / MAP did not increase in the ETR-P1 / fl group at 9 hours.
  • the change of the serum TNF- ⁇ level in the ETR-P1 / fl group ( ⁇ ), the CLP group ( ⁇ ), and the sham group ( ⁇ ) is shown.
  • the change of the amount of HMGB-1 in the serum of the ETR-P1 / fl group ( ⁇ ), the CLP group ( ⁇ ) and the sham group ( ⁇ ) is shown.
  • the change of the amount of NOx in the serum of the ETR-P1 / fl group ( ⁇ ), the CLP group ( ⁇ ), and the sham group ( ⁇ ) is shown.
  • the endothelin activity inhibitor is not particularly limited as long as it suppresses endothelin-1 activity, and more preferably suppresses endothelin-1 action via endothelin receptor type A. It is not particularly limited.
  • endothelin receptors particularly endothelin receptor type A, bosentan, sitaxsentan, ambrisentan, atrasentan, S-139, tezosentan BMS-193884
  • compounds such as drausentan and entrasentan are known.
  • the amino acid sequence deduced from the human endothelin precursor cDNA is compared with the amino acid sequence deduced from the human endothelin receptor cDNA and sensed at the amino acid level.
  • C1 VLNLCALSVDRYRAVASWSRVI (SEQ ID NO: 4)
  • C2 CALSVDRYRAVASWGIPLITAIEI (SEQ ID NO: 5)
  • C3 QGIGIPLITAIEI (SEQ ID NO: 6)
  • C4 IADNAERYSANLSSHV (SEQ ID NO: 7)
  • C5 FLGTTTLPPNLALP (SEQ ID NO: 8)
  • C6 LNRRNGSLRIALSEHLKNRREVA (SEQ ID NO: 9)
  • a peptide in which the terminal of the C1 peptide is an amide, and any one of the specific amino acids in the sequence of the terminal amide is substituted with glycine Peptides are mentioned.
  • peptides in which the N-terminal and / or C-terminal amino acids of the C1 peptide have been removed, and those in which a part of the amino acids in the sequence have been substituted are also preferably used.
  • the following sequences are exemplified.
  • the cysteines at positions 1 and 15 form a disulfide bond.
  • C18 VALSVDRYRAVASW-NH 2 (SEQ ID NO: 18)
  • a peptide of C11 (SEQ ID NO: 11) is particularly preferably used.
  • the administration method and administration route of the pharmaceutical composition of the present invention are not particularly limited, but enteral or oral forms, for example, as nasal forms such as sprays, sugar-coated tablets, gelatin capsules, emulsions, solutions or suspensions. Alternatively, it can be administered in a rectal form such as a suppository.
  • the pharmaceutical composition of the present invention may also be administered intravenously or intramuscularly as an injection.
  • the pharmaceutical composition of the present invention contains other physiologically acceptable components such as salts, diluents, carriers, buffers, binders, surfactants, and preservatives. May be.
  • Preparations for parenteral administration include sterile aqueous solutions or suspension ampoules with physiologically acceptable solvents, or sterile powders that can be used diluted with physiologically acceptable diluents, such as endothelin activity inhibitors.
  • physiologically acceptable diluents such as endothelin activity inhibitors.
  • the dose of the pharmaceutical composition of the present invention is not limited, and may be appropriately determined depending on the patient's age, weight, sex, symptom, desired effect, and the like.
  • the pharmaceutical composition of the present invention is suitably used for SIRS treatment.
  • SIRS Systemic Inflammatory Response“ Syndrome ”
  • SIRS Systemic Inflammatory Response“ Syndrome
  • pathological conditions diagnosed with SIRS those caused by infectious diseases are diagnosed as zepsis or sepsis. Whether or not the cause is an infection is determined by confirmation of an infection focus by a pathogenic microorganism or an abnormal increase in CRP (10 or more).
  • SIRS means various bacteremia, fungiemia, parasitemia, viremia, trauma, burns, pancreatitis, surgery, etc. that cause SIRS. This includes preventing the onset of SIRS in an invaded patient, reducing the degree of SIRS onset or preventing death due to the onset of SIRS. Furthermore, SIRS treatment includes preventing death in patients who have already developed SIRS according to the above diagnostic criteria.
  • the pharmaceutical composition of the present invention can be used not only for patients who develop SIRS, but also for patients who are predicted to develop SIRS, specifically bacteremia, fungiemia, parasites. It is suitably used for the treatment of SIRS in patients undergoing various invasions such as septicemia, viremia, trauma, burns, pancreatitis, and surgery. In addition, it is known that death due to highly toxic influenza virus infection is caused by developing SIRS. Therefore, the pharmaceutical composition of the present invention is suitably used for the treatment of patients infected with highly toxic influenza viruses regardless of the presence or absence of symptoms or their strength.
  • the present invention is also suitably used for the treatment of patients with highly toxic influenza virus infection.
  • strongly toxic influenza virus infection means an infection caused by influenza virus, which is known to have a high possibility of developing SIRS and dying due to influenza infection.
  • influenza viruses include H5N1 avian influenza virus that is currently known, and includes those viruses or other viruses that are highly toxic due to mutation.
  • the pharmaceutical composition of the present invention is useful for the treatment of a patient who is infected with a highly virulent influenza virus or a patient who is suspected of having the infection regardless of the onset of symptoms.
  • “Treatment of highly virulent influenza virus infection” includes any of suppression of symptoms caused by virulent influenza viruses, reduction of symptoms that develop, and treatment of symptoms that have already occurred. Furthermore, in this invention, it uses suitably also in order to rescue the patient from whom serious pneumonia resulting from a virulent influenza virus and SIRS symptom already developed from death.
  • the pharmaceutical composition of the present invention can be effectively used for treatment of birds infected with highly toxic influenza viruses such as H5N1 avian influenza virus infection and prevention of spread of infection.
  • highly toxic influenza viruses such as H5N1 avian influenza virus infection
  • a chicken infected with influenza virus occurs in a poultry house, it can be cured by administering the pharmaceutical composition of the present invention to the infected chicken to prevent the spread of infection.
  • the spread of influenza virus infection can be prevented by administering the pharmaceutical composition of the present invention in advance to chickens kept in the same chicken house where infected chickens are produced or in neighboring chicken houses.
  • ETR-P1 / f1 in neonatal porcine peritonitis model (1)
  • the effect of the pharmaceutical composition of the present invention was confirmed in a neonatal porcine peritonitis model that is close to the actual pathological condition of Zepsis infection.
  • the newborn pig was laparotomized under inhalation anesthesia, the ileocecal region was tagged, a hole was opened with an 18-gauge injection needle so that feces leaked out, the intestine was returned to the abdominal cavity, and the abdominal wall was sutured.
  • This treatment is a peritonitis model called CLP (Cecal Ligation and Puncture), and a newborn pig subjected to CLP develops a systemic inflammatory response syndrome (SIRS) and dies.
  • CLP Cercal Ligation and Puncture
  • SIRS systemic inflammatory response syndrome
  • From 30 minutes after this CLP treatment 0.05 mg / kg / hr of ETR-P1 / fl was continuously administered intravenously.
  • the survival period of each of the sham surgery group, the CLP surgery group, and the ETR-P1 / f1 administration group after the CLP surgery was examined. All animals were euthanized after 24 hours.
  • the test was conducted in 8 untreated groups, 10 CLP surgery groups, and 7 ETR-P1 / f1 administration groups after CLP surgery. The results are shown in FIG.
  • Newborn pigs were raised with mother pigs and isolated only during treatment. Newborn pigs weighed between 1450 and 2200 g with a median of 1715 g.
  • the piglet was administered intramuscularly with 10 mg / kg ketamine chloride. Thereafter, sodium pentobarbital in a 5% glucose aqueous solution was administered from the peripheral blood vessel at a rate of 5 mg / kg / h during the treatment period to maintain anesthesia.
  • Each piglet was intubated with a 4 mm inner diameter tube and ventilated with an infant ventilator (IV-100, Sechrist Industries, Anaheim, CA, USA).
  • the inspiratory / expiratory pressure at the start was 14/4 cmH 2 O, and the inspiratory time was 0.5 seconds.
  • Indoor air was used for artificial respiration. The respiratory pressure was then adjusted to maintain PaCO 2 at 30-50 mmHg.
  • a 4F polyvinyl catheter was inserted into the left femoral artery by the cut-down method and used for blood sample acquisition, mean arterial pressure (MAP) and heart rate (HR) measurement.
  • MAP mean arterial pressure
  • HR heart rate
  • a 4-Fr Bellman angiographic catheter (American Edwards Laboratories, Irvine, Calif., USA) was inserted through the fluoroscopic right lateral vein, placed in the main pulmonary artery, and used for mPAP measurement. Further, a 4F polyvinyl catheter was inserted from the left external jaw vein to reach the superior vena cava, and used for measuring the central vena cava pressure.
  • MAP Mean arterial pressure
  • mPAP mean pulmonary arterial pressure
  • HR heart rate
  • CLP treatment CLP group CLP treatment was performed as follows. An incision was made so that the cecum and terminal ileum (approximately 4 cm) could be exposed to the abdomen of the newborn pig. The ileocecal artery was confirmed and ligated near the cecum to stop blood flow to the distal end of the cecum. A cut of about 1 cm was made on the antimesenteric side. After gently squeezing feces from the cecum into the abdominal cavity, the abdominal incision was bound.
  • Siamese treatment (siamese group) In the sham group, after the abdominal incision of the newborn piglet, the cecum and the terminal ileum were exposed to the outside for 2 minutes and then returned to the abdominal cavity, and the abdominal incision was closed.
  • ETR-P1 / fl administration ETR-P1 / fl group
  • ETR-P1 / fl administration started 30 minutes after CLP treatment.
  • a solution of ETR-P1 / fl in 5% glucose was administered into the central vein so that the dose of ETR-P1 / fl was 0.05 mg / kg / h.
  • the solution volume for intravenous administration was adjusted to 5 mL / kg / h.
  • Test Protocol In each group, blood samples were collected from the femoral artery catheter under aseptic conditions to measure arterial blood gases, serum TNF- ⁇ , NO metabolites, and HMGB-1. Blood samples were taken before CLP or sham treatment and 1, 3, 6, and 9 hours after treatment. Sampling was also performed after 12 hours in the ETR-P1 / fl group and the Sham group. All samples were stored in sterile tubes.
  • HMGB-1 was measured using an ELISA kit for HMGB-1 (Shino-Test Corporation, Sagamihara, Japan).
  • Measurement of stroke volume M-mode echocardiography was performed using a SONOS500 (Hewlett Packard) with a 5.0 MHz transducer before CLP or sham treatment and 1, 3, 6 and 9 hours after treatment. .
  • the ETR-P1 / fl group and the sham group were also measured 12 hours after CLP / sham treatment.
  • Two-dimensional M-mode measurement of the left ventricle was performed from the apex long-axis image, and the stroke volume was calculated. The measurement was performed three times at each measurement, and the average value at each measurement was calculated.
  • the first and second survival targets were calculated as CLP group survival time and 2 ⁇ SEM, CLP group survival time and 2 ⁇ SD, respectively.
  • the third survival target was at the end of the study 24 hours after CLP surgery.
  • the 95% confidence interval was between 14.4 and 23.4 hours for the ETR-P1 / fl group and between 7.5 and 10.5 hours for the CLP group.
  • 83.3% (n 5)
  • 50% (n 3)
  • none of the CLP groups could reach the first survival target (FIG. 2). All animals in the sham group survived for 24 hours.
  • Relative mPAP / MAP values gradually increased in the CLP group and were significantly higher at 9 hours compared to the Sham group and the ETR-P1 / fl group: 0.73 ⁇ 0.08 (CLP group) vs. 0. 46 ⁇ 0.07 (sham group) and 0.50 ⁇ 0.058 (ETR-P1 / fl group), both p ⁇ 0.05 (FIG. 3C).
  • the relative mPAP / MAP value of the ETR-P1 / fl group was close to that of the Sham group.
  • heart rate increased at 3 hours after CLP treatment: 184.8 ⁇ 13.0 beats / minute [before CLP], 237.8 ⁇ 15 .5 beats / minute [1h], 272.2 ⁇ 14.0 beats / minute [3h], p ⁇ 0.05 and p ⁇ 0.01, respectively.
  • the relative cardiac output (CO) showed a significant decrease at 3, 6 and 9 hours, and was significantly lower than the sham group (Table 2).
  • the relative stroke volume of the ETR-P1 / fl group gradually decreased, the difference between the ETR-P1 / fl group and the sham group was not significant.
  • HMGB-1 showed detectable values at 3, 6 and 9 hours in the CLP group and was significantly higher than the ETR-P1 / fl group and the Siam group (FIG. 4B). Serum HMGB-1 was not detected at all in the Siam group, but there was no significant difference in serum HMGB-1 levels between the ETR-P1 / fl group and the Sham group.
  • Serum NOx levels in the ETR-P1 / fl group and the Siam group were comparable throughout the study period. In both groups, the same level of NOx as before the start of the test was maintained. On the other hand, NOx levels in the CLP group began to increase from 1 hour after CLP surgery and were higher than those in the ETR-P1 / fl group and the Sham group at 3, 6 and 9 hours (FIG. 4C).
  • the composition of the present invention is useful for treating SIRS.
  • the composition of the present invention suppresses an increase in both TNF- ⁇ , which is an inflammatory mediator that occurs early in zepsis, and HMGB-1, which is an inflammatory mediator that occurs in the late stage of zepsis.
  • TNF- ⁇ which is an inflammatory mediator that occurs early in zepsis
  • HMGB-1 which is an inflammatory mediator that occurs in the late stage of zepsis.
  • elevated plasma TNF- ⁇ correlates with the severity of endotoxemia.
  • HMGB-1 is a protein known only as a nuclear transcription factor, but is now thought to be a mediator associated with delayed endotoxin lethality and systemic inflammation.
  • HMGB-1 levels correlate with the severity of septic shock as well as TNF- ⁇ levels. Therefore, a decrease in the level of HMGB-1 is an index for measuring the effectiveness of sepsis treatment.
  • H5N1 influenza virus (H5N1 IV) preparation at 10 5.3 TCID 50 was the culture supernatant of MDCK cells infected in GIT medium.
  • ETR-P1 / f1 which is an endothelin activity inhibitor, has a strong therapeutic effect against H5N1 avian influenza virus infection, which is a highly toxic influenza virus.
  • Such a therapeutic effect also shows the use as a prevention method against the spread of highly pathogenic avian influenza virus H5N1 infection.

Abstract

Disclosed are a pharmaceutical composition for treating SIRS and a pharmaceutical composition for treating highly pathogenic influenza, each of which comprises an endothelin activity inhibitor. Preferably, the endothelin activity inhibitor to be used has the following sequence: ALSVD(X)Y(X)AVAS (wherein X represents an amino acid residue), particularly the following sequence: VLNLCALSVDRYRAVASWSRVI-NH2. The pharmaceutical compositions are useful for patients who have developed sepsis or SIRS including sepsis, patients who are in the environments where the development of SIRS is expected, patients who have been infected by highly pathogenic influenza, and patients who are more likely to be infected by highly pathogenic influenza.

Description

ＳＩＲＳまたは強毒性インフルエンザ感染症処置のための医薬組成物Pharmaceutical composition for treatment of SIRS or highly toxic influenza infection
　本発明は、ＳＩＲＳ処置のための組成物に関する。本発明はまた、強毒性インフルエンザ感染症、特にＨ５Ｎ１トリインフルエンザ感染症処置のための組成物に関する。本発明はさらに、ＳＩＲＳ並びに強毒性インフルエンザ感染症の処置のための方法に関する。 The present invention relates to a composition for SIRS treatment. The invention also relates to a composition for the treatment of highly toxic influenza infections, in particular H5N1 avian influenza infections. The invention further relates to a method for the treatment of SIRS as well as highly toxic influenza infections.
　ゼプシスを含む全身性炎症反応症候群(ＳＩＲＳ) は、北米において毎年300,000名の患者の死因である致死的な疾患であり、いまだに有効な治療剤が存在しない。米国では年間７５万人にゼプシスが発症し、２２万人が死亡しているが特効救命薬がない。活性化プロテインC（APC)を用いても３０％の死亡率を２４％に改善できるに過ぎない。ゼプシスでは既に体内に病原細菌が増加しており、抗生物質で細菌を抑制しても菌体内毒素（LPS）を取り除くことは出来ず、抗生物質投与とヘパリンやステロイドなどの対症療法での治療にとどまっているのが現状である。 Systemic Inflammatory Response Syndrome (SIRS) including zepsis is a fatal disease that causes 300,000 patients to die each year in North America, and there is still no effective therapeutic agent. In the United States, 750,000 people develop zepsis annually and 220,000 people die, but there is no special lifesaving medicine. Using activated protein C (APC) can only improve 30% mortality to 24%. In Zepsis, pathogenic bacteria have already increased in the body, and even if bacteria are suppressed with antibiotics, endotoxin (LPS) cannot be removed, and antibiotic treatment and treatment with symptomatic treatment such as heparin and steroids The current situation remains.
　ゼプシスは全身性炎症反応症候群(ＳＩＲＳ)の一種であり、ＳＩＲＳは播種性血管内凝固(DIC)および多臓器不全 (MOF)を引き起こし、これらは非常に致死的である。補体の第５成分(C5) から補体活性化の際に生じるC5 コンバターゼによって放出される74-アミノ酸ペプチドのC5a アナフィラトキシンのＳＩＲＳにおける中心的役割が提唱されている(非特許文献１および２)。 Zepsis is a type of systemic inflammatory response syndrome (SIRS), which causes disseminated intravascular coagulation (DIC) and multiple organ failure (MOF), which are very fatal. A central role in the SIRS of the C5a anaphylatoxin, a 74-amino acid peptide released by C5 タ ー ゼ convertase generated during complement activation from the fifth component (C5) of complement has been proposed (Non-patent Documents 1 and 2). ).
　ここで本発明者らが先に公表したC5aのアミノ酸37～53(RAARISLGPRCIKAFTE)（配列番号１）は、C5a 受容体 (C5aR)のアンチセンスホモロジーボックス(AHB) ペプチド(非特許文献３)に対するアンチセンスペプチドであり、PL37と称されている(非特許文献４)。C5aのこの領域はC5aR 刺激のための可能性のある部位であると推定されている(非特許文献５)。本発明者らはコンピュータプログラム、MIMETIC (非特許文献６－８）を用いて、PL37に対する相補性ペプチドASGAPAPGPAGPLRPMF（配列番号２）を作成し、これがC5a アナフィラトキシンに結合し、補体に媒介される致死的なショックをラットにおいて防止することを確認し、PepAと名付けた(特許文献１、非特許文献９) 。特許文献１の系では、全身症状の発症誘導前にPepAまたはPepAのN-末端アラニンのアセチル化したペプチドであるAcPepA（配列番号３）を投与してゼプシスの発症そのものを予防的に抑えることに成功している。 Here, amino acids 37 to 53 (RAARISLGPRCIKAFTE) (SEQ ID NO: 1) of C5a previously published by the present inventors are an anti-sense against the antisense homology box (AHB) peptide (Non-patent Document 3) of C5a receptor (C5aR). It is a sense peptide and is called PL37 (Non-Patent Document 4). This region of C5a is presumed to be a potential site for C5aR stimulation (Non-Patent Document 5). The present inventors created a complementary peptide ASGAPAPGPAGPLRPMF (SEQ ID NO: 2) to PL37 using a computer program, MIMETIC (6-8), which binds to C5a anaphylatoxin and is mediated by complement. After confirming that lethal shock is prevented in rats, it was named PepA (Patent Document 1, Non-Patent Document 9). In the system of Patent Document 1, PepA or AcPepA (SEQ ID NO: 3), which is an acetylated peptide of N-terminal alanine of PepA, is administered before the onset of systemic symptoms to prevent the onset of zepsis prophylactically. Has succeeded.
　インフルエンザウイルスにはＡ、ＢおよびＣの３つの型がある。世界的な流行を起こし、多くの死者が出るのは主にＡ型である。Ａ型インフルエンザウイルスは更にウイルス表面のタンパク質であるヘマグルチニンおよびノイラミニダーゼの抗原性により多くの亜型に分類される。これまでに少なくともＨＡに１６種類、ＮＡに９種類の変異が見つかっており、その組み合わせの数の亜型が存在し得る。これら亜型の違いはＨ１Ｎ１～Ｈ１６Ｎ９といった略称で表現されている。 There are three types of influenza viruses: A, B and C. It is type A that causes a global epidemic and causes many deaths. Influenza A viruses are further classified into a number of subtypes based on the antigenicity of hemagglutinin and neuraminidase, which are viral surface proteins. So far, at least 16 mutations have been found in HA and 9 mutations in NA, and there can be as many subtypes as there are combinations thereof. Differences between these subtypes are expressed by abbreviations such as H1N1 to H16N9.
　インフルエンザウイルスは、ヒトを含む哺乳類および鳥類に感染する。従来は種の壁があるためヒトにはヒトインフルエンザ、鳥類にはトリインフルエンザのみが感染すると考えられてきたが、近年ヒトおよびトリの両方に感染するインフルエンザウイルスが出現している。これらの中で、特にＨ５Ｎ１は高病原性トリインフルエンザウイルスと呼ばれ（日本では家畜伝染病予防法で規定されている）、これに感染した場合は、ヒトおよびトリの両者で極めて致死率が高い。例えばＨ５Ｎ１トリインフルエンザに感染し、インフルエンザを発症するとニワトリは２日以内に死亡するといわれている。 Flu virus infects mammals and birds including humans. Conventionally, due to the species barrier, it has been considered that only human influenza is infected in humans and avian influenza is infected in birds. In recent years, influenza viruses that infect both humans and birds have emerged. Among these, H5N1 is particularly called highly pathogenic avian influenza virus (in Japan, it is regulated by the Livestock Infectious Disease Prevention Law), and when infected, both humans and birds have extremely high mortality rates. . For example, chickens are said to die within 2 days if they become infected with H5N1 avian influenza and develop influenza.
　エンドセリンは、最初ブタ大動脈内皮細胞の培養上清から単離されたペプチドであり、前駆体から酵素で切断されて成熟ペプチドに変換されると強力な血管収縮作用を獲得する。これは既存の物質中最も強力な血管平滑筋収縮作用および血圧上昇作用であることが知られており、現在脚光を浴びている生理活性物質のひとつである。血管収縮作用だけではなく、強心作用、腎メサンギウム収縮作用など多岐にわたる生理作用を有することが報告されており、また、少なくとも３つのアイソフォーム（エンドセリン－１、エンドセリン－２、エンドセリン－３）が報告されている。
　一方、エンドセリンレセプターとしてはエンドセリン－１に特異的なＡ型受容体とエンドセリンの３種のイソペプチドに対して非特異的なＢ型受容体の２種類が知られており、ｃＤＮＡのクローニングによりそのアミノ酸配列も推定されている。
　しかし、かかる多岐にわたる生理活性を有するエンドセリンについては、ＳＩＲＳ並びに強毒性インフルエンザ感染症におけるその役割に関する報告は全く見当たらない。 Endothelin is a peptide that was first isolated from the culture supernatant of porcine aortic endothelial cells, and acquires a strong vasoconstrictive action when it is cleaved by an enzyme from a precursor and converted to a mature peptide. This is known to be the most potent vascular smooth muscle contraction and blood pressure raising action among existing substances, and is one of the physiologically active substances currently in the limelight. It has been reported to have not only vasoconstrictive action but also various physiological actions such as cardiotonic action and renal mesangial contractile action, and at least three isoforms (endothelin-1, endothelin-2, endothelin-3) have been reported. Has been.
On the other hand, two types of endothelin receptors are known: type A receptor specific for endothelin-1 and type B receptor nonspecific for three isopeptides of endothelin. The amino acid sequence has also been deduced.
However, there are no reports regarding the role of endothelin having such diverse physiological activities in SIRS and highly toxic influenza infection.
　本発明者等は先にエンドセリン受容体Ａ型のアンチセンスホモロジーボックスペプチドを同定し（非特許文献３、特許文献２）、該ペプチドがエンドセリン受容体Ａ型を介して生じるエンドトキシンショック、出血性ショック等の炎症応答を減弱化し、ヒスタミン放出等の作用を抑制することを報告している。今回、発明者らは、鋭意検討した結果、エンドセリン活性抑制剤がＳＩＲＳ並びに強毒性インフルエンザ感染症の処置に極めて有効であることを見出し、本発明を完成するに至った。 The present inventors have previously identified an antisense homology box peptide of endothelin receptor A type (Non-patent Documents 3 and 2), and endotoxin shock and hemorrhagic shock caused by the peptide via endothelin receptor A type. Have been reported to attenuate inflammatory responses such as histamine and to suppress the action of histamine release. As a result of intensive studies, the present inventors have found that endothelin activity inhibitors are extremely effective in treating SIRS and highly toxic influenza infections, and have completed the present invention.
特開２００４－２６９５２０号公報JP 2004-269520 A 特許３９２３５１５号公報Japanese Patent No. 3923515
　本発明は、ＳＩＲＳ処置のための医薬組成物を提供することを目的とする。本発明はまた、強毒性インフルエンザウイルス感染症の処置のための医薬組成物を提供することを目的とする。本発明は更に、強毒性インフルエンザウイルス感染症の処置並びにＳＩＲＳ処置のための方法を提供することを目的とする。 The object of the present invention is to provide a pharmaceutical composition for SIRS treatment. The present invention also aims to provide a pharmaceutical composition for the treatment of highly virulent influenza virus infection. The present invention further aims to provide a method for the treatment of highly virulent influenza virus infection as well as SIRS treatment.
　本発明は、エンドセリン活性抑制剤を含有する、ＳＩＲＳ処置のための医薬組成物を提供する。 The present invention provides a pharmaceutical composition for SIRS treatment containing an endothelin activity inhibitor.
　本発明はまた、エンドセリン活性抑制剤を含有する、強毒性インフルエンザウイルス感染症、例えば高病原性トリインフルエンザ感染症の処置のための医薬組成物を提供する。 The present invention also provides a pharmaceutical composition for the treatment of highly virulent influenza virus infection, for example, highly pathogenic avian influenza infection, containing an endothelin activity inhibitor.
　別の観点において、本発明はＳＩＲＳ処置の必要な患者へエンドセリン活性抑制剤の有効量を投与することを含む、ＳＩＲＳ処置方法を提供する。また、強毒性インフルエンザウイルス感染症の処置が必要な患者へエンドセリン活性抑制剤の有効量を投与することを含む、強毒性インフルエンザ感染症の処置方法を提供する。 In another aspect, the present invention provides a method for treating SIRS, comprising administering an effective amount of an endothelin activity inhibitor to a patient in need of SIRS treatment. Also provided is a method for treating a highly toxic influenza infection, comprising administering an effective amount of an endothelin activity inhibitor to a patient in need of treatment for a highly toxic influenza virus infection.
　更に別の観点において、本発明はエンドセリン活性抑制剤の、ＳＩＲＳ処置のための医薬組成物の製造における使用を提供する。本発明はまた、エンドセリン活性抑制剤の、強毒性インフルエンザ感染症処置のための医薬組成物の製造における使用を提供する。 In yet another aspect, the present invention provides the use of an endothelin activity inhibitor in the manufacture of a pharmaceutical composition for SIRS treatment. The present invention also provides the use of an endothelin activity inhibitor in the manufacture of a pharmaceutical composition for the treatment of highly toxic influenza infection.
　本発明において、エンドセリン活性抑制剤としては特に限定されないが、配列表の配列番号４～１８のペプチド、特に配列番号１１に記載のペプチドが好適に用いられる。 In the present invention, the endothelin activity inhibitor is not particularly limited, but the peptides of SEQ ID NOs: 4 to 18 in the sequence listing, particularly the peptide of SEQ ID NO: 11 is preferably used.
　本発明の医薬組成物により、ＳＩＲＳ並びに強毒性インフルエンザウイルス感染症を効果的に処置することが可能である。 The SIRS and highly toxic influenza virus infection can be effectively treated by the pharmaceutical composition of the present invention.
実施例１の結果を示すグラフである。3 is a graph showing the results of Example 1. 実施例２の生存期間を示すグラフである。ETR-P1/fl群（■）、CLP群（●）およびシャム群（点線）の生存期間を示す。平均生存期間はＥＴＲ－Ｐ１／ｆｌ群はＣＬＰ群より有意に長かった：１８．９±２．３（ＥＴＲ－Ｐ１／ｆｌ）対９．０±０．８（ＣＬＰ）ｐ＜０．００５， Mann-Whitney test。ＥＴＲ－Ｐ１／ｆｌ群の２匹（３３．３％）は２４時間の時点で生存していた。シャム群は全て２４時間生存した。3 is a graph showing the survival time of Example 2. The survival periods of the ETR-P1 / fl group (■), CLP group (●), and Siam group (dotted line) are shown. Mean survival was significantly longer in the ETR-P1 / fl group than in the CLP group: 18.9 ± 2.3 (ETR-P1 / fl) vs. 9.0 ± 0.8 (CLP) p <0.005 Mann-Whitney test. Two animals (33.3%) in the ETR-P1 / fl group were alive at 24 hours. All sham groups survived for 24 hours. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）のＭＡＰ(Mean Arterial Pressure)比の変化を示す。６時間、９時間においてＭＡＰはＥＴＲ－Ｐ１／ｆｌ群においてＣＬＰのみの群より有意に高い値を維持していた（ｐ＜０．０１）。図中、§：ｐ＜０．０５対シャム群、¶：ｐ＜０．０５対ＥＴＲ－Ｐ１／ｆｌ群、各値は平均±ＳＥＭにて示した。以下図３および図４において同じ記号を用いた。The change in the MAP (Mean Arterial Pressure) ratio of the ETR-P1 / fl group (Δ), CLP group (●), and Sham group (◯) is shown. At 6 hours and 9 hours, MAP maintained a significantly higher value in the ETR-P1 / fl group than in the CLP only group (p <0.01). In the figure, §: p <0.05 vs. Siam group, ¶: p <0.05 vs. ETR-P1 / fl group, each value is shown as mean ± SEM. Hereinafter, the same symbols are used in FIGS. 3 and 4. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）のｍＰＡＰ比の変化を示す。The change of the mPAP ratio of the ETR-P1 / fl group (Δ), the CLP group (●), and the sham group (◯) is shown. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）の心拍数（ＨＲ）の変化を示す。６時間、９時間において、ＣＬＰ群では他の群と比して有意に高い心拍数が認められた（ｐ＜０．０５）。Changes in heart rate (HR) of ETR-P1 / fl group (Δ), CLP group (●), and Sham group (◯) are shown. At 6 hours and 9 hours, a significantly higher heart rate was observed in the CLP group than in the other groups (p <0.05). ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）のｍＰＡＰ／ＭＡＰ比の変化を示す。ｍＰＡＰ／ＭＡＰは９時間においてＥＴＲ－Ｐ１／ｆｌ群では上昇しなかった。The change of the mPAP / MAP ratio of the ETR-P1 / fl group (Δ), the CLP group (●), and the sham group (◯) is shown. mPAP / MAP did not increase in the ETR-P1 / fl group at 9 hours. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）の血清中のＴＮＦ－αレベルの変化を示す。The change of the serum TNF-α level in the ETR-P1 / fl group (Δ), the CLP group (●), and the sham group (◯) is shown. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）の血清中のＨＭＧＢ－１量の変化を示す。The change of the amount of HMGB-1 in the serum of the ETR-P1 / fl group (Δ), the CLP group (●) and the sham group (◯) is shown. ＥＴＲ－Ｐ１／ｆｌ群（△）、ＣＬＰ群（●）およびシャム群（○）の血清中のＮＯｘ量の変化を示す。The change of the amount of NOx in the serum of the ETR-P1 / fl group (Δ), the CLP group (●), and the sham group (◯) is shown.
　本発明の医薬組成物において、エンドセリン活性抑制剤としては、エンドセリン－１の活性を抑制するものであればよく、より好ましくはエンドセリン受容体Ａ型を介するエンドセリン－１の作用を抑制するものであれば特に限定的ではない。 In the pharmaceutical composition of the present invention, the endothelin activity inhibitor is not particularly limited as long as it suppresses endothelin-1 activity, and more preferably suppresses endothelin-1 action via endothelin receptor type A. It is not particularly limited.
　エンドセリンとエンドセリン受容体で拮抗する化合物として、ＥＰ－Ａ－５５２４８９、ＥＰ－Ａ－５２８３１２、ＥＰ－Ａ－４９９２６６、ＷＯ９１／１３０８９、ＥＰ－Ａ－４３６１８９、ＥＰ－Ａ－４５７１９５、ＥＰ－Ａ－５１０５２６、ＷＯ９２／１２９９１、特開平４－２８８０９９、特開平４－２４４０９７、特開平４－２６１１９８、ＥＰ－Ａ－４９６４５２、ＥＰ－Ａ－５２６７０８、ＥＰ－Ａ－５２６６４２、ＥＰ－Ａ－５１０５２６、ＥＰ－Ａ－４６０６７９、ＷＯ９２／２０７０６、ＥＰ－Ａ－６２６１７４、ＥＰ－Ａ－６５５４６３、ＥＰ－Ａ－７１４９０９、特開平７－１７３１６１などが提案されている。また、エンドセリン受容体、特にエンドセリン受容体Ａ型の拮抗作用を有する物質として、ボセンタン(bosentan)、シタキセンタン(sitaxsentan)、アンブリセンタン(ambrisentan)、アトラセンタン（atrasentan）、S-139、テゾセンタン(tezosentan)、BMS-193884、ドラウセンタン（drausentan）およびエントラセンタン(entrasentan)等の化合物が知られている。 As compounds that antagonize endothelin and endothelin receptor, EP-A-552489, EP-A-528312, EP-A-499266, WO91 / 13089, EP-A-436189, EP-A-457195, EP-A-510526 , WO92 / 12991, JP-A-4-288099, JP-A-4-244097, JP-A-4-261198, EP-A-496452, EP-A-526708, EP-A-526642, EP-A-510526, EP-A -460679, WO92 / 20706, EP-A-626174, EP-A-655463, EP-A-714909, and JP-A-7-173161 have been proposed. In addition, as substances having an antagonistic action on endothelin receptors, particularly endothelin receptor type A, bosentan, sitaxsentan, ambrisentan, atrasentan, S-139, tezosentan BMS-193884, compounds such as drausentan and entrasentan are known.
　本発明の医薬組成物において特に好適に用いられるのは、ヒトのエンドセリン前駆体のｃＤＮＡから推定されるアミノ酸配列とヒトのエンドセリンレセプターのｃＤＮＡから推定されるアミノ酸配列とを比較し、アミノ酸レベルでセンスアミノ酸・アンチセンスアミノ酸の対応関係で少なくとも８０％相補的な関係になっている部分のヒトエンドセリンレセプターのアミノ酸配列を含有するペプチド、その類縁体である。かかるペプチドとしては特許第３９２３５１５号に開示されており、特に下記アミノ酸配列を含有するペプチドが例示される：
Ｃ１：ＶＬＮＬＣＡＬＳＶＤＲＹＲＡＶＡＳＷＳＲＶＩ（配列番号４）
Ｃ２：ＣＡＬＳＶＤＲＹＲＡＶＡＳＷＧＩＰＬＩＴＡＩＥＩ（配列番号５）
Ｃ３：ＱＧＩＧＩＰＬＩＴＡＩＥＩ（配列番号６）
Ｃ４：ＩＡＤＮＡＥＲＹＳＡＮＬＳＳＨＶ（配列番号７）
Ｃ５：ＦＬＧＴＴＬＲＰＰＮＬＡＬＰ（配列番号８）
Ｃ６：ＬＮＲＲＮＧＳＬＲＩＡＬＳＥＨＬＫＮＲＲＥＶＡ（配列番号９） In the pharmaceutical composition of the present invention, the amino acid sequence deduced from the human endothelin precursor cDNA is compared with the amino acid sequence deduced from the human endothelin receptor cDNA and sensed at the amino acid level. A peptide containing the amino acid sequence of the human endothelin receptor at least 80% complementary in the corresponding amino acid / antisense amino acid relationship, and its analogs. Such peptides are disclosed in Japanese Patent No. 3923515, and in particular, peptides containing the following amino acid sequences are exemplified:
C1: VLNLCALSVDRYRAVASWSRVI (SEQ ID NO: 4)
C2: CALSVDRYRAVASWGIPLITAIEI (SEQ ID NO: 5)
C3: QGIGIPLITAIEI (SEQ ID NO: 6)
C4: IADNAERYSANLSSHV (SEQ ID NO: 7)
C5: FLGTTTLPPNLALP (SEQ ID NO: 8)
C6: LNRRNGSLRIALSEHLKNRREVA (SEQ ID NO: 9)
　本願発明の医薬組成物において用いられる好ましいエンドセリン活性抑制剤としては、Ｃ１ペプチドの末端をアミド体としたペプチド、および当該末端アミド体の配列中、特定のアミノ酸のいずれか１つをグリシンで置換したペプチドが挙げられる。具体的には以下の配列が好適に用いられる：
Ｃ１１：ＶＬＮＬＣＡＬＳＶＤＲＹＲＡＶＡＳＷＳＲＶＩ－ＮＨ2（配列番号１１）
Ｃ１２：ＶＬＮＬＣＡＬＳＶＤＲＹＲＡＶＡＳＧＳＲＶＩ－ＮＨ2（配列番号１２）
Ｃ１３：ＶＬＮＬＣＡＬＳＶＤＲＹＧＡＶＡＳＷＳＲＶＩ－ＮＨ2（配列番号１３）
Ｃ１４：ＶＬＮＬＣＡＬＳＶＤＧＹＲＡＶＡＳＷＳＲＶＩ－ＮＨ2（配列番号１４）
Ｃ１５：ＶＬＮＬＧＡＬＳＶＤＲＹＲＡＶＡＳＷＳＲＶＩ－ＮＨ2（配列番号１５）
上記式および以下の式において、「－ＮＨ2」はＣ末端のカルボキシル基がアミド化された化合物を示す。また、Ｃ１ペプチドのＮ末端および／またはＣ末端のアミノ酸を除去したペプチド、およびその配列中のアミノ酸の一部を置換したものも好適に用いられる。具体的には以下に示す配列が例示される。
Ｃ１６：ＣＡＬＳＶＤＲＹＲＡＶＡＳＷ－ＮＨ２（配列番号１６）
Ｃ１７：ＣＡＬＳＶＤＲＹＲＡＶＡＳＷＣ－ＮＨ２（配列番号１７）但し、１位と１５位のシステインはジスルフィド結合を形成している。
Ｃ１８：ＶＡＬＳＶＤＲＹＲＡＶＡＳＷ－ＮＨ２（配列番号１８） As a preferred endothelin activity inhibitor used in the pharmaceutical composition of the present invention, a peptide in which the terminal of the C1 peptide is an amide, and any one of the specific amino acids in the sequence of the terminal amide is substituted with glycine Peptides are mentioned. Specifically, the following sequences are preferably used:
C11: VNLNLCALSVDRYRAVASWSRVI-NH 2 (SEQ ID NO: 11)
C12: VLNLCALSVSVRYRAVASGSRVI-NH 2 (SEQ ID NO: 12)
C13: VLNLCALSVSVRYGAVASWSRVI-NH 2 (SEQ ID NO: 13)
C14: VLNLCALSVDGYRAVASWSRVI-NH 2 (SEQ ID NO: 14)
C15: VLNLGALSVDRYRAVASWSRVI-NH 2 (SEQ ID NO: 15)
In the above formula and the following formula, “—NH 2 ” represents a compound in which the C-terminal carboxyl group is amidated. Further, peptides in which the N-terminal and / or C-terminal amino acids of the C1 peptide have been removed, and those in which a part of the amino acids in the sequence have been substituted are also preferably used. Specifically, the following sequences are exemplified.
C16: CALSVDRYRAVASW-NH 2 (SEQ ID NO: 16)
C17: CALSVDRYRAVASW-NH 2 (SEQ ID NO: 17) However, the cysteines at positions 1 and 15 form a disulfide bond.
C18: VALSVDRYRAVASW-NH 2 (SEQ ID NO: 18)
　上記Ｃ１～６並びにＣ１１～１８のペプチドはいずれも、エンドセリン－１によるマウス３Ｔ３細胞内カルシウムイオン濃度増加およびエンドセリン－１によるラット大動脈平滑筋血管収縮のいずれかの試験においてエンドセリン－１の作用を抑制することが確認されたものである（特許第３９２３５１５号）。 The above peptides C1-6 and C11-18 all inhibit endothelin-1 action in endothelin-1 in either mouse 3T3 intracellular calcium ion concentration increase or rat aortic smooth muscle vasoconstriction in endothelin-1 (Japanese Patent No. 3923515).
　即ちこれらのペプチドの骨格となる配列：
ＡＬＳＶＤ（Ｘ）Ｙ（Ｘ）ＡＶＡＳＷ（配列番号１０）
（式中、Ｘはいずれかのアミノ酸を示す）
を含有するペプチドはエンドセリンの活性を抑制する効果を示し、本願において好適に用いられる。 That is, the sequence that forms the backbone of these peptides:
ALSVD (X) Y (X) AVASW (SEQ ID NO: 10)
(Wherein X represents any amino acid)
Peptides containing lysine have the effect of suppressing the activity of endothelin and are preferably used in the present application.
　本発明においては、特にＣ１１（配列番号１１）のペプチドが好適に用いられる。 In the present invention, a peptide of C11 (SEQ ID NO: 11) is particularly preferably used.
　本発明の医薬組成物の投与方法並びに投与経路は特に限定されないが、経腸または経口形態で、たとえば錠剤、糖衣錠、ゼラチンカプセル、乳剤、液剤または懸濁剤として、噴霧剤などの経鼻形態で、あるいは坐剤などの経直腸形態で、投与できる。本発明の医薬組成物はまた、注射剤として、静脈投与または筋肉内投与してもよい。 The administration method and administration route of the pharmaceutical composition of the present invention are not particularly limited, but enteral or oral forms, for example, as nasal forms such as sprays, sugar-coated tablets, gelatin capsules, emulsions, solutions or suspensions. Alternatively, it can be administered in a rectal form such as a suppository. The pharmaceutical composition of the present invention may also be administered intravenously or intramuscularly as an injection.
　本発明の医薬組成物は、エンドセリン活性抑制剤に加えて、塩、希釈剤、担体、バッファー、結合剤、界面活性剤、保存剤のような生理的に許容される他の成分を含有していてもよい。非経口的投与製剤は、滅菌水溶液又は生理学的に許容される溶媒との懸濁液アンプル、または生理学的に許容される希釈液で用時希釈して使用しうる滅菌粉末、例えばエンドセリン活性抑制剤がペプチドである場合には通常ペプチド溶液を凍結乾燥して得られる粉末、を含有するアンプルとして提供される。 In addition to the endothelin activity inhibitor, the pharmaceutical composition of the present invention contains other physiologically acceptable components such as salts, diluents, carriers, buffers, binders, surfactants, and preservatives. May be. Preparations for parenteral administration include sterile aqueous solutions or suspension ampoules with physiologically acceptable solvents, or sterile powders that can be used diluted with physiologically acceptable diluents, such as endothelin activity inhibitors. When is a peptide, it is usually provided as an ampoule containing a powder obtained by lyophilizing a peptide solution.
　本発明の医薬組成物の投与量としては限定的でなく、患者の年齢、体重、性別、症状、所望の効果等により適宜決定すればよい。 The dose of the pharmaceutical composition of the present invention is not limited, and may be appropriately determined depending on the patient's age, weight, sex, symptom, desired effect, and the like.
　本発明の医薬組成物は、ＳＩＲＳの処置に好適に用いられる。 The pharmaceutical composition of the present invention is suitably used for SIRS treatment.
　本明細書および請求の範囲において「ＳＩＲＳ（Systemic Inflammatory Response Syndrome」とは、過剰な炎症反応が全身性に起こり、炎症により種々の臓器障害を誘起する致命的な病態を意味する。「ＳＩＲＳ」は、米国において下記診断基準を満たす症状を示すものと定義づけられており、本明細書においてもかかる定義に従うものとする： In the present specification and claims, “SIRS (Systemic Inflammatory Response“ Syndrome ”) means a fatal pathological condition in which an excessive inflammatory reaction occurs systemically and induces various organ disorders due to inflammation. , In the United States is defined as exhibiting symptoms that meet the following diagnostic criteria, and this specification shall also be followed:
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ＳＩＲＳと診断された病態のなかで感染症が原因となっているものがゼプシスあるいは敗血症であると診断される。感染症が原因であるか否かは病原微生物による感染巣の確認やＣＲＰの異常上昇（１０以上）により判定される。 Among the pathological conditions diagnosed with SIRS, those caused by infectious diseases are diagnosed as zepsis or sepsis. Whether or not the cause is an infection is determined by confirmation of an infection focus by a pathogenic microorganism or an abnormal increase in CRP (10 or more).
　本願明細書並びに請求の範囲において、「ＳＩＲＳの処置」とは、ＳＩＲＳの誘因となる菌血症、真菌血症、寄生虫血症、ウイルス血症、外傷、熱傷、膵炎、手術などの種々の侵襲を受けた患者においてＳＩＲＳの発症を予防すること、ＳＩＲＳ発症の程度を減弱化あるいはＳＩＲＳの発症による死亡を阻止することを含む。また、さらにＳＩＲＳの処置には既に上記診断基準によるＳＩＲＳが発症した患者において死亡を防止することを含む。 In the present specification and claims, “treatment of SIRS” means various bacteremia, fungiemia, parasitemia, viremia, trauma, burns, pancreatitis, surgery, etc. that cause SIRS. This includes preventing the onset of SIRS in an invaded patient, reducing the degree of SIRS onset or preventing death due to the onset of SIRS. Furthermore, SIRS treatment includes preventing death in patients who have already developed SIRS according to the above diagnostic criteria.
　従って、本発明の医薬組成物は、ＳＩＲＳを発症している患者のみならず、ＳＩＲＳを発症することが予測される状況にある患者、具体的には、菌血症、真菌血症、寄生虫血症、ウイルス血症、外傷、熱傷、膵炎、手術などの種々の侵襲を受けている患者におけるＳＩＲＳの処置に好適に用いられる。また、強毒性インフルエンザウイルス感染症による死亡はＳＩＲＳを発症することによるものであることが知られている。従って、本発明の医薬組成物は、症状の発現の有無、その強弱を問わず強毒性インフルエンザウイルスに感染した患者の処置に好適に用いられる。 Therefore, the pharmaceutical composition of the present invention can be used not only for patients who develop SIRS, but also for patients who are predicted to develop SIRS, specifically bacteremia, fungiemia, parasites. It is suitably used for the treatment of SIRS in patients undergoing various invasions such as septicemia, viremia, trauma, burns, pancreatitis, and surgery. In addition, it is known that death due to highly toxic influenza virus infection is caused by developing SIRS. Therefore, the pharmaceutical composition of the present invention is suitably used for the treatment of patients infected with highly toxic influenza viruses regardless of the presence or absence of symptoms or their strength.
　本発明はまた、強毒性インフルエンザウイルス感染症患者の処置に好適に用いられる。ここで「強毒性インフルエンザウイルス感染症」とは、インフルエンザ感染によりＳＩＲＳを発症して死亡する可能性が高いことが知られているインフルエンザウイルスによる感染症を意味する。かかるインフルエンザウイルスとして、現在知られているものではＨ５Ｎ１トリインフルエンザウイルスがあるが、該ウイルス、あるいはその他のウイルスが突然変異により強毒性となったものいずれも含むものとする。 The present invention is also suitably used for the treatment of patients with highly toxic influenza virus infection. Here, “strongly toxic influenza virus infection” means an infection caused by influenza virus, which is known to have a high possibility of developing SIRS and dying due to influenza infection. Such influenza viruses include H5N1 avian influenza virus that is currently known, and includes those viruses or other viruses that are highly toxic due to mutation.
　本発明の医薬組成物は、症状の発現の前後を問わず、強毒性インフルエンザウイルスに感染している患者、あるいは該感染の疑われる患者の処置に有用である。「強毒性インフルエンザウイルス感染症の処置」とは、強毒性インフルエンザウイルスに起因する症状の発症の抑制、発症する症状の軽減化並びに既に発症した場合の症状の治療のいずれをも含むものとする。さらに、本発明においては、強毒性インフルエンザウイルスに起因する重篤な肺炎やＳＩＲＳ症状が既に発症した患者を死から救うためにも好適に用いられる。 The pharmaceutical composition of the present invention is useful for the treatment of a patient who is infected with a highly virulent influenza virus or a patient who is suspected of having the infection regardless of the onset of symptoms. “Treatment of highly virulent influenza virus infection” includes any of suppression of symptoms caused by virulent influenza viruses, reduction of symptoms that develop, and treatment of symptoms that have already occurred. Furthermore, in this invention, it uses suitably also in order to rescue the patient from whom serious pneumonia resulting from a virulent influenza virus and SIRS symptom already developed from death.
　本発明の医薬組成物は強毒性インフルエンザウイルス、例えばＨ５Ｎ1トリインフルエンザウイルス感染症に感染した鳥類の治療並びに感染拡大防止のために有効に用いることができる。例えば、鶏舎内でインフルエンザウイルスに感染した鶏が発生した場合に、該感染鶏へ本発明の医薬組成物を投与することによって治癒させて感染拡大を防ぐことができる。また、感染鶏が生じた同一鶏舎や近隣の鶏舎に飼われている鶏に予め本発明の医薬組成物を投与することにより、インフルエンザウイルス感染拡大を防ぐことができる。 The pharmaceutical composition of the present invention can be effectively used for treatment of birds infected with highly toxic influenza viruses such as H5N1 avian influenza virus infection and prevention of spread of infection. For example, when a chicken infected with influenza virus occurs in a poultry house, it can be cured by administering the pharmaceutical composition of the present invention to the infected chicken to prevent the spread of infection. In addition, the spread of influenza virus infection can be prevented by administering the pharmaceutical composition of the present invention in advance to chickens kept in the same chicken house where infected chickens are produced or in neighboring chicken houses.
　本発明を実施例によりさらに詳細に説明する。
使用ペプチド：
エンドセリン活性抑制剤ＥＴＲ－Ｐ１／ｆ１
ＶＬＮＬＣＡＬＳＶＤＲＹＲＡＶＡＳＷＳＲＶＩ－ＮＨ2（配列番号１１）
AcePepA（参考）：
ＡＳＧＡＰＡＰＧＰＡＧＰＬＲＰＭＦ（配列番号３）N末端アセチル化
アナフィラトキシンＣ５ａ不活化ペプチド。ヒトＣ５ａのアミノ酸３７－５３位をターゲットとして得られたアンチセンスペプチドであり、ゼプシスショックを抑制する作用を有する（特開２００４－２６９５２０およびMol. Immunol. 45:4113(2008))。 The invention is explained in more detail by means of examples.
Peptides used:
Endothelin activity inhibitor ETR-P1 / f1
VLNLCALSVDRYRAVASWSRVI-NH 2 (SEQ ID NO: 11)
AcePepA (reference):
ASGAPAPGPAGPPLRPMF (SEQ ID NO: 3) N-terminal acetylated anaphylatoxin C5a inactivating peptide. It is an antisense peptide obtained by targeting amino acid positions 37-53 of human C5a, and has an action of suppressing zepsis shock (Japanese Patent Application Laid-Open No. 2004-269520 and Mol. Immunol. 45: 4113 (2008)).
新生仔ブタ腹膜炎モデルにおけるETR-P1/f1の作用（１）
　実際のゼプシス感染病態に近い、新生仔ブタ腹膜炎モデルにおいて本発明の医薬組成物の効果を確認した。
　新生仔ブタを吸入麻酔下で開腹し、回盲部を桔札して１８ゲージの注射針で孔を開け糞便が漏れ出るようにしたあと腸管を腹腔内に戻し、腹壁を縫合した。この処置はCLP (Cecal Ligation and Puncture) と呼ばれる腹膜炎モデルであり、ＣＬＰを施された新生仔ブタは全身性炎症反応症候群（ＳＩＲＳ:Systemic Inflammatory Response Syndrome）を起こして死亡する。
　このＣＬＰ処置３０分後から０．０５ｍｇ／ｋｇ／ｈｒの量のＥＴＲ－Ｐ１／ｆｌを静脈内に連続投与した。シャム手術群、ＣＬＰ手術群およびＣＬＰ手術後ＥＴＲ－Ｐ１／ｆ１投与群それぞれの生存期間を調べた。なお、全動物は２４時間後に安楽死させた。試験は無処置群８頭、ＣＬＰ手術群１０頭、ＣＬＰ手術後ＥＴＲ－Ｐ１／ｆ１投与群７頭にて行った。結果を図１に示す。
　ＣＬＰ手術のみ実施した群では１８時間までに全例が死亡し、平均生存期間はＣＬＰ処置後約１０時間（１０．２４±１ｈｒ）であった。一方ＥＴＲ－Ｐ１／ｆ１投与群では生存期間は術後２４時間目でも７頭中２頭が生存しており、平均生存期間は約１８時間（１８±２．０５ｈｒ）まで延長された。したがって、本発明の医薬組成物はＳＩＲＳを抑制することがわかる。 Action of ETR-P1 / f1 in neonatal porcine peritonitis model (1)
The effect of the pharmaceutical composition of the present invention was confirmed in a neonatal porcine peritonitis model that is close to the actual pathological condition of Zepsis infection.
The newborn pig was laparotomized under inhalation anesthesia, the ileocecal region was tagged, a hole was opened with an 18-gauge injection needle so that feces leaked out, the intestine was returned to the abdominal cavity, and the abdominal wall was sutured. This treatment is a peritonitis model called CLP (Cecal Ligation and Puncture), and a newborn pig subjected to CLP develops a systemic inflammatory response syndrome (SIRS) and dies.
From 30 minutes after this CLP treatment, 0.05 mg / kg / hr of ETR-P1 / fl was continuously administered intravenously. The survival period of each of the sham surgery group, the CLP surgery group, and the ETR-P1 / f1 administration group after the CLP surgery was examined. All animals were euthanized after 24 hours. The test was conducted in 8 untreated groups, 10 CLP surgery groups, and 7 ETR-P1 / f1 administration groups after CLP surgery. The results are shown in FIG.
In the group in which only CLP surgery was performed, all cases died by 18 hours, and the average survival time was about 10 hours (10.24 ± 1 hr) after CLP treatment. On the other hand, in the ETR-P1 / f1 administration group, 2 out of 7 animals survived even 24 hours after the operation, and the average survival time was extended to about 18 hours (18 ± 2.05 hr). Therefore, it turns out that the pharmaceutical composition of this invention suppresses SIRS.
新生仔ブタ腹膜炎モデルにおけるETR-P1/f1の作用（２）
　実施例１と同様の新生仔ブタ腹膜炎モデルにおいて本発明の医薬組成物の作用の詳細を観察した。 Action of ETR-P1 / f1 in neonatal porcine peritonitis model (2)
The details of the action of the pharmaceutical composition of the present invention were observed in a neonatal porcine peritonitis model similar to that in Example 1.
使用動物
　米国国立衛生研究所（ＮＩＨ）の実験動物取り扱い指針に従い試験を行った。試験プロトコルは名古屋市立大学大学院医学研究科の倫理委員会の承認を得た。１８匹の生後３日目の雑種新生仔ブタを近隣農家より入手した。新生仔ブタは６匹ずつ３群に分けた：
１）ＣＬＰ（Ｃｅｃａｌ Ｌｉｇａｔｉｏｎ ａｎｄ Ｐｕｎｃｔｕｒｅ）群
２）ＥＴＲ－Ｐ１／ｆl群：ＣＬＰ処置３０分後からＥＴＲ－Ｐ１／ｆｌを静脈内に連続投与。
３）シャム群：ＣＬＰ処置と同様に開腹し、一旦回盲部を引き出した後腹腔内に戻し腹壁を縫合した。 Animals used Tests were conducted in accordance with the National Animal Health Laboratory (NIH) laboratory animal handling guidelines. The test protocol was approved by the Ethics Committee of Nagoya City University Graduate School of Medicine. Eighteen newborn hybrid piglets on the third day of life were obtained from neighboring farmers. Newborn piglets were divided into 3 groups of 6 each:
1) CLP (Cecal Ligation and Puncture) group 2) ETR-P1 / fl group: ETR-P1 / fl was continuously administered intravenously 30 minutes after CLP treatment.
3) Sham group: The abdomen was opened in the same manner as the CLP treatment, the ileocecal region was once pulled out, and then returned to the abdominal cavity, and the abdominal wall was sutured.
　新生仔ブタは母ブタと共に飼育し、処置時にのみ隔離した。新生仔ブタの体重は１４５０～２２００ｇの間、中央値は１７１５ｇであった。 Newborn pigs were raised with mother pigs and isolated only during treatment. Newborn pigs weighed between 1450 and 2200 g with a median of 1715 g.
　仔ブタへ１０ｍｇ／ｋｇ塩化ケタミンを筋肉内投与した。その後５％グルコース水溶液中のペントバルビタールナトリウムを末梢血管より５ｍｇ／ｋｇ／ｈの速度で処置期間中投与して麻酔を維持した。各仔ブタは気管内へ内径４ｍｍの管を挿管して乳児用人工呼吸器（ＩＶ－１００，Sechrist Industries, Anaheim, CA, USA）にて人工呼吸を行った。開始時の吸気・呼気圧を１４／４ｃｍＨ２Ｏとし、吸気時間を０．５秒とした。人工呼吸には室内の空気を用いた。その後呼吸圧をＰａＣＯ２が３０－５０ｍｍＨｇを維持するよう調節した。カットダウン法により４Ｆポリビニルカテーテルを左側大腿動脈へ挿入して血液サンプルの取得、平均動脈圧（ＭＡＰ）および心拍数（ＨＲ）の測定に用いた。４－Ｆｒベルマン血管造影用カテーテル（American Edwards Laboratories, Irvine, CA, USA）を蛍光透視した右側外顎静脈より挿入し、肺主動脈へ設置してｍＰＡＰの測定に用いた。さらに４Ｆポリビニルカテーテルを左外顎静脈より挿入して上大静脈へ到達させ、中心大静脈圧の測定に用いた。平均動脈圧（ＭＡＰ）、平均肺動脈圧（ｍＰＡＰ）および心拍数（ＨＲ）は新生児用モニター（model 78801 B, Hewlett Packard, Andover, MA, USA）にて測定し、データをMacLab/8s system (ADI Instruments, Mountain View, CA, USA）にて集めた。体温は直腸温をモニターし、温熱パッドおよびポリビニルカバーを用いて維持した。 The piglet was administered intramuscularly with 10 mg / kg ketamine chloride. Thereafter, sodium pentobarbital in a 5% glucose aqueous solution was administered from the peripheral blood vessel at a rate of 5 mg / kg / h during the treatment period to maintain anesthesia. Each piglet was intubated with a 4 mm inner diameter tube and ventilated with an infant ventilator (IV-100, Sechrist Industries, Anaheim, CA, USA). The inspiratory / expiratory pressure at the start was 14/4 cmH 2 O, and the inspiratory time was 0.5 seconds. Indoor air was used for artificial respiration. The respiratory pressure was then adjusted to maintain PaCO 2 at 30-50 mmHg. A 4F polyvinyl catheter was inserted into the left femoral artery by the cut-down method and used for blood sample acquisition, mean arterial pressure (MAP) and heart rate (HR) measurement. A 4-Fr Bellman angiographic catheter (American Edwards Laboratories, Irvine, Calif., USA) was inserted through the fluoroscopic right lateral vein, placed in the main pulmonary artery, and used for mPAP measurement. Further, a 4F polyvinyl catheter was inserted from the left external jaw vein to reach the superior vena cava, and used for measuring the central vena cava pressure. Mean arterial pressure (MAP), mean pulmonary arterial pressure (mPAP) and heart rate (HR) were measured on a newborn monitor (model 78801 B, Hewlett Packard, Andover, MA, USA), and the data was measured using the MacLab / 8s system (ADI Instruments, Mountain View, CA, USA). Body temperature was monitored by rectal temperature and maintained using a thermal pad and polyvinyl cover.
ＣＬＰ処置（ＣＬＰ群）
　ＣＬＰ処置は下記のとおり行った。新生仔ブタの腹部へ盲腸および回腸末端（約４ｃｍ）を暴露可能なように切開部を作成した。回盲部動脈を確認し、盲腸近傍で結紮して盲腸の遠端への血流を止めた。約１ｃｍの切れ目を抗腸間膜側へ作成した。盲腸から糞便を腹腔内へ穏やかに絞り出した後、腹部切開部を綴じた。 CLP treatment (CLP group)
CLP treatment was performed as follows. An incision was made so that the cecum and terminal ileum (approximately 4 cm) could be exposed to the abdomen of the newborn pig. The ileocecal artery was confirmed and ligated near the cecum to stop blood flow to the distal end of the cecum. A cut of about 1 cm was made on the antimesenteric side. After gently squeezing feces from the cecum into the abdominal cavity, the abdominal incision was bound.
シャム処置（シャム群）
　シャム群においては、新生仔ブタの腹部切開後、盲腸および回腸末端を２分間外部へ暴露した後に腹腔内へ戻し、腹部切開部を閉じた。 Siamese treatment (siamese group)
In the sham group, after the abdominal incision of the newborn piglet, the cecum and the terminal ileum were exposed to the outside for 2 minutes and then returned to the abdominal cavity, and the abdominal incision was closed.
ＣＬＰ処置＋ＥＴＲ－Ｐ１／ｆｌ投与（ＥＴＲ－Ｐ１／ｆｌ群）
　ＥＴＲ－Ｐ１／ｆｌ投与はＣＬＰ処置の３０分後に開始した。ＥＴＲ－Ｐ１／ｆｌの５％グルコース中溶液をＥＴＲ－Ｐ１／ｆｌ投与量が０．０５ｍｇ／ｋｇ／ｈとなるよう、中心静脈内へ投与した。静脈投与の溶液量は５ｍＬ／ｋｇ／ｈとなるよう調節した。 CLP treatment + ETR-P1 / fl administration (ETR-P1 / fl group)
ETR-P1 / fl administration started 30 minutes after CLP treatment. A solution of ETR-P1 / fl in 5% glucose was administered into the central vein so that the dose of ETR-P1 / fl was 0.05 mg / kg / h. The solution volume for intravenous administration was adjusted to 5 mL / kg / h.
　各動物は死亡するまで、または２４時間観察した。２４時間生存した動物はナトリウムフェノバルビタール致死量を投与して殺した。 Each animal was observed for 24 hours until death. Animals that survived 24 hours were killed by administering a lethal dose of sodium phenobarbital.
　試験プロトコル
　各群において、血液サンプルを無菌下で大腿動脈カテーテルより採取して、動脈血液ガス(arterial blood gases)、血清ＴＮＦ－α、ＮＯ代謝物、ＨＭＧＢ－１を測定した。血液サンプルはＣＬＰまたはシャム処置前、および処置１、３、６および９時間後に採取した。サンプリングはＥＴＲ－Ｐ１／ｆｌ群およびシャム群では１２時間後にも行った。全てのサンプルは滅菌チューブに保存した。 Test Protocol In each group, blood samples were collected from the femoral artery catheter under aseptic conditions to measure arterial blood gases, serum TNF-α, NO metabolites, and HMGB-1. Blood samples were taken before CLP or sham treatment and 1, 3, 6, and 9 hours after treatment. Sampling was also performed after 12 hours in the ETR-P1 / fl group and the Sham group. All samples were stored in sterile tubes.
　測定
　動脈血液ガスは標準分析器（モデル１２４８； CIBA Corning, Medfield, MA, USA）を用いて測定した。血清ＴＮＦ－αはブタＴＮＦ－α用イムノアッセイキット（R&D Systems, Minneapolis, MN, USA)を用いて測定した。ＮＯ代謝物は血中NO2 - + NO3 - (NOx)濃度を測定して評価した。NOx濃度は全窒素並びに窒素ＥＬＩＳＡキット（R&D Systems）を用いて測定した。ＨＭＧＢ－１はＨＭＧＢ－１用ＥＬＩＳＡキット(Shino-Test Corporation, Sagamihara, Japan)を用いて測定した。 Measurement Arterial blood gas was measured using a standard analyzer (Model 1248; CIBA Corning, Medfield, MA, USA). Serum TNF-α was measured using an immunoassay kit for porcine TNF-α (R & D Systems, Minneapolis, MN, USA). NO metabolites were evaluated by measuring the blood NO 2 − + NO 3 − (NOx) concentration. The NOx concentration was measured using total nitrogen and a nitrogen ELISA kit (R & D Systems). HMGB-1 was measured using an ELISA kit for HMGB-1 (Shino-Test Corporation, Sagamihara, Japan).
　拍出量の測定
　Ｍ－モード心エコー試験を５．０ＭＨｚのトランスデューサーを有するＳＯＮＯＳ５００（ヒューレットパッカード社）を用いて、ＣＬＰまたはシャム処置前、および処置後１、３、６および９時間後に行った。ＥＴＲ－Ｐ１／ｆｌ群およびシャム群についてはＣＬＰ／シャム処置１２時間後にも測定を行った。心尖部長軸像より左心室の二次元Ｍモード測定を行い、拍出量を計算した。測定は各測定時にそれぞれ３回行い、各測定時の平均値を算出した。第１及び第２生存ターゲットをそれぞれＣＬＰ群の生存時間および２×ＳＥＭ、ＣＬＰ群の生存時間および２×ＳＤとして算出した。第３の生存ターゲットはＣＬＰ手術後２４時間の試験終了時とした。 Measurement of stroke volume M-mode echocardiography was performed using a SONOS500 (Hewlett Packard) with a 5.0 MHz transducer before CLP or sham treatment and 1, 3, 6 and 9 hours after treatment. . The ETR-P1 / fl group and the sham group were also measured 12 hours after CLP / sham treatment. Two-dimensional M-mode measurement of the left ventricle was performed from the apex long-axis image, and the stroke volume was calculated. The measurement was performed three times at each measurement, and the average value at each measurement was calculated. The first and second survival targets were calculated as CLP group survival time and 2 × SEM, CLP group survival time and 2 × SD, respectively. The third survival target was at the end of the study 24 hours after CLP surgery.
統計分析
　データの分布はShapiro-Wilk testにて分析した。ＣＬＰ手術後９時間迄は、各群の測定値の平均をそれぞれの測定時間においてＡＮＯＶＡ検定に基づいて比較し、その後Bonferroni post hoc testを行った。データが正規分布ではない場合にはKrusal-Wallis testを用い有意差が見出された場合にはMann-Whitney testを用いて分析した。９時間以降は非対象Student’s t-testを用いた。データが正規分布でない場合にはMann-Whitney test を用いてETR-P1/fl群のデータとシャム群のデータを比較した。３つの群間の生存期間の相違は Kaplan-Meier testを用いて計算し、Kruskal-Wallis test 次いでMann-Whitney testを用いて比較した。データは平均±SEMで示した。差が危険率０．０５未満の場合に有意差であると認定した。全てのデータ分析は市販の統計分析ソフトウエアパッケージSPSS(Statistical Package for Social Sciences, Chicago, Illinois, USA)を用いて行った。 Statistical analysis Data distribution was analyzed with the Shapiro-Wilk test. Up to 9 hours after the CLP operation, the average of the measurement values of each group was compared based on the ANOVA test at each measurement time, and then the Bonferroni post hoc test was performed. When data was not normally distributed, Krusal-Wallis test was used, and when a significant difference was found, analysis was performed using Mann-Whitney test. After 9 hours, non-target Student's t-test was used. When the data were not normally distributed, the data of the ETR-P1 / fl group was compared with the data of the Siam group using the Mann-Whitney test. Differences in survival between the three groups were calculated using the Kaplan-Meier test and compared using the Kruskal-Wallis test followed by the Mann-Whitney test. Data are shown as mean ± SEM. A difference was found to be significant when the risk factor was less than 0.05. All data analysis was performed using the commercially available statistical analysis software package SPSS (Statistical Package for Social Sciences, Chicago, Illinois, USA).
結果
　各測定結果を表２、表３、および図２～４に示す。
Figure JPOXMLDOC01-appb-T000002
 Results The measurement results are shown in Table 2, Table 3, and FIGS.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
動物の状態並びに生存期間
　ＣＬＰ群、シャム群、およびＥＴＲ－Ｐ１／ｆｌ群の体重に有意差は無く、それぞれ１６４６±８８ｇ、１７１８±５０ｇおよび１８５０±１０６ｇであった。 Animal status and survival time There was no significant difference in body weight between the CLP group, the Siam group, and the ETR-P1 / fl group, which were 1646 ± 88 g, 1718 ± 50 g, and 1850 ± 106 g, respectively.
　ＥＴＲ－Ｐ１／ｆｌ群の生存期間はＣＬＰ群より有意に長かった（１８．９±２．３ｈ対９．０±０．８ｈ、Ｐ＝０．００５）。９５％信頼区間として、ＥＴＲ－Ｐ１／ｆｌ群では１４．４時間から２３．４時間の間、ＣＬＰ群では７．５時間から１０．５時間の間であった。ＥＴＲ－Ｐ１／ｆｌ群では８３．３％（ｎ＝５）、５０％（ｎ＝３）および３３．３％（ｎ＝２）の動物がそれぞれ、第１、第２および第３生存ターゲットに到達した。対してＣＬＰ群では１匹も第１生存ターゲットに到達できなかった（図２）。シャム群の動物は全て２４時間生存した。 The survival period of the ETR-P1 / fl group was significantly longer than that of the CLP group (18.9 ± 2.3 h vs. 9.0 ± 0.8 h, P = 0.005). The 95% confidence interval was between 14.4 and 23.4 hours for the ETR-P1 / fl group and between 7.5 and 10.5 hours for the CLP group. In the ETR-P1 / fl group, 83.3% (n = 5), 50% (n = 3) and 33.3% (n = 2) animals were the first, second and third survival targets, respectively. Reached. In contrast, none of the CLP groups could reach the first survival target (FIG. 2). All animals in the sham group survived for 24 hours.
血液ｐＨおよび過剰塩基への作用
　シャム群では血液ｐＨ値の変化は無かったが、ＣＬＰ群では６から９時間の間に血液ｐHは顕著に下がった（表２）。ＥＴＲ－Ｐ１／ｆｌ群においては、血液ｐＨのレベルはシャム群のレベルに相当するものであった。６時間と９時間において、ＥＴＲ－Ｐ１／ｆｌ群の血液ｐＨレベルはＣＬＰ群と比し有意に高かった（それぞれ７．３５±０．０３対７．２５±０．０２、ｐ＝０．０２（６時間）；７．３３±０．０４対７．１８±０．０３、ｐ＝０．０１（９時間））（表２）。 Effect on blood pH and excess base There was no change in blood pH value in the sham group, but blood pH decreased significantly between 6 and 9 hours in the CLP group (Table 2). In the ETR-P1 / fl group, the blood pH level corresponded to that in the sham group. At 6 hours and 9 hours, the blood pH level of the ETR-P1 / fl group was significantly higher than that of the CLP group (7.35 ± 0.03 vs 7.25 ± 0.02, respectively, p = 0.02). (6 hours); 7.33 ± 0.04 vs 7.18 ± 0.03, p = 0.01 (9 hours)) (Table 2).
　ＣＬＰ群およびＥＴＲ－Ｐ１／ｆｌ群における動脈過剰塩基（ＢＥ）値は低下した。ＥＴＲ－Ｐ１／ｆｌ群のＢＥレベルはＣＬＰ群より高く維持されていたが、その差は有意ではなかった（表２）。 Arterial excess base (BE) values in the CLP group and the ETR-P1 / fl group decreased. The BE level in the ETR-P1 / fl group was maintained higher than that in the CLP group, but the difference was not significant (Table 2).
血圧への作用
　ＣＬＰ／シャム処置３時間後まで３つの群の間で相対平均動脈圧（ＭＡＰ）に差はなかった（図３―Ａ）。ＣＬＰ群では相対ＭＡＰ値が徐々に下がり６時間後には６０％、９時間では７５％下がった。ＣＬＰ群の動物の相対ＭＡＰ値はＣＬＰ処置前より有意に低かった（０．３７±０．０３［６ｈ］および０．２４±０．０１［９ｈ］いずれもｐ＜０．０１）が、シャム群の相対ＭＡＰは試験期間中ほとんど変化がなかった。ＥＴＲ－Ｐ１／ｆｌ群の動物における相対ＭＡＰ値においては有意ではないがゆっくりとした減少が認められ、６時間並びに９時間においてはＣＬＰ群より有意に高かった（０．７５±０．１０対０．３７±０．０３［６ｈ］、０．７６±０．１１対０．２４±０．０１［９ｈ］；いずれもｐ＜０．０１）（図３Ａ）。試験期間中、ＥＴＲ－Ｐ１／ｆｌ群とシャム群の間で相対ＭＡＰ値の差は無かった。 Effects on blood pressure There was no difference in relative mean arterial pressure (MAP) between the three groups until 3 hours after CLP / sham treatment (FIG. 3-A). In the CLP group, the relative MAP value gradually decreased, 60% after 6 hours, and 75% after 9 hours. Relative MAP values for animals in the CLP group were significantly lower than before CLP treatment (both 0.37 ± 0.03 [6h] and 0.24 ± 0.01 [9h] p <0.01), but sham The relative MAP of the group remained almost unchanged during the study period. There was a non-significant but slow decrease in the relative MAP values in animals in the ETR-P1 / fl group, which was significantly higher than the CLP group at 6 and 9 hours (0.75 ± 0.10 vs. 0). 37 ± 0.03 [6h], 0.76 ± 0.11 vs. 0.24 ± 0.01 [9h]; both p <0.01) (FIG. 3A). There was no difference in relative MAP values between the ETR-P1 / fl group and the Siam group during the study period.
　６時間後の相対平均肺動脈圧（ｍＰＡＰ）値はＣＬＰ群ではシャム群より高かった：１．３０±０．０９（ＣＬＰ群）対０．９０±０．０８（シャム群）、ｐ＜０．０５（図３Ｂ）。９時間後においてもＣＬＰ群の相対ｍＰＡＰ値はシャム群より高かったが、その差は有意なものではなくなった。ＥＴＲ－Ｐ１／ｆｌ群の相対ｍＰＡＰ値はシャム群に近いものであったが、ＣＬＰ群との有意差はなかった。 Relative mean pulmonary artery pressure (mPAP) values after 6 hours were higher in the CLP group than in the Sham group: 1.30 ± 0.09 (CLP group) vs. 0.90 ± 0.08 (Sham group), p <0. 05 (FIG. 3B). Even after 9 hours, the relative mPAP value of the CLP group was higher than that of the Sham group, but the difference was not significant. The relative mPAP value of the ETR-P1 / fl group was close to that of the Sham group, but there was no significant difference from the CLP group.
　相対ｍＰＡＰ／ＭＡＰ値はＣＬＰ群で徐々に上昇し、９時間においてはシャム群並びにＥＴＲ－Ｐ１／ｆｌ群に比して有意に高かった：０．７３±０．０８（ＣＬＰ群）対０．４６±０．０７（シャム群）および０．５０±０．０５８（ＥＴＲ－Ｐ１／ｆｌ群）、両者ｐ＜０．０５（図３Ｃ）。ＥＴＲ－Ｐ１／ｆｌ群の相対ｍＰＡＰ／ＭＡＰ値はシャム群と近いものであった。 Relative mPAP / MAP values gradually increased in the CLP group and were significantly higher at 9 hours compared to the Sham group and the ETR-P1 / fl group: 0.73 ± 0.08 (CLP group) vs. 0. 46 ± 0.07 (sham group) and 0.50 ± 0.058 (ETR-P1 / fl group), both p <0.05 (FIG. 3C). The relative mPAP / MAP value of the ETR-P1 / fl group was close to that of the Sham group.
心拍数、拍出量並びに体温への作用
　ＣＬＰ群において、心拍数（ＨＲ）はＣＬＰ処置後３時間において増加した：１８４．８±１３．０拍／分［ＣＬＰ前］、２３７．８±１５．５拍／分［１ｈ］、２７２．２±１４．０拍／分［３ｈ］、それぞれｐ＜０．０５およびｐ＜０．０１。そして、ＣＬＰ処置後１時間並びに３時間のＣＬＰ群のＨＲはシャム群並びにＥＴＲ－Ｐ１／ｆｌ群より高かった：［１ｈ＝２３７．８±１５．５拍／分（ＣＬＰ）１８５．５±１１．１拍／分（シャム群）１７６．９±１５．８拍／分（ＥＴＲ－Ｐ１／ｆｌ群）］、［３ｈ＝２７２．２±１４．０拍／分（ＣＬＰ）２１０．３±１５．６拍／分（シャム群）、２１０．２±２２．２拍／分（ＥＴＲ－Ｐ１／ｆｌ群）］（全件ｐ＜０．０５）（図３Ｄ）。試験期間を通じてＥＴＲ－Ｐ１／ｆｌ群のＨＲはシャム群と近い値を維持していた。 Effects on heart rate, stroke volume and body temperature In the CLP group, heart rate (HR) increased at 3 hours after CLP treatment: 184.8 ± 13.0 beats / minute [before CLP], 237.8 ± 15 .5 beats / minute [1h], 272.2 ± 14.0 beats / minute [3h], p <0.05 and p <0.01, respectively. And the HR of the CLP group at 1 hour and 3 hours after CLP treatment was higher than that of the Sham group and the ETR-P1 / fl group: [1h = 237.8 ± 15.5 beats / minute (CLP) 185.5 ± 11 .1 beat / minute (sham group) 176.9 ± 15.8 beat / minute (ETR-P1 / fl group)], [3h = 272.2 ± 14.0 beat / minute (CLP) 210.3 ± 15 .6 beats / minute (sham group), 210.2 ± 22.2 beats / minute (ETR-P1 / fl group)] (all cases p <0.05) (FIG. 3D). Throughout the test period, the HR of the ETR-P1 / fl group maintained a value close to that of the Sham group.
　ＣＬＰ群において、相対拍出量（ＣＯ）は３、６及び９時間においてかなりの低下を示し、またシャム群より有意に低かった（表２）。ＥＴＲ－Ｐ１／ｆｌ群の相対拍出量は徐々に低下したが、ＥＴＲ－Ｐ１／ｆｌ群とシャム群の間の差は有意とはならなかった。 In the CLP group, the relative cardiac output (CO) showed a significant decrease at 3, 6 and 9 hours, and was significantly lower than the sham group (Table 2). Although the relative stroke volume of the ETR-P1 / fl group gradually decreased, the difference between the ETR-P1 / fl group and the sham group was not significant.
　試験期間全体において３群間で体温については差は無かった（表３）。 There was no difference in body temperature between the three groups during the entire test period (Table 3).
炎症性メディエーター
　ＣＬＰ群の血清ＴＮＦ－αレベルは急速に上昇し（１ｈ）、１、３、６および９時間の何れの時点においてもシャム群より高かった：１ｈ＝２５３．２±４４．２ｐｇ／ｍＬ（ＣＬＰ群）９．２±３．０ｐｇ／ｍＬ（シャム群）、３ｈ＝１８７．７±２６．６ｐｇ／ｍＬ（ＣＬＰ群）７．０±１．８ｐｇ／ｍＬ（シャム群）、６ｈ＝１３８．７±２５．４ｐｇ／ｍＬ（ＣＬＰ群）、１３．６±２．６ｐｇ／ｍＬ（シャム群）、９ｈ＝１２６．０±３１．９ｐｇ／ｍＬ（ＣＬＰ群）３１．７±８．２ｐｇ／ｍＬ（シャム群）（全件ｐ＜０．０１）。ＥＴＲ－Ｐ１／ｆｌ群においてはこれに対し、血清ＴＮＦ－αレベルはシャム群と同程度の値を保ち、１，３および６時間の時点でＣＬＰ群より低かった：ｈ＝±０．０ｐｇ／ｍＬ，３ｈ＝２±１６．１ｐｇ／ｍＬ，６ｈ＝３５．５±１９．５ｐｇ／ｍＬ（全件ｐ＜０．０１）（図４Ａ）。 Serum TNF-α levels in the inflammatory mediator CLP group rose rapidly (1 h) and were higher than those in the Sham group at any time point of 1, 3, 6, and 9 hours: 1 h = 253.2 ± 44.2 pg / mL (CLP group) 9.2 ± 3.0 pg / mL (sham group), 3h = 187.7 ± 26.6 pg / mL (CLP group) 7.0 ± 1.8 pg / mL (sham group), 6h = 138.7 ± 25.4 pg / mL (CLP group), 13.6 ± 2.6 pg / mL (sham group), 9h = 126.0 ± 31.9 pg / mL (CLP group) 31.7 ± 8.2 pg / ML (siamese group) (all cases p <0.01). In contrast, in the ETR-P1 / fl group, serum TNF-α levels remained comparable to those in the Sham group and were lower than those in the CLP group at 1, 3 and 6 hours: h = ± 0.0 pg / mL, 3h = 2 ± 16.1 pg / mL, 6h = 35.5 ± 19.5 pg / mL (all cases p <0.01) (FIG. 4A).
　ＨＭＧＢ－１はＣＬＰ群において３、６および９時間において検出可能な値を示し、ＥＴＲ－Ｐ１／ｆｌ群およびシャム群より有意に高かった（図４Ｂ）。血清ＨＭＧＢ－１はシャム群では全く検出されなかったが、ＥＴＲ－Ｐ１／ｆｌ群およびシャム群の間で血清ＨＭＧＢ－１レベルの有意差は無かった。 HMGB-1 showed detectable values at 3, 6 and 9 hours in the CLP group and was significantly higher than the ETR-P1 / fl group and the Siam group (FIG. 4B). Serum HMGB-1 was not detected at all in the Siam group, but there was no significant difference in serum HMGB-1 levels between the ETR-P1 / fl group and the Sham group.
　ＥＴＲ－Ｐ１／ｆｌ群とシャム群の血清ＮＯｘレベルは試験期間を通して同程度であった。両群において、試験開始前と同程度のＮＯｘレベルを保っていた。ＣＬＰ群のＮＯｘレベルは一方で、ＣＬＰ手術後１時間より増加しはじめ、３，６および９時間ではＥＴＲ－Ｐ１／ｆｌ群並びにシャム群より高かった（図４Ｃ）。 Serum NOx levels in the ETR-P1 / fl group and the Siam group were comparable throughout the study period. In both groups, the same level of NOx as before the start of the test was maintained. On the other hand, NOx levels in the CLP group began to increase from 1 hour after CLP surgery and were higher than those in the ETR-P1 / fl group and the Sham group at 3, 6 and 9 hours (FIG. 4C).
　１２時間から２４時間の間にはＥＴＲ－Ｐ１／ｆｌ群とシャム群の間で相対ＭＡＰ、相対ｍＰＡＰおよびｍＰＡＰ／ＭＡＰについての差は無かった（表３）。 There was no difference in relative MAP, relative mPAP and mPAP / MAP between the ETR-P1 / fl group and the Siam group between 12 hours and 24 hours (Table 3).
　実施例１および２の結果より本願発明の組成物がＳＩＲＳの処置に有用であることがわかる。また、本発明の組成物がゼプシスの早い時期に生じる炎症性メディエーターであるＴＮＦ－α、およびゼプシス後期に生じる炎症性メディエーターであるＨＭＧＢ－１の両方の上昇を抑える。敗血症患者において、血漿中ＴＮＦ－αの上昇は内毒素血症の重篤度と相関する。ＨＭＧＢ―１は核転写因子としてのみ知られていたタンパク質であるが、現在は遅延型エンドトキシン致死および全身性炎症と関連するメディエーターであると考えられている。ＨＭＧＢ－１レベルはＴＮＦ－αレベルと同様敗血症性ショックの重篤度と相関する。従って、ＨＭＧＢ－１のレベルの低下は敗血症の治療の有効性を測る指標となる From the results of Examples 1 and 2, it can be seen that the composition of the present invention is useful for treating SIRS. In addition, the composition of the present invention suppresses an increase in both TNF-α, which is an inflammatory mediator that occurs early in zepsis, and HMGB-1, which is an inflammatory mediator that occurs in the late stage of zepsis. In sepsis patients, elevated plasma TNF-α correlates with the severity of endotoxemia. HMGB-1 is a protein known only as a nuclear transcription factor, but is now thought to be a mediator associated with delayed endotoxin lethality and systemic inflammation. HMGB-1 levels correlate with the severity of septic shock as well as TNF-α levels. Therefore, a decrease in the level of HMGB-1 is an index for measuring the effectiveness of sepsis treatment.
　Ｈ５Ｎ1トリインフルエンザウイルス感染症に対するETR-P1/flの効果
　Ｈ５Ｎ1 トリインフルエンザウイルス感染症に対するETR-P1/fl並びにAcePepAの効果を調べた。AcePepAがサルや豚でＳＩＲＳに対する救命効果を発揮することについては、本願発明者らが先に報告している（特開2004-269520およびPCT/JP2009/061872 ）。
　105.3 TCID50のH5N1インフルエンザウイルス（H5N1 IV）０．２ｍｌを雄の１０日齢のニワトリのヒナ（体重約８０ｇ）へ経鼻感染させた。
　AcePepA投与群では、ウイルス感染３０分後、１日後及び２日後に該ペプチド（２ｍｇ／ｍｌ生理食塩水中）０．０８ｍｌを筋肉内投与した。
　ETR-P1/f1投与群（１）では、ウイルス感染３０分後、該ペプチド（０．２ｍｇ／ｍｌ生理食塩中）０．０８ｍｌを筋肉内投与した。
　ETR-P1/f1投与群（２）では、ウイルス感染３０分間後、１日後、及び２日後に該ペプチド（０．２ｍｇ／ｍｌ生理食塩中）０．０８ｍｌを筋肉内投与した。結果を表４に示す。 Effect of ETR-P1 / fl on H5N1 avian influenza virus infection The effects of ETR-P1 / fl and AcePepA on H5N1 avian influenza virus infection were examined. The inventors of the present application have previously reported that AcePepA exerts a lifesaving effect on SIRS in monkeys and pigs (JP 2004-269520 and PCT / JP2009 / 061872).
10 5.3 0.2 ml of TCID 50 H5N1 influenza virus (H5N1 IV) was intranasally infected into male 10-day-old chicks (body weight approximately 80 g).
In the AcePepA administration group, 0.08 ml of the peptide (2 mg / ml in physiological saline) was intramuscularly administered 30 minutes, 1 day, and 2 days after virus infection.
In the ETR-P1 / f1 administration group (1), 0.08 ml of the peptide (in 0.2 mg / ml physiological saline) was intramuscularly administered 30 minutes after virus infection.
In the ETR-P1 / f1 administration group (2), 0.08 ml of the peptide (in 0.2 mg / ml physiological saline) was intramuscularly administered 30 minutes, 1 day, and 2 days after virus infection. The results are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
 * H5N1 influenza virus (H5N1 IV) preparation at 105.3 TCID50 was the culture supernatant of MDCK cells infected in GIT medium.
Figure JPOXMLDOC01-appb-T000004
 * H5N1 influenza virus (H5N1 IV) preparation at 10 5.3 TCID 50 was the culture supernatant of MDCK cells infected in GIT medium.
　無処置群並びにAcePepA投与群では２日目までに全例が死亡した。これに対して、ETR-P1/f1投与群では初日のみに投与した場合も初日、１日、２日目に投与した場合も同様に全例が３日目まで生存していた。 In the untreated group and the AcePepA administration group, all cases died by the second day. On the other hand, in the ETR-P1 / f1 administration group, all cases survived until the third day in the same manner when administered only on the first day or when administered on the first day, the first day, and the second day.
　上記結果より、エンドセリン活性抑制剤であるETR-P1/f1が強毒性インフルエンザウイルスであるＨ５Ｎ１トリインフルエンザウイルス感染症に対して強力な治療効果を有することが示される。かかる治療効果は、高病原性鳥インフルエンザウイルスＨ５Ｎ１の感染拡大に対する防疫法としての用途も併せて示すものである。 The above results indicate that ETR-P1 / f1, which is an endothelin activity inhibitor, has a strong therapeutic effect against H5N1 avian influenza virus infection, which is a highly toxic influenza virus. Such a therapeutic effect also shows the use as a prevention method against the spread of highly pathogenic avian influenza virus H5N1 infection.

Claims (18)

  1. エンドセリン活性抑制剤を含む、ＳＩＲＳ処置用医薬組成物。 A pharmaceutical composition for treating SIRS, comprising an endothelin activity inhibitor.
  2. エンドセリン活性抑制剤が、エンドセリン－１の活性を抑制する、請求項１記載の組成物。 The composition according to claim 1, wherein the endothelin activity inhibitor inhibits the activity of endothelin-1.
  3. エンドセリン活性抑制剤が、
    ＡＬＳＶＤ(Ｘ)Ｙ(Ｘ)ＡＶＡＳ　（配列番号１０）
    （式中、Ｘはいずれかのアミノ酸を示す）
    のアミノ酸配列を有するペプチドである、請求項２記載の組成物。     Endothelin activity inhibitor
    ALSVD (X) Y (X) AVAS (SEQ ID NO: 10)
    (Wherein X represents any amino acid)
    The composition according to claim 2, which is a peptide having the amino acid sequence of
  4. エンドセリン活性抑制剤が、配列番号４～９および１１～１８何れかに記載のペプチドである、請求項２記載の組成物。 The composition according to claim 2, wherein the endothelin activity inhibitor is the peptide according to any of SEQ ID NOs: 4 to 9 and 11 to 18.
  5. エンドセリン活性抑制剤が、配列番号１１記載のペプチドである、請求項４記載の組成物。 The composition according to claim 4, wherein the endothelin activity inhibitor is a peptide represented by SEQ ID NO: 11.
  6. ＳＩＲＳがゼプシスである、請求項１～５何れかに記載の組成物。 The composition according to any one of claims 1 to 5, wherein SIRS is zepsis.
  7. ＳＩＲＳが強毒性インフルエンザウイルス感染に起因するものである、請求項１～５何れかに記載の組成物。 The composition according to any one of claims 1 to 5, wherein SIRS is caused by infection with a virulent influenza virus.
  8. 強毒性インフルエンザウイルスがＨ５Ｎ１トリインフルエンザウイルスである、請求項７記載の組成物。 8. The composition of claim 7, wherein the virulent influenza virus is H5N1 avian influenza virus.
  9. ヒトの処置に用いられる、請求項１～８何れかに記載の組成物。 The composition according to any one of claims 1 to 8, which is used for human treatment.
  10. 鳥類の処置に用いられる、請求項１～８何れかに記載の組成物。 The composition according to any one of claims 1 to 8, which is used for treatment of birds.
  11. エンドセリン活性抑制剤を含む、強毒性インフルエンザウイルス感染症処置用組成物。 A composition for treating highly toxic influenza virus infection, comprising an endothelin activity inhibitor.
  12. エンドセリン活性抑制剤が、エンドセリン－１の活性を抑制する、請求項１１記載の組成物。 The composition according to claim 11, wherein the endothelin activity inhibitor inhibits endothelin-1 activity.
  13. エンドセリン活性抑制剤が、
    ＡＬＳＶＤ(Ｘ)Ｙ(Ｘ)ＡＶＡＳ　（配列番号１０）
    （式中、Ｘはいずれかのアミノ酸を示す）
    のアミノ酸配列を有するペプチドである、請求項１１記載の組成物。     Endothelin activity inhibitor
    ALSVD (X) Y (X) AVAS (SEQ ID NO: 10)
    (Wherein X represents any amino acid)
    The composition according to claim 11, which is a peptide having the amino acid sequence of
  14. エンドセリン活性抑制剤が、配列番号４～９および１１～１８何れかに記載のペプチドである、請求項１１記載の組成物。 The composition according to claim 11, wherein the endothelin activity inhibitor is the peptide according to any one of SEQ ID NOs: 4 to 9 and 11 to 18.
  15. エンドセリン活性抑制剤が、配列番号１１記載のペプチドである、請求項１１記載の組成物。 The composition according to claim 11, wherein the endothelin activity inhibitor is a peptide represented by SEQ ID NO: 11.
  16. 強毒性インフルエンザウイルスがＨ５Ｎ１トリインフルエンザウイルスである、請求項１１～１５何れかに記載の組成物。 The composition according to any one of claims 11 to 15, wherein the virulent influenza virus is H5N1 avian influenza virus.
  17. 鳥類を処置するためのものである、請求項１１～１６何れかに記載の組成物。 The composition according to any one of claims 11 to 16, which is for treating birds.
  18. ヒトを処置するためのものである、請求項１１～１６何れかに記載の組成物。 The composition according to any one of claims 11 to 16, which is for treating a human.
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