JP5686734B2 - Pharmaceutical composition for treatment of SIRS or highly toxic influenza infection - Google Patents

Description

  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.

  Systemic inflammatory response syndrome (SIRS), including zepsis, is a fatal disease responsible for the death of 300,000 patients 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.

  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 SIRS of C5a anaphylatoxin, a 74-amino acid peptide released by C5 convertase generated during complement activation from the fifth component of complement (C5) has been proposed (Non-Patent Documents 1 and 2). ).

  Here, amino acids 37 to 53 (RAARISLGPRCIKAFTE) (SEQ ID NO: 1) of C5a previously published by the present inventors are the 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 (Non-Patent Documents 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.

  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.

  Influenza viruses infect 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.

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, an A-type receptor specific to endothelin-1 and a non-specific B-type receptor 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.

  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

J. Immunol. 172, 6382-6387 (2004) Immunopharmacology, 38: 3-15 (1997) Nature Med. 1, 894-901 (1995) J. Immunol. 157, 4591-4601 (1996) Neuroscience 86, 903-911 (1998) Microbiol. Immunol. 46, 211-215 (2002) Biochem. Biophys. Res. Comm. 324, 236-240 (2004) Microbiol. Immunol. 47, 241-245 (2003) J. Immunol. 172, 6382-6387 (2004)

  The present invention aims 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 treating SIRS, which contains an endothelin activity inhibitor.

  The present invention also provides a pharmaceutical composition for the treatment of highly virulent influenza virus infections, such as highly pathogenic avian influenza infections, comprising an endothelin activity inhibitor.

  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.

  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.

  In the present invention, the endothelin activity inhibitor is not particularly limited, but peptides of SEQ ID NOs: 4 to 18 in the sequence listing, particularly peptides described in SEQ ID NO: 11 are preferably used.

  With the pharmaceutical composition of the present invention, it is possible to effectively treat SIRS as well as highly virulent influenza virus infection.

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 MAP (Mean Arterial Pressure) ratio of an ETR-P1 / fl group ((triangle | delta)), a CLP group ((circle)), and a sham group ((circle)) 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. The change of mPAP ratio of ETR-P1 / fl group ((triangle | delta)), CLP group (-), and sham group ((circle)) is shown. The change of the heart rate (HR) of an ETR-P1 / fl group ((triangle | delta)), a CLP group (-), and a sham group ((circle)) is 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 mPAP / MAP ratio of an ETR-P1 / fl group ((triangle | delta)), a CLP group (-), and a sham group ((circle)) is shown. mPAP / MAP did not increase in the ETR-P1 / fl group at 9 hours. The change of the TNF- (alpha) level in the serum of an ETR-P1 / fl group ((triangle | delta)), a CLP group (-), and a sham group ((circle)) is shown. The change of the amount of HMGB-1 in the serum of an ETR-P1 / fl group ((triangle | delta)), a CLP group (-), and a sham group ((circle)) is shown. 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.

  In the pharmaceutical composition of the present invention, the endothelin activity inhibitor may be anything that inhibits the activity of endothelin-1, more preferably one that inhibits the action of endothelin-1 via the endothelin receptor type A. It is not particularly limited.

  EP-A-552489, EP-A-528312, EP-A-499266, WO91 / 13089, EP-A-436189, EP-A-457195, EP-A-510526 as compounds that antagonize endothelin and endothelin receptor. , WO92 / 12991, JP-A-4-288099, JP-A-4-244097, JP-A-4-261198, EP-A-496445, 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. Further, as a substance having an antagonistic action on endothelin receptor, particularly endothelin receptor A type, bosentan, sitaxsentan, ambrisentan, atrasentan, S-139, tezosentan BMS-193884, compounds such as drausentan and entrasentan are known.

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)

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: VLNLCALSVDRYRAVASWSRVI-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 are 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, cysteines at the 1st and 15th positions form a disulfide bond.
C18: VALSVDRYRAVASW-NH ₂ (SEQ ID NO: 18)

  The peptides C1-6 and C11-18 all inhibit endothelin-1 action in any of the tests of endothelin-1 in mouse 3T3 intracellular calcium ion concentration and endothelin-1 in rat aortic smooth muscle vasoconstriction (Japanese Patent No. 3923515).

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.

  In the present invention, the 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.

  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.

  The dose of the pharmaceutical composition of the present invention is not limited, and may be appropriately determined depending on the age, weight, sex, symptom, desired effect, etc. of the patient.

  The pharmaceutical composition of the present invention is suitably used for the treatment of SIRS.

  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 various organ damages are induced by inflammation. “SIRS” , In the United States is defined as exhibiting symptoms that meet the following diagnostic criteria, and this specification shall also be followed:

  Among the pathological conditions diagnosed as SIRS, those caused by infection 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).

  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.

  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.

  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.

  The pharmaceutical composition of the present invention is useful for the treatment of a patient who is infected with a virulent influenza virus, or a patient 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. 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.

The invention is explained in more detail by means of examples.
Peptides used:
Endothelin activity inhibitor ETR-P1 / f1
VLNLCALSVSVRYRAVASWSRVI-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)).

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.

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.

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.

  Newborn pigs were raised with mother pigs and were isolated only during treatment. Newborn pigs weighed between 1450-2200 g and the median was 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. A 4-Fr Bellman angiographic catheter (American Edwards Laboratories, Irvine, Calif., USA) was inserted through the fluoroscopic right lateral vein and placed in the main pulmonary artery for measurement of mPAP. 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 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.

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.

  Each animal was observed until death or 24 hours. Animals that survived 24 hours were killed by administering a lethal dose of sodium phenobarbital.

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.

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).

Measurement of stroke volume M-mode echocardiography was performed using a SONOS 500 (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.

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).

Result Each measurement result is shown in Table 2, Table 3, and FIGS.

Animal status and survival time There was no significant difference in body weight between the CLP group, the Sham group, and the ETR-P1 / fl group, which were 1646 ± 88 g, 1718 ± 50 g, and 1850 ± 106 g, respectively.

  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.

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 of 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).

  Arterial excess base (BE) values in the CLP group and the ETR-P1 / fl group decreased. Although the BE level of the ETR-P1 / fl group was maintained higher than that of the CLP group, the difference was not significant (Table 2).

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. A non-significant but slow decrease in relative MAP values in animals in the ETR-P1 / fl group was observed and 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 test period.

  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 Siam group, but there was no significant difference from the CLP group.

  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.

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.

  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.

  There was no difference in body temperature between the three groups during the entire study period (Table 3).

Serum TNF-α levels in the inflammatory mediator CLP group rose rapidly (1 h) and were higher than 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 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 Sham 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 and Sham groups.

  Serum NOx levels in the ETR-P1 / fl group and the Sham 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).

  There were no differences in relative MAP, relative mPAP and mPAP / MAP between ETR-P1 / fl and Sham groups between 12 and 24 hours (Table 3).

  The results of Examples 1 and 2 show 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 late 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.

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.

* H5N1 influenza virus (H5N1 IV) preparation at 10^5.3 TCID₅₀ was the culture supernatant of MDCK cells infected in GIT medium.

* H5N1 influenza virus (H5N1 IV) preparation at 10 ^5.3 TCID ₅₀ was the culture supernatant of MDCK cells infected in GIT medium.

  All patients died by day 2 in the untreated group and the AcePepA-administered group. 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.

  From the above results, it is shown 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 (6)

ALSVD (X) Y (X) AVAS (SEQ ID NO: 10)
(Wherein X represents any amino acid)
A composition for treating highly virulent influenza virus infection, comprising a peptide having the amino acid sequence of: The composition according to claim 1, wherein the peptide is the peptide according to any one of SEQ ID NOs: 4-9 and 11-18. The composition according to claim 1, wherein the peptide is the peptide represented by SEQ ID NO: 11. The composition according to any one of claims 1 to 3, wherein the virulent influenza virus is H5N1 avian influenza virus. The composition according to any one of claims 1 to 4, which is for treating birds. The composition according to any one of claims 1 to 4, which is for treating a human.

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