MXPA96004207A - Novedous remedy for tractor pyrrato viral disease - Google Patents

Abstract

The present invention relates to a therapeutic or prophylactic agent for viral diseases whose active ingredient is antileucoprotease (ALP). Formulations containing ALP as an active ingredient have a particularly effective application against viral diseases, particularly viral diseases caused by viruses that are activated by clear tryptase, i.e., viruses with wall glycoproteins, which replicate by tract infection Respiratory, such as influenza virus, parainfluenza virus, RS virus, measles virus and paper virus

Description

NOVEDOUS REMEDY FOR VIRAL DISEASE OF THE RESPIRATORY TRACT FIELD OF THE INVENTION The present invention relates to a therapeutic or prophylactic agent for viral diseases whose active ingredient is antileucoprotease (hereinafter referred to as ALP). More specifically, the present invention relates to a therapeutic or prophylactic agent containing ALP as the active ingredient for viral diseases of the respiratory tract and caused by viruses that are activated by clear tryptase. BACKGROUND OF THE INVENTION ALP is present in external secretions such as bronchial mucus, saliva, seminal fluid, cervical mucus and nasal discharges, and is an inhibitor of serine protease with a molecular mass of 12 kDa, consisting of 107 amino acid residues. , which is considered to be the same substance as the secretory leukoprotease inhibitor (ILPS), bronchial mucus inhibitor, mucosal protease inhibitor and human seminal inhibitor. The amino acid sequence of ALP has been determined (Publication No. WO86 / 03497), and the protein gene has been isolated and sequenced (Publication No. O86 / 03519).

From homologous comparisons, it is known that ALP comprises two inhibitory fields. One is an N-terminal field that is postulated to inhibit several enzymes similar to trypsin, and the other is a C-terminal field from which by investigating crystal structure analysis by X-rays it is believed that it binds with the chymotrypsin and has an inhibitory activity of elastase. [M.G. Gruetter, The EMBO. Journal, Vol. 7, No. 2, pp. 345-351 (1988)]. Since ALP inhibits chymotrypsin-like enzymes such as leucoqito elastase and cathepsin G and also inhibits trypsin-like enzymes, such as trypsin, plasmin, kallikrein and thrombin, etc., it is known to be related to emphysema , arthritis, glomerulonephritis, periodontitis, muscle atrophy, tumor invasion (Publication No. WO86 / 03497), chronic bronchitis, and chronic cervical inflammation (Japanese Unexamined Patent Publication No. 62-259591). However, the role of ALP in viral diseases is still unknown. However, the role of ALP in viral diseases is still unknown. Viral infections occur through the stages of 1) binding of a virus to the membrane receptors in the target cells, 2) fusion of the membrane between the virus wall and the target cell membrane,? M and 3) transfusion of the viral genome into target cells. In step 2) of the membrane fusion, the glycoprotein precursors of the viral wall have to be converted to the mature form of the glycoproteins of the virus wall with a membrane fusion activity. After this conversion by means of proteolysis, the virus acquires membrane fusion activity between the walls of the virus and the membranes of the cell in the respiratory tract. The hemagglutinin (HA) of the influenza virus and CF of the 10 Sendai virus (parainfluenza paramyxovirus virus type I) are the precursors of the glycoprotein of the wall, and its proteolytic cleavage is essential for the expression of viral infectivity and for the viral replication. The present inventors have been successful in the isolation of a novel specific arginine serine protease from rat lungs, which has been H? ^. called "clear triptase" (The Journal of Biological Chemistry, Vol. 267, pp. 13573-13579, 1992). Clear tryptase separates FQ from the Sendai virus in two subunits F ^ _ and F2, and activates the infectivity of the virus in vitro in a dose-dependent manner. In addition, antibodies against clear tryptase are known to inhibit the proteolytic activation of Sendai virus in rat lungs, resulting in the suppression of viral replication and pathological changes in rat lungs (Journal of Virology, Vol. 66, pp. 7211-7216, 1992). In addition, clear tryptase also separates HA from influenza A / Aichi / 2/68 (H3N2) virus in HA-L and HA2 (The Journal of Biological Chemistry, Vol. 267, pp. 13573-13579, 1992). Taken together, it is believed that clear tryptase is a major host factor that determines the pathogenicity of such contagious viruses in the respiratory tract. The present inventors previously discovered that the pulmonary surfactant inhibits cleavage by the clear tryptase of the glycoprotein precursors of the virus wall, and therefore blocks viral infection of the bronchial mucosal epithelial cells and viral replication (Publication No. H ^ 'WO94 / 00181, Letters FEBS, 322, 115-119 (1993)): DESCRIPTION OF THE INVENTION As a result of continued investigation 20 by the present inventors, with the purpose of finding a substance that inhibits activation of viruses by clear tryptase, it was found that ALP is capable of markedly inhibiting the viral activation of clear tryptase and therefore inhibits the replication of viruses in virus-infected animals, and thus completing the present invention. The present invention provides a therapeutic and prophylactic agent for viral diseases containing ALP as the active ingredient thereof. The ALP used in accordance with the invention includes not only ALP isolated and purified from natural sources and ALP produced by genetic engineering methods (for example, by the methods described in the Publications of Patent O86 / 03519, WO89 / 06239, Japanese Unexamined Patent Publication No. 62-259591, No. 3-123490, etc.) but also proteins consisting of ALP with a substitution, deletion, insertion or addition in certain portion of the amino acid sequence, which presents the same activity as ALP. Viral diseases that must be attacked with the * ^ w ^. therapeutic or prophylactic agent of the invention, include viral diseases caused by influenza virus, paramyxovirus, respiratory syncytial virus (from hereinafter, "RS virus"), rhinovirus, coronavirus, reovirus, adenovirus, Coxsackie virus, ecovirus, herpes simplex virus, rotavirus, enterovirus, polio virus, cytomegalovirus, varicella zoster virus and HIV, but are preferably viral diseases in which the infection in the respiratory tract due to viruses triggered by clear tryptase, i.e., virus with wall, such as influenza virus, paramyxovirus, RS virus, measles virus and mumps virus. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibitory effect of ALP on the cleavage of the glycoprotein precursor of the Sendai FQ virus wall of clear tryptase. Figure 2 shows the inhibitory effect of ALP in the cleavage of the wall glycoprotein precursor of the HA influenza virus of clear tryptase. Figure 3 shows the inhibitory effect of ALP on infection with Sendai virus and influenza virus in cultured cells. The experiment of the infection with the Sendai virus is shown in, "A" and that of the influenza virus in "B". Figure 4 shows the effect of ALP on rats infected with influenza virus (influenza virus adapted to mouse A / Asia / l / 57 (H2N2) .The virus titer in the lung is shown in "A "and the extent of the damage in the lung "B". In "A", the solid line represents the virus title in the absence of ALP, and the dotted line represents the same virus title in the presence of ALP, while the open circles represent the total virus titer and the closed circles represent the title of the active virus. HE administered ALP at the points indicated by the arrows.

F? BEST MODALITY FOR CARRYING OUT THE INVENTION The effect of ALP on viral diseases will be described below. The following ALP, virus and triptases were used in each of the "experiments." 5 ALP Natural ALP was used, prepared in accordance with the method described in Japanese Unexamined Patent Publication No. 62-259591. Virus 10 The viruses used were Sendai virus and influenza virus [Influenza virus A / Aichi / 2/68 (H3N2) and influenza virus adapted to mouse A / Asia / l / 57 (H2N2)]. Sendai and influenza viruses cultured in the amniotic cavity of developing chicken eggs were suspended 15 in a calcium-free phosphate buffer solution in A ratio of 254 HAU / ml (haemagglutination unit / ml). Clear Tryptase Clear tryptase was prepared from rat lungs, in accordance with the Kido method (The Journal of 20 Biological Chemistry, Vol. 267, pp. 13573-13579, 1992). The rat lungs were washed with physiological saline and then minced with scissors, homogenized at a pH of 5.5, and centrifuged to obtain a supernatant solution which is used as the extract. The crude extract was then subjected to ion exchange column chromatography on a CM-52 cellulose column (trade name) and a Sephadex CM-52 column (trade name), and the fractions were collected with an activity measured with Boc-Gln -Ala-Arg-MCA 5 as a substrate. The active fractions collected were subjected to an affinity chromatography on an arginine-Sepharose column (trade name) which is a specific serine protease adsorbent. The enzyme similar to trypsin in the eluate was collected by specific adsorption. Finally, This enzyme solution was subjected to a gel filtration column and the active fractions were collected for isolation and purification to prepare clear tryptase. Test of the Inhibition of the Splitting of Viral Protein by ALP (Example 1 - Sendai Virus) The test was carried out in accordance with the method described in The Journal of Virology, Vol. 7211-7216, 1992. 20 Clear tryptase (50 μg / ml) and various concentrations of ALP (10 nM, 100 nM and 1 μM), dissolved in distilled water were pre-incubated for 5 minutes at 0 ° C, and then the unfolding of the protein FQ of the inactive Sendai virus labeled with glucosamine [^ H] was determined cultured in LLC-MK2 cells. The results of the analysis by i Wfi sodium dodecyl sulfate-polyacrylamide electrophoresis are shown in Figure 1. It was found that ALP inhibits cleavage of CF by the clear tryptase in the subunits F- | _ and F2 5 a dose-dependent manner, with a 100% inhibition at 1 μM. (Example 2 - Influenza Virus) The unfolding of the HA protein of the influenza virus (influenza A / Aichi / 2/68 virus ( H3N2)) in "A- ^ 10 and HA2 by the clear tryptase was investigated in accordance with the method described in The Journal of Biological Chemistry, Vol. 267, pp.13573-13579, 1992 and [Example 1]. is illustrated in Figure 2, ALP inhibits the unfolding of HA in the HA- ^ and HA2 subunits of clear tryptase in a dose-dependent manner.

ALP (in vitro) (Example 3 - Sendai Virus) 20 This test was carried out according to the method described in the publication W094 / 00131. Clear triptase (20 μg / ml) was preincubated, with various concentrations (0.1, , 100 and 1000 nM) of ALP in saline at 0-C for 20 minutes. Afterwards, inactive cultivated Sendai virus was treated in LLC-MK2 cells with the reaction mixture at 37 ° C for 5 minutes. The reaction was over. with aprotmma (100 μg / ml). The active Sendai virus obtained with this treatment was added again to the LLC-MK cells, which were then cultured for 15 hours, after which the UIC (Cell Infecting Unit) was measured by the immunofluorescence cell counting method. Viral infectivity was expressed as a UIC titer (log10UIC / ml). (Example 4 - Influenza virus: Influenza virus 10 A / Aichi / 2/68 (H3N2)). Treatment, measurement of UIC and determination of viral infectivity titer were carried out in the same manner as in Example 3, except that influenza virus (influenza virus A / Aichi / 2/68 (H3N2)) was used in place of Sendai virus and MDCK cells were used instead of LLC-MK2 cells. Figure 3 shows the inhibitory effect of ALP on infection with Sendai virus and influenza virus.

Inactivated viruses before treatment with clear tryptase 20 were activated (1 x 10 4 UlC / ml and 1.2 x 10 4 UlC / ml, respectively) with clear tryptase up to 4 x 10 6 UlC / ml and 1. 8 x 106 UlC / ml, respectively. ALP reduced both viral infectivities by addition of ALP (1 nM - 1 μM) in a dose dependent manner and inhibited the cleavage of the glycoprotein from the viral mL wall by the clear tryptase. Almost 100% inhibitory effect of infection was observed with ALP at 100 nM - 1 μM. Test of inhibitory effect of ALP on viral infection (in vivo) 5 (Example 5 - Experiments with Animals on the anti-viral effect) ALP influenza) The test was carried out in accordance with the method described in Journal of Virology, Vol. 66, pp. 7211-M 7216, 1992. 10 SD rats (3 weeks of age, body weight: 120 g, product of Japan Charles River Co.) were specifically infected intranasally with 1 x 10 4 plaque-forming units (PFU) of the plague virus. influenza (influenza virus adapted to mouse A / Asia / l / 57 (H2N2)). ALP was administered intranasally at 6 μg (50 μl) per rat once every 8 hours, after infection for a total of 15 times. The control was administered 50 μl of physiological saline solution. At the indicated time, three rats were sacrificed every 24 hours, and the titer of the total virus 20 and of the active virus were measured in the lung homogenates. The degree of inflammation of the total lung was expressed as a lung injury score based on visual observation. The lung injury score was a score of 5 levels from 0 to 4 representing the proportion of hepatization, that is, brown areas, with respect to the total area of the lung. For lung injury score, 0 indicates 0% heptization with respect to the total lung surface, 1 indicates 1-25%, 2 indicates 26-50%, 3 indicates 51-75% and 4 indicates 76-100% . 5 The results are summarized in Figure 4. Rats infected with influenza virus (influenza virus adapted to mouse A / Asia / l / 57 (H2N2)) and which were administered physiological saline wßfr exhibited a increase in the virus title of approximately 2,000 times on the fifth day of which almost 95% was active virus. Severe inflammation of the lung was observed, a maximum was reached for the seventh day and continued until the ninth day. In contrast, the rats to which they were administered ALP intranasally at 6 μg / rat for a total of 15 times exhibited only a small increase in the w ¥ virus titer of approximately 10 times by the fifth day, which represented an inhibition of the virus titer to a simple 0. 5% of the observed with the group not treated with ALP. Further, 95% of this slightly increased virus remained inactive. Inflammation of the lung reached a maximum on the fifth day with a score of 1, showing only a minimal advance. Therefore, ALP clearly inhibits the increase of lung lesions due to viral infection, demonstrating its effectiveness against the growth of viral infection. Method and Dosage A single dose of the therapeutic agent or Prophylactic for viral diseases according to the invention contains ALP at a concentration of 0.1 μg - 500 mg, preferably 1 μg - 100 mg and more preferably 10 μg - 10 mg for children and 0.5 μg - 1000 mg, ^ * preference 5 μg - 500 mg and more preferably 50 μg - 10 50 mg for adults. The dose is dissolved in an electrolytic solution such as aqueous or physiological saline solution, the concentration is adjusted to 0.1-500 mg / ml, preferably 0.5-200 mg / ml and more preferably 1-100 mg / ml, and infused or it is nebulized in the respiratory tract, or it is used in the form of a collusive agent, either before the viral infection or after the outbreak of the viral disease. The therapeutic or prophylactic agent of the invention may contain, if necessary, pharmaceutical additives such as stabilizers, preservatives, isotonic agents, buffering agents or suspending agents, or agents. Pharmaceuticals, such as bronchodilators, antitussive agents, antiallergic agents, antipyretic analgesics, antibiotics, synthetic antibacterial agents or other agents antivirals. The preparation may be a liquid form, or a powder or aerosol preparation that is suspended at the time of use and may be presented in a container, vial or other sealed container such as an antiseptic preparation. 5 Industrial Applicability Therefore, ALP notably inhibits the activation of viruses by clear tryptase and thus prevents viral replication in animals infected with these viruses. Consequently, the formulations that contain ALP as an effective ingredient have an effective application for diseases, particularly viral diseases caused by viruses that are activated by clear tryptase, i.e., viruses with wall glycoproteins, which replicate by respiratory tract infection, such as influenza viruses, parainfluenza virus, RS virus, measles virus and mumps virus.

Claims (13)

1. ^ CLAIMS 1. A clear tryptase inhibitor, characterized in that it contains antileucoprotease as an active ingredient.
2. 2. A therapeutic or prophylactic agent for 5 viral diseases characterized in that it contains antileucoprotease as an active ingredient.
3. 3. The therapeutic or prophylactic agent for viral diseases according to claim 2, characterized in that the virus is a virus with a 10 wall glycoprotein that replicates through the infection of the respiratory tract.
4. 4. The therapeutic or prophylactic agent for viral diseases according to claim 2 or claim 3, characterized in that the virus is a virus 15 which is activated by clear tryptase.
5. 5. The therapeutic or prophylactic agent for t * á * w viral diseases according to claim 2, claim 3 or claim 4, characterized in that the virus is influenza virus, parainfluenza virus, RS virus, measles virus, or mumps virus.
6. 6. A therapeutic method for viral diseases, characterized in that the patient is administered a medically effective dose of antileucoprotease.
7. 7. The therapeutic method according to claim 6, characterized in that the virus is a virus with a wall glycoprotein, which is replicated through infection of the respiratory tract.
8. 8. The therapeutic method according to claim 6 or claim 7, characterized in that the 5 virus is a virus that is activated by clear tryptase.
9. 9. The therapeutic method according to claim 6, claim 7 or claim 8, characterized in that the virus is influenza virus, parainfluenza virus, RS virus, measles virus or mumps virus.
10. 10. The use of antileucoprotease for the production of a composition of a therapeutic agent for viral diseases.
11. 11. The use according to claim 5, characterized in that the virus is a virus with a wall glycoprotein, which is replicated through respiratory tract infection.
12. 12. The use according to claim 10, or claim 11, characterized in that the virus is a virus that is activated by clear tryptase.
13. 13. The use according to claim 10, claim 11, or claim 12, characterized in that the virus is influenza virus, parainfluenza virus, RS virus, measles virus or mumps virus.