JP3061376B2 - Influenza virus infection protective agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a protective agent for influenza virus infection containing lactoferrin as an active ingredient.

[0002]

2. Description of the Related Art Viral diseases are one of the biggest problems left in the medical field at present. Although a number of studies have been conducted on antiviral drugs, treatment with drugs has been difficult because the virus multiplies by the proliferation function of cells. So far, what has been recognized as a drug showing a therapeutic effect on viral diseases, there is only encephalitis, acyclovir and vidarabine for strip rash amantadine, herpes simplex virus against influenza A ₂ type. Of these, amantadine has not been approved for use as an antiviral agent in Japan.

[0003] Recently, infections caused by HIV, so-called AIDS, have become a problem, and a large number of compounds have been subjected to screening. As a result, azidothymidine (AZT) has been developed.
Has been shown to have a life-prolonging effect on AIDS-infected individuals and has been used to treat HIV-infected individuals. However, these drugs are expensive, have strong side effects, have a limited therapeutic spectrum, and still have problems as antiviral agents.

On the other hand, interferon has recently attracted attention as an antiviral agent. Interferon was discovered in 1957 and has been studied as a substance that protects the virus from infecting cells. Interferon can be obtained by culturing leukocytes or fibroblasts, but recently, mass production by genetic recombination has become possible. As a method of using interferon as an antiviral agent, there have been reports of the application of nasal administration to the treatment of respiratory infections, for example, influenza, but the metabolism and dynamics in the body are unknown, The effectiveness has not yet been confirmed. Also, although mass production by genetic recombination has become possible, the production cost of interferon is expensive, and it is still expensive for use in the treatment of common viral diseases such as influenza and the use for protection against infection.

[0005] At present, administration of vaccines for preventing infection is the most widespread measure against viral diseases. These include live vaccines in which each virus is attenuated by some method, inactivated vaccines prepared by formalin treatment of viruses, and component vaccines in which only the antigen portion of the virus is purified. These vaccines have prevented most diseases. However, taking influenza, which is the most typical viral disease, as an example, it is difficult to prevent infection with a vaccine. Influenza virus has an antigen in a part called the envelope on the virus surface and uses this antigen as a vaccine, but this antigen part is often mutated. It is clear that no effect is exhibited. In addition, it is difficult to prevent infection with a vaccine, such as a virus such as HIV, for which the antigen as a vaccine is unknown, or a cytomegalovirus infection that often occurs when the immune function is reduced by administration of an immunosuppressant after organ transplantation. is there.

In recent years, research on virology has progressed, and it has become clear that in the case of virus infection, the virus binds to a virus receptor present on the cell surface and the virus enters the cell from this portion. Eg, HIV binds to CD ₄ receptor present on the surface of T ₄ lymphocytes. By administering Therefore CD ₄ in large quantities in blood, it is said that it is possible to prevent the onset of AIDS. Thus, research on viruses and virus receptors indicates the potential for the development of new virus therapeutics.

[0007] The influenza virus has an enzyme protein called hemagglutinin that agglutinates red blood cells on the surface of the virus. This hemagglutinin is an important factor determining viral antigens. The hemagglutinin general is, H _1, H _2, although H ₃ and three types are known, often this type is mutated. This reduces the effectiveness of the vaccine. When influenza infects cells, hemagglutinin recognizes and binds to sialic acid-linked sugar chains present on the cell membrane surface and starts invasion into cells. Can be evaluated as Therefore, a substance having a sugar chain structure that inhibits hemagglutination can inhibit the binding of influenza virus to cells, prevent infection, and prevent the spread of the influenza virus to other cells after infection.

The present inventors have already applied for a patent by obtaining a substance that protects against influenza virus infection, as disclosed in Japanese Patent Application Laid-Open No. 63-284133. In addition, Yamakawa et al. Isolated a substance that inhibits the hemagglutination of influenza virus from human blood cells and clarified that it is a protein having sialic acid as a constituent sugar (Yamakawa et al., “Biochemistry” 31). Volumes 416-421, 1959). But,
The regulation of viral receptor binding inhibition and glycoproteins has not yet been elucidated.

[0009]

As described above, important factors in protecting against virus infection include a cell surface receptor to which the virus binds and a substance that antagonizes it. In the case of influenza virus, various receptors have been identified. "Protein / Nucleic Acid / Enzyme vol. 32, pp. 117-135, 1988" discloses hemagglutinin and receptors. According to these, a certain rule cannot always be found for the sugar chain structures of glycoproteins and glycolipids and the role of receptors. The present invention have conducted intensive studies for infection defense virus, lactoferrin having a sialic acid and mannose sugar chain structure antagonize the receptor of various influenza Wie <br/> Angeles, preventing influenza virus infection I found to do. Accordingly, the present invention is La
It is an object to provide an influenza virus infection protective agent containing ctoferrin as an active ingredient.

[0010]

Means for Solving the Problems The feature of the present invention, inflation
It is to use lactoferrin as a protective agent for Luenza virus infection. Lactoferrin is generally an iron-binding protein isolated from mammalian milk, but in the practice of the present invention may be of any species or origin. If necessary, lactoferrin produced by genetic recombination can also be used. Current,
It is assumed that most inexpensive and readily available are those isolated from milk. When separating from milk,
A method using an anti-lactoferrin antibody disclosed in JP-A 61-145200 can be adopted.

Ovotransferrin is a glycoprotein with a molecular weight of about 77,000 to 87,000 contained in chicken egg white and is an iron-binding protein very similar to lactoferrin. In order to obtain ovotransferrin, known purification such as chromatography can be used. For example, the method using carboxymethylcellulose (Gallian et al.
Of Food Science, Vol. 45, p. 460, 1980), a method using metal-immobilized affinity chromatography (Alumashiki et al., Agricultural Biological Chemistry, Vol. 51, pp. 2881-2887, 1987) Can be adopted. Iron-binding proteins such as lactoferrin and ovotransferrin obtained as described above, which are partially hydrolyzed with a protease, can also be used in the present invention. In the case of enzymatic hydrolysis of lactoferrin and ovotransferrin, it is preferable to keep the degradation rate at 60% or less in order to maintain the ability to protect against virus infection and to exert the effect of enzymatic degradation.

Lactoferrin influenza as described above
Use as c-virus infection protective agent can be administered orally, transdermally, are possible administration of injections such, it is possible to formulate in accordance with each route of administration. Although it is shown as a reference, in the case of using lactoferrin cytomegalovirus infection or therapeutic purposes virus (CMV), dosing the composition 1g
Effectively, it is necessary that an amount of 0.1 μg or more is present. When lactoferrin is sterilized, 0.45
It can be sterilized by filtration with a membrane filter of μm or less. Lactoferrin, an iron-binding protein according to the present invention, is contained in milk and its safety has been confirmed. Next, a description will be given of a test example using a reaction (HI) for inhibiting hemagglutination caused by influenza virus, which shows the protective effect against influenza virus infection of the present invention.

[0013]

[Test Example 1] Injection with lactoferrin and ovotransferrin
Fluenza virus HI activity assay The following viruses were targeted as influenza viruses. Inactivated influenza virus obtained from Denka Seiken, A / Yamagata _{/ 12/86 (H 1 N} 1), A / Niigata / 102/81
(H ₃ N ₂ ), A / Sichuan / α / 87 / (H ₃ N ₂ ), A / Fukuoka / C29 / 8
5 (H ₃ N ₂ ), B / Nagasaki / 1/87, B / Singapore / 222
/ 79, and uninactivated influenza viruses A / PR / 8/34 (H ₁ N ₁ ) and A / Aichi / 2/68 (H ₃ N ₂ ), which were assigned by Shizuoka Pharmaceutical University.

Chick stabilized erythrocytes of these viruses
(HITACHI) activity against Takeda Pharmaceutical Co., Ltd. was measured. The HI activity was measured according to the method of Yamakawa et al. ("Biochemistry", vol. 31, p. 416-421). The samples used for measurement were bovine lactoferrin (bLf), human lactoferrin (hLf), goat lactoferrin (gLf), ovotransferrin (oTf), and these with trypsin.
Enzymatically degraded at 20%, 40% and 60% degradation rates.
The results were as shown in Table 1.

[0015]

[Table 1]

As shown in Table 1, all the samples showed strong HI activity. A / Yamagata / 120/86 did not agglutinate chick red blood cells. In addition, (A) of Table 1 targets chick-stabilized erythrocytes, and (B) targets human O-type erythrocytes.

[0017]

[Test Example 2] To erythrocytes of bLf, hLf, gLf, oTf and their enzymatic degradation products
Each sample used in the presence confirmation test in Test Example 1 of nonspecific adsorption was dissolved in physiological saline,
It was adjusted to a concentration of 0.5% (w / v). Test Example 1 was added to this solution.
The human O-type erythrocytes or chick-stabilized erythrocytes prepared in the above were added and suspended so that the erythrocyte concentration was 1% (v / v) or 10%. This was left at room temperature for 1 hour with occasional stirring, and then centrifuged at 1500 rpm for 10 minutes.
HI activity was measured in the same procedure as in Test Example 1. As shown in Table 2, no change was observed in the HI activity of each sample even after such treatment.

[0018]

[Table 2]

From the results of Test Examples 1 and 2, the iron-binding protein and its enzymatic degradation product inhibit erythrocyte agglutination by virus, and the effect is not caused by non-specific adsorption to erythrocytes. However, it was presumed to be caused by specific affinity between the virus hemagglutinin and each active ingredient. Furthermore, it was confirmed that the affinity between the iron-binding protein and the virus was not affected by the mutation of the virus antigen. Note that (A) in the table targets chick-stabilized erythrocytes, and (B) targets human O-type erythrocytes.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Preparation of bLf : Bovine lactoferrin (bLf) can be obtained from "Journal of Dilly Science", Vol. 20, pp. 752-759.
(1987) using an anti-bovine lactoferrin monoclonal antibody affinity column. The skim milk was loaded on an anti-bovine lactoferrin monoclonal antibody affinity column, and the bovine lactoferrin (b
Lf) was adsorbed, and then washed thoroughly with phosphate buffered saline (PBS) at pH 7.3. Then include 0.5M salt
After washing with a phosphate buffer of pH 7.3, the bLf adsorbed on the column was eluted with a 0.2 M sodium acetate buffer (pH 3.7, containing 0.15 M salt). After elution, adjust the pH to near neutral,
After dialysis against deionized water for 3 days, freeze-dried, bLf
I got The purity of the obtained bLf was confirmed by electrophoresis, but showed a single band.

[0021]

Example 2 Preparation of hLf : Human lactoferrin (hLf) was converted to heparin-
It was prepared from human milk using a Sepharose CL-6B column. The skimmed human milk is loaded on a heparin-Sepharose CL-6B column, and hLf in the skimmed human milk is adsorbed in the column.
Thereafter, the plate was washed with a 0.01 M phosphate buffer at pH 7.3. Next, hLf adsorbed in the column was eluted with a 0.01 M phosphate buffer at pH 7.3 containing 1.0 M salt. Further, the eluate was dialyzed against deionized water for 3 days, and then lyophilized to obtain hLf. The purity of the obtained hLf was confirmed by electrophoresis, but was 98% or higher.

[0022]

Example 3 Preparation of oTf : Protein was precipitated by adding ammonium sulfate to egg white to a concentration of 2.5M. Collect the precipitate by centrifugation,
Redissolved in M phosphate buffer and loaded on a diethylaminoethylcellulose column. After the oTf in the egg white was adsorbed on the column, the column was washed with a 0.01 M phosphate buffer at pH 6.0, and then the oTf was eluted with a phosphate buffer at pH 6.0 containing 0.1 M salt. The eluate was collected and dialyzed against deionized water for 3 days, and then lyophilized to obtain oTf. OTf obtained
Was confirmed by electrophoresis, and showed a purity of 95% or more.

[0023]

Example 4 Protection against Influenza Virus Infection : Inactivated Influenza Virus A / PR Used in Test Example 1
/ 8/34 and A / Aichi / 2/68 were serially diluted, and 5 chicken 10-day eggs were inoculated into the urine cavity in 0.1 ml portions. Three days later, 50 μl of the urine fluid of each egg was taken. This was mixed with 0.5% chick-stabilized erythrocytes, stirred, and the infection rate was determined by agglutination of erythrocytes. BLf obtained in Examples 1, 2 and 3 was added to 0.1 ml of the virus dilution at a dilution ratio showing 100% infection rate,
Dissolve 2.0 mg, 1.0 mg, and 0.5 mg of hLf and oTf, respectively, and incubate at room temperature for 30 minutes.
Eggs were inoculated into the urinary cavity in 0.1 ml portions each day. Three days later, 50 μl of the urine fluid of each egg was taken to determine the infection rate by hemagglutination. Using five eggs per group, the infection rate of each group was obtained.
In addition, the protective effect against infection was measured in the same manner for the enzymatically degraded products of each protein. The results are as shown in Table 3, and each sample showed a significantly strong protective effect against influenza virus.

[0024]

[Table 3]

[0025]

Example 5 Effect of bLf for Inhibiting Cytomegalovirus Infection : The effect of bLf obtained in Example 1 for inhibiting cytomegalovirus (CMV) infection was confirmed. bLf was added to MEM medium supplemented with 2% serum.
The solution was dissolved to a concentration of mg / ml and sterilized by filtration through a 0.45 μm filter to obtain a stock solution. This stock solution was diluted with a MEM medium supplemented with 2% serum as needed. After incubating human CMV (Towne strain) with a culture solution containing bLf at each concentration for 1 hour, human fetal fibroblasts (HEL
Cells). After culturing for 24 hours, the cells were fluorescently stained with human CMV-positive serum, and the ability to adsorb human CMV to cells was measured. As a result, as shown in Table 4, the CMV was 1 mg / ml b
It was confirmed that incubation with Lf for 1 hour completely lost the ability to adsorb and enter human CMV.

[0026]

[Table 4]

[0027]

Example 6 Inhibition Test of CMV Growth by hLf : After incubating hLf dissolved and diluted in the same manner as in Example 5 and human CMV for 1 hour, HEL cells were infected with CMV. Next, the above infected HEL cells were cultured in hLf-containing soft agar medium (0.8% soft agar, manufactured by Difco) adjusted to the same concentration as the hLf diluent, and plaques appearing on day 9 were counted and compared. The soft agar medium was layered every three days.

On the other hand, the same test was conducted under the condition that hLf was not contained in the soft agar medium, and the plaques formed were counted.

[0029]

[Table 5] As shown in Table 5, a medium containing hLf at a concentration of 1 mg / ml
After treatment with V, the cells were cultured in the same concentration of hLf soft agar medium, and a strong inhibition of CMV growth was observed.

[0030]

Example 7 CMV growth inhibitory effect of oTf : oTf obtained in Example 3 was treated in the same manner as described in Example 6, and plaques generated in oTf-containing soft agar medium were counted. As shown in Table 6,
0.5 mg / ml of oTf almost completely inhibited the growth of CMV.

[0031]

[Table 6]

[0032]

Example 8 Influence of bLf on human CMV infectivity : HEL cells were cultured on a 96-well plastic plate (Falcon), and human CMV was diluted 10-fold from 10 ^-1 to 10 ^-7. Was added, the cells were infected, and the titer was measured. In the same manner as in Example 5, the virus solution of each dilution series was added to each concentration series.
Incubate for 1 hour with bLf medium, then
HEL cells grown on a 96-well plastic plate were infected with a virus solution, and 24 weeks later, the number of wells in which infection was established was counted. As a result, CMV with a titer of 10 ⁷ is 0.
A titer of 1.8 × 10 ⁵ at 1 mg / ml bLf and a 0.5 mg / ml bLf
At a titer of 5.6 × 10 ¹ and at a bLf of 1.0 mg / ml a titer of 3.16
(Table 7).

[0033]

[Table 7]

[0034]

Example 9 Effect of Prevention and Treatment of CMV Infection in Animals : This example shows an example in which the effect of preventing CMV infection in animals and the effect of treating infected animals were confirmed. The bLf obtained in Example 1 was dissolved to a concentration of 1000 μg per ml of physiological saline solution,
After dissolution, the solution was sterilized by filtration through a 0.22 μm filter. On the other hand, 20 mice (C57B16J) from which the thymus was removed were divided into two groups, one group was administered with the above bLf solution for 7 days, 1 ml daily via vein, and one group was similarly administered with physiological saline. . Thereafter, mouse CMV was infected according to the method of Grundy et al. (Transbrandation, 37, 484-490, 1984).

That is, the mice were infected by intraperitoneal administration of salivary gland virus of the Smith species of mouse CMV. After breeding for an additional month, the amount of mouse CMV in urine was measured by enzyme linked immunoassay. All mice to which only the physiological saline solution was administered were positive for mouse CMV in urine, whereas two out of 10 mice were positive in the bLf administration group. In addition, 1 ml of this physiological saline solution was injected into a vein for 15 days.
Each group was administered daily, and one group was similarly administered only with a physiological saline solution. 15 days later, both groups were dissected after anesthesia, and the condition of the organs was observed.

[0036]

[Table 8] As shown in Table 8, only the other three mice were clearly recovered by CMV infection and clearly recovered.

[0037]

As described above, lactoferrin clearly protects against influenza virus infection and has a therapeutic effect, so that the present invention can be effectively used as an influenza virus infection protective agent. In addition, it is clear that the lactoferrin according to the present invention is not particularly affected by the change in antigen of influenza virus, and a broad preventive effect can be expected. Furthermore, the lactoferrin according to the present invention is a component contained in food, and can be supplied safely and at low cost. Therefore, by implementing the present invention, an influenza virus infection protective agent that is safe and has a protective effect against a wide range of influenza viruses can be supplied safely and inexpensively.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shunichi Dosako 5-15-39-616 Kitaurawa, Urawa City, Saitama Prefecture (72) Inventor Shigeaki Tanaka 1431-6 Kozono Ayase City, Kanagawa Prefecture (56) References Special Kaihei 1-233226 (JP, A) Journal of the Japanese Society of Pediatrics, Vol. 88, No. 7, 1984, p. 1581-1582, Abstract A-43 Cancer Research, Vol. 47 (1987), p. 4184-4188 (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 38/00-38/58 CA (STN)

Claims (1)

(57) [Claims] 1. An inflation containing lactoferrin as an active ingredient.
Luenza virus infection protective agent.