WO2021210070A1 - Pharmaceutical composition for adenoviral conjunctivitis - Google Patents

Abstract

The purpose of the present invention is to provide a pharmaceutical composition effective for adenoviral conjunctivitis.　Provided is a pharmaceutical composition for adenoviral conjunctivitis, containing an anti-adenovirus polyclonal antibody. The pharmaceutical composition is an ophthalmic solution. The pharmaceutical composition can be used for the treatment of adenoviral conjunctivitis. The pharmaceutical composition can be used for the prevention of adenoviral conjunctivitis in an uninfected eye.

Description

Pharmaceutical composition for adenovirus conjunctivitis

The present invention relates to a pharmaceutical composition for adenovirus conjunctivitis.

It is said that there is no effective drug for both prevention and treatment of adenovirus conjunctivitis, that is, epidemic keratoconjunctivitis.
For example, Non-Patent Document 1 describes epidemic keratoconjunctivitis.
"There is no effective drug for treatment / prevention of adenovirus in general. Anti-inflammatory agents are instilled as a symptomatic treatment, and if the cornea is inflamed and opaque, steroids are instilled. For the possibility, apply antibacterial agents. Pay attention to the handling and disposal of secretions of patients with eye diseases, wash hands and disinfect properly. Be careful not to contaminate eye drops with virus, and contaminate them. The equipment in the hospital is sterilized with an autoclave or disinfected with alcohol, iodine, etc. The basis of prevention is thorough prevention of contact infection. Items that come into contact with the eyes such as towels and eye drops are for personal use. do."
Is explained.

National Institute of Infectious Diseases Infectious Diseases Epidemiology Center, "What is Epidemic Keratoconjunctivitis?", [Online], April 1, 2014, National Institute of Infectious Diseases, [Search on April 8, 2020], Internet <URL: https://www.niid.go.jp/niid/ja/diseases/a/yellow-fever/392-encyclopedia/528-ekc.html> National Institute of Infectious Diseases Infectious Disease Epidemiology Center, “Pathogenic Microorganisms Detection Information”, Vol.38 No.7 (No.449), [online], July 2017, National Institute of Infectious Diseases, [April 8, 2020 Day search], Internet <https://www.niid.go.jp/niid/images/idsc/iasr/38/449.pdf>

An object of the present invention is to provide a highly effective pharmaceutical composition for adenovirus conjunctivitis.

The pharmaceutical composition of the present invention is for adenovirus conjunctivitis, which is characterized by containing an anti-adenovirus polyclonal antibody.

Since the pharmaceutical composition is an eye drop, it can be commercialized without being restricted by strict regulations as much as an internal medicine, and is highly practical.

Since the pharmaceutical composition is for the treatment of adenovirus conjunctivitis, it can contribute to the treatment of adenovirus conjunctivitis.

Since the pharmaceutical composition is for the prevention of eyes on the side not infected with adenovirus conjunctivitis, it can contribute to the prevention of adenovirus conjunctivitis.

According to the present invention, it is possible to provide a pharmaceutical composition that is very effective against adenovirus conjunctivitis.

It is a graph which shows the efficacy test result of adenovirus immune serum. It is a graph which shows the infection inhibition test result of adenovirus immune serum. It is a graph which shows the treatment experiment result with adenovirus immune serum in a rabbit. It is a graph which shows the ratio of the adenovirus serotype of epidemic keratoconjunctivitis.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
In the present invention, we have succeeded in preventing and treating adenovirus conjunctivitis, which is an infectious disease, by instilling a polyclonal antibody.

Specifically, the preventive and therapeutic effects on adenovirus conjunctivitis infection can be confirmed by intraventrally administering adenovirus antibody to mice and measuring the luciferase activity of adenovirus expressing a reporter gene (luciferase) against eye infection. rice field.

[Preventive experiment in mice]
[Materials and methods]
[Adenovirus and vector]
E1, E3-deficient adenovirus type 5 vectors expressing reporter genes such as green fluorescent protein (GFP) (Ad5-GFP) and luciferase (Ad5-Luci) were constructed. These adenovirus vectors were grown in HEK293 cells and purified by cesium chloride equilibrium density gradient centrifugation. A solution containing 10 mM Tris (pH 7.5), 1 mM MgCl2, and 10% glycerol was dialyzed using a Float-A-Lyzer G2 dialysis machine (MWCO: 20 kD, Spectrum Laboratories Inc.). Stored at -80 ° C until use. The concentration of virions in each preparation is as follows: 1 OD 260 = 1 × 10 ¹² virus particles (VP) / mL (formula 1)
Was calculated from the optical density at 260 nm (OD 260).

[Preparation of immune serum against adenovirus]

Anti-adenovirus immune sera were prepared using 8-week-old female ICR mice (Nippon SLC Co., Ltd.). On days 0 and 14, mice ^{were intramuscularly immunized with 10 10} VPs of Ad5, Ad7, Ad8, Ad19 and Ad37, and blood was drawn 3 months after the initial immunization.

[Antibody administration and infection by instillation of immune serum]
8-week-old female BALB / c mice (5 mice per group, Nippon SLC Co., Ltd.) were instilled with 5 μL of Ad5-specific immune serum, 10-fold or 100-fold diluted serum twice at 30-minute intervals. (Antibody administration). Thirty minutes after antibody treatment, 5 μL of 10 ¹⁰ VP Ad5-Luci was instilled into mice (infection). Serum was usually used as a negative control.

72 hours after infection, mouse eyes containing the conjunctiva were isolated, washed with PBS to remove blood, and 1.5 mL tube (Eppendorf AG) containing 300 μL of 1 × Cell Culture Lysis reagent (Promega Co., Ltd.). Soaked in. Tissues were homogenized with a 1.5 mL microtube pestle for a microcentrifuge tube (Scientific Specialties, Inc.), centrifuged at 8,000 g for 10 minutes, and the supernatant was used for luciferase assay. 4 μL of cytolysis was mixed with 20 μL of luciferase assay substrate and luciferase activity was measured with a luminometer.

[Neutralization experiment]
Immune sera against Ad5, Ad7, Ad8, Ad19 and Ad37 were incubated at 56 ° C. for 30 minutes. Continuous 2-fold dilutions were incubated with the corresponding virus at 37 ° C. for 1 hour each. Serum was usually used as a control. The virus-serum mixture was incubated with A547 cells (ovarian melanoma cell line) in 96-well plates at 37 ° C., 5% CO2 for 7-10 days. Neutralizing antibody titers were determined as the highest dilutions that resulted in a cytopathic effect (CPE).

[result]
1. 1. FIG. 1 shows the results for Ad5. The "normal serum" on the horizontal axis is the control group, which is the group of non-immunized serum, the "immunized serum" is the group of the above-mentioned immune serum, and the "1/10 serum" is a 10-fold dilution of the immune serum. It is a group of immune sera, and "1/100 serum" is a group of 100-fold diluted sera. The vertical axis is the relative luciferase activity with normal serum as "1". Looking at the results, immune sera and 10-fold diluted immune sera showed 86.6% and 62.1% inhibition of adenovirus infection compared to the control group (P = 0.0003 and 0.0116, respectively). However, this was not the case in the 100-fold diluted immune serogroup (19.9% inhibition, P = 0.448).

2. Neutralizing Antibodies: It was investigated whether immune sera have a similar ability to inhibit Ad virus infection against other types of adenoviruses such as Ad5, Ad7, Ad8, Ad19 and Ad37 that cause conjunctivitis. A neutralization test was performed.

The results are shown in FIG. “CTL” on the horizontal axis is a control group (control) of non-immunized normal serum, and “Ad5” to “Ad37” are a group of each adenovirus. The vertical axis shows the neutralizing antibody titer on a logarithmic scale (log2). As shown in FIG. 2, immunization of each adenovirus induced a neutralizing antibody titer similar to ^{Ad5 (2 13.4).} The results show that ^{the immune sera of Ad7 (2 12.8} ), Ad8 (2 ^12.5 ), Ad19 (2 ^11.6 ) and Ad37 (2 ^13.5 ) have similar capacity for corresponding adenovirus infections. Shown.

From the above results, it was shown that the pharmaceutical composition containing the anti-adenovirus polyclonal antibody of the present invention has a strong effect on the prevention of adenovirus conjunctivitis.

[Treatment experiment with rabbits]
[Infection and treatment]
[Infection] An 8-week-old, 1.3-1.6 kg, female New Zealand white rabbit was used. A total of 11 rabbits were anesthetized with Somnopentil (Kyoritsu Seiyaku Co., Ltd.), and on day 0, after rubbing the surface of the eye with 12 cross-hatching strokes with a sterile 25-gauge needle (Terumo Corporation), at 4-hour intervals. Twice, 10 ¹⁰ vg (vector genome) Ad5, 20 μL was inoculated into both eyes.

[Treatment: antibody serum group] Then, from the 1st to the 7th day, 6 rabbits were ^{injected with Ad5-specific mouse immune serum (Ad5 type vector 10 10} vg three times intramuscularly into mice, and then about 10 days later. Serum obtained by collecting blood, titer: 1: 1,000) was instilled at 50 μL / eye, 3 times / day.
[Control: Normal serum group] Five rabbits were used as controls, and 200-fold diluted normal mouse serum was similarly instilled.

[Collection] Elapsed days 0, 1, 2, 3, 5, 7, 9, 12, 14, and 16 days, on days of serum treatment, before the serum treatment, wipe the eyes with a cotton swab (tear fluid collection) ) Was performed. Cells extracted with each swab, along with antibiotics (because without antibiotics, cells are contaminated with germs when quantifying virus titers), 1.5 mL tube containing 0.5 mL DMEM medium I put it in. The tubes were vortexed to finely diffuse and the swab cultures were frozen and stored at -80 ° C until used in the plaque assay.

[Plaque assay]
The Ad5 titer was determined on A549 cells (human lung cancer cell line) by a standard plaque assay for TCID50 (http://www.virapur.com/protocols/TCID50%20Protocol.pdf). The cotton swab cultures frozen and stored above are serially diluted with DMEM medium supplemented with 2% FBS (fetal bovine serum) and antibiotics and placed in 96-well plates, followed by 3 × 10 ⁴ A549. Cells / wells were added and the plates were incubated at 37 ° C., 5% CO2 for 10 days. Then the virus titer was calculated.

[result]
The results are shown in FIG. The horizontal axis shows the number of days elapsed, and the vertical axis shows the average virus titer in each group on a logarithmic scale. Since it is a logarithmic scale, the error appears to be large with a small titer. High viral titers were detected on day 2. After that, a gradual decrease was observed in the antibody serum group, but not in the normal serum group. The P value from the 5th day to the 16th day was 0.05 or less, and a significant difference was observed between the normal serogroup and the antibody serogroup (see * mark). On the 14th and 16th, half of the samples in the antibody serogroup did not detect the virus, but in the normal serogroup, the virus was detected in all the samples.
From the above results, it was shown that the pharmaceutical composition containing the anti-adenovirus polyclonal antibody of the present invention is effective for the treatment of adenovirus conjunctivitis.

Injecting antibodies derived from other animals into humans often causes symptoms such as anaphylaxis, but instillation on the body surface is unlikely to cause such side reactions. Above all, it is highly beneficial that a strong virus neutralizing antibody acts directly on the affected area and acts immediately.

About 80% or more of human adenovirus epidemic keratoconjunctivitis is infected with type 3,4,5,19 / 64,37,53,54,56, and most of them are mixed with 8 kinds of antibodies. Ad conjunctivitis can be treated. Although a technique for producing a human monoclonal antibody is known, a monoclonal antibody costs about 1000 times as much as that of the present invention and cannot be put into practical use as an eye drop. As shown in FIG. 3, the neutralization of Ad by instillation of the antibody we prepared is the best. An antibody of Ad virus that can be produced at low cost has a high practical effect in the treatment and prevention of conjunctivitis.

There are more than 51 serotypes and more than 100 genotypes of adenovirus, and there are many types. Respiratory infections are types Ad3 and 7. Pharyngoconjunctivitis, which is called pool fever, mainly develops in types 4, 5, 7, 8, 19, and 37, mainly in type 3. Epidemic keratoconjunctivitis that occurs in summer is also common in types 3, 8, 19, 37, and 54. Types 19 and 37 are infected by sexual activity and cause urethritis and cervicitis.

About 80% of adenovirus epidemic keratoconjunctivitis is type 3,4,8,19 / 64,37,53,54,56, and if 8 kinds of animal-derived antibodies discovered by us are mixed, it is more than 85% viral. This is an important finding to prevent the exacerbation of conjunctivitis (Fig. 4, Non-Patent Document 2). Adenovirus-derived conjunctivitis is the most common conjunctivitis and is the second largest expression after influenza in children and the like, and in that respect, there is a great need as a therapeutic agent and a preventive agent.

In Japan, the above adenoviruses 3,4,8,19 / 64,37,53,54,56 can prevent more than 85% of human adenovirus infections.

All publications, patents and patent applications cited herein are incorporated herein by reference only.

Claims (4)

A pharmaceutical composition for adenovirus conjunctivitis, which comprises an anti-adenovirus polyclonal antibody. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is an eye drop. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is for the treatment of adenovirus conjunctivitis. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is for preventing an eye on the side not infected with adenovirus conjunctivitis.

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