KR20230108408A - Devices for treating dermatopathies caused by viruses - Google Patents

Abstract

The present invention provides a device for treating skin diseases, characterized in that it comprises a light ray having a blue light energy level for irradiating a disease lesion site caused by infection with a virus that causes skin diseases. According to the present invention, it is possible to irradiate the device to virus-infected lesions, such as skin and mouth mucosa, without killing host cells, reducing inflammatory reactions, and suppressing the proliferation or activity of viruses. Therefore, according to the present invention, it is possible to quickly improve the symptoms of the lesion site while simply and easily.

Description

Devices for treating skin diseases caused by viruses {Devices for treating dermatopathies caused by viruses}

The present invention is a device for treating a disease caused by infection with a virus that causes a skin disease, and can reduce the inflammatory response and inhibit the activity or proliferation of the virus without damaging the host cell when symptoms of the disease caused by the virus appear. Provides a device with

Viruses cannot be cultured in general nutrient media, but self-replicate and multiply selectively in living cells using enzymes in host cells. The surface protein of the virus attaches to the host cell receptor, penetrates into the host cell through the fusion of the viral envelope and the cell membrane, and the DNA/RNA of the virus is uncoated, and the virus mRNA is synthesized, viral protein is synthesized, replicated, and released out of the cell through assembly with capsid proteins to complete viral self-replication.

Viruses that cause skin diseases are typically Varicella zoster virus, Herpes simplex virus (HSV), Smallpox, Molluscum contagiosum, Human papillomavirus, These include Parvovirus, Rubella, Measles, and Coxsackie virus.

Due to the increase in stress due to modernization, the incidence of diseases caused by chronic infectious viruses is increasing, and diseases caused by varicella zoster virus or herpes simplex virus are particularly representative. Herpes simplex is a disease that causes small blisters forming clusters with erythema on the skin or mucous membranes caused by infection around the mouth, such as the lips, by HSV type 1 (DNA virus), a type of herpes virus. The virus is activated by various causes such as mental tension, trauma, and fatigue, progresses to pustules, and eventually scabs form and fall off. These skin lesions are healed within 2-3 weeks, but the infection rate is higher than 70-90% worldwide. As much as it shows, effective treatment and prevention of recurrence through suppression of viral replication/activation are required.

However, the virus is in a dormant state and is activated by various causes such as febrile illness, mental tension, trauma, and fatigue, resulting in recurrent herpes. In addition, as the blisters grow, they cause pain and discomfort and spread to the surroundings, or when the cause of infection is not removed or treated properly, the blisters burst and the dermis is exposed and secondary infection may occur.

The HSV-1 virus is an enveloped virus with a diameter of about 150 nm and composed of a core consisting of linear dsDNA in the center, a nucleocapsid, a tegument, and an envelope. In order to infect a cell, an entry process into the host cell is first necessary. Viral proteins, glycoproteins gB and gC, are attached to heparin sulfate chains on cell surface proteoglycans (HSPG) on the cell surface of the host cell membrane. When the gD protein binds to the gD receptor, the viral envelope fuses with the cell membrane and penetrates into the cell. Once inside the host cell, the nucleocapsid is transported to the nucleus, where the viral DNA is separated from the envelope proteins, and proteins within the viral particle inhibit host cell macromolecular synthesis. In the nucleus, proteins such as virus-specific DNA polymerase and DNA-binding protein are involved in viral DNA replication, and host cell RNA polymerase ), about 1/3 of the viral genome is transcribed, early mRNA encodes regulatory proteins that promote protein synthesis, and structural proteins that make viral particles are encoded in late mRNA, and nucleocapsids are transferred to the cytoplasm. is emitted Afterwards, the capsid binds to the tegument protein and is surrounded by a membrane derived from the endosome or the trans-Golgi network to form external viral glycoproteins and external envelope proteins. When the outer tegument protein comes and envelops it, it goes out of the cell and the replication process takes place.

To date, acyclovir (acyclic guanosine analogue) is a representative drug that selectively inhibits the replication of herpes simplex virus and varicella zoster virus, and targets viral polymerase and DNA replication. It inhibits viral DNA polymerase and blocks DNA polymerization of replicating viruses to prevent early viral growth and replication. However, there are various side effects such as increased mutation and resistance to antiviral agents and decreased efficacy.

On the other hand, hand, foot and mouth disease frequently occurs in children and is a clinical syndrome in which a characteristic rash appears, and is also caused by coxsackievirus A16 or enterovirus 71, EV-71, RNA virus. In particular, hand, foot and mouth disease caused by EV-71 in children causes neurological complications at a high rate, leading to brainstem encephalitis, neurogenic pulmonary edema, pulmonary hemorrhage, and sudden death from shock. The main symptoms include blisters on the tongue, oral mucosa, pharynx, palate, gums, and lips, which later form ulcers, and macular rash, blistering rash, and pustular rash appear on the hands, fingers, feet, buttocks or groin, especially It occurs most often in the hands. Lesions of the hands and feet are 3-7 mm in size and may appear on the backs of the hands, palms, and soles.

Enterovirus is an icosahedral virus that does not have an envelope. Recently, through phylogenetic analysis through RdRp (RNA dependent RNA polymerase) gene sequence, enterovirus genus HEV (human enterovirus) A-D were classified according to their genotype. HEV-A includes 11 serotypes of coxsackievirus group A (CVA) and EV-71, and HEV-B includes all coxsackievirus group B (CVB) and 38 serotypes of EV-69, EV-73, and CVA9. Eggplant serotypes are included. HEV-C includes 11 CVAs, and HEV-D includes two sera, EV-68 and EV-70.

EV-71 is a positive-sense, single-stranded RNA virus that does not have an envelope. In the early stages of infection in infants and children, mild cold symptoms such as fever, headache, sore throat, and Causes hand-foot-and-mouth disease. It can cause acute flaccid paralysis and encephalitis, often like polio, several days after infection, as described above. EV-71 virus is a mammalian cell, such as the scavenger receptor B2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL-1) of the host cell. It is fused using the entry receptor of the host cell, and the viral RNA entering the host cell is translated into a polyprotein through the process of translation and protein processing. viral protein is synthesized, and viral RNA replicated by RNA-dependent RNA polymerase is packaged/maturation process with viral protein The virus is then released out of the cell and the virus replicates.

Despite repeated viral infections, side effects such as resistance have been reported in the development of usable vaccines or effective antiviral treatments. Pleconaril binds to the capsid protein of the virus that binds to the sensitive cell receptor and prevents the virus from entering the cell, showing antiviral activity. It has not been approved, and resistant viruses have also been reported. Ribavirin has also been reported to exhibit drug resistance, although it exhibits broad antiviral activity against RNA viruses and DNA viruses.

In a situation where there is no cure for the EV-71 virus, it is possible to develop a substance or treatment that has an antiviral effect, establish a treatment system by identifying the mechanism of action of the antiviral activity, and apply it to various viruses that cause skin diseases. There is a demand for the development of a treatment system that can

Registered Patent No. 10-1352238 discloses a topical application of a substance that induces a DTH response in a patient at an HSV damaged area in order to induce a delayed-type hypersensitivity (DTH) response to the damaged area during one or more lesions caused by HSV infection. It proposes a treatment method comprising the step of administering, and Patent Publication No. 10-2018-0074113 proposes a pharmaceutical composition for preventing or treating herpes simplex virus infection containing licorice extract as an active ingredient did

On the other hand, a method of reducing the recurrence of HSV infectious disease and the pain period during HSV disease by irradiating light in the red and far-infrared wavelength range has been proposed. (de Paula Eduardo C, Aranha ACC, Sim es A, et al (2014) Laser treatment of recurrent herpes labialis: a literature review. Lasers Med Sci 29:1517-1529. doi: 10.1007/s10103-013-1311-8)

An object of the present invention is to provide a device for treating skin diseases caused by viruses that cause various skin diseases, and in particular, to provide a means that can quickly and easily improve the symptoms of a lesion site.

The present invention provides a device for treating skin diseases, characterized in that it comprises a light ray having a blue light energy level for irradiating a disease lesion site caused by infection with a virus that causes skin diseases.

The virus includes Varicella zoster virus, Herpes simplex virus (HSV), Smallpox, Molluscum contagiosum, Human papillomavirus, Parvovirus, Rubella, Measles, enterovirus or Coxsackie virus.

Preferably, the virus is Herpes simplex virus (HSV) or enterovirus 71 (EV-71).

The blue light has a wavelength in the range of greater than 430 nm and less than 500 nm.

The blue light is emitted from an LED or laser light source.

The blue light has an energy density in the range of 10 to 60 J/cm ² .

The blue light has an effect of inhibiting the proliferation or activity of viruses.

The blue light does not kill or damage host cells except for virus-infected lesions.

The blue light is irradiated at intervals of 4 to 8 hours.

The blue light is irradiated for 10 to 30 minutes.

The device for treating diseases caused by infection with a virus causing skin diseases of the present invention irradiates blue light to lesions such as skin and mouth mucous membranes infected with viruses, thereby reducing inflammatory reactions without damaging host cells, It is possible to inhibit the proliferation or activity of the virus.

Figure 1 shows the number of vDNA copies in the cell supernatant in Examples 1 and 2 and Comparative Example 3.
Figure 2 shows the results of observing plaque formation and the reduction rate in Examples 1 and 2 and Comparative Example 3.
Figure 3 shows the number of vDNA copies in the cell supernatant in Comparative Examples 1, 2 and 3.
4 is a photograph of the herpes simplex virus-infected lesion before, after 1 day and 2 days after treatment in Example 3.
5 shows changes in protein expression over time in Vero cells infected with HSV-1 virus in Examples 4 and 5 and Comparative Example 4.
Figure 6 shows the results of observing plaque formation and the reduction rate in Examples 6 and 7 and Comparative Example 5.

The present invention provides a means of irradiating light rays to virus-infected skin and mucous membranes of the mouth without killing host cells, reducing inflammatory reactions, and suppressing the proliferation or activity of infected viruses.

The virus causes skin diseases, such as Varicella zoster virus, Herpes simplex virus (HSV), Smallpox, Molluscum contagiosum, Human papillomavirus, It may be Parvovirus, Rubella, Measles, enterovirus or Coxsackie virus.

Preferably, the virus is Herpes simplex virus (HSV) or enterovirus 71 (EV-71).

The light beam used in the device of the present invention is blue light. Therefore, the present invention provides a device for treating a disease caused by a virus infection, characterized by comprising a light ray having a blue light energy level for irradiating the lesion site of a disease caused by a virus infection.

The light beam is one having a wavelength in the range of greater than 430 nm and less than 500 nm. Preferably, the light ray may have a wavelength in the range of 425 to 475 nm, more preferably 450 nm. When light rays with wavelengths in this range are irradiated to lesions showing clinical signs suspected of being caused by a virus, for example, in the case of a disease infected with the herpes simplex virus, blisters begin to be removed, followed by scabs (scabs). There is an effect of improving the symptoms.

This effect is very rapid improvement of symptoms as it appears in 1 to 2 days compared to the healing period of 10 days to 2 weeks in the case of symptoms of general diseases.

Moreover, in the process of improving these symptoms, it is confirmed that the virus-infected host cell is only effective in inactivating the virus without causing damage or death.

In addition, the blue light is not limited thereto, but is irradiated from an LED or laser light source.

In addition, the blue light has an irradiation amount in the range of 10 to 60 J/cm ² . When irradiated with blue light having an irradiation amount in the above range, the effect of inhibiting the growth of viruses appears, and when irradiated with a higher irradiation amount of light, there may be an effect on the host cell.

The blue light has an effect of inhibiting the proliferation or activity of viruses. On the other hand, the blue light does not kill or damage host cells except for virus-infected lesions.

Therefore, the device of the present invention can be used in the treatment process by irradiating blue light from the device to the lesion site for the treatment of skin diseases caused by viral infection.

For the effect of improving symptoms, it is preferable to irradiate blue light at intervals of 4 to 8 hours in the above procedure. In addition, it is preferable that the irradiation time for one irradiation is in the range of 10 to 30 minutes. When treating with such a time interval and irradiation time, the symptoms of the lesion site can be rapidly improved without adversely affecting the normal area around the lesion.

The present invention will be described in more detail through the following examples. However, it should not be considered that the present invention is limited thereto.

Example 1

Monkey normal kidney cells (Vero), the host cells, were put into a 12-well plate at a concentration of 1.4 X 10 ⁵ cells/ml, maintained at 5% CO ₂ for 24 hours, and incubated at 37°C in a CO ₂ incubator (Thermo Forma, USA). cultured. The next day, after confirming confluence of 95% or more, the cultured cells were washed once with PBS and then infected with HSV-1 virus at MOI (multiplicity of infection) = 1 for 1 hour. Thereafter, remaining viruses were removed with PBS, and replaced with overlay media (DMEM + 3% FBS + antibiotics + 0.4% agarose) containing agarose.

Blue light of 450 nm was irradiated at an irradiation dose of 10 J/cm ² , and a total of 30 J/cm ² was irradiated once for 20 minutes at 0 hpi (post-infection hour), 24 hpi, and 48 hpi on the day of virus infection.

Example 2

In Example 1, a total of 60 J/cm ² of blue light energy was irradiated at an interval of 8 hours twice a day for the number of blue light irradiations.

Comparative Example 1

Experiments were performed in the same manner as in Example 1 except that 550 nm green light was irradiated instead of 450 nm blue light.

Comparative Example 2

Experiments were performed in the same manner as in Example 2 except that 550 nm green light was irradiated instead of 450 nm blue light.

Comparative Example 3

After infecting Vero cells with the HSV-1 virus in the same manner as in Example 1, blue light was not irradiated.

[Rating 1]

In Examples 1, 2 and Comparative Example 3, 48 hours after virus infection, viral DNA (vDNA) was isolated from 200 ul of virus-infected cell culture medium using AccuPrep® Viral DNA/RNA Extraction Kit (Bioneer, K-3033) , 95°C 10 minutes (1 time), 95°C 15 seconds, 60°C 30 seconds (40 times) After performing PCR using ABI step one plus equipment, measure the quantification cycle (CT) value of vDNA did Each was measured three times, and the average value was calculated, and the results are shown in Table 1.

CT value average Example 1 22.90 22.82 22.78 22.77 Example 2 23.70 23.64 23.61 23.60 Comparative Example 3 21.59 21.54 21.50 21.52

Compared to Comparative Example 3, the quantification cycle (CT) value increased in Examples 1 and 2, and the increase was greater in Example 2, which was irradiated twice. That is, as a result of PCR, the quantification cycle (CT) value increased in Examples 1 and 2 compared to Comparative Example 3, indicating that the amount of viral DNA decreased after irradiation with blue light.

[Evaluation 2]

In Examples 1 and 2 and Comparative Example 3, the cell supernatant was recovered 48 hours after virus infection and the number of vDNA copies was measured. The results are shown in Figure 1.

The amount of vDNA decreased after blue light irradiation in Examples 1 and 2 compared to Comparative Example 3. In Example 1, where the number of times of irradiation was 1, it decreased by 65.9%, and in Example 2, where the number of times of irradiation was 2 times, it decreased by 83.4%, and the decrease was greater.

[Rating 3]

In Examples 1 and 2 and Comparative Example 3, the cells were fixed with a 3.7% formalin solution 72 hours after virus infection, washed with PBS, and stained with 0.5% crystal violet to observe plaque formation. Results are shown in FIG. 2 .

Compared to Comparative Example 3, it was confirmed that plaque formation was noticeably reduced in Examples 1 and 2. Looking at the plaque reduction rate, it decreased by 27.2% in Example 1 and by 61.9% in Example 2 compared to Comparative Example 3.

[Rating 4]

For Comparative Examples 1, 2, and 3, vDNA was isolated in the same manner as in Evaluation 2, and copies/ul were measured. The results are shown in Figure 3.

Looking at this, there was no difference in the amount of vDNA in Comparative Examples 1 and 2 irradiated with green light compared to Comparative Example 3 in which no light was irradiated. From this, it was possible to confirm the significant effect of blue light in the present invention.

Example 3

For patients infected with the herpes simplex virus and showing symptoms around the lips, 450 nm blue light is irradiated at 24 hour intervals at an irradiation dose of 10 J/cm ² for 20 minutes using the device of the present invention to the lesion site once for 20 minutes. treatment was carried out. Pictures of the lesions on the 1st and 2nd days after starting the treatment are shown in FIG. 4 .

As a result, it was confirmed that the blisters began to be removed on the first day after the start of treatment, and the symptoms were cured with scabs already forming on the second day. This is much faster than the general progression of symptoms at the lesion site infected with herpes simplex virus, and it was confirmed that the device and method of the present invention have a therapeutic effect by rapidly progressing symptoms at the lesion site caused by herpes simplex virus infection.

Example 4

Monkey normal kidney cells (Vero), the host cells, were put into a 12-well plate at a concentration of 1.4 X 10 ⁵ cells/ml, maintained at 5% CO ₂ for 24 hours, and incubated at 37°C in a CO ₂ incubator (Thermo Forma, USA). cultured. The next day, after confirming confluence of 95% or more, the cultured cells were washed once with PBS and then infected with HSV-1 virus at MOI (multiplicity of infection) = 1 for 1 hour. Then, the remaining virus was removed with PBS and replaced with DMEM media (DMEM + 3% FBS + antibiotics) containing a minimum concentration of FBS. Blue light of 450 nm was irradiated with an irradiation amount of 10 J/cm ² at 0 hpi for 20 minutes.

Example 5

In Example 4, 450 nm blue light was 20 J/cm ² .

Comparative Example 4

After infecting Vero cells with the HSV-1 virus in the same manner as in Example 4, blue light was not irradiated.

[Rating 5]

In Examples 4 and 5 and Comparative Example 4, changes in protein expression over time were observed. Cell destruction and protein extraction solution was added to Vero cells hourly from 0 to 4 hours after virus infection to extract proteins from the cells, and then cell debris and proteins were separated by centrifugation. After electrophoresis of the extracted protein, it is transferred to a membrane by electrical force using a kit, followed by phospho-mTOR, phospho-AKT, and phospho-p38. MAPK (phospho-p38 MAPK), phospho-ERK1/2 (phospho-ERK1/2), PERK (protein kinase RNA-like endoplasmic reticulum kinase), and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) antibodies were reacted and the luminescent material The attached secondary antibody was then reacted. Here, the ECL reagent (Dugen Bio, DG-WPAL120) that reacts with the luminescent material was reacted to measure the amount of luminescence using Fusion FX7 (Vilber lourmat), thereby measuring the amount of protein expression. The measurement results are shown in FIG. 5 .

The initial stage of HSV-1 virus infection is the process of fusion between HSV-1 and the host cell membrane followed by entry into the cell. The PI3K-AKT-MAPK signaling cascade is an important signaling mechanism involved in cell proliferation and survival when the virus enters the host cell. one of When the PI3K/AKT and MAPK cascades are activated, viruses alter the host cell cycle by evading host cell defense systems and promoting viral production. In FIG. 5, phosphorylation of AKT, p38 MAPK, and ERK1/2 increased after 4 hours of infection with HSV-1 virus, but phosphorylation of AKT and p38 MAPK after irradiation with blue light temporarily increased until 2 hours after infection, but 4 hours after infection, the control group decreased compared to In addition, phosphorylation of mTOR (mammalian target of rapamycin) signaling, which is known to be important for cell proliferation and survival, was increased 4 hours after infection after irradiation with blue light, indicating that one of the signal transduction mechanisms required for host cell survival was activated.

Endoplasmic reticulum (ER, endoplasmic reticulum) is a cell organelle involved in lipid and protein synthesis, protein modification and folding, and calcium ion regulation. It plays a role in virus replication/growth by synthesizing proteins necessary for virus replication during viral infection. In the process, ER stress occurs, in which misfolded or unfolded proteins accumulate, and the unfolded protein response (UPR) system is activated. Representatively, there is PERK protein, and PERK-dependent eIF2a phosphorylation induces protein translation inhibition. After HSV-1 infection, PERK protein expression increased over time, but at 4 hours after blue light irradiation, PERK expression increased more than the control group. This shows that by inducing continuous ER stress after blue light irradiation, protein synthesis necessary for viral replication is inhibited in host cells, and consequently, viral replication inhibition and inactivation are induced.

As a result, the above experiments showed that blue light irradiation in HSV-1 virus-infected host cells promotes mTOR phosphorylation and the expression of PERK, an ER stress protein, and regulates MAPK and PI3K/AKT/mTOR signal transduction mechanisms. The effect of cell survival and virus inactivation was shown fragmentarily.

Example 6

Experiments were performed under the same infection conditions as in Example 1, except that Enterovirus-71 virus was used instead of HSV-1 virus and Vero E6 cells were used as host cells. Blue light of 450 nm was irradiated at an irradiation amount of 10 J/cm ² , and a total amount of 20 J/cm ² was irradiated once for 20 minutes at 0 hpi (post-infection hour) and 24 hpi on the day of virus infection.

Example 7

In Example 5, a total of 40 J/cm ² of blue light energy was irradiated at an interval of 8 hours, twice a day, for the number of blue light irradiations.

Comparative Example 5

After infecting Vero E6 cells with Enterovirus-71 virus in the same manner as in Example 6, blue light was not irradiated.

[Rating 6]

In Examples 6 and 7 and Comparative Example 5, the cells were fixed with a 3.7% formalin solution 72 hours after viral infection, washed with PBS, and stained with 0.5% crystal violet to observe plaque formation. Results are shown in FIG. 6 .

Compared to Comparative Example 5, it was confirmed that plaque formation was noticeably reduced in Examples 6 and 7. Looking at the plaque reduction rate, it decreased by 29.8% in Example 6 and by 63% in Example 7 compared to Comparative Example 5.

Claims (10)

An apparatus for treating a skin disease, characterized in that it comprises a light beam having a blue light energy level for irradiating a lesion site of a disease caused by infection with a virus that causes a skin disease.
In claim 1,
The virus includes Varicella zoster virus, Herpes simplex virus (HSV), Smallpox, Molluscum contagiosum, Human papillomavirus, Parvovirus, A device for treating skin diseases, characterized in that it is Rubella, Measles, enterovirus or Coxsackie virus.
In claim 1,
The virus is a herpes simplex virus (Herpes simplex virus, HSV) or enterovirus 71 (EV-71), characterized in that, a device for treating skin diseases.
In claim 1,
The blue light is characterized in that it has a wavelength in the range of more than 430nm and less than 500nm, skin disease treatment device.
In claim 1,
The blue light is characterized in that irradiated from the LED or laser light source, skin disease treatment device.
In claim 1,
The blue light is characterized in that having an energy density in the range of 10 to 60 J / cm ² , skin disease treatment device.
In claim 1,
The blue light is a device for treating skin diseases, characterized in that there is an effect of inhibiting the proliferation or activity of viruses.
In claim 1,
The blue light is a device for treating skin diseases, characterized in that it does not kill or damage host cells except for lesions infected with viruses.
In claim 1,
Characterized in that the blue light is irradiated at intervals of 4 to 8 hours, a device for treating skin diseases.
In claim 1,
The blue light is characterized in that irradiated for 10 to 30 minutes, skin disease treatment device.