KR20220111620A - A pharmaceutical composition for use of prevention or treatment of COVID-19 infection - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising thymomodulin and a pharmaceutically acceptable carrier, capable of preventing or treating COVID-19. The pharmaceutical composition of the present invention can safely and effectively alleviate, prevent or treat COVID-19 infection or symptoms associated with infection.

Description

A pharmaceutical composition for use of prevention or treatment of COVID-19 infection

The present invention relates to a pharmaceutical composition capable of preventing or treating Covid-19 infection.

The 2019 novel Corona virus (2019 novel Corona virus; severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) is the causative virus (2019-nCoV) of the novel coronavirus infection that occurred in Wuhan, China in December 2019. This new virus is named 'COVID-19' or 'COVID-19', meaning that it was first confirmed in late 2019. Epidemic diseases caused by the novel coronavirus are transmitted through the respiratory tract, and are highly contagious in the early stages of infection. After infection, it is a serious respiratory disease that can develop into pneumonia and even death through symptoms such as sore throat, high fever, cough, and shortness of breath. Relying on conservative treatment such as respiratory tract.

On the other hand, thymomodulin is marketed as an adjuvant treatment for bacterial and viral infectious diseases such as hepatitis and respiratory diseases, and is a purified product of protein extracted from calf thymus. The molecular weight is 1 kD to 10 kD, and is known to include thymosin alpha 1 and thymosin beta 4 .

Korean Patent Application Publication No. 10-2020-0063275 Korean Patent Application Publication No. 10-2007-0072919

Pediatr Med Chir. 1988; 10(6): 603-7 Pediatr Med Chir. 1990; 12(3): 229-32 Drugs Exp Clin Res, 1985, 11(9), 665-9 Minerva Med. 1987 Sep15: 78(17): 1281-9

Accordingly, one object of the present invention is to provide a pharmaceutical composition that can safely and effectively prevent or treat Covid-19 and a method for preparing the same.

Another object of the present invention is to provide a novel use of chymomodulin as a therapeutic or prophylactic agent for Covid-19.

Another object of the present invention is to provide a pharmaceutical composition capable of inhibiting the proliferation of the Covid-19 virus.

One aspect of the present invention relates to a pharmaceutical composition for use in the prevention or treatment of Corona 19 virus (COVID-19, Covid-19) infection comprising chimomodulin and a pharmaceutically acceptable carrier.

The active substance, thymomodulin, is an acid or enzymatic digest of a protein extracted from calf thymus, and is a mixture of several peptides corresponding to thymosin fragment 5. The molecular weight of chymomodulin is 1 kD to 10 kD, and its CAS number is 90803-92-2. It contains thymosin alpha 1, thymosin beta 4 and thymosin gamma and is currently marketed as an adjuvant treatment for bacterial and viral infections such as hepatitis and respiratory diseases. The inventors of the present invention found that chymomodulin not only inhibits the proliferation of Covid-19, but also prevents or alleviates weight loss due to Covid-19 infection, and alleviates all pathological consequences such as inflammation, congestion, bleeding, and tissue injury. has been found to be able to alleviate

Since chimomodulin is known to have few side effects, the novel use of chimomodulin for the treatment or prevention of Covid-19 infection provided by the present invention means that it can safely and effectively treat or prevent Covid-19. .

Based on the total weight of the pharmaceutical composition, chymomodulin may be included in an amount of 5 to 35% by weight, specifically 10 to 30% by weight, more specifically 20 to 30% by weight.

The dosage of chymomodulin for the prevention or treatment of Covid-19 virus infection may vary depending on the age, sex, weight, and severity of the infection of the patient, but ranges from 40 mg to 800 mg per day. More specifically, the daily dose of chymomodulin is 80 mg, 160 mg or 240 mg. The administration method of the pharmaceutical composition for the prevention or treatment of Covid-19 is to administer 80 mg to 800 mg of chimomodulin once a day or twice a day, for 1 week to 24 weeks. Specifically, a daily dose of 80 mg to 240 mg of chimomodulin once or twice a day is administered for 4 to 20 weeks. More specifically, a daily dose of 80 mg to 160 mg of chimomodulin once or twice a day is administered for 4 to 12 weeks. In particular, for the prevention of Corona 19 virus (COVID-19) infection, a daily dose of 80 mg to 800 mg of chimomodulin to a healthy person, the elderly, or an underlying disease, once a day or twice a day, 4 weeks to It may be administered for 24 weeks, and specifically, a daily dose of 80 mg to 240 mg of chimomodulin once or twice a day may be administered for 4 to 20 weeks. In particular, for the treatment of Corona 19 virus (COVID-19) infection, a daily dose of 80 mg to 800 mg of zymomodulin is administered to a person infected with Corona 19 virus, once a day or twice a day, for 1 week to 24 weeks. and specifically 80 mg to 240 mg of chimomodulin once or twice a day may be administered for 2 to 20 weeks.

The pharmaceutical composition may be for oral administration. Accordingly, the pharmaceutical composition may be formulated in the form of pills, powders, liquids, tablets or capsules, and in embodiments may be formulated as liquids, capsules or tablets.

Examples of the pharmaceutically acceptable carrier include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition may further include a filler, an extender, a binder, a wetting agent, a disintegrant, and the like.

The pharmaceutical composition is effective for inhibiting the proliferation of Covid-19 or preventing or treating an infection thereof. Symptoms of Covid-19 infection are fever, malaise, cough, dyspnea, pneumonia, sore throat, headache, diarrhea, nausea, weight loss, etc., and when the pharmaceutical composition of the present invention is repeatedly administered, one or more symptoms are relieved or can disappear

The pharmaceutical composition may be used in combination with other therapeutic agents, vaccines, or alleviation agents for symptoms of infection that are effective in preventing, treating, or relieving symptoms of Covid-19. Such therapeutic agents or alleviating symptoms of infection include plasma therapy using plasma from a patient infected with the virus, monoclonal antibody therapy, ribavirin, fenciclovir, nitacosanide, nafamostat, remdesivir, favipiravir, chloroquine, kaletra, Avigan, Arbidol, Galidesivir, Darunavir and the like are mentioned.

The pharmaceutical composition of the present invention can safely and effectively alleviate, prevent or treat Covid-19 infection or symptoms caused by infection.

1 shows from the 3rd to the 7th day after the Covid-19 inoculation, G1 (normal group: untreated control), G2 (infected group: Covid-19 infection and no treatment), G3 (chimomodulin administered group: Covid- 19 Infection and administration of chymomodulin) are photographs of the lungs of hamsters for comparison of gross bleeding and damage levels.
Figure 2 is a graph showing the rate of change based on the body weight before the virus inoculation of G1, G2, G3 on the 4th, 5th, 6th, and 7th days after the Covid-19 inoculation.
3 is a graph showing the level of inflammatory cell infiltration in the alveoli of G1, G2, and G3 on the 3rd, 5th, and 7th days after covid-19 inoculation.
4 is a graph showing the level of inflammatory cell infiltration in the stroma of G1, G2, and G3 on the 3rd, 5th, and 7th days after covid-19 inoculation.
5 is a graph showing the relative expression rate of the RNA gene of FAM (E gene) isolated from the autopsied lung tissue of G2 and G3 on the 3rd, 5th, and 7th days after covid-19 inoculation.
6 is a graph showing the relative expression rate of the RNA gene of RED (RdRP gene) isolated from the autopsied lung tissue of G2 and G3 on the 3rd, 5th, and 7th days after Covid-19 inoculation.
7 is a graph showing the relative expression rate of the RNA gene of Cy5 (N gene) isolated from the autopsied lung tissue of G2 and G3 on the 3rd, 5th, and 7th days after Covid-19 inoculation.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Experimental Example 1

33 4-week-old males (weight 80-100 g) of Syrian hamster (producer and supplier: JANVIRER LABS (France)/Saron Bio) were treated with a temperature of 23±3 ℃, a relative humidity of 55±15 %, and the number of ventilation 10 to 20 times/ hr, illumination time of 12 hours (lights from 8 am to 8 pm), and illumination was set at 150-300 Lux. During the breeding period, environmental conditions such as temperature/humidity, ventilation frequency, and illuminance of the animal room were measured regularly. As a result of environmental measurement, no abnormalities that could adversely affect the results of the test were observed. Feed was freely ingested, and purified water was freely ingested using a polycarbonate drinking bottle.

G1 (normal group: untreated control group), G2 (infected group: covid-19 infection and no treatment), G3 (chimomodulin administered group: covid-19 infection and chymomodulin administration) as shown in the table below. were randomly divided into:

[Table 1]

Composition of the test group

Chimomodulin administered to group G3 was prepared by the following method: After weighing 5 g of chimomodulin powder (supplier: Samik Pharmaceutical Co., Ltd.), 100 mL of distilled water was added, followed by stirring for 10 minutes.

Virus infection was performed by subculturing the test virus SARS-CoV-2 (NCCP43326) and using a virus of 10 ⁴ TCID50/mL in PBS solution, and 200 μL of this virus solution was administered to the left nasal cavity of the hamster to infect ( G2 and G3 groups).

Group G3 was orally administered chimomodulin at the above dose once a day for a total of 8 weeks, and after infection with Covid-19, administration was continued for an additional 1 week at the same dose, and chimomodulin was administered for a total of 9 weeks.

The G2 group was infected with Covid-19 at the same time and in the same manner as the G3 group.

Experimental Example 2 : Observation items and test methods and results

1. Observation of general symptoms

Survival rate and weight change were observed as general symptoms every day for 7 days immediately after virus inoculation. Only in the case of abnormalities, abnormal symptoms, severity, and observation time were recorded on the clinical symptom observation record sheet. As a result of the observation of general symptoms, no deaths were observed during the entire experimental period, and no specific symptoms were observed due to the administration of the test substance.

2. Weighing

Each individual body weight was measured and recorded using an electronic scale every day for 7 days from the day of virus inoculation, and the rate of change of body weight compared to DPI 0 is shown in Table 2 below:

[Table 2]

As a result of body weight measurement, the body weight levels of the infected group (G2) and the SI-TM200508 (chimomodulin) administered group (G3) were significantly lower than those of the normal group (G1) from day 1 to day 5 after virus inoculation (p<0.001). , p<0.01 or p<0.05), the body weight level of G2 was significantly lower than that of G1 from day 6 to day 7 after virus inoculation (p<0.05). From the 4th to the 7th day after virus inoculation, the body weight of G3 was significantly higher than that of G2 (p<0.001, p<0.01, or p<0.05) (see FIG. 2 ).

The above results mean that weight loss due to Covid-19 infection can be significantly alleviated by administration of chymomodulin.

3. Autopsy and histopathology

Autopsies were performed on designated days immediately after virus inoculation. On the 3rd day after virus inoculation, 3 mice in each group in the G1 and G2 groups, 3 mice on the 5th day, and 3 mice on the 7th day were necropsied. Part of the lesion visible in the lung tissue was fixed in NBF, followed by H&E staining and microscopic examination. In the case of the G3 group, 5 mice on the 3rd day, 5 mice on the 5th day, and 5 mice on the 7th day after virus inoculation were autopsied. Some of the lesions visible in the lung tissue were fixed in NBF, followed by H&E staining and microscopic examination. The degree of inflammatory cell infiltration of the alveoli and interstitium was observed, and the results are shown in Tables 3 to 4 below (see FIGS. 3 to 4):

[Table 3]

[Table 4]

In G1, no abnormal lesions were observed in histopathology results during the entire autopsy period, and on the 7th day after virus inoculation, the level of inflammatory cell infiltration in the alveoli of G2 was significantly higher than that of G1 (p<0.01), and alveolar inflammation of G3 The level of cell invasion was significantly lower than that of G2 (p<0.05) [see Table 3]. On the 7th day after virus inoculation, the level of G2 interstitial inflammatory cell infiltration was significantly higher than that of G1 (p<0.05), and the level of G3 interstitial inflammatory cell infiltration was significantly lower than that of G2 (p<0.05) [see Table 4]. ]. In addition, G3 had lower levels of congestion and bleeding in lung tissue compared to G2.

These results mean that pathological consequences such as alveolar and interstitial inflammatory cell infiltration, congestion and hemorrhage caused by Covid-19 infection can be reduced or alleviated by chymomodulin treatment.

Additionally, macroscopic findings of autopsied lung tissues were evaluated on the 3rd, 5th, and 7th days after virus inoculation. In G1, no abnormal lesions were observed in gross findings during the entire autopsy period. From the 3rd to the 7th day after virus inoculation, it can be seen from the lung photographs of FIGS. 1A to 1C that the level of gross hemorrhage and damage of G3 was less than that of G2. Figure 1a is a photograph of the lungs of the G1, G2, and G3 groups on the 3rd day after virus inoculation, Figure 1b is a photograph of the lungs of the G1, G2, and G3 groups on the 5th day after the virus inoculation, and Figure 1c is the G1, These are pictures of the lungs of groups G2 and G3.

4. Real-time RT-PCR

Part of the lesion seen in the lung tissue collected at the time of autopsy was RNA extracted and the remaining amount of virus was evaluated using the Seegene Allplex 2019-nCoV assay kit (Cat no. RP10243X). The evaluation method is as follows:

A part of the lesion site (about 1 g) was placed in EP-TUBE and RNA prep was performed. GeneAll's kit was used, and the method was performed according to the manufacturer's method (GeneAll hybrid-R RNA purification kit; GeneAll, 3033522).

The isolated RNA was quantified using nanodrop (Take3 Multi-Volume plate, BioTeK, Instruments, VT, USA), and then diluted to the same concentration (100 ng/μL) for each experimental group.

Using the same amount of diluted RNA template, Seegene Allplex 2019-nCoV assay kit (Cat no. RP10243X) was used to confirm the expression of each gene at FAM, HEX, RED, and Cy5 wavelengths. Real time RT-PCR conditions were performed according to the manufacturer's method enclosed in the kit. FAM (E gene), RED (RdRP gene) and Cy5 (N gene) were used to confirm the double virus-related gene, and the reliability of real-time PCR was secured by checking the expression of the internal control gene using HEX. In addition, using the ct value of the internal control gene in the subsequent result confirmation section, the ct value of the virus gene was normalized to confirm and compare the gene expression level.

The FAM, RED, and Cy5 are protein genes constituting Covid-19 and are used in a diagnostic test to confirm whether or not Covid-19 infection is present. If the presence of each or all of them is confirmed, the infection is diagnosed as positive.

Check the viral gene expression level of RNA isolated from the lung tissue autopsied on the 3rd, 5th and 7th day after virus inoculation (FAM: E gene, RED: RdRP gene, Cy5: N gene), and the results are shown in Table 5 to Table 7 (see the results of Figures 5 to 7).

[Table 5]

[Table 6]

[Table 7]

As a result of real time RT-PCR, the FAM level of G3 was significantly lower than that of G2 on the 5th and 7th days after virus inoculation. (p<0.01/p<0.05). And on the 3rd, 5th, and 7th days after virus inoculation, the level of Cy5 of G3 was significantly lower than that of G2 (p<0.001/p<0.01/p<0.05). In addition, the level of RED in G3 on the 3rd and 7th days after virus inoculation was significantly lower than that of G2 (p<0.001/p<0.01). From the above results, it can be seen that the Covid-19 virus proliferation is effectively inhibited by chymomodulin administration.

Statistical processing of the results assumed normality of the data, and was analyzed using parametric multiple comparison procedures or non-parametric multiple comparison procedures. If the parametric one-way ANOVA result was significant, a post hoc test was performed using Dunnett's multiple comparison test. If the nonparametric Kruskal-Wallis'H-test analysis result was significant, the Mann Whitney U test A post-hoc test was performed using

Preparation Example 1: Preparation of chymomodulin-containing capsules

The preparation of the tablet according to this embodiment was based on the primary mixing of the active material and the excipient, followed by secondary mixing with magnesium stearate, a lubricant, to fill the capsule.

Silicon dioxide 5mg as an excipient to 80mg of chymomodulin, a pharmacologically active ingredient (manufactured after processing by collecting thymus from healthy cattle from Korea, New Zealand, China, Brazil, etc. to prevent contagious spongiform encephalopathy infection) Cellulose 30 mg, carboxymethyl cellulose calcium 120 mg, and calcium hydrogen phosphate hydrate 10 mg were put into a drum-type or V-type mixer and homogeneously mixed for 25±1 minutes. After that, 5 mg of magnesium stearate was added as a lubricant, and the mixture was homogeneously secondarily mixed for 5±1 minutes.

The mixed powder was filled in capsules to prepare chymomodulin-containing capsules.

Preparation Example 2: Preparation of chymomodulin-containing tablets

The preparation of the tablet according to this example was based on the primary mixing of the active material and the excipient, followed by secondary mixing with magnesium stearate, a lubricant, and direct tableting.

Silicon dioxide 5mg as an excipient to 80mg of chymomodulin, a pharmacologically active ingredient (manufactured after processing by collecting thymus from healthy cattle from Korea, New Zealand, China, Brazil, etc. to prevent contagious spongiform encephalopathy infection) 52 mg of cellulose, 120 mg of calcium carboxymethylcellulose, and 10 mg of calcium hydrogen phosphate hydrate were put into a drum-type or V-type mixer and homogeneously mixed for 25±1 minutes. Then, 3 mg of magnesium stearate as a lubricant was added and homogeneously mixed for 5±1 minutes.

Tablets were prepared by tableting the mixed powder to a total weight of 270 mg and an average hardness of 10±5 Kp using a rotary tableting machine.

Preparation Example 3: Preparation of an oral solution containing chymomodulin

Pharmacologically active ingredient chymomodulin (to prevent infectious spongiform encephalopathy infection, thymus from healthy cattle from Korea, New Zealand, China, Brazil, etc. is collected and processed after processing) 400 mg of purified water 80 mL, concentrated glycerin 10 mL and Add an appropriate amount of ethanol and stir for about 1 hour to dissolve, add appropriate amounts of 70% D-sorbitol solution and citric acid hydrate, and add sodium benzoate or methyl paraoxybenzoate or protyl paraoxybenzoate as a preservative in an appropriate amount to prevent microbial propagation After stirring for about 30 minutes, the mixture was adjusted to 100 mL with purified water to prepare a solution.

Claims (11)

A pharmaceutical composition for use in the prevention or treatment of Corona 19 virus (COVID-19) infection comprising chimomodulin and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 1, wherein the chimomodulin is included in an amount of 5 wt% to 35 wt%, based on the total weight of the pharmaceutical composition. According to claim 1, wherein the chimomodulin inhibits the corona 19 virus proliferation, the pharmaceutical composition. The pharmaceutical composition of claim 1 , which is administered orally. The pharmaceutical composition according to claim 4, which is a tablet, capsule or liquid. According to claim 1, wherein the pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, At least one selected from the group consisting of cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, a pharmaceutical composition. The pharmaceutical composition according to any one of claims 1 to 6, wherein a daily dose of 80 mg to 800 mg of chymomodulin is administered once a day or twice a day, for 1 week to 24 weeks. The method according to claim 7, wherein the pharmaceutical composition is administered in a daily dose of 80 mg to 800 mg of chimomodulin to a healthy person, the elderly or an underlying disease, once a day or A pharmaceutical composition, administered twice a day, for 4 to 24 weeks. The method of claim 7, wherein the pharmaceutical composition is a daily dose of 80 mg to 800 mg of chymomodulin to a corona 19 virus infected person for the treatment of Corona 19 virus (COvid-19) infection, once a day or 2 times a day Administered once, for 1 week to 24 weeks, the pharmaceutical composition. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition is administered together with other therapeutic agents, vaccines or infection symptom relievers effective for preventing, treating or alleviating symptoms of Covid-19. composition. 11. The method of claim 10, wherein the therapeutic agent is plasma treatment using plasma from a patient infected with a virus, monoclonal antibody therapy, ribavirin, penciclovir, nitacosanide, nafamostat, remdesivir, favipiravir, chloroquine, kaletra, avigan, arbidol, gallidesivir, or darunavir, a pharmaceutical composition.

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