KR20220081311A - Pharmaceutical composition comprising Niclosamide for intramuscular and/or subcutaneous administration - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for intramuscular and/or subcutaneous administration of niclosamide comprising niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof, wherein a high blood concentration is maintained for a long time. It can exhibit the effect that can be, and can be effectively used for the prevention or treatment of virus-related diseases.

Description

Pharmaceutical composition comprising niclosamide for intramuscular and/or subcutaneous administration

The present invention relates to a pharmaceutical composition for intramuscular and/or subcutaneous administration comprising niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, a use thereof, and a method for administering the same.

Numerous studies and techniques have been reported to utilize various pharmacological effects through systemic exposure of niclosamide.

First, oral and intravenous administration and improved in vivo drug exposure were reported by solubilizing niclosamide or applying various formulation methods.

According to the literature reported to Carbohydrate Polymers by Anurag Lodagekar et al., it was reported that in vitro anticancer activity and bioavailability in mice were increased by inclusion of niclosamide with cyclodextrin. However, even in this case, the half-life of the niclosamide/cyclodextrin inclusion compound presented in the literature was only 4.6 hours.

According to the literature reported in the Journal of Microencapsulation by Xingwang Zhang et al., when niclosamide was loaded into a submicron lipid emulsion, it was able to exhibit superior release rate compared to niclosamide in vitro and maintain the stability of the emulsion for a long time. However, when it was orally administered to rats, the observed half-life was only 2.6 to 3.2 hours.

According to the literature reported in Drug Development and Industrial Pharmacy by Yanghuan Ye et al., nanocrystals with a size of about 235 nm could be prepared by wet milling niclosamide with an appropriate surfactant. This was a formulation capable of intravenous administration of niclosamide, but the obtained half-life when administered intravenously to rats was only 1.36 hours.

According to the literature reported in 2018 by Maqsood Ur Rehman et al., when niclosamide is prepared as a solid lipid nanoparticle with lipids, the particle size can be prepared at a level of about 200 nm, and the dissolution rate of the drug is increased. It was confirmed that exposure to the body was increased, but a significant increase in half-life was not observed.

As such, when niclosamide is formulated in various ways and administered orally or intravenously, a commercially effective drug exposure profile has not been achieved.

Second, a method for synthesizing structural analogues by partially changing the structure of niclosamide has been reported because it is difficult to achieve significant improvement even when various formulations are applied to niclosamide.

According to the literature reported by Yi-Wei Chang et al. in the Journal of food and drug analysis in 2006, niclosamide exhibits very rapid elimination when administered intravenously, and also shows rapid elimination when administered orally. activity could not be achieved. Therefore, the authors of this document tried to overcome this by synthesizing a number of analogs with some changes in the structure of niclosamide. However, as is known in the art, when developing a substance using this structural analog (analog), it is considered a new drug substance completely different from niclosamide, so the same toxicity test and clinical test as the new drug are required, so the development period of at least 10 years This is necessary, and there is a possibility that the toxicity will increase compared to niclosamide, which has been safe for decades.

Similarly, the literature reported to ACS Medicinal Chemistry Letters by Haijun Chen et al. also discloses the possibility of application as anticancer by designing and synthesizing O-Alkylamino-tethered analogs to improve the inappropriate biokinetics of niclosamide. As in the above literature, this suggests the difficulty of improvement using niclosamide itself and the necessity of developing a new drug substance through the synthesis of structural analogues.

Third, when niclosamide is developed as a treatment for lung disease or pulmonary infection, a study of delivering niclosamide drug particles directly to the lung, the site of action, has been reported.

Ashlee D. Brunaugh et al. recently reported the development of inhalable particles by encapsulating niclosamide in human lysozyme.

As seen in the prior art, there was no technique for intramuscularly and/or subcutaneously administering a drug without changing the chemical structure of niclosamide, and also a technique showing an excellent blood half-life profile like the composition disclosed in the present invention. not reported

On the other hand, in order to commercially utilize niclosamide's strong antiviral activity and various reported in vitro activities (anti-inflammatory, anticancer, etc.), it is necessary to maintain the drug concentration in the body. To this end, various attempts have been made around the world.

In addition, when niclosamide is orally administered, it is known that a large amount of microbial reduction and glucuronic acid inclusion reaction occur in the intestine. In addition, various gastrointestinal side effects have been reported during oral administration, which is attributed to the properties of the drug distributed in large quantities in the gastrointestinal tract after oral administration.

The present invention solves the problems of oral and intravenous administration of niclosamide, and simplifies the administration method by administering niclosamide a single time intramuscularly and/or subcutaneously, and is a pharmaceutical that can maintain effective blood concentration in the body It aims to provide a composition.

The present invention relates to a muscle of niclosamide comprising niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, and administering 24 mg to 1500 mg of niclosamide in a single total dose. Provided is a pharmaceutical composition for intra- and/or subcutaneous administration.

The present invention also provides the use of the pharmaceutical composition for intramuscular and/or subcutaneous administration of niclosamide and a method for intramuscular and/or subcutaneous administration of the pharmaceutical composition to a subject in need thereof. .

Through the present invention, when niclosamide is administered intramuscularly and/or subcutaneously, superior bioavailability can be achieved compared to oral administration, and the blood concentration of niclosamide can be maintained at a drug concentration higher than the virus inhibitory concentration for a long time.

It does not require hospitalization facilities for intravenous administration, so it can be a great help in saving important medical personnel and facilities in case of an infectious disease outbreak, and has the advantage that various gastrointestinal side effects that may occur during oral administration do not appear.

Through the present invention, it is possible to apply the wide range of antiviral activity of niclosamide in literature to the human body, and it is possible to secure an antiviral drug that can be used preferentially during an epidemic as well as industrial development and profit.

1 shows a PK graph of IV administration and IM administration according to Example 2.
2 is a graph showing the PK values of IM administration according to Example 2 and IC50 concentrations of 55.6 ng/mL and 91.6 ng/mL.
Figure 3 shows a PK graph of IM administration and SC administration by GMP according to Example 3.
Figure 4 shows the PK graph of the phase 1 clinical trial in India according to Example 4.
5 shows a simulated PK graph for each dose of IM administration according to Example 5;
6 shows a PK graph simulating administration of 960 mg IM according to Example 5.
7 shows a PK graph simulated by multiple administration of niclosamide according to Example 6 (a total of 840 mg for 2 days with 1.0 cc (240 mg) of the arm + 2.5 cc (600 mg) of the hip).
FIG. 8 shows a PK graph simulated by multiple administration of niclosamide according to Example 6 (total of 960 mg for 2 days with 1.0 cc (240 mg) of the arm + 3.0 cc (720 mg) of the hip).
9 shows a PK graph simulating multiple administration of niclosamide according to Example 6 (2.0cc (480mg) on both sides of the arm or hip for 2 days).
10 shows a PK graph measured for each dose of niclosamide according to Example 7-1-1.

Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description.

However, this is not intended to limit the present invention to specific embodiments, and it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

The present invention relates to a muscle of niclosamide comprising niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof, and administering 24 mg to 1500 mg of niclosamide in a single total dose. Provided is a pharmaceutical composition for intra- and/or subcutaneous administration.

The pharmaceutical composition comprising niclosamide may be administered in a single dose or in multiple doses. Single or multiple administration, for example, does not mean the number of injections, but means whether the administration cycle of the pharmaceutical composition containing niclosamide is single or multiple. For example, even if divided administration of the pharmaceutical composition is performed several times, if the administration cycle corresponds to one time, it means "single". As can be seen in the Examples of the present invention, it may be desirable to administer a single total dose in divided doses for the pharmaceutical composition containing niclosamide, but such divided administration is not considered to be one time in the administration cycle. Even if a single total dose is divided into several parts of the body, if it is included within the same administration cycle, it is considered to be a single dose.

The single total dose refers to the sum of the divided doses when a single dose is divided into a single dose of the pharmaceutical composition according to the present invention.

The single total dose may be administered intramuscularly and/or subcutaneously at one site, or dividedly administered at multiple sites intramuscularly and/or subcutaneously.

The single total dose may be 96 mg to 1500 mg, or 96 to 1200 mg. More specifically, it may be 96 mg or more, 100 mg or more, 120 mg or more, 144 mg or more, 150 mg or more, and 1500 mg or less, 1400 mg or less, 1300 mg or less, 1200 mg or less. The single total dose may be 300 to 1200 mg, or 800 to 1200 mg.

Depending on the age, condition, or symptom level of the patient, the dosage may be increased from 1/2 to 1/10, specifically 1/2, 1/3, 1/4, 1/5, or 10 It can be administered by adjusting the dosage by one minute. For example, if the dosage is 960 mg, one-half of 480 mg may be administered, one-third 320 mg, one quarter 240 mg, one fifth 192 mg, and one tenth 96 mg can be administered.

The concentration of niclosamide in the pharmaceutical composition may be 200 to 300 mg/ml. More preferably, it may be 200 to 280 mg/ml, 200 to 270 mg/ml, 210 to 270 mg/ml, or 220 to 260 mg/ml, and more specifically 240 mg/ml.

In one aspect of the present invention, the body part of the intramuscular and/or subcutaneous administration may be an arm, hip, thigh and/or abdomen, and more specifically, both arms, both hips, both thighs, and/or abdomen. However, the present invention is not limited thereto.

In one embodiment of the present invention, it may be administered more than once to the same or different parts of the body. Specifically, it may be administered 1 to 6 times in the same or different parts of the body.

In one embodiment of the present invention, the single administration of niclosamide is once per arm; 1 in the butt; 1 time on the thigh; 1 in the abdomen;

2 times, 1 on each arm; 2 times, 1 on each hip; 2 times, 1 on each thigh; 2 times, 1 on the arm and 1 on the hip; 2 times, 1 on the arm and 1 on the thigh; 2 times, 1 on the arm and 1 on the abdomen; 2 times, 1 on the hip and 1 on the thigh; 2 times, 1 in the hip and 1 in the abdomen; 2 times, 1 on the thigh and 1 on the abdomen;

3 times, 1 on each arm and 1 on the hip; 3 times, 1 on each arm and 1 on the thigh; 3 times, 1 on each arm and 1 on the abdomen; 3 times, 1 on each hip and 1 on each arm; 3 times, 1 on each hip and 1 on the thigh; 3 times, 1 on each hip and 1 on the abdomen; 3 times, 1 on each thigh and 1 on each arm; 3 times, 1 on each thigh and 1 on the buttocks; 3 times, once on each thigh and once on the abdomen; 3 times, 1 on the arm, 1 on the hip, and 1 on the thigh; 3 times, 1 time on the arm, 1 time on the thigh, and 1 time on the abdomen; 3 times, 1 on the arm, 1 on the hip, and 1 on the abdomen; 3 times, 1 time on the buttocks, 1 time on the thighs, and 1 time on the abdomen;

4 times, 1 on each arm and 1 on each hip; 4 times, 1 time on each arm and 1 time on each thigh; 4 times, 1 on each hip and 1 on each thigh; 4 times, 1 on each arm, 1 on the hip, and 1 on the thigh; 4 times, 1 on each arm, 1 on the hip and 1 on the abdomen; 4 times, 1 time on each arm, 1 time on the thigh and 1 time on the abdomen; 4 times, 1 on each hip, 1 on the arm, and 1 on the thigh; 4 times, 1 on each hip, 1 on the arms and 1 on the abdomen; 4 times, 1 on each hip, 1 on the thigh, and 1 on the abdomen; 4 times, 1 on each thigh, 1 on the arm, and 1 on the hip; 4 times, once on each thigh, once on the arms and once on the abdomen; 4 times, 1 time each on both thighs, 1 time on the buttocks and 1 time on the abdomen; 1 time on the arm, 1 time on the hip, 1 time on the thigh, 1 time on the abdomen, a total of 4 times;

5 times, 1 time on each arm, 1 time on each hip, 1 time on the thigh; 5 times, 1 time on each arm, 1 time on each hip, 1 time on the abdomen; 5 times, 1 time on each arm, 1 time on both thighs, 1 time on the buttocks; 5 times, 1 time on each arm, 1 time on both thighs, 1 time on the abdomen; 5 times, 1 time on each hip, 1 time on both thighs, 1 time on the arms; 5 times, 1 time on each hip, 1 time on both thighs, 1 time on the abdomen; 1 time on each arm, 1 time on the buttocks, 1 time on the thigh, 1 time on the abdomen, a total of 5 times; 5 times, 1 time on each hip, 1 time on the arms, 1 time on the thighs, and 1 time on the abdomen; 1 time on each thigh, 1 time on the arms, 1 time on the buttocks, 1 time on the abdomen, a total of 5 times;

6 times, 1 time on each arm, 1 time on each hip, 1 time on both thighs; 6 times, 1 time on each arm, 1 time on both hips, 1 time on the thigh, 1 time on the abdomen; 6 times, 1 time on each arm, 1 time on both thighs, 1 time on the buttocks and 1 time on the abdomen; or 1 time on each hip, 1 time on both thighs, 1 time on the arms, 1 time on the abdomen for a total of 6 times; can be administered.

The dosage of the pharmaceutical composition for each administration site may be 0.1ml to 5ml. Specifically, it may be 0.2ml to 4.5ml, 0.3ml to 4.3ml, or 0.5ml to 4ml.

In one example, when the administration site is the arm, the dosage of the pharmaceutical composition may be 2ml or less, 1.5ml or less, 1.2ml or less, 1ml or less, 0.1ml or more, 0.3ml or more, 0.5ml or more, specifically , may be 0.1ml to 2ml, 0.3ml to 1.5ml, 0.5ml to 1.5ml, 0.5ml to 1.2ml, or 0.6ml to 1.1ml, preferably 0.8ml.

In another example, when the administration site is the buttocks, the dosage of the pharmaceutical composition may be 5ml or less, 4.5ml or less, 4ml or less, 0.5ml or more, 1ml or more, specifically, 0.5ml to 5ml, It may be 0.5ml to 4.5ml, 1ml to 4ml, 1ml to 3ml, 1ml to 2ml, or 1ml to 1.5ml, preferably 1.2ml.

The correlation between the dose (ml) of the pharmaceutical composition, the concentration of niclosamide (mg/ml), and the dose (mg) of niclosamide is ‘dose of the pharmaceutical composition (ml) X concentration of niclosamide (mg). (mg/ml) = administration dose of niclosamide (mg)' can be confirmed by the formula, and each numerical value can be appropriately adjusted with the formula. In one example, if the concentration of niclosamide is 240 mg/ml, for example, if the dosage of the pharmaceutical composition is 0.1 ml to 5 ml for each administration site, the dosage of niclosamide for each administration site is 24 mg to 1200 mg.

The pH of the pharmaceutical composition may be 4 to 7, specifically, may be pH 6. The higher the pH, the higher the solubility, which may decrease the formulation stability of niclosamide. The present formulation can be properly maintained at a pH of 4 to 7 without using a separate pH adjusting agent.

The pharmaceutical composition may include niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or a solvate thereof as an active ingredient, and a stabilizer, an isotonic agent and a buffer as an excipient.

Here, the pharmaceutical composition may be a liquid formulation or a freeze-dried formulation. Here, the liquid agent may be a suspension. In addition, the freeze-dried formulation can be used by rehydrating it with water for injection before administration to make a suspension.

The stabilizer may include an anionic polysaccharide cellulose derivative or a salt thereof. More specifically, the anionic polysaccharide may be a carboxy polysaccharide, and the anionic polysaccharide cellulose derivative may be carboxymethyl cellulose, carboxyethyl cellulose, carboxymethyl chitosan or carboxymethyl dextran, but is not limited thereto. More specifically, the anionic polysaccharide cellulose derivative or a salt thereof may be carboxymethyl cellulose or a salt thereof, but as an anionic polysaccharide cellulose derivative, it is applied as an excipient of a pharmaceutical preparation to exhibit the effect of the present invention. .

Buffers may further include phosphate and sodium salts. The phosphate and sodium salt may be derived from sodium phosphate monobasic monohydrate, but is not limited thereto.

The tonicity agent may additionally include mannitol, but is not limited thereto, and as long as it is an excipient that can be applied to the injection formulation as a sugar alcohol, it can be changed and applied as needed.

The pharmaceutical composition may be used to prevent or treat various virus-related diseases.

[Various antiviral activity]

The potent activity of niclosamide against SARS-CoV was first reported in the literature reported by Chang-Jer Wu et al. in Antimicrobial Agents and Chemotherapy. The IC50 inhibitory concentration was reported to be at the level of 1.56 uM. Since then, various antiviral tests using niclosamide have been reported in vitro.

It was found by Gassen et al. that niclosamide increased viral autophagy in host cells through SKP2 inhibition, and it was reported that it exhibited potent activity against MERS-CoV. In addition, in 2020, it has been reported from multiple research groups that niclosamide exhibits strong activity against SARS-CoV-2.

As above, niclosamide has been reported to have strong activity against beta-coronaviruses such as SARS-CoV, SARS-CoV-2, and MERS-CoV.

The antiviral activity of niclosamide has also been reported against Flavivirus to which Zika virus, dengue virus, West Nile virus, yellow fever virus and Japanese encephalitis virus belong.

Xu et al. Nat. Med. According to the literature reported in 2016, 22, 1101-1107, it was found that niclosamide exhibited an IC50 value of 0.37 Um level against Zika virus and had a strong antiviral effect.

Li et al., Cell Res. According to the literature reported in 2017, 27, 1046-1064, it is known that niclosamide directly inhibits Flavivirus replication by interfering with the NS2B-NS3 interaction during the viral replication process of Flavivirus. Therefore, this study suggested that niclosamide would be effective against dengue, West Nile, yellow fever, and Japanese encephalitis viruses.

In addition, Fang et. Al is PLoS ONE. The activity of niclosamide against Japanese encephalitis virus was also reported in the literature reported in 2013, 8, e78425.

It was reported by Stachulski et al. that niclosamide exhibits potent antiviral activity at a level of 0.16 uM against human hepatitis C virus (HCV) (J. Med. Chem. 2011, 54, 8670-8680).

Activity against the Ebola virus, which shows a very high fatality rate during infection, and causes a serious level of infection in Africa, has also been reported. Madrid et al. ACS Infect. Dis. According to the literature reported in 2015, 1, 317-326., niclosamide has an EC50 value of 1.5 uM for Ebola virus, and its efficacy in animal models has not yet been demonstrated.

The antiviral activity of niclosamide and its mechanism have also been reported on the antiviral activity of niclosamide against the rhinovirus, which causes various types of cold in humans and shows a fatality rate when infected with asthma or chronic obstructive pulmonary disease. Jurgeit et al. PLoS Pathog. According to the literature reported in 2012, 8, e1002976., it was reported that niclosamide exhibited an IC50 value of less than 1 uM for rhinovirus, and that niclosamide interfered with the formation of an acidic environment necessary for viral infection/replication. In addition, this literature reported that niclosamide exhibited an IC50 value of 0.83 uM for influenza.

For the Chikungunya virus, which is characterized by fever and joint pain upon infection and is known to be actively transmitted by female mosquitoes, Wang et al., Antiviral Res. Antiviral activity and mechanism by niclosamide were reported in 2016, 135, 81-90. The EC50 value of niclosamide against chikungunya virus was 0.95 uM, and it was reported that niclosamide prevented not only cell penetration of chikungunya virus but also cell-to-cell virus transmission.

For adenoviruses for which currently developed antiviral agents do not exist, Marrugal-Lorenzo et al. Antiviral Res. 2016, 135, 81-90. reported the antiviral activity of niclosamide. Specifically, it was reported that niclosamide exhibited an EC50 value of 0.6 uM, and niclosamide inhibited intracellular migration of human adenovirus particles.

Among human herpes viruses known to infect most of the world's population, activity against Epstein-Barr Virus (EBV) has also been reported. Huang et al. Antiviral Res. According to the literature reported in 2017, 138, 68-78., niclosamide inhibited EBV replication in lymphocytes and epithelial cells, and the inhibitory mechanism was a disruption of the mammalian target of rapamycin (mTOR) signaling pathway, which is important in EBV replication in vivo. .

According to the literature reported by Niyomdecha et al. in Heliyon 6 (2020) e04050, it was reported that niclosamide inhibited the replication of human inmmunodeficiency virus type 1 (HIV1) through inhibition of the mTORC1 signaling pathway.

As above, niclosamide is known to exhibit various antiviral activities regardless of the type of virus, but a method for maintaining long-term antiviral activity concentration by effectively systemic exposure to niclosamide for several decades has not been reported.

Through the present invention, it is possible to develop various antiviral therapeutic agents using niclosamide, and in addition to the antiviral activity reported above, it can be applied to almost all viral diseases, and to respond to newly generated viruses and virus variants. It is possible.

In one embodiment of the present invention, the pharmaceutical composition, the pharmaceutical composition is influenza virus (Influenza virus), flavivirus (Flavivirus), adenovirus (Human adenovirus, HAdV), coronavirus severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), MERS-CoV, coronavirus disease 2019 (COVID) -19)), Herpes virus, Zica virus, Japanese encephalitis virus (JEV), Epstein-Barr virus (EBV), Ebola virus (EBOV) ), rhinovirus, Chikungunya virus (CHIKV), hepatitis C virus (HCV), hepatitis B virus (HBV), hepatitis A virus , HAV), rotavirus, Astrovirus, Hantavirus, Dengue virus, SFTS virus (severe fever with thrombocytopenia syndrome virus), HIV virus, West Nile Virus (WNV) , and it may be for the prevention or treatment of one or more viral infections selected from the group consisting of yellow fever virus.

In one embodiment of the present invention, the pharmaceutical composition may be for preventing or treating coronavirus disease 2019 (COVID-19).

In addition, the pharmaceutical composition of the present invention may be administered not only to a patient exhibiting infection symptoms or signs of infection, but also to a patient likely to become infected.

The blood concentration in the body for 3 to 14 days after administration of the pharmaceutical composition may exceed the IC ₅₀ value for coronavirus infection-19 (Covid-19) of niclosamide.

Here, the IC ₅₀ of niclosamide for coronavirus infection-19 (Covid-19) may be 45 to 100 ng/ml, 47 to 97 ng/ml, 50 to 95 ng/ml, or 53 to 93 ng/ml. have.

On the other hand, after the initial administration of the pharmaceutical composition, a pharmaceutical composition comprising niclosamide or a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof is additionally administered at a dose of 24 mg to 1500 mg 1 to 4 times more. can be administered.

Here, the administration site of the initial administration of niclosamide and the additional administration may be the same or different.

In one embodiment of the present invention, the additional administration of niclosamide may be further administered 1 to 4 times for one month, 3 weeks, or 2 weeks after the initial administration.

In a further aspect of the present invention, the additional administration of niclosamide is once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, during the treatment period after the initial administration. , once every 7 days, once every 8 days, or once every 9 days can be administered 1 to 3 more times in total. The dosage regimen can be designed so that the blood concentration of niclosamide can be maintained, for example, for 7 to 14 days, above the IC50 concentration of niclosamide for the target virus. For example, the dosage regimen may be varied by administering at least 436 mg or more once in 2 days, administering at least 600 mg or more once in 3 days, for a total of 3 times, and at least 960 mg or more once in 4 days for a total of 3 administrations, etc. can be set.

Additional administration of niclosamide may be administered by the same administration method as the administration method of single administration of niclosamide described above.

The present invention also includes niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof for intramuscular and/or subcutaneous administration of niclosamide, and a single total dose of 24 mg to 1500 mg. Use of a pharmaceutical composition characterized in that niclosamide is administered may be provided.

The present invention may also provide a method for intramuscularly and/or subcutaneously administering the pharmaceutical composition to a subject in need thereof.

<Example>

"DWRX2003" used in the following examples and drawings means niclosamide.

Example 1: Confirmation of SARS-CoV-2 virus treatment effect in hamsters following administration of niclosamide

(1) Hamster preparation and test group composition

The species and strains of the hamsters were Syrian hamster, RjHan:AURA, and 27 males were classified into the following G1 (untreated control group), G2 (infected group), and G3 (test group) 9 each.

gender Number of animals (number of animals) Infection presence test substance route of administration Dosage (mg/kg) Dosing volume (mL/kg) G1 M 9 - - - - - G2 M 9 + - - - One G3 M 9 + niclosamide intramuscular administration 240 One

(2) Virus preparation and infection SARS-CoV-2 was subcultured to prepare a virus of 10 ⁴ TCID 50/ml in a PBS solution, and 200ul of this solution was administered to the left nasal cavity of the hamster.

(3) administration

Just before viral infection, niclosamide was administered once, and the total dose was divided into two and administered to the left and right thighs of the hamster.

(4) Experimental results

Survival rates and body weight changes were observed daily for 7 days immediately after virus inoculation and niclosamide administration, and 3 animals in each test group were necropsied on the 3rd, 5th and 7th days after virus inoculation and niclosamide administration. Part of the lesion seen from the lung tissue collected at the time of autopsy was extracted with RNA, and virus gene amplification test was performed through RT-PCR using the Seegene Allplex 2019-nCoV assay kit (Cat no. RP10243X), and the remaining amount of virus was evaluated.

1) Death and weight measurement

No dead animals were observed during the entire experimental period, and no other specific symptoms due to niclosamide administration were observed. As a result of body weight measurement, the body weight level of the G3 group (test substance administered group) was significantly higher than that of the G2 group (infected group) from the 2nd to 7th days after virus inoculation.

Weight (g) DPI G1 G2 G3 0 111.09 ± 16.70 137.92 ± 24.41** 130.17 ± 10.13 One 113.60 ± 15.99 130.41 ± 21.52 122.93 ± 10.06 2 116.52 ± 15.98 126.06 ± 21.22 120.93 ± 11.10 3 118.80 ± 16.05 120.75 ± 19.60 118.87 ± 11.77 4 118.16 ± 17.21 (6) 117.85 ± 12.35 (6) 117.51 ± 5.46 (6) 5 119.28 ± 16.76 116.16 ± 11.19 120.07 ± 6.71 6 123.02 ± 24.65 (3) 117.42 ± 13.42 (3) 121.42 ± 6.49 (3) 7 123.61 ± 25.07 117.53 ± 14.06 122.56 ± 5.66 N 9 9 9

DPI is the number of days after virus infection, the number in parentheses is the number of surviving individuals, and ** indicates a significant difference in p<0.01 compared to the G1 group.

2) Virus gene amplification test through RT-PCR

Virus gene expression levels of RNA isolated from lung tissue autopsied on the 3rd, 5th and 7th days after virus inoculation were confirmed. The E gene means the envelope in the structure of the virus, and plays a role in protecting the genetic material when moving between host cells. The N gene refers to the nucleocapsid in the structure of the virus and plays an important role in RNA replication and new virus assembly. RdRP gene refers to viral RNA-dependent RNA polymerase.

As a result, the G2 group had the lowest cq value, which is the viral replication cycle value, in all items (FAM, RED, and Cy5) during the entire test period. This means that there are the most viral genes in the infected lung tissue, and the relative expression value of the G3 group was calculated based on this. On the 3rd, 5th, and 7th days after virus inoculation, the relative expression level of the G3 group was significantly lower than that of the G2 group in all items (FAM, RED and Cy5).

FAM (E gene), unit: cq DPI G1 G2 G3 3 - 22.43 ± 0.69 24.48 ± 0.24 5 - 18.52 ± 4.59 26.84 ± 1.63 7 - 21.61 ± 5.00 30.79 ± 0.48 N 9 9 9

RED (RdRP gene), unit: cq DPI G1 G2 G3 3 - 18.64 ± 2.81 19.64 ± 0.80 5 - 21.79 ± 2.86 26.61 ± 2.00 7 - 20.35 ± 3.40 29.59 ± 0.87 N 9 9 9

Cy5 (N gene) , unit: cq DPI G1 G2 G3 3 - 22.43 ± 0.69 24.48 ± 0.24 5 - 18.52 ± 4.59 26.84 ± 1.63 7 - 21.61 ± 5.00 30.79 ± 0.48 N 9 9 9

3) Macroscopic findings The macroscopic findings of the autopsied lung tissue were evaluated on the 3rd, 5th, and 7th days after virus inoculation. In the G1 group, no abnormal lesions were observed in gross findings during the entire autopsy period. In the case of gross findings in the G2 group, petechiae were observed on the 3rd, 5th, and 7th days of virus inoculation, but the G3 group showed a more relaxed result compared to the G2 group. In the case of the G3 group, petechiae and lung tissue damage on the 3rd day of virus inoculation were delayed compared to the G2 group, and less gross damage was observed on the 5th and 7th days of virus inoculation.

4) Histopathological examination

After virus inoculation, the autopsied lung tissues on the 3rd, 5th, and 7th days were evaluated by H&E staining after fixing with a fixative. Each photograph was evaluated by scoring according to the evaluation criteria of Table 6 below, and the results are shown in Tables 7 to 11 below.

In the G1 group, no abnormal lesions were observed in histopathological results during the entire autopsy period. In the case of G3, as a result of histopathology on the 3rd day of virus inoculation, weak interstitial inflammatory cell infiltration appeared, but it was confirmed that it was relieved compared to the G2 group on the 5th day of virus inoculation. On the 7th day of virus inoculation, weak interstitial inflammatory cell infiltration was observed in the G3 group. It seems that lung tissue damage caused by the infected virus had a significant effect on the initial stage of infection, but niclosamide alleviated or delayed lung tissue damage by initially inhibiting virus proliferation. In addition, as a result of statistical analysis of histopathology scoring (Scoring), it can be confirmed that the G3 group has lower values than the G2 group on the 3rd, 5th, and 7th days after virus inoculation.

INFLAMMATORY CELL INFILTRATION, ALVEOLAR SPACE DPI G1 G2 G3 3 0.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 5 0.0 ± 0.0 2.3 ± 0.6 0.7 ± 0.6 7 0.0 ± 0.0 0.7 ± 0.6 0.3 ± 0.6 N 9 9 9

INFLAMMATORY CELL INFILTRATION, INTERSTITIAL DPI G1 G2 G3 3 0.0 ± 0.0 2.0 ± 0.0 1.7 ± 0.6 5 0.0 ± 0.0 2.0 ± 1.7 1.3 ± 0.6 7 0.0 ± 0.0 1.3 ± 1.2 0.7 ± 0.6 N 9 9 9

INFLAMMATORY CELL INFILTRATION, PERVASCULAR DPI G1 G2 G3 3 0.0 ± 0.0 2.3 ± 0.6 1.3 ± 0.6 5 0.0 ± 0.0 2.3 ± 0.6 1.3 ± 0.6 7 0.0 ± 0.0 2.0 ± 1.3 0.7 ± 0.6 N 9 9 9

HISTOPATHOLOGICAL EXAMINATION DPI G1 G2 G3 3 0.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 5 0.0 ± 0.0 0.7 ± 0.6 1.0 ± 0.0 7 0.0 ± 0.0 0.7 ± 0.6 1.0 ± 0.0 N 9 9 9

HEMORHAGE DPI G1 G2 G3 3 0.0 ± 0.0 0.7 ± 1.2 1.0 ± 0.0 5 0.0 ± 0.0 1.0 ± 1.0 1.0 ± 0.0 7 0.0 ± 0.0 1.3 ± 0.7 1.0 ± 0.0 N 9 9 9

Therefore, when niclosamide was administered intramuscularly during viral infection, it was possible to confirm weight recovery, suppression of viral gene expression level, and alleviation of tissue damage.

Example 2. Confirmation of PK (pharmacokinetic data) results of intravenous and intramuscular administration of niclosamide to hamsters

Two groups of 5 hamsters were prepared, one group was administered with niclosamide by intravenous administration (IV) and the other group by intramuscular administration (IM). Thereafter, pharmacokinetic evaluation was performed for each hamster, and the results are shown in Tables 12 and 13 and FIGS. 1 and 2 below.

When niclosamide is administered intravenously, it is rapidly eliminated from the body and it can be seen that most of the niclosamide in the body is decomposed in less than 24 hours. However, when administered intramuscularly, it was confirmed that the concentration in the body can be maintained at a high level even after 300 hours after administration.

Example 3. Confirmation of PK (pharmacokinetic data) results after intramuscular and subcutaneous administration of niclosamide to SD rats

The same composition was administered intramuscularly and subcutaneously in rats to observe pharmacokinetic changes. First, niclosamide prepared by the GMP process was intramuscularly administered to three rats, and niclosamide prepared by the GMP process was intramuscularly administered to the other three rats. Thereafter, pharmacokinetic evaluation was performed for each rat group, and the results are shown in Tables 14, 15 and 3 below. (Table 14 is niclosamide IM administration prepared by GMP process, Table 15 is GMP It shows administration of niclosamide SC prepared by the process.) As a result, it can be seen that, like IM administration, SC administration has a very long half-life, and the drug is slowly released and exposure to the body is increased.

Example 4. Confirmation of PK (pharmacokinetic data) results according to intramuscular administration of niclosamide in humans

A phase 1 clinical trial was conducted in healthy adults to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of niclosamide by dose in India. Eight patients per dose group (6 patients in the niclosamide group, 2 patients in placebo) were assigned, and after the safety and tolerability of the previous dose group were confirmed, a step-by-step escalation test proceeded to the next dose group.

As shown in Table 16 below, each dose group was injected intramuscularly once into a total of 4 regions of the arm and hip muscles. After administration, blood samples were collected from the subjects and the concentration of niclosamide in plasma of the drug was measured by HPLC-MS/MS. The blood concentration curve according to time after intramuscular injection of DWRX2003 144mg, 432mg, 960mg, and 1200mg is shown in FIG.

capacity group arms (both sides) buttocks (both sides) Cohort 1 - 144 mg 0.1 mL/site*2 sites 0.2 mL/site*2 sites Cohort 2 - 432　mg 0.3 mL/site *2 sites 0.6 mL/site*2 sites Cohort 3 - 960 mg 0.8 mL/site*2 sites 1.2 mL/site*2 sites Cohort 4 - 1200 mg 1.0 mL/site *2 sites 1.5 mL/site*2 sites

Example 5. Dosage setting of niclosamide in humans The expected effective plasma concentration of niclosamide calculated as IC50 from the results reported for SARS-CoV-2 inhibitory efficacy is 55.6 to 91.56 ng/mL (Jeon et al. al., 2020 in vitro inhibition of SARS-CoV-2 (S type) virus infection using Vero cells; Gassen et al., 2020 in vitro inhibition of SARS-CoV-2 (G type) virus infection using Vero cells; See Ko et al., 2020 in vitro inhibition of SARS-CoV-2 (S type) virus infection using Calu-3 cells)

In addition, based on the results of Examples 1 to 4, the effective dose capable of representing the effective blood concentration was predicted based on the body concentration of niclosamide according to time in humans at the administration doses of 144 mg, 432 mg, 960 mg, and 1200 mg. The results are shown in FIG. 5 . As can be seen in FIG. 5 , the time in which the blood concentration is maintained at the IC50 in vitro is predicted to be at least 3 days to a maximum of 14 days depending on the dose.

The administration dose in humans showing similar exposure to 240 mg/kg administration, which is the administration dose in the hamster model according to Example 1, was predicted to be 960 mg, which is summarized in Table 17, and a simulation graph is shown in FIG. 6 .

capacity group human hamster Dose (mg) 960 31.2 (240 mg/kg) Cmax (ng/mL) 695.6 420.9 AUC _0-168h (ng h/mL) 23,745 28,687 AUC _0-336h (ng h/mL) 31,453 36,553

A phase 2 clinical trial was designed with a dose of 960 mg of niclosamide. Specifically, a single intramuscular administration is performed by dividing into a test group and a control group as shown in Table 18 below to a mild or severe patient confirmed with COVID-19 or a person who has developed one or more symptoms within 7 days, and, accordingly, a patient negative to the virus Check whether it shows the effect of treatment for COVID-19, such as the rate and the proportion of those who recover from symptoms.

Example 6. Simulation of multiple doses of niclosamide

In addition to single administration of niclosamide, it is possible to maintain the in-vitro IC50 concentration (55.6 to 91.56 ng/mL) or higher for about 14 days by administering multiple doses (eg, administration over several days). As the repeat dosing interval increases, the required dose may also increase proportionally.

Three simulations were performed to confirm the multiple administration of niclosamide. In the first simulation, a total of 840 mg of niclosamide was administered repeatedly for 2 days to 1.0 cc (240 mg) of the arm + 2.5 cc (600 mg) of the hip, and the results are shown in FIG. 7 . In the second simulation, a total of 960 mg was repeatedly administered for 2 days to 1.0 cc (240 mg) of the arm + 3.0 cc (720 mg) of the hip, and the results are shown in FIG. 8 . In addition, in the third simulation, niclosamide was administered to each side of the arm or hip for 2 days at 2.0cc (480mg), and the results are shown in FIG. 9 .

It can be confirmed that the effective blood concentration in the body can be maintained high even when niclosamide is administered multiple times by dispersing it twice or more at a total dosage similar to single administration of niclosamide.

Experimental Example 7. Results of phase 1 clinical trial of niclosamide

Experimental Example 7-1. Results of phase 1 clinical trial of niclosamide in Korea

A randomized, double-blind, placebo-controlled, single-dose, dose escalation phase 1 clinical trial was conducted in healthy volunteers. In Part 1, a total of 16 test subjects were assigned to two cohorts, and the test drug and control drug were randomly assigned to each in a ratio of 6:2. Also, in Part 2, a total of 12 test subjects were randomly assigned to two cohorts at a ratio of 1:1 and administered.

The cohorts, doses, and administration sites of Parts 1 and 2 are shown in Table 19 below.

test group total capacity Administration site:
Dose per administration site, volume xn sites Part 1 cohort 1 96 mg Both buttocks:
48 mg/0.2 mL x 2 sites cohort 2 432 mg Both arms:
72 mg/0.3 mL x 2 sites
Both buttocks:
144 mg/0.6 mL x 2 sites part 2 Cohort A 144 mg Both arms:
72 mg/0.3 mL x 2 sites Cohort B 144 mg Both buttocks:
72 mg/0.3 mL x 2 sites

Experimental Example 7-1-1. Evaluation of the pharmacokinetics of niclosamide

Pharmacokinetic parameters were obtained for each subject by a noncompartmental analysis method using Phoenix WinNonlin 8.3 (CERTARA USA Inc.) and SAS ^® (Version 9.4, SAS Institute Inc. NC. USA), and the values were collected for each cohort. Pharmacokinetic values were summarized for each, and the results are shown in Table 20.

cohort 1 Cohort A Cohort B cohort 2 administration group DWRX200396 mg DWRX2003
144 mg
(both arms) DWRX2003
144 mg
(both hips) DWRX2003
432 mg N 6 6 6 6 AUC _last ^*
(h*ng/mL) 3854.499
[486.462] 8606.962
[4787.455] 8872.195
[2543.036] 20135.89
[3800.854] C _max
(ng/mL) 25.973
[6.194] 147.803
[64.478] 131.111
[80.171] 132.665
[41.593] AUC _inf (h*ng/mL) 7710.584
[2766.575] 9342.763
[1.633] 10434.278
[2890.693] 22348.26
[4683.097] T _max ^**
(h) 3.000
[2.000 - 47.969] 9.000
[ 6.000 - 10.002] 5.500
[2.004 - 48.000] 2.500
[2.000 - 12.000] t _1/2
(h) 630.553
[515.116] 247.420
[49.743] 354.064
[266.511] 273.390
[108.699] MRT
(h) 268.895
[30.633] 172.567
[50.979] 237.509
[109.979] 314.160
[24.446] CL/F (mL/h) 13910.020
[5379.515] 18950.562
[8558.508] 14872.512
[4914.741] 20011.860
[3926.432] Vz/F (mL) 10042509.000
[4709531] 6598659.018
[2663824] 6886281.076
[5003271] 7537771.000
[2012177] Metabolic ratio ^***
(h*ng/mL)/
(h*ng/mL) 0.25277342
[0.040] 0.248035797
[0.073] 0.188943525
[0.078] 0.261913946
[0.062]

^* AUC _last : 0 - 648 hours for DWRX2003 96 mg, 0 - 984 hours for other doses

^** T _max : Median [min - max]

^*** Metabolic ratio: AUC _{last_metabolites} /AUC _{last_Niclosamide}

Experimental Example 7-1-2. Safety evaluation of niclosamide

In this clinical trial, it was confirmed whether there were any adverse reactions in test subjects administered with Cohorts 1 and 2 and Cohorts A and B. As a result, 47 adverse reactions were confirmed in 8 out of 16 patients treated with cohorts 1 and 2, and 24 adverse events were confirmed in 7 out of 12 patients treated in cohorts A and B.

The observed adverse events and the number of cases are shown in Table 21 below.

cohort 1 Cohort A Cohort B cohort 2 administration group DWRX200396 mg DWRX2003
144 mg
(both arms) DWRX2003
144 mg
(both hips) DWRX2003
432 mg N 6 6 6 6 Systemic adverse reactions and local adverse reactions other than the site of administration One 6 4 3 Local adverse reactions at the site of administration 2 8 6 41

However, among the above adverse events, there were no serious adverse reactions (TESAE) or adverse reactions of special interest (TEAESI) that occurred after administration, and no adverse reactions collected due to drug hypersensitivity were confirmed.

From the comparison of Cohorts 1 and 2, it was confirmed that the incidence of adverse reactions increased clinically significantly as the dose of niclosamide increased.

In cohort A, it was confirmed that one local adverse reaction at the administration site had severity (grade 2), but all other adverse reactions were very mild in severity and there were no significant problems, confirming that safety and tolerability could be secured. . In addition, from the comparison of cohorts A and B, it can be confirmed that there is no significant difference in safety and tolerability according to the difference in administration site (both arms and both hips).

Experimental Example 7-2. Results of phase 1 clinical trial of niclosamide in Australia

A randomized, double-blind, placebo-controlled, single dose, dose escalation phase 1 clinical trial was conducted in 31 healthy volunteers.

Random doses of 288 mg, 432 mg, 672 mg, and non-randomized doses of 672 mg DWRX2003 were administered to test subjects as a single dose through 4 intramuscular injections, that is, 1 injection in each of the left and right arms and hips, and pharmacokinetics and safety evaluations were conducted.

Experimental Example 7-2-1. Evaluation of the pharmacokinetics of niclosamide

The results of pharmacokinetic parameters for the randomized doses of 288 mg, 432 mg, 672 mg and non-randomized 672 mg DWRX2003 are shown in Table 22 below.

Experimental Example 7-2-2. Safety evaluation of niclosamide

Of the 31 participants, 28 (90.3%) reported 287 post-dose adverse events (TEAEs), of which 275 of 28 (90.3%) of 31 participants were adverse drug reactions (ADRs) and 31 patients 260 of 26 participants (83.9%) were injection site reactions.

Overall, there was no increase in the incidence of TEAEs with increasing dose of DWRX2003, and the incidence of TEAEs was higher among participants receiving DWRX2003 (of 25 participants) compared to placebo (4 of 6 participants [66.7%]). 24 [96.0%]).

In addition, 210 adverse reactions were mild (grade 1) in 28 [90.3%] of 31 participants who confirmed adverse reactions. Slightly higher (Grade 2) TEAEs were identified in 15 of 31 [48.4%] TEAEs, randomized 288 mg: 2 of 6 [33.3%], 432 mg: 3 of 6 participants [42.9%], 672 mg: 5 of 6 participants [83.3%], non-randomized - 672 mg: 5 of 6 participants [83.3%]), which increased with increasing DWRX2003 dose.

Severe (Grade 3) TEAEs were found in randomized - 672 mg: 2 of 6 participants [33.3%] and non-randomized - 672 mg: 3 of 6 participants [50.0%], with the highest dose of DWRX2003 was administered only, and no moderate or severe TEAEs were reported in participants receiving placebo.

Therefore, although adverse reactions were confirmed with the administration of niclosamide, it can be confirmed that safety and tolerability were secured as most of the symptoms were mild.

Claims (20)

An intramuscular and/or A pharmaceutical composition for subcutaneous administration. [Claim 2] The pharmaceutical composition according to claim 1, wherein the single total administration dose is administered to a single site intramuscularly and/or subcutaneously or dividedly administered to multiple sites of muscle and/or subcutaneous administration. The pharmaceutical composition according to claim 1, wherein the single total dose is 96 to 1200 mg. The pharmaceutical composition according to claim 1, wherein the single total dose is 300 to 1200 mg. The pharmaceutical composition according to claim 1, wherein the single total dose is 800 to 1200 mg. The pharmaceutical composition according to claim 1, wherein the concentration of niclosamide in the pharmaceutical composition is 200 to 300 mg/ml. The pharmaceutical composition according to claim 1, wherein the body parts for intramuscular and/or subcutaneous administration are arms, hips, thighs and/or abdomen. The pharmaceutical composition according to claim 7, wherein the intramuscular and/or subcutaneous administration is administered in 1 to 6 divided doses in the same or different parts of the body. The pharmaceutical composition according to claim 1, wherein the dosage of the pharmaceutical composition for each administration site is 0.1 ml to 5 ml. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises niclosamide or a pharmaceutically acceptable salt thereof, a hydrate or solvate thereof as an active ingredient, and a stabilizer, an isotonic agent and a buffer as an excipient. composition. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is a liquid formulation or a lyophilized formulation. The pharmaceutical composition according to claim 11, wherein the liquid agent is a suspension. The pharmaceutical composition according to claim 10, wherein the stabilizer is carboxymethylcellulose or a salt thereof. According to claim 1, wherein the pharmaceutical composition is influenza virus (Influenza virus), flavivirus (Flavivirus), adenovirus (Human adenovirus, HAdV), coronavirus severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV) -1), type 2 severe acute respiratory syndrome coronavirus (SARS-CoV-2), MERS-CoV, coronavirus disease 2019 (COVID-19), herpes virus ( Herpes virus, Zica virus, Japanese encephalitis virus (JEV), Epstein-Barr virus (EBV), Ebola virus (EBOV), rhinovirus, Chikungunya virus (CHIKV), hepatitis C virus (HCV), hepatitis B virus (HBV), hepatitis A virus (HAV), rotavirus, astro Astrovirus, Hantavirus, Dengue virus, severe fever with thrombocytopenia syndrome virus, HIV virus, West Nile Virus (WNV), and Yellow fever virus ) a pharmaceutical composition for the prevention or treatment of one or more viral infections selected from the group consisting of. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is for the prevention or treatment of coronavirus infection-19 (Covid-19). The pharmaceutical composition according to claim 15, wherein the blood concentration in the body for 3 to 14 days after administration of the pharmaceutical composition exceeds the IC ₅₀ value of niclosamide for coronavirus infection-19 (Covid-19). The pharmaceutical composition according to claim 16, wherein the IC ₅₀ of niclosamide against coronavirus infection-19 (Covid-19) is 45-100 ng/ml. The method according to claim 1, wherein after the initial administration of the pharmaceutical composition, the pharmaceutical composition comprising niclosamide or a pharmaceutically acceptable salt thereof, hydrate or solvate thereof is administered at a dose of 24 mg to 1500 mg once. A pharmaceutical composition for intramuscular and/or subcutaneous administration of niclosamide, characterized in that it is further administered to 4 times. The pharmaceutical composition according to claim 18, wherein the administration site of the initial administration of niclosamide and the administration site of the additional administration are the same or different. A method for intramuscularly and/or subcutaneously administering the pharmaceutical composition according to any one of claims 1 to 19 to a subject in need thereof.

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