Synergistic antiviral drug composition containing targeted nanoparticles

Abstract

The present invention provides an antiviral pharmaceutical composition containing targeted nanoparticles that exhibit synergistic effects. [Solution] The present invention provides two-agent nanoparticles in which a first agent, namely remdesivir, which is a pharmaceutically acceptable active molecule, and a second agent, or a combination thereof, are co-encapsulated in nanoparticles and delivered simultaneously, and supported by an amphiphilic polysaccharide macromolecule, as well as pharmaceutical compositions containing such nanoparticles. The disclosure also relates to the use of nanoparticles and pharmaceutical compositions in antiviral treatment.

Description

[Technical Field]

[0001] The present invention relates to an antiviral pharmaceutical composition comprising targeted nanoparticles that provides a synergistic effect. [Background technology]

[0002] Treating viruses has become a major challenge in clinical practice. Vaccines are recognized as one of the definitive and ultimate means of resolving human virus-related diseases. Coronaviruses, such as SARS-CoV-1 from 2002-2003 and SARS-CoV-2 from 2019-2021, have threatened human lives and lifestyles worldwide since their outbreak in 2019.

[0003] The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in Wuhan, China in December 2019 and has since spread worldwide. As of the end of April 2020, more than 3 million cases and over 230,000 deaths have been reported in more than 200 countries. This new beta-coronavirus is similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Based on its genetic proximity, it is thought to have originated from a bat-derived coronavirus and spread to humans via an unknown intermediate mammalian host. The SARS-CoV-2 viral genome was rapidly sequenced, enabling diagnostic testing, epidemiological tracking, and the development of prevention and treatment strategies. Currently, there is no strong, clinically conclusive evidence from randomized clinical trials that any possible treatment improves outcomes for hospitalized patients suspected or confirmed to have COVID-19.

[0004] With a desperate need for a cure or better treatment for COVID-19, or more commonly known as coronavirus, many attempts have been made in various countries to effectively slow the rate of infection spread and, more importantly, reduce or stop mortality in the current pandemic situation. These attempts involve a second-line drug consisting of one of several drugs, such as favipiravir or avigan, hydroxychloroquine, carfilzomib, darunavir, pitavastatin, lamivudine, lopinavir, nelfinavir, ritonavir, darunavir, ledipasvir, telaprevir, rosuvastatin calcium, atovaquone, moexipril, azithromycin, curcumin, dexamethasone, or artemisinin. However, serious side effects that result in lower or higher mortality rates are a concern, as has been clinically reported. For example, chloroquine and hydroxychloroquine are effective against malaria, rheumatoid arthritis, and lupus, but are not considered effective against COVID-19 and have caused deaths due to cardiac side effects. Remdesivir is a broad-spectrum antiviral drug, and as of 2020, it is being tested as a specific treatment for COVID-19 and has been approved by the U.S. Food and Drug Administration (FDA) under Emergency Use Authorization (EUA) for the emergency treatment of patients hospitalized with serious illness. However, it has caused serious liver and kidney damage or dysfunction in hospitalized patients. While combinations of these drugs have been shown to have synergistic therapeutic effects, they also produce side effects.

[0005] A new clinical need has emerged to find better synergistic solutions using drug candidates, including or not limited to autophagy inhibitors, protease inhibitors, antibiotics, or herbal medicines. [Overview of the project]

[0006] Therefore, the present invention provides a good synergistic solution for using antiviral drug candidates encapsulated in AGO particles. As is well recognized in many antiviral strategies, drug combinations, either by sequential or simultaneous delivery, exhibit excellent therapeutic synergies against viruses, particularly coronaviruses. In the process, it is well known that encapsulating single or multiple drugs using nanotechnology is expected to significantly improve therapeutic efficacy while simultaneously reducing side effects caused by the toxicity of the free drug itself and the additives used in combination with drugs to facilitate administration. Thus, co-encapsulation and simultaneous delivery of multiple drugs can provide synergistic effects against coronaviruses, such as SARS-CoV-2.

[0007] In one embodiment, the present invention provides nanoparticles comprising a first antiviral agent and a second antiviral agent, wherein the first and second agents are encapsulated in alginate-oleic acid (AGO) particles.

[0008] In some examples of the present invention, the AGO particles include fatty acid-modified alginates and molecules selected from the group consisting of oleic acid-modified alginates, omeg-3-modified alginates, and oleic acid-modified alginates.

[0009] In one embodiment of the present invention, the first antiviral drug is remdesivir.

[0010] In one embodiment of the present invention, the second antiviral agent is an autophagy inhibitor, a protease inhibitor, an antibiotic, or a herbal medicine.

[0011] In one example of the present invention, the second antiviral agent is selected from the group consisting of favipiravir, avigan, hydroxychloroquine, carfilzomib, darunavir, pitavastatin, lamivudine, lopinavir, nelfinavir, ritonavir, darunavir, ledipasvir, telaprevir, rosuvastatin calcium, atovaquone, moexipril, moexipril, moexipril, azithromycin, curcumin, artemisinin, and combinations thereof.

[0012] In one example of the present invention, the particles are remdesivir-curcumin particles.

[0013] In one embodiment, AGO particles are conjugated with a human angiotensin-converting enzyme 2 (ACE2) antibody on the surface of the particles.

[0014] In another embodiment, the present invention provides a pharmaceutical composition comprising particles according to the present invention.

[0015] In a further embodiment, the present invention provides a method for treating a viral infection in a subject, comprising administering a pharmaceutical composition according to the present invention to the subject.

[0016] According to the present invention, the particle size is in the range of 50 nm to 600 nm, more preferably 50 nm to 300 nm; most preferably 50 nm to 100 nm.

[0017] Furthermore, the present invention provides a pharmaceutical composition containing the above-mentioned particles.

[0018] Furthermore, the present invention provides a method for treating a viral infection in a subject, comprising administering the above-mentioned pharmaceutical composition to the subject.

[0019] In one example of the present invention, the virus is a coronavirus, particularly SARS-CoV-2.

[0020] The present invention will be further illustrated by the following embodiments. However, it should be understood that the following embodiments are for illustrative purposes only and should not be construed as actually limiting the present invention. [Brief explanation of the drawing]

[0021] The above summary and the following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, currently preferred embodiments are shown in the drawings.

[0022] [Figure 1] Shows remdesivir and dexamethasone released from the two-drug-loaded AGO nanoparticles produced in Example 4.

[0023] [Figure 2] Shows the spherical shape of the two-drug-loaded AGO nanoparticles produced in Example 4.

[0024] [Figure 3] Shows the histopathological findings of a pilot test of the acute oral toxicity of sodium alginate-mOA (AGO) in female rats; no significant changes in the adrenal gland (A), heart (B), kidney (C), liver (D), lung (E) and spleen (F) were seen in the 2000 mg / kg AGO group (animal code: 1). HE staining. 400x magnification.

Mode for Carrying Out the Invention

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] The present invention provides two-drug-containing nanoparticles designed for antiviral treatment, with or without targeting ability, wherein the two drugs include a first drug, namely remdesivir, and a second drug selected from the group consisting of favipiravir, avigan, hydroxychloroquine, carfilzomib, darunavir, pitavastatin, lamivudine, lopinavir, nelfinavir, ritonavir, darunavir, rezipravir, telaprevir, rosuvastatin calcium, atovaquone, moexipril, moexipril, moexipril, azithromycin, curcumin and artemisinin.

[0027] Remdesivir

[0028] Remdesivir is recognized by the FDA as a promising antiviral drug approved for emergency use only. It is a prodrug converted to GS-441524 monophosphate by the action of esterases and phosphoramidases. This is then further phosphorylated to triphospho GS-441525 by nucleotide kinases. As an adenosine nucleotide triphosphate analog, it interferes with the action of viral RNA-dependent RNA polymerase, evading proofreading by viral exoribonuclease (ExoN) and causing a decrease in viral RNA production. In some viruses, such as respiratory multinuclear viruses, it can suspend RNA-dependent RNA polymerase, but its primary effect (in the case of Ebola) is to induce irreversible chain arrest. Unlike many other chain terminators, this is not mediated by preventing the addition of subsequent nucleotides, but rather occurs delayed, after further bases have been added to the growing RNA chain. Furthermore, co-encapsulating remdesivir with other inhibitors, antibiotics, and / or herbal medicines such as curcumin using polymer nanoparticles for subsequent medical purposes is a possible combination.

[0029] Other antiviral drugs

[0030] According to the present invention, the second drug may be an autophagy inhibitor, protease inhibitor, antibiotic, or herbal medicine exhibiting antiviral activity. In one embodiment, the second drug is selected from the group consisting of favipiravir, avigan, hydroxychloroquine, carfilzomib, darunavir, pitavastatin, lamivudine, lopinavir, nelfinavir, ritonavir, darunavir, ledipasvir, telaprevir, rosuvastatin calcium, atovaquone, moexipril, moexipril, moexipril, azithromycin, curcumin, artemisinin, and combinations thereof. As is well recognized in many anti-cancer strategies, combinations of drugs delivered sequentially or concurrently exert excellent therapeutic synergistic effects against malignant tumors.

[0031] Alginate-oleate (AGO) particles

[0032] Alginate-oleate (AGO) particles, also known as "AGO," are particles made from a novel macromolecule disclosed in PCT / CA2021 / 050293, filed on March 5, 2021 (which, by attribution, is part of this specification as a whole). The AGO polymer consists of alginate and oleate linked by a spacer, and alginate-oleate (AGO) particles are formed from the AGO macromolecule. AGO particles possess biofunctional properties such as controlled cytocompatibility and controlled degradation (by renal metabolism), which are introduced into modified alginate without altering clinically advantageous properties, such as cell-specific compatibility, non-immunogenicity, and structural stability, for end use in medical settings.

[0033] Examples of AGO particles include fatty acid-modified alginates and molecules selected from the group consisting of oleic acid-modified alginates, omeg-3-modified alginates, and oleic acid-modified alginates.

[0034] The pharmaceutical composition according to the present invention can be formulated into a dosage form suitable for oral administration using techniques well known to those skilled in the art. Examples of dosage forms include, but are not limited to, injections (e.g., sterile aqueous solutions or dispersions), sterile powders, tablets, lozenges, pills, and capsules.

[0035] The pharmaceutical composition according to the present invention may be administered by one or more routes, such as nasal spray, intramuscular injection, and subcutaneous injection.

[0036] In certain embodiments, the pharmaceutical composition is formulated into dosage forms suitable for oral administration and subcutaneous injection.

[0037] The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier widely used in the field of pharmaceutical formulation. For example, the pharmaceutically acceptable carrier may comprise one or more substances such as solvents, emulsifiers, suspending agents, decomposing agents, binders, additives, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption retarders, plasticizers, excipients, disintegrants, surfactants, and / or thickeners.

[0038] The dosage and frequency of administration of the pharmaceutical composition according to the present invention may vary depending on the severity of the disease being treated, the route of administration, and the weight, age, physical condition, and response of the subject being treated. For example, the daily dose of the pharmaceutical composition according to the present invention is for a body surface area of ​​1 m². 2 The dose may range from 50 mg to 150 mg per dose and can be administered once or several times over a period of one day or one week.

[0039] Please understand that both the general description above and the detailed description below are merely illustrative and descriptive, and do not limit the present invention.

[0040] The present invention is further illustrated by the following embodiments, which are presented for demonstrative purposes only and are not limiting. [Examples]

[0041] Example 1. Production of remdesivir-curcumin-containing nanoparticles

[0042] A remdesivir stock solution with a remdesivir concentration of 20 mg / mL and a curcumin concentration of 10 mg / mL was prepared by dissolving 1.0 mg of remdesivir powder (purchased from Selleckchem) and 0.5 mg of curcumin (purchased from Sigma) in 0.5 mL of dimethyl sulfoxide (DMSO) and DI aqueous solution. 5 μL of the remdesivir-curcumin stock solution was added to 95 μL of DI aqueous solution to obtain a diluted remdesivir-curcumin solution with a remdesivir concentration of 1 mg / mL and a curcumin concentration of 0.5 mg / mL.

[0043] 0.5 mg of oleic acid-modified alginate (AGO) (i.e., amphiphilic alginate) powder (purchased from Nuecology Biomedical Inc., Canada) was added to an Eppendorf tube, followed by 50 μL of diluted remdesivir-curcumin solution. 0.95 mL of 1X PBS solution was added to the resulting mixture to form a 1 mL mixed solution (pH 7.4) with an initial AGO concentration of 0.5 mg / mL, an initial free remdesivir concentration of 50 μg / mL, and a free curcumin concentration of 25 μg / mL. The above procedure was repeated once to form another mixed solution with the same initial AGO concentration and the same initial free remdesivir and curcumin concentrations. The two mixed solutions were then stirred at 4°C for 12 hours to form two dual AGO nanoparticle solutions.

[0044] Example 2. Determination of drug encapsulation rate of remdesivir-curcumin-containing AGO nanoparticles.

[0045] The drug encapsulation rate of the remdesivir-curcumin-supported nanoparticle solution obtained in Section A of this example was determined as follows.

[0046] Standard solutions of remdesivir and curcumin were obtained by serial dilution of stock solutions of remdesivir and curcumin, respectively. The absorbances of the standard solutions of remdesivir and curcumin were determined using ultra-high-performance liquid chromatography (UPLC-UV).

[0047] Furthermore, each of the remdesivir-curcumin nanoparticle solutions was centrifuged at 12,000 rpm at 4°C for 10 minutes. This yielded 500 μL of supernatant, which was added to 500 μL of DMSO solution to form 1 mL of the test solution. The test solution was then subjected to HPLC analysis, and the unencapsulated remdesivir and curcumin concentrations (i.e., the remaining free drug concentrations) were determined by comparing the test solution to a calibration curve. The drug encapsulation rate of the test solution was calculated using the following formula (1): A = [(BC) / B] × 100 (1) A = Drug encapsulation rate (%) B = Initial free drug concentration C = Unsealed drug concentration in each test solution

[0048] The obtained experimental data is expressed as mean ± SD (standard deviation) or as the mean.

[0049] The drug encapsulation rates of the remdesivir-curcumin nanoparticle solutions prepared by stirring for 12 and 24 hours were 75.4% ± 0.35% and 64% ± 0.25%, respectively. In other words, the nanoparticle concentrations (i.e., the concentrations of encapsulated remdesivir and curcumin) of the nanoparticle solutions prepared by stirring for 12 and 24 hours were approximately 50.7 μg / mL and approximately 40 μg / mL, respectively.

[0050] Example 3. Determination of the physical properties of nanoparticles according to the present invention.

[0051] An appropriate amount of AGO powder used in Example 1 was dissolved in 1 mL of deionization (DI) solution to form an AGO nanoparticle solution with an AGO concentration of 1.0 mg / mL.

[0052] The drug encapsulation rate was 60-75% and the remdesivir-curcumin nanoparticle concentration was 30 μg / mL. The remdesivir-curcumin nanoparticles in the nanoparticle solution obtained in Section A of this example were conjugated with an anti-ACE2 antibody. Specifically, 1 μL of anti-human ACE2 antibody (1 mg / mL in double-distilled water) was added to the remdesivir-curcumin nanoparticle solution and stirred at 4°C for 1 hour. 0.05 mL of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution (0.1%, w / v) was added to the resulting solution and allowed to stand for at least 4 hours. Thus, an anti-human ACE2 antibody conjugate particle solution containing anti-ACE2 AGO nanoparticles was formed. The drug encapsulation rate of the anti-ACE2 antibody conjugate nanoparticle solution was determined according to the method generally used in this example.

[0053] The hydrodynamic diameters of AGO nanoparticles, remdesivir-curcumin nanoparticles, and anti-ACE2 remdesivir-curcumin nanoparticles were measured twice in distilled water using dynamic light scattering (DLS) with a BI-200SM goniometer (Brookhaven Inc., Holtsville, NY).

[0054] The zeta potentials of AGO nanoparticles, remdesivir-curcumin nanoparticles, and anti-ACE2 remdesivir-curcumin nanoparticles were measured twice in distilled water using a laser Doppler anemometer system (Beckman Coulter Inc., USA) to investigate the surface potentials of these particles.

[0055] Furthermore, AGO nanoparticles and remdesivir-curcumin nanoparticles were observed at 20,000x magnification using a Jeol 2100 Transmission Electron Microscope (Jeol Ltd., Japan) to determine the particle size and confirm the encapsulation of ganetespib by CHC and the conjugation of anti-EGFR antibodies. The obtained experimental data are expressed as mean ± SD (standard deviation) or mean.

[0056] The drug encapsulation rate in the antibody-conjugate nanoparticle solution was 55-70%. In other words, the concentration of anti-ACE2 remdesivir-dexamethasone nanoparticles in the antibody-conjugate nanoparticle solution was 35 μg / mL.

[0057] The hydrodynamic diameters and zeta potentials of AGO nanoparticles, remdesivir-dexamethasone AGO nanoparticles, and anti-ACE2 remdesivir-dexamethasone nanoparticles are shown in Table 1 below. [Table 1]

[0058] As shown in Table 1, the hydrodynamic diameter of the remdesivir-dexamethasone nanoparticles was larger than that of the AGO particles, indicating that both drugs could be encapsulated in the drug carrier AGO to form nanoparticles. Furthermore, the hydrodynamic diameter of the anti-ACE2 dual-agent AGO nanoparticles was larger than that of the dual-agent AGO nanoparticles, indicating that the anti-ACE2 antibody could be conjugated into nanoparticles containing both remdesivir and curcumin encapsulated by AGO.

[0059] In light of the above, AGO can simultaneously encapsulate both remdesivir and curcumin to form nanoparticles, which can then be further conjugated with biologically functional molecules.

[0060] Example 4. Determination of drug encapsulation rate of remdesivir-dexamethasone-containing AGO nanoparticles.

[0061] A remdesivir-dexamethasone-supported AGO nanoparticle solution was prepared based on the manufacturing conditions shown in Example 2, and the drug encapsulation rate was determined as follows.

[0062] Standard solutions of remdesivir and dexamethasone were obtained by serial dilution of stock solutions of remdesivir and dexamethasone, respectively. The absorbances of the standard solutions of remdesivir and dexamethasone were determined using ultra-high-performance liquid chromatography (UPLC).

[0063] Furthermore, each of the remdesivir-dexamethasone AGO nanoparticle solutions was centrifuged at 10,000 rpm at 4°C for 10 minutes. 500 μL of the supernatant was obtained and added to 500 μL of DMSO solution to form 1 mL of the test solution. The test solution was then subjected to UPLC analysis, and the unencapsulated remdesivir and dexamethasone concentrations (i.e., the remaining free drug concentrations) were determined by comparing the test solution to a calibration curve. The drug encapsulation rate of the test solution was calculated using the following formula (1): A = [(BC) / B] × 100 (2) A = Drug encapsulation rate (%) B = Initial free drug concentration C = Unsealed drug concentration in each test solution

[0064] The obtained experimental data is expressed as mean ± SD (standard deviation) or as the mean.

[0065] The drug encapsulation rates of the remdesivir-dexamethasone AGO nanoparticle solution prepared by 24 hours of stirring were 71.8% ± 0.75% and 89.2% ± 0.54% for remdesivir and dexamethasone, respectively (Table 2). In other words, the nanoparticle concentrations (i.e., the concentrations of encapsulated remdesivir and dexamethasone) of the nanoparticle solution prepared by 24 hours of stirring were approximately 40 μg / mL and approximately 20 μg / mL, respectively. [Table 2]

[0066] Example 5. Drug dissolution test of remdesivir-dexamethasone AGO nanoparticles

[0067] An in vitro drug release test was performed by suspending the two-drug-supported AGO nanoparticles prepared in Example 4 in pH 4 PBS. The suspension was divided into 1.5 mL Eppendorf tubes. The tubes were gently shaken in an orbital shaker incubator at 37°C. At predetermined times (4, 8, 12, 24, 36, and 48 hours), the released (free) drugs were separated from the drug-loaded nanoparticles by centrifugation. For remdesivir and dexamethasone, the samples were centrifuged at 12,000 rpm for 4 minutes, and the pellets consisting of remdesivir and dexamethasone released by precipitation were analyzed. The concentrations of remdesivir and dexamethasone were quantified by UPLC. The percentage of remdesivir and dexamethasone released at each time was calculated using the following formula: Released drug (%) = [(Released drug) / (Initial amount of drug)] × 100 (2)

[0068] The 72-hour drug release profiles are shown in Figure 1. While remdesivir was clearly depleted in approximately 48 hours, dexamethasone showed a slower release profile, reaching 70% release by the 72-hour dissolution time. This drug release study demonstrated that both drugs can be co-released with different release kinetics from as-manufactured dual-drug AGO nanoparticles. This study also indicates potential therapeutic effects due to the synergistic effect of the drugs, but the as-manufactured nanoparticles act as antiviral agents in vivo, which may be beneficial for clinical interpretation.

[0069] Example 6. Structural morphology of remdesivir-dexamethasone AGO nanoparticles

[0070] The morphological structure of the two-component AGO nanoparticles was investigated using a scanning electron microscope, as shown in Figure 2. The as-manufactured two-component supported particles exhibited a spherical shape with a size range of approximately 100–250 nm, suggesting that the self-assembly behavior of AGO molecules allows for efficient processing under mild, neutral aqueous solutions with high drug encapsulation efficiency. This finding also suggests superior processing advantages compared to conventional high-energy emulsification processes such as liposome formation for encapsulating and delivering target drugs.

[0071] Example 7. In vivo evaluation of the biosafety of remdesivir-dexamethasone-supported AGO nanoparticles by oral administration.

[0072] (1) Rearing and testing systems: All animal experiments and rearing were approved by the Institutional Animal Care and Use Committee (IACUC) of the Agricultural Technology Research Institute (IACUC No. 109064).

[0073] Animal Care: The animals were housed in an ATRI AAALAC-certified facility. In the animal rooms, polycarbonate cages were used to house animals of the same sex and treatment group. Temperature: 22±4℃ Relative humidity: 30-70% Light cycle: 12 hours light, 12 hours dark Diet: Autoclaved laboratory 5053-PicoLab(registered trademark) Rodent Diet 20 (LabDiet, Richmond, IN, USA) Water: Autoclaved RO water was provided free-flowing through water bottles attached to the cages. Identification: Animals were identified by ear notches, and each cage was labeled with cage number, test number, IACUC number, sex, treatment, and animal ID number. (2) Test system: Seed and source: Sprague Dawley seeds / BioLASCO Taiwan Co. Ltd. Age and body weight: At the start of the study, the rats were approximately 8 weeks old, and their pre-administration body weight ranged from 177.43g to 210.66g. Gender / Number: Female / 3 Acclimatization: Animals are isolated in the GLP animal facility at the ATRI Animal Center and acclimatized in the laboratory for 6 days before administration.

[0074] (3) Preparation of the test substance: "AGO" was prepared and processed according to Example 4. The test substance was weighed, mixed and dissolved in normal physiological saline, and diluted to 200 mg / mL before administration.

[0075] (4) Experimental procedure: The animals were fasted for 16-18 hours before administration and for 3-4 hours after administration. The drug was administered using a syringe and feeding tube at a dose of 10 mL / kg, based on the body weight measured on the day of administration. Clinical observation: Animals were continuously observed for 10 minutes after administration, and again 30 minutes and 4 hours after administration on the day of administration. Animals were observed once daily from day 1 to day 14 after administration. Weight measurement: Weight was measured using an electronic balance 0 days after administration (before administration), 7 days after administration, and 14 days after administration. Gross necropsy: Surviving animals were subjected to gross necropsy 14 days after administration. Surviving animals were euthanized by bleeding from the abdominal aorta under isoflurane anesthesia. For all animals, the external surface of the body, all orifices, subcutaneous tissue, skull, abdominal and pelvic cavities and their contents were observed. Blood collection and procedure: Blood samples were obtained from the abdominal aorta and collected without anticoagulants. Serum was collected by centrifugation at 3,500 rpm for 15 minutes in a refrigerated centrifuge, then at 4°C for up to 1 hour. Each serum sample was stored at -30°C. Tissue sampling and organ weighing: For all animals, the heart, lungs, spleen, liver, kidneys, and adrenal glands were removed and weighed using an electronic balance. The hearts, lungs, spleen, liver, kidneys, and adrenal glands were preserved in 10% neutralized buffered formalin.

[0076] result [Table 3] (1) Mortality / mortality rate: No animal deaths were observed during the study period. (2) Clinical observations: No clinically adverse signs were observed in any of the animals during the study period. (3) Body weight: No abnormalities were observed in any of the animals during the study period. (4) Gross anatomical findings: No abnormal findings were observed in any of the animals on the day of dissection. (5) Organ weight: The organ weights of each animal were observed and are shown in Table 3. No abnormal proliferation or changes were observed. Table 3

[0077] Example 8. Histopathological analysis of vital organs prepared from Example 7.

[0078] This study was commissioned by Nuecology Biomedical Inc. (Vancouver, BC, Canada). The study evaluated pathological changes induced by the test substance, sodium alginate-mOA (AGO), via forced oral administration in female rats. Three 8-week-old female Sprague Dawley rats were force-administered 2,000 mg / kg of AGO in double-distilled H2O solution. All rats were sacrificed on day 14. The hearts, kidneys, lungs, livers, and spleens were collected and subjected to histopathological evaluation. No significant lesions of the heart, kidneys, liver, lungs, or spleen were observed in the AGO-treated females during histopathological evaluation.

[0079] In conclusion, a pilot study of the acute oral toxicity of 2,000 mg / kg sodium alginate-mOA did not cause significant lesions of the heart, kidneys, liver, lungs, or spleen in female rats, according to histopathological examination.

[0080] Three 8-week-old female Sprague Dawley rats were force-administered 2,000 mg / kg of sodium alginate-mOA(AGO) in a double-distilled H2O solution. All rats were sacrificed on day 14.

[0081] The heart, kidneys, lungs, liver, and spleen were collected and subjected to histopathological evaluation (Table 3). For histopathological evaluation, the tissues were further processed, embedded in paraffin, cut into 3 μm sections using microtones, stained with hematoxylin-eosin (HE), and evaluated under a light microscope (BX-53, Olympus, Tokyo, Japan).

[0082] The severity of the lesions was graded according to the method described by Shackelford et al. (Toxicologic Pathology 30: 93-96, 2002). The degree of the lesions was graded from 1 to 5 according to severity: 1 = minimum (<1%); 2 = very small (1-25%); 3 = moderate (26-50%); 4 = moderate-severe (51-75%); 5 = severe / high (76-100%). The pathological terminology for each organ is shown in Table 4. [Table 4]

[0083] result:

[0084] Histopathological findings:

[0085] Heart, kidneys, liver, lungs, or spleen: No significant lesions of the heart, kidneys, liver, lungs, or spleen were observed in the 2,000 mg / kg AGO treatment group (Figure 3; Table 5).

[0086] Conclusion:

[0087] Three 8-week-old female Sprague Dawley rats were force-administered 2,000 mg / kg of sodium alginate-mOA (AGO) in double-distilled H2O solution. All rats were sacrificed on day 14. The hearts, kidneys, lungs, livers, and spleens were collected and subjected to histopathological evaluation.

[0088] Histopathological evaluation revealed no significant lesions in the heart, kidneys, liver, lungs, or spleen in female rats treated with AGO 2,000 mg / kg (Table 6).

[0089] In conclusion, a pilot study of the acute oral toxicity of 2,000 mg / kg sodium alginate-mOA did not cause significant lesions of the heart, kidneys, liver, lungs, or spleen in female rats, according to histopathological examination.

[0090] Example 9. In vivo evaluation of the acute toxicity of remdesivir-supported ACE2-coated AGO nanoparticles administered by subcutaneous injection.

[0091] A remdesivir-supported ACE2-coated AGO nanoparticle solution was prepared according to the manufacturing conditions shown in Example 2, and its acute toxicity by in vivo subcutaneous injection was determined.

[0092] Fifteen 7-week-old female ICR mice were divided into three groups, each receiving subcutaneous (sc) injection of RAGO gel at concentrations of 7.5 mg / kg(L), 15 mg / kg(M), and 25 mg / kg(H) in PBS-buffered water. Each group contained five female mice that received a single subcutaneous injection of the test substance. The injection volume was 100 μL / 20 g body weight. All mice were sacrificed on day 14.

[0093] The heart, kidneys, lungs, liver, and spleen were collected and subjected to histopathological evaluation (Table 8). For histopathological evaluation, the tissues were further processed, embedded in paraffin, cut into 3 mm sections using microtones, stained with hematoxylin-eosin (HE), and evaluated under a light microscope (BX-53, Olympus, Tokyo, Japan). [Table 5] [Table 6] [Table 7] [Table 8]

[0094] The severity of the lesions was graded according to the method described by Shackelford et al. (Toxicologic Pathology 30: 93-96, 2002). The degree of the lesions was graded from 1 to 5 according to severity: 1 = minimum (<1%); 2 = very small (1-25%); 3 = moderate (26-50%); 4 = moderate-severe (51-75%); 5 = severe / high (76-100%). The pathological terminology for each organ is shown in Table 9.

[0095] result:

[0096] (1) Histopathological findings: Heart, kidneys, liver, lungs, or spleen: No significant cardiac, renal, hepatic, pulmonary, or spleen lesions were observed in the 7.5 mg / kg RAGO, 15 mg / kg, and 25 mg / kg dose groups (Table 10).

[0097] (2) Non-specific findings: kidney: - Lesionary tubular cyst Only one female mouse in the 25 mg / kg RAGO gel group developed focal, minute tubular cysts in the kidney (Figure 3A-C). The incidence was 1 / 5 in the H-RAGO group. No significant treatment-related effects from the test substance were observed in the kidney (Table 10). liver: -Multifocal lipid changes Female mice treated with M-RAGO gel showed multifocal, minute fatty changes in the liver (Figures 1D-F). The incidence was 1 / 5 in M-RAGO gel mice. No significant treatment-related effects from the test substance were observed in the liver (Table 10). lung: - Diffuse artificial alveolar collapse All groups of female mice treated with RAGO gel showed minimal to moderate / severe artificial alveolar collapse in the lungs (Figures 3G-I). The incidence rates were 5 / 5, 5 / 5, and 5 / 5 in female mice treated with RAGO gel. No significant treatment-related effects from the test material were observed in the lungs (Table 10).

[0098] Conclusion:

[0099] Fifteen 7-week-old female ICR mice were divided into three groups, each receiving subcutaneous (sc) injection of RAGO gel at concentrations of 7.5 mg / kg(L), 15 mg / kg(M), and 25 mg / kg(H) in PBS-buffered aqueous solution. Each group contained five female mice that received a single subcutaneous dose of the test substance. All mice were sacrificed on day 14. The heart, kidneys, lungs, liver, and spleen were collected and subjected to histopathological evaluation.

[0100] Histopathological evaluation revealed no significant lesions of the heart, kidneys, liver, lungs, or spleen in female mice administered RAGO gel at doses of 7.5 mg / kg (L), 15 mg / kg (M), and 25 mg / kg (H) via subcutaneous (sc) injection. Several nonspecific or artificial lesions were found, including minute tubular cysts in the kidneys and multifocal adipose tissue in the liver. Diffuse alveolar collapse was observed in the alveolar spaces of the lungs, possibly related to improper fixation. Microscopic examination showed no significant treatment-related effects on organs by the test material. These observations are shown in tables and figures and were considered nonspecific or artificial lesions and incidental findings. In conclusion, toxicity studies of remdesivir-loaded RAGO gel at doses of 7.5, 15, or 25 mg / kg administered subcutaneously to female mice did not cause significant lesions of the heart, kidneys, liver, lungs, or spleen, as determined by histopathological examination. [Table 9] [Table 10]

[0101] Example 10. Remdesivir-curcumin-containing AGO TM Manufacturing of nanoparticles

[0102] 1.0 mg of remdesivir powder (purchased from Selleckchem) and 0.5 mg of methotrexate (MTX, purchased from Sigma) were dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) and DI aqueous solution, respectively, to prepare remdesivir stock solutions with a remdesivir concentration of 20 mg / mL and an MTX concentration of 10 mg / mL. 5 μL of the remdesivir-methotrexate stock solution was added to 95 μL of DI aqueous solution to obtain a diluted remdesivir-methotrexate solution with a remdesivir concentration of 1 mg / mL and a methotrexate concentration of 0.5 mg / mL.

[0103] 0.5 mg oleate-modified alginate (AGO TM)(i.e., amphiphilic alginic acid) powder (Nuecology Biomedical Inc., Canada) was added to an Eppendorf tube, and then 50 μL of diluted remdesivir-methotrexate solution was added. 0.95 mL of 1X PBS solution was added to the resulting mixture to form an initial AGO TM mixed solution (pH 7.4) of 1 mL with an initial AGO concentration of 0.5 mg / mL, an initial free remdesivir concentration of 40 μg / mL, and a free methotrexate concentration of 40 μg / mL. The above procedure was repeated once to form another mixed solution (Table 11) with the same initial AGO TM concentration and different ratios of initial free remdesivir and methotrexate concentrations. Then, two mixed solutions with drug concentrations in the range of 1:1, 2:1, and 5:1 were stirred at 4 °C for 12 hours to form a two-drug AGO TM nanoparticle solution.

Table 11

[0104] After encapsulating these two drugs into AGO TM nanoparticles, the physical properties of the resulting two-drug AGO TM nanoparticles were characterized for zeta potential and particle size as shown in Table 12.

Table 12

[0105] The "absolute" zeta potential of the obtained remdesivir-MTX AGO TM nanoparticles showed a range of 20 - 32 mV, indicating two-drug-loaded AGO TM nanoparticles with high colloidal stability in water-based solutions such as saline, blood, and body fluids. This suggests the potential for use in pharmaceutical applications for antiviral purposes. Moreover, the obtained two-drug AGO TM nanoparticles showed a size range of 650 - approximately 900 nm, which is also suitable for many administration routes including IV injection, SC injection, oral, and nasal spray.

[0106] This specification contains many details, which should not be construed as limitations on the scope of the invention or the claims, but rather as descriptions of features specific to particular embodiments or examples of the invention. Certain features described herein in relation to separate embodiments or examples may also be implemented in combination in a single embodiment.

Claims

1. Nanoparticles comprising a first antiviral agent and a second antiviral agent, wherein the first and second agents are encapsulated within alginate-oleic acid (AGO) particles.

2. The nanoparticles according to claim 1, wherein the AGO particles include fatty acid-modified alginates and molecules selected from the group consisting of oleic acid-modified alginates, omeg-3-modified alginates, and oleic acid-modified alginates.

3. The nanoparticle according to claim 1, wherein the first antiviral drug is remdesivir.

4. The nanoparticle according to claim 1, wherein the second antiviral agent is an autophagy inhibitor, a protease inhibitor, an antibiotic, or a herbal medicine.

5. The nanoparticles according to claim 1, wherein the second drug is selected from the group consisting of favipiravir, avigan, hydroxychloroquine, carfilzomib, darunavir, pitavastatin, lamivudine, lopinavir, nelfinavir, ritonavir, darunavir, ledipasvir, telaprevir, rosuvastatin calcium, atovaquone, moexipril, moexipril, moexipril, azithromycin, curcumin, artemisinin, and combinations thereof.

6. The nanoparticles according to claim 1, which are remdesivir-curcumin particles.

7. The nanoparticles according to claim 1, wherein the AGO particles are conjugated with a human angiotensin-converting enzyme 2 (ACE2) antibody on the surface of the particles.

8. The nanoparticles according to claim 1, wherein the particle size is 50 nm to 600 nm.

9. The nanoparticles according to claim 8, wherein the particle size is 50 nm to 300 nm.

10. A pharmaceutical composition comprising the particles described in any one of claims 1 to 9.

11. The pharmaceutical composition according to claim 10, comprising a first antiviral agent and a second antiviral agent, wherein the first agent is remdesivir and the second agent is curcumin.

12. The pharmaceutical composition according to claim 10, which is effective against coronavirus.

13. The pharmaceutical composition according to claim 10, which is effective against SARS-CoV-2.

14. The pharmaceutical composition according to claim 11, which is effective against coronavirus.

15. The pharmaceutical composition according to claim 11, which is effective against SARS-CoV-2.

16. A method for treating a viral infection in a subject, comprising administering a pharmaceutical composition according to any one of claims 10 to 15 to the subject.

17. The method according to claim 16, wherein the virus is a coronavirus.

18. The method according to claim 16, wherein the virus is SARS-CoV-2.

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