Treatment using meta arsenite

Abstract

The present invention relates to the use of sodium meta arsenite or potassium meta arsenite in methods for a) reducing an inflammatory response caused by a viral infection, b) treating or preventing an inflammatory condition caused by a viral infection, or c) treating or preventing hypercytokinemia caused by a viral infection. The present invention also relates to methods for treating or preventing a viral infection in a subject.

Description

[Technical Field]

[0001] This application claims priority to Australian Provisional Patent Application No. 2020900433, filed February 16, 2020, and Australian Provisional Patent Application No. 2021900204, filed January 29, 2021. The entire contents of Australian Provisional Patent Application Nos. 2020900433 and 2021900204 are incorporated herein by reference.

[0002] The present invention relates to methods for reducing an inflammatory response caused by a viral infection in a subject, and for treating or preventing an inflammatory condition caused by a viral infection in a subject. [Background technology]

[0003] The inflammatory response occurs in the body in response to injury, infection, and other insults. The inflammatory response involves a cascade of both pro-inflammatory and anti-inflammatory cytokines. The balance between these cytokines often determines the outcome after inflammation or injury.

[0004] As a successful consequence of inflammation or injury, the production of pro-inflammatory cytokines leads to the recruitment of blood leukocytes, activation of tissue macrophages, and production of immune mediators.

[0005] However, in some situations, such as sepsis, or following infection with an infectious agent such as a virus, including avian influenza or certain strains of coronavirus (e.g., SARS-CoV and SARS-CoV-2), the inflammatory response to the infection can result in an acute inflammatory state with unregulated production of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). Unregulated production of such pro-inflammatory cytokines can lead to pneumonia and / or multiple organ failure, which can be fatal in susceptible individuals.

[0006] Excessive, sometimes unregulated, secretion and / or production of proinflammatory cytokines is often a factor that can lead to the rapid spread of disease symptoms in some viral infections. For example, coronaviruses (CoVs), a large family of viruses that cause illnesses ranging from the common cold to more severe illness, are known to cause increased, sometimes unregulated, secretion of proinflammatory cytokines. Examples of coronaviruses include MERS-CoV, SARS-CoV, and SARS-CoV-2. Common signs of coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, and breathing difficulties. In more severe cases, infection can cause pneumonia, severe acute respiratory syndrome, kidney failure, and death.

[0007] Thus, there is a need for improved pharmaceutical compositions for use in the treatment or prevention of inflammatory conditions in which viral infection results in the unregulated production of pro-inflammatory cytokines. Summary of the Invention

[0008] The inventors have developed sodium meta arsenite (O=As-O - Na + ) (SMA) or potassium meta arsenite (O=As-O - K + ) (KMA) can reduce or inhibit the production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 from macrophages.

[0009] Accordingly, a first aspect is a method of reducing an inflammatory response caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0010] A further first aspect relates to sodium meta arsenite (O=As-O) for use in reducing an inflammatory response caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing an inflammatory response caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0011] The inventors envision that SMA and KMA may be used to treat or prevent conditions that result in an inflammatory response to viral infection (inflammatory conditions caused by viral infection).

[0012] Accordingly, a second aspect is a method of treating or preventing an inflammatory condition caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0013] A further second aspect relates to sodium meta arsenite (O=As-O) for use in the treatment or prevention of an inflammatory condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing an inflammatory condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0014] A third aspect is a method for treating or preventing hypercytokinemia due to a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0015] A further third aspect relates to sodium meta arsenite (O=As-O) for use in the treatment or prevention of hypercytokinemia due to viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing hypercytokinemia caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0016] A fourth aspect is a method of treating a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0017] A further fourth aspect relates to sodium meta arsenite (O=As-O) for use in treating a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0018] A fifth aspect is a method of treating a coronavirus infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0019] A further fifth aspect relates to sodium meta arsenite (O=As-O) for use in treating a coronavirus infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating a coronavirus infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0020] A sixth aspect is a method of reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0021] A further sixth aspect relates to sodium meta arsenite (O=As-O) for use in reducing TNF-α, IL-1β and / or IL-6 production in a subject suffering from an inflammatory condition caused by a viral infection. - Na + ) or potassium meta arsenite (O=As-O - K +), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing TNF-α, IL-1β and / or IL-6 production in a subject suffering from an inflammatory condition caused by a viral infection. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0022] A seventh aspect is a method of treating coronavirus SARS-CoV-2 infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0023] A further seventh aspect relates to a compound comprising sodium meta arsenite (O=As-O) for use in the treatment of coronavirus SARS-CoV-2 infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating coronavirus SARS-CoV-2 infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0024] An eighth aspect is a method of treating or preventing a disease or condition mediated by elevated TNF-α, IL-1β and / or IL-6 levels due to a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0025] A further eighth aspect relates to sodium meta arsenite (O=As-O) for use in the treatment or prevention of a disease or condition mediated by elevated TNF-α, IL-1β and / or IL-6 due to a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing a disease or condition mediated by elevated TNF-α, IL-1β and / or IL-6 due to a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0026] A ninth aspect relates to sodium meta arsenite (O=As-O) when used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection. - Na + ) or potassium meta arsenite (O=As-O - K + (I) a pharmaceutical composition comprising:

[0027] A tenth aspect is a pharmaceutical composition for oral administration, when used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, The pharmaceutical composition is provided wherein the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[0028] An eleventh aspect is a pharmaceutical composition for oral administration, when used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer Including, pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; The coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. Pharmaceutical compositions are provided.

[0029] A twelfth aspect is a method of treating a disease or symptom caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0030] A further twelfth aspect relates to a compound comprising sodium meta arsenite (O=As-O) for use in treating a disease or condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ), or sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating a disease or condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) is provided.

[0031] The present invention provides the following: 1. A method for reducing an inflammatory response caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to a subject. 2. The method according to item 1, wherein the viral infection is a coronavirus infection. 3. The method according to item 2, wherein the coronavirus is SARS-CoV-2. 4. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 2. The method of claim 1, wherein the compound is orally administered. 5. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 2. The method of claim 1, wherein the medicament is administered at a dose ranging from 2 mg per day to 20 mg per day. 6. A method for treating or preventing an inflammatory condition caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na +) or potassium meta arsenite (O=As-O - K + ) to a subject. 7. The method according to item 6, wherein the viral infection is a coronavirus infection. 8. The method according to item 7, wherein the coronavirus infection is caused by SARS-CoV-2. 9. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 7. The method of claim 6, wherein the compound (I) is orally administered. 10. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 7. The method according to item 6, wherein the medicament is administered at a dose ranging from 2 mg per day to 20 mg per day. 11. A method for treating or preventing hypercytokinemia caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O = As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to a subject. 12. The method according to item 11, wherein the viral infection is an infection caused by a coronavirus. 13. The method according to item 12, wherein the coronavirus is SARS-CoV-2. 14. A method of treating a viral infection in a subject, comprising administering to a subject an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to a subject. 15. The method according to item 14, wherein the viral infection is caused by infection with a coronavirus. 16. The method according to item 15, wherein the coronavirus is SARS-CoV-2. 17. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 15. The method of claim 14, wherein the compound is orally administered. 18. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + Item 15. The method of item 14, wherein the medicament is administered at a dose ranging from 2 mg per day to 20 mg per day. 19. A method of treating a coronavirus infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to a subject. 20. The method according to item 19, wherein the coronavirus infection is caused by SARS-CoV-2. 21. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 20. The method of claim 19, wherein the compound is orally administered. 22. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 20. The method of claim 19, wherein the medicament is administered at a dose ranging from 2 mg per day to 20 mg per day. 23. A method for reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to a subject. 24. Sodium meta arsenite (O=As-O- Na + ) or potassium meta arsenite (O=As-O - K + 24. The method of claim 23, wherein the compound is orally administered. 25. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + 24. The method of claim 23, wherein the medicament is administered at a dose ranging from 2 mg per day to 20 mg per day. 26. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) but the following: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer administered by a composition comprising 26. The method according to any one of items 1 to 25, wherein the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 27. Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) but the following: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (V) optionally a binder in the range of 0 to about 30% w / w a solid core, and (b) an enteric coating containing an enteric polymer administered by a composition comprising pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; 26. The method according to any one of items 1 to 25, wherein the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 28. A pharmaceutical composition for oral administration, when used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, A pharmaceutical composition, wherein the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 29. A pharmaceutical composition for oral administration, when used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer Including, pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; A pharmaceutical composition, wherein the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 30. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing an inflammatory response caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 31. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing an inflammatory condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 32. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing hypercytokinemia caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 33. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 34. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 35. The use according to any one of items 30 to 34, wherein the viral infection is a coronavirus infection. 36. Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating coronavirus infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use. 37. The use according to item 35 or 36, wherein the coronavirus infection is caused by SARS-CoV-2. 38. The use according to any one of items 30 to 37, wherein the medicament is formulated for oral administration. 39. Medicines that: (a) Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer A pharmaceutical composition comprising: The weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 39. The use according to any one of items 30 to 38, comprising a pharmaceutical composition. 40. Medicines that: (a) Sodium meta arsenite (O=As-O -Na + ) or potassium meta arsenite (O=As-O - K + ), as well as the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer A pharmaceutical composition comprising: pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; The coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. 39. The use according to any one of items 30 to 38, comprising a pharmaceutical composition. 41. A pharmaceutical composition for oral administration comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; or A pharmaceutical composition for use in treating a coronavirus infection in a subject. 42. A pharmaceutical composition for oral administration, comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer Including, pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; or A pharmaceutical composition for use in treating a coronavirus infection in a subject. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1A-C: Graphs showing the mean (±SEM) cytotoxicity and viability of cultured rat primary macrophages incubated for 24 h in culture medium containing 100 ng / mL lipopolysaccharide (LPS) and sodium meta arsenite (A; 30, 10, 7, 5, 3, 1, 0.3, and 0.1 μM) or control (B and C). n=3. [Figure 2] Figure 2A-F. Graphs showing mean (±SEM) TNF-α (A), IL-1β (C), or IL-6 (E) secretion and viability of cultured rat primary macrophages incubated for 24 hours in culture medium containing 100 ng / mL LPS and various concentrations of sodium meta arsenite (30, 10, 7, 5, 3, 1, 0.3, and 0.1 μM) compared to positive (celecoxib) and negative (vehicle) controls (B, D, and F). n=3. Values not sharing a common letter are significantly different (p≦0.05). [Figure 3] Figure 3A and B A. Graphs showing carbon monoxide produced by RAW264.7 cells after stimulation with LPS and treatment with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on carbon monoxide production (iNOS assay)). B. Graphs showing cell viability after treatment with LPS and sodium meta arsenite (*: p<0.01 compared to control (LPS+)). [Figure 4]1 is a graph showing prostaglandin E2 (PGE2) production by RAW264.7 cells after stimulation with LPS and treatment with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on PGE2 production (PGE2 assay); *: p<0.01 compared to control (LPS+)). [Figure 5] 1 is a Western blot showing iNOS and COX-2 protein expression in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on iNOS and COX-2 protein expression). [Figure 6] 1 is a Western blot showing TNF-α and IL-1β protein expression in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on TNF-α and IL-1β protein expression). [Figure 7] 1 is an image of an electrophoresis gel showing the mRNA expression, as determined by RT-PCR, of iNOS and COX-2 in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on iNOS and COX-2 gene expression). [Figure 8] 1 is a graph showing iNOS mRNA expression in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on iNOS mRNA expression (real-time PCR); *: p<0.01 compared to control (LPS+)). [Figure 9]1 is an image of a gel electrophoresis of RT-PCR products showing TNF-α, IL-1β, and IFN-β mRNA expression in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on TNF-α, IL-1β, and IFN-β gene expression). [Figure 10] 1 is a graph showing NF-κB transcription activity in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on LPS-induced NF-κB transcription activity; *: p<0.01 compared to control (LPS+)). [Figure 11] 1 is a Western blot showing NF-κB (p50) and (p65) protein expression in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite (i.e., showing the effect of sodium meta arsenite on NF-κB protein expression). [Figure 12] 1 is a Western blot showing protein expression of IκB and IKK in RAW264.7 cells treated with LPS with and without various concentrations of sodium meta arsenite. [Figure 13] 1 shows the AUC (area under the curve) values of TNF-α levels in bronchoalveolar lavage fluid of a mouse model of ARDS after treatment with PAX-1 (SMA), dexamethasone, or no treatment. Data are expressed as mean ± 95% confidence interval (*: p<0.05, **: p<0.005). [Figure 14] 1 shows the area under the curve (AUC) values of IL-6 levels in bronchoalveolar lavage fluid of a mouse model of ARDS after treatment with PAX-1 (SMA), dexamethasone, or no treatment. Data are expressed as mean ± 95% confidence interval (*: p<0.05, **: p<0.005). [Figure 15]1 shows the AUC (area under the curve) values of IL-1β levels in bronchoalveolar lavage fluid of a mouse model of ARDS after treatment with PAX-1 (SMA), dexamethasone, or no treatment. Data are expressed as mean ± 95% confidence interval (*: p<0.05, **: p<0.005). [Figure 16] FIG. 1 is a graph showing mouse survival in an ARDS mouse model after treatment with PAX-1 (SMA), dexamethasone, or no treatment (G1 (negative control, 0 mg / kg), G2 (PAX-1, 1.03 mg / kg), G3 (PAX-1, 1.54 mg / kg), G4 (PAX-1, 2.05 mg / kg), G5 (dexamethasone, 3 mg / kg); **p<0.005, significant difference from negative control (G1) by log-rank (Mantel-Cox) test; ***p<0.0005, significant difference from negative control (G1) by log-rank (Mantel-Cox) test; ****p<0.0001, significant difference from negative control (G1) by log-rank (Mantel-Cox) test; n=10). [Figure 17] Graph showing inhibition of SARS-CoV-2 replication by chloroquine, remdesivir, lopinavir, PAX-1(SMA) in DMSO ("Komipharm(DMSO)"), and PAX-1(SMA) in PBS ("Komipharm(PBS)"). DETAILED DESCRIPTION OF THE INVENTION

[0033] Preferred embodiments of the present invention are described below by way of example only.

[0034] definition Unless otherwise defined herein, the following terms are understood to have the general meanings that follow: The terms referenced below have the general meanings that follow when the term is used alone and when the term is used in combination with other terms, unless otherwise indicated.

[0035] As used herein, "treating" means affecting a subject, tissue, or cell to achieve a desired pharmacological and / or physiological effect, including inhibiting a condition, i.e., preventing the onset of the condition, or reducing or ameliorating the effects of a condition, i.e., reversing or regressing the effects of a condition.

[0036] As used herein, "preventing" means preventing a condition from occurring in a cell, tissue, or subject that may be at risk of having the condition, but does not necessarily mean that the condition will eventually develop or that the subject will eventually develop the condition. Preventing includes delaying the onset of the condition in a cell, tissue, or subject.

[0037] As used herein, "reducing the inflammatory response caused by viral infection" refers to reducing the severity of the inflammatory response to viral infection compared to the severity of untreated inflammatory response.Reducing severity can involve, for example, reducing the severity or number of symptoms that appear compared to the severity or number of symptoms that appear in untreated response, or reducing the serum level of one or more pro-inflammatory cytokines compared to the serum level of one or more pro-inflammatory cytokines in untreated response.

[0038] As used herein, "inflammatory conditions caused by viral infection" refers to conditions resulting from an inflammatory response to a viral infection. Typically, inflammatory conditions are caused, at least in part, by increased, and in some cases uncontrolled, levels of one or more pro-inflammatory cytokines during viral infection. During viral infection, pro-inflammatory immune cells migrate to the site of infection and respond by secreting large amounts of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, particularly IL-1β and IL-6. The secretion of such pro-inflammatory cytokines promotes the migration of additional immune cells to the site of infection. As a result of the rapid influx of immune cells, further secretion of pro-inflammatory cytokines, and destruction of infected cells, fluid accumulates in the affected area and tissue damage occurs. For example, coronaviruses are respiratory viruses that infect the lungs of a subject. The inflammatory response to coronaviruses causes respiratory inflammation, leading to fluid accumulation in the alveoli and, in severe cases, shortness of breath and pneumonia. Over time, the fluid from the inflammation can consolidate, causing pulmonary fibrosis and, in some cases, death. Even if the subject survives, the inflammation can result in a decrease in lung function.

[0039] As used herein, "reducing TNF-α, IL-1β, and / or IL-6 levels" refers to reducing the amount of TNF-α, IL-1β, and / or IL-6 secreted from immune cells, typically macrophages. The amount of TNF-α, IL-1β, and / or IL-6 secreted from immune cells can be determined, for example, by determining the serum levels of TNF-α, IL-1β, and / or IL-6 in the subject.

[0040] As used herein, the term "subject" refers to a mammal. A mammal can be human or non-human. Examples of non-humans include primates, livestock (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). Typically, the mammal is a human or a primate. More typically, the mammal is a human.

[0041] The term "composition" encompasses compositions and formulations comprising an active pharmaceutical ingredient ("API") and an excipient or carrier, and also encompasses compositions and formulations having an encapsulating material as a carrier to provide a capsule in which the active pharmaceutical ingredient (with or without other carriers) is surrounded by the encapsulating carrier. In pharmaceutical compositions, the excipient or carrier is "pharmaceutically acceptable," meaning that it is not undesirable for biological or other reasons; i.e., the material can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components in the composition in which it is contained. Supplementary active ingredients can also be incorporated into the composition.

[0042] "Pharmaceutically acceptable," as in the enumeration of "pharmaceutically acceptable salts" or "pharmaceutically acceptable excipients or carriers," as used herein means a substance that is not undesirable for biological or other reasons, i.e., the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious way with any of the other components in the composition in which it is contained.

[0043] The term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical active ingredient sufficient to produce a desired therapeutic response without undue adverse side effects (e.g., toxicity, irritation, or allergic response) when used in the methods of the present invention, commensurate with a reasonable benefit / risk ratio. This amount may, for example, be effective in reducing TNF-α, IL-1β, and / or IL-6 production by a subject's immune cells, more typically macrophages. The specific effective or therapeutically effective amount will vary depending on factors such as the particular condition being treated, the subject's age, weight, general health, physical condition, sex, and diet, the duration of treatment, the nature of concomitant therapy (if any), and the severity of the particular condition.

[0044] As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agent, isotonic and absorption delaying agent, buffer, carrier solution, suspension, colloid, etc. The use of such media and agents in pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

[0045] As used herein, "administration" or "administering" or "administering" refers to dispensing, applying, or tendering two or more agents (e.g., sodium meta arsenite and / or arsenic trioxide, and cisplatin, adriamycin, and / or a taxane, e.g., paclitaxel or docetaxel) to a subject. Administration can be carried out using any of several methods known in the art. For example, "administering" as used herein means via injection (intravenous administration (iv)), parenteral, and / or oral administration. "Parenteral" means intravenous, subcutaneous, and intramuscular administration. It is understood that the preferred method and order of actual administration will vary depending, inter alia, on the particular formulation of SMA or KMA utilized. The method and order of administration of SMA or KMA in a given setting can be determined by one of ordinary skill in the art using conventional techniques and in view of the information provided herein.

[0046] As used herein, the term "about" refers to a slight variation of the specified value, preferably within 10% of the specified value. In any case, the term "about" can mean that a relatively high degree of variation is allowed, for example, depending on the experimental techniques used. Such variation of the specified value is understood by those skilled in the art and falls within the context of the present invention. Furthermore, in order to provide a more concise description, some of the quantitative expressions presented herein are not qualified with the term "about." It is understood that, regardless of whether the term "about" is explicitly used, any quantity presented herein is intended to refer to an actual specified value, and also to refer to an approximate value that can be reasonably estimated based on ordinary skill in the art for such a specified value, including equivalent values and approximate values resulting from experimental and / or measurement conditions for such a specified value.

[0047] Unless otherwise stated, all amounts are expressed herein as weight percent (% w / w).

[0048] Of course, any material used in preparing the pharmaceutical compositions described herein should be pharmaceutically pure and substantially non-toxic in the amounts employed.

[0049] Inflammatory response One embodiment is a method for reducing an inflammatory response caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0050] An inflammatory response caused by a viral infection is an immune response to a viral infection, in which pro-inflammatory cytokines are secreted by immune cells, typically macrophages, in response to the viral infection. In one embodiment, the pro-inflammatory cytokines include TNF-α, IL-1β, and / or IL-6. In some embodiments, the inflammatory response includes hypercytokinemia (also known as a "cytokine storm").

[0051] Inflammatory responses caused by viral infections can be either acute or chronic. Acute inflammation typically lasts only a few days. In contrast, chronic inflammation typically lasts for weeks, months, or even indefinitely and can cause tissue damage.

[0052] One embodiment is a method of treating or preventing an inflammatory condition caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + The inflammatory condition caused by a viral infection is a condition resulting from an inflammatory response caused by a viral infection.

[0053] In one embodiment, the inflammatory condition is systemic inflammatory response syndrome (SIRS) caused by a viral infection. In one embodiment, the viral infection is caused by an RNA virus.

[0054] In one embodiment, the inflammatory condition is caused by influenza infection. In one embodiment, the influenza is avian influenza. In one embodiment, the inflammatory condition caused by influenza infection is pneumonia.

[0055] In one embodiment, the inflammatory condition is caused by a coronavirus infection. In one embodiment, the coronavirus is selected from the group consisting of 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV-2. In one embodiment, the coronavirus is MERS-CoV. In one embodiment, the coronavirus is SARS-CoV. In one embodiment, the coronavirus is SARS-CoV-2 (also known as the "2019 novel coronavirus").

[0056] In various embodiments, the inflammatory condition is selected from Middle East Respiratory Syndrome (MERS - caused by MERS-Cov) and Severe Acute Respiratory Syndrome (SARS - caused by SARS-CoV), or a condition caused by the 2019 novel coronavirus (SARS-CoV-2) (e.g., COVID-19).

[0057] In one embodiment, the inflammatory condition is pneumonia caused by COVID-19.

[0058] The methods described herein involve administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered.

[0059] As described in the Examples, the present inventors have found that sodium meta arsenite can reduce or inhibit the production and / or secretion of the pro-inflammatory cytokines TNF-α, IL-1β and / or IL-6 from macrophages.

[0060] One embodiment is a method of reducing levels, typically serum levels, of TNF-α, IL-1β and / or IL-6 in a subject suffering from an inflammatory condition caused by a viral infection, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0061] In one embodiment, the method reduces TNF-α levels in a subject. Typically, the method reduces TNF-α serum levels in a subject.

[0062] In one embodiment, the method reduces IL-1β levels in the subject. Typically, the method reduces IL-1β serum levels in the subject.

[0063] In one embodiment, the method reduces the levels of TNF-α and IL-1β in the subject. Typically, the method reduces the serum levels of TNF-α and IL-1β in the subject.

[0064] In one embodiment, the method reduces IL-6 levels in the subject. Typically, the method reduces IL-6 serum levels in the subject.

[0065] In one embodiment, the method reduces the levels of IL-1β and IL-6 in the subject. Typically, the method reduces the serum levels of IL-1β and IL-6 in the subject.

[0066] In one embodiment, the method reduces the levels of TNF-α, IL-1β, and IL-6 in the subject. Typically, the method reduces the serum levels of TNF-α, IL-1β, and IL-6 in the subject.

[0067] One embodiment is a method of treating or preventing a disease or condition mediated by elevated TNF-α, IL-1β and / or IL-6 levels due to a viral infection in a subject, comprising administering to a subject an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0068] In one embodiment, the disease or condition is pneumonia. In one embodiment, the viral infection is a coronavirus infection. In one embodiment, the coronavirus is SARS-CoV-2.

[0069] In one embodiment, the disease or condition is MEARS or SARS.

[0070] In one embodiment, the disease or condition is hypercytokinemia. In one embodiment, the viral infection is a coronavirus infection. In one embodiment, the coronavirus is SARS-CoV-2.

[0071] One embodiment is a method of treating a disease or symptom caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + (Illegible text - likely OCR error)

[0072] In one embodiment, the disease or condition is a malady such as fever, chills, flu-like symptoms, inflammation, or brain fog, or a combination thereof. Thus, in one embodiment, the disease or condition is fever. In another embodiment, the disease or condition is chills. In another embodiment, the disease or condition is flu-like symptoms. In another embodiment, the disease or condition is inflammation. In another embodiment, the disease or condition is brain fog.

[0073] Flu-like symptoms include headache, fever, cough, shortness of breath (dyspnea), difficulty breathing, phlegm production, chest tightness, fatigue, sore throat, runny nose, loss of appetite, and aches and pains (including muscle and body aches).

[0074] In one embodiment, the viral infection is a coronavirus infection. In one embodiment, the coronavirus is SARS-CoV-2.

[0075] In one embodiment, the disease or condition is treated with sodium meta arsenite (O=As-O) via anti-inflammatory mechanisms and / or via suppression of viral replication. - Na + ) or potassium meta arsenite (O=As-O - K + In one embodiment, the disease or condition is treated with sodium meta arsenite (O=As-O) via an anti-inflammatory mechanism. - Na + ) or potassium meta arsenite (O=As-O - K + In one embodiment, the disease or condition is treated with sodium meta arsenite (O=As-O) through the inhibition of viral replication. - Na + ) or potassium meta arsenite (O=As-O - K + In one embodiment, the disease or condition is treated with sodium meta arsenite (O=As-O) through anti-inflammatory mechanisms and suppression of viral replication. - Na +) or potassium meta arsenite (O=As-O - K + ) is treated.

[0076] Typically, sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) and a pharmaceutically acceptable carrier.

[0077] In some embodiments, the carrier is a non-naturally occurring carrier.

[0078] Pharmaceutical Composition As described above, sodium meta arsenite (O=As-O) is typically used in the methods and uses described herein. - Na + ) or potassium meta arsenite (O=As-O - K + ) is sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) and a pharmaceutically acceptable carrier.

[0079] Pharmaceutical compositions may contain other drugs or additional active agents as described above, and may be formulated, for example, by using conventional solid or liquid vehicles or diluents and pharmaceutical additives (e.g., excipients, binders, preservatives, stabilizers, flavorings, etc.) of a type appropriate to the desired mode of administration, in accordance with well-known pharmaceutical formulation techniques (see, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., 2005, Lippincott Williams & Wilkins), or other techniques.

[0080] The pharmaceutical compositions may be in a form suitable for intravenous, oral, nasal, topical (including dermal, buccal and sublingual), or parenteral (including intramuscular, subcutaneous and intravenous) administration, as well as administration by inhalation or insufflation.

[0081] Thus, the compounds described herein can be incorporated into pharmaceutical compositions and unit dosage forms together with pharmaceutically acceptable carriers. Pharmaceutical compositions can be solid, such as tablets or filled capsules, for oral administration, or liquid, such as solutions, suspensions, emulsions, elixirs, or filled capsules. Pharmaceutical compositions can be liquid, such as solutions, suspensions, or emulsions, for intravenous administration.

[0082] Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range employed.

[0083] For preparing pharmaceutical compositions from the compounds described herein, pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules, cachets, lozenges (solid or chewable), suppositories, and dispersible granules.Solid carriers can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.

[0084] Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, etc. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

[0085] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in saline, water, or aqueous polyethylene glycol solution.

[0086] Sterile liquid form compositions include sterile solutions, suspensions, emulsions, syrups, and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier such as sterile water, sterile organic solvent, or a mixture of both.

[0087] In one embodiment, the sodium meta arsenite and potassium meta arsenite are formulated for oral administration. Compositions for oral administration can be solid or liquid formulations.

[0088] In one embodiment, the composition for oral administration is a solid formulation.

[0089] Sodium meta arsenite and potassium meta arsenite can be synthesized from arsenic trioxide (As2O3). For example, sodium meta arsenite can be synthesized by reacting arsenic trioxide (As2O3) with aqueous sodium hydroxide to form trivalent sodium meta arsenite (top left side of Scheme 1 below). The solution is cooled, the sodium meta arsenite is filtered, and the water is evaporated. The formed sodium meta arsenite is then washed with methanol to remove water, filtered under vacuum, and then dried. Potassium meta arsenite can be prepared in a similar manner to sodium meta arsenite, using aqueous potassium hydroxide instead of aqueous sodium hydroxide.

[0090] However, meta arsenite (O=As-O - The main complications in the speciation chemistry of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + The ability of sodium meta arsenite (O=As-O) to convert into several different forms in solution, such as when an oral dosage form containing sodium meta arsenite (O=As-O) dissolves in the stomach. - Na + ) is readily soluble in strong acids, strong bases, and neutral conditions. The form present depends on the solution pH and the oxidation tendency of sodium meta arsenite (Scheme 1 below). Potassium meta arsenite behaves similarly to sodium meta arsenite. In general, neutral to alkaline conditions tend to favor the formation (or retention) of As(III) (arsenite), while acidic conditions (especially in the presence of chloride ions, such as in the stomach) tend to favor the formation of As(V) (arsenate).

[0091] [ka]

[0092] In addition, meta arsenite (O=As-O -) can be oxidized to meta arsenite during storage in the presence of chloride, metal ions, or moisture (e.g., in the dissolution medium or excipients, excipients with metal ions, especially ferric ions, can catalyze oxidation), or atmospheric oxygen. Oxidation of meta arsenite can occur very rapidly at low pH. Sodium meta arsenite (O=As-O - Na + ) and potassium meta arsenite (O=As-O - K + ) are both hygroscopic.

[0093] In solution, the main decomposition product of sodium meta arsenite is pentavalent meta arsenate (AsO4 3- or As(V)) species. This is hypothesized to proceed as shown in Box 1 below, although in theory, oxidation (change in valence) could occur without absorbing evolved oxygen (e.g., by interaction with excipients or by reaction with sodium meta arsenite or metal ions present in the composition).

[0094] [ka]

[0095] Sodium meta arsenite (O=As-O) in the stomach - Na + ) or potassium meta arsenite (O=As-O - K + A further complication arising from the dissolution of meta arsenite is the formation of arsenic(III) chloride (AsCl3) from chloride ions in the stomach. Oxidation of meta arsenite can occur more rapidly in the presence of chloride. Arsenic(III) chloride is toxic to humans and can cause severe adverse effects.

[0096] In some embodiments, where the composition is for oral administration, an enteric-coated solid pharmaceutical composition is provided that comprises sodium meta arsenite or potassium meta arsenite, is suitable for oral administration, passes through the stomach, and begins to dissolve in the small intestine (where the acidity is pH 6.5-7.5). The risk of oxidation of the meta arsenite form to the meta arsenate form (in the stomach or during storage) and the risk of formation of toxic arsenic(III) chloride from chloride ions in the stomach are minimized by using appropriate excipients and carriers and an appropriate enteric coating of appropriate thickness. Dissolution of the enteric-coated solid pharmaceutical composition in the small intestine can occur rapidly or over an extended period of time (e.g., 0.5, 0.75, 1, 2, 3, 4, 5, or 6 hours, preferably within 2 hours).

[0097] Preferred embodiments of the pharmaceutical composition for oral administration are described below: The pharmaceutical composition for oral administration can be prepared by the effective methods described below.

[0098] Oral administration of pharmaceutical compositions In one embodiment, a pharmaceutical composition suitable for oral administration comprises: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, Here, the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[0099] For example, in the above embodiments, the one or more pharmaceutically acceptable excipients can be selected from a filler or diluent, a disintegrant, a glidant, a lubricant, and a binder. In some embodiments, the solid core can include two or more of these excipients, three or more of these excipients, four or more of these excipients, or all of these excipients. Thus, in some embodiments, the solid core includes a filler or diluent, a disintegrant, a glidant, a lubricant, and a binder.

[0100] In one embodiment, a pharmaceutical composition suitable for oral administration comprises: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w a solid core, and (b) an enteric coating containing an enteric polymer Including, pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; A pharmaceutical composition is provided, wherein the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[0101] The pharmaceutical composition may be in the form of an enteric-coated tablet or an enteric-coated capsule. In some embodiments, the pharmaceutical composition is an enteric-coated tablet. In some embodiments, the pharmaceutical composition is an enteric-coated capsule.

[0102] In the pharmaceutical composition, the active pharmaceutical ingredient (API) is sodium meta arsenite or potassium meta arsenite.

[0103] Sodium meta arsenite and potassium meta arsenite can be obtained commercially in high purity (>98% As(III) and minimal levels of As(V)). Sodium meta arsenite and potassium meta arsenite are hygroscopic.

[0104] The inorganic compounds sodium meta arsenite and potassium meta arsenite, respectively, are typical tablet excipients (typical tablet excipients usually have a loading of approximately 1.2–1.6 g / cm 3 The higher particle (true) density (e.g., sodium meta arsenite has a density of approximately 2.1-2.3 g / cm) compared to organic materials (e.g., sodium meta arsenite has a density of approximately 2.1-2.3 g / cm). 3 , and about 8.76 g / cm for potassium meta arsenite. 3 )

[0105] Due to the difference in particle size between the API and the excipients, the possibility of API segregation in the composition is high. Those skilled in the art will appreciate that using an API with a preferred particle size is advantageous because it results in improved powder mixing and blend uniformity, minimizes or eliminates powder segregation during compression, and achieves sufficient uniformity of the contents in the composition.

[0106] In some embodiments of compositions for oral administration, the particle size of the API is about 50-150 microns, in some embodiments, the particle size of the API is about 70-120 microns, in some embodiments, the particle size of the API is about 90-100 microns.

[0107] In some embodiments, the API is sodium meta arsenite.

[0108] In some embodiments, the API is potassium meta arsenite.

[0109] In some embodiments, the amount of API in the solid core of the pharmaceutical composition for oral administration is about 0.1 to 5.0% w / w of the solid core, preferably about 0.5 to 3.0% w / w of the solid core, more preferably about 1.0 to 2.5% w / w of the solid core, even more preferably about 1.5 to 2.0% w / w of the solid core, and most preferably about 1.6 to 1.8% w / w of the solid core, for example, about 1.65% w / w, about 1.66% w / w, about 1.67% w / w, about 1.68% w / w, about 1.69% w / w, about 1.70% w / w, about 1.71% w / w, about 1.72% w / w, about 1.73% w / w, about 1.74% w / w, or about 1.75% w / w of the solid core.

[0110] In some embodiments, the particle size of the API and the particle size of the pharmaceutically acceptable excipient are similar.Advantageously, the use of API and excipients with similar particle sizes can result in improved powder mixing and blend uniformity, can minimize or eliminate powder segregation during compression, and can achieve sufficient uniformity of the contents in the composition.

[0111] In some embodiments, the API is micronized. It will be appreciated by those skilled in the art that reducing the particle size of the API by micronization can improve blend uniformity and content uniformity in dosage forms (e.g., tablets) when the API is present at low levels.

[0112] In some embodiments, the API is not micronized. It will be appreciated by those skilled in the art that micronizing hygroscopic APIs (e.g., sodium meta arsenite and potassium meta arsenite) may increase the risk of degradation due to higher surface area and reactivity.

[0113] In one embodiment, in addition to sodium meta arsenite or potassium meta arsenite, the oral pharmaceutical composition comprises one or more pharmaceutically acceptable excipients selected to minimize oxidation of meta arsenite to meta arsenate.

[0114] In some embodiments, the pharmaceutically acceptable excipient is selected such that after storage at room temperature for at least about 1 month, preferably at least about 2 months, more preferably at least about 3 months, even more preferably at least about 4 months, and most preferably at least about 6 months, less than about 10% w / w, preferably less than about 5% w / w, more preferably less than about 2% w / w, even more preferably less than about 1% w / w, and most preferably less than about 0.5% w / w of the sodium meta arsenite or potassium meta arsenite oxidizes to sodium meta arsenate or potassium meta arsenate.

[0115] In another embodiment, in addition to sodium meta arsenite or potassium meta arsenite, the pharmaceutical composition for oral administration contains one of the following pharmaceutically acceptable excipients: (i) a filler or diluent; (ii) a disintegrant, (iii) glidants; (iv) a lubricant, and (v) optionally a binder Includes:

[0116] It is understood by those skilled in the art that some excipients have multiple functions. When an excipient contained in a pharmaceutical composition has multiple functions, the pharmaceutical composition is considered to contain the excipients having these functions. For example, when an excipient acts as both a binder and a disintegrant, the pharmaceutical composition is understood to contain both a binder and a disintegrant.

[0117] Generally, one or more pharmaceutically acceptable excipients are compatible with sodium meta arsenite or potassium meta arsenite. Preferably, the pharmaceutically acceptable excipients have a low moisture level or low water activity to minimize the possibility of oxidation of meta arsenite to meta arsenite. Thus, preferably, oral pharmaceutical compositions do not contain excipients with high moisture levels or high water activity (such excipients, e.g., excipients with metal ions, particularly iron ions, can catalyze oxidation). However, those skilled in the art will understand that this has limited practicality for oral pharmaceutical compositions, since some available moisture is required for adequate compression.

[0118] In some embodiments, the particle size of the API and the particle size of the pharmaceutically acceptable excipient are similar.Advantageously, the use of API and excipients with similar particle sizes can result in improved powder mixing and blend uniformity, can minimize or eliminate powder segregation during compression, and can achieve sufficient uniformity of the contents in the solid core.

[0119] In some embodiments, where possible, the primary high density excipient is selected to match the density of sodium meta arsenite or potassium meta arsenite (sodium meta arsenite has an estimated true density of approximately 2.1-2.3 g / cm). 3 Potassium meta arsenite has an estimated true density of approximately 8.76 g / cm 3 Typical tablet excipients are organic substances with a loading of approximately 1.2-1.6 g / cm 3 It has a density of

[0120] The filler or diluent may be selected from, for example, dibasic calcium phosphate anhydrous, partially pregelatinized starch, silicified microcrystalline cellulose, microcrystalline cellulose, calcium sulfate dihydrate, lactose, calcium hydrogen phosphate, calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, or mixtures thereof. In some embodiments, the filler or diluent is dicalcium phosphate anhydrous, partially pregelatinized starch, or mixtures thereof. In some embodiments, the filler or diluent is dicalcium phosphate anhydrous. In some embodiments, the filler or diluent is partially pregelatinized starch. In some embodiments, the diluent may be a compressible diluent, for example, silicified microcrystalline cellulose, microcrystalline cellulose, or partially pregelatinized starch.

[0121] The filler or diluent may be present in the solid core of the oral pharmaceutical composition in an amount of about 5-95% w / w of the solid core. In some embodiments, the filler or diluent is present in the solid core of the pharmaceutical composition in an amount of about 10-90% w / w of the solid core, e.g., about 10% w / w of the solid core, about 15% w / w of the solid core, about 20% w / w of the solid core, about 25% w / w of the solid core, about 30% w / w of the solid core, about 35% w / w of the solid core, about 40% w / w of the solid core, about 45% w / w of the solid core, about 50% w / w of the solid core, about 55% w / w of the solid core, about 60% w / w of the solid core, about 65% w / w of the solid core, about 70% w / w of the solid core, about 75% w / w of the solid core, about 80% w / w of the solid core, about 85% w / w of the solid core, or about 90% w / w of the solid core.

[0122] The disintegrant may be selected from, for example, L-hydroxypropyl cellulose, partially pregelatinized starch, crospovidone, potato starch, corn starch, sodium starch glycolate, and alginic acid. Sodium starch glycolate and crospovidone are super disintegrants. In some embodiments, the disintegrant is L-hydroxypropyl cellulose, partially pregelatinized starch, sodium starch glycolate, or a mixture of two or more thereof. In some embodiments, the disintegrant is L-hydroxypropyl cellulose. In some embodiments, the disintegrant is partially pregelatinized starch. In some embodiments, the disintegrant is sodium starch glycolate.

[0123] The disintegrant may be present in the solid core of the oral pharmaceutical composition in an amount of about 10-90% w / w, e.g., about 10-50% w / w of the solid core. In some embodiments, the disintegrant is present in the solid core of the oral pharmaceutical composition in an amount of about 15-85% w / w, e.g., about 15% w / w of the solid core, about 20% w / w of the solid core, about 25% w / w of the solid core, about 30% w / w of the solid core, about 35% w / w of the solid core, about 40% w / w of the solid core, about 45% w / w of the solid core, about 50% w / w of the solid core, about 55% w / w of the solid core, about 60% w / w of the solid core, about 65% w / w of the solid core, about 70% w / w of the solid core, about 75% w / w of the solid core, about 80% w / w of the solid core, or about 85% w / w of the solid core.

[0124] The glidant may be selected from, for example, colloidal silicon dioxide and talc. In some embodiments, the glidant is colloidal silicon dioxide. In some embodiments, the glidant is talc.

[0125] The glidant may be present in the solid core of the oral pharmaceutical composition in an amount of about 0.1-5% w / w of the solid core. In some embodiments, the glidant comprises about 0.3-4% w / w of the solid core, e.g., about 0.3% w / w of the solid core, about 0.4% w / w of the solid core, about 0.5% w / w of the solid core, about 0.6% w / w of the solid core, about 0.7% w / w of the solid core, about 0.8% w / w of the solid core, about 0.9% w / w of the solid core, about 1.0% w / w of the solid core, about 1.1% w / w of the solid core, about 1.2% w / w of the solid core, about 1.3% w / w of the solid core, about 1.4% w / w of the solid core, about 1.5% w / w of the solid core, about 1.6% w / w of the solid core, about 1.7% w / w of the solid core, about 1.8% w / w of the solid core, about 1.9% w / w of the solid core, about 2.0% w / w of the solid core, or about 2.5% w / w of the solid core. 1% w / w, about 2.2% w / w of solid core, about 2.3% w / w of solid core, about 2.4% w / w of solid core, about 2.5% w / w of solid core, about 2.6% w / w of solid core, about 2.7% w / w of solid core, about 2.8% w / w of solid core, about 2.9% w / w of solid core, about 3.0% w / w of solid core, about 3.1% w / w of solid core, solid core about 3.2% w / w of the solid core, about 3.3% w / w of the solid core, about 3.4% w / w of the solid core, about 3.5% w / w of the solid core, about 3.6% w / w of the solid core, about 3.7% w / w of the solid core, about 3.8% w / w of the solid core, about 3.9% w / w of the solid core, or about 4.0% w / w of the solid core.

[0126] The lubricant may be selected from, for example, sodium stearyl fumarate, magnesium stearate, stearic acid, talc, and silica. In some embodiments, the lubricant is sodium stearyl fumarate. In some embodiments, the lubricant is magnesium stearate. In some embodiments, the lubricant is stearic acid. In some embodiments, the lubricant is talc. In some embodiments, the lubricant is silica.

[0127] The lubricant may be present in the solid core of the oral pharmaceutical composition in an amount of about 0.1-5% w / w of the solid core. In some embodiments, the lubricant is present in the solid core in an amount of about 0.3-4% w / w of the solid core, such as about 0.3% w / w of the solid core, about 0.4% w / w of the solid core, about 0.5% w / w of the solid core, about 0.6% w / w of the solid core, about 0.7% w / w of the solid core, about 0.8% w / w of the solid core, about 0.9% w / w of the solid core, about 1.0% w / w of the solid core, about 1.1% w / w of the solid core, about 1.2% w / w of the solid core, about 1.3% w / w of the solid core, about 1.4% w / w of the solid core, about 1.5% w / w of the solid core, about 1.6% w / w of the solid core, about 1.7% w / w of the solid core, about 1.8% w / w of the solid core, about 1.9% w / w of the solid core, about 2.0% w / w of the solid core, about 2.1 %w / w, about 2.2%w / w of solid core, about 2.3%w / w of solid core, about 2.4%w / w of solid core, about 2.5%w / w of solid core, about 2.6%w / w of solid core, about 2.7%w / w of solid core, about 2.8%w / w of solid core, about 2.9%w / w of solid core, about 3.0%w / w of solid core, about 3.1%w / w of solid core, solid core about 3.2% w / w of the solid core, about 3.3% w / w of the solid core, about 3.4% w / w of the solid core, about 3.5% w / w of the solid core, about 3.6% w / w of the solid core, about 3.7% w / w of the solid core, about 3.8% w / w of the solid core, about 3.9% w / w of the solid core, or about 4.0% w / w of the solid core.

[0128] When present, the binder may be selected from, for example, silicified microcrystalline cellulose, microcrystalline cellulose, partially pregelatinized starch, L-hydroxypropyl cellulose (low-substituted hydroxypropyl cellulose), hydroxypropyl cellulose, copovidone (polyvinylpyrrolidone), pregelatinized maize starch, hydroxypropyl methylcellulose, starch, acacia, maize starch, and gelatin. In some embodiments, the binder is L-hydroxypropyl cellulose (low-substituted hydroxypropyl cellulose). In some embodiments, the binder is a mixture of L-hydroxypropyl cellulose (low-substituted hydroxypropyl cellulose) and hydroxypropyl cellulose. In some embodiments, the binder is partially pregelatinized starch.

[0129] The binder may be present in the solid core of the oral pharmaceutical composition in an amount of about 0-30% w / w of the solid core. In some embodiments, the binder may be present in the solid core of the oral pharmaceutical composition in an amount of about 1-30% w / w of the solid core, e.g., about 5-25% w / w of the solid core. For example, the binder may be present in the solid core of the pharmaceutical composition in an amount of about 5% w / w of the solid core, about 10% w / w of the solid core, about 15% w / w of the solid core, about 20% w / w of the solid core, about 25% w / w of the solid core, or about 30% w / w of the solid core.

[0130] The orally administered pharmaceutical composition may optionally include an antioxidant in the core. Antioxidants function as reducing agents by (a) lowering the redox potential, (b) scavenging oxygen, or (c) terminating free radical reactions (acting as free radical inhibitors). Mechanisms (a) and (b) are most relevant to the decomposition of sodium or potassium meta arsenite to sodium or potassium meta arsenate. Advantageously, the antioxidant acts to reduce or prevent the oxidation of As(III) to As(V) in the composition.

[0131] Examples of antioxidants that may be used in the solid core include sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium sulfate, sodium thiosulfate, cysteine hydrochloride, ascorbic acid, propyl gallate, butylhydroxytoluene (BHT), and butylhydroxyanisole (BHA).

[0132] The antioxidant can be present in the solid core in an amount of about 0.01 to 0.2% w / w of the solid core, e.g., 0.01% w / w, 0.02% w / w, 0.03% w / w, 0.04% w / w, 0.05% w / w, 0.06% w / w, 0.07% w / w, 0.08% w / w, 0.09% w / w, 0.10% w / w, 0.11% w / w, 0.12% w / w, 0.13% w / w, 0.14% w / w, 0.15% w / w, 0.16% w / w, 0.17% w / w, 0.18% w / w, 0.19% w / w, or 0.20% w / w.

[0133] Of course, one skilled in the art will understand that the amount of API (sodium meta arsenite or potassium meta arsenite), excipients, and other ingredients in the solid core will be adjusted to comprise 100% of the solid core.

[0134] Advantageously, the solid core of the pharmaceutical composition for oral administration has good blend uniformity and content uniformity by using the appropriate excipients described above.

[0135] In some embodiments, the solid core of the oral pharmaceutical composition does not include any one or more of the following: silicified microcrystalline cellulose, microcrystalline cellulose, calcium sulfate dihydrate, copovidone (polyvinylpyrrolidone), crospovidone, stearic acid, talc, and sodium metabisulfite.

[0136] The pharmaceutical composition for oral administration may include an enteric coating comprising an enteric polymer. The enteric coating may be applied using any suitable coating technique known in the art. The enteric coating material may be dispersed or dissolved in either water or a suitable organic solvent.

[0137] As enteric coating polymers, for example, one or more of the following can be used, either separately or in combination: copolymers of acrylic acid and its esters or methacrylic acid or its esters, polysorbates, cellulose acetate phthalate polymers, hydroxypropylmethylcellulose phthalate polymers, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethylethylcellulose, shellac, or solutions or dispersions of other suitable enteric coating polymers.

[0138] In some embodiments, the enteric coating is a methacrylate-based coating, such as one comprising a copolymer of methacrylic acid and ethyl acrylate.Several useful products are commercially available.

[0139] Enteric coating polymer products are commercially available from Rohm GmbH & Co., Darmstadt, Germany, under the trade name EUDRAGIT®, and include L100, 100-55, and S100. Examples of useful EUDRAGIT® products include EUDRAGIT L100-55, EUDRAGIT S100, and EUDRAGIT L30D-55. EUDRAGIT L100-55 is a poly(methacrylic acid-co-ethyl acrylate) (1:1). EUDRAGIT S100 is a methacrylic acid-methyl methacrylate copolymer (1:2). EUDRAGIT L30D-55 is an aqueous dispersion of a pH-dependent polymer that becomes soluble at pH 5.5 or above for targeted delivery to the duodenum. EUDRAGIT L30D-55 methacrylic acid copolymer is a 1:1 copolymer of methacrylic acid and ethyl acrylate, with the formula (C5H2O2·C4H6O2) x It has.

[0140] Acryl-EZE® from Colorcon is an aqueous acrylic enteric system that is dispersible in water for application of enteric film coatings to solid dosage forms such as tablets, granules, and beads. Examples of useful Acryl-EZE® products include Acryl-EZE II white (493Z180022) and Acryl-EZE Green (93O11863).

[0141] The enteric coating may further contain a pharmaceutically acceptable plasticizer to achieve desirable mechanical properties, such as flexibility and hardness, of the enteric coating. Examples of such plasticizers include, but are not limited to, triacetin, citrate esters, phthalate esters, dibutyl sebacate, cetyl alcohol, polyethylene glycol, polysorbate, or other plasticizers. Anti-tacking agents, such as magnesium stearate, titanium dioxide, and talc, and other additives may also be included in the enteric coating.

[0142] In some embodiments, the enteric coating results in a weight gain of about 7-17% w / w of the solid core, e.g., about 8-14% w / w of the solid core. In some embodiments, the enteric coating results in a weight gain of about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% w / w, about 13% w / w, about 13.5% w / w, or about 14% w / w. In some embodiments, the enteric coating results in a weight gain of about 12% w / w of the solid core.

[0143] In some embodiments, the solid core may be sub-coated before being coated with the enteric coating using polymers known in the art to be suitable for sub-coating.

[0144] Pharmaceutical compositions for oral administration are in one embodiment solid and enterically coated and suitable for oral administration, for example enteric coated tablets or enteric coated capsules.

[0145] In some embodiments, the oral pharmaceutical composition is an enteric-coated tablet having a solid core with a diameter of about 5 to 8 mm. The diameter is the diameter of the widest part of the solid core. In some embodiments, the diameter of the solid core is about 5.5 to 7.5 mm. In some embodiments, the diameter of the solid core is about 6.0 to 7 mm, e.g., about 6 mm, about 6.5 mm, or about 7 mm. Preferably, the oral pharmaceutical composition is an enteric-coated tablet having a solid core with a diameter of 6.5 mm. More preferably, the pharmaceutical composition of the present invention is an enteric-coated tablet having a solid core with a diameter of 6.5 mm and containing sodium meta arsenite.

[0146] In some embodiments, the thickness of the solid core of an enteric-coated tablet can be about 2 mm to 6 mm, for example, about 2 mm to 5 mm. The thickness of the solid core of an enteric-coated tablet is the depth of the solid core, i.e., the height of the solid core measured when the solid core is placed on a flat surface. In some embodiments, the thickness of the solid core of an enteric-coated tablet is about 3 mm to 4.5 mm. In some embodiments, the thickness of the solid core of an enteric-coated tablet is about 3.1 mm to 4.2 mm, for example, about 3.1 mm, about 3.2 mm, about 3.3 mm, about 3.4 mm, about 3.5 mm, about 3.6 mm, about 3.7 mm, about 3.8 mm, about 3.9 mm, about 4.0 mm, about 4.1 mm, or about 4.2 mm. Preferably, the thickness of the solid core of an enteric-coated tablet is about 3.4 mm, about 3.5 mm, about 3.6 mm, about 3.7 mm, about 3.8 mm, or about 3.9 mm.

[0147] In some embodiments, the pharmaceutical composition for oral administration is an enteric-coated capsule having a solid core having a length of about 8.0 to 16 mm. In some embodiments, the length of the solid core is about 8.5 to 15 mm. In some embodiments, the length of the solid core is about 8.5 to 14.5 mm, for example, about 8.5 mm, about 9.0 mm, about 9.5 mm, about 10.0 mm, about 10.5 mm, about 11.0 mm, about 11.5 mm, about 12.0 mm, about 12.5 mm, about 13.0 mm, about 13.5 mm, about 14 mm, or about 14.5 mm. Preferably, the pharmaceutical composition for oral administration is an enteric-coated capsule having a solid core having a length of about 14.3 mm. More preferably, the pharmaceutical composition of the present invention is an enteric-coated capsule having a solid core having a length of about 14.3 mm and containing sodium meta arsenite.

[0148] In some embodiments, the thickness of the solid core of the enteric-coated capsule can be about 3 mm to 8 mm, for example, about 4.0 mm to 7.0 mm. The thickness of the solid core of the enteric-coated capsule is the depth of the solid core, i.e., the height of the solid core measured when the solid core is placed on a flat surface. In some embodiments, the thickness of the solid core is about 4.5 to 6.5 mm, for example, about 4.5 mm, about 4.6 mm, about 4.7 mm, about 4.8 mm, about 4.9 mm, about 5.0 mm, about 5.1 mm, about 5.2 mm, about 5.3 mm, about 5.4 mm, about 5.5 mm, about 5.6 mm, about 5.7 mm, about 5.8 mm, about 5.9 mm, about 6.0 mm, about 6.1 mm, about 6.2 mm, about 6.3 mm, about 6.4 mm, or about 6.5 mm. Preferably, the thickness of the solid core of the enteric-coated capsule is about 5.31 mm.

[0149] In some embodiments, the hardness of the solid core is about 50 N to about 200 N, e.g., about 50 to about 150 N, or about 70 to about 120 N. In some embodiments, the hardness of the solid core is about 80 N to about 115 N, e.g., about 85 N, about 90 N, about 95 N, about 100 N, about 105 N, or about 110 N. In some embodiments, the hardness of the solid core is at least about 50 N, at least about 55 N, at least about 60 N, at least about 65 N, at least about 70 N, at least about 75 N, at least about 80 N, at least about 85 N, at least about 90 N, at least about 95 N, at least about 100 N, at least about 105 N, at least about 110 N, at least about 115 N, at least about 120 N, at least about 125 N, at least about 130 N, at least about 135 N, at least about 140 N, at least about 145 N, at least about 150 N, at least about 155 N, at least about 160 N, at least about 165 N, at least about 170 N, at least about 175 N, at least about 180 N, at least about 185 N, at least about 190 N, at least about 195 N, or about 200 N. Preferably, the hardness of the solid core is at least about 85 N, more preferably at least about 90 N, even more preferably at least about 100 N, and most preferably at least about 110 N. Typically, the hardness of the solid core does not exceed about 210 N.

[0150] In some embodiments, the friability of the solid core is less than about 0.5%, preferably less than about 0.45%, more preferably less than about 0.4%, even more preferably less than about 0.35%, and most preferably less than about 0.3%. In some embodiments, the friability of the solid core is less than about 0.25%. In some embodiments, the friability of the solid core is less than about 0.2%. In some embodiments, the friability of the solid core is less than about 0.15%. In some embodiments, the friability of the solid core is less than about 0.1%, for example, about 0.08%.

[0151] In some embodiments, the mass of the solid core is about 50 mg to 250 mg. In some embodiments, the mass of the solid core is about 80 mg to 220 mg. In some embodiments, the mass of the solid core is about 100 mg to 200 mg. In some embodiments, the mass of the solid core is about 120 mg to 180 mg. In some embodiments, the mass of the solid core is about 140 mg to 160 mg, for example, about 140 mg, about 145 mg, about 150 mg, about 155 mg, or about 160 mg. Preferably, the mass of the solid core is 150 mg.

[0152] In some embodiments, the oral pharmaceutical composition comprises a solid core selected from: A solid core, comprising sodium meta arsenite, dicalcium phosphate anhydrous, L-hydroxypropyl cellulose, hydroxypropyl cellulose, colloidal silicon dioxide, and sodium stearyl fumarate A solid core, including sodium meta arsenite, dicalcium phosphate anhydrous powder, partially pregelatinized starch, dicalcium phosphate anhydrous, sodium starch glycolate, colloidal silicon dioxide, and sodium stearyl fumarate. A solid core, containing sodium meta arsenite, dicalcium phosphate anhydrous powder, dicalcium phosphate anhydrous, L-hydroxypropyl cellulose, sodium starch glycolate, colloidal silicon dioxide, and sodium stearyl fumarate a solid core comprising sodium meta arsenite, dicalcium phosphate anhydrous, partially pregelatinized starch, sodium starch glycolate, colloidal silicon dioxide, and sodium stearyl fumarate; Solid core, containing sodium meta arsenite, dicalcium phosphate anhydrous, silicified microcrystalline cellulose, sodium starch glycolate, colloidal silicon dioxide, and sodium stearyl fumarate.

[0153] In some embodiments, the oral pharmaceutical composition is an enteric coated tablet comprising 1.67% w / w sodium meta arsenite of the solid core, having a solid core diameter of about 6.5 mm, a solid core mass of 150 mg, and an enteric coating adding about 12% w / w of the solid core.

[0154] In some embodiments, the oral pharmaceutical composition is an enteric-coated tablet comprising 1.67% w / w sodium meta arsenite of the solid core, having a solid core diameter of about 6.5 mm, a solid core mass of 150 mg, and an enteric coating having a coating thickness of about 0.2 mm.

[0155] In some embodiments, after administration of the pharmaceutical composition by oral administration, the pharmaceutical composition has the following dissolution characteristics: 75% or more in 45 minutes, preferably 75% or more in 30 minutes.

[0156] In some embodiments, dissolution of the pharmaceutical composition of the present invention and release of the API in the small intestine occurs rapidly or occurs over an extended period of time (e.g., 0.5, 0.75, 1, 2, 3, 4, 5, or 6 hours, preferably 2 hours or less).

[0157] In some embodiments, once the enteric coating dissolves, the solid core disintegrates in less than about 10 minutes, preferably less than about 8 minutes, more preferably less than about 6 minutes, even more preferably less than about 5 minutes, and most preferably less than about 4 minutes.

[0158] The pharmaceutical composition for oral administration is preferably provided in unit dosage form. The unit dosage form may be a packaged preparation, where the package contains discrete amounts of the pharmaceutical composition, such as packeted tablets or capsules. The unit dosage form may also be a tablet or capsule itself, or any suitable number of these may be packaged. The packaged form may be, for example, made of metal or plastic foil, such as a blister pack, for example, an oxygen-impermeable or low-permeable Alu-Alu blister. The packaged form may be accompanied by instructions for administration.

[0159] In some embodiments, oral pharmaceutical compositions can be stored at ambient or room temperature for at least 3 months, preferably at least 6 months, more preferably at least 1 year, and most preferably 18-24 months. In some embodiments, oral pharmaceutical compositions can be refrigerated (e.g., at about 2-8°C).

[0160] The pharmaceutical composition can be prepared by the method disclosed in WO 2019 / 178643.

[0161] Compositions for parenteral administration In certain circumstances, it is desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally.The solution of active compound as free base or pharmacologically acceptable salt can be prepared by water appropriately mixed with surfactant such as hydroxypropyl cellulose.Dispersion can also be prepared by glycerol, liquid polyethylene glycol, and their mixture, and oil.Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0162] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be enhanced by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0163] For parenteral administration using aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards required by government and local agency biological product standards.

[0164] Sterile injectable solution is prepared by incorporating the active compound of the required amount in suitable solvent with some other components as listed above if necessary, and then filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains basic dispersion medium and other components as listed above.For the case of sterile powder that is used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces the powder of active component and any additional desired component from the above-mentioned sterile filtered solution.

[0165] The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

[0166] Therapeutic agents can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Such preparations are sterile. Injectable preparations can be presented in unit dosage form, for example, in ampoules or multi-dose containers with added preservatives. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0167] In addition to the formulations described above, compound can also be formulated as depot preparation.Such long-acting preparations can be administered by implantation (for example, subcutaneous or intramuscular) or by intramuscular injection.Thus, for example, compound can be formulated with suitable polymeric or hydrophobic material (for example, emulsion in acceptable oil) or ion exchange resin, or as poorly soluble derivative, for example, poorly soluble salt.Liposome and emulsion are well-known examples of delivery vehicle or carrier for hydrophilic drugs.

[0168] Suitable pharmaceutically acceptable carriers and diluents used in the pharmaceutical preparations of the present invention are well known to those skilled in the art of formulating compounds into pharmaceutical compositions. Pharmaceutical preparations of the present invention suitable for parenteral administration can be formulated for intravenous infusion or injection using pharmaceutically acceptable carriers by a number of means well known to those skilled in the art. In certain embodiments, such pharmaceutical preparations are in the form of unit dosage forms of freeze-dried mixtures of active ingredients prepared by conventional techniques, which can be reconstituted with water or other suitable injection fluids at the time of administration.

[0169] Dosage The appropriate dosage of sodium meta arsenite or potassium meta arsenite can be readily determined by one of skill in the art.

[0170] Suitable dosage levels of sodium meta arsenite or potassium meta arsenite administered to a subject are generally about 0.01-0.8 mg / kg of the subject's body weight per day, e.g., about 0.05-0.7 mg / kg of the subject's body weight per day, about 0.1-0.6 mg / kg of the subject's body weight per day, or about 0.2-0.5 mg / kg of the subject's body weight per day, and can be administered in single or multiple doses per day.

[0171] For example, a suitable dosage level of sodium meta arsenite or potassium meta arsenite administered to a patient (e.g., a patient suffering from a coronavirus infection, such as a SARS-CoV-2 infection) can be about 2.0-30 mg / day / person, e.g., about 2.5-20.0 mg / day / person or about 2.5-17.5 mg / day / person. Preferably, the dosage level of sodium meta arsenite or potassium meta arsenite administered is about 5.0 to 12.5 mg / day / person, more preferably about 10.0 to 12.5 mg / day / person, such as 5.0 mg / day / person, 5.5 mg / day / person, 6.0 mg / day / person, 6.5 mg / day / person, 7.0 mg / day / person, 7.5 mg / day / person, 8.0 mg / day / person, 8.5 mg / day / person, 9.0 mg / day / person, 9.5 mg / day / person, 10.0 mg / day / person, 10.5 mg / day / person, 11.0 mg / day / person, 11.5 mg / day / person, 12.0 mg / day / person, or 12.5 mg / day / person. In some embodiments, the dosage level of sodium meta arsenite or potassium meta arsenite administered is 7.5 mg per day.

[0172] It is understood that the specific dose level and frequency of administration for any particular subject may vary and will depend on a variety of factors, including the subject's age, weight, general health, sex and diet, mode and time of administration, rate of excretion, drug combination, and the severity of the particular condition.

[0173] The pharmaceutical composition of the present invention can be taken before (e.g., 30 minutes before), during, or after (e.g., 30 minutes after) a meal. Preferably, the pharmaceutical composition of the present invention is taken immediately after a meal.

[0174] An example of a dosing regimen for a tablet of the present invention having 2.5 mg of sodium meta arsenite (SMA) is set forth below: - 5.0 mg SMA intake: 1 tablet immediately after breakfast and 1 tablet immediately after dinner; - 7.5 mg SMA intake: 2 tablets immediately after breakfast and 1 tablet immediately after dinner; - 10.0 mg SMA intake: 2 tablets immediately after breakfast and 2 tablets immediately after dinner.

[0175] Administration with other drugs In some embodiments, the pharmaceutical compositions can be used in combination with one or more other drugs.

[0176] For example, the pharmaceutical compositions described herein can be administered with other therapeutic agents such as analgesics, anesthetics, antifungals, antibiotics, antihistamines, antihypertensives, antimalarials, antibacterials, antiseptics, antiarthritics, antithrombins, antituberculous drugs, antitussives, antivirals, cardioactive drugs, expectorants, immunosuppressants, sedatives, sympathomimetics, toxins (e.g., cholera toxin), tranquilizers, and drugs for urinary tract infections.

[0177] The sequential or substantially simultaneous administration of each therapeutic agent can be accomplished by any suitable route, including, but not limited to, oral, intravenous, intramuscular, direct absorption through mucosal tissue, and combinations thereof. The therapeutic agents can be administered by the same or different routes. For example, the first therapeutic agent of the selected combination can be administered by intravenous injection, such as cisplatin or arsenic trioxide, while the other therapeutic agent, such as sodium meta arsenite, can be administered orally. Alternatively, for example, both or all therapeutic agents can be administered by intravenous injection or infusion. The order in which the therapeutic agents are administered is not important.

[0178] kit The present invention also provides kits for carrying out the therapeutic regimens of the present invention. Such kits contain a therapeutically effective amount of SMA or KMA in a pharmaceutically acceptable form in one or more containers. The SMA or KMA in the vial of the kit of the present invention can be in the form of a pharmaceutically acceptable solution, for example, combined with sterile saline, dextrose solution, buffer solution, or other pharmaceutically acceptable sterile liquid. Alternatively, the SMA or KMA can be lyophilized or desiccated; in this case, the kit optionally further contains a pharmaceutically acceptable solution (e.g., saline dextrose solution, etc.), preferably saline, in a container to reconstitute the complex to form a solution for injection. The kit also contains an appropriate amount of another therapeutic agent for treating pain and / or inflammation. Such other therapeutic agent can be formulated as a combination with the SMA or KMA contained in the kit, or can be formulated separately.

[0179] The invention is further described below with reference to the following non-limiting examples. [Example]

[0180] [Example 1] Inhibition of pro-inflammatory cytokine secretion

[0181] material and method All materials used in the preparation of the pharmaceutical compositions exemplified below were purchased from commercial suppliers.

[0182] Macrophage Growth Medium Primary rat peritoneal macrophages were grown in high-glucose, pyruvate-containing DMEM (Invitrogen Catalog No. 11995) supplemented with heat-inactivated fetal bovine serum (Invitrogen Catalog No. 10099-141) at a final concentration of 10%, penicillin / streptomycin (Invitrogen Catalog No. 15140-122) at a final concentration of 100 U / mL / 100 μg / mL, glutamax (Invitrogen Catalog No. 35050-061) at a final concentration of 2 mM, and MEM NEAA (Invitrogen Catalog No. 11140-050) at a final concentration compatible with MEM medium (Invitrogen Catalog No. 11095).

[0183] THP-1 cells and THP-1 macrophages were grown in ATCC modified RPMI (Invitrogen catalog no. A10491-01) supplemented with heat-inactivated fetal bovine serum (Invitrogen catalog no. 10099-141) to a final concentration of 10% and 2-mercaptoethanol to a final concentration of 0.05 mM.

[0184] All cells were incubated in a humidified atmosphere at 37°C and 5% CO2.

[0185] Cell cytotoxicity and viability (MTT-based) assays The cytotoxicity and viability of cultured primary macrophages incubated with a range of NaAsO2 concentrations compared to vehicle control were determined 24 hours after treatment using the CytoTox-GLO kit (cytotoxicity) and MTT assay (viability).

[0186] On day 1, 1 x 10 cells were cultured in growth medium. 6 Macrophage cells were seeded onto 96-well plates by adding 125 μL of 10% poly-L-lysine-coated solution to each well. Non-adherent cells were removed after 3 hours.

[0187] On day 2, the growth medium was gently replaced with 100 μL / well of fresh serum-free DMEM medium for 3 hours. The serum-free medium was replaced with 63 μL of medium containing a range of NaAsO concentrations (30, 10, 7, 5, 3, 1, 0.3, 0.1, and 0 μM), 100 ng / ml LPS, or a control, and incubated for 24 hours.

[0188] On the third day, 1. Cytotoxicity was determined using the CytoTox-GLO kit according to the manufacturer's instructions. 2. DMEM was reconstituted with MTT to a final concentration of 5 mg / mL. 3. 24 hours after treatment, 6.3 μL of reconstituted MTT solution was added to each well and incubated in a CO2 incubator for 4 hours. 4.70 μL of MTT solubilizing solution was added to each well and the resulting formazan crystals were dissolved by re-pipetting 10 times. 5. The absorbance of each well was measured at a wavelength of 570 nm using a spectrophotometer, and the background absorbance at 690 nm was subtracted.

[0189] Cytokine secretion To investigate the effect of NaAsO2 on cytokine secretion from primary cultures of rat macrophages, supernatants from macrophage wells incubated with various concentrations of NaAsO2 and LPS for 24 h were analyzed for pro-inflammatory cytokine concentrations using the MesoScale Discovery V-PLEX kit.

[0190] Day 1 1 x 10 in growth medium 6 Macrophage cells were seeded onto 24-well plates by adding 323 μL of the medium (at 10% poly-L-lysine) to each well. Non-adherent cells were removed after 3 hours, and the medium was replaced with 500 μL of fresh DMEM medium per well.

[0191] Day 2 1. The growth medium was gently replaced with 500 μL / well of fresh serum-free DMEM medium for 3 hours. 2. The serum-free medium was replaced with 250 μL of growth medium containing a range of NaAsO2 concentrations (30, 10, 7, 5, 3, 1, 0.3, 0.1, and 0 μM) and 100 ng / ml LPS (to induce an inflammatory state) or a control, and incubated for 24 hours.

[0192] Day 3 1. 24 hours after treatment, cell supernatants were collected and stored at -80°C. 2. Pro-inflammatory cytokines were measured using the MesoScale Discovery V-PLEX kit according to the manufacturer's instructions.

[0193] Differentiation of THP-1 cells Day 1 2 x 10 cells in THP-1 growth medium containing 1 µL / mL of phorbol 12-myristate 13-acetate (PMA). 5 THP-1 cells were seeded into 96-well plates by adding 250 μL of cells / mL to each well of a 10% poly-L-lysine coated 96-well plate.

[0194] Day 2 The growth medium was gently replaced with 100 μL / well of fresh serum-free DMEM medium for 2 hours. The serum-free medium was then replaced with 63 μL of growth medium containing a range of NaAsO concentrations (30, 10, 7, 5, 3, 1, 0.3, 0.1, and 0 μM) and 100 ng / ml LPS (to induce an inflammatory state) or control, and incubated for 24 hours.

[0195] Day 3 After 24 hours of treatment, MTT assays were performed as detailed above.

[0196] Data analysis Data are expressed as mean (±SEM) and differences from primary macrophage cytokine secretion controls were determined using ANOVA with post-hoc Tukey's multiple comparison test. Prism version 6.05 was used for all data figures, statistical analysis and IC 50 The criterion for statistical significance used in the calculations was p≦0.05.

[0197] result Primary peritoneal macrophages were harvested from rats and incubated with LPS (100 ng / mL) and a range of concentrations (0.1–30 μM) of sodium meta arsenite for 24 hours. CytoTox-GLO and MTT assay kits were then used to assess cytotoxicity and cell viability (Figure 1A). Digitonin, a cytotoxic detergent for cells, caused higher toxicity compared to vehicle in the CytoTox-GLO kit (Figure 1B). Additionally, Triton-X, another detergent, caused lower cell viability compared to vehicle in the MTT assay (Figure 1C). When incubated with sodium meta arsenite, there was a concentration-dependent increase in cytotoxicity and a corresponding concentration-dependent decrease in viability. EC 50 and IC 50 The cytotoxicity and cell viability used to derive the values showed a similar but reciprocal relationship, suggesting that the decrease in viability during sodium meta arsenite incubation may be due to cell death rather than a simple failure of intracellular machinery.

[0198] I C 50 There was a concentration-dependent decrease in the secretion of TNF-α, IL-1β, and IL-6, with IC values of 2.3, 0.8, and 0.5 μM, respectively (Figure 2A, C, and E). Importantly, the IC 50 The IC value was as high as 5.7 μM, indicating that sodium meta arsenite significantly increased cell viability. 50These results suggest that sodium meta arsenite inhibits the secretion of cytokines TNF-α, IL-1β, and IL-6 from cultured rat macrophages at concentrations that do not kill the cells. Importantly, celecoxib (10 μM) successfully inhibited the secretion of all three pro-inflammatory cytokines (Figure 2B, D, and E).

[0199] summary There was a concentration-dependent decrease in the secretion of pro-inflammatory cytokines, such that incubation of cells with 3 μM sodium meta arsenite induced a complete inhibition of the secretion of IL-1β and IL-6 release from the cells in the absence of significant cell death.

[0200] There was significant inhibition of TNF-α, IL-1β, and IL-6 secretion from macrophages at concentrations of sodium meta arsenite that did not significantly reduce cell viability.

[0201] In conclusion, the in vitro data herein demonstrate that incubation of cultured primary rat macrophages with sodium meta arsenite for 24 hours results in a concentration-dependent inhibition of the secretion of pro-inflammatory cytokines.

[0202] [Example 2] In this study, we investigated whether sodium meta arsenite suppresses lipopolysaccharide (LPS)-induced inflammatory responses in murine macrophage Raw264.7 cells. Lipopolysaccharide-activated macrophages produce numerous molecules and proteins, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and free radicals, associated with acute inflammation. The response was mediated by the NF-κB pathway, an intracellular cascade. Therefore, regulation of this pathway is crucial for controlling inflammation.

[0203] cell culture The murine macrophage cell line RAW264.7 (American Type Culture Collection, ATCC; Manassas, VA, USA) was grown in DMEM supplemented with 10% heat-inactivated FBS and antibiotics / antimycotics (100 U / ml penicillin G sodium, 100 μg / ml streptomycin sulfate, and 0.25 mg / ml muphotericin). RAW264.7 cells stably transfected with the pNF-κB-SEAP-NPT plasmid (SEAP-RAW cells) were kindly provided by Dr. Yeong Shik Kim (Seoul National University, Korea). SEAP-RAW cells were maintained in DMEM containing 500 μg / ml G418. All cells were incubated at 37°C in a humidified atmosphere with 5% CO2.

[0204] Nitric oxide (NO) assay The RAW264.7 macrophage cell line was incubated with lipopolysaccharide (LPS, an endotoxin from Escherichia coli), followed by measurement of COX-2- and iNOS-induced NO levels. Cytotoxicity was determined using the sulforhodamine B assay or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

[0205] Measurement of PGE2 accumulation To evaluate the inhibitory activity of test substances against COX-2, RAW264.7 cells were incubated with 1 μg / ml LPS. After an additional 20 hours of incubation, the medium was removed and analyzed by PGE2 enzyme-linked immunosorbent assay (PGE2-ELISA). In these assays, activity was defined as the difference between PGE2 accumulation in the absence and presence of sodium meta arsenite.

[0206] COX-2 enzyme activity assay To measure the inhibitory activity of overexpressed COX-2 enzyme, RAW264.7 cells were treated with LPS (1 μg / ml) for 20 hours, then treated with sodium meta arsenite for 30 minutes. Subsequently, the cells were treated with a COX-2 substrate (arachidonic acid, 10 μM), and PGE2 levels were determined using PGE2-ELISA.

[0207] RT-PCR analysis RAW264.7 cells were pretreated with sodium meta arsenite for 30 min, followed by treatment with LPS (1 μg / ml) for 5 h to extract total RNA. The expression of iNOS, COX-2, mRNA, and cytokine genes was determined by reverse transcription polymerase chain reaction (RT-PCR).

[0208] Western blot analysis RAW264.7 cells were pretreated with sodium meta arsenite for 30 minutes, cultured for 16 hours, and then treated with LPS (1 μg / ml). Protein concentrations from disrupted cells were determined using a BSA assay. The effects of sodium meta arsenite on the protein expression of iNOS, COX-2, cytokines, and NF-κB, Akt were determined by Western blot analysis.

[0209] Reporter gene assay SEAP-RAW cells were pretreated with sodium meta arsenite for 2 hours, and then incubated with LPS (1 μg / ml) for 18 hours. The collected supernatants were heated at 65°C for 5 minutes, and SEAP assay buffer (2 M diethanolamine, 1 mM MgCl2, 500 μM 4-methylumbelliferyl phosphate (MUP)) was added for 1 hour at 37°C in the dark. Fluorescence from the SEAP / MUP product was measured using a 96-well microplate fluorometer at 360 nm excitation and 449 nm emission and normalized to protein concentration. Data are expressed as a percentage of sodium meta arsenite-treated cells relative to vehicle-treated control cells without LPS.

[0210] result Effect of sodium meta arsenite on nitric oxide (NO) production Nitric oxide (NO) is a well-known pro-inflammatory mediator in the pathogenesis of inflammation. Most NO is synthesized by inducible nitric oxide synthase (iNOS), an enzyme closely associated with inflammatory responses and cancer formation. NO production by iNOS has been reported to affect the activity and expression of COX-2. To investigate whether sodium meta arsenite has NO inhibitory activity, NO production was determined in LPS-induced RAW264.7 mouse macrophage cells in the presence of 0.625–10 μM sodium meta arsenite.

[0211] NO production was significantly and concentration-dependently attenuated by sodium meta arsenite (at concentrations of 10, 5, 2.5, 1.25, and 0.625 μM) by 100.2, 77.2, 42.2, 21.5, and 12.5%, respectively. The IC50 value of sodium meta arsenite for inhibition of NO production was approximately 2.87 μM (Figure 3A).

[0212] Effect of sodium meta arsenite on PGE2 production iNOS is highly expressed in macrophages and contributes to organ destruction in some inflammatory and autoimmune diseases. COX-2 is also a pro-inflammatory enzyme that converts arachidonic acid to prostaglandins, producing prostaglandin E2 (PGE2). PGE2 is another important mediator produced by COX-2-catalyzed arachidonic acid metabolites in inflammatory responses. Under basal conditions, iNOS and COX-2 products, including NO and prostaglandins, are involved in regulating cellular function and homeostasis.

[0213] To investigate whether sodium meta arsenite can regulate COX-2-mediated PGE2 production, PGE2 production was measured in RAW264.7 cells after treatment with sodium meta arsenite (2.5, 5, 7.5, and 10 μM). Sodium meta arsenite inhibited PGE2 production in a dose-dependent manner. At the maximum dose of sodium meta arsenite (10 μM), PGE2 production was inhibited by 20% (Figure 4).

[0214] Assessment of sodium meta arsenite on protein expression Effect of sodium meta arsenite on the protein expression of iNOS and COX-2 To evaluate the inhibitory effect of sodium meta arsenite on iNOS-induced COX-2 production, iNOS and COX-2 protein levels were analyzed by Western blot analysis. Raw264.7 cells were pretreated with 2.5, 5, 7.5, or 10 μM sodium meta arsenite for 30 minutes and then stimulated with 1 μg / ml LPS for 16 hours. As shown in Figure 5, iNOS expression was significantly inhibited by sodium meta arsenite in a concentration-dependent manner. COX-2 expression was slightly inhibited by sodium meta arsenite.

[0215] Effect of sodium meta arsenite on protein expression of TNF-α and IL-1β The proinflammatory cytokine tumor necrosis factor-α (TNF-α) is considered a central mediator of inflammatory responses. It also mediates inflammatory responses by secreting various proinflammatory mediators, including IL-1β and PGE2, in response to LPS. IL-1β is a notable proinflammatory cytokine with a wide range of functions. The effect of sodium meta arsenite on TNF-α and IL-1β protein levels was analyzed by Western blot analysis. Raw264.7 cells were pretreated with 2.5, 5, 7.5, or 10 μM sodium meta arsenite for 30 min and then stimulated with 1 μg / ml LPS for 8 h. TNF-α and IL-1β expression was significantly inhibited by sodium meta arsenite in a concentration-dependent manner (Figure 6).

[0216] Assessment of sodium meta arsenite on gene expression Effect of sodium meta arsenite on iNOS and COX-2 mRNA expression The effect of sodium meta arsenite on iNOS and COX-2 mRNA expression was investigated by RT-PCR. Raw264.7 cells were pretreated with 2.5, 5, 7.5, or 10 μM sodium meta arsenite for 30 minutes and then stimulated with 1 μg / ml LPS for 8 hours. 1 μg of the resulting total RNA was then used for RT-PCR.

[0217] iNOS expression was significantly inhibited by sodium meta arsenite in a concentration-dependent manner (FIGS. 7 and 8). COX-2 expression was not affected by sodium meta arsenite (FIG. 7).

[0218] This was confirmed by the fact that sodium meta arsenite exhibited an inhibitory effect on iNOS but not COX-2, suggesting that sodium meta arsenite potently inhibits inflammatory responses through modulation of iNOS expression (Fig. 6). iNOS and COX-2 gene expression was analyzed by Western blot analysis. iNOS mRNA levels were measured by real-time PCR and were significantly inhibited by sodium meta arsenite in a concentration-dependent manner (Fig. 8).

[0219] Effect of sodium meta arsenite on TNF-α and IL-1β mRNA expression The proinflammatory cytokine TNF-α is considered a central mediator in the inflammatory response. It mediates the inflammatory response by secreting various proinflammatory mediators, including TNF-α, IL-1β, and PGE2, in response to LPS. Among proinflammatory cytokines, IL-1β or IFN-β has the greatest potential to cause damage to host tissues. Indeed, various mechanisms are dedicated to restricting its intracellular activity by carefully regulating its transcription and processing during the inflammatory response. Therefore, the effects of sodium meta arsenite on the mRNA levels of TNF-α, IL-1β, and IFN-β were analyzed by RT-PCR analysis.

[0220] Raw264.7 cells were pretreated with 2.5, 5, 7.5, or 10 μM sodium meta arsenite for 30 minutes and then stimulated with 1 μg / ml LPS for 5 hours. One μg of the resulting total RNA was then used for RT-PCR. TNF-α mRNA levels were significantly reduced by sodium meta arsenite in a concentration-dependent manner, whereas sodium meta arsenite had no effect on the expression of IL-1β and IFN-β mRNA in RAW264.7 macrophages (Figure 9).

[0221] Effect of sodium meta arsenite on the transcriptional activity of nuclear factor kappa B (NF-κB) The NF-κB transcription factor has been shown to play a significant role in LPS-induced expression of pro-inflammatory mediators, including iNOS. The promoter region of the iNOS-encoding gene contains an NF-κB binding motif, suggesting that NF-κB binding to the NF-κB site upstream of the iNOS promoter plays an important role in LPS-induced upregulation of the iNOS gene. To investigate the molecular mechanism underlying sodium meta arsenite-mediated inhibition of NF-κB transcription, NF-κB transcription activity was investigated using a reporter gene assay system. RAW264.7 cells were stably transfected with the pNF-κB secreted alkaline phosphatase (SEAP)-NPT plasmid, which contains four copies of the κB sequence fused to SEAP as a reporter. The pNF-κB-SEAP-NPT plasmid, which contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells, was constructed and transfected into RAW264.7 macrophages. Aliquots of the culture medium were heated and then reacted with 4-methylumbelliferyl phosphate (MUP). SEAP activity was measured as relative fluorescence units (RFU). LPS treatment of transfected cells for 18 hours increased SEAP expression approximately three-fold compared to basal levels in control cells without LPS. Treatment of cells with sodium meta arsenite significantly inhibited LPS-induced SEAP expression in a concentration-dependent manner (Figure 10).

[0222] To investigate whether sodium meta arsenite modulates the NF-κB signaling pathway, RAW264.7 macrophages were treated with LPS (1 μg / mL) for 15 min in a 30-min sodium meta arsenite (2.5, 5, 7.5, or 10 μM) pretreatment, and the levels of p65, p50, IκB, and IKK were also analyzed by Western blot analysis.

[0223] Sodium meta arsenite significantly decreased NF-κB protein levels in a concentration-dependent manner (FIG. 11). Sodium meta arsenite significantly inhibited IκB degradation in a concentration-dependent manner (FIG. 12).

[0224] [Example 3] Preparation of Oral Compositions Oral Composition

[0225] Sodium meta arsenite ("SMA") was obtained from Sigma Aldrich Fine Chemicals. As supplied, the SMA drug substance exhibited very high purity (>98% As(III)) and minimal levels of As(V). Table 1 below presents the properties of the SMA drug substance as supplied.

[0226] [Table 1]

[0227] 2.5 mg sodium meta arsenite ("SMA") enteric coated tablets were prepared using the materials listed in Table 2 below. When possible, high density primary excipients were selected to maximize the SMA (approximately 2.1-2.3 g / cm). -3 The density of the PEG-1000 was matched to that of the PEG-1000 (an inorganic substance with an estimated true density of 1000 mg / kg, which is very dense compared to most excipients).

[0228] [Table 2]

[0229] The equipment listed in Table 3 below was used in the preparation and analysis of the SMA enteric coating compositions.

[0230] [Table 3]

[0231] [Production Example 1] The enteric coated tablets of Formulation Examples 1.1-1.4 containing sodium meta arsenite ("SMA") as the active pharmaceutical ingredient (API) were prepared according to the procedure described below.

[0232] Generally, sodium meta arsenite ("SMA") and excipients were blended together (via a three-stage blending process without water or solvents) to form a powder blend, as described in detail below. The powder blend was then compressed to form a solid tablet core. The solid tablet core was then coated with an enteric coating.

[0233] blend The blending process described below was used to blend the ingredients.

[0234] The API and other ingredients of the composition were dispensed and weighed. Because the concentration of the API was very low, a three-stage blending process (utilizing an "API premix" and a "main mix") was utilized to improve blend uniformity.

[0235] The API was screened through a 200 μm sieve (hand screening), and the sieving time was between 5 and 8 minutes.

[0236] A premix containing the API ("API premix") was prepared by blending the screened API with a few grams (20 g for 500 g batch size and 30 g for 700 g batch size) of filler in a suitable container (100 ml container for 500 g batch size and 150 ml for 700 g batch size) in a Turbla blender at 49 rpm for 10 minutes.

[0237] The glidant (colloidal silicon dioxide) was screened through a 500 μm sieve to deagglomerate it, and then all other dispensed ingredients, including the sieved glidant, except for the lubricant (sodium stearyl fumarate), were added to a 2 L glass Turbula jar, and the API premix was sandwiched in the middle of the powder mass.

[0238] The resulting mixture ("main mix") was blended using a Turbula blender at 49 rpm for about 10 to about 20 minutes to form a blended powder ("main blend").

[0239] The lubricant (sodium stearyl fumarate) was co-screened with a small amount of the main blend using a 500 μm sieve, and then the co-screened mixture was added to the main blend. This lubrication step was performed separately to avoid potential complications from over-lubrication (e.g., reduced tablet hardness or dissolution problems).

[0240] The resulting mixture was mixed in a Turbula blender at 49 rpm for 2 minutes, thereby forming a powder blend. The powder blend was characterized for flow properties.

[0241] compression The powder blend was compressed to a target tablet weight of 150 mg in a Manesty F3 single-punch tablet press using a 6.5 mm vertical concave (NCCP) tooling. The Manesty F3 only has universal units (AU) for compression force, making it impossible to directly measure the applied force. The target hardness level was greater than 90 N.

[0242] Enteric coating An enteric coating dispersion at 20% w / w solids was prepared by dispersing Acryl-EZE II white (493Z180022) in deionized water. The dispersion was stirred using a paddle stirrer for 45 minutes before use and throughout the coating process. The dispersion was screened through a 250 μm sieve before use.

[0243] A 15" coating pan (Thai Coater) was equilibrated to set the spot temperature before adding the solid tablet cores. Due to the small batch size, "bulking inerts" were added to the API solid cores to meet the loading requirements of the coating pan. The solid tablet cores were equilibrated in the drying pan for 10 minutes before coating. The same temperature and airflow were used for the heating, coating, and drying steps. The coated tablets were dried in the pan for 10 minutes after coating. Samples were collected after weight gains of 8, 10, and 12% w / w.

[0244] Dissolution test Dissolution testing was performed using 500 mL of media and USP Method 2 (paddle), initially at a paddle speed of 100 rpm. A single set of six enteric-coated tablets (n=6) was tested. Samples of the dissolution media were withdrawn after 2 hours in acid to determine the level of sodium meta arsenite and assess resistance to gastric juices. The media was replaced with pH 6.8 phosphate buffer, and samples were withdrawn at 15-minute intervals to generate dissolution profiles.

[0245] This method is based on the Pharmacopoeial method for enteric-coated dosage forms (EP.2.9.3 and USP <711> ) and are shown in Table 4 below.

[0246] [Table 4]

[0247] Formulation A solid pharmaceutical composition (P63) containing sodium meta arsenite ("SMA") as the active pharmaceutical ingredient (API) was prepared using the method described in Preparative Example 1 above.

[0248] The compositions were prepared at a 700 g scale. Blend uniformity and content uniformity samples were collected to assess homogeneity after 20 minutes of main blending time.

[0249] Table 5 below presents the composition of the tablet solid core (before the coating step) containing 2.53 mg of sodium meta arsenite. (Table 5.1 below presents another possible composition of the tablet solid core (before the coating step) containing 2.50 mg of sodium meta arsenite.)

[0250] [Table 5]

[0251] After the blending step, the powder blend demonstrated good flow properties as indicated by the Carr index (29.3%). The powder blend before compression had the following properties: Aerated density: 0.64g / cm 3 Tapped density: 0.91g / cm 3 ·Carr index: 29.3% ·Hausner ratio: 1.30

[0252] The powder blend compressed very well, with no weight variation and / or visible separation observed throughout the run. High tablet hardness (104.8 N) and low friability (0.08%) were achieved, and the disintegration time (34 seconds) was relatively fast. The average thickness of the solid core of the tablets was 3.63 mm.

[0253] Blend uniformity samples were taken after 20 minutes of blending, and content uniformity samples were collected at the beginning, middle, and end of the compression run. Blend uniformity results showed excellent uniformity with a relative standard deviation (RSD) % value of 1.3. Content uniformity of the tablet solid core during the compression run (beginning, middle, and end) showed good uniformity, achieving a maximum acceptable value (AV) of <7.4 (AV values of <15 are acceptable).

[0254] After the compression step, the solid tablet cores were coated with Acryl-EZE II white (493Z180022) enteric coating polymer system, prepared as described in Manufacturing Example 1. The coating parameters are shown in Table 6 below.

[0255] [Table 6]

[0256] The enteric-coated tablets exhibited an acceptable dissolution profile (500 ml medium, paddle speed 100 rpm). After 120 minutes, the composition was intact in acidic medium (pH 1.0) and released 0% of the API. After 135 minutes at pH 6.8, 91% of the API was released. After 150 minutes at pH 6.8, 98% of the API was released. After 165 minutes at pH 6.8, 100% of the API was released.

[0257] The enteric-coated tablets demonstrated sufficient gastric resistance and met the proposed preliminary specification of 75% or greater release in 45 minutes for enteric-coated dosage forms.

[0258] Table 5.1 below presents alternative possible compositions for the solid core of a tablet (before the coating step) containing 2.50 mg of sodium meta arsenite. Solid cores having the components set forth in Table 5.1 can be prepared in a manner similar to that described above for solid cores having the components set forth in Table 5.

[0259] [Table 7]

[0260] Formulation Example 1.2 A solid pharmaceutical composition (P23) containing sodium meta arsenite ("SMA") as the active pharmaceutical ingredient (API) was prepared using the method described in Preparative Example 1 above.

[0261] The compositions were prepared at a 500 g scale. Blend uniformity samples were collected after 10, 15, and 20 minutes of main blending time. The blends were compressed to form solid tablet cores, which were then coated.

[0262] Table 7 below presents the composition of the tablet solid core (before the coating step) containing 2.50 mg of sodium meta arsenite.

[0263] [Table 8]

[0264] After the blending step, the powder blend demonstrated good flow properties as indicated by the Carr index (26.37%). The powder blend prior to compression had the following properties: Loose density: 0.67g / cm 3 Packed density: 0.91g / cm 3 ·Carr index: 26.37% ·Hausner ratio: 1.36 ·Angle of repose: 24.32°

[0265] Blend uniformity samples were collected after blending for 10, 15, and 20 minutes of the main blending time. The composition showed good uniformity at the 20 minute blending time.

[0266] Compression was performed in a Manesty F3 single punch machine using a 6.5 mm NCCP tool. The solid cores had an average hardness of 94.3 N, an average thickness of 3.62 mm, a friability of 0.33%, and a disintegration time of 39 seconds.

[0267] The weight of the solid cores remained constant throughout the compression run, producing acceptable solid cores. No visible separation was observed. Samples (10 solid cores in duplicate) were collected at the beginning, middle, and end of the compression run and submitted for content uniformity testing.

[0268] After the compression step, the solid tablet cores were coated with Acryl-EZE II white (493Z180022) enteric coating polymer system, prepared as described in Manufacturing Example 1, and samples were collected after weight gains of 8, 10, and 12% w / w. The coating parameters are shown in Table 8 below.

[0269] [Table 9]

[0270] Enteric coated tablets with 8%, 10% and 12% w / w weight gain were subjected to dissolution testing (500 ml dissolution medium, paddle speed 75 rpm) to identify the appropriate level of enteric coating. The dissolution results are presented in Figure 9 below.

[0271] [Table 10]

[0272] The enteric-coated tablets remained intact in acidic media after 120 minutes. The enteric-coated tablets demonstrated sufficient gastric resistance and met the tentative proposed specification of 75% or greater release in 45 minutes for enteric-coated dosage forms.

[0273] Based on the dissolution results, 12% w / w was found to be the optimum coating weight gain.

[0274] Formulation Example 1.3 A solid pharmaceutical composition (P31) containing sodium meta arsenite ("SMA") as the active pharmaceutical ingredient (API) was prepared using the method described in Preparative Example 1 above.

[0275] The compositions were prepared at a 500 g scale. Blend uniformity samples were collected after 10, 15, and 20 minutes of main blending time. The blends were compressed to form solid tablet cores, which were then coated. L-hydroxypropyl cellulose (L-HPC, low-substituted hydroxypropyl cellulose LH-B1 grade) was used because it acts as a binder and disintegrant. Since L-HPC is insoluble in water, this was expected to result in hard tablets.

[0276] Table 10 below presents the composition of the tablet solid core (before the coating step) containing 2.50 mg of sodium meta arsenite.

[0277] [Table 11]

[0278] After the blending step, the powder blend demonstrated good flow properties as indicated by the Carr index (23.68%). The powder blend prior to compression had the following properties: Loose density: 0.58g / cm 3 Packed density: 0.76g / cm 3 ·Carr index: 23.68% ·Hausner ratio: 1.31 ·Angle of repose: 27.96°

[0279] Blend uniformity samples were collected after blending at 10, 15, and 20 minutes of the main blend time. The composition showed good uniformity at the 20 minute blend time.

[0280] Compression was performed in a Manesty F3 single punch machine using a 6.5 mm NCCP tool. The solid cores had an average hardness of 104.3 N, an average thickness of 3.52 mm, a friability of 0.23%, and a disintegration time of 30 seconds.

[0281] The weight of the solid cores remained constant throughout the compression run, producing acceptable solid cores. No visible separation was observed. Samples (10 solid cores in duplicate) were collected at the beginning, middle, and end of the compression run and submitted for content uniformity testing.

[0282] After the compression step, the solid tablet cores were coated with Acryl-EZE II white (493Z180022) enteric coating polymer system, prepared as described in Manufacturing Example 1, and samples were collected after weight gains of 8, 10, and 12% w / w. The coating parameters are shown in Table 11 below.

[0283] [Table 12]

[0284] Enteric coated tablets with 8%, 10% and 12% w / w weight gain were subjected to dissolution testing (500 ml dissolution medium, paddle speed 75 rpm) to identify the appropriate level of enteric coating. The dissolution results are presented in Figure 12 below.

[0285] [Table 13]

[0286] The 8% w / w weight gain enteric-coated tablets failed the acid resistance test. The 10% w / w weight gain enteric-coated tablets and the 12% w / w weight gain enteric-coated tablets demonstrated sufficient gastric resistance and met the provisional proposed specification of 75% or greater release in 45 minutes for enteric-coated dosage forms.

[0287] Based on the dissolution results, 12% w / w was found to be the optimum coating weight gain.

[0288] Formulation Example 1.4 A solid pharmaceutical composition (P66) containing sodium meta arsenite ("SMA") as the active pharmaceutical ingredient (API) was prepared using the method described in Preparative Example 1 above.

[0289] The compositions were prepared at a 700 g scale. Blend uniformity and content uniformity samples were collected to assess homogeneity after 20 minutes of main blending time.

[0290] Table 13 below presents the composition of the tablet solid core (before the coating step) containing 2.53 mg of sodium meta arsenite.

[0291] [Table 14]

[0292] After the blending step, the powder blend demonstrated good flow properties as indicated by the Carr index (25.74%). The powder blend prior to compression had the following properties: Loose density: 0.75g / cm 3 Packed density: 1.01g / cm 3 ·Carr index: 25.74% ·Hausner ratio: 1.35

[0293] The powder blend compressed very well, with no weight variations and / or visible separation observed throughout the run. High solid core hardness (87.4 N) and low friability (0.11%) were achieved, and the disintegration time (2 minutes 52 seconds) was relatively fast. The average thickness of the solid core was 3.66 mm.

[0294] Blend uniformity samples were taken after 20 minutes of blending, and content uniformity samples were collected at the beginning, middle, and end of the compaction run. Blend uniformity results showed excellent uniformity with a relative standard deviation (RSD) % value of 2.1. Content uniformity of the solid core during the compaction run (beginning, middle, and end) showed good uniformity, achieving a maximum acceptable value (AV) of <6.3 (AV values of <15 are acceptable).

[0295] After the compression step, the solid tablet cores were coated with Acryl-EZE II white (493Z180022) enteric coating polymer system, prepared as described in Manufacturing Example 1. The coating parameters are shown in Table 14 below.

[0296] [Table 15]

[0297] The enteric-coated tablets exhibited acceptable dissolution profiles (500 ml medium, paddle speed 100 rpm). After 120 minutes, the composition was intact in acidic medium (pH 1.0) and released 0% of the API. After 135 minutes at pH 6.8, it released 21% of the API. After 150 minutes at pH 6.8, it released 86% of the API. After 165 minutes at pH 6.8, it released 96% of the API. After 195 minutes at pH 6.8, it released 98% of the API.

[0298] The enteric-coated tablets demonstrated sufficient gastric resistance and met the provisional proposed specification of 75% or greater release in 45 minutes for enteric-coated dosage forms.

[0299] [Production Example 2] Table 15 below provides the composition of the enteric coated tablets containing 2.5 mg of sodium meta arsenite as the active pharmaceutical ingredient (API). The enteric coated tablets were prepared using the method described below.

[0300] [Table 16]

[0301] Generally, sodium meta arsenite ("SMA") and excipients were blended together (via a two-stage blending process without water or solvents) to form a powder blend, as described in detail below. The powder blend was then compressed to form a solid tablet core. The solid tablet core was then coated with an enteric coating.

[0302] blend The blending process described below was used to blend the ingredients.

[0303] The API and other ingredients of the composition were dispensed and weighed. Because the concentration of the API was very low, a two-stage blending process (utilizing an "API premix" and a "main mix") was utilized to improve blend uniformity.

[0304] The API was screened through a 106 μm sieve (sieving time was about 5-8 min).

[0305] A portion of the calcium phosphate dibasic was added to the sieved API and the resulting mixture was blended for 30 minutes to provide the "API premix."

[0306] The API premix was then blended with the remaining calcium phosphate dibasic, and other excipients (silicified microcrystalline cellulose, sodium starch glycolate, colloidal silicon dioxide, and sodium stearyl fumarate) to provide the "main mix." The main mix was blended with an intensifier bar for 4 minutes to provide the powder blend.

[0307] compression The powder blend was compressed to a target tablet weight of 150 mg ± 5% (range 142.5 to 157.5 mg) on a Key International tablet press using 0.25 inch tooling. The solid cores were de-dusted.

[0308] The final solid core exhibited no significant attrition rate (0.00%) and a hardness of 156.9 N (16 kp).

[0309] Enteric coating An enteric coating dispersion at 25% w / w solids was prepared by dispersing Acryl-EZE green powder in deionized water. The dispersion was stirred for approximately 30 minutes (until homogeneous).

[0310] The de-dusted solid cores were spray coated (350 g / min) with the dispersion at a weight gain of approximately 10-12% w / w. The pan speed was approximately 6-8 rpm. The coated tablets were dried after coating.

[0311] [Example 4] Inhibitory effect of a single dose of SMA in an LPS-induced ARDS model in BALB / c mice This study evaluated the ability of substances to control acute respiratory distress syndrome (ARDS) induced by intratracheal administration of LPS to house mice (Mus musculus) (BALB / c) in an ARDS model by measuring cytokine levels in bronchoalveolar lavage fluid (BALF) after oral administration of the test substance, SMA, and the positive control substance, dexamethasone.

[0312] There were five groups of mice, G1 to G5: (G1) negative control, (G2) 1.03 mg / kg SMA, (G3) 1.54 mg / kg SMA, (G4) 2.05 mg / kg SMA, and (G5) 3 mg / kg dexamethasone, a positive control. There were 10 mice in each group.

[0313] The test substance, SMA, was orally administered once 2 hours before the induction of ARDS, and the positive control substance, dexamethasone, was orally administered once 1 hour before the induction of ARDS.

[0314] General symptoms were observed once one day after the end of the isolation adaptation period. The animals' weights were measured twice, before acquisition and at the start of the study. No abnormalities caused by the administration of the substance were observed in any group until the end of the study.

[0315] For group allocation, body weight was measured for all animals and animals were randomly assigned to each group, and there was no statistical significance in the weight of animals in all groups.

[0316] Survival analysis showed that survival was prolonged by administration of the test substances with statistical significance (G3: p<0.005 (48 hours after LPS treatment); G4: p<0.0005 (48 hours after LPS treatment); G5: p<0.0001 (48 hours after LPS treatment)).

[0317] TNF-α analysis showed that measurements were above the limit of quantitation (LoQ) at all time points and that target expression was statistically significantly suppressed by test substance administration (G4: p<0.005 (1, 2, 6, and 12 hours after LPS administration); G5: p<0.0005 (4 hours after LPS administration) and p<0.0001 (1, 2, 6, and 12 hours after LPS administration)) except before (0 hour) and 24 hours after LPS administration.

[0318] IL-6 analysis found that measurements were above the LoQ at all time points and that target expression was statistically significantly suppressed by administration of the test substances except before LPS administration (0 h) (G4: p<0.05 (2 and 4 h after LPS administration), and p<0.005 (6 and 12 h after LPS administration); G5: p<0.05 (1, 2, 4, and 24 h after LPS administration), p<0.0005 (6 h after LPS administration), and p<0.0001 (12 h after LPS administration)).

[0319] IL-β analysis revealed that IL-β measurements were above the LoQ at 4 and 6 hours, and target expression was found to be statistically significantly suppressed by test substance administration (G4: p<0.005 (4 and 6 hours after LPS administration); G5: p<0.0005 (4 and 6 hours after LPS administration)) except before LPS administration (0 hour), 1 hour, 2 hours, 12 hours, and 24 hours. Expression was found to be statistically significant 12 hours after LPS administration (G4: p<0.005, G5: p<0.0005), but was excluded because measurements did not exceed the LoQ.

[0320] IFN-gamma data were excluded from the analysis as they failed to exceed the LoQ at all measurement time points.

[0321] As a result of GM-CSF analysis, all measurements were excluded from the analysis because they did not exceed the LoQ, except for group G1, where measurements were taken 6 hours after LPS administration. Expression 4 hours after LPS administration was analyzed as statistically significant (G2: p<0.05, G3: p<0.05, G4: p<0.05, G5: p<0.05), but was excluded because measurements did not exceed the LoQ.

[0322] This study was conducted to examine the inhibitory ability of the test substance SMA on LPS-stimulated pro-inflammatory mediators in an ARDS model induced by repeated intratracheal administration of LPS to house mice (Mus musculus) (BALB / c). In this study, the effects of the test substance and positive control substance on LPS-stimulated pro-inflammatory mediators were evaluated in groups administered with the test substance or positive control substance. Levels of cytokines known to be major mediators of ARDS (TNF-α, IL-6, and IL-1β) were found to be significantly inhibited in the test substance and positive control groups when analyzed using BALF.

[0323] The test substance SMA was determined to dose-dependently inhibit LPS-stimulated TNF-α and IL-6 production at specific measurement time points, demonstrating the efficacy of SMA as a therapeutic agent to prevent ARDS.

[0324] In the case of IFN-gamma, GM-CSF and IL-1β, the analytical values were outside the analytical range due to the LoQ at some measurement time points, but the test substance SMA was found to inhibit the production of IL-1β in a dose-dependent manner at some measurement time points.

[0325] In conclusion, SMA exerts a rapid inhibitory effect on the production of pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β and thus can be used to alleviate acute respiratory syndrome and prolong survival.

[0326] In the figures and tables of Example 4, SMA is referred to as "PAX-1."

[0327] 4.1 Experimental Overview This study was conducted by assessing the ability of the test substance SMA and the positive control dexamethasone to control acute respiratory distress syndrome (ARDS) by measuring cytokine release in bronchoalveolar lavage fluid after oral administration in an LPS-induced ARDS model via intratracheal administration to house mice (Mus musculus) (BALB / c).

[0328] 4.2. Study Materials and Procedures 4.2.1 Test Substances

[0329] [Table 17]

[0330] 4.2.2 Positive control substances

[0331] [Table 18]

[0332] 4.2.3 Vehicle

[0333] [Table 19]

[0334] 4.2.4 Preparation of Test Substances and Analysis of Formulations Test substances (SMA) were prepared by weighing the components to dosage concentrations of 1.03, 1.54 and 2.05 mg / kg. 4.2.5 Generation of Acute Respiratory Distress Syndrome (ARDS) Model 4.2.5.1 Inducers

[0335] [Table 20]

[0336] 4.2.5.2 Preparation and Treatment Methods Preparation (BALF) On the day of LPS treatment, the required volume based on the animal's weight was prepared by weighing 100 μg of LPS and adding 500 μL of water for injection. The tube containing the mixture was thoroughly mixed using a vortex mixer and kept on ice until treatment.

[0337] [Table 21]

[0338] Preparation (survival) On the day of LPS treatment, the required volume based on the animal's weight was prepared by weighing 48 mg of LPS and adding 12 mL of water for injection. The tube containing the mixture was mixed thoroughly using a vortex mixer and kept on ice until administration.

[0339] [Table 22]

[0340] 4.2.6 Test animals Species and varieties BALB / cAnNTac Manufacturer: DaehanBiolink Co., Ltd., Korea Age: 8 weeks DOB February 10-12, 2020 (BALF) DOB April 1-3, 2020 (survival) Gender Male (BALF), female (survival) Purchase location: DaehanBiolink Co., Ltd., Korea Sex, number of animals, age and weight range (BALF) at the time of enrollment Male, 360 mice, 8 weeks old, 19.2g - 25.0g Sex, number of animals, and weight range at time of enrollment (survival) 60 female mice, 6 weeks old, 18.2g-21.3g

[0341] Induction of ARDS BALF One day after the isolation acclimation period, the mice were weighed and then 50 μL of the LPS mixture was administered intratracheally to the anesthetized mice at a dose of 10 μg / 50 μL / mouse using a disposable pipette tip. After administration, the mice were examined while waking up from anesthesia. The mice were treated with LPS twice, on days 1 and 5. The treated mice were observed daily for general symptoms.

[0342] survival One day after the isolation acclimation period was completed, the mice were weighed and then intraperitoneally injected with the LPS mixture at a concentration of 20 mg / kg using a disposable syringe (1 mL, 26 G). Treated mice were observed hourly for general symptoms and examined for dead mice.

[0343] Group assignment BALF Primary LPS-treated mice that were healthy before the second dose (boosting) were divided into five groups with 70 mice per group, as uniform as possible based on body weight.

[0344] survival After the quarantine acclimation period, healthy animals were divided into a total of 5 groups with 10 mice per group, with each group being as uniform as possible based on body weight.

[0345] 4.2.7 Treatment Treatment pathway Study substance: Oral (gastric gavage) LPS (BALF): Forced administration into the bronchi (intratracheal injection) LPS (survival): intraperitoneal

[0346] Treatment method and frequency Treatments were administered once using disposable syringes (BD 1 ml syringes, catalogue: REF301321, lot: 9326990 BD, USA), and for each study substance, treatment was based on the time it took to induce acute respiratory distress syndrome (LPS treatment). SMA (test substance) 2 hours before Dexamethasone (positive control) 1 hour before

[0347] 4.2.8 Group Composition and Treatment Dose 4.2.8.1 Group composition (BALF)

[0348] [Table 23]

[0349] 4.2.8.2 Group composition (survival)

[0350] [Table 24]

[0351] 4.2.8.3 Therapeutic Dose Setting Therapeutic doses of the test substance (SMA) are planned to be 5, 7.5, and 10 mg and will be applied in a clinical setting in healthy adults weighing 60 kg. The human equivalent dose (HED) is calculated based on FDA guidelines using body surface area. * The doses were calculated by substituting the body surface area of the test animals (mice) according to the calculation method described above, and were set at 1.03, 1.54, and 2.05 mg / kg.

[0352] * Extract from Guidance for industry, estimating the maximum safe starting dose for early clinical trials of therapeutic drugs in healthy adult volunteers.

[0353] [Table 25]

[0354] 4.2.9 Observation and weighing Observing general symptoms During the observation period, animals were examined daily for general symptoms such as appearance, behavior, and feces, and any dead animals were examined.

[0355] Disposal of dead animals A total of eight deaths occurred during the observation period and were excluded from the analysis.

[0356] Weight measurement Body weights were measured on the day of cell line implantation, weekly, and on the day of sacrifice. If body weights were measured on the day of treatment, they were measured before administration.

[0357] 4.2.10 BALF Sampling and Cytokine Analysis Bronchoalveolar lavage fluid (BALF) sampling The airway of the anesthetized animal was incised, exposing the bronchus, and a disposable 22-gauge catheter (BD, Catalog No. REF382423, USA) was inserted into the bronchus. The catheter and bronchus were secured with sutures (AILEE, Catalog No. SK521, Lot No. 7908772U, Korea) to prevent leakage of the infusion solution. The lungs were then slowly lavaged twice through the catheter with 600 μL of PBS (Welgene, Catalog No. ML008-01, Lot No. ML08200201, Korea) loaded into a disposable syringe (BD 1 ml syringe, BD, Catalog No. REF301321, Lot No. 9326990, USA). The lavage solution was transferred to a microtube (SPL, Catalog No. 60015, Lot No. LAOC16A60015, Korea). The transferred BALF (bronchoalveolar lavage fluid) was immediately centrifuged (Hanil, HI_SM-13 / A2.0, Korea) to separate the cells from the supernatant, which was then transferred to a new tube, freshly frozen using liquid nitrogen, and stored in an ultra-low temperature freezer until cytokine analysis.

[0358] [Table 26]

[0359] 4.2.11 Survival analysis Animals that died were examined every hour up to 24 hours after LPS treatment and at 48 hours.

[0360] 4.2.12 Statistical analysis of data Cytokine analysis from bronchoalveolar lavage fluid (BALF) obtained from the study was performed using Prism (Graphpad, version 7).

[0361] Equal variance tests were performed using the D'Agostino-Pearson omnibus normality test. For the sample analysis results, with the exception of body weight data, the test for equal variance was rejected due to lack of sample quality. For body weight analysis, if variances were equal, a one-way analysis of variance (ANOVA, significance level: 0.05) was performed. If significance was observed, a Dunnett's multiple t-test was performed to confirm the significance between each test group (G2–G5) relative to the negative control group (G1) (significance levels: one-tailed 0.05 and 0.01). If the study was rejected based on the results of several weight measurement time points and cytokine analysis, a Kruskal-Wallis test (significance level: 0.05) was performed. If significance was observed, a Dunn's multiple test was performed to confirm the significance between each test group (G2-G5) relative to the negative control group (G1) (significance levels: one-sided 0.05 and two-sided 0.1).

[0362] For the survival analysis results, a log-rank (Mantel-Cox) test was performed to confirm the significance (significance level: one-sided 0.05 and two-sided 0.1) between each test group (G2-G5) relative to the negative control group (G1).

[0363] 4.3. Results and Discussion 4.3.1 Assessment of cytokine production and inhibition 4.3.1.1. Analysis of Cytokine Production (Figures 13-15, Tables 16-19) Multiplex (Liminex, Austin, TX, USA), which measures mean fluorescence intensity (MFI), was used to analyze TNF-α, IL6, IL-1β, IFN-gamma, and GM-CSF. Samples were sorted by group and BALF collection time point, resulting in a total of seven sets for analysis, with one sample from each group assigned to each set.

[0364] All analyses were calculated by substituting the measured MFI with the standard curve equation for each set, calculated by a fourth-order polynomial.2 The value was confirmed to be 1 in all analyses, confirming that the measurement data were highly reliable.

[0365] Those samples that were excluded from the analysis due to the limit of quantitation were diluted using the reagents in the study protocol.

[0366] When dilution ratios were applied to analyze LPS-stimulated high-level cytokine release, it was confirmed that those with low values were measured below the limit of quantification and included as inaccurate measurements. These were excluded from the analysis due to amplification by the dilution ratio, but tables and figures were generated including all data.

[0367] Analysis of TNF-α (Figure 13, Table 16) TNF-α expression in all groups was measured and found to be 2 pg / mL and 11–12 pg / mL before (0 h) and 24 h after LPS treatment, respectively. However, because these levels were below the limit of quantification for MFI, they were excluded from data analysis. Analyses were performed 1, 2, 6, and 12 h after LPS treatment.

[0368] [Table 27]

[0369] The changes in TNF-α production in the negative control group (G1) started at 1,997 pg / mL, then 1,417 pg / mL, 1,036 pg / mL, 735 pg / mL, and 249 pg / mL, and it was observed that LPS-induced TNF-α production increased and then decreased in a time-dependent manner.

[0370] The changes in TNF-α production in the group treated with 1.03 mg / kg SMA (G2) were 1,303 pg / mL, 1,004 pg / mL, 794 mg / mL, 502 pg / mL, and 174 pg / mL. It was observed that LPS-induced TNF-α production increased and then decreased in a time-dependent manner, but there was no statistical significance (p<0.05) when comparing the time-dependent TNF-α production with that of the negative control group (G1).

[0371] The changes in TNF-α production in the group treated with 1.54 mg / kg SMA (G3) were 1,062 pg / mL, 707 pg / mL, 611 mg / mL, 407 pg / mL, and 120 pg / mL. It was observed that LPS-induced TNF-α production increased and then decreased in a time-dependent manner, but there was no statistical significance (p<0.05) when comparing the time-dependent TNF-α production with that of the negative control group (G1).

[0372] The changes in TNF-α production in the group treated with 2.05 mg / kg SMA (G4) were 729 pg / mL, 559 pg / mL, 539 mg / mL, 303 pg / mL, and 38 pg / mL. LPS-induced TNF-α production was observed to increase and then decrease in a time-dependent manner, and at some time points, the TNF-α values were statistically significant (p<0.005, 1 hour, 2 hours, 6 hours, and 12 hours) when compared with those in the negative control group (G1).

[0373] The changes in TNF-α production in the group treated with 3 mg / kg of the positive control substance, dexamethasone (G5), were 508 pg / mL, 394 pg / mL, 338 mg / mL, 204 pg / mL, and 23 pg / mL. It was observed that LPS-induced TNF-α production increased and then decreased in a time-dependent manner, and at some time points, the TNF-α values were statistically significant (p<0.0005 at 4 hours, p<0.0001 at 1, 2, 6, and 12 hours) when compared with those in the negative control group (G1).

[0374] The changes in TNF-α production in all groups (G1-G5) over time were plotted graphically (data not shown), from which the overall reduction in TNF-α was obtained by calculating the AUC (area under the curve) values for each group (Figure 13). Statistically significant reductions in TNF-α production were evident in all groups treated with SMA or dexamethasone compared to the negative control group (G1) (p<0.005: G2-G5). Table 19 below provides the numerical and statistical analysis of the AUC values for each drug treatment group.

[0375] Analysis of IL-6 (Figure 14, Table 17) Before LPS treatment (time 0), IL-6 expression in all groups was measured and was found to be 12–13 pg / mL. However, because these values were below the quantification limit of MFI, they were excluded from the data analysis. Data from 1, 2, 4, 6, 12, and 24 hours after LPS treatment were used for the analysis.

[0376] [Table 28]

[0377] The changes in IL-6 production in the negative control group (G1) started from 3,663 pg / mL, then 10,238 pg / mL, 13,015 pg / mL, 10,298 pg / mL, 8,169 pg / mL, and 3,513 pg / mL, and it was observed that LPS-induced IL-6 production increased and then decreased in a time-dependent manner.

[0378] The changes in IL-6 production in the group treated with 1.03 mg / kg SMA (G2) were 2,984 pg / mL, 10,188 pg / mL, 12,871 mg / mL, 8,954 pg / mL, 7,276 pg / mL, and 3,402 pg / mL. LPS-induced IL-6 production increased and then decreased in a time-dependent manner, but the time-dependent IL-6 production was not statistically significant (p<0.05) when compared with that of the negative control group (G1).

[0379] The changes in IL-6 production in the group treated with 1.54 mg / kg SMA (G3) were 3,152 pg / mL, 7,107 pg / mL, 9,842 mg / mL, 6.814 pg / mL, 5,094 pg / mL, and 3,623 pg / mL. LPS-induced IL-6 production increased and then decreased in a time-dependent manner, but the time-dependent IL-6 production was not statistically significant (p<0.05) when compared with that of the negative control group (G1).

[0380] The changes in IL-6 production in the group treated with 2.05 mg / kg SMA (G4) were 2,193 pg / mL, 4,701 pg / mL, 8,209 mg / mL, 4,341 pg / mL, 2,629 pg / mL, and 2,096 pg / mL. LPS-induced TNF-α production increased and then decreased in a time-dependent manner, and at some time points, the IL-6 levels were statistically significant (p<0.05 at 2 and 4 hours, p<0.005 at 6 and 12 hours) when compared with those in the negative control group (G1).

[0381] The changes in IL-6 production in the group treated with 3 mg / kg of the positive control substance, dexamethasone (G5), were 2,172 pg / mL, 4,411 pg / mL, 7.727 mg / mL, 2.064 pg / mL, 1.294 pg / mL, and 1,804 pg / mL. LPS-induced IL-6 production increased and then decreased in a time-dependent manner, and at some time points, the IL-6 values were statistically significant when compared with those in the negative control group (G1) (p<0.05 at 1, 2, 4, and 24 hours, p<0.0005 at 6 hours, p<0.0001 at 12 hours).

[0382] The changes in IL-6 production in all groups (G1-G5) over time were plotted graphically (data not shown), from which the overall reduction in IL-6 was obtained by calculating the AUC (area under the curve) value for each group (Figure 14). Statistically significant reductions in IL-6 production were evident in several groups treated with SMA or dexamethasone compared to the negative control group (G1) (p<0.005: G3, G5). Table 19 below provides the numerical and statistical analysis of the AUC values for each drug treatment group.

[0383] Analysis of IL-1β (Figure 15, Table 18) IL-1β expression in all groups was measured before LPS treatment (hour 0) and after 1, 2, and 24 hours of LPS treatment, ranging from 203 to 295 pg / mL. However, because these values were below the MFI quantification limit, they were excluded from the data analysis. Data from 4, 6, and 12 hours after LPS treatment were used for the analysis.

[0384] [Table 29]

[0385] The change in IL-1β production in the negative control group (G1) started at 510 pg / mL, then increased to 686 pg / mL and then decreased to 414 pg / mL. It was observed that LPS-induced IL-1β production increased and then decreased in a time-dependent manner.

[0386] The changes in IL-1β production in the group treated with 1.03 mg / kg SMA (G2) were 468 pg / mL, 600 pg / mL, and 405 pg / mL. LPS-induced IL-1β production increased and then decreased in a time-dependent manner, but the time-dependent IL-1β production was not statistically significant (p<0.05) when compared with that in the negative control group (G1).

[0387] The changes in IL-1β production in the group treated with 1.54 mg / kg SMA (G3) were 413 pg / mL, 517 pg / mL, and 382 pg / mL. LPS-induced IL-1β production increased and then decreased in a time-dependent manner, but the time-dependent IL-1β production was not statistically significant (p<0.05) when compared with that in the negative control group (G1).

[0388] The changes in IL-1β production in the group treated with 2.05 mg / kg SMA (G4) were 374 pg / mL, 483 pg / mL, and 335 pg / mL. LPS-induced IL-1β production was observed to increase and then decrease in a time-dependent manner, and at some time points, the IL-1β values were statistically significant (p<0.005: 4 hours, 6 hours, and 12 hours) when compared with those in the negative control group (G1).

[0389] The changes in IL-1β production in the group treated with 3 mg / kg of the positive control substance, dexamethasone (G5), were 350 pg / mL, 458 pg / mL, and 318 pg / mL. LPS-induced IL-1β production was observed to increase and then decrease in a time-dependent manner, and at some time points, the IL-1β values were statistically significant (p<0.0005: 4, 6, and 12 hours) when compared with those in the negative control group (G1).

[0390] The changes in IL-1β production in all groups (G1-G5) over time were plotted graphically (data not shown), from which the overall reduction in IL-1β was obtained by calculating the AUC (area under the curve) value for each group (FIG. 15). Statistically significant reductions in IL-1β production were evident in several groups treated with SMA or dexamethasone compared to the negative control group (G1) (p<0.005: G3, G5). Table 19 below provides the numerical and statistical analysis of the AUC values for each drug treatment group.

[0391] [Table 30]

[0392] Analysis of IFN-gamma Analysis of INF-gamma was excluded from the data analysis because its MFI was below the limit of quantitation at all time points.

[0393] Analysis of GM-CSF The analysis of GM-CSF was excluded from the data analysis because its MFI was below the limit of quantitation at all time points.

[0394] 4.3.1.2 Production rate analysis The inhibition rate of cytokine production in the groups treated with the test substance and the positive control substance was calculated, taking the normalized negative control group as 100%.

[0395] Analysis of TNF-α TNF-α production rate analysis was performed using data from 1, 2, 4, 6 and 12 hours, excluding data from 0 and 24 hours.

[0396] The inhibition rates of TNF-α production were 65%, 71%, 77%, 68% and 70% in the group treated with 1.03 mg / kg SMA (G2), and no significant differences (p<0.05) were observed in all statistical analyses.

[0397] The inhibition rates of TNF-α production were 53%, 50%, 59%, 55%, and 48% in the group treated with 1.54 mg / kg SMA (G3), and no significant differences (p<0.05) were observed in any statistical analysis.

[0398] The inhibition rates of TNF-α production were 37%, 39%, 52%, 41%, and 15% in the group treated with 2.05 mg / kg SMA (G4). At several time points, the reducing effect of SMA was observed to be statistically significant (p<0.005: 1 hour, 2 hours, 6 hours, and 12 hours).

[0399] The inhibition rates of TNF-α production were 25%, 28%, 33%, 28%, and 9% in the group (G5) treated with 3 mg / kg of the positive control substance dexamethasone. At several time points, the reducing effect of dexamethasone was statistically significant (p<0.0005 at 4 hours, p<0.0001 at 1, 2, 6, and 12 hours).

[0400] Analysis of IL-6 IL-6 production rate analysis was performed using data from 1, 2, 4, 6, 12 and 24 hours, excluding data from 0 hours.

[0401] The inhibition rates of IL-6 production were 81%, 100%, 99%, 87%, 89% and 97% in the group treated with 1.03 mg / kg SMA (G2), and no significant differences (p<0.05) were observed in any statistical analysis.

[0402] The inhibition rates of IL-6 production were 86%, 69%, 76%, 66%, 62% and 103% in the group treated with 1.54 mg / kg SMA (G3), and no significant differences (p<0.05) were observed in any statistical analysis.

[0403] The inhibition rates of IL-6 production were 60%, 46%, 63%, 42%, 32%, and 60% in the group treated with 2.05 mg / kg SMA (G4). At some time points, the reduction effect of SMA was statistically significant (p<0.05 at 2 and 4 hours, p<0.005 at 6 and 12 hours).

[0404] The inhibition rates of IL-6 production were 59%, 43%, 59%, 20%, 16%, and 51% in the group (G5) treated with 3 mg / kg of the positive control substance dexamethasone. At some time points, the reducing effect of dexamethasone was statistically significant (p<0.05 at 1, 2, 4, and 24 hours, p<0.0001 at 6 and 12 hours).

[0405] Analysis of IL-1β IL-1β production rate analysis was performed using the 4-, 6-, and 12-hour data, excluding the 0-, 1-, 2-, and 24-hour data.

[0406] The inhibition rates of IL-1β production were 92%, 87%, and 98% in the group treated with 1.03 mg / kg SMA (G2), and no significant differences (p<0.05) were observed in any statistical analysis.

[0407] The inhibition rates of IL-1β production were 81%, 75% and 92% in the group treated with 1.54 mg / kg SMA (G3), and no significant differences (p<0.05) were observed in all statistical analyses.

[0408] The inhibition rates of TNF-1β production were 73%, 70%, and 81% in the group treated with 2.05 mg / kg SMA (G4). At several time points, the reduction effect of SMA was observed to be statistically significant (p<0.05: 4 hours, 6 hours, and 12 hours).

[0409] The inhibition rates of IL-1β production were 69%, 67%, and 77% in the group (G5) treated with 3 mg / kg of the positive control substance dexamethasone. At several time points, the reducing effect of dexamethasone was observed to be statistically significant (p<0.0001: 4, 6, and 24 hours).

[0410] Analysis of IFN-gamma Analysis of INF-gamma was excluded from the production rate analysis because the MFI was below the limit of quantification at all time points.

[0411] Analysis of GM-CSF Analysis of GM-CSF was excluded from the production rate analysis because the MFI was below the limit of quantification at all time points.

[0412] 4.3.2 Survival analysis 4.3.2.1 Survival analysis (Figure 16) Dead mice were examined hourly after treatment with 20 mg / kg LPS, and survival rates were statistically significantly increased (G3: p<0.005, G4: p<0.0005, G5: p<0.0001) compared to the negative control group (G1).

[0413] 4.3.3 Weight and general symptoms body weight The mean weight of all mice was 22.3 g at enrollment and 24.1 g at group assignment. Normal weight gain was observed during the isolation acclimation period.

[0414] Group allocation was performed so that all groups had average body weights. There was no statistical significance (p<0.05) when compared with the negative control group (G1).

[0415] General symptoms No abnormalities were observed during the isolation adaptation period, during which general observations were carried out daily.

[0416] During the study period, a total of eight deaths occurred due to the intratracheal administration of LPS. Respiratory distress symptoms occurred immediately after the end of the intratracheal administration. Transient respiratory distress was observed in all LPS-treated mice; however, this was likely due to the volume of the LPS administered and not LPS-induced respiratory distress syndrome. Nonetheless, eight mice did not appear to recover from the symptoms. Cardiopulmonary resuscitation and temperature maintenance were performed on eight mice with severe respiratory distress, but they died and no samples were obtained for analysis.

[0417] 4.4. Conclusion This study was conducted to confirm the inhibitory effect of the test substance SMA on LPS-induced pro-inflammatory mediators in a model of acute respiratory distress syndrome (ARDS) in house mice (Mus musculus) (BALB / c) induced by repeated LPS treatment via intratracheal administration. The results of this study confirmed the inhibitory effect of the test substance and positive control substance on LPS-induced pro-inflammatory mediators, and showed that the expression of cytokines known to be major mediators of acute respiratory distress syndrome (TNF-α, IL-6, IL-1β) was significantly suppressed in bronchoalveolar lavage fluid (BALF).

[0418] This study confirmed that the test substance SMA inhibits the production of TNF-α and IL-6 at specific time points and that the test substance SMA is effective as a preventive treatment for acute respiratory syndrome.

[0419] Although the assay values for IFN-gamma, GM-CSF, and IL-1β were below the limit of quantitation at some time points and were therefore excluded from the analysis, the test substance SMA dose-dependently inhibited the production of IL-1β at some time points.

[0420] In conclusion, SMA exerts a rapid inhibitory effect on the production of pro-inflammatory mediators such as TNF-α, IL-6 and IL-1β and can therefore be used to alleviate acute respiratory syndrome and prolong survival.

[0421] [Example 5] In vitro experiments demonstrating SMA exerts a viral suppressive effect against SARS-CoV-2 The in vivo experiments described in Example 4 above confirmed that PAX-1 (SMA) is effective in inhibiting pro-inflammatory cytokines, similar to dexamethasone, a drug approved for use in Europe as a treatment for SARS-CoV-2-associated pneumonia.

[0422] Example 5 describes in vitro studies that revealed that PAX-1 exhibits virus-suppressing effects similar to those of the antiviral drug remdesivir. PAX-1 possesses antiviral and anti-inflammatory properties and is effective in treating diseases such as viral infection-induced pneumonia. Treatment with PAX-1 is expected to significantly reduce recovery time to as little as one week. The progression of COVID-19-associated disease can be prevented when PAX-1 is administered during the early stages of infection.

[0423] 5.1 Mechanisms of viral suppression / killing and inhibition of inflammatory cytokines The mechanism of action of PAX-1 involves its specific binding to telomeres in solid human tumor cell lines, resulting in telomere-associated DNA damage, telomere erosion, and cell death (Phatak P, Dai F, Butler M, et al. (2008) KML001 Cytotoxic Activity Is Associated with Its Binding to Telomeric Sequences and Telomere Erosion in Prostate Cancer Cells. Cancer Therapy: Preclinical 14(14): 4593-4603). PAX-1 also suppresses cancer cell proliferation by reducing the expression of transcription factors involved in the transcription of telomerase mRNA. Furthermore, binding of PAX-1 to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leads to the translocation of the telomerase catalytic subunit, called telomerase reverse transcriptase (hTERT), into the cytoplasm, thereby inhibiting telomerase activity and ultimately killing the cancer cells. Recent studies have found structural and functional similarities between the hTERT domain and viral RNA-dependent RNA polymerases (RdRPs) through the conserved reverse transcriptase motif consisting of a right-hand structure (finger, thumb, and palm domains) (Machitani M, Yasukawa M, Nakashima J, Furuichi Y, Masutomi K. RNA-dependent RNA polymerase, RdRP, a promising therapeutic target for cancer and potentially COVID-19. Cancer Sci. 2020 Aug 17;111(11):3976-84. doi: 10.1111 / cas.14618). Viral RdRPs play essential roles in viral genome transcription and replication, making their inhibition one of the primary targets for antiviral drugs.Considering the proven inhibitory effect of PAX-1 on hTERT and the structural similarity between viral RdRPs and the hTERT RdRP domain, it seems reasonable to propose that PAX-1-mediated inhibition of hTERT RdRP activity could be applied to inhibit coronavirus RdRP activity. Furthermore, the antiviral properties of PAX-1 are not limited to coronaviruses but are applicable to a wide range of viruses, demonstrating PAX-1's versatile therapeutic potential for anticancer and antiviral treatments (Machitani M, Yasukawa M, Nakashima J, Furuichi Y, Masutomi K. RNA-dependent RNA polymerase, RdRP, a promising therapeutic target for cancer and potentially COVID-19. Cancer Sci. 2020 Aug 17;111(11):3976-84. doi: 10.1111 / cas.14618).

[0424] Sodium meta arsenite has been shown to be a potent inhibitor of human telomerase.

[0425] It has been widely accepted that the overproduction of cytokines in response to viral infection is the primary cause of COVID-19-induced pneumonia (inflammation).

[0426] Viral infection is followed by aberrant cellular activation of gene expression, which leads to excessive cytokine release, which then induces inflammation (pneumonia). Example 4 shows that PAX-1 inhibits or reduces the production / secretion of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6.

[0427] 5.2 In vitro evaluation of the antiviral effect of PAX-1 on SARS-CoV-2-infected cells Overview The objective of this study was to examine the antiviral effect of PAX-1 against SARS-CoV-2. The antiviral efficacy of the compound was determined by dose-response curve (DRC) experiments in a SARS-CoV-2 cell infection model. Infected cells were imaged via immunofluorescence using an antibody specific for the viral nucleocapsid (N) protein, and the acquired images were analyzed using Columbus software (Perkin Elmer).

[0428] Experiments conducted at the Pasteur Institute have shown that PAX-1 has an antiviral effect (IC 50 = 4.25 μM) is that of remdesivir (IC 50 = 5.27 μM), indicating that PAX-1 has antiviral properties similar to remdesivir.

[0429] 5.2.2 Materials and Methods 5.2.2.1 Viruses and cell lines SARS-CoV-2 was provided by the Korea Centers for Disease Control and Prevention (KCDC), and Vero cells were obtained from ATCC (ATCC-CCL81).

[0430] 5.2.2.2 Reagents Chloroquine, lopinavir, and remdesivir were used as reference compounds and were purchased from Sigma-Aldrich, SelleckChem, and MedChemExpress, respectively. Primary antibodies specific for SARS-CoV-2 N protein were purchased from Sino Biological, and secondary antibodies Alexa Fluor 488 goat anti-rabbit IgG and Hoechst 33342 were purchased from Molecular Probes.

[0431] 5.2.2.3 Dose-response curve analysis by immunofluorescence 1.2 x 10 cells per well in a 384-well tissue culture plate 4Vero cells were inoculated. 24 hours after seeding, 10 different concentrations of compounds were serially diluted in DMSO and PBS and treated with the compound, with the highest concentration being 50 μM. One hour after drug treatment, the cells were infected with SARS-CoV-2 (MOI 0.0125) in a BSL3 facility and incubated at 37°C for 24 hours. Cells were then fixed with 4% paraformaldehyde (PFA) and subsequently permeabilized. Cells were then stained with anti-SARS-CoV-2 nucleocapsid (N) primary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody, and Hoechst 33342. Fluorescence images of infected cells were captured using the Operatta image analysis device (Perkin Elmer).

[0432] 5.2.2.4 Image analysis The acquired images were analyzed using Columbus software. The total number of Hoechst-stained cells per well was counted and used as the total cell number. The number of cells expressing viral N protein was used as the total number of infected cells. The infection rate was calculated as the number of cells expressing N protein / total number of cells.

[0433] The degree of infection per well was normalized to the average infectivity of wells of uninfected cells (mock) on the same plate and to the average infectivity of wells of infected cells treated with 0.5% DMSO (v / v).

[0434] The cytotoxicity of the compounds was normalized by normalizing the number of cells in each well to the average number of cells in the mock wells, and represented on the graph as "cell number relative to mock."

[0435] Response curves derived from each drug concentration, as well as IC 50 and CC 50 The value was calculated using the formula Y = nadir + (peak - nadir) / (1 + (IC 50 / X) ヒルスロープ ) were used for induction. All IC 50 and CC 50The values were calculated by fitting dose-response curves from two replicates of independent experiments, and the selectivity index (SI) values were calculated using CC 50 / I C 50 It was calculated by

[0436] 5.2.3. Results - Compound Dose-Response Curve (DRC) Analysis This study explored the antiviral effect of PAX-1 on the replication of SARS-CoV-2 (COVID-19 virus) in Vero cells, as well as its possible cytotoxic effects, in comparison with remdesivir (i.e., the first antiviral drug approved for use during the COVID-19 pandemic) and lopinavir (i.e., a drug currently under evaluation for the antiviral treatment of COVID-19 in combination with ritonavir).

[0437] Vero cells are a widely used and accepted cellular model for replicating and isolating SARS-CoV-2. Briefly, Vero cells (ATCC-CCL81) were infected with SARS-CoV-2 (obtained from the Korea Disease Control and Prevention Agency) at a multiplicity of infection (MO) of 0.0125 in the presence of various concentrations of test drugs or DMSO / PBS (control). Infected cells were fixed 24 hours post-infection and stained with anti-SARS-CoV-2 nucleocapsid antibody and Hoechst 33342, and the total number of infected cells was determined using immunofluorescence staining. Image analysis was performed using Operatta (Perkin Elmer). The half-maximal inhibitory concentration (IC) of each drug was calculated. 50 ) and half-maximal cytotoxic concentration (CC 50 ) values were determined using fitted dose-response curves.

[0438] The results are shown in Figure 17. Blue dots indicate the compounds' inhibition of SARS-CoV-2 infection, and red squares indicate the compounds' cytotoxicity.

[0439] As shown in Figure 17, SARS-CoV-2 replication was inhibited by PAX-1 ("Komipharm (PBS)" in Figure 17) to the same extent as remdesivir and more efficiently than lopinavir. IC of PAX-1 inhibition of SARS-CoV-2 infection 50 The IC value was 4.25 μM, i.e., the same order of magnitude as remdesivir (IC 50 = 5.27 μM), which is an order of magnitude lower than lopinavir (IC 50 = 13.11 μM). 50 The value was 21.05 μM compared to over 50 μM for both remdesivir and lopinavir. CC1 showed slightly greater inhibition of cell viability compared to the other two compounds, despite PAX-1 showing slightly greater inhibition of cell viability compared to the other two compounds. 50 / I C 50 The SI of antiviral activity relative to cytotoxicity, calculated using the SI, was comparable to that of lopinavir. Thus, PAX-1 effectively inhibited SARS-CoV-2 replication in vitro.

[0440] 5.2.4. Discussion The cytotoxic concentration of PAX-1 against normal cells (CC 50 ) was 21.05 μM, which is the antiviral activity index (IC 50 , 4.25 μM), indicating the safety of the drug. 50 There is no need to take PAX-1 at concentrations five times higher than the normal values.

[0441] Recent studies of PAX-1 toxicity have involved testing both remdesivir and lopinavir simultaneously for comparison. Experimental results have shown that lopinavir has the following index: IC 50 =13.11 μM, CC 50 It showed a potent cytotoxicity of >50 μM and an SI value of 3.81 compared to PAX-1. Lopinavir is currently undergoing clinical trials led by the US FDA for the treatment of COVID-19.

[0442] Considering the above factors, PAX-1 can also be used as an antiviral agent without causing side effects or severe adverse effects during treatment.

[0443] PAX-1 binds to telomere sequences, which are proliferative potentials attached to the ends of chromosomes. The concentrations of PAX-1 used to inhibit inflammatory cytokines do not have cytotoxic effects on normal immune cells. Furthermore, PAX-1 is completely metabolized and excreted from the body 72 hours after ingestion.

[0444] Some of the patients in clinical trials of PAX-1 (472 patients have been involved in the trials so far) were given 20 mg / day (8 tablets per day). No deaths due to drug toxicity have been reported, indicating that PAX-1 is a very safe substance.

[0445] Growing evidence shows that coronavirus antibodies weaken rapidly and that coronaviruses from animal species can mutate and cross-pollinate with humans, creating the risk of reinfection. Clearly, there is a great need for the rapid development of antiviral drugs against COVID-19.

[0446] [Example 6] COVID-19 patient diary (treated with SMA) Example 6 describes the daily life log of a 59-year-old woman (with no pre-existing condition) infected with SARS-CoV-2 and treated with SMA. This example demonstrates that SMA is effective in alleviating or treating symptoms of SARS-CoV-2 infection, such as chest tightness, difficulty breathing, shortness of breath (dyspnea), fever, loss of appetite, runny nose, cough, phlegm production, and pain.

[0447] [Table 31-1]

[0448] [Table 31-2]

[0449] [Table 31-3]

[0450] Where any prior art publication is referred to herein, it should be understood that such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or any other country.

[0451] In the claims that follow and in the description of the invention above, unless otherwise required by context, either by express language or by necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., they specify the presence of stated features but do not exclude the presence or addition of further features in various embodiments of the invention. The present invention includes the following aspects. [1] 1. A method for reducing an inflammatory response caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject. [2] The method according to [1], wherein the viral infection is a coronavirus infection. [3] The method described in [2], wherein the coronavirus is SARS-CoV-2. [4] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is orally administered. [5] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day. [6] 1. A method for treating or preventing an inflammatory condition caused by a viral infection in a subject, comprising administering to a subject an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject. [7] The method according to [6], wherein the viral infection is a coronavirus infection. [8] The method described in [7], wherein the coronavirus infection is caused by SARS-CoV-2. [9] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is orally administered.

[10] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day.

[11] A method for treating or preventing hypercytokinemia caused by a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject.

[12] The method according to

[11] , wherein the viral infection is an infection caused by a coronavirus.

[13] The method described in

[12] , wherein the coronavirus is SARS-CoV-2.

[14] 1. A method of treating a viral infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject.

[15] The method according to

[14] , wherein the viral infection is caused by infection with a coronavirus.

[16] The method described in

[15] , wherein the coronavirus is SARS-CoV-2.

[17] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is orally administered.

[18] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day

[14] .

[19] A method of treating a coronavirus infection in a subject, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject.

[20] The method described in

[19] , wherein the coronavirus infection is caused by SARS-CoV-2.

[21] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is orally administered.

[22] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day

[19] .

[23] 1. A method for reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection, comprising administering an effective amount of sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) to said subject.

[24] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is orally administered.

[25] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day.

[26] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) but the following: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer administered by a composition comprising The method according to any one of [1] to

[25] , wherein the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[27] Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) but the following: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer administered by a composition comprising the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition; The method according to any one of [1] to

[25] , wherein the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[28] 1. When used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, a pharmaceutical composition for oral administration comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, A pharmaceutical composition, wherein the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[29] 1. When used to reduce an inflammatory response caused by a viral infection and / or to treat or prevent an inflammatory condition caused by a viral infection, a pharmaceutical composition for oral administration comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer Including, the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition; A pharmaceutical composition, wherein the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[30] Sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing an inflammatory response caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O -K + ) use.

[31] sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing an inflammatory condition caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use.

[32] Use of sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating or preventing hypercytokinemia caused by a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use.

[33] Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating a viral infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use.

[34] Sodium meta arsenite (O=As-O) in the manufacture of a medicament for reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection. - Na + ) or potassium meta arsenite (O=As-O - K + ) use.

[35] The use according to any one of

[30] to

[34] , wherein the viral infection is a coronavirus infection.

[36] Sodium meta arsenite (O=As-O) in the manufacture of a medicament for treating coronavirus infection in a subject. - Na + ) or potassium meta arsenite (O=As-O - K + ) use.

[37] The use according to

[35] or

[36] , wherein the coronavirus infection is caused by SARS-CoV-2.

[38] The use according to any one of

[30] to

[37] , wherein the medicament is formulated for oral administration.

[39] The medicament is selected from the group consisting of: (a) Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) and one or more pharmaceutically acceptable excipients, wherein said one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer A pharmaceutical composition comprising: The use according to any one of

[30] to

[38] , comprising a pharmaceutical composition in which the weight percentage of the enteric coating is about 6% w / w to about 20% w / w relative to the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition.

[40] The medicament is selected from the group consisting of: (a) Sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ), as well as the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer A pharmaceutical composition comprising: the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; The coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition. The use according to any one of

[30] to

[38] , including a pharmaceutical composition.

[41] 1. A pharmaceutical composition for oral administration comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating containing an enteric polymer Including, the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; or A pharmaceutical composition for use in treating a coronavirus infection in a subject.

[42] 1. A pharmaceutical composition for oral administration comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-95% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating containing an enteric polymer Including, the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; or A pharmaceutical composition for use in treating a coronavirus infection in a subject.

Claims

1. reducing the inflammatory response caused by a viral infection in a subject; Treating or preventing an inflammatory condition caused by a viral infection in a subject; Treating a viral infection in a subject; reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition caused by a viral infection; or Treating or preventing hypercytokinemia caused by viral infection in a subject Sodium meta arsenite (O=As-O) in the manufacture of a drug for － Na ＋ ) or potassium meta arsenite (O=As-O － K ＋ ) use of The use, wherein the viral infection is a coronavirus infection, and the coronavirus infection is caused by SARS-CoV-2.

2. 2. The use of claim 1, wherein the replication of SARS-CoV-2 is reduced.

3. 3. The use of claim 1 or 2, wherein one or more symptoms of SARS-CoV-2 are alleviated or treated, and the one or more symptoms of SARS-CoV-2 are selected from chest tightness, difficulty breathing, shortness of breath (dyspnea), fever, loss of appetite, runny nose, cough, sputum production, and pain.

4. 4. The use according to any one of claims 1 to 3, wherein the medicament is formulated for oral administration.

5. The sodium meta arsenite (O=As-O － Na ＋ ) or potassium meta arsenite (O=As-O － K ＋ 5. The use according to any one of claims 1 to 4, wherein said agonist is administered in a dose ranging from 2 mg per day to 20 mg per day.

6. The pharmaceutical composition is selected from the group consisting of: (a) Sodium meta arsenite (O=As-O － Na ＋ ) or potassium meta arsenite (O=As-O － K ＋ ) and one or more pharmaceutically acceptable excipients, wherein said one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating comprising an enteric polymer A pharmaceutical composition comprising:

6. The use according to any one of claims 1 to 5, comprising a pharmaceutical composition wherein the weight percentage of the enteric coating is from about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition, and the coating thickness is from about 6.5% to about 15% of the thickness of the pharmaceutical composition.

7. The pharmaceutical composition is selected from the group consisting of: (a) Sodium meta arsenite (O=As-O － Na ＋ ) or potassium meta arsenite (O=As-O － K ＋ ), as well as the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-85% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating comprising an enteric polymer A pharmaceutical composition comprising: the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; The use according to any one of claims 1 to 5, comprising a pharmaceutical composition.

8. 8. The use according to claim 6 or 7, wherein the amount of sodium meta arsenite or potassium meta arsenite in the solid core is about 0.1 to 5.0% w / w of said solid core.

9. The use according to claim 7 or 8, which satisfies one or more of the following: (a) the filler or diluent is present in an amount of about 10-85% w / w of said solid core in the pharmaceutical composition; (b) the filler or diluent is selected from dibasic calcium phosphate anhydrous, partially pregelatinized starch, silicified microcrystalline cellulose, microcrystalline cellulose, calcium sulfate dihydrate, lactose, calcium hydrogen phosphate, calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, or mixtures thereof; (c) the disintegrant is present in an amount of about 10-50% w / w of said solid core in the pharmaceutical composition; (d) the disintegrant is selected from L-hydroxypropyl cellulose, partially pregelatinized starch, crospovidone, potato starch, corn starch, sodium starch glycolate, and alginic acid; (e) the glidant is present in an amount of about 0.3-4.0% w / w of said solid core in the pharmaceutical composition; (f) the glidant is selected from colloidal silicon dioxide and talc; (g) the lubricant is present in an amount of about 0.3-4.0% w / w of said solid core in the pharmaceutical composition; (h) the lubricant is selected from sodium stearyl fumarate, magnesium stearate, stearic acid, talc, and silica; (i) the pharmaceutical composition further comprises a binder in an amount of about 1-30% w / w of said solid core, said binder being selected from silicified microcrystalline cellulose, microcrystalline cellulose, partially pregelatinized starch, L-hydroxypropyl cellulose (low substituted hydroxypropyl cellulose), hydroxypropyl cellulose, copovidone (polyvinylpyrrolidone), pregelatinized maize starch, hydroxypropyl methylcellulose, starch, acacia, maize starch, and gelatin; (j) the enteric coating provides a weight gain of about 7-17% w / w of the solid core; (k) The enteric coating is selected from copolymers of acrylic acid and its esters or methacrylic acid or its esters, polysorbates, cellulose acetate phthalate polymers, hydroxypropyl methylcellulose phthalate polymers, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, and shellac, and combinations thereof.

10. 1. A pharmaceutical composition for oral administration comprising: (a) a solid core comprising sodium meta arsenite or potassium meta arsenite and one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients are selected to minimize oxidation of meta arsenite to meta arsenate; and (b) an enteric coating comprising an enteric polymer Including, the weight percentage of the enteric coating is about 6% w / w to about 20% w / w based on the total weight of the pharmaceutical composition, and the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; or for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; The pharmaceutical composition, wherein the viral infection is a coronavirus infection, and the coronavirus infection is caused by SARS-CoV-2.

11. 1. A pharmaceutical composition for oral administration comprising: (a) sodium meta arsenite or potassium meta arsenite, and the following pharmaceutically acceptable excipients: (i) a filler or diluent in the range of about 5-85% w / w; (ii) a disintegrant in the range of about 10-90% w / w; (iii) a glidant in the range of about 0.1-5% w / w; (iv) a lubricant in the range of about 0.1-5% w / w, and (v) optionally a binder in the range of 0 to about 30% w / w Contains a solid core and (b) an enteric coating comprising an enteric polymer Including, the pharmaceutically acceptable excipient is selected to minimize oxidation of meta arsenite to meta arsenate; the weight percentage of the enteric coating is about 6% w / w to about 20% w / w, based on the total weight of the pharmaceutical composition; the coating thickness is about 6.5% to about 15% of the thickness of the pharmaceutical composition; for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; or for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; The pharmaceutical composition, wherein the viral infection is a coronavirus infection, and the coronavirus infection is caused by SARS-CoV-2.

12. 12. The pharmaceutical composition of claim 10 or 11, wherein the amount of sodium meta arsenite or potassium meta arsenite in the solid core is about 0.1 to 5.0% w / w of said solid core.

13. 13. The pharmaceutical composition according to any one of claims 10 to 12, which satisfies one or more of the following: (a) the filler or diluent is present in an amount of about 10-85% w / w of said solid core in the pharmaceutical composition; (b) the filler or diluent is selected from dibasic calcium phosphate anhydrous, partially pregelatinized starch, silicified microcrystalline cellulose, microcrystalline cellulose, calcium sulfate dihydrate, lactose, calcium hydrogen phosphate, calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, or mixtures thereof; (c) the disintegrant is present in an amount of about 10-50% w / w of said solid core in the pharmaceutical composition; (d) the disintegrant is selected from L-hydroxypropyl cellulose, partially pregelatinized starch, crospovidone, potato starch, corn starch, sodium starch glycolate, and alginic acid; (e) the glidant is present in an amount of about 0.3-4.0% w / w of said solid core in the pharmaceutical composition; (f) the glidant is selected from colloidal silicon dioxide and talc; (g) the lubricant is present in an amount of about 0.3-4.0% w / w of said solid core in the pharmaceutical composition; (h) the lubricant is selected from sodium stearyl fumarate, magnesium stearate, stearic acid, talc, and silica; (i) the pharmaceutical composition further comprises a binder in an amount of about 1-30% w / w of said solid core, said binder being selected from silicified microcrystalline cellulose, microcrystalline cellulose, partially pregelatinized starch, L-hydroxypropyl cellulose (low substituted hydroxypropyl cellulose), hydroxypropyl cellulose, copovidone (polyvinylpyrrolidone), pregelatinized maize starch, hydroxypropyl methylcellulose, starch, acacia, maize starch, and gelatin; (j) the enteric coating provides a weight gain of about 7-17% w / w of the solid core; (k) The enteric coating is selected from copolymers of acrylic acid and its esters or methacrylic acid or its esters, polysorbates, cellulose acetate phthalate polymers, hydroxypropyl methylcellulose phthalate polymers, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, and shellac, and combinations thereof.

14. 14. The pharmaceutical composition of any one of claims 10 to 13, wherein the replication of SARS-CoV-2 is reduced.

15. 15. The pharmaceutical composition of any one of claims 10 to 14, wherein one or more symptoms of SARS-CoV-2 are alleviated or treated, wherein the one or more symptoms of SARS-CoV-2 are selected from chest tightness, difficulty breathing, shortness of breath (dyspnea), fever, loss of appetite, runny nose, cough, sputum production, and pain.

16. Sodium meta arsenite (O=As-O － Na ＋ ) or potassium meta arsenite (O=As-O － K ＋ 16. The pharmaceutical composition of any one of claims 10 to 15, wherein said agonist is administered at a dose ranging from 2 mg per day to 20 mg per day.

17. A pharmaceutical composition comprising sodium meta arsenite or potassium meta arsenite, for use in reducing an inflammatory response caused by a viral infection in a subject; for use in treating or preventing an inflammatory condition caused by a viral infection in a subject; for use in treating or preventing hypercytokinemia caused by a viral infection in a subject; for use in treating a viral infection in a subject; or for use in reducing TNF-α, IL-1β and / or IL-6 levels in a subject suffering from an inflammatory condition due to a viral infection; The pharmaceutical composition, wherein the viral infection is a coronavirus infection, and the coronavirus infection is caused by SARS-CoV-2.

18. The pharmaceutical composition of claim 17, wherein the replication of SARS-CoV-2 is reduced.

19. The pharmaceutical composition of claim 17 or 18, wherein one or more symptoms of SARS-CoV-2 are alleviated or treated, and the one or more symptoms of SARS-CoV-2 are selected from chest tightness, difficulty breathing, shortness of breath (dyspnea), fever, loss of appetite, runny nose, cough, sputum production, and pain.

20. The pharmaceutical composition of any one of claims 17 to 19, wherein the pharmaceutical composition is formulated for oral administration.

21. The pharmaceutical composition of any one of claims 17 to 20, wherein the sodium meta arsenite (O=As-O - Na + ) or potassium meta arsenite (O=As-O - K + ) is administered at a dose ranging from 2 mg per day to 20 mg per day.

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