“INHALATION COMPOSITION OF ARTEMISININ OR ITS DERIVATIVES FOR USE IN CORONAVIRUS DISEASE”

FIELD OF INVENTION:

The present invention relates to an inhalation composition comprising artemisinin or its derivatives to a patient (e.g., a human). The present invention further relates to method of treating Coronavirus disease by administering artemisinin or derivatives thereof. The present invention also relates to use of the inhalation composition comprising artemisinin or its derivatives for the treatment of Coronavirus disease.

BACKGROUND OF INVENTION:

Coronaviruses are a large family of viruses which may cause illness in animals or humans. In humans, several coronaviruses are known to cause respiratory infections ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The most recently discovered coronavirus causes coronavirus disease Coronavirus disease. Coronavirus disease is the infectious disease caused by the most recently discovered coronavirus. The most common symptoms of Coronavirus disease are fever, dry cough, and tiredness. Other symptoms that are less common and may affect some patients include aches and pains, nasal congestion, headache, conjunctivitis, sore throat, diarrhea, loss of taste or smell or a rash on skin or discoloration of fingers or toes. Around 1 out of every 5 people who gets Coronavirus disease becomes seriously ill and develops difficulty breathing. Older people, and those with underlying medical problems like high blood pressure, heart and lung problems, diabetes, or cancer, are at higher risk of developing serious illness. There is multiple ongoing research about numerous antiviral agents, immunotherapies, and vaccines which are being investigated and developed as potential therapies for this serious Coronavirus disease disease. Artemisinin is a widely used drug for malaria infections due to Plasmodium falciparum. Artesunate is a semisynthetic derivative of artemisinin, an antimalarial drug, which is obtained from the plant Artemisia annua L. Artemisia annua L. is a plant that has been used in traditional Chinese medicine for centuries. Artemisinin has also been defined by the World Health Organization as “the best hope for malaria treatment”. It is also known that artemisinin inhibits endocytosis more strongly than chloroquine, and unlike chloroquine, did not cause inhibition of transport vesicle- vacuole fusion. Artesunate may inhibit NF-kB (Nuclear Factor kappa B) downregulation and viral protein synthesis, disrupting the early phase of viral replication. Artesunate has the highest antiviral activity against HCMV (Human Cytomegalovirus) among the derivatives of artemisinin. Artemisinin/artesunate. has been shown to inhibit the reproduction of hepatitis B virus in vitro. Artemisinin also inhibits the replication of hepatitis C replicon, which, just like SARS-CoV-2, is a single-stranded RNA virus.

A recent study (Joon-Yong Bae et al.) clearly shows the in vitro anti-viral efficacy of Artesunate against SARS-CoV-2. According to this study, Artesunate in Vero cells showed an IC50 of 53.06 pM, a CC50 of higher than 100 pM (> 100 pM) and an SI of > 1.885. The inhibitory effects of artesunate in Calu-3 cells at 24 hours post infection was: IC50 1.76 pM, CC50>100 pM, and SI > 56.82; and 48 hours post infection: IC50 0.453 pM, CC50> 100 pM, and SI > 220.8 were notably better than those of Artesunate in Vero cells.

Coronavirus disease primarily infects the lungs in the affected individuals and in severe cases causes death due to ARDS and pneumonia. As, Coronavirus disease primarily infects the upper respiratory tract, delivery of drugs via the inhalation route appear to provide a quick and targeted delivery. As therapeutic agents are delivered directly to the lungs, the inhalation route offers a more rapid onset of action, allows smaller doses to be used and has a better efficacy to safety ratio compared to systemic therapy. WO20 16083827 discloses composition of artesunate for intranasal and pulmonary delivery for use in malaria.

WO2010110747 discloses inhalation composition of artemisinin for use in asthma and chronic obstructive pulmonary disease.

However, there is a need in the art for inhalation composition comprising an artemisinin derivative for use in Coronavirus disease.

The present invention relates to inhalation composition comprising artemisinin or derivatives thereof for use in the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering artemisinin or derivatives thereof. The present invention further relates to inhalation composition comprising artesunate for use in treatment of Coronavirus disease.

OBJECT OF THE INVENTION

An object of the present invention is to provide an inhalation composition comprising artemisinin or its derivatives to a patient (e.g., a human) for use in treatment of Coronavirus disease.

Another object of the present invention is to provide an inhalation composition comprising artemsinin or its derivatives and pharmaceutically acceptable excipients.

One more object of the present invention is a method of treatment of Coronavirus disease by administering artemisinin or derivatives thereof.

Yet another object of the present invention is to provide an inhalation composition which is a propellant containing metered dose aerosol comprising artemisinin or its derivatives thereof. The aerosol composition may be administered for the treatment of Coronavirus disease.

A further object of the present invention is to provide an inhalation composition which is a nebulization composition comprising artemisinin or its derivatives thereof. The nebulization composition may be administered for the treatment of Coronavirus disease.

An object of the present invention is to provide an inhalation composition which is a dry powder composition comprising artemisinin or its derivatives thereof. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

One more object of the present invention is to provide an inhalation composition which is a nasal spray composition comprising artemisinin or its derivatives thereof. The nasal spray composition may be administered for the treatment of Coronavirus disease.

Yet another object of the present invention is to provide an inhalation composition which is a propellant containing metered dose aerosol comprising artesunate. The aerosol composition may be administered for the treatment of Coronavirus disease.

A further object of the present invention is to provide an inhalation composition which is a nebulization composition comprising artesunate. The nebulization composition may be administered for the treatment of Coronavirus disease.

An object of the present invention is to provide an inhalation composition which is a dry powder composition comprising artesunate. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease. One more object of the present invention is to provide an inhalation composition which is a nasal spray composition comprising artesunate. The nasal spray composition may be administered for the treatment of Coronavirus disease.

SUMMARY OF THE INVENTION:

The present invention relates to an inhalation composition comprising artemisinin or its derivatives to a patient (e.g., a human). The inhalation composition comprises artemsinin or its derivatives and pharmaceutically acceptable excipients. The inhalation composition may be a solution, suspension or a powder. The inhalation composition may be filled within a container suitable for inhalation. The inhalation composition may be administered for the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering artemisinin or derivatives thereof.

In one embodiment, the inhalation composition is a propellant containing metered dose aerosol comprising artemisinin or its derivatives thereof. The aerosol composition may be administered for the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nebulization composition comprising artemisinin or its derivatives thereof. The nebulization composition may be administered for the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a dry powder composition comprising artemisinin or its derivatives thereof. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nasal spray composition comprising artemisinin or its derivatives thereof. The nasal spray composition may be administered for the treatment of Coronavirus disease. In one embodiment, the inhalation composition is a propellant containing metered dose aerosol comprising artesunate. The aerosol composition may be administered for the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nebulization composition comprising artesunate. The nebulziation composition may be administered for the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a dry powder composition comprising artesunate. The dry powder inhalation composition may be administered for the treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nasal spray composition comprising artesunate. The nasal spray composition may be administered for the treatment of Coronavirus disease.

In one embodiment, the inhalation composition of the present invention is free of preservative.

The inhalation composition may contain about 0.01% w/w to about 90% w/w of artemisinin, such as from about 0.01% w/w to about 70% w/w, preferably about 10% w/w to about 50% w/w of artemisinin.

The inhalation composition may contain about 0.01% w/w to about 90% w/w of artesunate, such as from about 0.01% w/w to about 70% w/w, preferably about 10% w/w to about 50% w/w of artesunate.

In one more embodiment, the inhalation composition of the present invention provided herein has a long shelf life, i.e., it is stable during long term storage. The pharmaceutical composition or solution may contain greater than about 80%, such as greater than about 85%, greater than about 90%, greater than about 95% or greater than about 98% of the initial amount of artemisinin or its derivative in the inhalation composition after being stored for 3 or 6 months or 1, 2 or 3 years at 25° C.

In an embodiment, the inhalation composition of the present invention have a pH of about 1 about 9. For example, the preferred pH range of the inhalation composition is about 1 to about 9, preferably about 4 to about 8, more preferably about 5 to about 7.

Another embodiment is a premeasured, prepackaged, premixed inhalation composition. Preferably, the inhalation composition is a ready-to-use composition which does not require any mixing or dilution by the subject prior to administration. The inhalation composition may be administered for the treatment of Coronavirus disease.

Yet another embodiment is a method of administering artesunate or its derivatives thereof by inhalation for use in treatment of Coronavirus disease.

One more embodiment is a kit comprising a inhalation device, instructions for using the device and the container containing the inhalation compositions of the present invention.

Other objects, features and advantages of the present invention will be apparent to those of ordinary skill in the art in view of the following detailed description of the invention and accompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an inhalation composition comprising artemisinin or its derivatives to a patient (e.g., a human). The inhalation composition comprises artemsinin or its derivatives and pharmaceutically acceptable excipients. The inhalation composition may be a solution, suspension or a powder. The inhalation composition may be filled within a container suitable for inhalation. The inhalation composition may be administered for the treatment of Coronavirus disease. The present invention also relates to process of preparing the inhalation compositions.

The term "artemisinin or its derivatives thereof " includes "artesunate", "artemether", "dihydroartemisinin", "artemisone", "arteether", "artenimol", "artesunic acid", "artelinic acid", "deoxoaretmisinin", "artemotil", "artemiside" and their pharmaceutically acceptable salts, esters, solvates, hydrates, enantiomers, and polymorphs.

The term “inhalation” in the present context encompasses administration by inhalation route including oral and nasal inhalation route and includes compositions meant to be administered as dry powder inhalation, metered dose inhalation, nebulization, nasal spray, or insufflations.

Artemisinin

Artemisinin is a widely used drug for malaria infections due to Plasmodium falciparum. Artemisinin is chemically a sesquiterpene lactone containing a peroxide bridge. The structure of artemisinin is as given below:

Artemisinin derivatives such as "artesunate", "artemether", "dihydroartemisinin", "artemisone", "arteether", "artenimol", "artesunic acid", "artelinic acid", "deoxoaretmisinin", "artemotil", "artemiside" have also demonstrated enhanced efficacy in severe malaria infections. The chemical structures of the derivatives are as given below:

The inhalation composition may contain about 0.01% w/w to about 90% w/w of artemisinin, such as from about 0.01% w/w to about 70% w/w, preferably about

10% w/w to about 50% w/w of artemisinin.

The inhalation composition may contain about 0.01% w/w to about 90% w/w of artesunate, such as from about 0.01% w/w to about 70% w/w, preferably about 10% w/w to about 50% w/w of artesunate. The artemisinin or its derivatives may be in micronized form. Suitable micronisation techniques such as dry milling, wet milling, air jet milling, sieving, homogenizing using a homogenizer such as rotor-stator and/or high pressure homogenizer such as a microfluidizer can be used for micronisation of the artemisinin or derivatives thereof. Alternately, the artemisinin or its derivatives may be in unmicronized form.

Inhalation composition

The inhalation compositions of the present invention are formulated for intranasal or pulmonary delivery. For example, the inhalation compositions may be in the form of solutions, suspensions, drops, inhalation powder. The inhalation compositions may be administered by any conventional means, using a metered dose inhaler (MDI), a dry powder inhaler (DPI), a nebulizer, or a nasal spray device.

The inhalation compositions of the present invention comprise artemisinin or its derivatives thereof. Preferably, the inhalation composition of the present invention comprises artesunate and pharmaceutically acceptable excipient thereof. Suitable pharmaceutically acceptable excipients may include, but not limited to carrier, a solvent, a thickening agent, propellants, bulking agents, preservatives, a pH regulator, a tonicity agent, a complexing agent, or combinations thereof. The inhalation compositions can be used in the treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a single unit dose composition. In another embodiment, the inhalation composition is a multiple dose composition.

In an embodiment, the inhalation composition of the present invention have a pH of about 1 about 9. For example, the preferred pH range of the inhalation composition is about 1 to about 9, preferably about 4 to about 8, more preferably about 5 to about 7. The inhalation composition may contain about 0.01% w/w to about 90% w/w of artemisinin, such as from about 0.01% w/w to about 70% w/w, preferably about 10% w/w to about 50% w/w of artemisinin.

The inhalation composition may contain about 0.01% w/w to about 90% w/w of artesunate, such as from about 0.01% w/w to about 70% w/w, preferably about 10% w/w to about 50% w/w of artesunate.

The invention also relates to a kit comprising a inhalation device, instructions for using the device and the container containing the inhalation compositions of the present invention

The inhalation compositions of the present invention can be administered by a suitable inhalation device.

The term "Geometric Standard Deviation" is the geometric breadth of the best- fitted log-normal function to the particle size data.

The term "Mass Median Aerodynamic Diameter" is the median aerodynamic size of a plurality of particles, typically in a polydisperse population. The "aerodynamic diameter" is the diameter of a unit density sphere having the same settling velocity, generally in air, as a powder and is therefore a useful way to characterize an aerosolized powder or other dispersed particle or particle formulation in terms of its settling behavior. The aerodynamic diameter encompasses particle or particle shape, density, and physical size of the particle or particle. MMAD is determined herein by cascade impaction, unless the context indicates otherwise.

The term "Fine particle dose" is the dose, expressed in pg or the percentage of the total dose, of the aerosolized drug particles with an aerodynamic diameter < 5 micron. The term "Fine particle fraction" is the ratio of Fine particle dose to the total recovered dose.

The term "DIO" is the particle diameter value that 10% of the population of particles lies below.

The term "D50" is the particle diameter value that 50 % of the population lies below and 50% of the population lies above.

The term "D90" is the particle diameter value that 90 % of the population lies below.

The inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a D10 of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5.

The inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron.

The inhalation composition of the present invention exhibit a GSD of about 1 to about 5.

The fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Nebulization compositions

The inhalation composition of the present invention may be a nebulization composition. In one embodiment, the inhalation composition is a nebulization composition comprising artemisinin or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a nebulization composition comprising artesunate and pharmaceutically acceptable excipients. In one embodiment, the artemisinin or its derivative thereof is in dissolved form. In another embodiment, the artemisinin or its derivative thereof is in suspended form.

Suitable pharmaceutical excipients include, but are not limited to stabilizers, complexing agent, isotonic agent or tonicity adjusting agent, pH modifier, buffer, vehicle, and preservatives.

The stabilizers or complexing agents may comprise, but are not limited to, edetic acid (EDTA) or one of the known salts thereof, e.g. sodium EDTA or disodium EDTA dihydrate (sodium edetate), trisodium edetate, edetate calcium disodium edetic acid, citric acid or its hydrate salt, sodium metabisulfite, potassium metabisulfite, ascorbic acid, ascorbyl palmitate, alpha tocopherol, nitrilotri acetic acid, fumaric acid, malic acid, maltol, cyclodextrins such as sulfobutylether- 0- cyclodextrin (SBECD), pentetic acid and the salts or combinations thereof. The nebulization composition may contain from about 1% to about 50% of stabilizer or complexing agent.

Tonicity agents, that may be used, comprise sodium chloride, ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium edetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisultite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate. The tonicity agents are used in the nebulization composition in an amount from about 0.05% to 1.0 % w/w.

The pH may be adjusted by the addition of pharmacologically acceptable acids. Pharmacologically acceptable inorganic acids or organic acids may be used for this purpose. Examples of preferred inorganic acids are selected from the group consisting of hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid and phosphoric acid. Examples of particularly suitable organic acids are selected from the group consisting of ascorbic acid, citric acid, malic acid, tartaric acid, maleic acid, succinic acid, fumaric acid, acetic acid, formic acid and propionic acid and the like or combinations thereof.

Suitable surfactants that may be used include, but are not limited to, C5-20-fatty alcohols, C5-20-fatty acids, C5-20-fatty acid esters, lecithin, glycerides, propyleneglycol esters, polyoxyethylenes, polysorbates, sorbitan esters and/or carbohydrates. C5-20-fatty acids, propyleneglycol diesters and/or triglycerides and/or sorbitans of the C5-20-fatty acids are preferred, while oleic acid and sorbitan mono-, di- or trioleates are particularly preferred. The nebulization composition may contain about 0% to about 1% surfactant.

In one embodiment, the nebulization composition may be a liposome composition. The liposomal composition may be a ready to use liquid composition or may be a powder composition obtained by a lyophilization or spray drying process. Suitable lipids which may be used include, but are not limited to, neutral lipids, positively-charged lipids, negatively-charged lipids, amphoteric lipids such as phospholipids, and cholesterol are advantageously used. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine including, but limited to, l,2-diacyl-glycero-3- phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingogly colipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1,2-diacyl-sn- glycero-3 -phosphoethanolamine, including, but not limited to, 1,2- di oleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1,2-di stearoylphosphatidyl choline (DSPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), and 1,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated l,2-diacyl-sn-glycero-3 -phosphocholines, l-acyl-2-acyl-sn-glycero-3- phosphocholines, l,2-diheptanoyl-SN-glycero-3 -phosphocholine) derivatives of the lipids. The nebulization composition may contain about 0.01% w/w to about 2%w/w of lipids.

In another embodiment of the present invention, the nebulization composition has a pH from about 2.0 to about 6.0. In another embodiment, the composition has a pH from about 2.0 to about 4.0.

The nebulization composition may optionally include a buffer. General and biological buffers in the pH range of about 2.0 to about 8.0 include, but are not limited to, citric acid/phosphate, acetate, barbital, borate, Britton-Robinson, cacodylate, citrate, collidine, formate, maleate, Mcllvaine, phFosphate, Prideaux- Ward, succinate, citrate-phosphate-borate (Teorell-Stanhagen), veronal acetate, MES (2-(N-morpholino)ethanesulfonic acid), BIS-TRIS (bis(2- hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA (N-(2-acetamido)-2- iminodiacetic acid), ACES (N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)- 2-hydroxypropanesulfonic acid), BISTRIS PROPANE (1,3- bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (N-(2- hydroxyethyl)-piperazine-N'-(2-eth-anesulfonic acid), DIPSO (3-(N,N-bis(2- hydroxyethyl)amino)-2-hydroxypropan-esulfonic acid), MOBS (4-(N- morpholino)-butanesulfonic acid), TAPSO (3-(N- tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid), TRIZMA® (tris(hydroxymethylaminomethane), HEPPSO (N-(2-hydroxyethyl)piperazine-N'- (2-hydroxy-propanesulfonic acid), POPSO (piperazine-N,N'-bis(2- hydroxypropanesulfonic acid)), TEA (triethanolamine), EPPS (N-(2- hydroxyethylpiperazine-N'-(3-propanesulfon-ic acid), TRICINE (N-tris(hydroxy- methyl)methylglycine), GLY-GLY (glycylglycine), BICINE (N,N-bis(2- hydroxyethyl)glycine), HEPBS (N-(2 -hydroxy ethyl)piperazine-N'-(4- butanesulfonic acid)), TAPS (N-tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid), AMPD (2-amino-2-methyl- 1,3 -propanediol), and/or any other buffers known to those of skill in the art.

The preservative may comprise one or more of benzalkonium chloride, benzoic acid, benzoates such as sodium benzoate and the like or combinations thereof and such other preservatives which may be known to the person having a skill in the art.

The nebulization compositions of the present invention are formulated with a pharmacologically acceptable vehicle for the dissolution of the artemisinin or derivatives thereof to facilitate nebulization and delivery of the actives into the lungs of a patient. Pharmacologically suitable vehicles include, but are not limited to water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol and polyoxyethylene alcohols or combination thereof.

In another embodiment, the inhalation composition is a nebulization composition comprising artesunate, a complexing agent, optionally a surfactant, optionally a pH adjusting agent, and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising artesunate, sulfobutylether-P-cyclodextrin, Polysorbate 80, optionally a pH adjusting agent, and water. In one more embodiment, the nebulization composition is a lyophilized composition. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nebulization composition comprising artesunate, a buffer, optionally a pH adjusting agent, and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nebulization composition comprising artesunate, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous optionally a pH adjusting agent, and water. In one more embodiment, the nebulization composition is a lyophilized composition. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In an embodiment, the inhalation composition is a nebulization composition comprising artesunate, a lipid, a surfactant, and water. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

In one embodiment, the inhalation composition is a nebulization composition comprising artesunate, dipalmitoyl phosphatidylcholine, Polysorbate 80, and water. In one more embodiment, the nebulization composition is a dry composition obtained by spray drying. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease. In yet another embodiment, the inhalation composition is a nebulization liposomal composition comprising artesunate, dipalmitoyl phosphatidylcholine, cholesterol, Polysorbate 80, and water. In an embodiment, the nebulization composition is a lyophilized composition. One embodiment relates to the use of the nebulization composition for treatment of Coronavirus disease.

The present invention will now be explained with reference to the following nonlimiting examples.

Example 1

Manufacturing Process:

1. Sulfobutylether-P-cyclodextrin (SBECD), Polysorbate 80 and sodium chloride were dissolved in water for Injection in a suitable stainless-steel vessel and continuous mixing.

2. Artesunate was added and dissolved to the solution of step 1.

3. pH was adjusted using hydrochloric acid and or sodium hydroxide to the desired pH value. (5.0-6.0). Keep nitrogen flushing throughout the process.

4. Solution was filtered through 0.2p filter and filled into respules.

Example 2

Manufacturing Process:

1. Sulfobutylether-P-cyclodextrin (SBECD), Polysorbate 80 and sodium chloride were added and dissolved in Water for Injection in a suitable stainless-steel vessel under continuous mixing.

2. Artesunate was added and dissolved in the solution of step 1.

3. pH was checked and adjusted using hydrochloric acid and or sodium hydroxide to the desired pH value. (6.0-7.0).

4. Keep nitrogen flushing throughout the process.

5. The solution was filtered through 0.2p filter and vials were subjected to the lyophilization process.

6. The lyophilized vials were then sealed.

Example 3

Manufacturing Process

1. Sodium phosphate monobasic monohydrate and sodium phosphate dibasic anhydrous were added and dissolved in required quantity of water for injection by continuous overhead stirring.

2. Artesunate was added to the solution of step 2 and dissolved using continuous stirring.

3. pH of the solution was adjusted to 6.5 ± 0.5 using Orthophosphoric acid/ sodium hydroxide.

4. Volume make up was done using water for injection. The solution was then filtered under pressure using a 0.45-mm prefilter and 0.22-mm filter into a staging glass tank.

5. The required quantity of the filtered solution was then filled aseptically into respules.

Example 4

Manufacturing Process:

1. Artesunate, and Dipalmitoyl Phosphatidyl choline were added and dispersed in an aseptic manner in a mixture of Polysorbate 80 in Water for Injection in a suitable vessel. Nitrogen flushing is performed throughout the process.

2. The suspension of step (1) obtained was subjected to size reduction by homogenization to obtain a suspension of desired particle size range. 3. The suspension obtained in step (2) was subjected to spray drying process to remove the solvent.

4. The dried particles obtained were then filled in glass vials and sealed.

Example 5

Manufacturing process (aseptic processing)

1. Artesunate, Cholesterol and Dipalmitoyl phosphatidylcholine (DPPC) were dissolved in a mixture of chloroform and methanol and passed through 0.2p filter.

2. The solvent was then removed by evaporation and a thin film of lipids was obtained.

3. The lipid membrane was then hydrated with aqueous solution of polysorbate 80 in water for injection.

4. The resultant suspension was then heated and homogenized to obtain an emulsion.

5. The emulsion was then filled in a respules.

Example 6

Manufacturing process (aseptic processing)

1. Artesunate, Cholesterol and Dipalmitoyl phosphatidylcholine (DPPC) were dissolved in a mixture of chloroform and methanol and passed through 0.2p filter.

2. The solvent was then removed by evaporation and a thin film of lipids was obtained.

3. The lipid membrane was then hydrated with aqueous solution of polysorbate 80 in water for injection.

4. The resultant suspension was then heated and homogenized to obtain an emulsion.

5. The emulsion was then filled in a suitable vials, partially sealed and subjected to a lyophilization process to obtain a dry power. The vials were then sealed.

The nebulization composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a DIO of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Dry powder inhalation compositions

The inhalation composition of the present invention may be a dry powder inhalation composition. In one embodiment, the inhalation composition is a dry powder inhalation composition comprising artemisinin or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a dry powder inhalation composition comprising artesunate and pharmaceutically acceptable excipients.

In one embodiment, the dry powder inhalation composition is a single unit dose composition. Such a composition may be packaged into capsules. In another embodiment, the dry powder inhalation composition is a multiple dose composition. Such a composition may be packaged into blisters.

Example of a suitable excipient includes a bulking agent. Such bulking agents may include, but are not limited to, lactose, mannitol, trehalose, raffinose, and maltodextrins. In some embodiments, it may be desirable to include a bulking agent to improve the physical stability of the composition. Furthermore, in some embodiments, the bulking agent may also improve the chemical stability of the inhalation composition. Other additives known to those of ordinary skill in the art may also be included. Generally, bulking agents suitable for use may be included in an amount of about 80% or less by weight of the composition, about 50% or less by weight of the composition, 30% or less by weight of the composition, or 10% or less by weight of the composition.

The dry powder compositions may further comprise pharmaceutically acceptable excipients, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium chloride, potassium chloride, calcium chloride, sodium acetate, sodium lactate, etc. In another embodiment, the inhalation composition is a dry powder inhalation composition comprising artesunate, and a bulking agent.

In one embodiment, the inhalation composition is a dry powder inhalation composition comprising artesunate and lactose monohydrate. One embodiment relates to the use of the dry powder composition for treatment of Coronavirus disease.

The present invention will now be explained with reference to the following nonlimiting examples.

Manufacturing process (aseptic processing)

1. Micronized artesunate and Lactose monohydrate were sifted through a suitable mesh are blended in a suitable blender.

2. The blend was filled in capsules of suitable size and packed in a blister.

The dry powder inhalation composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Propellant containing metered dose aerosols The inhalation composition of the present invention may be a metered dose inhalation composition. In one embodiment, the inhalation composition is a metered dose inhalation composition comprising artemisinin or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a metered dose inhalation composition comprising artesunate and pharmaceutically acceptable excipients.

Suitable pharmaceutical excipients include, but are not limited to propellant, a cosolvent, a surfactant, a buffer/pH adjusting agent, a preservative, a complexing agent, antioxidants or combinations thereof.

Suitable propellants include hydrocarbons such as n-propane, n-butane or isobutane or mixtures of two or more such hydrocarbons such as monofluorotrichloromethane, dichlorodifluoromethane and halogen-substituted hydrocarbons, for example fluorine-substituted methanes, ethanes, propanes, butanes, cyclopropanes or cyclobutanes, particularly 1,1,1,2-tetrafluoroethane (HFA134a) and 1,1,1,2,3,3,3-heptafhioropropane (HFA227) or mixtures of two or more such halogen- substituted hydrocarbons. In one embodiment, the artemisinin or its derivative thereof is in dissolved form. In another embodiment, the artemisinin or its derivative thereof is in suspended form. The metered dose inhalation composition may contain propellant in an amount to sufficiently propel plurality of doses.

Examples of suitable cosolvents include alcohols, ethers, and glycols. Preferably, the cosolvent is a short chain polar alcohol. More preferably, the cosolvent is an aliphatic alcohol having from one to six carbon atoms, such as ethanol or isopropanol. Examples of suitable ethers include dimethyl ether and diethyl ether. The metered dose inhalation composition may contain about 0.1% w/w to about 10%w/w of co-solvents.

The surfactants may be selected from oils such as corn oil, olive oil, cottonseed oil & sunflower oil, mineral oil like liquid paraffin, oleic acid, phospholipids such as lecithin and citric acid, sorbitan trioleate, glycerol, glycol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyethylene glycol, propoxyiated polyethylene glycol, polyoxyethylene lauryl ether, and purified diethylene glycol monoethyl ether. The metered dose inhalation composition may contain about 0.1% w/w to about 10% w/w of surfactant.

Suitable buffers include, but not limited to, acetate, citrate, glutamate, phosphate, benzoate, lactate, ascorbate, tartarate, succinate, glycine, triethanolamine, diethanolamine, tromethamine or suitable mixture thereof.

The inhalation composition of the present invention may contain a preservative. Suitable preservatives include, but not limited to benzalkonium chloride or benzoates such as sodium benzoate, sorbic acid or sorbates such as potassium sorbates and the like.

Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof..

Suitable antioxidants which may be used in the inhalation compositions of the invention, include, but are not limited to, glycine, a-tocopherol, a-tocopherol Polyethylene Glycol Succinate (Vitamin E TPGS), ascorbic acid, propyl gallate, Butylated Hydroxy Anisole (BHA), Butylated Hydroxy Toluene (BHT),

In one embodiment, the inhalation composition is a metered dose inhalation composition comprising artemisinin or its derivatives thereof, a propellant, a cosolvent, and optionally a surfactant. The inhalation composition is used in the treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a metered dose inhalation composition comprising artemisinin or its derivatives thereof, HFA-134a (1,1, 1,2- tetrafluoroethane), and ethanol. The inhalation composition is used in the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a metered dose inhalation composition comprising artesunate, a propellant, a co-solvent, and optionally a surfactant. The inhalation composition is used in the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a metered dose inhalation composition comprising artesunate, HFA-134a (1,1,1,2-tetrafluoroethane), and ethanol. The inhalation composition is used in the treatment of Coronavirus disease.

The metered dose inhalation composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a DIO of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Nasal spray compositions

The inhalation composition of the present invention may be a nasal spray composition. In one embodiment, the inhalation composition is a nasal spray composition comprising artemisinin or its derivatives thereof and pharmaceutically acceptable excipients. In another embodiment, the inhalation composition is a nasal spray composition comprising artesunate and pharmaceutically acceptable excipients. In one embodiment, the artemisinin or its derivative thereof is in dissolved form. In another embodiment, the artemisinin or its derivative thereof is in suspended form.

Suitable pharmaceutical excipients include, but are not limited to vehicle, preservative, suspending agent, wetting agent, complexing agent tonicity agent.

Pharmacologically suitable vehicle for use herein include, but are not limited to, polar solvents, or compounds that contain hydroxyl groups or other polar groups. Solvents include water or alcohols, such as ethanol, isopropanol, and glycols including propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether, glycerol and polyoxyethylene alcohols. Polar solvents also include protic solvents, including, but not limited to, water, aqueous saline solutions with one or more pharmaceutically acceptable salt(s), alcohols, glycols or a mixture thereof.

The nasal spray composition may optionally include a buffer. General and biological buffers in the pH range of about 2.0 to about 8.0 include, but are not limited to, citric acid/phosphate, acetate, barbital, borate, Britton-Robinson, cacodylate, citrate, collidine, formate, maleate, Mcllvaine, phosphate, Prideaux- Ward, succinate, citrate-phosphate-borate (Teorell-Stanhagen), veronal acetate, MES (2-(N-morpholino)ethanesulfonic acid), BIS-TRIS (bis(2- hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA (N-(2-acetamido)-2- iminodiacetic acid), ACES (N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MOPSO (3-(N-morpholino)- 2-hydroxypropanesulfonic acid), BISTRIS PROPANE (1,3- bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-hydroxyethyl)-2- aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES (N-(2- hydroxyethyl)-piperazine-N'-(2-eth-anesulfonic acid), DIPSO (3-(N,N-bis(2- hydroxyethyl)amino)-2-hydroxypropan-esulfonic acid), MOBS (4-(N- morpholino)-butanesulfonic acid), TAPSO (3-(N- tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid), TRIZMA® (tris(hydroxymethylaminomethane), HEPPSO (N-(2-hydroxyethyl)piperazine-N'- (2-hydroxy-propanesulfonic acid), POPSO (piperazine-N,N'-bis(2- hydroxypropanesulfonic acid)), TEA (triethanolamine), EPPS (N-(2- hydroxyethylpiperazine-N'-(3-propanesulfon-ic acid), TRICINE (N-tris(hydroxy- methyl)methylglycine), GLY-GLY (glycylglycine), BICINE (N,N-bis(2- hydroxyethyl)glycine), HEPBS (N-(2 -hydroxy ethyl)piperazine-N'-(4- butanesulfonic acid)), TAPS (N-tris(hydroxymethyl)methyl-3- aminopropanesulfonic acid), AMPD (2-amino-2-methyl- 1,3 -propanediol), and/or any other buffers known to those of skill in the art.

Preservatives include, but are not limited to, phenylethyl alcohol, benzalkonium chloride or benzoic acid, or benzoates such as sodium benzoate and phenylethyl alcohol. The nasal spray composition may contain about 0.005% w/w to about 0.2% w/w of a preservative.

Suspending agents which can be used in the nasal spray composition of the present invention include, but are not limited to polyoxyethylene sorbitan fatty esters or polysorbates, including, but not limited to, polyethylene sorbitan monooleate (Polysorbate 80), polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 65 (polyoxyethylene (20) sorbitan tristearate), polyoxyethylene (20) sorbitan mono-oleate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate; lecithins; alginic acid; sodium alginate; potassium alginate; ammonium alginate; calcium alginate; propane- 1,2-diol alginate; agar; carrageenan; locust bean gum; guar gum; tragacanth; acacia; xanthan gum; karaya gum; pectin; amidated pectin; ammonium phosphatides; microcrystalline cellulose; methylcellulose; hydroxypropylcellulose; hydroxypropylmethylcellulose; ethylmethylcellulose; carboxymethylcellulose; sodium, potassium and calcium salts of fatty acids; mono-and di-glycerides of fatty acids; acetic acid esters of mono- and di- glycerides of faty acids; lactic acid esters of mono-and di-glycerides of fatty acids; citric acid esters of mono-and di-glycerides of fatty acids; tartaric acid esters of mono-and di-glycerides of fatty acids; mono-and diacetyltartaric acid esters of mono-and di-glycerides of fatty acids; mixed acetic and tartaric acid esters of mono-and di-glycerides of fatty acids; sucrose esters of fatty acids; sucroglycerides; polyglycerol esters of fatty acids; polyglycerol esters of polycondensed fatty acids of castor oil; propane- 1,2-diol esters of faty acids; sodium stearoyl-21actylate; calcium stearoyl-2-lactylate; stearoyl tartrate; sorbitan monostearate; sorbitan tristearate; sorbitan monolaurate; sorbitan monooleate; sorbitan monopalmitate; extract of quillaia; polyglycerol esters of dimerised fatty acids of soya bean oil; oxidatively polymerised soya bean oil; and pectin extract. In some embodiments, the nasal spray formulations comprise polysorbate 80, microcrystalline cellulose, carboxymethylcellulose sodium and/or dextrose. The nasal spray composition may contain about 0.01% w/w to about 5% of suspending agent.

Suitable surfactants that may be used include, but are not limited to, C5-20-fatty alcohols, C5-20-fatty acids, C5-20-fatty acid esters, lecithin, glycerides, propyleneglycol esters, polyoxyethylenes, polysorbates, sorbitan esters and/or carbohydrates. C5-20-fatty acids, propyleneglycol diesters and/or triglycerides and/or sorbitans of the C5-20-fatty acids are preferred, while oleic acid and sorbitan mono-, di- or trioleates are particularly preferred. The nasal spray composition may contain about 0% to about 1% surfactant.

Complexing agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as the disodium salt, citric acid, nitrilotriacetic acid and the salts thereof. The nasal spray composition may contain about 0.0005%w/w to about 0.5% w/w of complexing agent.

Tonicity agents, that may be used, comprise sodium chloride, ammonium carbonate, ammonium chloride, ammonium lactate, ammonium nitrate, ammonium phosphate, ammonium sulfate, ascorbic acid, bismuth sodium tartrate, boric acid, calcium chloride, calcium disodium edetate, calcium gluconate, calcium lactate, citric acid, dextrose, diethanolamine, dimethylsulfoxide, edetate disodium, edetate trisodium monohydrate, fluorescein sodium, fructose, galactose, glycerin, lactic acid, lactose, magnesium chloride, magnesium sulfate, mannitol, polyethylene glycol, potassium acetate, potassium chlorate, potassium chloride, potassium iodide, potassium nitrate, potassium phosphate, potassium sulfate, propylene glycol, silver nitrate, sodium acetate, sodium bicarbonate, sodium biphosphate, sodium bisulfite, sodium borate, sodium bromide, sodium cacodylate, sodium carbonate, sodium citrate, sodium iodide, sodium lactate, sodium metabisulfite, sodium nitrate, sodium nitrite, sodium phosphate, sodium propionate, sodium succinate, sodium sulfate, sodium sulfite, sodium tartrate, sodium thiosulfate, sorbitol, sucrose, tartaric acid, triethanolamine, urea, urethan, uridine and zinc sulfate.

In one embodiment, the inhalation composition is a nasal spray composition comprising artemisinin or its derivatives thereof, suspending agent, preservative, tonicity adjusting agent, complexing agent, buffer, surfactant and water. The inhalation composition is used in the treatment of Coronavirus disease.

In a further embodiment, the inhalation composition is a nasal spray composition comprising artemisinin or its derivatives thereof, xanthan gum, benzalkonium chloride, sodium chloride, disodium EDTA, phosphate buffer, Polysorbate 80 and water. The inhalation composition is used in the treatment of Coronavirus disease.

In another embodiment, the inhalation composition is a nasal spray composition comprising artesunate, suspending agent, preservative, tonicity adjusting agent, complexing agent, buffer, surfactant and water. The inhalation composition is used in the treatment of Coronavirus disease.

In one more embodiment, the inhalation composition is a nasal spray composition comprising artesunate, xanthan gum, benzalkonium chloride, sodium chloride, disodium EDTA, phosphate buffer, Polysorbate 80 and water. The inhalation composition is used in the treatment of Coronavirus disease.

The nasal spray composition can be administered by suitable devices commonly known in the art.

In one embodiment, the inhalation composition when administered from an inhalation device exhibits a droplet size distribution comprising a DIO of 5-30 microns, a D50 of 20-60 microns, a D90 of 40-150 microns, a SPAN of not more than 5. In another embodiment, the inhalation composition of the present invention exhibit a MMAD of below about 10 micron, between about 2 micron to 10 micron. In a further embodiment, the inhalation composition of the present invention exhibit a GSD of about 1 to about 5. In a further embodiment, the fine particle fraction (FPF) obtained by administering the inhalation composition may be about 10% to about 80%.

Method of treatment

The inhalation compositions comprising artemisinin or derivatives thereof can be used for the treatment of Coronavirus disease. The present invention further relates to method of treating Coronavirus disease by administering artemisinin or derivatives thereof. In one embodiment, the inhalation composition is a metered dose composition. In another embodiment, the inhalation composition is a dry powder composition. In one more embodiment, the inhalation composition is a nebulization composition. In a further embodiment, the inhalation composition is a nasal spray composition.

The inhalation compositions of the present invention comprising artesunate can be used for the treatment of Coronavirus disease. In one embodiment, the inhalation composition is a metered dose composition. In another embodiment, the inhalation composition is a dry powder composition. In one more embodiment, the inhalation composition is a nebulization composition. In a further embodiment, the inhalation composition is a nasal spray composition.

Pre-clinical studies:

Rabbit studies comparing the pharmacokinetic profile of intra-nasal Artesunate vs I.V Artesunate.

The objective of this study was to assess the pharmacokinetic as well as brain penetration profile of Artesunate formulations (solution and powder) in male New Zealand White Rabbits following single intravenous injection (IV) and intranasal administration (IN) at dose of 6.25 mg/kg body weight.

Method:

The pharmacokinetics and brain penetration of Artesunate was assessed by intravenous and intranasal administration at a dose of 6.25 mg/kg body weight using three male New Zealand White Rabbits in each group. For pharmacokinetic and brain penetration profile, rabbits from group 1 and 3 were dosed intravenously with artesunate solution while rabbits from group 2 and 4 were dosed intranasally with artesunate powder.

Administration of Test Formulations:

Rabbits from group 1 and group 3 were received intravenous injection of test item solution through marginal ear vein. Group 2 and group 4 animals were received the test item formulation (powder) through intranasal route. The powder formulation was intranasally administered using an indigenously made experimental device.

Blood Sample Collection for Pharmacokinetic Study:

Approximately 0.5 mL of blood sample from each rabbit from all dose groups was collected from the marginal ear vein into centrifuge tubes containing saturated solution of Sodium fluoride/ Potassium oxalate (30pL) at predetermined time points. Plasma was separated by centrifuging blood samples at 5000 rpm for 10 min at 4 °C. The plasma samples were labeled and stored at -70 °C. The labels included the details such as study number, dose group, animal number, time point, type of sample and day of sampling. All the samples were analyzed by a fit-for- purpose LC-MS/MS method.

Serial Blood PK Sampling at Predetermined Time Points from Each Animal Following Single Intravenous or Intranasal Dose Administration:

Blood, CSF and Brain Sample Collection for Brain Penetration Study:

Blood was collected; plasma was separated and stored at pre-defined time points. Immediately after blood collection, rabbits were anesthetized using isoflurane and CSF was collected at pre-defined time points by percutaneous cisternal puncture into pre-labeled tubes and stored at -70 °C. The rabbits were sacrificed immediately by inhalation anesthesia overdose. Brain was removed as quickly as possible and rinsed with PBS to remove superficial blood and CSF. After weighing, the brain was placed in a 50 mL centrifuge tube containing PBS with saturated solution of Sodium fluoride/ Potassium oxalate (60pL/mL of PBS) required for preparing 20% homogenate. The homogenate was then centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant homogenate samples were collected, labeled and stored at -70 °C. All the plasma, CSF and homogenate samples were analyzed by a fit-for-purpose LC-MS/MS method.

Blood, CSF and Brain Collection at Predetermined Time Points from Each Animal Following Single Intravenous or Intranasal Dose Administration:

Bio Analysis:

All rabbit plasma, CSF and brain tissue samples throughout the study were initially stored at -70 °C. Sample analysis for the test item was carried out using fit for research LC-MS/MS method.

Pharmacokinetic Parameter Evaluation:

Based on the individual plasma concentration pharmacokinetic parameters were calculated by non-compartmental analysis by using Phoenix TM WinNonlin®Ent- Version 6.3 (Pharsight Corporation, USA). Mean plasma concentrations (N=3) were used to determine the pharmacokinetic parameters. Pharmacokinetic parameters including Peak plasma concentration (Cmax), time to reach the peak plasma concentration (Tmax), Half-life (ti/2), AUCo-t was evaluated (Appendix- VII). For brain penetration study samples i.e. plasma, CSF and brain tissue samples, AUCo-twas calculated.

Following is the summary of plasma pharmacokinetic parameters in G1 and G2:

It was found that the plasma PK parameters of intravenous and intra nasal administered Artesunate are identical. A high degree of similarity observed between the CSF levels and brain penetration in the IV and intra nasal route of administration.

Artemisinin and Artesunate have been also been evaluated for In vitro cytotoxicity studies, permeability studies as well as the cellular uptake studies, as given below:

In vitro cytotoxicity studies for Artemisinin and Artesunate in human nasal epithelial (RPMI2650) cells and human alveolar epithelial (A549) cells Material and Method

Reagents

Cell line: A549

Cell type: Lung epithelial cells

Reviving Passage no: 44

Media: MEM Alpha media with 10% FBS (complete media)

Cell line: RPMI 2650 Cell type: Nasal epithelial cell

Reviving Passage no: 20

Media: MEM Alpha media with 10% FBS

Identification: MEM Alpha media

Supplier: Gibco

Batch No.: 2217403

Physical Description: Pink colored solution

Storage Conditions: 4-8°C

Identification: Fetal Bovine serum

Supplier: Gibco

Batch No.: 42F1383K

Physical Description: Yellowish brown solution

Storage Conditions: 4-8°C

Identification: Trypsin

Supplier: Hi media

Batch No.: 0000381648

Physical Description: Pink colored solution

Storage Conditions: 4-8°C

Identification: Antibiotic Antimycotic solution

Supplier: Hi media

Batch No.: 0000360171

Physical Description: Colourless liquid

Storage Conditions: 4-8°C

Methods: Cell proliferation assay- A549 and RPMI2650 cells were seeded in 96-well plate at a cell density of 0.5xl0 ⁶ cells per plate. After seeding the plates were cultured overnight at 37 °C, 5 % CO2. 24hr post seeding cytotoxicity was assessed by using 300,100, 70, 30, 10, 1 and 0.1 pMol/L Artemisinin and Artesunate. Cells were incubated for 24h and 48h timepoint. At the end of incubation period, MTS reagent was added (20 pL per well) and absorbance was measured at 460 nm. Data was acquired using Spectramax microplate reader (Molecular devices, CA, USA). Data was analysed to determine the % proliferation rate at each time point and concentration tested.

Stock preparation:

100 mM stock was prepared in DMSO, as shown below in table.1. Working stock was prepared in complete media. 10X working stocks are prepared and serial dilutions is performed. 20 ul/well of each lOx working stock was added in respective wells giving the desired concentrations, as mentioned in the method section.

Results:

In alveolar derived epithelial cells, Artemisinin demonstrated less toxicity of 8- 13% cytotoxicity at the highest test concentration of 300 pM. Interestingly, Artesunate showed ~50 % inhibition at 300 pM and 100% proliferation starting from 100 pM at 24h timepoint. However, this effect was further increased at 48hr timepoint with ~50 % inhibition observed at 100 pM and 73 - 88% proliferation at remaining range of concentration in the below table. Remarkably, in nasal derived cell line RPMI 2650, Artemisinin and Artesunate showed less cytotoxic effect. Artemisinin showed same response with cytotoxic effect while Artesunate induced 8-22% cytotoxicity at 24hr and 48hr timepoint, respectively. % proliferation observed in A549 treated with Artemisinin and Artesunate

% proliferation observed in RPMI2650 treated with Artemisinin and Artesunate

Using the alveolar and nasal derived epithelia cells, Artemisinin has nominal cytotoxic effect at highest test concentration of 300 pM while artesunate had safety tested till 100 pM in lung derived cells and >300 M in nasal cells. Thus higher safety window for using higher concentration that can easily affect the virus clinically.

Thus the tested concentration is much higher than the effect dose of the drug which are safe enough to be effective, to be use in vitro studies. .Also, the permeability data for Artemisinin and its derivative showed Papp value range above 200 nm/s indicating high permeability for the drug using Caco-2 cells. This itself will validate the use of these drugs which will have increase bioavailability which will tested in-house.

Permeability study using bronchial epithelial cells or nasal derived cells:

Cell line: A549, Calu-3 and RPMI 2650

Cell density: 2.5 x 10 ⁵ cells/mL per well (n=2/ concentration)

Compounds: Artemisinin and Artesunate

Drug concentration: pM and - pM

Incubation: 0 min, 5min, 15 min, 30 min, 45min, 60min, 90min & 120min

1. Alveolar and bronchial epithelial cell lines were cultured in freshly prepared MEM media containing NEAA along with 1 mM sodium pyruvate, and 2 mM L-Glutamine. For 50 mL of complete culture media, 5 mL of filter sterile fetal bovine serum, 100 pL Antibiotic solution in 45 mL MEM and filter sterilized using 0.2-micron filter and sterile 20 mL syringe was added.

2. Cells were seeded using 6-well transwell culture insert having a surface area of 4.5 cm ² , at the density of 2xl0 ⁵ cells/ml.

3. 1 ml cell suspension containing 2xl0 ⁵ cells/ml to 2.5xl0 ⁵ cells/ml was seeded to the apical (A) side of the hanging cell culture inserts and 2 ml of complete media is added to all the basal (B) wells. For a 24 well culture inserts (having a surface area of 0.3 cm ² ) 400 pL of cell suspension was seeded to the apical and 800 pL of complete media to all the basal wells.

4. Plates were incubated and maintained at 37 °C, 5 % CO2 till the day of the assay (7 - 12 days) with alternate day media change, from the basal side post 24 hrs of seeding while apical side is kept empty. On the day of the assay, fresh buffer was prepared as follows: Modified HBSS buffer with 10 mM HEPES (i.e. 1191.1 mg of HEPES in 500 mL of commercially available HBSS), pH to 7.4 (± 0.05). Monolayer integrity was tested by using the Trans epithelial electrical resistance (TEER) before initiating the assay by immersing electrodes in each well of the 6 well or 24 well plate so that the shorter arm is dipped into the apical side and the longer arm into the basal side of the well. Wells with TEER values > 300 ohms. cm ² are used for the assay. TEER values of bronchial epithelial cells, in general, are achieved above 300 Q*cm ² from day 10 and sustain at 400 Q*cm ² till day 12. Thus, the assay is scheduled in between day 7 to day 12 post seeding. Unidirectional assay (direct drug dosing) is performed after taking the TEER readings. For 6 well culture inserts 1 ml of HBSS buffer is added to the apical side and 2 ml to the basal side of the wells or add 400 pL of HBSS buffer to the apical side of the wells and 800 pL to the basal side if 24 well cell culture inserts are used. 1ml or 450 pL of the desired concentrations of the test compounds (Artemisinin and Artesunate) in HBSS buffer (pH 7.4 ) were added to the respective assigned wells while 2 ml or 800 pL of HBSS buffer to the basal side of all the wells to initiate the assay. 50 pL sample from the apical side as a 0 min sample (A0) was collected as a mass balance sample This is replaced with 50 pL of HBSS buffer (pH 7.4) followed which the plate was transferred on an incubator shaker set at 70-90 rpm at 37°C in between each timepoints and sample collection. In following time points, 100 pL samples from the basal side at different time points was collected till the final sample collection timepoint is reached, eg. 120 min. At every time point after collecting the sample the well was immediately replenished with 100 pL of HBSS buffer. 13. After collecting the last time point sample, 50 pL sample from the apical side (A120) of the wells was collected as a mass balance sample.

14. At the end of the assay, TEER value is re-captured to confirm if the cell layer integrity is retained throughout the course of the experiment along with Sodium Fluorescence (Na Flu) permeability.

15. The test drug (Artemisinin and Artesunate) will be considered as a high permeable or low permeable depending upon the Papp calculated, using the formula as mentioned. Papp formula: Papp = (dQ/dt)*VR/(A*C0) Where, dQ/dt is the cumulative amount in the receiver compartment versus time in pmoles/s, VR is the volume in receiver well, A is the area of the cell monolayer, CO is the initial concentration of the dosing solution.

The permeability data with an Papp value range above 200 nm/s indicating high permeability for the drug using Caco-2 cells can be implicit, thus validating the use of these drugs with increase bioavailability for elucidating the therapeutic effect against the coronavirus.

Intracellular cellular uptake assay

Cell line: A549 and RPMI 2650

Cell density: 0.5 million per well

Compounds: Artemisinin and Artesunate

Drug concentration: pM and - pM

Incubation: 15 min, 30 min, Ihr, 2hr & 4hr post addition for all the timepoints. The plates to be incubated at 37°C, 5% CO2 incubator.

Study protocol: a) A549 and RPMI 2650 cells were seeded in a 6-well plate at a density of 0.5-1 x 10 ⁶ cells / mL. Post seeding the cells were incubated overnight at 37°C, 5% CO ₂ . b) After incubation, media from the wells was removed and replaced with fresh media containing various concentrations of Artemisinin and Artesunate. c) The cells were incubated with the drugs for different time points raining from 15 mins to 24 hrs. d) After incubation the supernatant from individual wells was collected in 2 mL tubes and centrifuged. After centrifugation the supernatant collected and stored till quantification for determining the extracellular drug concentrtion. e) For intracellular levels detection, cells were scraped using a cell scraper from all wells. This cell suspension from individual wells were collected in labelled 2 mL tubes and centrifuged at 500g for 10 mins. f) Supernatant was discarded and 500 pL of ice cold acidic methanol was added to the pellet obtained in all the tubes followed by incubation for 10- 15 minutes for cell lysis. g) At the end of the incubation time, the tubes were centrifuged at 500g for 20 mins, supernatant was colected and stored at - 80°C., till further analysis h) The above procedure was repeated for all the mentioned timepoints.

Based on the preliminary invitro data it is clearly suggestive that the working concentration is likely to be achieve the intracellular levels, which are therapeutic concentration that are inhibitory for virus infection and efficiently sensitive towards coronavirus.

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and application of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as described.