AMINOQUINOLINES FOR TREATING CORONAVIRUS INFECTIONS

The present invention relates to methods for the treatment of coronaviruses, especially to SARS-CoV-2, and to compositions and compounds for use in such methods.

Background

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease was first identified in December 2019 in Wuhan, the capital of China's Hubei province, and has since spread globally, resulting in the ongoing 2019-20 coronavirus pandemic. Common symptoms include fever, cough, and shortness of breath. Other symptoms may include muscle pain, sputum production, diarrhoea, sore throat, loss of smell, and abdominal pain. While the majority of cases result in mild symptoms, some progress to viral pneumonia and multi organ failure. As of 4 April 2020, more than 1 ,140,000 cases of have been reported in more than two hundred countries and territories, resulting in over 60,800 deaths. There are presently no known therapies that are effective against SARS-Cov-2, and no vaccine is available. Accordingly, there is an urgent need for antiviral agents that can control or prevent SARS-Cov-2 in infected individuals, and that can prevent SARS-Cov-2 from spreading.

The primary way that SARS-Cov-2 appears to spread is by close person-to-person contact. Most cases of SARS-Cov-2 have involved people who cared for or lived with someone with SARS-Cov-2, or had direct contact with infectious material (for example, respiratory secretions) from a person who has SARS-Cov-2. Potential ways in which SARS- Cov-2 can be spread include touching the skin of other people or objects that are contaminated with infectious droplets followed by touching of eye(s), nose, or mouth. This can happen when someone who is sick with SARS-Cov-2 coughs or sneezes droplets onto themselves, other people, or nearby surfaces. It also is possible that SARS-Cov-2 can be spread more broadly through the air or by other ways that are currently not known.

The lungs are the organs most affected by SARS-Cov-2 because the virus accesses host cells via the enzyme ACE2, which is most abundant in the type II alveolar cells of the lungs. The virus uses a special surface glycoprotein called a "spike" (peplomer) to connect to ACE2 and enter the host cell. The density of ACE2 in each tissue correlates with the severity of the disease in that tissue and some have suggested that decreasing ACE2 activity might be protective, though another view is that increasing ACE2 using angiotensin II receptor blocker medications could be protective and that these hypotheses need to be tested. As the alveolar disease progresses, respiratory failure might develop and death may follow.

Summary of the Invention

In a first embodiment, the invention provides a method for treating a coronavirus infection in an animal comprising administering to the lungs of an animal an effective amount of an anti-malarial compound.

In a second embodiment, the invention provides a kit comprising an anti-malarial compound in combination with a device that effects local administration of said anti-malarial agent. In a third embodiment, the invention provides an anti-malarial compound for use in a method for treating a coronavirus infection in an animal comprising administering to the lungs of an animal an anti-inflammatory effective amount of such a compound.

Brief Description of the Figures

Figure 1 is a graph. Figure 2 is a graph.

Detailed Description of the Preferred Embodiments

Accordingly, the present invention relates generally to the treatment of a coronavirus infection by administration to the lungs of an effective amount of an anti-malarial agent. By anti-malarial, as used herein, it is meant that the drug has been historically belonged to the class of drugs known as anti-malarials. Preferred antimalarials include aminoquinolines especially 8 and 4-aminoquinolines, acridines, e.g., 9-amino acridines and quinoline methanols, e.g., 4-quinolinemethanols.

Compounds used in the Invention

Compounds suitable for the present invention are anti-malarial agents that have immunomodulatory and anti-inflammatory effects. Anti-malarial agents are well known in the art. Examples of anti-malarial agents can be found, for example, in GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, chapters 45-47, pages 1029-65 (MacMillan Publishing Co. 1985), hereby incorporated by reference. The preferred anti-malarial compounds are quinine based or are aminoquinolines, especially 4- and 8-amino quinolines. An especially preferred class of antimalarials has a core quinoline structure (examples are mefloquine and quinine) which is usually substituted at one or more positions, typically at least at the 4- an d/or 8-positions. One skilled in the art would understand that such agents could be administered in derivatized forms, such as pharmaceutically acceptable salts, or in a form that improves their pharmacodynamic profiles, such as esterification of acid or alcohol substituents with lower alkyls (e.g., C1-6) or lower alkanoyloxy , respectively, wherein R ₂ o is lower alkyl. Another class of antimalarials, exemplified by quinacrine, is based on an acridine ring structure, and may be substituted in the manner described above.

Especially preferred compounds for use in the present invention are aminoquinolines, including 4-amino and 8-aminoquinolines and their derivatives (collectively, "aminoquinoline derivatives") and aminoacridines, especially 9-amino acridines. The preferred 4- and 8 aminoquinolines and 9-amino acridines are described by the following formula: or pharmaceutically acceptable salts thereof, wherein R ₂ and R ₃ are independently hydrogen, or lower alkyl or R ₂ and R ₃ taken together with the carbon atoms to which they are attached form an aryl ring, which ring may be unsubstituted or substituted with an electron withdrawing group or an electron donating group, one of Ri and Ri2 is NHR _I3 while the other is hydrogen; Ri ₃ is

R15 is

R ₇

(Rg) (CH ₂ ) _n1 N

R ₈

R _4J R _IO , R _{I I} and R _I4 are independently hydrogen or an electron donating group or electron withdrawing group;

R ₅ and Re, are independently hydrogen or lower alkyl which may be unsubstituted or substituted with an electron withdrawing or electron donating group;

Ry and Re are independently hydrogen or lower alkyl, which may be unsubstituted or substituted with an electron withdrawing or electron donating group; Ar is aryl having 6-18 ring carbon atoms;

Rg is hydrogen or hydroxy or lower alkoxy or

O

OCR25 j

R25 is lower alkyl or hydrogen; and n and ni are independently 1-6.

As used herein, the terms "electron donating groups" and "electron withdrawing groups" refer to the ability of a substituent to donate or withdraw an electron relative to that of hydrogen if the hydrogen atom occupied the same position in the molecule. These terms are well understood by one skilled in the art and are discussed in Advanced Organic Chemistry, by J. March, John Wiley & Sons, New York, NY, pp. 16-18 (1985) and the discussion therein is incorporated herein by reference. Electron withdrawing groups include halo, including bromo, fluoro, chloro, iodo and the like; nitro; carboxy; carbalkoxy; lower alkenyl; lower alkynyl; formyl; carboamido; aryl; quaternary ammonium compounds, and the like. Electron donating groups include such groups as hydroxy; lower alkoxy; including methoxy; ethoxy and the like; lower alkyl, such as methyl; ethyl, and the like.; amino; lower alkylamino; di-loweralkylamino; aryloxy, such as phenoxy and the like; arylalkoxy, such as benzyl and the like; mercapto, alkylthio, and the like. One skilled in the art will appreciate that the aforesaid substituent may have electron donating or electron withdrawing properties under different chemical conditions.

The term alkyl, when used alone or in conjunction with other groups, refers to an alkyl group containing one to six carbon atoms. It may be straight-chained or branched. Examples include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl and the like.

Lower alkoxy refers to an alkyl group which is attached to the main chain by an oxygen bridging atom. Examples include methoxy, ethoxy, and the like.

Lower alkenyl is an alkenyl group containing from 2 to 6 carbon atoms and at least one double bond. These groups may be straight chained or branched and may be in the Z or E form. Such groups include vinyl, propenyl, 1 -butenyl, isobutenyl, 2-butenyl, 1 -pentenyl, (Z)- 2-pentenyl, (E)-2-pentyl, (Z)-4-methyl-2-pentenyl, (E)-4-methyl-2-pentenyl, allyl, pentadienyl, e.g., 1 ,3 or 2, 4-pentadienyl, and the like. It is preferred that the alkenyl group contains at most two carbon-carbon double bonds; and most preferably one carbon-carbon double bond.

The term alkynyl include alkynyls containing 2 to 6 carbon atoms. They may be straight chain as well as branched. It includes such groups as ethynyl, propynyl, 1 -butynyl, 2- butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, and the like.

The term aryl refers to an aromatic group containing only carbon ring atoms which contains up to 18 ring carbon atoms and up to a total of 25 carbon atoms and includes the polynuclear aromatic rings. These aryl groups may be monocyclic, bicyclic, tricyclic, or polycyclic, and contain fused rings. The group includes phenyl, naphthyl, anthracenyl, phenanthranyl, xylyl, tolyl and the like.

The aryl lower alkyl groups include, for example, benzyl, phenethyl, phenpropyl, phenisopropyl, phenbutyl, diphenylmethyl, 1 ,1-diphenylethyl, 1 ,2-diphenylethyl and the like.

The term halo includes fluoro, chloro, bromo, iodo and the like.

The preferred values of F and R ₃ are independently hydrogen or alkyl containing 1-3 carbon atoms. It is most preferred that R ₃ is hydrogen. It is most preferred that R ₂ is hydrogen or alkyl containing 1-3 carbon atoms, especially methyl or ethyl. It is most preferred that R ₂ is hydrogen or alkyl containing 1-3 carbon atoms or hydrogen and R ₃ is hydrogen.

Alternatively, if R ₂ and R ₃ are taken together with the carbon atoms to which they are attached, it is most preferred that they form a phenyl ring. The phenyl ring is preferably unsubstituted or substituted with lower alkoxy, hydroxy, lower alkyl or halo.

It is preferred that R ₄ is an electron withdrawing group, more specifically, halo, especially chloro, or is hydroxy or lower alkoxy. It is even more preferred that when Ri is NHR _I3 , R ₄ is substituted on the 7-position of the quinoline ring. It is most preferred that when Ri is NHR _I3 , R ₄ is halo.

However, when R _I2 is NHR _I3 , it is preferred that R is an electron donating group, such as hydroxy or alkoxy. More specifically, it is preferred that R is methoxy or ethoxy when R _I2 is NHR _I3 . It is even more preferred that R ₄ is on the 6-position of the quinoline ring when R _I2 is NHR _I3 .

It is preferred that one of R ₅ and R ₆ is hydrogen and the other is lower alkyl. It is even more preferred that R ₅ is hydrogen and R ₆ is lower alkyl, especially alkyl containing 1-3 carbon atoms and most preferably methyl.

The preferred value of R _? is lower alkyl, especially alkyl containing 1-3 carbon atoms and most preferably methyl and ethyl.

Preferred values of R ₈ is lower alkyl containing 1-3 carbon atoms, and most preferably methyl and ethyl. However, it is preferred that the alkyl group is unsubstituted or if substituted, is substituted on the omega (last) carbon in the alkyl substituent. The preferred substituent is lower alkoxy and especially hydroxy. The preferred R ₉ is lower alkoxy and especially hydroxy.

Rii is preferably an electron withdrawing group, especially trifluoromethyl. It is preferably located on the 8-position of the quinoline ring.

Ri4 is preferably an electron withdrawing group, and more preferably trifluoromethyl. It is preferably present on the 2-position of the quinoline ring.

It is preferred that R _I5 is wherein R ₇ and R ₈ are independently alkyl containing 1 -3 carbon atoms and Ar is phenyl.

In both Ri3 and R _I5 , it is preferred that R and R ₈ contain the same number of carbon atoms, although one may be unsubstituted while the other is substituted. It is also preferred that R and R ₈ are the same.

The preferred value of n is 3 or 4 while the preferred value of n1 is 1 .

Preferred antimalarials include the 8-aminoquinolines, 9-aminocridines and the 7- chloro-4-aminoquinolines. Examples include pamaquine, primaquine, pentaquine, isopentaquine, quinacrine salts, 7-chloro-4-aminoquinolines, such as the chloroquines, hydroxychloroquines, sontoquine, amodiaquine and the like.

Another class of preferred antimalarial are cinchono alkaloids and 4-quinoline methanols, such as those having the formula:

wherein one of R _I8 and R _I9 is hydroxy or loweralkylcarbonyloxy or hydrogen, and the other is H, and R ₂ o is hydrogen or loweralkoxy and R _2I is hydrogen or CH=CH ₂ .

Examples include rubane, quinine, quinidine, cinchoidine, epiquinine, epiquinidine, cinchonine, and the like. The most preferred anti-malarials include mefloquinine, and chloroquine and its congeners, such as hydroxychloroquine (HCQ), amodiaquine, pamaquine and pentaquine and pharmaceutically acceptable salts thereof

The most preferred anti-malarial agents for the invention are chloroquine (I) and hydroxychloroquine (II), shown below, or a pharmaceutically suitable salt thereof, such as chloroquine phosphate and hydroxychloroquine sulphate.

(I) (II)

The antimalarials are commercially available or are prepared by techniques known in the art. Antimalarial agents like chloroquine (I) and hydroxychloroquine (II) exert several pharmacological actions which may be involved in their therapeutic effect in the treatment of rheumatic disease, but the role of each is not known. These include interaction with sulphydryl groups, interference with enzyme activity (including phospholipase, NADH- cytochrome C reductase, cholinesterase, proteases and hydrolases), DNA binding, stabilisation of lysosomal membranes, inhibition of prostaglandin formation, inhibition of polymorphonuclear cell chemotaxis and phagocytosis, possible interference with interleukin 1 production from monocytes and inhibition of neutrophil superoxide release.

Hydroxychloroquine (HCQ) sulfate, a derivative of Chloroquine (CQ), was first synthesized in 1946 by introducing a hydroxyl group into CQ (I) and was demonstrated to be much less (-40%) toxic than CQ in animals. More importantly, HCQ (II) is still widely used for the treatment of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Since CQ (I) and HCQ (II) share similar chemical structures and mechanisms of acting as a weak base and immunomodulator, the inventors hypothesised that HCQ (II) may be a potent candidate to treat infection by SARS-CoV-2.

Both CQ (I) and HCQ (II) are weak bases that are known to elevate the pH of acidic intracellular organelles, such as endosomes/lysosomes, essential for membrane fusion. In addition, CQ (I) could inhibit SARS-CoV entry through changing the glycosylation of ACE2 receptor and spike protein. Time-of-addition experiments confirmed that HCQ (II) effectively inhibited the entry step, as well as the post-entry stages of SARS-CoV-2, which was also found upon CQ (I) treatment (Xueting Y, Fei Y, Miao Z, Cheng C, Baoying H, Peihua N, Xu L, Li Z, Erdan D, Chunli S, Siyan Z, Roujian L, Haiyan L, Wenjie T, Dongyang L; In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Clin Infect Dis. 2020 Mar 9; 237.)

A therapeutic composition within the present invention is formulated for localized (targeted) delivery and includes at least one anti-malarial agent, as described above. As previously emphasized, the present invention contemplates administration of the anti- malarial compounds to the lungs, by local or targeted delivery. "Local or targeted delivery" and "locally administering" are used in this description to denote direct delivery to the site, such that the therapeutic agent acts directly on affected tissue or the area of a disease affecting the lung. Local delivery contrasts with methods by which a therapeutic agent is administered orally, or otherwise systemically, and is absorbed into the circulation for distribution throughout the patient's body. Examples of local delivery include inhalation, and nasal spray. It is to be noted that the anti-malarial compound is not injected intravenously, that is, into the circulatory blood of the patient.

As one having ordinary skill in the art would understand, they may be prepared essentially as detailed in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. , (Mack Publishing Co. 1990) ("Remingtons"), which is hereby incorporated by reference.

The compounds of the present invention are present in the pharmaceutical compositions in effective amounts. The anti-malarial compounds used in the present invention are administered in an amount which depends upon the condition of the subject, the type infection from which the subject suffers, the timing of the administration of the subject, the route of administration, the particular formulation and the like. However, unlike oral dosing for antimalarial purposes which takes usually about a month before there is a noticeable or measurable onset of action, onset of action from local or targeted administration to the lung of the anti-malarials for treatment of coronavirus is noticed or observed within 10 days after initial administration. Effective amounts of the anti-malarial compounds, hereinafter known as drug, is that amount which provides the observable onset of action within 10 days, and more preferably within 7 days after administration. Significantly less amount of drug is given locally than by systemic administration to achieve efficacious results, and the onset of action, as indicated hereinabove, is much faster by local administration. It is preferred that the drug is administered to the lung at a dosage of about 0.020 to about 2 mg/kg animal weight and more preferably from about 0.100 to about 1 mg/kg and most preferably from about 0.200 to about 0.650 mg/kg.

For pulmonary delivery, a therapeutic composition of the invention is formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system.

Solid or liquid particulate forms of the active compound prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size are within the respirable range. The therapeutic composition containing the anti-malarial compounds are preferably administered by direct inhalation into the respiratory system for delivery as a mist or other aerosol or dry powder. Particles of non-respirable size which are included in the aerosol tend to be deposited in the throat and swallowed; thus the quantity of non-respirable particles in the aerosol is preferably minimized.

The dosage of active compound via this route will vary depending on the condition being treated and the state of the subject, but generally may be an amount sufficient to achieve dissolved concentrations of anti-malarial compound on the airway surfaces of the subject. Depending upon the solubility of the particular formulation of active compound administered, the daily dose may be divided among one or several unit dose administrations. The daily dose by weight will depend upon the age and condition of the subject. Such a daily dose of the anti-malarial compound ranges from about 0.20 mg/kg per day to about as 2.0 mg per day, and more preferably from about 0.1 to about 1 mg/kg and most preferably from about 0.200 mg/kg to about 0.650 mg/kg. In the most preferred embodiments, only one dose is administered to the patient per day.

The doses of the active compounds may be provided as one or several pre packaged units. In the manufacture of a formulation according to the invention, the anti- malarial compounds or the pharmaceutically acceptable salts are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation. One or more drugs may be incorporated in the formulations of the invention, which formulations may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the drug with the other various components described hereinbelow present therein.

Aerosols of liquid particles comprising the anti-malarial compounds may be produced by any suitable means, such as inhalatory delivery systems. A preferred mode of administration is via a nebulizer. The airborne particles are generated by a jet of air from either a compressor or compressed gas cylinder-passing through the device (pressure driven aerosol nebulizer). In addition, newer forms utilize an ultrasonic nebulizer by vibrating the liquid at speed of up to about 1 MHz. See, e.g., U.S. Pat. No. 4,501 ,729, the contents of which are incorporated by reference. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.

Mesh nebulizers (one type of which uses a "flat plate" geometry of a piezoelectric element and a mesh to produce aerosol) are commonly used to generate aerosols in such drug delivery apparatus. U.S. Patent 5,478,378 describes a nebulizer in which the aerosol is formed using a mesh plate instead of a porous solid body, thereby to lessen or eliminate the foregoing shortcomings. The mesh plate has a plurality of orifices for the liquid in a reservoir. The orifices are tapered outwardly toward the outlet for the liquid. The liquid or the nozzle assembly is vibrated ultrasonically by a piezoelectric element to nebulize the liquid. The liquid reservoir is preferably permanently filled with liquid and maintained at a slight negative pressure.

Jet, ultrasonic or mesh nebulizers may be used to administer the compositions according to the invention, in particular the nebulizer formulations described below.

Suitable formulations for use in nebulizers (“nebulizer formulations”) consist of the active ingredient in a pharmaceutically acceptable liquid vehicle. The vehicle is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic but may be hypertonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the flavouring agents, volatile oils, buffering agents and surfactants, which are normally used in the preparation of pharmaceutical compositions.

Preferably, the pH of the nebulizer formulation is between 4 and 9. Preferably, the pH of the nebulizer formulation is between 6 and 8. More preferably, pH of the nebulizer formulation is between 7 and 8, such as around pH 7.4. In an alternative embodiment, the pH is below 7, i.e. the nebulizer formulation is acidic; preferably, the pH is between 3 and 5, such as around 4. The pH may be adjusted by the use of buffer systems known in the art, such as citrate, phosphate and acetate buffers; alternatively, the formulation is not buffered, but the pH is adjusted by the addition of a strong acid (e.g. hydrochloric or sulphuric acid) and/or strong base (e.g. sodium or potassium hydroxide).

The tonicity of the nebulizer formulation should range between 250 and 450 mOsm/l, preferably between 260 and 400, even more preferably between 280 and 350 mOsm/l, most preferably 280-300 mOsm/l; it can be adjusted by using any physiologically acceptable salt or non-volatile compounds.

In order to achieve an optimum therapeutic profile, the particle size distribution of the nebulized formulation has been found to be significant. In some embodiments, the nebulizer provides a Geometric Standard Deviation (GSD) of emitted droplet size distribution of the solution administered with a nebulizer of about 1.1 to about 2.1 , about 1 .2 to about 2.0, about 1.3 to about 1.9, at least about 1 .4 to about 1.8, at least about 1 .5 to about 1.7, about 1.4, about 1.5, or about 1 .6. In this respect, an additive such as ethanol has found to be beneficial in attaining the optimum distribution. When present, ethanol may be used in an amount of up to 1 % w/w of the nebulizer formulation.

In some embodiments, administration of the anti-malarial compounds with the high nebulizer provides a Mass Median Aerodynamic Diameter (MMAD) of droplet size of the solution emitted with the high efficiency nebulizer of about 0.5 pm to about 6 pm, about 0.75 pm to about 5 pm, or about 1 pm to about 3 pm.

In some particular embodiments, the nebulizer provides droplets having a particular combination of MMAD and GSD, for example: an MMAD of less than about 5 pm and a GSD of about 1.1 to about 2.1 ; an MMAD of less than about 4.5 pm and a GSD of about 1.1 to about 2.1 ; an MMAD of about 1 pm to about 5 pm and a GSD of about 1.1 to about 2.1 ; an MMAD of about 1 to about 3 pm and a GSD of about 1.1 to about 2.1 ; an MMAD of less than about 5 pm and a GSD of about 1.1 to about 2.0; an MMAD of less than about 4.5 pm and a GSD of about 1.1 to about 2.0; an MMAD of about 1 pm to about 5 pm and a GSD of about 1.1 to about 2.0; an MMAD of about 1 to about 3 mhi and a GSD of about 1.1 to about 2.0; an MMAD of less than about 5 mhi and a GSD of about 1.1 to about 1.9; an MMAD of less than about 4.5 mhi and a GSD of about 1.1 to about 1.9; an MMAD of about 1 mhi to about 3 mhi and a GSD of about 1.1 to about 1.9; an MMAD of about 1 to about 3 mhi and a GSD of about 1.1 to about 1.9; an MMAD of less than about 5 mhi and a GSD of about 1.1 to about 1.8; an MMAD of less than about 4.5 mhΊ and a GSD of about 1.1 to about 1 .8; an MMAD of about 1 pm to about 5 mhi and a GSD of about 1.1 to about 1.8; or an MMAD of about 1 to about 3 pm and a GSD of about 1.1 to about 1.8.

In a typical administration protocol for the nebulizer formulations of the invention, the nebulizer is loaded with a volume of the nebulizer formulation, referred to herein as the “loaded volume”. Typically, nebulizers include a reservoir or medication cup for this purpose. The volume of the nebulizer formulation used per administration is preferably from 0.05 to 50 mL, more preferably from 0.1 to 25 ml_, more preferably from 0.5 to 10 ml_, most preferably 1 to 5 mL such as about 5 mL. In preferred embodiment 0.1 to 2 mL, such as about 1 mL of the nebulizer formulation is used in an administration to a patient.

The loaded volume contains an amount of anti-malarial compound, dependent on the dose it is intended to deliver to the patient. In preferred embodiments, the amount is between 1 and 100 mg, preferably between 2 and 50 mg, more preferably between 4 and 40 mg, most preferably between 5 and 20 mg. In an alternative preferred embodiment, the amount is between 2 and 25 mg, such as about 4 mg, about 10 mg, or about 20 mg.

The nebulizer formulation is preferably administered on a daily basis. In one preferred embodiment, the nebulizer formulation is administered twice per day. In an alternative preferred embodiment, the nebulizer formulation is administered once per day.

The Emitted Dose (ED), or Delivered Dose (DD), of anti-malarial compound administered to a patient is the portion of volume of liquid filled into the nebulizer, i.e. the fill volume, which is actually emitted from the mouthpiece of the device. The difference between the nominal dose and the ED is the amount of volume lost primarily to residues, i.e. the amount of fill volume remaining in the nebulizer after administration, or lost in aerosol form. The ED of the anti-malarial compound is to be tested under simulated breathing conditions using a standardized bench setup, which are known to one of skill in the art. In some embodiments, the ED of the anti-malarial compound of the present invention is between 0.5 and 10 mg/ml, preferably between 1 and 5 mg/ml, more preferably between 1.5 and 4 mg/ml, still more preferably between 2 and 3 mg/ml, such as about 2.5 mg/ml. The respirable dose (RD) is calculated by multiplying the DD, obtained in breath simulation experiments, by the percentage of aerosol particles less than 5 pm (RF<5 pm) determined by laser diffraction.

The Respirable Dose Delivery Rate (RDDR) is the speed at which a respirable dose of the anti-malarial compound is nebulized, administered, and delivered to a patient's lungs. RDDR, measured as a function of pg/min, is determined by dividing the RD (in pg) by the amount of time necessary for inhalation. The amount of time necessary for inhalation is measured as the amount of time from the first moment of administration of the emitted droplet from the nebulizer until the emitted droplet of respirable diameter is delivered to the lung, as measured using a standardized bench setup simulating breathing conditions. In some embodiments, the RDDR is at least about 100 pg/min, at least about 150 pg/min, at least about 200 pg/min, about 100 pg/min to about 5,000 pg/min, about 150 pg/min to about 4,000 pg/min or about 200 pg/min to about 3,500 pg/min. In some embodiments, administration of the anti-malarial compound to the patient with an aqueous inhalation device provides an RDDR of at least about 100 pg/min or about 100 pg/min to about 5,000 pg/min.

Together, the loaded volume and the RDDR will determine the delivery time during an administration of nebulizer formulation, i.e. the time over which the patient inhales the nebulized medication. Preferably, this will be between 1 and 60 minutes. More preferably, this will be from 2 to 30 minutes. Still more preferably, this will be from 3 to 15 minutes. In an alternative preferred embodiment, the patient inhales the nebulized medication over 15 to 30 minutes.

Several anti-malarial compound agents, including hydroxychloroquine (HCQ) are known to have an extremely bitter taste. In the case of pulmonary delivery, bitter actives have a pronounced tendency to induce the cough reflex, which leads to dose variability, patient compliance issues, and safety concerns in those suffering from respiratory ailments.

In contrast to the field of oral delivery, there exists no specific solution, formulations or methodologies for pulmonary delivery that have been devised to minimize bitter or otherwise unpleasant taste or to suppress or diminish cough. Very surprisingly, it has been found the compositions and methods of the present invention substantially avoid these issues, such that there is substantially no bitter taste or cough reflex. In one embodiment of the invention, there is provided a method of treatment of a patient suffering from SARS-CoV-2 comprising administering a nebulized formulation of hydroxychloroquine or a pharmaceutically acceptable salt thereof so as to acheive concentration of hydroxychloroquine in the lower respiratory tract of from 0.1 to 2 mM. At these concentrations, viral RNA increase is inhibited by at least 50%, at least 80%, at least 90% or at least 99%.

A further aspect of the invention relates to a filled ampoule containing the nebulizer formulation as described herein. Preferably, the ampoule contains between 0.1 and 10 ml of the nebulizer formulation, more preferably between 0.5 and 5 ml, most preferably between 1 and 4 ml such as about 1 ml, about 2.5 ml, or about 3 ml. In use, the contents of the ampoule are poured into the chamber of the nebulizer and the patient then breathes the vapour generated until the ampoule contents are used. The invention further provides a method of manufacturing sterile nebulizer formulations, by combining an anti-malarial compound with a pharmaceutically acceptable liquid vehicle under nitrogen gas before filling into ampoules. The nebulizer formulations can be filled into ampoules using blow fill seal technology to yield ampoules with the required extractable volume of formulation. By sterile, it is meant that the resultant pharmaceutical composition meets the requirements of sterility enforced by medicine regulatory authorities, such as the FDA in the US, the EMA in the European Union or the MHRA in the UK. Tests are included in current versions of the compendia, such as the US Pharmacopoeia, European Pharmacopoeia (Ph. Eur.) 10th Edition and the British Pharmacopoeia.

Formulations of the invention are suitable for filling into ampoules using "blow fill seal" (BFS) methods. The principle is that a plastic parison is extruded from polymer, formed into a container, filled and sealed in a single aseptic operation. BFS is now the preferred method for aseptic manufacture of ampoules due to the flexibility in container design, overall product quality, product output and low operational costs. Fill accuracies of better than ±5% have been demonstrated for container volumes as small as 0.5ml and hence BFS is suitable for manufacture of ampoules according to the invention. One BFS operation includes the multi-step process of blow moulding, aseptic filling and hermetic sealing of liquid products with fill volumes ranging from 0.1 ml to 1 ,000ml, though for ampoules volumes in the range 0.5ml to 5ml are more common. A variety of polymers may be used in the process; low and high-density polyethylene and polypropylene are preferred.

Aerosols of solid particles comprising the anti-malarial compound may likewise be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump.

The powder employed in the insufflator consists either solely of the active ingredient or of a powder blend comprising the anti-malarial compound, a suitable powder diluent, such as lactose, and an optional surfactant. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the anti-malarial compound in a liquified propellant. During use these devices discharge the formulation through a valve, adapted to deliver a metered volume, from 10 to 22 microliters to produce a fine particle spray containing the anti-malarial compound. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavouring agents.

Any propellant may be used in carrying out the present invention, including both chlorofluorocarbon-containing propellants and non-chlorofluorocarbon-containing propellants. Fluorocarbon aerosol propellants that may be employed in carrying out the present invention including fluorocarbon propellants in which all hydrogen are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Examples of such propellants include, but are not limited to: CF ₃ CHFCF ₂ , CF ₃ CH ₂ CF ₂ H, CF ₃ CHFCF ₃ , CF ₃ CH ₂ CF ₃ , CF ₃ CHCI-CF ₂ CI, CF ₃ CHCI-CF ₃ , CF ₃ CHCI-CH ₂ CI, GF ₃ CHF-CF ₂ CI, and the like. A stabilizer such as a fluoropolymer may optionally be included in formulations of fluorocarbon propellants, such as described in U.S. Patent No. 5,376,359 to Johnson.

Compositions containing respirable dry particles of micronized anti-malarial compounds may be prepared by grinding the dry active compound, with e.g., a mortar and pestle or other appropriate grinding device, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. The aerosol, whether formed from solid or liquid particles, may be produced by the aerosol generator at a rate of from about 10 to 150 litres per minute. Aerosols containing greater amounts of medicament may be administered more rapidly. Typically, each aerosol may be delivered to the patient for a period from about 30 seconds to about 20 minutes, with a delivery period of about 1 to 5 minutes being preferred.

The particulate composition comprising the anti-malarial compound may optionally contain a carrier which serves to facilitate the formation of an aerosol. A suitable carrier is lactose, which may be blended with the active compound in any suitable ratio.

For example, chloroquine phosphate is a colourless crystalline solid which is readily soluble in water. Inhaled liquid forms may be formulated to contain such additives as are typically used in such pharmaceutical preparations, including, but not limited to an acceptable excipient and/or surfactant. A therapeutic composition of chloroquine phosphate may be pre-formulated in liquid form, or prepared for the addition of a suitable carrier, like sterile water or physiological saline, immediately prior to use. The aerosol containing chloroquine phosphate typically contain a propellant especially a fluorocarbon propellant.

See Remington's, chapter 92. A particularly useful composition of chloroquine phosphate is formulated in a nebulizer, for the treatment of a variety of pulmonary conditions. For the preparation of HCQ in inhaled powder form, the compound is finely divided, or micronized to enhance effectiveness, and admixed with a suitable filler.

Inhaled powders may contain a bulking agent and/or stabilizer, as described hereinabove. Id., chapter 88. An insufflator (powder blower) may be employed to administer the fine powder.

The anti-malarial compounds may, where appropriate, be conveniently present in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound, i.e., the anti-malarial compound with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, shaping the product into the desired delivery system. Methods for admixing a pharmaceutical with a carrier are known in the art and are applicable to the present formulation.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical 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. More than one anti-malarial compound can also be incorporated into the pharmaceutical compositions.

It is especially advantageous to formulate local compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of anti-malarial compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the anti-malarial compound utilized and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an anti-malarial compound for the treatment of anti-inflammatory conditions in living subjects having a diseased condition in which bodily health is impaired as hereinbelow disclosed.

Therapeutic Rationale

The inventive methods, detailed below, may be applied by the clinician to treat a variety of coronavirus infections. Preferably, the coronavirus is selected from the group of Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (HCoV-HKlH), Human coronavirus 229E (HCoV-229E), Human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), Middle East respiratory syndrome-related coronavirus (MERS-CoV), Severe acute respiratory syndrome coronavirus (SARS-CoV or "SARS-classic"), and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More preferably, the coronavirus is SARS-CoV-2.

Although the inventor does not wish to be bound by any theory of mechanism of the invention, it is believed that the therapeutic approach of the present invention effectively inhibits or attenuates at least one of the viral enzymatic processes. The inhibition or attenuation of one or more of the underlying causative or exacerbating processes is effected by the anti-malarial agents that have anti-viral effect, thus results in an effective treatment of a variety of pulmonary conditions.

As noted previously, conventional therapies, i.e., systemic deliveries of anti-malarials, especially by oral administration, suffer from significant failings. For example, when HCQ is delivered through the conventional systemic routes, there is a significant delay in the onset of the anti-viral action, due to active concentration of the therapeutic agent in certain organs, which are often not the target organ. Moreover, long-term high dose use has been shown to carry a risk of serious side effects, including retinal damage.

Nevertheless, it was thought heretofore that systemic delivery was necessary to achieve a therapeutic effect. Thus, like other anti-inflammatory pharmaceuticals, anti- malarial compounds have uniformly been prescribed systemically, typically by oral dosing.

The present invention is the first to demonstrate that targeted delivery of an anti- malarial compound to the lung is effective in treating coronavirus related conditions. The inventor has found, unexpectedly, that localized delivery of anti-malarial compounds to the lung maintains or improves therapeutic value, while avoiding the problems associated with conventional antiviral regimes.

Also unexpected is the inventor's demonstration that locally delivered anti-malarial compounds, such as HCQ, have potent anti-viral effects. Inhaled anti-malarial compounds, such as HCQ, are well tolerated as evidenced by the lack of increase in airway resistance after inhalation, demonstrating that the nebulized form is not a bronchial irritant and, hence, is suitable for administration via inhalation. Moreover, the local administration of the anti- malarial compounds significantly reduces and/or eliminates the toxic side effects of these compounds which are manifested when given by systemic administration, such as by oral administration. The major toxicity of this class of pharmaceuticals when given systemically is related to the selective accumulation of the drug in the retina and subsequent binding of melanin which may lead to retinal photoreceptor damage. This may lead to retinal damage. Other side effects associated with chloroquine therapy include nausea, anorexia, diarrhoea, pruritus, urticaria, increased skin pigmentation, exfoliative dermatitis, headache, and scotomata. By administering the antimalarial, such as chloroquine or hydroxychloroquine locally, such by inhalation, less drug is required and therefore either the patient does not experience the aforementioned side effects or, if experienced, they are significantly less severe.

Relative to systemic administration, such as oral administration, the present invention has significant benefits over available oral or systemically administered routes, such as a more rapid accumulation of therapeutic amounts of compound in the lung which is targeted, as well as a rapid onset of action. Localized delivery results in reduced dosage requirements, both daily and cumulatively, and minimizes side effects. The low-dose, targeted, and organ-oriented approach of the instant invention minimizes all the drawbacks of the systemic approach, such as increased cost for the medicine and inconvenience to the patients, resulting from prolonged and high dose usage.

The anti-malarial compounds of the invention, especially, aminoquinoline derivatives, are, without wishing to be bound, believed to be particularly effective because they are multi factorial inhibitors, blocking both humoral and cell-mediated/delayed response immune systems. Anti-malarial compounds, such as aminoquinoline derivatives, appear to exert their pharmacologic effects due to several underlying properties. For example, see MacKenzie,

1983, Am J Medicine 75:1 A:5-10; Fox, 1993, Sem . Arthritis Rheumatism 23:82-91 .

These properties seem to result from the unique effect on membranes of compounds in this group. Without wishing to be bound, it is believed that anti-malarial compounds, such as aminoquinoline derivatives, are able to elevate intravesical pH by intracellularly concentrating in acidic cytoplasmic vesicles in a variety of immune function cells. Since several processes critical for the generation of immune response depends on neutral or acidic pH environments, this action inhibits viral reproduction.

Interference with vesical fusion decreases secretion and release of intracellular products, such as immunoglobins including IgE (allergic antibody), interleukins and cytokines used in signalling and augmenting immune responses (e.g., IL-1 , IL-6, TNF-a, ICAM-1 , IL-4), exocytosis of lysosomal products such as superoxides in neutrophils and macrophages. Similarly, such interference also decreases efficiency of phagolysosomal system by inhibiting production of superoxides in neutrophils. Furthermore, increases of lysosomal pH interfere with lysosomal acid hydrolases. As a consequence of these actions, antigen processing is inhibited.

The depletion of cell surface markers has been reported to reduce the transmission of a number of viruses including rhinovirus and adenovirus. In part, this may be due to known reduction of membrane receptors, such as ICAM-1 , which are critical for viral uptake. Interference with the phagolysosomal system may help explain an observed decrease in viral replication for other viruses including HIV and influenza. This in turns explains the effects of anti-malarial compounds on coronaviruses observed by the present inventor.

Cumulatively, the result of anti-malarial compound, including aminoquinoline derivatives, inhibition of antigen presentation and T-cell activation is a reduction in delayed hypersensitivity (cell-mediated) responses, modulation of humoral responses, decreases in viral uptake and replication, modulation and/or suppression of early and late phase allergic response, and inhibition of inflammatory effector cell function. Finally, derivatives of anti- malarials, including aminoquinolines, are reported to block viral replication and transmission (for example, of rhinovirus and adenovirus) and to have anti-brochospastic effects.

The inventor has found that each of the effects noted above can be accomplished via localized delivery, for example, by application of the anti-malarial compounds to the lung and uptake.

In sum, anti-malarial compounds such as the aminoquinoline derivatives have beneficial effects with respect to a wide range of coronavirus induced diseases. For each of these illnesses, localized delivery has the same advantages, including more rapid onset of action at less risk due to lower cumulative and daily doses.

Therapeutic Methodology

In accordance with the present invention, a therapeutic composition as described above, typically is applied to patients suffering from a coronavirus induced ailment.

Thus, a patient in this context often will suffer from a disorder characterized by one or more of the foregoing signs of a coronavirus. By the same token, the present invention entails localized administration, to a patient in need, of an anti-malarial compound, formulated along the lines detailed above, in an amount that alleviates or ameliorates a symptom or the underlying pathology of a coronavirus ("an effective amount" or "anti inflammatory effective amount").

In a particular embodiment of the invention, there is disclosed a method of treating a patient population suffering from a coronavirus induced ailment, especially SARS-CoV-2. In a preferred embodiment, the methods of treatment include the step of measuring the peripheral capillary oxygen saturation as measured by pulse oximeter (Sp0 ₂ ), and categorizing patients as not hypoxemic, hypoxemic, and severely hypoxemic. As used herein, “not hypoxemic” refers to a patient having an Sp0 ₂ of above 95%, “hypoxemic” refers to a patient having an Sp0 ₂ of from 80% to 95%, and “severely hypoxemic” refers to a patient having an Sp0 ₂ of below 80 %.

In some embodiments, the invention relates to a method of treatment of a coronavirus, especially SARS-CoV-2, comprising administering to the lungs of a hypoxemic, or severely hypoxemic patient an effective amount of an anti-malarial compound. In some embodiments, the patient is hypoxemic. In some embodiments, the patient is severely hypoxemic.

In alternative embodiments, the invention relates to a method of treatment of a coronavirus, especially SARS-CoV-2, comprising administering to the lungs of a patient that is not hypoxemic an effective amount of an anti-malarial compound.

In a particular embodiment of the invention, there is disclosed a method of treating a patient suffering from a coronavirus induced ailment, especially SARS-CoV-2, which involves a step of measuring the level of a biomarker associated with inflammation, and then administering an effective amount of an anti-malarial compound. Preferably, the biomarker is selected from the group consisting of IL-6, IL-17, IL-1 b, and TNF-a. Preferably, the biomarker is IL-6.

In some embodiments, the invention relates to a method of treatment of a coronavirus, especially SARS-CoV-2, comprising administering to the lungs of a patient having an elevated level of a biomarker an effective amount of an anti-malarial compound.

In some embodiments, the biomarker is selected from the group consisting of IL-6, IL-17, IL- 1 b, and TNF-a. Preferably, the biomarker is IL-6. Preferably, the patient having an elevated level of a biomarker has an IL-6 level of over 50 pg/ml, preferably over 65 pg/ml, more preferably over 80 pg/ml.

The compound may be administered by any suitable means, as described hereinabove depending on the condition being treated.

As used herein, the plural signifies the singular and vice-versa.

Moreover, in the chemical formula described hereinabove, if not specifically drawn, it is to be understood that if a central atom does not have all the valences, the remaining bonds are to hydrogen atoms.

The following examples are given to illustrate the present invention. It should be understood, however, that the invention is not to be limited to the specific conditions or details described in these examples. In addition, throughout the specification, any and all references to publicly available documents are specifically incorporated by reference.

Examples

Example 1 Animal Studies of the Effect of Nebulized chloroquine on SARS-CoV-2

Using an animal model of SARS-CoV-2 employing hACE2 transgenic mouse strain (J Virol. 2007 Jan; 81(2): 813-821), the contents of which are incorporated by reference, the inventor conducts animal experiments investigating the effect of nebulized chloroquine phosphate on early and late SARS-CoV-2 infected mice. These studies confirm that targeted and localized delivery of chloroquine phosphate has potent anti-viral effects. In addition, quite unexpectedly, local delivery, in this case administered via nebulized aerosol, results in more rapid onset of drug effect than in oral administration and at significantly lower dosage levels, both daily and cumulatively. Inhaled chloroquine phosphate partially blocks immediate viral-mediated bronchostrictive responses, virtually eliminates late-phase responses which occur on four to 12 hours post virus challenge, and moreover, shows continued effect at blocking the bronchial hyperresponsiveness even twenty-four hours later after local administration of the anti- malarial compound. Example 2

Example 1 is repeated, using hydroxychloroquine sulphate instead of chloroquine phosphate. Good control of viral infection and symptoms is observed.

Example 3

Preparation of a nebulizer formulation of hydroxychloroquine sulphate. The composition refers to 1 unit-dose vial (10 ml)

Hydroxychloroquine sulphate 10 mg; Sodium chloride 9 mg; ethanol 0.1 ml_; sterile pyrogen- free water to 9 ml_. The pH is adjusted to 7.4 with 0.1 M NaOH and 0.1 M HCI, and the volume made up to 10 ml_ with water. Final active ingredient concentration 2 mg/ml_.

The resulting solution is mixed for 15 minutes. The solution is filtered through one 0.45 pm Nylon filter, and through two 0.2 pm Nylon filters. The solution is distributed in 10 ml polyethylene unit dose vials under nitrogen purging.

Example 4

Administration of nebulizer formulation of Example 3. 10 mL of nebulizer formulation of hydroxychloroquine sulphate prepared according to Example 3 is loaded into the medication cup of an Omron NE-U780 UltraAir Pro Nebuliser (Omron Corporation, Japan). The formulation is nebulized and administered over 10 minutes to a patient suffering from SARS-CoV-2. Example 5

Preparation of a nebulizer formulation of hydroxychloroquine sulphate. The composition refers to 1 unit-dose vial (10 ml)

The procedure of preparation is as follows: 1. Weigh required amount of HCQ, transfer to suitable vessel.

2. Add appropriate amount of isotonic agent.

3. Dissolve in Water for injection up to 100 g while stirring.

4. Determine pH and adjust to 7.35-7.45 with NaOH solution.

5. Filter through a membrane filter with a pore size of 0.22 urn. 6. Fill into 10 mL glass ampoules.

7. Autoclave.

Example 6

Further formulations according to the invention are prepared in accordance with Table 1 below. Qualitative and quantitative composition of formulations A, B and C

Sodium hydroxide 0.2M q.s. pH corrector Ph. Eur.

Water for injections 2969 2964.8 2956 98.97 98.83 98.53 Solvent Ph. Eur.

Total 3 mL 100

Table 1

Description of manufacturing process and process controls

The preparation of the formulation consists of the following steps: 1. Weighing of active substance and isotonic agent

2. Dissolving of both components in water for injections while stirring

3. pH adjustment

4. Membrane filtration (pore size 22 urn)

5. Filling into 10 mL glass ampoules 6. Autoclaving

All components used for the formulation are of Ph. Eur. quality.

The container closure system is 10 mL clear glass ampoules with rubber stopper and aluminium seal (supplier Dr. Kulich Pharma). The container is single-dose, i.e. one dose will be withdrawn from each ampoule. For the sterilization step, autoclaving at 121 °C for 20 minutes was employed. After such a sterilization process, the preparation remains sterile for at least 1 month. The objective of the formulation development was to verify the stability of the formulation after autoclaving, and its photostability with regards to the selected clear glass container. Example 7

The most important parameter to characterize the preparation for nebulization and to derive the actual inhaled dose is the aerodynamic assessment of nebulised aerosol (Ph. Eur. 2.9.44). The general-purpose nebulizer Aerogen® Pro System used to study this objective. The same device will be used in the study. Two methodologies have been used for the assessment of the particle size distribution, laser diffraction by Malvern and impactor method by NGI. The results (presented below in table 2 and Figure 1 , table 3 and Figure 2) indicate that around 50% of the particles are below 5um, i.e. effective in the inhalation delivery. Table 2 - particle size distribution by Malvern technique

Table 3. Aerodynamic assessment by NGI Example 8

This is an open-label, single ascending dose multi-cohort clinical trial under fasting conditions. Each subject receives one dose of the study drug. 18 volunteers are included in this study. The study population is divided into three cohorts with an ascending dose of hydroxychloroquine:

• Cohort 1 (6 subjects) 4 mg 15 min nebulization (according to Examples 6 or 7)

• Cohort 2 (6 subjects) 10 mg 15 min nebulization (according to Examples 6 or 7)

• Cohort 3 (6 subjects) 20 mg 15 min nebulization (according to Examples 6 or 7)

Subjects in all cohorts are nebulized via continuous nebulization of 3 ml of solution for nebulization over 15 min using Aerogen® Pro System (Aerogen, Galway Business Park, Dangan, Galway, H91 HE94 Ireland). Dosing in each cohort is performed in one day. It will be performed stepwise, in subgroups, with a maximum 2 subjects in parallel.

On Day 1 , a peripheral intravenous catheter is inserted into a forearm vein shortly before collection of pre-dose sample. 4 ml of whole blood is collected into K2EDTA vacutainer tubes. Blood samples are gently shaken in vacutainers immediately after collection. Blood samples are withdrawn by the catheter until approx. 8-12 hours post-dose. Other samples are collected by direct venepuncture. The blood samples are collected by direct venepuncture also if subject rejects insertion of the catheter. Exact time (hh:mm) of each blood sampling is recorded in written in source documentation. All significant sampling time deviations will be justified.

The blood samples are taken before, during and after the 15 min course of HCQ nebulization at: pre-dose and 2, 5, 10 and 15 min (end of inhalation) and 20, 25, 30, 45, 60 min, 1.25,

1.5, 2, 2.5, 3, 4, 8, 12, 24 and 48 h after the start of nebulization (20 samples total).

The quantitative determination of hydroxychloroquine is performed using a sensitive and selective HPLC/MS/MS method using isotopically labelled internal standard. The lower limit of quantification (LLOQ) of hydroxychloroquine in plasma is 1 pg/mL

Example 9

Suppression of bitter taste/cough reflex. Cohort 1 (2 subjects) 4 mg 15 min nebulization

The cohort was nebulized according to the method of Example 8. An absence of bitter taste was reported by the subjects, and no coughing was observed.