WO2022034226A1

WO2022034226A1 - Avermectin and milbemycin compositions for inhalation

Info

Publication number: WO2022034226A1
Application number: PCT/EP2021/072635
Authority: WO
Inventors: Carlos Javier CHACCOUR DÍAZ; Juan Manuel Irache Garreta; Fernando María MARTÍNEZ GALÁN; Ana Luisa Martínez López; Felix Georg Michael HAMMANN
Original assignee: Universidad De Navarra; Universität Bern
Priority date: 2020-08-14
Filing date: 2021-08-13
Publication date: 2022-02-17

Abstract

The invention relates to avermectins or milbemycins suitable for administration to the lungs, and to their use in the local and systemic treatment of disorders and diseases through said route of administration.

Description

AVERMECTIN AND MILBEMYCIN COMPOSITIONS FOR INHALATION

FIELD OF THE INVENTION

The present invention relates to the field of medicine, and more particularly to the treatment of diseases or disorders of the respiratory system.

BACKGROUND OF THE INVENTION

Avermectins are 16-membered macrocyclic lactones obtained by fermentation of soil actinomycete Streptomyces avermitilis. The compounds were identified by Merck, Sharp & Dohme and The Kitasato Institute towards the end of the last century, such as in Burg etal., Antimicrobial agents and chemotherapy, 1979, 15(3):361 -367 or in US Patent 4,310,519. Shortly thereafter, 22,23- unsaturated derivatives of the compounds were reported, such as in US Patent 4,199,569, thus expanding the avermectin family.

The avermectins were originally reported as potent anthelmintic, insecticidal, ectoparasiticidal and acaracidal agents. In fact, Ivermectin, which is the most notable member of the avermectin family, was FDA-approved for human use in 1987. The very closely related milbemycins are also known as possessing the same pharmacological activity (Shoop et al., Veterinary Parasitology, Volume 59, Issue 2, September 1995, Pages 139-156).

However, further research on the avermectins since revealed their potential for treating additional diseases, such as cancer (see Juarez et al., Am J Cancer Res, 2018, 8(2):317-331 ); viral diseases, especially those caused by RNA viruses, such as AIDS, Dengue, Zika or COVID-19 (see Wagstaff et al., Biochem J, 2012, 443:851 -856; Barrows et al., Cell Host Microbe, 2016, 20:259- 270; Caly et al., Antiviral Research, 2020, 178: 104787), NF-KB-related diseases or disorders (see Zhang et al., 2008, Inflamm Res, 57:524-529; or Ci et al., 2009, Fundam Clin Pharmacol, 23:449-455); or a7 nAChR-related diseases or disorders (see Krause et al., Molecular Pharmacology, 1998, 53(2):283-94; Collins, Mol Pharmacol, 2010, 78(2): 198-204). However, the use of avermectins - and of specifically ivermectin - as well as of milbemycins in the art is generally limited to the oral and topical route, and additionally to the parenteral route in the case of veterinary medicine. In addition to the general desire to be able to develop alternative routes of administration for any given treatment, many of the above mentioned diseases might particularly benefit from localized action of the compounds at the lungs.

SUMMARY OF THE INVENTION

It has now surprisingly been found that avermectins and milbemycins can be delivered by inhalation to the lower respiratory system to achieve significant levels of the compounds both in the lungs and plasma, and are thus well suited for treating diseases mentioned elsewhere herein, in particular diseases affecting the lungs.

Thus, in a first aspect of the invention, the invention relates to an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in a form suitable for inhalation into the lungs.

In a second aspect, the invention provides a pharmaceutical composition suitable for inhalation into the lungs, wherein the pharmaceutical composition comprises:

- an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and

- at least one pharmaceutically acceptable inhalation excipient.

In a third aspect, the invention provides an inhaler device comprising a precursor composition as described elsewhere herein.

Similarly, in a further aspect, the present invention relates to an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, as defined in the first aspect of the invention, or a pharmaceutical composition as defined in the second aspect of the invention, or a device as defined in the third aspect of the invention, for use in medicine, and in particular for use in the prevention or treatment of a disease or disorder of the lungs. Similarly, in a further aspect, the present invention relates to a method for preventing or treating a disease or disorder, in particular of the lungs, the method comprising administering a compound as defined in the first aspect of the invention, a pharmaceutical composition as defined in the second aspect of the invention to a subject in need of said prevention or treatment.

Similarly, in a further aspect, the present invention relates to the use of a compound as defined in the first aspect of the invention, a pharmaceutical composition as defined in the second aspect of the invention, or a device as defined in the third aspect of the invention, in the manufacture of a medicament for the prevention or treatment of a disease or disorder, in particular of the lungs.

In particular, the disease or disorder of the lungs is a parasitic infection, cancer, a viral infection, an NF-KB-related disease or disorder, or an a7 nAChR- related disease or disorder.

These aspects as well as preferred embodiments thereof are further described hereinafter in the detailed description and in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Graphical timeline depiction of the in vivo biological testing carried out for a pharmaceutical composition suitable for lung inhalation according to the present invention.

Figure 2. Lung concentrations of avermectin in the lungs following in vivo administration of a pharmaceutical composition according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As set out in the brief summary of the invention, the present invention provides a pharmaceutical composition suitable for inhalation into the lungs, wherein the pharmaceutical composition comprises:

- at least one pharmaceutically acceptable inhalation excipient. The term “avermectin or milbemycin” refers to a compound comprising the following 16-membered lactone structural core:

wherein the wavy line represents a linkage to the remainder of the molecule. In particular, the compound possesses said core, and is a compound produced by a bacterium of the Streptomyces genus, or a derivative of such compound, such as those reviewed in Gilbert et al., Comprehensive Molecular Insect Science, Elsevier, 2005, Vol 5, 25-52. In a particular embodiment, the bacterium is Streptomyces avermitilis, hygroscopicus, cyanogriseus or thermoarchaensis. Non-limiting examples of such avermectins or milbemycins are abamectin, ivermectin, eprinomectin, doramectin, selamectin, milbemectin, moxidectin, nemadectin, emamectin or milbemycin oxime.

In a particular embodiment, the avermectin or milbemycin is a compound of formula (la)

(la), wherein

Ri is H or hydroxyl, which is single bonded to the core of the molecule; or an N- ORox oxime group which is double bonded to the core of the molecule, wherein

Rox is H or a C-i-Ce alkyl group; R2 is C1-C6 alkyl or C2-C6 alkenyl group; R3 is a hydroxyl or C-i-Ce alkoxy, which is single bonded to the core of the molecule; or an N-ORox oxime group which is double bonded to the core of the molecule, wherein Rox is H or a C1-C6 alkyl group; X is H or -OR4, wherein R4 is H, a-L- oleandrosyl, 4'-O-C2-C6 alkanoyl-a-L-oleandrosyl (i.e. O-C2-C6 alkanoyl replaces 4’-OH group), 4'-(amino)-4'-deoxy-a-L-oleandrosyl, 4'-(C2-Ce alkanoylamino)-4'- deoxy-a-L-oleandrosyl, 4'-(a-L-oleandrosyl)-a-L-oleandrosyl, 4"-O-C2-Ce alkanoyl-4'-(a-L-oleandrosyl)-a-L-oleandrosyl, 4"-(amino)-4"-deoxy-4'-(a-L- oleandrosyl)-a-L-oleandrosyl, 4"-(C2-Ce alkanoylamino)-4"-deoxy-4'-(a-L- oleandrosyl)-a-L-oleandrosyl; and the dashed bond is a single or double bond.

In a preferred embodiment, the avermectin or milbemycin is an avermectin. As used in the present invention, the term “avermectin” refers to any of the 16-membered lactone compounds produced by Streptomyces avermitilis, or to synthetic derivatives thereof, in particular to 22,23-unsaturated derivatives thereof. Avermectins may be isolated from Streptomyces avermitilis by the methods disclosed in US Patents 4,310,519 or 4,199,569; or synthesized according to the teachings of Yamashita et al., The Journal of Antibiotics, 2016, 69:31-50; or EP 0214731. In a more particular embodiment, the avermectin is a compound of formula (lb):

(lb), wherein Ri is H or hydroxy; R2 is C1-C6 alkyl; R3 is hydroxy or C-i-Ce alkoxy; R4 is H, a-L- oleandrosyl, 4'-O-C2-C6 alkanoyl-a-L-oleandrosyl, 4'-(amino)-4'-deoxy-a-L- oleandrosyl, 4'-(C2-Ce alkanoylamino)-4'-deoxy-a-L-oleandrosyl, 4'-(a-L- oleandrosyl)-a-L-oleandrosyl, 4"-O-C2-Ce alkanoyl-4'-(a-L-oleandrosyl)-a-L- oleandrosyl, 4"-(amino)-4"-deoxy-4'-(a-L-oleandrosyl)-a-L-oleandrosyl, 4"-(C2-Ce alkanoylamino)-4"-deoxy-4'-(a-L-oleandrosyl)-a-L-oleandrosyl; and the dashed bond is a single or double bond.

In a preferred embodiment, R1 is H. In a preferred embodiment, R2 isopropyl, sec-butyl or cyclohexyl, more preferably it is iso-propyl or sec-butyl. In a preferred embodiment, R3 is hydroxy. In a preferred embodiment, X is H. In a preferred embodiment, R4 is 4'-(a-L-oleandrosyl)-a-L-oleandrosyl. In a preferred embodiment, the dashed bond is a single bond.

In a preferred embodiment, the avermectin is ivermectin. Ivermectin is a mixture of 22,23-dihydroavermectins B1 a (R1 is H; R2 is sec-butyl; R3 is hydroxy; R4 is 4'-(a-L-oleandrosyl)-a-L-oleandrosyl; and dashed bond is single bond) and B1 b (R1 is H; R2 is iso-propyl; R3 is hydroxy; R4 is 4'-(a-L-oleandrosyl)-a-L- oleandrosyl; and dashed bond is single bond). In an embodiment, the weight ratio of B1 a to B1 b compound is from about 75:25 to about 99: 1 , and more particularly is typically about 4:1. In a particular embodiment, the avermectin is 22,23- dihydroavermectin B1 a. In another particular embodiment, the avermectin is 22,23-dihydroavermectin B1 b. Ivermectin is readily commercially available, such as from Sigma-Aldrich (Ref. I8898). In any embodiment described herein, the compound is preferably ivermectin.

In another embodiment, the avermectin or milbemycin is a milbemycin. As used in the present invention, the term “milbemycin” refers to any of the 16- membered lactone compounds produced by Streptomyces hygroscopicus, cyanogriseus or thermoarchaensis, or to a synthetic derivative thereof. Milbemycins may be produced or synthetically modified as reviewed in Gilbert et al., Comprehensive Molecular Insect Science, Elsevier, 2005, Vol 5, 25-52.

The term “alkoxy” refers to a group of formula -ORa wherein R_a is an alkyl group. A preferred alkoxy group is ethoxy or methoxy, and even more preferably it is methoxy. The term “alkyl” refers to a straight, branched or cyclic hydrocarbon chain radical containing no unsaturation (double or triple bond), and which is attached to the rest of the molecule by a single bond. The term “alkenyl” refers to an alkyl group comprising at least one C-C double bond.

The term “alkanoyl” refers to the group defined as -(C=O)Ra, where R_a is an alkyl group. A preferred alkanoyl group is the acetyl group, i.e. a C2 alkanoyl (Ra is methyl). The term “aminoalkanoyl” refers to the group defined as - NRb(C=O)R_a (i.e. an amide), where Ra is an alkyl group and Rb is H or an alkyl group. A preferred aminoalkanoyl group is the acetylamino group, i.e. a C2 aminoalkanoyl wherein R_a is methyl and Rb is H.

The terms oleandrosyl and 4'-(oleandrosyl)-oleandrosyl respectively refer to the following structures:

The term “salt” must be understood as any form of an avermectin or milbemycin according to the present invention in which said compound is in ionic form, or is in ionic form and coupled to a counter-ion (a cation or anion). Preferably, the salt is a pharmaceutically acceptable salt, i.e. a salt that is tolerated physiologically (preferably meaning that it is not toxic, particularly, as a result of the counter-ion) when used in an appropriate manner (i.e. in reasonable medical doses) for a treatment according to the present invention. Preferably, the salt is pharmaceutically acceptable to the lungs. Examples of pharmaceutically acceptable salts are acid addition salts such as the hydrochloride addition salt, or base addition salts such as the sodium hydroxide addition salt.

The term “stereoisomer” must be understood as an enantiomer, diastereomer, or a mixture thereof, such as a racemate, of an avermectin or milbemycin according to the present invention. Likewise, the term also encompasses geometric isomers about any double bonds present in avermectin or milbemycin, i.e. (E)-isomers and (Z)-isomers (trans and cis isomers). Furthermore, avermectin or milbemycin may exist as atropisomers. Furthermore, any avermectin or milbemycin may exist as tautomers. Specifically, the term tautomer refers to one of two or more structural isomers of an avermectin or milbemycin that exist in equilibrium and are readily converted from one isomeric form to the other. Common tautomeric pairs are amine-imine, amide-imidic acid such as lactam-lactim, or keto-enol.

In a preferred embodiment, the avermectin or milbemycin core has the following structure:

In a preferred embodiment, the avermectin is of formula (lb’):

(lb’), wherein Ri, R2, R3, R4 and the dashed bond have the same meanings as described above.

More preferably, the avermectin is ivermectin as described above, with the following formula:

wherein R2 is one of the following two groups, or a mixture of two compounds of this formula each with a different one of the following two groups:

The term “solvate” in accordance with this invention should be understood as meaning any avermectin or milbemycin according to the present invention in which said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates, like for example, methanolate. A preferred solvate is the hydrate. Preferably, the solvate is a pharmaceutically acceptable solvate, i.e. a solvate that is tolerated physiologically (preferably meaning that it is not toxic, particularly, as a result of the solvating molecule) when used in an appropriate manner (i.e. in reasonable medical doses) for a treatment according to the present invention. Preferably, the salt is pharmaceutically acceptable to the lungs.

The pharmacological outcome achieved with a compound or pharmaceutical composition that targets one area of the respiratory tract can vary considerably with respect to the outcome achieved when employing a same or similar - in terms of chemical composition - compound or pharmaceutical composition targeting a different area of the respiratory system.

The compounds and pharmaceutical compositions of the present invention are suitable for inhalation specifically into the lungs. More specifically, they are in a form for delivering therapeutically effective amounts of avermectin or milbemycin into the lungs. The present inventors have unveiled that delivery of the avermectin or milbemycin or of a pharmaceutical composition comprising it to specifically the lungs, and more specifically the alveoli, allows achieving therapeutically effective concentrations of the avermectin or milbemycin locally at the lungs and systemically by absorption of the avermectin or milbemycin through the lungs, or both, during an extended period (at least 7 days) following inhalation.

Chemical species suitable for inhalation possess different mass median aerodynamic diameters (MMAD) depending on which area of the respiratory system is targeted. Larger particles or droplets, typically with a MMAD greater than 6 micron, deposit in the upper respiratory system. Lung delivery is therefore typically achieved employing MMADs of 6 micron or lower, typically of between about 0.5 to 6 micron. Targeted delivery within the lungs again usually depends on MMAD. For instance, the smaller particles or droplets within the latter range, typically those from about 0.5 to 2 micron, are particularly suitable for alveolar deposition. Alveolar deposition is preferred in the context of the present invention. Thus, in an embodiment, the compounds or the pharmaceutical composition according to the present invention have a particle MMAD of about 6 micron or lower, typically of between about 0.5 to 6 micron. In another embodiment, the MMAD is of about 5 micron or lower, typically of between about 0.5 to 5 micron. In another embodiment, the MMAD is of about 3 micron or lower, typically of between about 0.5 to 3 micron. Preferably, the MMAD is of 2 micron or lower, more preferably of between about 0.5 to 2 micron. In further embodiments, in any of the above embodiments, the lower MMAD limit is 0.1 micron. In further embodiments, in any of the above embodiments, the lower MMAD limit is 0.9 micron.

In an embodiment, the particle size geometric standard deviation (GSD), preferably for any of the above MMAD embodiments, is of about 3 micron or lower, more preferably of about 2 micron or lower, and even more preferably it is monodisperse or as close as possible to monodispersity. Monodisperse GSD can be attained by methods known in the art, such as those described in Usrnani et al., J Appl Physiol (1985), 2003 Nov;95(5):2106-12.

In the context of the present invention, the MMAD and GSD can be measured using an impactor as described in United States Pharmacopeia Convention, Inc., Rockville, MD at General Chapter<1601 > Products for nebulization — characterization tests; USP35-NF30 Page 942; or <601 > Aerosols, nasal sprays, metered-dose inhalers, and dry powder inhalers; USP35-NF30 Page 232.

The pharmaceutical compositions suitable for inhalation of the present invention possess the above described MMAD values. However, it is possible that a pharmaceutical composition (which includes excipients such as a dispersion medium) comprises compound particles that possess a mean size lower than the actual MMAD value of the pharmaceutical composition particles. For instance, nebulized droplets may possess a MMAD of 1 micron, but the compound particles within said droplets may be of a smaller mean size. As described hereinbelow, the present inventors have found that compound particles in the submicron range can be obtained.

Thus, in an embodiment, the precursor composition or the pharmaceutical composition suitable for inhalation of the invention comprises particles of avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, possessing a mean size of between 0.1 and 2 micron, in particular of between 0.1 and 1 micron, more particularly of between 0.1 and less than 0.5 micron, such as between 0.1 and 0.2 micron. In an embodiment, these mean sizes refer to the size of the particles comprising or consisting of the compound and the solubilizing aid, such as the complex formed by the compound and the cyclodextrin and/or amphiphilic agent (in micelle or non-micelle form), as described further bellow. In a particularly preferred embodiment, these mean sizes refer to the size of the particles comprising or consisting of the compound, the cyclodextrin and the amphiphilic agent. In a most particularly preferred embodiment, these mean sizes refer to the size of the particles comprising or consisting of ivermectin, HP-[3-CD and lecithin. Mean sizes can be calculated by photon correlation spectroscopy as described in the Examples.

In an embodiment, the pharmaceutical compositions of the invention, or their precursor compositions, comprise an amount of avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in the range from 0.01 wt% to 95 wt%, preferably from 0.5 wt% to 50 wt%, with respect to the total weight of the composition.

In the context of the present invention, a precursor composition refers to a composition which can be aerosolized into the compounds or pharmaceutical compositions of the present invention. Thus, in another aspect, the invention is also directed to a pharmaceutical composition according to the present invention which is not in a form suitable for inhalation, and more particularly not suitable for inhalation into the lungs. Thus, the precursor composition comprises the same components as the pharmaceutical composition, but they are in a form not suitable for inhalation to the lungs.

The pharmaceutical compositions of the invention may be in any form suitable for inhalation into the lungs, but are typically solids, such as powders; or liquids (typically in the form of droplets), such as suspensions, emulsions or solutions; in the form of an aerosol. Aerosolization may be achieved in a variety of manners, such as through the use of an inhaler device. Non-limiting examples of suitable inhaler devices are nebulizers, powder inhalers (aka dry powder inhalers or DPIs), or metered- dose inhalers (MDIs, aka pressurized metered dose inhalers or pMDIs).

In a preferred embodiment, aerosolization is achieved through the use of a nebulizer. Nebulizers are capable of generating droplets of the compound or pharmaceutical composition of the present invention from a liquid precursor composition in a form not suitable for inhalation. Non-limiting examples of nebulizers suitable for the purposes of the present invention are jet, ultrasonic, or vibrating mesh nebulizers.

Jet nebulizers are based on Venturi’s principle which states that fluid pressure decreases as its passes through a narrow sectional area. In these nebulizers, air stream moves through a small capillary tube at high velocity creating a low pressure that drives the liquid to be aerosolized up the capillary tube. The high velocity blast of air carrying the droplets will bump into baffles placed in different numbers and positions depending on the design of the jet nebulizer. The impaction of large droplets on these baffles either break them into smaller sized droplets that will leave the nebulizer or will retain them in the device to be re-nebulized until their size is small enough to leave the nebulizer. Baffles also reduce the velocity of the aerosol cloud emitted from the nebulizer, which reduces impaction in the oropharyngeal region when inhaled by the patient.

In ultrasonic nebulizers, sound waves are created due to the vibration of piezoelectric crystals at high frequency, creating crests that break the liquid into small droplets.

Vibrating mesh nebulizers have a mesh plate that, when it vibrates through the action of a piezoelectric element, breaks the liquid into very fine droplets.

In another embodiment, aerosolization is achieved through the use of a metered-dose inhaler. MDIs are capable of generating droplets of the compound or of the pharmaceutical composition of the present invention from a liquid precursor composition in a form not suitable for inhalation.

An MDI comprises three major components: the canister which is produced in aluminium or stainless steel by means of deep drawing, where the formulation resides; the metering valve, which allows a metered quantity of the formulation to be dispensed with each actuation, and an actuator (or mouthpiece) which allows the patient to operate the device and directs the aerosol into the patient's lungs. To use the inhaler the patient presses down on the top of the canister, with their thumb supporting the lower portion of the actuator. Actuation of the device causes the precursor formulation to decompress within the metering valve, which leads to an explosive generation of aerosol droplets according to the present invention. The propellant typically evaporates after aerosol formation.

When aerosolized with an MDI, the precursor composition of the invention comprises a propellant instead of a pharmaceutically acceptable inhalation excipient. Since the propellant typically evaporates upon aerosolization, the aerosol becomes an aerosol consisting of the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, suitable for inhalation, in particular into the lungs, and a gas, such as air, suitable for inhalation, in particular into the lungs; such an aerosol is also meant to be encompassed in the present invention. In any of these embodiments, the precursor composition and the aerosol can still comprise a pharmaceutically acceptable inhalation excipient, which is additional to the propellant or the aerosol gas. It is further understood that in any of the embodiments described herein, an aerosol necessarily requires the presence of a gas, such as air, which in particular is suitable for carrying the particles of the compounds or pharmaceutical compositions of the invention. In a preferred embodiment, the MDI comprises an aerochamber, valve holding chamber or spacer. The use of an aerochamber, valve holding chamber or spacer can minimize the amount of particles of the aerosol that are off-target. Non-limiting examples of suitable propellants include chlorofluorocarbons (CFCs) such as dichlorodifluoromethane, trichlorofluoromethane, and dichlorotetrafluoroethane; hydrofluoroalkanes (HFAs), such as 1 ,1 , 1 ,2- tetrafluoroethane, 1 ,1 ,1 ,2,3,3,3-heptafluoro-n-propane; and carbon dioxide; or mixtures thereof.

However, in a preferred embodiment, the precursor composition, the pharmaceutical composition or the inhaler device of the invention does not comprise a propellant.

In another embodiment, aerosolization is achieved through the use of a powder inhaler. DPIs are capable of generating particles of the compound or of the pharmaceutical composition of the present invention from a powder precursor composition in a form not suitable for inhalation, typically in the form of aggregates comprising particles of the avermectin or milbemycin. When using a DPI, the pharmaceutical composition does not necessarily comprise a pharmaceutically acceptable inhalation excipient, hence DPIs are also capable of generating a particle consisting of avermectin or milbemycin as described elsewhere herein in a form suitable for inhalation or an aerosol consisting of such particles and a gas such as air, suitable for inhalation, in particular to the lungs.

As regards the aerosolization process, the operator puts the mouthpiece of the inhaler into their mouth and takes a sharp, deep inhalation to entrain the precursor composition powder from the device and subsequently break-up the powder particle aggregates into particles that are small enough to reach the lungs.

Inhaler devices of the kinds described above suitable for providing the pharmaceutical composition of the present invention are described in WO2006125132; Ibrahim et al., Medical Devices: Evidence and Research, 2015, 8:131 — 139; or Gardenhire et al., A Guide to Aerosol Delivery Devices for Respiratory Therapists, 4th Edition, 2017, the American Association for Respiratory Care. Inhaler devices suitable for generating aerosols in the lower MMAD particle range are commonly referred to as ultra-fine or extra-fine, or similar, in the art.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable inhalation excipient. The term “pharmaceutically acceptable inhalation excipient” refers to an excipient which is tolerated physiologically by, and is preferably not toxic to, the respiratory system, in particular the lungs, when inhaled in a form suitable for inhalation, e.g. when aerosolized. Unless specifically defined, a pharmaceutically acceptable inhalation excipient is not necessarily in a form suitable for inhalation.

The term “excipient” refers to components of the pharmaceutical composition other than the active ingredient (herein the avermectin or milbemycin, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, or simply “the compound”). They preferably include a "carrier, adjuvant and/or vehicle". Carriers improve the delivery of the active ingredient to the target site of action, herein the lungs. An adjuvant is a substance added to the pharmaceutical composition that affects the action of the active ingredient in a predictable way. Vehicle is a substance, preferably without therapeutic action, used as a medium to give bulk to the pharmaceutical composition (Stedman's Medical Spellchecker© 2006 Lippincott Williams & Wilkins).

Examples of pharmaceutically acceptable inhalation excipients are disclosed in Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) and include diluents such as lactose, sucrose, dicalcium phosphate; lubricants such as magnesium stearate; binders such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose, cellulose derivatives; wetting agents, emulsifying agents, solubilizing agents, and/or pH buffering agents.

When the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, emulsion or suspension, the composition comprises the avermectin or milbemycin, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, dissolved in a solvent or dispersed in an emulsion or suspension medium. It is understood that the solvent or emulsion or suspension medium must be a pharmaceutically acceptable inhalation excipient. The pharmaceutical composition of the invention will typically also comprise said solvent or emulsion or suspension medium following aerosolization.

The solvent or emulsion or suspension medium may comprise an aqueous fraction, such as water or aqueous saline, and an organic fraction such as an alcohol such as ethanol, isopropyl alcohol, benzyl alcohol, glycerol, a glycol such as propylene glycol or polyethylene glycol, dimethyl sulfoxide (DMSO) or mixtures thereof; or the solvent or emulsion or suspension medium may consist of either of said two fractions, i.e. it is aqueous or organic. Preferably, the solvent or emulsion or suspension medium comprises an alcohol as described above or DMSO. More preferably, the solvent or emulsion or suspension medium comprises an alcohol as described above. Preferably the alcohol is ethanol or benzyl alcohol, and more preferably it is ethanol. In a particular embodiment, the solvent or emulsion or suspension medium comprises benzyl alcohol. In a particular embodiment, the solvent or emulsion or suspension medium comprises DMSO. In another preferred embodiment, the solvent or emulsion or suspension medium comprises a glycol, such as propylene glycol, in an amount ranging from 1 to 20%, more preferably from 5 to 15% with respect to the total volume of the solvent or medium, and optionally the remainder of the volume comprises or consists of an aqueous fraction, such as water.

In an embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises the avermectin or milbemycin, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, in an amount ranging from 1 to 500 mg/10 mL, more preferably from 5 to 300 mg/10 mL, even more preferably from 5 to 150 mg/10 mL.

In another embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a solid, such as a powder, and comprises the avermectin or milbemycin, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, in an amount ranging from 0.1 to 40% wt., particularly from 1 to 30% wt., more particularly from 5 to 25% wt., even more particularly from 8 to 21 % wt., with respect to the total weight of the composition.

Preferably, the avermectin or milbemycin, or pharmaceutically acceptable salt, solvate or stereoisomer thereof, is dissolved in the solvent. Dissolution of the avermectin or milbemycin can be achieved either by employing a solvent which itself dissolves the avermectin or milbemycin; or by employing a solvent which itself cannot dissolve or poorly dissolves the avermectin or milbemycin, and to which further pharmaceutically acceptable inhalation excipients are added to enhance the dissolution of the avermectin or milbemycin in said solvent.

In a preferred embodiment, the precursor composition of the invention comprises an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an organic solvent as described above, preferably an alcohol or DMSO, more preferably an alcohol, which is preferably ethanol or benzyl alcohol and is more preferably ethanol. In another embodiment, the precursor composition of the invention consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an organic solvent as described above, optionally mixed with an aqueous fraction.

In a preferred embodiment, the pharmaceutical composition suitable for inhalation of the invention comprises an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an organic solvent as described above, preferably an alcohol or DMSO, more preferably an alcohol, which is preferably ethanol or benzyl alcohol and is more preferably ethanol. In another embodiment, the pharmaceutical composition of the invention consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an organic solvent as described above, optionally mixed with an aqueous fraction. In such compositions, the solvent comprises less than 40%, preferably less than 10%, more preferably less than 1 %, even more preferably less than 0.1 % by weight aqueous fraction as described above, or the solvent does not comprise an aqueous fraction. In an embodiment the aqueous fraction is water. In an embodiment, the solvent is devoid of aqueous fraction.

In such compositions, the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, can be partly or fully dissolved without the aid of further pharmaceutically acceptable inhalation excipients that enhance the dissolution of the avermectin or milbemycin in said solvent.

Preferably, the amount by weight of the avermectin or milbemycin with respect to the total weight of such compositions is between 0.01 and 20 wt%, more preferably between 0.1 and 5 wt%, and even more preferably between 0.5 and 2 wt%. Preferably, the pharmaceutical composition is in the form of an aerosol, and in particular the aerosol is obtained by nebulization of the precursor composition.

In another preferred embodiment, the precursor composition of the invention comprises or consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and a cyclodextrin. In an embodiment, the precursor composition is a liquid, such as a solution, or emulsion or suspension, wherein the avermectin or milbemycin is dissolved or dispersed. The liquid must be suitable for aerosolization into the pharmaceutical composition of the invention. The solvent, or emulsion or suspension medium may be selected from those described above, but is preferably aqueous, and is even more preferably water. In this embodiment, the cyclodextrin acts as solubilizing aid.

Thus, in a preferred embodiment, the pharmaceutical composition suitable for inhalation of the invention comprises or consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and a cyclodextrin, and optionally a solvent, or emulsion or suspension medium as described above. In these compositions, the avermectin or milbemycin is complexed to the cyclodextrin, in particular they form of an inclusion complex, in particular wherein the avermectin or milbemycin resides within the interior of the cyclodextrin. In these compositions, the avermectin/milbemycin-cyclodextrin complex is dissolved or dispersed in the solvent, or emulsion or suspension medium, and more preferably it is dissolved in the solvent.

Cyclodextrins suitable in the context of the present invention are a-, [3- or y-cyclodextrins or cyclodextrins comprising by more than eight units of a-1 ,4- glucopyranose, such as up to 32 units. The cyclodextrin may be unsubstituted, or it may be substituted, in particular by derivatizing the -OH groups of the cyclodextrin. Non-limiting examples of substituted cyclodextrins are derivatives wherein the -OH groups of the cyclodextrin are partly or entirely replaced by -OR groups wherein R is selected from C1-6 alkyl- such as methyl-, which may be unsubstituted or substituted such as in hydroxy-Ci-e alkyl- such as hydroxyethyl- or hydroxypropyl-, or in carboxy-Ci-6 alkyl- such as carboxymethyl-; acyl- such as acetyl-, succinyl-, benzoyl-, palmityl-; sulfonyl- such as toluenesulfonyl-; or by a phosphate or sulfate group, which may be C1-6 alkylated; or by a saccharide, such as glucosyl- or maltosyl-.

Preferably, the cyclodextrin is a a-, [3- or y-cyclodextrin, more preferably it is (3-cyclodextrin.

Preferably, the substituted cyclodextrin is selected from derivatives wherein the -OH groups of the cyclodextrin are partly or entirely replaced by -OR groups wherein R is selected from C1-6 alkyl- or hydroxy-Ci-e alkyl derivative. In a preferred particular embodiment, the cyclodextrin is hydroxypropyl-[3-cyclodextrin (HP-[3-CD) or methyl-(3-cyclodextrin (M-[3-CD), more preferably it is HP-[3-CD. In a particular embodiment, it is 2-hydroxypropyl-[3-cyclodextrin. It was found that HP-[3-CD was particularly exceptional at solubilizing the compounds of the invention.

Cyclodextrins of the kind described above are well-known to the person skilled in the art and are reported inter alia in Rezanka, Cyclodextrin Fundamentals, Reactivity and Analysis (Environmental Chemistry for a Sustainable World Book 16), Springer, 2018, 57-103; or are commercially available such as from Sigma-Aldrich (Ref. no. C4642, C4680, C4767, C4805, C4892, C4930, H107, C0926, H5784, H125, C4555 or M7439).

The precursor composition of the present invention comprising the avermectin or milbemycin and the cyclodextrin can be prepared by mixing the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, the cyclodextrin, and any pharmaceutically acceptable inhalation excipient. The mixing may be performed in the presence or absence of a solvent or emulsion or suspension medium.

When performed in the absence of a solvent or emulsion or suspension medium, the mixing can be by dry grinding or kneading. Grinding methods for attaining ultrafine solids of a size suitable for inhalation into the lungs are known in the art, such as from US 2001016467. The obtained ultrafine solids are suitable for use as precursor compositions in powder inhalers as described above.

However, the mixing is preferably in the presence of a solvent or emulsion or suspension medium. In such processes, the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, the cyclodextrin, and any other pharmaceutically acceptable inhalation excipient are added to the solvent or emulsion or suspension medium and mixed until the formation of cyclodextrin-avermectin/milbemycin complex is detected by standard analytical techniques such as HRMS, ¹H NMR or DSC. Where the starting materials are not initially dissolved, e.g. as is the case of avermectin or milbemycin in aqueous media, the mixing is continued until a solution is achieved. If the solvent employed is a pharmaceutically acceptable inhalation excipient, the obtained solution may be directly used as a precursor composition, such as in nebulizers or MDIs. Alternatively, if the solvent is not a pharmaceutically acceptable inhalation excipient, or if the formed cyclodextrin- avermectin/milbemycin complex is to be stored until future use, the solvent may be eliminated, such as by freeze-drying, spray-drying, filtration or evaporation. The obtained powder may be ground to ultrafine sizes as described above and used as precursor composition for aerosolization such as with a powder inhaler, or re-dissolved or dispersed in a pharmaceutically acceptable inhalation solvent or medium for use as precursor composition for aerosolization such as with a nebulizer or MDI. Methods that are suitable for forming such precursor compositions are those described in EP 0930077 B1 . Solvents suitable for these processes can be any of those mentioned elsewhere herein, such as alcohols or mixtures thereof with water as described above, such as 90% ethanol. Additional agents which are pharmaceutically acceptable inhalation excipients and further aid in the dissolution of the avermectin or milbemycin, such as saccharides such as glucose or lactose, may be added to the reaction mixture. The aerosolization provides the pharmaceutical composition of the invention.

In an embodiment, the molar ratio of cyclodextrin to avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, is in the range from 1 :10 to 20: 1 , preferably in the range 1 :1 to 10:1 , in particular in the range 1 :1 to 4: 1 , more particularly it is 1 : 1 .

In an embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises the cyclodextrin in an amount ranging from 0.01 to 20% w/v, particularly from 0.01 to 15% w/v, more particularly from 0.04 to 10% w/v. % w/v refers to grams of the indicated substance per 100 mL of the liquid composition.

In an embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a solid, such as a powder, and comprises the cyclodextrin in an amount ranging from 40 to 95% wt., particularly from 60 to 85%, with respect to the total weight of the composition.

In a particularly preferred embodiment, the precursor composition or the pharmaceutical composition suitable for inhalation comprising a cyclodextrin as described in any embodiment herein further comprises an amphiphilic agent. In an embodiment, this precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, wherein the avermectin or milbemycin is dissolved or dispersed. The solvent, or emulsion or suspension medium may be selected from those described above, but is preferably aqueous, and is even more preferably water.

In another embodiment, this precursor composition or pharmaceutical composition suitable for inhalation is a solid, such as a powder. As explained above, the precursor composition can be in a form not suitable for inhalation to the lungs, for instance it may be in the form of a powder comprising particles of compound or of compound-cyclodextrin complex or of compound-cyclodextrin- amphiphilic agent complex possessing a mean size greater than those required to achieve a MMAD suitable for inhalation. In such cases, the precursor composition in powder form can be converted into the pharmaceutical composition of the invention by different means described elsewhere herein, such as by using grinding methods, such as that described in US 2001016467; or by dissolving or dispersing the powder in a solvent, or emulsion or suspension medium; and aerosolizing. It has been observed that the production of the pharmaceutical composition suitable for inhalation in liquid form can be directly and efficiently produced from such compositions in powder form with larger than required particles when said powder comprises the amphiphilic agent and has been obtained by a method as described below. Such powder compositions therefore represent an additional aspect of the present invention. Likewise, precursor or pharmaceutical compositions suitable for inhalation in liquid form obtained from said powders are also part of the present invention. Thus, in a particular embodiment, the precursor composition or the pharmaceutical composition suitable for inhalation of the invention comprises particles comprising or consisting of the compound, the cyclodextrin and the amphiphilic agent, possessing a mean size of between 0.5 and 2 micron, in particular of between 0.9 and 1 .6 micron,

In an embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises the amphiphilic agent in an amount ranging from 0.001 to 5% w/v, particularly from 0.01 to 1 % w/v, more particularly from 0.04 to 0.2% w/v. In a particular embodiment, the amount of amphiphilic agent is below the critical micelle concentration (CMC).

In another embodiment, the amount ratio in w/v% of cyclodextrin to amphiphilic agent ranges from 0.1 :1 to 200: 1 , such as from 10:1 to 200: 1 .

In another embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a solid, such as a powder, and comprises the amphiphilic agent in an amount ranging from 0.1 to 25% wt., particularly from 1 to 20% wt., more particularly from 5 to 15 wt%., with respect to the total weight of the composition.

Typically, the amphiphilic agent is a surfactant, a block co-polymer, or a mixture thereof.

In an embodiment, the amphiphilic agent is a surfactant, which is preferably a phospholipid. In phospholipids, a relatively hydrophilic moiety comprises a glycerol group attached to a phosphate group (phosphoether linkage), and more particularly to a phosphate group which may in turn be attached to a further alcohol group (phosphoether linkage), such as to a choline, ethanolamine or serine group. A relatively hydrophobic moiety of the phospholipid comprises a fatty acid residue, which is attached to the glycerol (ester linkage). Typically, the glycerol portion of the phospholipid is attached to two fatty acid residues and a phosphate group as described above. Typically, the fatty acid residue is a long chain fatty acid residue, such as a Cs-26 fatty acid residue, such as a lauric acid, myristic acid, palmitic acid, or oleic acid residue.

Non-limiting examples of suitable phospholipids are 1 ,2-dimyristoyl-sn- glycero-3-phosphocholine, 1 ,2-dilauroyl-sn-glycero-3-phosphocholine, 1 ,2- distearoyl-sn-glycero-3-phosphocholine, 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dipalmitoyl-sn-glycero-3-phosphorcholine, 1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3- phosphate monosodium salt, 1 ,2-dimyristoyl-sn-glycero-3-[phospho-L-serine], or 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-glutaryl sodium salt.

Phospholipids are well-known in the art and are commercially available, such as from Sigma-Aldrich (e.g Refs P2663, P5693, or 429415).

In a particularly preferred embodiment, the phospholipid is 1 ,2-dipalmitoyl- sn-glycero-3-phosphocholine, aka DPPC. DPPC is one of the main components of pulmonary surfactant and is recognized as a generally recognized as safe (GRAS) excipient for pulmonary drug delivery by the US Food and Drug Administration. DPPC is commercially available, such as from Sigma-Aldrich (Ref. P4329; CAS 63-89-8); or may be synthesized in the laboratory such as described in Singh, Journal of Lipid Research, 1990, 31 :1522-1525.

In a most preferred embodiment, the phospholipid is lecithin, such as soybean lecithin and more particularly soybean L-a-lecithin.

Other surfactants that may be employed in the context of the present invention are sphingolipids or Vitamin E TPGS.

However, in an embodiment, the precursor or pharmaceutical composition of the present invention does not comprise a polysorbate, in particular it does not comprise polysorbate 80 (aka polyoxyethylene (20) sorbitan monooleate), and more particularly it is not an emulsion comprising of any of these, even more particularly it is not an emulsion comprising any of these in an aqueous emulsion medium.

In an embodiment, the amphiphilic agent is a block co-polymer. Amphiphilic block copolymers comprise a relatively hydrophobic polymeric block and a relatively hydrophilic polymeric block. The block co-polymer comprises at least two polymeric blocks, and is typically a di- or tri-block copolymer.

In an embodiment, the polymeric blocks can be selected from; poly(alkylene oxide), such as polyethylene oxide) (herein also meant to comprise polyethyleneglycol) or polypropylene oxide); poly(alkylene amine), such as polyethylene imine); polyester such as polycaprolactone or poly(lactic acid); poly(A/-vinylpyrrolidone); polyacrylate, in particular polyalkylacrylate or an alkyl ester thereof, such as poly(methylmethacrylate); poly(amino acid), such as poly(aspartic acid); or polysaccharides, such as chitosan.

Non-limiting examples of suitable amphiphilic block copolymers are poly(ethylene glycol)-poly(lactic acid), poly(ethylene glycol)-poly(s- caprolactone), poly(lactic acid)-chitosan, poly(ethylene glycol)-poly(ethylene imine); or poloxamers. Preferably, the amphiphilic block copolymer is a poloxamer.

Such blocks and polymers can be prepared by standard polymerization chemistry from the monomeric building blocks, which are readily commercially available such as from Sigma-Aldrich (Refs. L1750, A9256) or directly commercially acquired, such as from Sigma-Aldrich (Refs. 440744, 181986).

In a particularly preferred embodiment, the cyclodextrin is a (3-cyclodextrin, in particular a hydroxy-Ci-6 alkyl (3-cyclodextrin, such as hydroxypropyl-[3- cyclodextrin and more particularly 2-hydroxypropyl-[3-cyclodextrin; and the amphiphilic agent is a phospholipid, in particular the amphiphilic agent is lecithin, such as soybean lecithin and more particularly soybean L-a-lecithin.

The present invention also provides a method for obtaining said compositions comprising or consisting of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; a cyclodextrin; and an amphiphilic agent; comprising the steps of: i) Preparing a solution of cyclodextrin, preferably in an aqueous or hydroalcoholic solvent, more preferably in water; ii) Adding, to the solution of cyclodextrin obtained in step i, the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, to obtain a compound-cyclodextrin solution or dispersion; iii) Preparing a solution or dispersion of an amphiphilic agent, preferably in an aqueous or hydroalcoholic solvent or dispersion medium, more preferably in water; iv) Combining the compound-cyclodextrin solution or dispersion with the solution or dispersion of the amphiphilic agent.

In an embodiment, the hydroalcoholic solvent employed in step i) is a water and ethanol mixture, such as a solvent comprising between 10 and 100 mg/dL of ethanol in water, in particular 50 mg/dL of ethanol in water.

In an embodiment, step ii) comprises firstly dissolving the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in a glycolic solvent such as propylene glycol, and then mixing the obtained glycolic solution with the solution of cyclodextrin obtained in step i),

In an embodiment, obtaining the compound-cyclodextrin solution or dispersion of step ii) comprises stirring, such as for a time of between 1 minute and a day, more preferably between 5 minutes and an hour, particularly for about 30 minutes.

In an embodiment, a dilution medium is added after step iv). This allows attaining the desired concentration of the different components in the composition. The dilution medium is preferably water.

In an embodiment, as was mentioned further above, the composition obtained in step iv) is dried to remove solvent or dispersion or dilution medium therefrom, preferably until a powder is obtained. It has surprisingly been found that producing a pharmaceutical composition suitable for inhalation of the present invention from such powder allows administering high amounts of the compound of the invention.

The drying is performed by means known in the art such as by freeze- drying, spray-drying, filtration or evaporation, preferably by spray-drying, and the spray-drying preferably comprises adding a protecting agent to the composition obtained in step iv) before or during drying. The protecting agent acts as a bulking agent to facilitate the drying of particles in an efficient way. Non-limiting examples of protecting agents are saccharides, such as lactose, mannitol, sucrose, maltodextrin, glucose, sorbitol; preferably, the protecting agent is mannitol.

Thus, in an embodiment, the precursor composition or pharmaceutical composition suitable for inhalation of the invention further comprises a protecting agent.

In an embodiment, the precursor or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises the protecting agent in an amount ranging from 1 to 100 mg/20 mL, particularly from 30 to 70 mg/20 mL, more particularly from 45 to 55 mg/20 mL.

In another embodiment, the precursor or pharmaceutical composition suitable for inhalation is a solid, such as a powder, and comprises the protecting agent in an amount ranging from 0.1 to 20% wt., particularly from 1 to 10% wt., more particularly from 2 to 6 wt%., with respect to the total weight of the composition.

These compositions comprising or consisting of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; a cyclodextrin; and an amphiphilic agent; have been found to be particularly stable upon storage. Furthermore, these compositions have been found to comprise particles comprising compound, cyclodextrin and amphiphilic agent, wherein said particles possess greater porosity and specific surface area than particles of compound alone.

Thus, in an embodiment, these compositions comprise particles comprising compound, cyclodextrin and amphiphilic agent, wherein said particles possess a specific surface area of greater than 1500 m²/g, preferably greater than 1750 m²/g; such as from any of these values to 2500 m²/g, in particular to 2000 m²/g. Specific surface area can be calculated according to the Brunauer, Emmett and Teller (BET) volumetric method described in the Examples.

In an embodiment, these compositions comprise particles comprising compound, cyclodextrin and amphiphilic agent, wherein said particles possess a pore mean diameter of from 0.5 to 0.6 pm. Pore mean diameter can be calculated with a mercury porosimeter as described in the Examples.

In an embodiment, these compositions possess a polydispersity index (PDI) lower than 0.3, such as between 0.3 and 0.06. PDI can be calculated by photon correlation spectroscopy as described in the Examples.

In an embodiment, these compositions possess a negative zeta potential, such as ranging from -40 to -60 mV. Zeta potential can be calculated by electrophoretic laser Doppler anemometry as described in the Examples.

In a preferred particular embodiment, the precursor composition or pharmaceutical composition suitable for inhalation comprises ivermectin or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; HP-[3-CD; and lecithin. More particularly, the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises:

- Ivermectin or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in an amount ranging from 5 to 150 mg/10 mL;

- HP-[3-CD, in an amount ranging from 0.01 to 15% w/v;

- Lecithin, in an amount ranging from 0.01 to 1 % w/v.

In a particular embodiment, the precursor composition or pharmaceutical composition suitable for inhalation is a liquid, such as a solution, or emulsion or suspension, and comprises:

- Ivermectin or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, in an amount ranging from 5 to 40 mg/10 mL;

- HP-[3-CD, in an amount ranging from 5 to 15% w/v;

- Lecithin, in an amount ranging from 0.01 to 0.2% w/v.

In another preferred embodiment, the precursor composition of the invention comprises or consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an amphiphilic agent. In a particular embodiment, the amphiphilic agent is in the form of a micelle. In an embodiment, the precursor composition is a liquid, such as a solution, or emulsion or suspension, wherein the avermectin or milbemycin is dissolved or dispersed. The liquid must be suitable for aerosolization into the pharmaceutical composition of the invention. The solvent, or emulsion or suspension medium may be selected from those described above, but is preferably aqueous, and is even more preferably water. In this embodiment, the micelle acts as solubilizing aid.

Thus, in a preferred embodiment, the pharmaceutical composition suitable for inhalation of the invention comprises or consists of an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and an amphiphilic agent which is optionally in the form of a micelle, and optionally a solvent, or emulsion or suspension medium as described above.

In these compositions comprising a micelle, the avermectin or milbemycin is complexed to the micelle, and in particular it is found within the micelle. When the micelle is in or prepared from an aqueous solvent or dispersion medium, the hydrophilic heads of the amphiphilic agent(s) forming the micelle form an outer layer or shell which is in contact with the aqueous medium, whereas the hydrophobic tails of said amphiphilic agent(s) assemble away from the aqueous environment into an oily core, at which the avermectin or milbemycin is found. In these compositions, the avermectin/milbemycin-micelle complex is dissolved or dispersed in the solvent or emulsion or suspension medium, and is preferably dissolved therein. Thus, a micelle according to the present invention comprises or consists of the amphiphilic agent which forms a shell, wherein the amphiphilic agent comprises a relatively hydrophilic moiety and a relatively hydrophobic moiety (relative to each other), wherein the hydrophilic moiety faces the outside of the micelle, and the hydrophobic moiety faces the inside of the micelle, forming a hydrophobic or oily core. The micelle preferably comprises the amphiphilic agent in an amount of at least 80% wt., preferably at least 90% wt., with respect to the total weight of the micelle.

In these embodiments, the term amphiphilic agent has the same meaning as was described further above. The precursor composition comprising the avermectin or milbemycin and the micelle can be prepared by different methods that are well known to the skilled person, such as direct dissolution, dialysis, dry-down, or lyophilization. Suitable methods are reported in Cholkar et al., Recent Patents on Nanomedicine, 2012, 2(2):82-95; or in Batrakova, Nanoparticulates as Drug Carriers, Imperial College Press, 2016, 57-93.

In a preferred embodiment, the precursor composition is prepared by dialysis or dry-down.

Dialysis comprises dissolving the block copolymer in an organic solvent such as an alcohol as described above, DMSO, N,N-dimethylformamide (DMF), acetonitrile, tetrahydrofuran (THF), or acetone; adding the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; and then slowly adding, such as adding dropwise, an aqueous fraction as described above. The formed micelles are then dialyzed against water, such as deionized water, to eliminate the organic solvent. The organic solvent is preferably an alcohol as described above or DMSO, and the alcohol is preferably ethanol.

Dry-down processes (aka solvent evaporation processes) comprise dissolving the avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, and the block copolymer in a solvent, preferably an organic solvent such as an alcohol as described above; evaporating the solvent under stirring to form an avermectin/milbemycin-copolymer film; and then adding an aqueous solvent as described above, such as phosphate buffer, which is preferably warmed to above room temperature (above 25°C), to reconstitute (i.e. hydrate) the film, preferably under agitation or sonication, to thus form the micelles.

In either of the above two processes, the obtained aqueous solution of avermectin/milbemycin-comprising micelles may be used as a precursor composition, such as in nebulizers or MDIs. Alternatively, if the micelles are to be stored until future use, the aqueous medium may be eliminated, such as by freeze-drying, or spray-drying. The obtained powder may be ground to ultrafine sizes as described above and used as precursor composition for aerosolization such as with a powder inhaler, or re-dissolved or dispersed in a pharmaceutically acceptable inhalation solvent or medium for use as precursor composition for aerosolization such as with a nebulizer or MDI.

It is understood that, in any of the methods for preparing micelles described above, the amphiphilic agent is added in amounts sufficiently high to enable constituting the micelle, i.e. in amounts equal to and usually above the critical micelle concentration (CMC). What constitutes the CMC will depend on the specific amphiphilic agent, solvents, and technique employed in each case, and can be easily determined by simple experimentation or reference to similar systems.

In another aspect, the present invention is directed to an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or to a pharmaceutical composition or inhaler device, according to the present invention; for use in medicine.

The use in medicine is particularly for preventing or treating a parasitic infection, cancer, viral infection, an NF-KB-related disease or disorder, or an a7 nAChR-related disease or disorder.

The treatment of parasitic infections is not limited to pulmonary infections or to infections of other sites of the body which depend on a pulmonary invasion step in the life cycle of the parasite causing the infection. This is because the present invention demonstrates that the avermectin or milbemycin can reach the systemic circulation following the pulmonary administration of the ivermectin.

Thus, in an embodiment, the parasitic infection is a systemic, intestinal or pulmonary parasitic infection. Preferably, it is a systemic or pulmonary infection. More preferably, it is a pulmonary infection.

In an embodiment, the parasitic infection is an infection which requires a pulmonary invasion step in the life cycle of the parasite causing the infection. In an embodiment, the parasite is selected from nematodes, such as Ancylostoma duodenale, Necator americanus, Ascaris lumbricoides, Strongyloides stercoralis, Brugia malayi, Loa loa, Toxocara genus; trematodes, such as Paragonimus, Schistosoma genus; and Cestodes, such as Equinococcis genus.

In an embodiment, the medical use is the prevention or treatment of eosinophilia secondary to a parasitic infection. Examples of such diseases include Loeffler's syndrome and tropical pulmonary eosinophilia.

In an embodiment, the parasitic infection is an ectoparasitic infestation, in particular caused by an ectoparasite selected from arthropods, such as mosquitos, preferably of the Anopheles and Aedes genus; mites, such as trombiculid or sarcoptes genus; triatomine bugs, blackflies, sandflies, botflies, Tunga fleas, ticks or head lice. The term ectoparasite encompasses both species which themselves are the parasite, such as haematophages or histophages, and species which are vectors of disease-causing pathogens.

In an embodiment, the cancer is selected from those susceptible to treatment or adjuvant therapy by inducing mitochondrial dysfunction, oxidative stress, modulation of P2X4/P2X7/Pannexin-1 to extracellular ATP, WNT-TCF pathway, pharmacoenhancement via P-gp or CYP interaction.

In particular, the cancer is selected from a carcinoma, sarcoma, melanoma and leukemia. More particularly, it is selected from prostate cancer, glioblastoma, glioma, ovarian cancer, breast cancer, colon cancer, pancreatic cancer, prostate cancer, renal cancer, lung cancer, intestine cancer and head and neck cancer.

In an embodiment, the viral disease is caused by a virus the replication of which relies on the interaction between integrase protein (IN) and the importin (IMP) a/[31 heterodimer. More particularly, it is the importation of the viral preintegration complex into the nucleus that is dependent on said interaction. Nonlimiting examples of such viruses include the HIV, SV40, Dengue, West Nile, Venezuelan equine encephalitis, Zika, SARS-CoV or PRV virus. Preferably, the virus is an RNA-virus, even more preferably it is a single-stranded RNA virus. In an embodiment, the viral disease is caused by a virus the replication of which relies on a helicase. Non limiting examples include Semliki Forest virus, SARS-CoV-2, Japanese encephalitis virus or West Nile virus.

In an embodiment, the NF-KB-related inflammatory or immune disease or disorder is one which is mediated by NF-KB dysfunction, preferably by NF-KB overactivation. Preferably, the NF-KB-related disease or disorder is one involving overactivation of the NF-KB non-canonical pathway. NF-KB is a heterodimeric transcription factor which is present in the cytosol of cells. When activated, NF- KB migrates into the nucleus of the cell and controls the expression of multiple genes involved in inflammatory and immune responses. Examples of such genes are those encoding proinflammatory cytokines TNF-a, IL-1 (3, IL-6 or IL-8. More particularly, the disease or disorder is an inflammatory or an autoimmune or alloimmune disease or disorder. More particularly, the NF-KB-related disease or disorder is selected from rheumatoid arthritis; multiple sclerosis; asthma; inflammatory bowel disease; colonic inflammation; chronic obstructive pulmonary disease; diabetes and obesity, in particular diabetes- or obesity-associated inflammation; transplantation rejection, in particular GVHD; liver injury and liver fibrosis; dermatitis; systemic lupus erythematosus; and psoriasis.

In an embodiment, the a7 nAChR-related disease or disorder is one that can be prevented or treated by allosteric modulation of said receptor. More particularly, the NF-KB-related disease or disorder is selected from Alzheimer’s disease, Parkinson’s disease and schizophrenia.

Preferably, the use in medicine is for the prevention or treatment of parasitic infections, cancer, viral infections, or NF-KB-related diseases or disorders, of the lungs.

Examples of parasitic infections of the lungs are pulmonary amebiasis, pulmonary leishmaniasis, pulmonary malaria, pulmonary babesiosis, pulmonary toxoplasmosis, ascariasis, hookworm, strongyloidiasis, syngamosis, dirofilariasis, tropical pulmonary eosinophilia, visceral larva migrans, trichinella, schistosomiasis, paragonimiasis, hydatid disease or rhinosporidiosis. Preferably, the parasitic infection is ascariasis, dirofilariasis or strongyloidiasis. Examples of viral infections of the lungs are those caused by adenoviruses, hantaviruses, SARS coronaviruses, cytomegaloviruses, HSV- zoster viruses, influenza viruses, measles viruses, parainfluenza viruses, respiratory syncytial viruses, human metapneumoviruses, nipah viruses, hemorrhagic fever viruses. In particular, the viral disease is caused by a virus the replication of which relies on the interaction between integrase protein (IN) and the importin (IMP) a/|31 heterodimer. More particularly, it is the importation of the viral pre-integration complex into the nucleus that is dependent on said interaction. Preferably, the virus is an RNA-virus, even more preferably it is a single-stranded RNA virus. Preferably, the virus is selected from hemorrhagic fever viruses, such as dengue virus. Most preferably, the virus is selected from SARS coronaviruses, in particular SARS-CoV-2. Thus, the present invention is useful in the prevention or treatment of COVID-19.

Examples of NF-KB-related diseases or disorders of the lungs are asthma; or chronic obstructive pulmonary disease.

In the context of the present invention, the avermectin or milbemycin is understood to be present or administered in therapeutically effective amounts. What is therapeutically effective depends on the clinical context. For instance, where a diseased state depends on attaining a specific viral or parasitic load threshold, a reduction or prevention of viral o parasitic spread to an extent keeping the load below said threshold will be therapeutically effective. Each disease is subject to specific clinical tests known to the person skilled in the art of medicine aimed at determining whether a therapeutic outcome has been achieved.

In an embodiment, the avermectin or milbemycin is administered by inhalation in a daily dose or in total daily doses in the range of between 1 mg/kg and 1000 mg/kg, preferably between 80 mg/kg and 150 mg/kg. These doses are those administered to Sprague Dawley rats, such as those of about 200 to 400 g weight. The present invention covers said doses as well as the equivalent doses and concentrations in humans. The skilled person knows how to convert rat doses into human doses, for instance following the Human Equivalent Dose Calculations established in the FDA’s “Guidance for Industry - Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” of July 2005, which is herein incorporated by reference. The physician will determine the most suitable dosage regime of the compounds and this will vary with the dosage form and the particular compound that is chosen, and it will furthermore vary with the patient undergoing treatment, e.g. with the age or medical history of the patient, or with the type of disease or condition that it being treated. Furthermore, the physician will determine which inhaler device is most suitable, and whether the use of an aerochamber is adequate or not, under the clinical circumstances.

As used herein, the term “treatment” or derivations thereof include the eradication, reversion, or control of the disease or disorder. The term “prevention” or derivations thereof refer to the avoiding or minimizing of the onset of the disease or disorder, such as through transmission from one subject to another; or its recurrence.

The subject which is treated is a human or animal, preferably a human subject. In a particular embodiment, the subject is a male. In another particular embodiment, the subject is a female.

Pulmonary administration by inhalation can be performed by inhalation into the lungs either via the mouth, the nose, or both. In an embodiment, the avermectin or milbemycin is administered locally to the lungs. In another embodiment, it is administered systemically via the lungs. In an embodiment, administration by inhalation lasts between 10 seconds and 1 hour, such as between 1 minute and 30 minutes, and more particularly between 5 and 15 minutes.

In a particular embodiment, the compounds and pharmaceutical compositions of this invention may be used together with other drugs to provide a combined therapy. The other drugs may form part of the same composition, or be provided as a separate composition for administration at the same time or at different time. The present invention is further understood by reference to the following examples which are presented as an illustration and are not intended to limit the present invention.

EXAMPLES

The following methods were employed in the different analyses mentioned in the following examples:

Ivermectin HPLC quantification

Ivermectin was quantified in an Agilent models 1100 series (Agilent Technologies, Waldbronn, Germany). The separation of ivermectin was carried out in a Kinetex® C18 column (150 x 46 mm, particle size 5 pm; Phenomenex, Inc.), at 37°C. The mobile phase was a mixture of acetonitrile:methanol:water:acetic acid (56:36:7.5:0.5 by vol.; pH 4.35) at a flow rate of 0.7 ml/min. Detection was carried out at 245 nm. Calibration curves were constructed in the range between 0.1 -1000 pg/ml (R2 > 0,999). The quantitation limit was 1 pg/mL with a standard deviation of 4%.

Particle size and polydispersity and zeta potential of particulates in water

The particle size and polydispersity index (PDI), and also zeta-potential, were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry respectively, using a Zetasizer analyser system (Brookhaven Instruments Corporation, New York, USA). The diameter of the nanoparticles was determined after dispersion in ultrapure water (1/10) and measured at 25°C by dynamic light scattering angle of 90°C. The zeta potential was determined as follows: 200 pL of the samples were diluted in 2 mL of a 0.1 mM KCI solution adjusted to pH 7.4.

Particle size in the solid state

The size distribution of particulates was evaluated by optical microscopy in a MORPHOLOGI G3 apparatus (Malvern).

Porosity and specific surface The porosity of solid samples was analysed in a mercury porosimeter Autopore IV 9500 (Micromeritics), using a specific penetrometer for powdered samples operating in a pressure range between 0.0015 and 207 MPa. The specific surface of samples was analysed by nitrogen adsorption isotherms, at the temperature of liquid nitrogen (77 K), using an ASAP2020 apparatus (Micromeritics). In all cases, samples were degassed at 80°C for 5 hours prior analysis. The specific surface was calculated by the BET volumetric method.

Example 1 : Preparation of avermectin composition suitable as precursor composition for aerosolization

Ivermectin powder was dissolved in pure ethanol to reach three different concentrations, 7mg/mL, 10 mg/ mL and 14 mg/ mL, and kept in opaque flasks at 5°C. All solutions were used as precursor compositions for nebulization in biological testing within one week of preparation.

Example 2: Preparation of avermectin composition suitable as precursor composition for aerosolization

[3-cyclodextrin and ivermectin are dissolved in ethanol 90% (10 mL) at a molar ratio of 1 :1. The mixture is stirred at room temperature in a magnetic agitator during 30 min. Then, the mixture is dried by either lyophilization or Spraydrying. By lyophilization, 1 mL of a lactose aqueous solution (3% w/v) is added to the ivermectin:ciclodextrin solution and the mixture is dried in a Telstar LyoBeta mini apparatus. By spray-drying, the mixture cyclodextrin: ivermectin is dried in a Buchi Mini Spray Drier B-290 apparatus (Buchi Labortechnik AG, Switzerland).

In all cases, the obtained powders may be dissolved in an aqueous composition before nebulization.

Example 3: Preparation of avermectin composition suitable as precursor composition for aerosolization

DPPC is added to the solutions of Example 1 and kept in a rotary evaporator under reduced pressure (Buchi Rotavapor R-144; Buchi, Postfach, Switzerland) and allowing the solvent to evaporate until a uniform dry film is formed. The dried film is then hydrated with warm phosphate buffer under agitation (Buchi Rotavapor R-144; Buchi, Postfach, Switzerland) to obtain the aqueous precursor composition comprising avermectin micelles ready for nebulization.

Example 4: Preparation of avermectin composition suitable as precursor composition for aerosolization

This composition was prepared by dissolving ivermectin (9.3 mg) in 5 mL of an aqueous solution of HP-[3-CD (17.52%). After 30 min of agitation at room temperature, 1 mL water containing soya lecithin (to yield a final concentration of 0.05% w/v) was added to the mixture of cyclodextrin and ivermectin. Finally, the volume was adjusted to 10 mL by adding water. The final composition of this ivermectin solution was as follows:

Example 5: Preparation of avermectin composition suitable as precursor composition for aerosolization

This composition was prepared by dispersing ivermectin (25 mg) in 5 mL of an aqueous solution of HP-[3-CD (19.4%). After 30 min of agitation at room temperature, 1 mL water containing soya lecithin (to yield a final concentration of 0.1 % w/v) was added to the mixture of cyclodextrin and ivermectin. Finally, the volume was adjusted to 10 mL by adding water. The final composition of this ivermectin suspension was as follows:

The resulting aqueous suspension presented a mean size of 157±1 nm with a polydispersity index of 0.069 ± 0.024. Moreover, the aqueous suspension of ivermectin was found to be stable for at least 10 days, when stored at room temperature. Example 6: Preparation of avermectin composition suitable as precursor composition for aerosolization

This composition was prepared by dissolving ivermectin (50 mg) in 1 mL propylene glycol. In parallel, an aqueous solution of HP-[3-CD (for a final concentration of 0.05% w/v) was prepared by dissolving the cyclodextrin in 5 mL water. Then both volumes were mixed under agitation. After 30 min of agitation at room temperature, 1 mL water containing soya lecithin (to yield a final concentration of 0.1 % w/v) was added to the composition containing ivermectin. Finally, the volume was adjusted to 10 mL by adding water. The final composition of this ivermectin suspension was as follows:

The resulting suspension presented a mean size of 135±2 nm, with a polydispersity index of 0.209 ± 0.014. Moreover, the aqueous suspension of ivermectin was found to be stable for at least 5 days, when stored at room temperature. In the absence of cyclodextrin, the composition presented a mean size of 176±2 nm, and was stable for 2 days, when stored at room temperature.

Example 7: Preparation of avermectin composition suitable as precursor composition for aerosolization

In a first step, ivermectin (either 100 mg or 280 mg) and HP-[3-CD (949 mg) were dissolved in 8 mL ethanol 50%. To this composition, 2 mL of an aqueous solution of soya lecithin was added. The mixture was stirred at room temperature during 30 min and, then, 10 mL water were added in order to produce ivermectin nano-Zmicroparticles. Then, 50 mg mannitol was added and dissolved under agitation. Finally, the milky dispersion was dried in a Spray-drying apparatus (Buchi Mini Spray Drier B-290; Buchi Labortechnik AG, Switzerland). The drying conditions were as follows: (i) inlet temperature of 90 °C; (ii) outlet temperature of 60 °C; (iii) air pressure of 4-6 bar; (iv) pumping rate of 5 mL/min; (v) spray-flow of 400-500 L/h; (vi) and aspirator at 80 % of the maximum capacity. The theoretical composition of ivermectin powder formulations (IVM-8 and IVM-20) is as follows:

The size distribution of particulates was evaluated by optical microscopy in a MORPHOLOGI G3 apparatus (Malvern). The amount of ivermectin in the solid formulation was quantified by HPLC after dispersing 5 mg of formulation in 1 mL ethanol. Samples were filtered before introduction in the HPLC system.

The main physico-chemical characteristics of the ivermectin powder formulations were as follows:

The pore size distribution of ivermectin samples was analysed in a mercury porosimeter apparatus. IVM-8 and IVM-20 presented pore distribution curves significantly different to those observed for pure ivermectin, suggesting an important modification of the macroscopic structure homogenous pores with a mean size ranging between 0.5 and 0.6 micrometres. Finally, the specific surface of the ivermectin formulations was characterised by nitrogen adsorption. Ivermectin formulations presented specific surfaces significantly higher than for the pure drug (about 34% and 40% higher for IVM-8 and IVM- 20, respectively), corroborating the higher porosity determined by mercury porosimeter. Specific surface of ivermectin samples calculated by the BET method where as follows:

As expected from porosity and specific surface data, ivermectin samples were easily and rapidly dispersed in water, yielding homogeneous dispersions of particulates in the nano/micrometre range. In the first case (IVM-8), the ivermectin formulation presented homogeneous nanoparticles of about 940 nm, whereas IVM-20 yielded microcrystals of 1.5 micrometres. Likely, in both cases, homogeneous and stable suspensions for at least 3 days were obtained by dispersion of up 100 mg ivermectin powder formulation per mL water. The physico-chemical characteristics of ivermectin formulations after dispersion in water were as follows:

Example 8: In vivo pulmonary administration of ivermectin

Methods

The compositions of Example 1 were administered to adult, 12-weeks old, Sprague-Dawley rats (ENVIGO RMS Spain S.L, Sant Feliu de Codines, Spain) that were kept in groups of 2-3 subjects of the same sex per cage at the animal research facilities of the University of Navarra. Light, temperature, humidity and feeding conditions followed the local standard operating procedures. Rats in the intervention groups were identified as TM/TF (males/females) plus a correlative number 1 -6. Rats in the control group were identified as VCM or VCF for male and female respectively.

For the nebulization of the precursor compositions of Example 1 , a Micro Cirrus TM device (Intersurgical® Berkshire, UK) with a driving oxygen flow of 8 L/min which can deliver aerosol particles of 0.5-2 microns was employed. A volume of 3 mL of the precursor compositions was used. A 22- mm elbow piece that covered the rats’ nose and mouth was used to deliver the aerosol. The oxygen flow was kept until the reservoir was empty at visual inspection for an average nebulizing time of 9 minutes. The reservoirs were weighed before and after the nebulization to confirm the final weight was the same as the empty one, before the procedure.

The rats for this study were randomly assigned into three groups: (a) lower dose, aiming at twice the lower boundary of the LCso for their sex (as reported by Merck Research Laboratories to the FDA for oral Mectizan); (b) higher dose, aiming at twice the upper boundary of the LCso for their sex and (c) controls receiving only ethanol vehicle. The sample was chosen according to usual practice in dose-range finding pilots. Treatment groups included three males and three females each, rats from each sex were housed in separate cages. The first three males received the lower dose, the first three females received the lower dose, the remaining three in each cage were assigned to the missing dosing group and the last rat in each cage remained untreated. The intervention was administered in dose-groups under anaesthesia with ketamine (Imalgene 1000. Merial) and diazepam (Roche Pharma) 75/5 mg/kg, intraperitoneal, single dose. Post-administration, the rats were monitored until recovery for 60-90 minutes on a thermic blanket and then monitored daily using a modified Irwin test until euthanasia. The weight of all subjects was recorded at baseline, day 3 and at the day of euthanasia. Additionally, a full blood count and a biochemistry toxicology panel including total serum proteins, albumin, AST, ALT, bilirubin, total cholesterol, glucose, creatinine, urea, CPK and LDH was performed in one subject per sex/group at euthanasia.

Plasma samples for determination of ivermectin levels were obtained at 1 , 4 and 24 hours after administration and at the time of euthanasia (days 3, 5 or 7). Blood samples of 0.5 ml were obtained under brief induction anaesthesia with inhaled isofluorane 1.5-2% (Isovet, Braun) from the retro orbital plexus, centrifuged and the plasma frozen at -80°C until processing.

At the end of their corresponding study period (see Figure 1 ), subjects were euthanized using a CO2 chamber. A macroscopic necropsy was performed in all subjects including external and in situ examination of all organs. The lungs and livers were extracted, measured and weighted separately. One lung and one liver sample from every subject was preserved in formaldehyde and sent to Patconsult LAB (Barcelona, Spain) for histopathological examination.

Ivermectin plasma levels were determined using a variation of a previously described HPLC-FLD (Eraslan, et al., Food Chem Toxicol, 2010, 48:2181-2185) with a detection limit of 0.1 ng/ml.

Lung tissue (0.5 mg) was homogenized in 1 ml of acetonitrile using an ultra-turrax homogenizer and then sonicated for 15 minutes, 100 mcl of the homogenized lung were processed in the HPLC-FLD using the plasma methods.

The adipose weight of the rats according to sex was calculated using the formula described by Ferrel and Koong (Ferrell, et al., J Nutr, 1986, 116: 2525-2535). Comparison of baseline characteristics was done using the Wilcoxon Rank Sum test in Stata 16 Software (StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC). Body weigh-PK by sex graphs were done in Microsoft Excel (Microsoft corporation, 2018).

Data set checkout and visualizations of pharmacokinetic data were performed in GNU R (R Core Team [2020] R: A language and environment for statistical computing, version 3.6.3, R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org/.). Non-compartmental analysis (NCA) was performed using Pkanalix (Monolix version 2019R2. Antony, France: Lixoft SAS, 2019. http://lixoft.com/products/monolix/). Peak plasma concentration Cmax), and time to peak plasma concentration (Tmax) were read directly from the profiles. Terminal elimination half-life (t1/2) was calculated as t1/2=(2)/ z after estimating z from the final two measurements of each profile. The area under the plasma concentration-time curve (AUC) from 0 to 168h post-dose (AUCO- GSh) was calculated using the linear up/log down method. Mean plasma concentrations from 0 to 168h post-dose are given as Cavg.,o-i68h. Lung tissue concentrations were back-calculated from lung densities reported by El-Khatib et al. (el-Khatib et al., Int J Radiat Oncol Biol Phys, 1989, 16:745-754) in Sprague-Dawley female rats aged 15-90 weeks. At 15 weeks of age, the average lung density was about 0.4 g/cm³, and therefore 1 g of lung tissue was converted to a volume of 2.2 mL.

Results

The baseline characteristics of the rats by sex is presented in the table below.

Male rats had an approximate median weight 100 grams (50%) higher than females (p-value = 0.002) and different body composition resulted in females having three times the estimated adipose weight (median 92.9 vs 29.1 grams, p-value 181 = 0.002).

The recommended minimal volume to generate nebula with the device used is of three mL, given that our formulation is ethanol based, this volume set the minimal ethanol dose given with the intervention in 2.3 grs (equivalent to 3 mL). All animals received the intervention uneventfully and recovered from anaesthesia within 90 minutes. At the time of the first blood sample (one hour after the intervention), they showed slight instability and lethargy which was attributed to the alcohol dose and had recovered fully by the time of the second blood sample, four hours after the intervention. The animals in the lower dose group received a median dose of 89 mg/kg of ivermectin, ranging 86-98 mg/kg for males and 88-95 mg/kg for females which correlates well with the target of twice the lower boundary of the oral LDso. The animals in the higher dose group received a median dose of 121 mg/kg of ivermectin, ranging 108-116 mg/kg for males and 126-141 mg/kg for females which represents 2.0 to 2.2-fold the upper boundary of the oral LDso for males and 2.3 to 2.6-fold the upper boundary of the LD50 for females. However, the significantly different adipose weight resulted in male rats receiving much higher doses of ivermectin per gram of fat than females (3.2 to 4-fold in the higher dose group, and 3.9 to 5.7-fold in the lower dose group), weights and doses of all animals are presented in the tables below.

*Ferrel and Koong method

** below quantification limit

All rats received 3 mL (2.3 g) of inhaled ethanol, which resulted in weight-adjusted ethanol doses of 6.1 -7.7 g/kg for males and 9.4-11.1 g/kg for females which is lower than or overlapping with the oral ethanol LDso described in the literature for rats of a similar age (10.0-11.2 g/kg). All animals had a normal behaviour and central nervous system assessment throughout the study according to the modified Irwin procedure. All animals had a normal weight for their age at baseline, except for one male (TM1 ) who was 17 grams 206 (5%) below the lower limit of the normality for 12 weeks (321 grams). All males except one in the lower dose group had lost weight at day three post intervention, ranging from 2 to 4.2% of their baseline weight. The control male, nebulized with ethanol vehicle only, lost 3.5% of its baseline weight by day three. All males except one in the higher dose group that lost further 0.6%, had started to gain weight or recovered their baseline value at the time of euthanasia. All females except two (one in the lower dose group and one in the higher dose group) had lost weight at day three post intervention, ranging from 0.4% to 3.4% of their baseline weight. The control female lost 0.4% of its baseline weight by day three. All females had started to gain weight or recovered their baseline value at the time of euthanasia.

Male rats had no significant haematological changes regardless of ivermectin dose group or ethanol dose adjusted by weight. All but two female rats had a slight anaemia with haemoglobin levels 0.1 to 1.7 g/dL below the minimum normal value. Additionally, all females had total red blood cells below the normal limit (median 0.8x106/ul) and slightly increased mean corpuscular values (median 3.5 fl above the upper normal limit).

Animals in both groups presented a delayed increase (2 to 3-fold the upper level of normality) in creatine kinase (CPK) and lactate dehydrogenase (LDH), this effect was seen earlier in females (all euthanized after hours post administration) than in males (only those euthanized at 168 hours). Except for one female in the higher ivermectin dose which had AST/ALT values 1.5-fold the upper limit, liver enzymes and bilirubin were normal or below the lower limit in all rats. Two males and all but one female had slightly increased creatinine values. All blood biochemistry results are presented in Supplementary Tables S1 and S2 for males and females respectively.

One male in the higher dose group (TM6) had a patchy pattern in both lungs. There were no macroscopic findings in the thoracic or abdominal organs of any other subject. The absolute and relative-to-body weights of lungs and livers from all subjects where within normal values. There were no pathologic changes in the histology of liver and lungs samples examined, including the lung sample from the male rat with a macroscopic patchy pattern.

Individual plasma profiles by dose group and sex are shown the table above. Secondary pharmacokinetic parameters in plasma are given in the tables below.

Concentrations in lung tissue remained detectable in all animals at time of necropsy (72-168 h, Figure 2). Although the total and weight-adjusted ivermectin doses were comparable between males and females in both dose groups, the higher estimated adipose weight of female rats resulted in a 3 to 4-fold dose of ivermectin per gram of fat. Ivermectin accumulates in adipose tissue, this deep compartment affects systemic concentrations of the drug in subjects with different body composition and sex-based differences have been well described before. In this experiment there was a direct relationship between the dose per gram of fat and the plasma levels reached at 1 , 4 and 24 hours. Nebulized ivermectin reached a Cmax twice as high and had an AUC 4 to 6-fold higher in male rats regardless of the dosing group. There was also a tendency for lung levels of male rats to be higher regardless of the dosing group. The haematological changes observed in most female rats, regardless of dosing group (mild macrocytic anaemia and reduced total RBC) are already present in animals euthanized 72 hours after the intervention and could either precede the intervention or be the consequence of the proportionally larger blood loss due sampling in this group. The elevated CPK and LDH observed 5-7 days after the intervention seem to be of muscular origin given normal values of bilirubin and liver enzymes in all rats; we hypothesize these changes are in relationship with the high ethanol dose received since they are also present in the control animals that did not receive any ivermectin; this is also compatible with the slight increase in creatinine values seen in most animals and the described initial weight loss and posterior recovery. The anaesthesia used during the intervention and for the PK sampling could have played a role in the CPK/LDH.

The plasma concentrations achieved in this study are above the nicotinergic acetylcholine receptor (nAChR) ICso for ivermectin as estimated in the literature (Krause, et al., Mol Pharmacol, 1998, 53:283-294). Lung tissue concentrations in male rats in the high-dose arm were well above this concentration after 72-168h. As far as the present inventors are aware of, this is the first report of a pharmaceutical composition of avermectin suitable for inhalation to the lungs which achieves therapeutically effective concentrations of the drug both locally at the lungs as well as systemically, furthermore up to one week post-administration.

Claims

52

CLAIMS A compound which is an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer of the avermectin or milbemycin; wherein the compound is in a form suitable for inhalation into the lungs; and wherein the mass median aerodynamic diameter of the particles of the compound is of 2 micron or lower. Pharmaceutical composition comprising:

- a therapeutically effective amount of a compound which is an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer of the avermectin or milbemycin; and

- at least one pharmaceutically acceptable inhalation excipient; wherein the pharmaceutical composition is in a form suitable for inhalation into the lungs; and wherein the mass median aerodynamic diameter of the particles of the pharmaceutical composition is of 2 micron or lower. Compound according to claim 1 or pharmaceutical composition according to claim 2, wherein the avermectin or milbemycin is selected from abamectin, ivermectin, eprinomectin, doramectin, selamectin, milbemectin, moxidectin, nemadectin, emamectin or milbemycin oxime. Compound or pharmaceutical composition according to claim 3, wherein the avermectin or milbemycin is ivermectin. Compound according to any one of claims 1 or 3 to 4, or pharmaceutical composition according to any one of claims 2 to 4, wherein the mass median aerodynamic diameter of the particles is between 0.5 and 2 micron. Pharmaceutical composition according to any one of claims 2 to 5, wherein the at least one pharmaceutically acceptable inhalation excipient is a 53 cyclodextrin, and the compound forms an inclusion complex with the cyclodextrin. Pharmaceutical composition according to any one of claims 2 to 6, further comprising an amphiphilic agent. Pharmaceutical composition according to any one of claims 2 to 5, wherein the at least one pharmaceutically acceptable inhalation excipient is a micelle, and the micelle comprises the compound. Pharmaceutical composition according to any one of claims 2 to 5, wherein the at least one pharmaceutically acceptable inhalation excipient is an alcohol, and the compound is dissolved in the alcohol; wherein the alcohol is preferably ethanol. Compound according to any one of claims 1 or 3 to 5, or pharmaceutical composition according to any one of claims 2 to 9, in the form of an aerosol. Inhaler device comprising a compound as defined in any one of claims 1 or 3 to 5 or 10, or a pharmaceutical composition as defined in any one of claims 2 to 10. Inhaler device according to claim 11 , which is a nebulizer, a powder inhaler, or a metered dose inhaler. Compound as defined in any one of claims 1 or 3 to 5 or 10, or a pharmaceutical composition as defined in any one of claims 2 to 10, or device as defined in any one of claims 11 to 12, for use in medicine. Compound, pharmaceutical composition or device for use according to claim 13, for use in the prevention or treatment of parasitic infections, cancer, viral infections, or NF-KB-related diseases or disorders; of the lungs. Compound, pharmaceutical composition or device for use according to claim 13, for use in the prevention or treatment of COVID-19. 54

16. Composition comprising:

- a therapeutically effective amount of a compound which is an avermectin or milbemycin, or a pharmaceutically acceptable salt, solvate or stereoisomer of the avermectin or milbemycin;

- at least one pharmaceutically acceptable inhalation excipient, wherein the at least one pharmaceutically acceptable inhalation excipient is a cyclodextrin, and the compound forms an inclusion complex with the cyclodextrin; and - an amphiphilic agent.

17. Composition according to claim 16, wherein the amphiphilic agent is a phospholipid. 18. Composition according to claim 16, wherein the amphiphilic agent is lecithin.

19. Composition according to any one of claims 16 to 18, in the form of a powder.