EP4701628A1 - New clofoctol formulation - Google Patents

Abstract

The invention concerns a new galenic formulation of CFT (clofoctol) allowing the administration of this antibiotic in aerosol form with the objective of treating pulmonary infections (COVID-19, influenza), cancer and inflammation thus targeting the diseased tissue while avoiding the problems of solubility of CFT and toxicity associated with this drug. This new formulation allows to answer these problems and concerns the development of polymeric nanoparticles (Nanoparticles) in suspension in an aqueous phase intended to be administrated in a form of aerosol or spray, said Nanoparticles comprising PLGA and PLGA-PEG polymers, allowing to obtain an effective encapsulation of CFT and a controlled release of CFT at the pulmonary level.

Description

NEW CLOFOCTOL FORMULATION Field of the invention The present invention relates to a formulation of clofoctol encapsulated into polymer nanoparticles, in particular PLGA/PLGA-PEG nanoparticles, and the use of such nanoparticles for the treatment of pulmonary diseases, such as infections, cancer, inflammations, in particular infection and inflammation caused by SARS-CoV-2 (Covid19). Background of the invention Clofoctol (CFT) (2-(2,4-dichlorobenzyl)-4-(1,1,3,3 -tetramethylbutyl)phenol, CAS number 37693-01-9) is a bacteriostatic antibiotic, indicated for the treatment of infections caused by Gram-positive bacteria. It was first described in French patent application FR2101076. In particular, CFT used as a suppository (Octofen®) has been shown to be effective to treat bacterial respiratory infections (Scaglione et al., 2012). In 2020, patent EP3922312A1 described the interest of CFT to treat COVID-19. Indeed, CFT strongly reduces SARS-CoV-2 replication both in cell culture and in animal models (Belouzard et al., 2022). In international application WO2022238263, CFT has been described to reduce inflammation in two models that recapitulate severe inflammation, one induced by the virus SARS-CoV-2, and the other induced by LPS. The data suggested that CFT could be suitable for the treatment of inflammation in general. In addition, studies have demonstrated that CFT has antitumor effects (for a review, Bailly and Vergoten, 2021). CFT has also been described to activate the unfolded protein response pathway, making it a potential drug candidate for the treatment of prostate cancer (Wang et al., 2014). CFT has also been shown to suppress glioma stem cell proliferation by activating KLF13 (Hu et al., 2019). To sum up, CFT is an active molecule with great potential to treat infections, inflammation and cancer, so that it deserves to be repositioned. For example, it could be used to fight against pulmonary infections (COVID-19, influenza...). It is also of interest to fight cancer or other diseases. Unfortunately, the in vivo administration of CFT faces various challenges: ^ Insolubility in aqueous media (~ 36 µg/L in water), requiring the use of additives or solvents with potential toxic effects to allow in vivo administration; ^ Toxicity (Belouzard et al., 2022); intraperitoneal administration of CFT in mice showed a high toxicity. ^ Low pulmonary biodisponibility; poor exposition of CFT in the lungs of hamsters after oral or intravenous administration. To treat lung diseases, a nasal or pulmonary administration of CFT would allow to target the organ of interest (lungs) while sparing the rest of the body. This would also allow for a reduction in the administered doses, and therefore, for a reduction in potential adverse effects associated with CFT. The problem to be solved is thus to develop a new formulation of CFT which improves its solubility and allow the administration by spray in the respiratory tract. The formulation should also allow a controlled release to afford a prolonged exposure as compared to free CFT. The formulation would advantageously possess a modified surface (such as a polyethylene glycol (PEG) coating) to control the interactions with the living media (biomolecules and cells such as lung macrophages). The formulation could be administered as a spray or under the form of dried powder, after freeze drying. Alternatively, such formulation could be administered by the intravenous route or other route, depending on the disease to be treated. Summary of the invention The invention is defined by the claims. The invention concerns a new galenic formulation of CFT (clofoctol) allowing the administration of this active molecule preferentially in aerosol form with the objective of treating pulmonary diseases (COVID- 19, influenza, cancer...). This strategy allows targeting the drug to the lung tissue and avoids the problems of solubility and toxicity associated with CFT. This new formulation addresses these problems and concerns the development of polymeric nanoparticles (NPs) in suspension in an aqueous phase intended to be administrated in a form of aerosol or spray, said nanoparticles comprising PLGA and PLGA-PEG polymers, allowing to obtain an effective encapsulation of CFT and a controlled release of CFT at the pulmonary level. Said PEG in the nanoparticles’ composition would allow a reduced uptake by the macrophages, whenever needed. Detailed description of the invention In a first aspect, the present disclosure relates to Clofoctol (CFT) encapsulated into polymer nanoparticles. Thus, the present disclosure relates to polymer nanoparticles comprising encapsulated Clofoctol (CFT). As used herein, the term clofoctol (CFT) refers to 2-(2,4-dichlorobenzyl)-4-(1,1,3,3 - tetramethylbutyl)phenol, CAS number 37693-01-9. As used herein, the term “nanoparticle” refers to any particle having a mean hydrodynamic diameter of less than 1000 nm. The CFT encapsulated nanoparticles of the present disclosure includes one, two, three or more biocompatible and/or biodegradable polymers, referred herein as polymer nanoparticles. In specific embodiments, said nanoparticles of encapsulated CFT comprise at least 50 wt% of polymer, preferably PLGA and PLGA-PEG, by weight of the nanoparticles (including the clofoctol). In particular, said nanoparticles of encapsulated CFT comprise at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt%, preferably between at least 50 wt% and 90 wt% of polymer by weight of the nanoparticles (including CFT). In particular, said nanoparticles of encapsulated CFT essentially comprise polymers PLGA and PLGA-PEG. In some embodiments, polymers may be polyesters, including copolymers comprising lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers comprising glycolic acid units, referred to herein as "PGA," and lactic acid units, such as poly-L-lactic acid ("PLLA"), poly-D-lactic acid ("PDLA"), poly-D,L- lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA". In some embodiments, exemplary polymers include, for example, polyhydroxyacids; PEGylated polymers and copolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylated PLGA), and derivatives thereof. In some embodiments, polymers include, for example, polyanhydrides, poly(ortho ester), PEGylated poly(ortho ester), poly( ^-caprolactone), PEGylated poly( ^-caprolactone), polylysine, PEGylated polylysine, poly(ethylene imine), PEGylated poly(ethylene imine), poly(L-lactide- co-L-lysine), poly(serine ester), poly(4-hydroxy-L proline ester), poly[ ^-(4-aminobutyl)-L-glycolic acid], and derivatives thereof. In specific embodiments, said polymer is PLGA. PLGA, also named PLG or poly(lactic-co-glycolic acid), is a copolymer of lactic (LA) and glycolic acid (GA), which is used in a host of Food and Drug Administration (FDA) approved therapeutic applications, owing to its biodegradability and biocompatibility. PLGA is typically synthesized by means of ring-opening co-polymerization of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid (GA) and lactic acid (LA). The molar ratio between LA and GA in PLGA is typically between 75:25 and 25:75. In particular, the molar ratio is 50:50. PLGA may be terminated by a carboxyl group, more particularly it is PLGA terminated by a carboxyl group and having a low molecular weight such as comprised between 10 and 20 kDa. In preferred embodiments, said polymer composition as used for the nanoparticle is a mix of PLGA and pegylated PLGA, that is to say polyethylene glycol (PEG) bound to PLGA (PLGA-PEG) forming a diblock copolymer with the general formula: where y, x and n are the numbers of the glycolic acid, lactic acid and ethylene glycol units, respectively. Y, x and n are integer higher than zero. In some embodiments, PEG has a molar mass between 2 and 20 KDa, that is to say, n is in between around 45 and 450. In specific embodiments, n is in between 45 and 114. In preferred embodiments, n is around 45. The glycolic and lactic acid units are randomly distributed in PLGA part of PEG-PLGA copolymer. In some embodiments, x and y are between around 7 and 500. In specific embodiments, x and y are between around 50 and 250. In a preferred embodiment, x=y, x and y are around 150, and the molar mass of the diblock PEG-PLGA copolymer is in the range 30-80 kDa. The PEG-PLGA copolymer can be blended with PLGA to form nanoparticles with various PEG contents, resulting in various PEG densities at their surface. In another embodiment, as will be appreciated by the skilled person in the art, the nanoparticles can be prepared by blending two or more copolymers of the type PEG-PLGA, where the molar masses of the PEG blocks are different, that is to say, with n comprised in between around 45 and 450. In another embodiment, multiblock copolymers can be used, comprising several PEG and PLGA blocks linked together by covalent bonds. Thus, the present disclosure relates to encapsulated CFT into nanoparticles comprising PLGA and PLGA-PEG. In one embodiment, the present disclosure relates to polymer nanoparticles comprising encapsulated CFT, said polymer nanoparticles comprising PLGA and PLGA-PEG. In particular, the weight ratio of the two types of polymers is between 100:0 to 0:100 (w/w). Typically, said ratio PLGA:PLGA-PEG is 100:0 (w/w) to 50:50 (w/w), particularly 80:20 (w/w) to 20:80 (w/w), 80:20 (w/w) to 50:50 (w/w), 75:25 (w/w) to 25:75 (w/w), in particular 75:25 (w/w) or 50:50 (w/w). The mean hydrodynamic diameter of the nanoparticles according to the present disclosure may range between 100 and 500 nm. In particular, these diameters are between 100 and 350 nm, particularly between 140 and 280 nm. Methods to measure the mean hydrodynamic diameter of the nanoparticles are described for example in Filipe et al. One of the key parameters is drug loading, which is defined here as the mass ratio of drug to polymer in the nanoparticles. In specific embodiments, the CFT loading may comprise between 10 and 30 wt%, by weight of the PLGA and PLGA-PEG in the nanoparticle, in particular between 15 and 20 wt%. In one embodiment, the calculated distance between the PEG chains grafted to PLGA is between 2 and 3 nm, in particular said distance is 2 nm, 2.25 nm, 2.5 nm, 2.75 nm or 3 nm. Methods for determining the distance of PEG chain in the nanoparticle are provided in the Examples section. In one embodiment, PLGA-PEG may bear a targeting ligand. For example, said ligand may be a mannose moiety. Mannose could be coupled to the chain end of PEG in PLGA-PEG. This copolymer can potentially enhance cell uptake. Alternatively, other ligands such as folic acid, biotin or ligand targeting cancer cells may be used. Indeed, PLGA-PEG may be functionalized with a ligand targeting cancer cells, including small molecules, peptides, antibodies, engineered proteins, or nucleic acid aptamers, as for example folic acid, mannose, monoclonal antibody. The present disclosure also relates to encapsulated CFT as described above wherein CFT is encapsulated with at least one other active molecule. Thus, in one embodiment, the present disclosure deals with polymer nanoparticles comprising encapsulated CFT as described above wherein CFT is encapsulated with at least one other active molecule. Said at least one other active molecule may be an anti-cancerous agent. In one embodiment, the present disclosure deals with encapsulation of CFT derivatives. Examples of such derivatives are given in Bailly and Vergoten (2021). Other examples of such derivatives are disclosed in WO 2023/201712A1 and present the general formula: wherein L 1 is selected from a linear alkyl group having 1 to 10 C atoms or a branched or cyclic alkyl group having 3 to 10 C atoms and R1 is selected from a guanidine group, an amino group, an arginine group, a histidine group, a lysine group or a combination of these groups. These derivatives display increased activity and are capable of effectively avoiding the generation of bacterial drug resistance. These derivatives could be efficiently encapsulated in the polymeric nanoparticles by the methods described in the present disclosure. For example, R1 is selected among the following groups:

5 In particular, the CFT derivatives are selected from the following structures:

Preparation of the nanoparticles Another aspect of the disclosure is directed to method of making nanoparticles encapsulating CFT. The present disclosure therefore relates to a manufacturing process of CFT encapsulated into nanoparticles comprising PLGA and PLGA-PEG, said process comprising a method of emulsification- solvent evaporation. The present disclosure thus deals with a manufacturing process of nanoparticles comprising encapsulated CFT, said nanoparticles comprising PLGA and PLGA-PEG. Said process comprising a method of emulsification-solvent evaporation. In particular, said process comprises the following steps: 1. preparing an aqueous solution of a surfactant at about 0.5% w/v, 2. solubilizing PLGA, PLGA-PEG and CFT at a ratio PLGA/PLGA-PEG : CFT about 6:1 w/w in a solvent, and adding it into the aqueous surfactant solution, 3. optionally adding ethanol, in particular 1-5% v/v, 4. sonicating the resulting mixture, 5. evaporating the solvent. In the sense of the present disclosure, “about” means more or less than 10%. Said process can be adapted according to the volume to be prepared. The solvent may be dichloromethane, acetone, a mix of dichloromethane-acetone or ethyl acetate. Surfactant may be poly(vinyl alcohol) (PVA), Pluronic^®, cholate (bile salt), Tween^®, or others. In particular, the volume ratio solvent: surfactant solution at step 3. is 1.5: 4. In another embodiment, PLGA-PEG bearing a targeting ligand can be used for nanoparticles formulation, by replacing PLGA-PEG or a fraction of it. For example, mannose could be coupled to the chain end of PEG in PLGA-PEG. Alternatively, folic acid, biotin or other ligands to target cancer cells could be used. In another embodiment, the nanoparticles can be prepared using a microfluidic device or by high- pressure homogenization. In another embodiment, as will be appreciated by the skilled person in the art, the PLGA nanoparticles can be coated with a lipid layer, for instance, by fusion with liposomes. Pharmaceutical compositions The present disclosure also deals with a pharmaceutical composition comprising encapsulated CFT as described above in suspension in an additional pharmaceutically acceptable excipient. Therefore, according to a second aspect, the disclosure relates to a pharmaceutical composition comprising a suspension of polymer nanoparticles comprising encapsulated CFT as described above, in an additional pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, or formulation auxiliary of any type. As will be appreciated by the skilled person in the art, the pharmaceutically acceptable excipients and/or carriers will be chosen based on the route of administration as described below, the location of the targeted tissue, the time course of delivery of the drug, etc. There are numerous examples of excipients that can be added to the pharmaceutical composition: Examples include without limitation, phospholipids, and more particularly 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC); water-soluble derivatives or copolymers of PEG or ethylene oxide, such as Tween^®, Pluronic^®, Carfil^®, Kolliphor^®; salts (such as sodium chloride, phosphates, etc.); sugars (mannose, glucose, trehalose, etc.); polysaccharides (dextran, hyaluronic acid; chitosan, etc.). Said excipients can be added to the pharmaceutical composition, after the preparation of the nanoparticles. Alternatively, excipients can be added during the preparation of the nanoparticles, for example, PEG or ethylene oxide derivatives can serve as surface-active ingredients to produce the nanoparticles. Examples of pharmaceutical compositions comprising polymers are given here. However, as will be appreciated by the skilled person in the art, CFT formulations can also be prepared using polymers and lipids, or lipids alone. For instance, solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) can be prepared in the absence of organic solvents. They are typically produced by sonication, high-pressure homogenization or high-speed stirring. The solid matrices of SLNs and NLCs contribute to increase the stabilities of active ingredients and are considered as safe carriers since they are produced from physiological and biodegradable lipids (e.g., triglycerides, partial glycerides, waxes, steroids, and fatty acids) and other materials generally recognized as safe (GRAS). If polymers and lipids are used together, nanocapsules will be formed, as described for instance in a review (Lima et al.). The pharmaceutical compositions of this disclosure can be administered to a patient by any means known in the art including oral and parenteral routes. The term "patient," as used herein, refers to humans as well as non-humans, including, for example, mammals, birds, reptiles, amphibians, and fish. For instance, the non-humans may be mammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). In certain embodiments, parenteral routes are desirable, since they avoid contact with the digestive enzymes that are found in the alimentary canal. According to such embodiments, the compositions may be administered by injection (e.g., intravenous, subcutaneous or intramuscular, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as by sprays). In preferred embodiments, said encapsulated CFT nanoparticles and said pharmaceutical composition are intended to be administered to the subject in need by inhalation routes. Thus, the pharmaceutical compositions of the present disclosure can be formulated in the form of a spray, aerosol or a mist. In particular, said encapsulated CFT nanoparticles and said pharmaceutical composition are compatible with the nebulizer-inhalers already on the market. They also can be formulated in the form of a dry powder. Indeed, the encapsulated CFT and the pharmaceutical composition can also be freeze dried with additives to obtain a dry powder for an administration with a dry powder inhaler. This is well described in the literature (Scherlieβ et al.). Methods of use The present disclosure also deals with the encapsulated CFT as described above or the pharmaceutical composition as described above, for use as a medicament. The disclosure deals with the encapsulated CFT as described above or the pharmaceutical composition as described above, for use in a therapeutic method. In particular, it deals with the encapsulated CFT as described above or the pharmaceutical composition as described above, for use in the treatment of a lung inflammation. Therefore, according to a third aspect, the disclosure relates to polymer nanoparticles comprising encapsulated CFT as described above or the pharmaceutical composition as described above, for use as a medicament. The disclosure relates to polymer nanoparticles comprising encapsulated CFT as described above or the pharmaceutical composition as described above, for use in a therapeutic method. In particular, it relates to polymer nanoparticles comprising encapsulated CFT as described above or the pharmaceutical composition as described above, for use in the treatment of a lung inflammation. As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. As used herein, the expression “lung inflammation”, also called pulmonary inflammation, refers to accumulation of inflammatory cells in airway tissue. As used herein, an agent is said to exert an anti- inflammatory effect if, when administered to the subject, the agent is capable of reducing said accumulation. Because the lung is a vital organ for gas exchange, excessive inflammation can be life threatening. This inflammation thus can render difficult for oxygen to pass through the alveoli into the bloodstream. Microscopically, inflammation is characterized by redness, swelling, heat, pain, and loss of function. Microscopically, it is exhibited by vasodilation, increased vascular permeability, and inflammatory cell infiltration. Lung inflammation can occur from: i) infectious causes, and ii) noninfectious causes, such as sarcoidosis, pneumonitis, or a type of allergic reaction. In particular, the lung inflammation is caused by a lung infection which could have various origins: viral, bacterial, fungal or parasite. These microorganisms can cause pneumonias. Thus, the present disclosure also deals with the encapsulated CFT as described above or the pharmaceutical composition as described above, for use in the treatment of a lung infection. For example, the lung infection may be caused by a bacterium selected from the group consisting of Streptococcus pneumoniae (also referred to as pneumococcus), Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Serratia marcescens, Burkholderia cepacia, Burkholderia pseudomallei, Bacillus anthracis, Bacillus cereus, Bordetella pertussis, Stenotrophomonas maltophilia, a bacterium from the citrobacter family, a bacterium from the ecinetobacter family, and Mycobacterium tuberculosis or Mycobacterium abscessus. For example, the lung infection may be caused by a fungus selected from the group consisting of selected from the group consisting of Histoplasma capsulatum, Cryptococcus neoformans, Coccidioides immitis, Candida albicans, and Pneumocystis jirovecii (which causes pneumocystis pneumonia (PCP), also called pneumocystosis). For example, the lung infection may be caused by a virus selected from the group consisting of influenza virus (e.g., Influenza virus A, Influenza virus B), respiratory syncytial virus, adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus and smallpox. More particularly, the present disclosure relates to the treatment of a disease caused by a coronavirus or influenza. Coronaviruses belong to the family Coronaviridae (order Nidovirales) and include viruses with a single- strand, positive-sense RNA genome approximately 26-32 kilobases in size. The Coronaviridae family includes Alpha-coronavirus (alphaCoV), Beta-coronavirus (betaCoV), Delta-coronavirus (deltaCoV) and Gamma-coronavirus (gammaCoV). Bats and rodents are thought to be the reservoir for alphaCoV and betaCoV. Currently, it is less clear which animals serve as the reservoir for deltaCoV and gammaCoV. Coronaviruses are named according to their appearance under the electron microscope, the viruses look like they are covered with pointed structures that surround them like a corona or crown due to the presence of spike glycoproteins on their envelope. Three coronaviruses have crossed the species barrier to cause deadly pneumonia in humans since the beginning of the 21^st century: Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and SARS-CoV2 (also known as 2019-nCoV). SARS-CoV2 is an enveloped, positive- sense, single-stranded RNA virus which belongs to the betaCoV genus, which genus also includes SARS-CoV and MERS-CoV. SARS-CoV2 shares 89% nucleotide identity with bat SARS-like CoV and 82% identity with human SARS-CoV. In a particular embodiment, the encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above, for use in the treatment of a disease caused by a coronavirus, wherein the coronavirus is SARS- CoV, MERS-CoV or SARS-CoV2. Typically, the coronavirus is SARS-CoV2, and the corresponding disease is COVID-19. In another embodiment, the lung inflammation can occur from noninfectious causes, such as pneumonitis, or a type of allergic reaction. For example, lungs inflammations may be caused by repeated exposure to airborne irritants and toxins, or by respiratory infections, and lung diseases such as asthma or chronic bronchitis. Pneumonitis can be acute (rapidly occurring and severe) or chronic (persistent or recurrent) and symptoms may include wheezing, chest pain, shortness of breath, and coughing. The present disclosure also deals with the encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above, for use in the treatment of a cancer. Cancer may be lung cancer, small cell lung cancer, cervical cancer, breast cancer, prostate cancer, colorectal cancer, skin cancer or pancreas cancer. Especially, it may be lung cancer or small cell lung cancer. As lung inflammation can lead to metastasis (Mazzela et al.) treatment with a CFT encapsulated into nanoparticles according to the disclosure able to fight both cancer and inflammation would be a must. Lung cancer is nowadays associated with the highest mortality. The encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above may also find potential applications for intravenous administration to fight a variety of cancers, such as breast, prostate, colorectal, skin, pancreas cancers. In one embodiment, CFT is co-administered with another active molecule. The nanoparticles comprising PLGA and PLGA-PEG could be loaded with CFT and at least one other active molecule. Thus, the present disclosure also deals with the encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above wherein CFT is encapsulated with at least one other active molecule. Alternatively, the nanoparticles may be administered in combination with at least one other active molecule. So, the administration of nanoparticles and the at least one other active principle can be simultaneous, sequential, or over a period of time. In one embodiment, the at least one other active principle is selected from: abemaciclib, almitrine bismesylate, amodiaquine, anakinra, angiotensin 1-7, anidulafungin, apixaban, aspirin, avatrombopag, azithromycin, baricitinib, bazedoxifene, berbamine, brexpiprazole, bromhexine, camostat, canakinumab, cepharanthine, ceritinib, cetylpyridinium, chloroquine, chlorpromazine, ciclesonide, clomifene citrate, cobicistat, croconazole, cyclosporine, danoprevir, darunavir, digitoxin, digoxin, dihydroartemisinine, dihydrogambogic acid, diltiazem, dronedarone, drotaverine, ebastine, eltrombopag, emapalumab, ethaverine, favipiravir, fingolimod, flunarizine, gilteritinib, harringtonine, hematoporphyrin, hexachlorophene, hydroxychloroquine, hydroxyprogesterone caproate, IMU-838, interferon Beta-1A, interferon Beta-1B, isoosajin, isopomiferin, isotretinoin, ivacaftor, ivermectin, JS016, ketoconazole, lidoflazine, loperamide, lopinavir, loratadine, loteprednol etabonate, lusutrombopag, LY- CoV555, mefloquine, nafamostat, niclosamide, nitazoxanide, omacetaxine mepesuccinate, osajin, oseltamivir osimertinib, otilimab, ouabain, oxiconazole, oxyclozanide, ozanimod, papaverine, pegylated interferon lambda, perhexiline maleate, pexidartinib, phenazopyridine, polydocanol, posaconazole, recombinant human interferon α1β, recombinant Interferon Alfa-2b, regorafenib, remdesivir, ribavirin, ritonavir, ruxolitinib, sitagliptin, sofosbuvir, sonidegib, sorafenib, tamoxifen, thalidomide, thimerosal, thioridazine, thymosin alpha 1, tilorone, tioguanine, tocilizumab, toremifene, triparanol, tyrphostin, umifenovir. Said at least one other active molecule may exert a synergic effect to treat the above-mentioned diseases. Said at least one other active molecule may be an antibiotic chosen among the antibiotic agents known from the state-of-the-art belonging to the penicillin group, cephalosporins, macrolides, tetracyclins, quinolones, polypeptides, and other antibiotics such as metronidazole, cotrimaxazole, clindamycin. Said at least one other active molecule may be an anti-inflammatory agent chosen among the agents known from the state of the art such as phenylbutazone, indomethacin, sulindac, aceclofenac, tiaprofenic acid, alminophène, diclofenac, etodolac, phenoprophene, flurbiprophene, ibuprophene, ketoprophene, ketorolac, nabumetone, naproxene, niflumic acid, mefenamic acid, piroxicam, meloxicam, tenoxicam, nimesulid, celecoxib, parecoxib. Said at least one other active molecule may be an antiviral agent chosen among the agents known from the state of the art to treat influenza, such as oseltamivir, zanamivir, amantadine and rimantadine, or neuraminidase inhibitors. Said at least one other active molecule may be an antiseptic agent chosen among the agents known from the state of the art such as chlorhexidine or benzalkonium chloride, ethanol, hexamidine, betadine, chlorine derivatives, and triclocarban. In particular, CFT may be encapsulated with at least one other active compound such as a metal nanoparticle, for example, a silver nanoparticle (size: 5-20 nm) with well-documented antibacterial action. In particular, said at least one other active molecule may be chosen among the anti-cancer agents known from the state of the art such as cisplatin, sorafenib, paclitaxel and docetaxel. For example, an improved effect of CFT and cisplatin has been demonstrated for the cancer treatment (Cheng et al.), as well as the co-administration of CFT and sorafenib (Fan et al.). Whereas each free molecule reduces tumor growth by around 30%, the combination reduces it by almost 100%. It is therefore reasonably expected that drug co-incorporation or combination of nanoparticles with these molecules, and administration to the lungs could be very effective to treat lung cancer. Sorafenib or other drug molecules could be easily co-encapsulated by the methodology presented here. A co- encapsulation or a combination with paclitaxel to treat cancer could also be contemplated. Indeed, the encapsulation of this last molecule in PLGA-PEG has been documented, but no study so far dealt with the co-incorporation of CFT-paclitaxel, which should be feasible in the frame of the invention. Such nanoparticles may be used in the treatment of cancer, such as lung cancer or small cell lung cancer. In another embodiment, such polymeric nanoparticles could be used for the treatment of human neuroglioma. For example, EP3459537 discloses that glioma stem cell treated with clofoctol exhibit significantly reduced self-renewal. In vivo, clofoctol showed efficacy to treat neuroglioma. For best efficacy, the nanoparticles loaded with clofoctol disclosed here could be administered by IV, nasal or pulmonary routes. Advantageously, the CFT-loaded nanoparticles disclosed here could be administered locally, after tumor exeresis. The present disclosure also deals with the encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above wherein PLGA-PEG is functionalized with a ligand targeting cancer cells, and their use in the treatment of cancer, such as lung cancer or small cell lung cancer. In one embodiment, the subject to be treated has one or more of the following conditions or diseases: overweight, obesity, diabetes, hyperlipidaemia, arterial hypertension, cardiovascular disease, chronic kidney disease, immunosuppression. In particular, the encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above, are administered by inhalation route. The inhalation route comprises oral inhalation and nasal inhalation. This allows to directly target the respiratory tract. In particular, they can be conveniently delivered in the form of an aerosol or a spray presentation from pressurized packs, or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). When the nanoparticles of CFT are administered in combination with at least one other active molecule, the administration of each may be done by different routes. In a particular embodiment, the encapsulated CFT or the polymer nanoparticles comprising encapsulated CFT as described above, are administered at a daily dose between 5 and 100 mg/kg, in particular between 5 and 75 mg/kg, more particularly between 20 and 50 mg/kg. Administrations could be repeated daily if necessary. The encapsulated CFT as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or the pharmaceutical composition as described above, also can be administered by intravenous or intraperitoneal route. The present disclosure also deals with a method of treatment of lung inflammation, lung infection or cancer which comprises administering encapsulated CFT into polymer nanoparticles as described above or a pharmaceutical composition comprising said encapsulated CFT as described above, to a subject in need thereof. The present disclosure also relates to a method of treatment of lung inflammation, lung infection or cancer which comprises administering the polymer nanoparticles comprising encapsulated CFT as described above, or a pharmaceutical composition comprising said encapsulated CFT as described above, to a subject in need thereof. Lung inflammation, lung infection and cancers are as described above in the application. In particular, said lung inflammation is chosen from the group consisting of a disease caused by a coronavirus or influenza. In particular, the lung inflammation is a disease caused by a coronavirus, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV2. Typically, the coronavirus is SARS-CoV2, and the corresponding disease is COVID-19. Thus, in a particular embodiment, the method is a method for treating COVID-19. The subject to be treated may have one or more of the following conditions or diseases: overweight, obesity, diabetes, hyperlipidaemia, arterial hypertension, cardiovascular disease, chronic kidney disease, immunosuppression. In particular, the encapsulated CFT as described above or the pharmaceutical composition as described above, are administered by inhalation route. The inhalation route comprises oral inhalation and nasal inhalation. This allows to directly target the respiratory tract. In particular, they can be conveniently delivered in the form of an aerosol or a spray presentation from pressurized packs, or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In a particular embodiment, the encapsulated CFT or the polymer nanoparticles comprising encapsulated CFT as described above, are administered at a daily dose between 5 and 100 mg/kg, in particular between 5 and 75 mg/kg, more particularly between 20 and 50 mg/kg. In one embodiment, CFT is co-administered with another active molecule. The nanoparticles may be loaded with CFT and at least one other active molecule. Thus, the present disclosure also deals with a method for the treatment of lung inflammation, lung infection or cancer which comprises administering encapsulated CFT or the polymer nanoparticles comprising encapsulated CFT as described above, and at least one other active molecule into polymer nanoparticles as described above or a pharmaceutical composition comprising said encapsulated CFT as described above, to a subject in need thereof. The present disclosure also deals with a method for the treatment of lung inflammation, lung infection or cancer which comprises administering encapsulated CFT into polymer nanoparticles as described above, the polymer nanoparticles comprising encapsulated CFT as described above, or a pharmaceutical composition comprising said encapsulated CFT as described above, to a subject in need thereof, in combination with at least one other active molecule. So, the administration of nanoparticles and the at least one other active principle can be simultaneous, sequential or over a period of time. Said at least one other active molecule is as previously described in the present application. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. Brief description of figures Figure 1. Illustration of the hypothesized mechanism of action of PEG to prevent protein adsorption on the surface of nanoparticles. Figure 2. Size distribution profiles of batches prepared by nanoprecipitation: EXPANSORB® 10P019: PLGA, 50:50 (lactide:glycolide), carboxyl-terminated (Mw: 10–20 kDa): A. [CFT] = 0 mg/mL: 220 ± 80 nm B. [CFT] = 0.5 mg/mL: 200 ± 60 nm C. [CFT] = 2.5 mg/mL: 185 ± 80 nm Figure 3. Size distribution profiles of batches prepared by emulsification-solvent evaporation: EXPANSORB® 10P019: PLGA, 50:50, carboxyl-terminated (Mw: 10–20 kDa): A. [CFT] = 0 mg/mL: 200 ± 50 nm B. [CFT] = 0,1 mg/mL: 175 ± 65 nm C. [CFT] = 2.5 mg/mL: 255 ± 70 nm D. [CFT] = 10 mg/mL: 210 ± 85 nm Figure 4. Size distribution profiles of batches prepared by emulsification-solvent evaporation: A. EXPANSORB® 10P019: PLGA, 50:50, carboxyl-terminated (Mw: 10–20 kDa): 255 ± 70 nm B. EXPANSORB® 10P024: PLGA, 50:50, ester-terminated (Mw: 10–20 kDa): 215 ± 70 nm C. EXPANSORB® 10P016: PLGA, 50:50, ester-terminated (Mw: 72–91 kDa): 215 ± 85 nm D. EXPANSORB® 10P006: PLA, carboxyl-terminated (Mw: 6–10 kDa): 220 ± 70 nm Figure 5. TEM images of different batches of nanoparticles, labelled with 2% PTA (scale bar = 1 µm): A. PLGA (15 mg/mL) + CFT (2.5 mg/mL) B. PLGA-PEG (15 mg/mL) + CFT (2.5 mg/mL) C. PLGA (11.25 mg/mL) + PLGA-PEG (3.75 mg/mL) + CFT (2.5 mg/mL) D. PLGA (7.5 mg/mL) + PLGA-PEG (7.5 mg/mL) + CFT (2.5 mg/mL) Figure 6. Kinetic study of CFT release from PLGA, PLGA-PEG, PLGA:PLGA-PEG (75:25), and PLGA:PLGA-PEG (50:50) nanoparticles. Release studied at 37°C in PBS including 20% FBS. Figure 7. Release study of CFT from PLGA-PEG nanoparticles over 24 h as a function of the percentage of FBS in the degradation medium. Figure 8. Profiles obtained by NTA before and after nebulization of PLGA and PLGA-PEG nanoparticles by PARI eFlow® rapid. Figure 9. In vitro activity of free CFT and CFT incorporated in PLGA nanoparticles. Vero 81 cells were pre-incubated with free CFT (Vhcl: DMSO) or CFT incorporated in PLGA nanoparticles (Vhcl: PLGA alone) before being infected with SARS-CoV2 (MOI 0.25). At 24 h post-infection, the viral load assessment was performed using the TCID50 technique. Figure 10. Pulmonary and plasma pharmacokinetics of CFT in mice. C57BL/6 mice received 50 mpk of CFT in its free form intraperitoneally (A) or CFT (5 mpk) incorporated into PLGA intranasally (B). At the different times indicated, the amount of CFT in plasma and ground lung was determined by LC-MS/MS. Figure 11. Antiviral efficacy of CFT-PLGA in K18-hACE2 mice. C57BL/6 mice transgenic for the human ACE2 receptor were infected intranasally with 5x10² virus particles. At 1 h, 8 h, 24 h, and 32 h post- infection, mice were treated with a PLGA formulation (control) or a CFT (5 mpk)-PLGA formulation intranasally (50 µL). At 2 days post-infection, lung viral load was assessed by TCID50 (A). Animal survival was also monitored (B) (*p < 0.05; **p < 0.01). Figure 12. Anti-inflammatory activity of CFT-PLGA in mice infected with SARS-CoV-2. C57BL/6 mice transgenic for the human ACE2 receptor were infected intranasally with 5x10² virus particles. At 1 h, 8 h, 24 h, and 32 h post-infection, mice were treated with a PLGA formulation (control) or a CFT (5mpk)- PLGA formulation intranasally (50 µL). At 2 days post-infection, the inflammatory level of the lung tissue was assessed by qRT-PCR. Figure 13. Determination of the maximum tolerated dose in mice. The animals were treated with 1 intranasal administration of PLGA-CFT or PLGA-PEG -CFT (50 µL at different concentrations). The animals were weighed 1-day post-administration to determine which doses are not tolerated. Figure 14. Tolerance assay of CFT-PLGA in hamsters. Animals were treated twice a day with 100 µL of PLGA-CFT or PLGA-PEG-CFT. The animals were weighed daily to monitor the toxicity of the treatment. Figure 15. Antiviral efficacy of CFT-PLGA in hamsters. Animals were infected intranasally with 2×10⁴ virus particles. At 1 h, 8 h, 24 h, 32 h, 48 h, and 56 h post-infection, mice were treated with a PLGA formulation (control) or a CFT (5 mpk)-PLGA formulation intranasally (100 µL). At 4 days post-infection, the lung viral load was assessed by TCID50 (** p < 0.01). Figure 16. MTT assays using PLGA-CFT nanoparticles on HeLa (A) and A549 (B) cells. Cells were plated in 96-well plates (5×10³ cells/well) and treated with various concentrations (from 6.25 µM to 200 µM) of free (white bars) or encapsulated (black bars) CFT for 24 h. Finally, cells were treated with MTT in order to assess cell viability (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). Figure 17. PLGA-PEG-CFT are less taken up by alveolar macrophages than PLGA-CFT in mouse lung. Fluorescent nanoparticles (labeled with rhodamine) were administered intranasally in mice.4 h post- administration, lung cells were isolated for flow cytometry analysis. The study of the percentage of alveolar macrophages (CD11C+ SiglecF+ F4/80+) positive for the fluorescent signal of the nanoparticles shows that the addition of PEG to the PLGA-CFT decreases their capacity to be engulfed. Examples Material & Methods 1. Preparation method: nanoprecipitation One of the NP preparation methods tested is nanoprecipitation. For a PLGA concentration of 15 mg/mL and CFT of 2.5 mg/mL, the typical protocol is as follows: first, an aqueous solution (Milli-Q water) of PVA (poly(vinyl alcohol)) (0.5% w/v) is prepared. The solution is left under magnetic stirring until complete dissolution. Then, 150 mg of PLGA and 25 mg of CFT are weighed into a vial and solubilized in 1 mL of pure acetone. In another vial, 1 mL of the PVA solution is introduced and placed under magnetic stirring. 100 µL of the solution containing PLGA and CFT are added dropwise to the PVA solution under magnetic stirring. Finally, the NP suspension is left under magnetic stirring overnight (550 rpm) to let acetone evaporate. The reagent masses can be changed to vary the properties of the nanoparticles. 2. Preparation method: emulsification-evaporation A second method for NP preparation is emulsification-solvent evaporation. For a theoretical concentration of PLGA of 15 mg/mL and CFT of 2.5 mg/mL, the standard protocol is as follows: as described in 1., an aqueous solution (Milli-Q water) of PVA (0.5% w/v) is prepared. Next, 60 mg of PLGA and 10 mg of CFT are weighed in a vial. The compounds are solubilized in 1.5 mL of DCM (dichloromethane). In the same vial, 4 mL of the PVA solution are added, before vortexing for 20 seconds. The suspension is immediately sonicated for 1.5 minute at 20% power (using a Bandelin Sonoplus probe), then for 30 seconds at 10% power. The vial is then left under magnetic stirring to let DCM evaporate. 3. Characterization by NTA, TEM and HPLC The physicochemical properties of the nanoparticles obtained are then characterized by various techniques. First, the nanoparticles, diluted to a concentration of 0.4 µg/mL, are characterized by NTA (nanoparticle tracking analysis) using a NanoSight LM10 from Malvern Panalytical, which allows to determine the mean hydrodynamic diameter, the size distribution, and the concentration of the sample. In order to observe them with an optimal resolution, nanoparticles are then studied by TEM (transmission electron microscopy). The preparations, diluted to a concentration of 750 µg/mL, are labeled for 30 seconds with 2% PTA (phosphotungstic acid), and observed with a JEOL JEM-1400 microscope at 80 kV. Loading and yield are determined by HPLC (high performance liquid chromatography). The undiluted nanoparticles are centrifuged for 5 minutes at 10000 rpm. Incorporated CFT is found within the nanoparticles in the pellet, while non-incorporated CFT is found in the supernatant. The supernatant is removed, and the pellet is dissolved in ACN (acetonitrile) to extract the CFT. Samples are diluted to a theoretical CFT concentration of 20 µg/mL in a 50:50 H2O:ACN mixture. Analysis is performed using a C18 column, at a flow rate of 1 mL/min, with a mobile phase containing 5% H2O and 95% ACN. The signal associated with CFT is detected after 5 minutes of a 7-minute analysis, running at a wavelength of 284 nm. The less signal associated with CFT in the supernatant, the greater the yield and encapsulation. 4. Release studies by HPLC The release of CFT over time from the nanoparticles is also studied by HPLC, according to the same parameters described previously. The nanoparticles are incubated at 37°C in PBS (phosphate buffer saline) with a variable amount of FBS (fetal bovine serum) for different kinetic points (from 0.5 to 24 h), before the samples are removed from the incubator and centrifuged. The proteins present in the FBS make it impossible to analyze the supernatant; therefore, it is not possible to study the supernatant during release studies. The pellet is dissolved and diluted before analysis. 5. Nebulization trials with PARI eFlow® rapid In order to demonstrate the potential of nanoparticles on an industrial scale, they are nebulized using a commercial nebulizer-inhalator (PARI eFlow® rapid, already used for the administration of amikacin by the pulmonary route (Arikace®)).3 mL of suspension are deposited in the chamber before switching on the system, and approximately 1 mL is collected in a vial before being analyzed by NTA to verify the proper preservation of the physicochemical properties of nanoparticles. 6. Internalization of nanoparticles by macrophages In order to assess intracellular internalization of nanoparticles, formulations with different PEG contents were incubated with J774 macrophages (5×10⁴ cells/well) for 4 h in 96-well plates. The nanoparticles were firmly labelled with rhodamine B, which was grafted to the polymer endgroup in order to study the fate of the nanoparticles in biological media. After the incubation, cell culture supernatants were harvested, diluted and directly studied by NTA to estimate the NP concentration in the said supernatants (Bourguignon et al., 2021). By knowing the NP concentration at the beginning of the experiment, it was possible to calculate the percentage of internalized nanoparticles. 7. In vitro tests To study the antiviral activity of CFT (incorporated or not), Vero 81 cells (ATCC, CCL-81) were incubated with different concentrations of CFT and PLGA before being infected with SARS-CoV2 (BetaCoV/France/IDF0372/2020 strain).24 h post-infection, the viral load was assessed by the TCID50 technique. 8. Pharmacokinetics in mice Eight-week-old C57BL/6 mice were administered the CFT-PLGA formulation intranasally (5 mpk CFT in 50 µL). At 30 min, 1 h, 2 h, and 4 h post-infection, animals were sacrificed to assess the concentration of CFT present in plasma and lung tissue. Lungs were mechanically ground (Tissue Lyser II) to extract the molecule with absolute ethanol (ratio 1:50). The extracts were analyzed by LC-MS/MS. 9. In vivo tests Eight-week-old C57BL/6 K18 mice expressing the human ACE2 receptor were infected intranasally with 5×10² viral particles. At 1 h, 8 h, 24 h, and 32 h post-infection, mice were treated with a PLGA formulation (control) or a CFT (5 mpk)-PLGA formulation intranasally (50 µL). At 2 days post-infection, the lung viral load was assessed by TCID50, and the inflammatory level of the tissue was assessed by qRT-PCR. The survival of the animals was also measured. 10. In vitro MTT assay to test the anticancer activity In order to assess the potential anticancerous properties of CFT-loaded nanoparticles, HeLa and A549 cells were seeded in 96-well plates (5×10³ cells/well). After 24 h of incubation, they were treated with CFT either as a free drug or in its encapsulated form, with concentrations ranging from 6.25 µM to 200 µM.24 h later, 20 µL of MTT (5 mg/mL) were added into the wells. After 2 h of incubation, supernatants were discarded and replaced with 100 µL of DMSO. The plates were read using a wavelength of 600 nm. Results 1. Preliminary results In a first step, the incorporation of CFT into cyclodextrin-based carriers (CDs) was experimented. Among the natural CDs, only γ-CD shows an affinity for CFT. However, it does not significantly increase the apparent solubility of CFT. Regarding solubility in CDs and their derivatives, the best results were obtained with β-methyl-cyclodextrin (40 mg/L = 0.04 mg/mL), an increase in solubility by a factor of 1000. This CD is the most hydrophobic and has therefore the best affinity for CFT. However, the amounts incorporated are not sufficient for pulmonary administration in mice. Liposomes also appear to be unsuitable, as the hydrophobicity of CFT implies that the latter would be concentrated at the periphery (lipidic bilayer) of the vesicles, thus limiting the possibilities of encapsulating the molecule. In this context, it was focused on the PLA/PLGA family of biodegradable polymers and explored the most suitable incorporation strategy (nanoprecipitation versus emulsification-solvent evaporation). 2. Determination of the optimal preparation method of nanoparticles The first test is done by nanoprecipitation, by employing a PLGA concentration of 15 mg/mL and varying the CFT concentration from 0 mg/mL to 10 mg/mL. However, regardless of the experimental conditions, precipitates and/or aggregates floating in the suspension were obtained. Thus, it appears that nanoprecipitation does not allow the elaboration of nanoparticles with such a high polymer concentration. We repeat the experiment using this time a theoretical PLGA concentration of only 7.5 mg/mL (Fig.2). It was observed that the active molecule disrupts the formulation of the nanoparticles, and increasingly so as its theoretical concentration increases. Based on the NTA studies, the 2.5 mg/mL CFT sample displays an extremely polydisperse population. In addition, the three other concentrations tested (5 mg/mL, 7.5 mg/mL, and 10 mg/mL) also result in batches with aggregates floating in water. In conclusion, nanoprecipitation is not a suitable method for incorporating CFT into these nanoparticles. New batches were then formulated using this time the emulsification-evaporation technique. Again, we test different theoretical CFT concentrations, going up to 10 mg/mL, and keeping the PLGA concentration at 15 mg/mL (Fig.3). While the test for a theoretical CFT concentration of 2.5 mg/mL (or lower) is conclusive (obtaining a monodisperse population), it appears that a CFT concentration too high disturbs the preparation of nanoparticles and results in an extremely polydisperse population. It was thus chosen a concentration of 15 mg/mL for PLGA, and 2.5 mg/mL for CFT. 3. Determination of the optimal polymer Other polymers are tested in order to determine the characteristics of the obtained nanoparticles (Fig. 4). PLGA terminated not by a carboxyl group but by an ester group was used, and of a more or less important molar mass. In addition to PLGA, PLA was also used. Three conclusions can be drawn from these tests: - The nature of the PLGA endgroup (carboxyl or ester) does not seem to have a major impact on the characteristics of the prepared nanoparticles. - Neither does the use of PLGA or PLA, although the population of nanoparticles obtained with PLGA is more monodisperse. - On the other hand, it appears that a higher molar mass disturbs the formation of nanoparticles, probably due to an increase of viscosity of the organic solution. In conclusion, the best size distributions are obtained with carboxyl-terminated, low molar mass-PLGA. In addition, hybrid formulations (mixing PLGA and PLGA-PEG with different ratios) were prepared in order to modify the density of PEG on the surface of the nanoparticles, and in fine, the kinetics of CFT release. To do this, at the beginning of the protocol, during the weighing of the reagents, one part of PLGA and one part of PLGA-PEG are weighed, the sum of the two remaining equal to 60 mg. A mixture of 75% PLGA and 25% PLGA-PEG (45 mg and 15 mg, respectively) and a mixture of 50% PLGA and 50% PLGA-PEG (30 mg and 30 mg) are prepared. The obtained nanoparticles are spherical in shape (Fig.5). Surprisingly, the inventors found that nanoparticles coated with a dense PEG "brush" did not release all their CFT content (about 50% of CFT remaining trapped in the core of the nanoparticles), and that the release of the active molecule was progressive (no "burst release"). It was then hypothesized that serum proteins played a major role in CFT release. The nanoparticles were thus coated with PEG "brushes" of different densities to modulate the interactions with proteins (Fig.1). The distance between the PEG chains grafted at their ends to the hydrophobic surface of PLGA nanoparticles determines the interactions with proteins, which can only adsorb if this distance is at least about 2 nm (Gref et al., 2000). If the PEG-PEG distance is below this threshold, the PEG "brush" will act as a repellent and prevent protein adsorption. It is possible to estimate the distance between two terminally-attached PEG chains on the surface of the nanoparticles using a formula (Gref et al., 2000): d = with : ^ d, the distance between two PEG molecules. ^ MPEG, the molecular weight of the PEG chains. ^ S, the surface of nanoparticles. ^ NA, the Avogadro number. ^ f, the fraction of PEG in relation to the mass of PLGA and PLGA-PEG. ^ and ρ, the density of nanoparticles. As described in Fig.1, the higher the density of PEG on the surface of the nanoparticles, the shorter the distance between two PEG molecules, and the more difficult it will be for proteins or other biomolecules in the medium to adsorb to the surface of nanoparticles. The results concerning the encapsulation of CFT according to the different formulations are reported in the following table (Table 1). With a material (polymer) concentration of around 15 mg/mL, the apparent solubility of CFT is increased by a factor of 100000 compared to its solubility in water (40 µg/L). The drug entrapment yield for PLGA (10P019) and PLGA-PEG (10P037) nanoparticles is above 95% (sometimes reaching 100%, depending on the experimental conditions), which makes them better carriers for CFT than nanoparticles prepared with alternative polymers (10P006, 10P016 and 10P024). The loading is also increased, reaching 16% for the most satisfactory batches. Table 1. Summary of the physicochemical characteristics of the different formulations prepared by emulsification-solvent evaporation for the encapsulation of CFT. To summarize the results obtained so far, the optimal formulation therefore involves carboxyl-terminated PLGA of low molar mass, which achieves a yield of over 95% and a loading of over 15%. It is also possible to mix PLGA and PLGA-PEG, which does not alter the CFT incorporation results. 3. Stability and storage In addition, a batch of PLGA-CFT nanoparticles was stored at 4°C for 3 months before being centrifuged and analyzed by HPLC to assess the incorporated drug. It was determined by HPLC that the CFT incorporation yield of the formulation remains 95%. This is a good result regarding the stability of the prepared nanoparticles under these conditions. 4. Release studies For the release kinetics study of CFT, the nanoparticles are first incubated at 37°C in pure PBS. After 24 h of incubation, an extremely low proportion of CFT (about 5%) is released and detected. The experiment is then repeated using 20% FBS in PBS, still at 37°C. The four batches observed by TEM are tested (Fig.6). The results indicate that for PLGA nanoparticles, there is a very important "burst release" (70% release of CFT in 0.5 h, ending up at 80% in 24 h). On the other hand, PLGA-PEG nanoparticles show a much more spread out release in time, with a lower final release percentage (less than 50% in 24 h). Another interesting fact is that PLGA and PLGA-PEG nanoparticles achieve a final release percentage close to that of PLGA nanoparticles, while delaying CFT release during the first kinetic points of the study. The hypothesis is that the proteins/biomolecules in the FBS interact with CFT, allowing its release. As shown in Fig.1, the PEG brushes on the surface of nanoparticles would prevent the interaction of the proteins with the molecule, which would explain why the release of CFT is increasingly extended as the proportion of PLGA-PEG in the composition of the nanoparticles increases. The results of another experiment are consistent with this hypothesis (Fig.7). PLGA-PEG nanoparticles were incubated at 37°C in PBS for 24 h with a FBS ratio ranging from 10% to 80%, before the samples were centrifuged and analyzed by HPLC. It appears that the release of CFT increases (until reaching more than 90%) with the increase of the FBS percentage compared to the PBS percentage. This tends to confirm that the release of the active molecule is due to an interaction with the proteins and other compounds present in the medium. Furthermore, PEG also plays a major role regarding interactions with macrophages in vitro. Indeed, internalization assays showed that PEGylated nanoparticles were not internalized (0%) after 4 h of incubation with J774 cells, whereas non-coated nanoparticles were taken up (20 ± 3%). Interestingly, nanoparticles with a distance between two PEG chains of around 2 nm (50% PLGA, 50% PLGA-PEG) showed an intermediate internalization percentage of 11.5 ± 2%. 5. Nebulization assays In addition, the possibility that the formulation can be used in a commercial nebulization system was investigated. The PARI eFlow® rapid system is used to nebulize PLGA and PLGA-PEG nanoparticles. The batches are analyzed by NTA before and after nebulization (Fig.8). The information obtained by NTA indicates not only that the nanoparticles maintain their hydrodynamic diameter, but also that their concentration remains the same after nebulization. Thus, the nanoparticles would neither be altered or lost during the process. This suggests the feasibility to administer the nanoparticles as a spray by nebulization. 6. Conclusion To summarize, the emulsification-solvent evaporation method allows to obtain PLGA(-PLGA-PEG)-CFT nanoparticles with a CFT incorporation yield higher than 95%, a loading higher than 15%, a stability of at least 3 months if stored at 4°C, and a compatibility with a commercial nebulizer-inhalator. Varying the proportion of PLGA-PEG in the formulation (for a distance between two PEG molecules on the surface of the nanoparticles of 2 nm to 3 nm) allows to delay the release of CFT during the first four hours. 7. Biological validation of PLGA nanoparticles incorporating CFT - In vitro tests To ensure that the incorporation of CFT into PLGA nanoparticles does not alter its antiviral activity, the efficacy of the formulation was compared with the free form of the molecule in vitro. Vero 81 cells were pre-incubated with the compounds at different concentrations (5, 10 and 20 µM) before being infected with SARS-Cov-2 (Fig.9). Evaluation of the viral load after 24 h of infection shows that incorporation of CFT into PLGA does not alter the activity of the molecule. It was also observed that PLGA particles alone were not cytotoxic (not shown) and had no negative impact on viral replication (Vhcl). This data shows that the use of PLGA nanoparticles to incorporate CFT is an interesting strategy that allows the solubilization of the molecule in aqueous medium while preserving its biological properties. - Pharmacokinetics in mice after pulmonary administration Before testing the efficacy of the nanoparticles formulation in infected mice, the pulmonary and plasma pharmacokinetics of CFT were studied after administration via the upper respiratory tract. The results were compared with those generated following intraperitoneal administration of CFT in its free form (Belouzard et al., 2022). The results show that pulmonary administration (intranasal route) of 5 mpk (mg/kg) of incorporated CFT (CFT-PLGA) (Fig.10. B) results in exposure in the lungs similar to that observed during intraperitoneal administration of 10-fold CFT in its free form (Fig.10. A). The obtained concentrations are about 100 µM during the first hours of exposure. Interestingly, plasma analysis reveals a very low systemic exposure after pulmonary administration. This last observation suggests that the administration of CFT-PLGA decreases the risks of peripheral toxicity while allowing an optimal pulmonary exposure. This strategy also allows to decrease the quantities of administered CFT. - Antiviral activity of CFT-PLGA in infected mice The antiviral efficacy of the formulation was studied in mice (K18-hACE2) infected with 5x10² TCID50 of SARS-CoV2. At 1 h, 8 h, 24 h, and 32 h post-infection, mice received a pulmonary administration of 5 mpk of CFT incorporated into PLGA nanoparticles. Control animals received the same volume of a PLGA formulation alone. Lung viral load was assessed 2 days post-infection (Fig.11. A). The results show a 1.5-log decrease in the treated mice compared with the control group. Follow-up of animal survival also showed that treatment with CFT-PLGA significantly increased the life span of the animals (Fig.11. B). These results show us that pulmonary administration of CFT-PLGA allows a good control of the viral infection. The effects observed are more interesting than those obtained with intraperitoneal administration of 10 times more active ingredient in its free form (Belouzard et al., 2022). - Anti-inflammatory activity of CFT-PLGA in infected mice The anti-inflammatory activity of the formulation was then evaluated in the lungs of mice infected with 5x10² TCID50 of SARS-CoV-2. At 1 h, 8 h, 24 h, and 32 h post-infection, mice received pulmonary administration of 5 mpk CFT incorporated into PLGA nanoparticles. At 2 days post-infection, the inflammatory level in the lung tissue was assessed by qRT-PCR (Fig.12). The results show that infection induces a high production of transcripts encoding the cytokines IL-6, TNFa, IL12p40, IFNg and IFNb compared to the group of non-infected mice (mock). The CFT-PLGA treatment completely blocks the infection-induced inflammatory response. - In vivo CFT-PLGA tolerance after intranasal administration To determine the maximal tolerated concentration in mice, different concentrations of the PLGA-CFT and PLGA-PEG-CFT formulations were administered intranasally in mice (Fig. 13). After 1 administration, the weight of the animals was assessed to determine the dose that does not induce weight loss. The results show that mice tolerate very well the doses administered up to 1.4 mpk. In hamsters (Fig.14), repeated administrations (twice a day for 3 days – 2.5 mpk) are very well tolerated, since the animals do not show significant weight loss. - Antiviral activity of CFT-PLGA in infected hamsters The antiviral efficacy of the formulation was studied in hamsters infected with 2x10⁴ TCID50 of SARS- CoV2. At 1 h, 8 h, 24 h, 32 h, 48 h, and 56 h post-infection, animals received a pulmonary administration of 2.5 mpk of CFT incorporated into PLGA nanoparticles. Control animals received the same volume of a PLGA formulation alone. Lung viral load was assessed 4 days post-infection (Fig.15). The results show a 1-log decrease in the treated hamster compared with the control group. 8. Anticancerous properties of PLGA-CFT nanoparticles Preliminary studies suggest the potential of PLGA-CFT nanoparticles against cancerous cells. HeLa and A549 cells (cervix and lung cancers, respectively) were treated with CFT, either in its free form or encapsulated in PLGA nanoparticles, for 24 h. Results show that the viability decreases for both cell lines as the CFT concentration increases (Fig. 16). Furthermore, the observed effect is even more important with loaded nanoparticles compared to the free drug, suggesting that nanoparticles enhance the anticancerous potential of CFT. For example, for HeLa cells, the IC50 appears to be between 25 µM and 50 µM for loaded nanoparticles, while it is between 50 µM and 100 µM for free CFT. 9. PLGA-CFT and PLGA-PEG-CFT accumulation in alveolar macrophages The addition of PEG to the nanoparticles is supposed to reduce their capture by macrophages. This was evaluated on alveolar macrophages from mouse lungs. Mice were intranasally administered with a fluorescent PLGA-CFT (rhodamine) formulation comprising 0, 50 or 100% of PLGA-PEG. After 4h of administration, the mice were euthanized and the percentage of rhodamine positive alveolar macrophages (F4/80+ CD11c+ SiglecF+) was quantified by flow cytometry (Fig.17). The results show that in the absence of PEG almost 90% of alveolar macrophages are positive for rhodamine while this decreases to 85 and 70% for PLGA-PEG(50%)-CFT and PLGA-PEG(100%)-CFT, respectively. This clearly shows that the addition of PEG reduces the capture of nanoparticles by the alveolar macrophages. Depending on the application, we can therefore distinguish whether the CFT should be released mainly in the macrophages (e.g., intracellular infection) or outside of them (e.g., epithelial inflammation or cancer treatment). Bibliography Bailly, Vergoten. A new horizon for the old antibacterial drug clofoctol. Drug Discov. Today 2021;26(5):1302-1310. Belouzard et al. Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice. PLoS Pathog.2022;18(5):e1010498. Bourguignon et al. 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Lima et al. Polymeric nanocapsules: A review on design and production methods for pharmaceutical purpose. Methods 2022;199:54-66. Mazzella et al. Systemic inflammation and lung cancer: is it a real paradigm? Prognostic value of inflammatory indexes in patients with resected non-small cell lung cancer. Cancers 2023;15(6):1854. Scaglione et al. In vitro and in vivo pharmacokinetic/pharmacodynamic activity of clofoctol. J. Chemother.2012:24(4);201-206. Scherlieβ et al. Particle engineering in dry powders for inhalation. Eur. J. Pharm. Sci.2022;172:106158. Wang et al. Identification of an old antibiotic clofoctol as a novel activator of unfolded protein response pathways and an inhibitor of prostate cancer. Br. J. Pharmacol.2014;171:4478-4489.

Claims

CLAIMS 1. Polymer nanoparticles comprising encapsulated clofoctol (CFT), said polymer nanoparticles comprising a polylactic-co-glycolic acid copolymer (PLGA) and pegylated PLGA (PLGA -PEG).

2. Polymer nanoparticles comprising encapsulated CFT according to claim 1 wherein the ratio by weight between PLGA and PLGA-PEG is 100:0 (w/w) to 50:50 (w/w), particularly 80:20 (w/w) to 20:80 (w/w), 80:20 (w/w) to 50:50 (w/w), 75:25 (w/w) to 25:75 (w/w), in particular 75:25 (w/w) or 50:50 (w/w).

3. Polymer nanoparticles comprising encapsulated CFT according to claim 1 or 2 wherein the charge of CFT loading into the nanoparticles is between 10 and 30 wt%, in particular between 15 and 20 wt%.

4. Polymer nanoparticles comprising encapsulated CFT according to any one of claims 1 to 3 wherein the mean hydrodynamic diameter of the nanoparticles is comprised between 100 and 500 nm, in particular between 100 and 350 nm, more particularly between 140 and 280 nm.

5. Pharmaceutical composition comprising a suspension of polymer nanoparticles according to any one of claims 1 to 4, and an additional pharmaceutical acceptable excipient.

6. Polymer nanoparticles comprising encapsulated CFT according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5, wherein said encapsulated CFT or said pharmaceutical composition are in the form of a spray, an aerosol or a dry powder.

7. Polymer nanoparticles comprising encapsulated CFT according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5, for use as a medicament.

8. Polymer nanoparticles comprising encapsulated CFT according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 5, for use in the treatment of lung inflammation, lung infection or cancer.

9. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to claim 8, wherein the lung inflammation is a disease caused by a coronavirus.

10. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to claim 9, wherein the coronavirus is SARS-CoV, MERS-CoV or SARS-CoV2.

11. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to claim 9 or 10 wherein the coronavirus is SARS-CoV2 and the corresponding disease is COVID-19.

12. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to any one of claim 7 to 11, wherein the polymer nanoparticles comprising encapsulated CFT or the pharmaceutical composition is administered by inhalation route, such as oral inhalation and nasal inhalation.

13. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to any one of claim 7 to 11, wherein the encapsulated CFT or the pharmaceutical composition is administered by intravenous route.

14. Polymer nanoparticles comprising encapsulated CFT or a pharmaceutical composition for use according to any one of claim 7 to 12, wherein the polymer nanoparticles comprising encapsulated CFT or the pharmaceutical composition is administered in a dose comprised between 5 and 100 mg/kg.

15. Manufacturing process of polymer nanoparticles comprising encapsulated clofoctol (CFT), said polymer nanoparticles comprising PLGA and PLGA-PEG, comprising the following steps: - preparing an aqueous solution of a surfactant at about 0.5% w/v, - solubilizing PLGA, PLGA-PEG and CFT at a ratio PLGA/PLGA-PEG : CFT about 6:1 w/w in a solvent and adding it in the aqueous surfactant solution, - sonicating of the resulting mixture, - evaporating the solvent.