WO2021191266A1 - Aerosolization of hdl for the treatment of lung infections - Google Patents

Abstract

Lung infections remain a substantial concern, even in wealthy countries. For instance, the emergence of a new betacoronavirus SARS-CoV-2 has led to a major health-related crisis associated with a significant mortality in intensive care units, due to the pulmonary complications of COVID-19. The inventors aim at developing a new therapeutic approach based on the local delivery of apolipoprotein A1 nanoparticles into the lung by aerosolization. These particles may display antiviral properties that could be improved by loading them with small interference RNAs specifically targeting SARS-CoV2 genome and antiviral drugs (such as the hydrophobic chloroquine). Their preliminary results have shown in preclinical mouse models that aerosolization allowed apoA1 nanoparticle uptake and persistence (up to 72h) in pulmonary cells. In addition to the potential intrinsic antiviral activity of apoA1, these particle display important anti-inflammatory effects that may limit the excessive immuno-inflammatory host response that is deleterious for the lung. Thus, the present invention relates to aerosolization of HDL for the treatment of lung infections.

Description

AEROSOLIZATION OF HDL FOR THE TREATMENT OF LUNG INFECTIONS

FIELD OF THE INVENTION:

The present invention is in the field of infectiology.

BACKGROUND OF THE INVENTION:

Lung infections remain a substantial concern, even in wealthy countries. Of infectious diseases, acute lower respiratory infections cause the most deaths and are the largest burden of disease. Subpopulations are especially prone to pneumonia, such as the elderly and those with comorbidities. For those older than 65 years, pneumonia hospitalizations are increasing, and hospitalization for pneumonia carries a significantly increased risk of mortality compared with other hospitalizations. At the other end of the age spectrum, pneumonia is the most common reason that children become hospitalized.

For instance, a wide range of viruses inducing upper and lower respiratory tract infections have been identified as causes of significant morbidity and mortality among infants and adults. These respiratory viruses include members of the Pneumoviridae family, including human respiratory syncytial virus (hRSV) type A and B, and human metapneumovirus (hMPV) type A and B; members of the Paramyxoviridae family, including parainfluenza virus type 3 (PIV-3) and measles virus; and members of the Coronaviridae family, including endemic human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKUl); severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERS-CoV).

In particular, in late December 2019, a new betacoronavirus SARS-CoV-2 has emerged in Wuhan China [1-3] The World Health Organization has named the severe pneumonia caused by this new coronavirus COVID-19 (for Corona Virus Disease 2019, WHO, 2020). Since its emergence, the SARS-CoV-2 has spread to 159 countries across the five continents causing, at the time of the writing, about 213,254 human infections with 81,238 cases in China (ECDC, March 19 2020). Europe has recently become the epicenter of COVID-19 epidemics with 82,869 confirmed cases; the majority of them being reported in Italy with 35,713 cases and 2978 deaths. In France, the number of confirmed cases is increasing with about 10,995 and 372 deaths on mid-march 2019 (Sante Publique France). To fight against the COVID-19 pandemic in a long term, in addition to the containment measures implemented in many countries, several projects have been launched around the world to understand the viral evolution and the pathophysiological consequences of the infection in order to identify therapeutic targets and to implement innovative therapies.

High-density lipoproteins (HDLs) are composed of their major protein apoAl (apolipoprotein Al) and a phospholipid layer forming particles able to transport cholesterol in its free or esterified form as well as triglycerides. In addition to apoAl, HDL particles contain major apolipoproteins including apoAII, Apo-CI, Apo-CII, Apo-CIII and key enzymes involved in their remodeling. Since the development of proteomic approaches and their blooming early 2000, a large variety of proteins have been identified, being associated with HDL particles. These proteins are linked to lipid metabolism but also belong to the acute-phase- response protein family, known to be modulated under acute and chronic inflammation conditions. Other proteins were related to complement pathway and its regulation as well as specific apoAl complexes that help kill pathogens suggested that HDLs may have a role in innate immune response. More than 100 different proteins have been identified in HDLs suggesting a multiplicity of functions for HDL particles [4] However, all protein species cannot be present in one HDL particles or even in subspecies, suggesting that only a fraction of the particles carry specific proteins [5]

Lipids account for about half of HDL particle mass and comprise more that 200 species identified including phospholipids, cholesterol (free or esterified), sphingolipids and triglycerides [6] Lipids contained in HDLs may display anti-infectious activity. For example, gangliosides have been used in reconstituted HDLs (rHDLs) to binds polymeric cholera toxin and protect cells from this biological toxin [7] Other gangliosides presents in HDL particles may also be relevant to protection from infection but need to be further investigated [8] Sphingosine-1 -phosphate (SIP) is a bioactive lipid mainly transported by HDLs in the blood stream that regulates pathophysiological processes involved in sepsis progression [9], in particular pro-inflammatory cytokine release, endothelial permeability, and vascular tone.

In addition to proteins and lipids, HDL particles are able to carry nucleic acids such as small RNAs including miRNA, tRNA, snRNA, etc. [10] MicroRNA (miR) are viewed as extracellular messenger that can be transported by lipoproteins and particularly by HDL particles [11] HDL-associated miR profiles have been identified mainly in dyslipidemic and atherosclerotic patients [12]

In addition to their function of reverse transport of cholesterol from peripheral tissues back to the liver, these nanoparticles display pleiotropic effects including antioxidant, anti inflammatory, anti-apoptotic functions particularly beneficial for endothelial cells [13] HDLs have been reported to exert anti-infectious properties such as lipopolysaccharide (LPS) binding and subsequent inactivation [14] HDL levels are decreased under septic conditions [15,16] and injection of reconstituted HDL was protective in three different models of sepsis [17] HDL antiviral capacity is less documented; HDLs may display antiviral effects by neutralizing both DNA and RNA viruses with or without an envelope [18] HDL-mediated antiviral activity could be due to apoAl interference with viral entry into the cell, or during the fusion with the target cell [19] HDLs may also induce direct viral inactivation [20] Van Lenten and coll have reported that D4F, an apoAl mimetic peptide could attenuate lung inflammation in mice infected with influenza A. This peptide also displayed antiviral activity, leading to a reduction of influenza titre by more than 50% PFU (plaque forming units/mg tissue) [21,22] These studies suggest that apoAl -based therapies may represent a potential therapeutic approach to fight influenza A-induced pneumonia. Furthermore, apoAl KO mice have increased inflammatory cell infiltration and impaired pulmonary vasodilatation [23], whereas ABCA1 KO mice (lacking this HDL receptor) also display decreased pulmonary functions. HDLs and ApoAl are acceptors for respectively ABCA1 and ABCG1, respectively, which are notably expressed in various cell types of the alveolus including alveolar epithelial type I and II cells (ATI and ATII) as well as alveolar macrophages (see figure adapted from [24]). However aerosolization of HDL for the treatment of lung infections has never been investigated.

SUMMARY OF THE INVENTION:

As defined by the claims, the present invention relates to aerosolization of HDL for the treatment of lung infections.

DETAILED DESCRIPTION OF THE INVENTION:

The emergence of a new betacoronavirus SARS-CoV-2 has led to a major health-related crisis associated with a significant mortality in intensive care units, due to the pulmonary complications of COVID-19. The inventors aim at developing a new therapeutic approach based on the local delivery of HDLs into the lung by aerosolization. These particles may display antiviral properties that could be improved by loading them with small interference RNAs specifically targeting SARS-CoV2 genome and antiviral drugs (such as the hydrophobic chloroquine). Their preliminary results have shown in preclinical mouse models that aerosolization allowed apoAl nanoparticle uptake and persistence (up to 72h) in pulmonary cells. In addition to the potential intrinsic antiviral activity of apoAl, these particle display important anti-inflammatory effects that may limit the excessive immuno-inflammatory host response that is deleterious for the lung. Accordingly, the first object of the present invention relates to a method of treating a lung infection in a patient in need thereof comprising administering to the patient’s lungs a therapeutically effective amount of HDL by aerosolization.

As used herein, the term “lung infection” has its general meaning in the art and means the invasion of lung tissues of a patient by disease-causing microorganisms, their multiplication and the reaction of lung tissues to these microorganisms and the toxins that they produce.

In some embodiments, the patient suffers from a chronic lung infection. As used herein, the term “chronic infection” refers to a long-term infection which may be an apparent, unapparent or latent infection.

In some embodiments, the patient suffers from an acute lung infection. As used herein, the term “acute lung infection” has its general meaning in the art and refers to a disease of the lungs characterized by inflammation and consolidation followed by resolution and caused by infection from viruses, fungi, or bacteria. The term is also known as “pneumonia”. Typically acute lung infection is associated with lung inflammation that is the rapid onset of progressive malfunction of the lungs, and is usually associated with the malfunction of other organs due to the inability to take up oxygen.

As used herein the term “patient” refers to a mammalian to which the present invention may be applied. Typically said mammal is a human, but may concern other mammals such as primates, dogs, cats, pigs, sheep, cows. In particular, the term “patient" refers to a mammalian patient, such as a human, who is confirmed to have a lung infection or who may be classified as having a probable or suspected case of a lung infection based on epidemiological factors. Patients include those who are diagnosed with a lung infection, those who test positive for infection by an infectious agent (pathogen) associated with a lung infection (e.g., SARS-CoV), those who are suspected of having a lung infection based on epidemiological factors, or those who are at an imminent risk of contracting a lung infection (e.g., one who has been exposed or will likely be exposed to a lung infection in the near future).

In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant.

In some embodiments, the patient may suffer from a lung disease such as Chronic Obstructive Pulmonary Disease (COPD) or Cystic Fibrosis.

As used herein, the term "treatment" or "treat" refer 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 patient 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 patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, the lung infection is a bacterial infection, such as bacterial pneumonia. In some embodiments, the bacterial infection is 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, Bordatella pertussis, Stenotrophomonas maltophilia., a bacterium from the citrobacter family, a bacterium from the ecinetobacter family, and Mycobacterium tuberculosis.

In some embodiments, the lung infection is a fungal infection. In some embodiments, the fungal infection is caused by a fungus selected from the group consisting of Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis jiroveci, Coccidioides immitis, Candida albicans , and Pneumocystis jirovecii (which causes pneumocystis pneumonia (PCP), also called pneumocystosis).

In some embodiments, the lung infection is a viral infection, such viral pneumonia. In some embodiments, the viral infection is 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 (SARS-coronavirus such as SARS-Covl or SARS-Cov 2), and smallpox. In some embodiments, the viral lung infection may be due to a member of the Pneumoviridae , Paramyxoviridae and/or Coronaviridae families are in particular selected from the group consisting of upper and lower respiratory tract infections due to: human respiratory syncytial virus (hRSV), type A and type B, human metapneumovirus (hMPV) type A and type B; parainfluenza virus type 3 (PIV-3), measles virus, endemic human coronaviruses (HCoV-229E, -NL63, -OC43, and -HKU1), severe acute respiratory syndrome (SARS) and Middle-East respiratory syndrome (MERS) coronaviruses. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of COVID-19.

In some embodiments, the lung infections include but are not limited to pneumonia (including community-acquired pneumonia, nosocomial pneumonia (hospital-acquired pneumonia, HAP; health-care associated pneumonia, HCAP), ventilator-associated pneumonia (VAP)), ventilator-associated trachebronchitis (VAT), and bronchitis.

As used herein, the term “HDL” encompasses native HDL or reconstituted HDL.

In some embodiments, the HDL is a native HDL. As used herein, the term “native HDL” refers to HDL purified from human healthy donors. Typically, HDL can be isolated by two different methods: ultracentrifugation and immunoabsorption. Isolation of HDL by immunoabsorption is performed using anti-apoAl column prepared by crosslinking goat polycolonal antibodies directed against apoAl to Sepharose beads. Isolation of HDL by ultracentrifugation is performed by classical double-step ultracentrifugation in KBr density gradient interval of 1.063-1.210 g/ml. It falls within the ability of the skilled man to carry out such methods.

In some embodiments, the HDL is a reconstituted HDL. As used herein, the terms “reconstituted HDL”, “rHDL” or “synthetic HDL” refer to a particle structurally analogous to native HDL, composed of a lipid or lipids in association with at least one of the proteins of HDL, preferably apoAl, and which exhibits all of the known physiological functions of HDL. The term is also named “apoAl nanoparticles”. Typically, the components of reconstituted HDL may be derived from blood, or produced by recombinant technology. Typically, reconstituted HDL may be prepared by complexation of apoAl to phospholipids. Methods for obtaining reconstituted HDL are disclosed in EP 1 425 031 and US 5,652,339. Typically, suitable lipids for the preparation of rHDL are phospholipids, preferably phosphotidylcholine, for example l-palmitoyl-2-linoleoyl phosphatidylcholine (PC) or 1,2-dipalmitoyl PC. Optionally, rHDL contains other lipids, for example cholesterol, cholesterol esters, triglycerides, or other lipids. The lipids may be synthetic, naturally occurring lipids or combinations thereof. Methods for preparing reconstituted HDL are well known in the art and typically those described in Other methods include those described in Matz CE, Jonas A. J Biol Chem. 1982 Apr 25;257(8):4535-40 or Kim Y, Fay F, Cormode DP, Sanchez-Gaytan BL, Tang J, Hennessy EJ, Ma M, Moore K, Farokhzad OC, Fisher EA, Mulder WJ, Langer R, Fayad ZA. ACS Nano. 2013 Nov 26;7(11):9975-83

Typically, native HDL or reconstituted HDL have a molar ratio of phospholipid/apoAl from 2 to 250, preferably from 10 to 200, more preferably from 20 to 100, more preferably 20 to 50 and most preferably from 30 to 40.

In some embodiments, rHDL may optionally contain additional lipids such as cholesterol, cholesterol esters, triglycerides and/or sphingolipids, preferably in a molar ratio of lipid /apoAl up to 20.

In some embodiments, the HDL is loaded with an agent. Typically, the agent comprised in the HDL according the invention has either lipophilic properties or an affinity for some proteins constituting the HDL. Alternatively, the agent may be chemically modified to improve its association with HDL. Typically, the agent is selected from the group consisting of anti- infective agents, anti-inflammatory agents, bronchodilators, enzymes, expectorants, leukotriene antagonists, leukotriene formation inhibitors, and mast cell stabilizers.

In some embodiments, the agent is an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a corticosteroid. As used, the term “corticosteroids” has its general meaning in the art and refers to class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with an anti-inflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (11b, 17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxy cortisone, dexamethasone (21 -(acetyloxy)-9-fluoro- 1 b, 17-dihydroxy- 16a-m-ethylpregna- 1 ,4-diene-3 ,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9- chloro-11-b, 17,21, tri hydroxy- 1 όb-methylpregna- 1 ,4 diene-3, 20-dione 17,21 -dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort and betamethasone corticosteroids, for example, cortisone, hydrocortisone, methylprednisolone, prednisone, prednisolone, betamethesone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.

In some embodiments, the agent is an antibiotic agent. Antibiotic include, but are not limited to, amoxicillin, b- lactamases, aminoglycosides, betalactam (glycopeptide), bΐhoΐhihhbeb, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sultonamide, tetracyclines, trimtethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In some embodiments, the agent is an anti-viral compound. Preferably, the anti-viral compound is preferably an antibody (e.g., monoclonal , polyclonal, chimeric, etc.), an inhibitor of viral RNA-dependent RNA polymerase, an inhibitor of a virus-encoded protease that affects processing of a viral RNA-dependent RNA polymerase, an inhibitor of virus budding or release from infected cells, such as one that affects the activity of hemagglutinin-esterase, an inhibitor of virus binding to a specific cell surface receptor, or an inhibitor of receptor-induced conformational changes in virus spike glycoprotein that are associated with virus entry and combinations thereof. Examples of anti-viral compounds include antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, aptamers, antibodies, peptides, small molecules, and other polypeptides. In some embodiments, the agent is a siRNA suitable for blocking the expression of a viral protein. In some embodiments, anti- viral compounds include nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (Pis) for example. Example of antiviral agents include but are not limited to acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and/or oseltamavir; a protease inhibitor, such as indinavir, nelfmavir, ritonavir and/or saquinavir; a nucleoside reverse transcriptase inhibitor, such as didanosine, lamivudine, stavudine, zalcitabine, zidovudine; a non-nucleoside reverse transcriptase inhibitor, such as nevirapine, efavirenz. In some embodiments, the anti-viral compound is hydroxychloroquine. As used herein, the term "hydroxychloroquine" or “HCQ” has its general meaning in the art and refers to 2-[[4- [(7-Chloro-4-quinolyl) amino] pentyl] ethylamino] ethanol sulfate (1:1). Methods of synthesis for hydroxychloroquine are disclosed in U.S. Pat. No. 2,546,658, herein incorporated by reference.

The person skilled in the art would be aware of the conditions for carrying out the loading of the agent. Typically, the loading is performed by ultracentrifugation of the HDLs that were previously incubated with an amount of the agent. For example, if the agent has a low affinity with the HDL, the HDL will be incubated with a higher concentration of said agent and for a longer time, than if the agent had a natural and high affinity for the HDL. In addition, the person skilled in the art is able to select the appropriate Molecular Weight Cutoff of the centrifugal device for carrying out the above mentioned filtrations.

As used herein, the expression ’’effective amount” means an amount of HDL used in the present invention sufficient to result in the desired therapeutic response i.e. the treatment of the lung infection.

As used herein, the term "aerosolization" refers to a process whereby a liquid formulation is converted to an aerosol. Typically, aerosolization is performed by a nebulizer.

As used herein, the term “nebulizer” or “aerosol generator” has its general meaning in the art and refers to a device that converts a liquid into an aerosol of a size that can be inhaled into the respiratory tract. Pneumonic, ultrasonic, electronic nebulizers, e.g., passive electronic mesh nebulizers, active electronic mesh nebulizers and vibrating mesh nebulizers are amenable for use with the invention if the particular nebulizer emits an aerosol with the required properties, and at the required output rate. The process of pneumatically converting a bulk liquid into small droplets is called atomization. The operation of a pneumatic nebulizer requires a pressurized gas supply as the driving force for liquid atomization. Ultrasonic nebulizers use electricity introduced by a piezoelectric element in the liquid reservoir to convert a liquid into respirable droplets. Various types of nebulizers are described in Respiratory Care, Vol. 45, No. 6, pp. 609-622 (2000), the disclosure of which is incorporated herein by reference in its entirety. The terms “nebulizer” and “aerosol generator” are used interchangeably throughout the specification. “Inhalation device”, “inhalation system” and “atomizer” are also used in the literature interchangeably with the terms “nebulizer” and “aerosol generator”.

For instance, the device can include a ventilator, optionally in combination with a mask, mouthpiece, mist inhalation apparatus, and/or a platform that guides users to inhale correctly and automatically deliver the drug (i.e. the HDL) at the right time in the breath. Representative aerosolization devices that can be used in accordance with the methods of the present invention include but are not limited to those described in U.S. Patent Nos. 6,357,671 ; 6,354,516; 6,241 ,159; 6,044,841 ; 6,041 ,776; 6,016,974; 5,823,179; 5,797,389; 5,660,166; 5,355,872; 5,284,133; and 5,277,175 and U.S. Published Patent Application Nos. 20020020412 and 20020020409.

Using a jet nebulizer, compressed gas from a compressor or hospital airline is passed through a narrow constriction known as a jet. This creates an area of low pressure, and liquid medication from a reservoir is drawn up through a feed tube and fragmented into droplets by the air stream. Only the smallest drops leave the nebulizer directly, while the majority impact on baffles and walls and are returned to the reservoir. Consequently, the time required to perform jet nebulization varies according to the volume of the composition to be nebulized, among other factors, and such time can readily be adjusted by one of skill in the art.

A metered dose inhalator (MDI) can be used to deliver a composition of the invention in a more concentrated form than typically delivered using a nebulizer. For optimal effect, MDI delivery systems require proper administration technique, which includes coordinated actuation of aerosol delivery with inhalation, a slow inhalation of about 0.5-0.75 liters per second, a deep breath approaching inspiratory capacity inhalation, and at least 4 seconds of breath holding. Pulmonary delivery using a MDI is convenient and suitable when the treatment benefits from a relatively short treatment time and low cost.

Aerosolized HDL of the invention comprise droplets that are a suitable size for efficient delivery within the lung. Preferably, the formulation is effectively delivered to lung bronchi, more preferably to bronchioles, still more preferably to alveolar ducts, and still more preferably to alveoli. Thus, aerosol droplets are typically less than about 15 pm in diameter, and preferably less than about 10 pm in diameter, more preferably less than about 5 pm in diameter, and still more preferably less than about 2 pm in diameter. For efficient delivery to alveolar bronchi of a human subject, an aerosol composition preferably comprises droplets having a diameter of about 1 pm to about 5 pm. Droplet size can be assessed using techniques known in the art, for example cascade, impaction, laser diffraction, and optical pattemation. See McLean et al. (2000) Anal Chem 72:4796-804, Fults et al. (1991 ) J Pharm Pharmacol 43:726-8, and Vecellio None et al. (2001 ) J Aerosol Med 14:107-14.

The compositions of the present invention may comprise one or more pharmaceutically acceptable excipients, in particular selected from the group of an HFC/HFA propellant, a co solvent, a bulking agent, a non-volatile component, a buffer/pH adjusting agent, a surfactant, a preservative, a complexing agent, or combinations thereof. Suitable propellants are those which, when mixed with the solvent(s), form a homogeneous propellant system in which a therapeutically effective amount of HDL can be dissolved. The HFC/HFA propellant must be toxicologically safe and must have a vapor pressure which is suitable to enable the HDL to be administered via a pressurized MDI. According to the present invention, the HFC/HFA propellants may comprise, one or more of 1,1,1,2-tetrafluoroethane (HFA- 134(a)) and 1,1,1,2,3,3,3,-heptafluoropropane (HFA-227), HFC-32 (difluoromethane), HFC-143(a) (1,1,1- trifluoroethane), HFC-134 (1,1,2,2-tetrafluoroethane), and HFC-152a (1,1-difluoroethane) or combinations thereof and such other propellants which may be known to the person having a skill in the art.

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.

FIGURE:

Figure 1. Human ApoAl is detected in mouse blood after HDL inhalation.

Blood was harvested from mice before (HO) and 2, 6, 24, 72 hours or 7 days after F1DL nasal administration. Plasma ApoAl concentration was measured using a commercial kit (Human apolipoprotein AI ELISA Kit SimpleStep, ref ab 189576, Abeam). Results are given in median ± interquartile range.

EXAMPLE 1

Methods:

Isolation of High-Density Lipoproteins

HDLs (density between 1.063 and 1.21 kg/1) were isolated from plasma of healthy normocholesterolemic volunteers by ultracentrifugation. Plasma density was adjusted to d = 1.22 with KBr. The resulting solution was overlaid with KBr saline solution (d = 1.063) and by water (d = 1). Ultracentrifugation was performed at 100, 000 g for 24 hours at 10°C. The HDL fraction was recovered as a single band, and was then dialyzed in buffered saline for 24h to eliminate KBr. For HDL concentration, we used a centrifugal concentrating device (Vivaspin 5 kD molecular weight cut-off; Sartorius, Les Ulis, France). Contamination by albumin was verified using electrophoresis in reducing conditions as described below. Protein quantification (essentially ApoAl) was performed using the Bradford method. HDLs were stored at 4°C and used within 2 weeks for experiments. HDL labelling with carbocyanines

Plasma from healthy donor was incubated at 37°C overnight with a fluorescent solution containing 10 mg/mL DilCis carbocyanines (Invitrogen©) followed by HDL isolation using ultracentrifugation as described elsewhere. Unbound DilCis was eliminated during this step. Labelled HDL were harvested and kept at 4°C in the dark for further utilization (less than 15 days after HDL isolation).

Dot blot

We used Dot blot protocol allowed detection and semi-quantification of AAT after spotting onto a nitrocellulose membrane and subsequent immunologic revelation. Fifty pL of 1 pg/pL purified AAT (Zemaira, CSL Behring) was blotted onto the membrane, with dilutions from 0.1 pg/pL to 0.03 pg/pL. Fifty pL of HDL enriched or not with AAT (concentration to be determined) were also blotted. An aspiration device (dot blot 96 system, Biometra©) allowed sample transfer onto the nitrocellulose membrane. Ponceau S solution staining was used to visually assess correct blotting. After washing with TBS-tween, the nitrocellulose membrane was saturated for 20 minutes at room temperature with 5% BSA. Primary antibody (goat pAb to a- 1 -antitrypsin conjugated to FERP, ref ab7635, Abeam) diluted to 1/5000 in BSA 1% was incubated for a 1 hour at RT. Three washing steps using TBS-Tween were used to eliminate unbound antibodies. Revelation was performed by applying a commercial kit (DAB peroxidase substrate kit, 3,3’ -diaminobenzidin, Vector laboratories©) following manufacturer’ s protocol.

HDL nasal administration, fluorescence microscopy and immunohistology

C57B1/6 mice, aged 10 weeks, were provided by Charles River laboratories. Standard laboratory chow and drinking water were provided ad libitum. Local ethic committee approved all animal experimental work. All mice were anesthetized using intraperitoneal (i.p.) medetomidine and ketamine (approx. 300 pg/kg and 25 mg/kg respectively). Intranasal administration was initiated 30 minutes after i.p. injection. A solution of 40 pL was disposed in the nostril entrance drop by drop, while mouse was maintained in standing position. Mice were then warmed and watched until wake-up. When indicated, mice were sacrificed by exsanguination after isoflurane anaesthesia.

Three sets of experiments were conducted.

1- For the first set, 2 mice (one male, 27.7g and one female, 22.4g, tolerance group) were administered twice 40pL of FIDLs concentrated to lOmg/mL (total dose approx. 30 mg/kg). These mice were monitored three times a week and were euthanized if they showed signs of respiratory distress, which included abnormal breathing, lack of mobility or lethargy.

2- For the second set, 8 male mice (24-27 g) were aerosolised with 40 pL of F1DL (5 mg/mL, approx. 7 mg/kg) labelled with carbocyanines (DilCis). Blood was harvested by submandibulary puncture (approx. 20 mΐ, in EDTA) before inhalation and at 2 hours, 6 hours, 24 hours, 72 hours and at day 7, centrifuged and frozen at -80°C for further analysis. Two mice were sacrificed by exsanguination at 6 hours, 24 hours, 72 hours and at day 7. Vascular compartment was washed with isotonic saline. Lungs, liver and kidneys were harvested, immersed in Optimal Cutting Temperature (OCT) medium and immediately frozen at -80°C. Twelve pm-sections were later obtained using a cryotome and nuclei were stained with 4’,6- diamidino-2-phenylindole (DAPI) for 15 minutes (60 pg per ml of mounting medium, Vectashield©). Observation was conducted with a fluorescence microscope.

3- For the third set of experiments, 6 mice (3 females, 3 males, 21-32g) were used for intranasal administration. Experiments were conducted each time in duplicate using 1 male and 1 female: 2 mice received 40 pL of native HDLs (6.4 mg/mL), 2 mice received 40 pL of HDL enriched with AAT (respectively 6.4 mg/mL total proteins and 0.1 pg/pL AAT)) and 2 mice received AAT in PBS (0.1 pg/pL). Mice were sacrificed on the second day following inhalation. The vascular compartment was washed with saline and infused with 4% paraformaldehyde (PFA); lungs were then fixed in 4% PFA overnight, kept for 2 days in a 30% sucrose solution and then frozen in OCT at -80°C. Frozen sections (12 pm) were obtained using a cryotome. OCT was rinsed with PBS, followed by 5 minutes incubation with proteinase K (Proteinase K Ready to use, ref S3020, Dako©) allowing proteolytic digestion of formalin- fixed tissues prior to immunohi stochemi cal procedures. After 20 minutes blocking with 5% bovine serum albumin (BSA), primary antibodies (8.1 pg/mL, rabbit pAb anti-human ot-1 antitrypsin, Dako©) or non-relevant antibodies (8.1 pg/mL negative control rabbit Ig fraction, Dako©) were incubated for 1 hour at room temperature (RT). After 3 washing steps with PBS, secondary antibodies (dilution 1/200, goat anti-rabbit conjugated to Alexa 594, Dako©) were incubated at RT during an additional hour. Images were observed on a fluorescent microscope after adding DAPI for nucleus staining as described above.

ApoAl quantification by ELISA Blood was harvested from mice on EDTA before (HO) and 2, 6, 24, 72 hours or 7 days after HDL nasal administration (second set of experiments). After 2000g centrifugation for 10 minutes, plasma was recovered and -80°C frozen for further analyses. Plasma ApoAl concentration was measured using a commercial kit (Human apolipoprotein AI ELISA Kit SimpleStep, ref ab 189576, Abeam©) following manufacturer’ s instructions. Results are given in median ± interquartile range.

Nebulization

Nebulization of HDL was performed with two commercial devices: a perforated oscillating membrane nebulizer (Eflow rapid, PARI©) and a pneumatic nebulizer (InnoSpire Elegance, Respironics, Philips©).

In brief, each device was loaded with HDLs at a concentration of 1 mg/mL in PBS. Five mL were disposed in the tank and nebulization was conducted with a constant depression of 100 mBar to mimic spontaneous inspiration. Nebulized product was then recovered in an impinger and kept at 4°C for further analyses (data not shown).

Electrophoretic migration

Two types of electrophoretic migration were performed:

1- For determining albumin contamination of HDLs after isolation, we performed a SDS-10% PAGE under reducing conditions using b-mercapto-ethanol. Polyacrylamide gel consisted in a 4% acrylamide stacking part and a 12% acrylamide concentration part, both containing sodium dodecyl sulphate (SDS). Sample were loaded in wells after 10 minutes denaturation at 95°C with 20% b-mercapto-ethanol, 2% SDS, 10% glycerol and Coomassie blue. One well was loaded with Color burst electrophoresis molecular weight marker (Sigma). Migration was conducted at 50 mA for 3 hours. Staining was obtained by incubation with a solution containing 30% propanolol, 10% acetic acid with 0.45% Coomassie blue R and water, for 1 hour followed by incubation overnight under gentle shaking in a destaining solution (30% ethanol, 10% acetic acid, water).

2- For qualitative HDL lipid profile analysis, we used the Lipoprint HDL subfraction test (Quantimetrix©) according to the protocol supplied by the manufacturer. Briefly, samples (25 mΐ) were mixed with Lipoprint loading gel containing Sudan black dye which binds to cholesterol. This mix was placed on the top of a supplied tube containing a 3% polyacrylamide gel. After photopolymerisation, electrophoresis was performed for 50 minutes at 3mA per tube. Gels were then scanned and analysed using the Lipoware© software. This method allows migration in non-reducing condition and provides ten subfractions of HDL according to their size. These subfractions are grouped in three categories: small, intermediate or large.

Total cholesterol dosing

We used a colorimetric assay (Total cholesterol colorimetric assay kit, Biovision©) to quantify total cholesterol in HDLs before and after aerosolisation. Briefly, total cholesterol in HDL solution was submitted to hydroxylation by cholesterol esterase to form cholesterol esters and then oxidized by cholesterol oxidase. This oxidation releases H2O2 proportionally to cholesterol esters and H₂O₂ can be quantified adding a cholesterol probe that produces red color (maximal absorbance at 570 nm).

Results:

A single ultracentrifugation step for HDL isolation from plasma

Classical HDL isolation protocols use two ultracentrifugation steps to separate HDLs from other lipoproteins and to avoid plasma protein contamination ( Ortiz-Munoz G, et al. (2009) HDL antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property. The FASEB Journal 23(9):3129-3139.). For practical purpose, we tried to recover HDLs by creating 3 layers of different densities adjusted with KBr, followed by a single ultracentrifugation step. HDLs were harvested in the middle layer (d= 1.21). SDS- PAGE and subsequent staining by Coomassie blue showed no major albumin contamination (data not shown).

HDL inhalation is harmless and leads to stable lung deposit and low systemic passage

After HDL isolation by ultracentrifugation, KBr was removed by dialysis and FEDLs were concentrated to 10 mg/mL using a centrifugal filter device (Vivaspin 5 kD molecular weight cut-off; Sartorius, Les Ulis, France). To test mice tolerance, 80 pL of HDL solution were administered (30 mg/kg) by inhalation to 2 mice. These mice behaved normally and were still alive at D14.

After inhalation of HDLs labelled with DilCis, a lipophilic dye, fluorescence microscopy showed a pulmonary deposit of HDL-DiICi₈ at 2 hours, 6 hours, 24, hours, 72 hours and up to 7 days (data not shown). A weak fluorescent signal was detected at day 7, suggesting that HDL and/or its label have being metabolized. No fluorescence was detected by histology in other organs (spleen, kidneys or liver).

After inhalation of HDL in mice (concentration 5mg/ml, namely approx. 7 mg/kg), human ApoAl was quantified in plasma by ELISA (Figure 1). We found that ApoAl was able to cross the lung epithelial barrier and was observed in plasma as soon as H2 (median 1.16 ng/ml; [interquartile range (IQR) 0.22-2.10]). HDL concentration peaked at H24 (68.59 ng/ml [IQR 54.92-82.26]) and then rapidly decreased. Blood concentration was negligible at D7 (0.44 ng/ml [IQR 0.01-0.87]).

HDLs are weakly modified by aerosolisation

After aerosolisation of 5 ml of native HDLs diluted to 1 mg/mL, 1.4 mL and 1.7 mL were harvested with Eflow© and Inno Spire Elegance© device respectively. Due to nebulization characteristics, aerosolized HDLs were recovered earlier with Eflow (8 min) than with InnoSpire Elegance (15 min) device. Protein concentration remained unchanged. Cholesterol concentration decreased with both nebulizers, in a comparable fashion (1.70 g/L in native HDL solution and 0.69 or 0.79 g/L in HDLs aerosolized by InnoSpire Elegance© or Eflow© devices respectively).

Lipid profiles assessed by the non-reducing electrophoretic Lipoprint method (Quantimetrix Systems) were not altered by any of the two devices, suggesting that no aggregate of HDLs was formed during aerosolisation (data not shown). Large, intermediate and small HDL subclasses (%) were distributed as follows: 49,46,5 for native HDLs, 55,42,4 for HDLs aerosolised by InnoSpire Elegance© and 54,41,4 for HDLs aerosolised by Eflow©.

Discussion:

Our in vivo study shows that HDL inhalation in mice is well tolerated and leads to a rapid and stable/sustainable lung deposit along with delayed transient systemic passage. Moreover, in vitro results suggest that HDL aerosolization is feasible, does not produce aggregates and that HDLs resist the physical constraints due to aerosolization.

With their size of about 10 nm, HDLs are the smallest lipoproteins in circulation. This small size allows penetration into the lungs but might preclude deposit because of wash out during expiration (Scheuch G, Siekmeier R (2007) Novel approaches to enhance pulmonary delivery of proteins and peptides. J Physiol Pharmacol 58 Suppl 5(Pt 2):615-625.). Our study showed that inhaled HDLs could reach the alveoli, cross the lung and finally reach the bloodstream. The droplets formed by nasal inhalation, mimicking those formed after aerosolization, may represent an explanation for this observation. Indeed, several animal studies focusing on aerosolization use natural halting mouse respiration to create droplets that reach lung alveoli (Koussoroplis SJ, et al. (2014) PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract. Journal of Controlled Release 187(C):91-100.). Nevertheless, the MicroSprayer® aerosoliser that was initially paned in our study is no more commercially available.

The presence of ApoAl in the bloodstream indicates that HDLs are able to cross the lung barrier. With a peak concentration observed at 24 hours, transcytosis is conceivable but further studies are warranted to confirm this hypothesis and to describe potential receptors or carriers involved in the pathway. In a study exploring pharmacokinetics of inhaled monoclonal antibodies (Herve V, et al. (2014) VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations. mAbs 6(6): 1638-1648.), authors suggested a receptor-mediated transport associated with a possible lymphatic passage. Because we did not use intravenous administration, we were not able to compare plasma HDL bioavailability between pulmonary and systemic administration. When present in the vascular compartment, HDLs use SR-B1 or ABCGl receptors to cross the endothelial barrier and reach the intima (Rohrer L, et al. (2009) High-Density Lipoprotein Transport Through Aortic Endothelial Cells Involves Scavenger Receptor BI and ATP -Binding Cassette Transporter Gl. Circulation Research 104(10): 1142-1150.). The pathway allowing HDLs to access the blood compartment after lung deposit is currently unknown.

Inhalation methods are characterised by a variable lung reaching and pulmonary deposit ( Moller W, et al. (2011) Nasally inhaled pulsating aerosols: lung, sinus and nose deposition. Rhinology 49(3):286-291.). Nasal route is even used for drug administration because of high permeability and vascularisation of the nasal mucosa ( Fortuna A, Alves G, Serralheiro A, Sousa J, Falcao A (2014) Intranasal delivery of systemic-acting drugs: Small-molecules and biomacromolecules. European Journal of Pharmaceutics and Biopharmaceutics 88(l):8-27.). Considering these data, it is therefore consistent to note fluctuating blood concentration following nasal administration. Even with recent aerosolisation devices, lung deposit after inhalation is highly dependant on nebulizer characteristics nebulisation configuration and drug administered, with variability reported to be in a range between 10 to 58% ( Knoch M, Keller M (2005) The customised electronic nebuliser: a new category of liquid aerosol drug delivery systems. Expert Opinion on Drug Delivery 2(2):377-390.). These results could be an explanation for the high interquartile ranges of ApoAl concentration in mice blood observed in our study. Similar results have been obtained with pulmonary administration of antibodies, where plasma biovailability is known to be around 10% compared to the same dose administered i.v. (2). Isotopic tracers could be used to better describe HDL pulmonary distribution following inhalation.

Aerosolisation of drugs has two theoretical main aims. First, airway administration allows topical delivery of a drug in pulmonary diseases, with a high local concentration (31). Second, it may allow systemic administration in a non-invasive fashion, taking advantage of a huge absorption area (approx. 100m2, with blood supply close to cardiac output), low amount of metabolizing enzymes and no first-pass effect compared to gastro-intestinal route.

In order to determine the potential application of HDL administration via the pulmonary route in human, we assessed the properties of HDLs after nebulization. In this study we tested two commercial devices, commonly used in clinical practice, to aerosolize HDL particles. Eflow© inhaler works with a perforated oscillating membrane whereas InnoSpire Elegance© nebulizer is a pneumatic device using the Venturi effect to enhance aerosolization. According to manufacturers' technical data, Eflow© and InnoSpire Elegance© devices allow production of particles with a Mass Median Diameter (MMD) of 4,1 pm and 4,54 pm respectively. In this study, both allowed HDL aerosolization without major lipoprotein modification that could have been induced by mechanical stress. Electrophoretic migration in denaturing and reducing conditions did not show any HDL polymer as attested by the unique band at 28 kDa (corresponding to ApoAl molecular weight). Studies addressing HDL aggregation use dithiothreitol, a strong reducing agent to separate aggregates. Nevertheless, polymers may have been separated by b-mercapto-ethanol; we thus submitted our samples to electrophoresis under non-reducing conditions to confirm this data. This experiment shows a comparable migration profile, demonstrating that aerosolization does not produce HDL aggregation.

Surprisingly, we found less total cholesterol in aerosolized HDL compared to native HDL. Adsorption of HDL particles to the aerosolization device or recovery system is conceivable.

HDL particles can be divided into several subfractions, depending on the isolation method. Most studies distinguish two HDL classes: HDL2 being larger and less dense (density < 1.125 kg/1) and than HDL3 (density > 1.125 kg/1). These HDL subclasses have been proven to contain different protein cargoes and to exert different effects with HDL3 possessing stronger antioxidant capacities. Because we used whole HDL for our experiments, we were unable to distinguish potential specific subclass modification induced by aerosolisation. However, the Lipoprint profiles are similar and suggest that both aerosolised and native HDLs display the same repartition between small, intermediate and large HDL particles.

Conclusion:

Pulmonary route is an attractive way for reaching lung structures and a particular interest for the treatment of lung infections. Pulmonary administration supposes aerosolization to form small particles able to reach the alveoli. High-density lipoproteins are involved in lung patho physiology and have pleiotropic effects that could potentially be used to cure lung infection diseases. Our results show that HDL inhalation is possible and allows vectorization toward the lung epithelium with a maximum accumulation reached at 48 hours. We also demonstrated a blood passage of HDLs after pulmonary administration with a peak concentration of ApoAl at 24 hours. Altogether, these results are promising and supportive of testing HDL-mediated lung therapy in animal models of lung infection. Additionally, we demonstrated that HDLs could be subjected to aerosolization without major physical modifications.

EXAMPLE 2 In vitro

ApoAl nanoparticles can either be made in-house or available commercially from CSL Behring: CSL111, (already used to limit sepsis in our previous work (Tanaka, S.; Geneve, C.; Zappella, N.; Yong-Sang, L; Planesse, C.; Louedec, L.; Viranaicken, W.; Bringart, M.; Montravers, P.; Denamur, E., et al. Reconstituted High-density Lipoprotein Therapy Improves Survival in Mouse Models of Sepsis. Anesthesiology 2020, 10.1097/ALN.0000000000003155). To perform test of ApoAl nanoparticles- virus interactions in cellulo , each SARS-CoV strain at 2 x 10⁵ TCID50 are incubated with ApoAl nanoparticles (50 to 400 pg/mL) at 37°C for 1 hour. Then the Apol nanoparticles-virus mixtures are transferred to suitable cell lines with 2 ^c 10⁵ cells per well (as above). After incubation at 37°C with a 5% C02 atmosphere until cytopathic effect, the viral inhibitory activity of ApoAl nanoparticles are calculated by RT-PCR using primers targeting the nsp4 and nucleoprotein genes as described (School of Public Health. Hong Kong University). The SARS-CoV progeny production is determined by measuring the quantity of infectious virus particles released into the supernatant of infected cells using PFA.

In vivo ApoAl nanoparticles are administered by intranasal route to mice (n=8/group) 3 days after the initial viral infection. Briefly, 80 microL of particles (40 mg apoAl/kg mouse) are administered. After 24 hours, mice are sacrificed and the same protocol as described for the in vitro study is used. The efficacy of ApoAl nanoparticle treatment is evaluated by histology, viral charge and cytokine production. The presence of ApoAl is evaluated in the different tissues by immunostaining.

EXAMPLE 3

Two strategies is used to improve the antiviral capacity of ApoAl nanoparticles: direct targeting of SARS Cov-2 RNA using siRNAs combined with apoAl nanoparticles loading with hydrophobic antiviral molecules in order to improve their pulmonary delivery

Design and delivery of si RNA by ApoAl nanoparticles

RNA silencing mechanisms are mobilized by vertebrate hosts in response to exogenous viral infections. siRNAs have been used against SARS-CoV both to clear viral infection in cell lines {Zhang, Y.; Li, T; Fu, L.; Yu, C.; Li, Y; Xu, X; Wang, Y; Ning, H.; Zhang, S.; Chen, W., et al. Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference. FEBS Lett 2004, 560, 141-146) and as prophylactic and therapeutic assays in non-human primates {Li, B.J.; Tang, Q.; Cheng, I). ; Qin, C.; Xie, F.Y.; Wei, Q.; Xu, J; Liu, Y.; Zheng, B.J.; Woodle, M.C., et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 2005, 11, 944-951). Here, siRNA development for SARS-CoV- 2 inhibition was assessed based on a recent review for MERS coronavirus [Sohrab, S.S.; El- Kafrawy, S.A.; Mirza, Z.; Kamal, M.A.; Azhar, E.I. Design and Delivery of Therapeutic siRNAs: Application to MERS-Coronavirus. Curr Pharm Des 2018, 24, 62-77\. The complete genome sequence of SARS-CoV-2 Wuhan-Hu-1 isolate (NC_045512.2) served as reference for all queries. We used the small interfering RNA software (DSIR http://biodev.extra.cea.fr/) to predict 21-nt siRNA target sequences in all coding regions of the virus. After filtering all non specific sequences, the top-20 scoring siRNA molecules all fell within the Orfla region of the SARS-CoV-2 genome. These 20 siRNA are synthesized (Genecust, Boynes, France) and preliminary screened for antiviral activity by direct transfection of suitable cells infected by HCoV-229E/GFP. The potent inhibitor siRNA candidates are fused with ApoAl nanoparticles for further assays against SARS-CoV-2. To that purpose, selected siRNA are incorporated in ApoAl nanoparticles as previously described by Rui et al. [ Rui , M; Tang, H.; Li, Y.; Wei, X; Xu, Y. Recombinant high density lipoprotein nanoparticles for target-specific delivery of siRNA. Pharm Res 2013, 30, 1203-1214 ]. To evaluate the antiviral activity, four modalities are tested: siRNA against SARS-CoV-2 alone

ApoAlnanoparticles alone (apolipoprotein Al combined with phosphatidylcholines)

ApoAl nanoparticles containing siRNA directed against SARS-CoV2

- NC scrambled

Enrichment of apoAl nanoparticles by antiviral hydrophobic molecules

It is expected that hydrophobic molecules will get incorporated into apoAl nanoparticles, in particular chloroquine, which may display antiviral properties. After dialysis, apoAl nanoparticle content is analyzed by mass spectrometry in order to quantify antiviral drug-loading in antiviral drugs. For both siRNA and antiviral molecule enrichment of apoAl nanoparticles, in cellulo and in vivo studies follow the same protocol as described in EXAMPLE 2. Cytopathic effect (MTS assay) is measured in presence of different non cytotoxic concentrations of drugs. The antiviral effect is evaluated with RT-PCR, PFA, immunofluoresence and cytokine assays. A number of mice is set at n=8/group according to the data available in the litterature, showing no sign of mortality for the different treatments used in our study. All animal protocols are submitted to our local ethics committee before any experiment. The total number of mice used in the project is 112 (n=8/group, ApoAl, Mock, ApoAl -siRNA, siRNA, siRNA scrambled, ApoAl -chloroquine, chloroquine, with or without infection by SARS-CoV2).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method of treating a lung infection in a patient in need thereof comprising administering to the patient’s lungs a therapeutically effective amount of HDL by aerosolization.

2. The method of claim 1 wherein the patient suffers from a chronic lung infection.

3. The method of claim 1 wherein the patient suffers from an acute lung infection.

4. The method of claim 1 wherein the patient suffers from a bacterial infection.

5. The method of claim 1 wherein the patient suffers from a fungal infection.

6. The method of claim 1 wherein the patient suffers from a viral infection.

7. The method of claim 6 wherein the viral infection is caused by a virus selected from the group consisting of influenza virus, respiratory syncytial virus, metapneumovirus, cytomegalovirus, parainfluenza virus, rhinovirus, adenovirus, coxsackie virus, echo virus, herpes simplex virus, coronavirus and smallpox.

8. The method of claim 7 wherein that patient suffers a Severe Acute Respiratory Syndrome (SARS).

9. The method of claim 7 wherein the patient suffers from COVID-19.

10. The method of claim 1 wherein the HDL is a native HDL or a reconstituted HDL.

11. The method of claim 1 wherein the HDL is loaded with an agent selected from the group consisting of anti-infective agents, anti-inflammatory agents, bronchodilators, enzymes, expectorants, leukotriene antagonists, leukotriene formation inhibitors, and mast cell stabilizers.

12. The method of claim 11 wherein the agent is an antibiotic.

13. The method of claim 11 wherein the agent is an anti-viral compound.

14. The method of claim 11 wherein the anti-viral compound is a siRNA suitable for blocking the expression of a viral protein.

15. The method of claim 11 wherein the anti- viral compound is hydroxychloroquine.