DESC:4. DESCRIPTION

FIELD OF INVENTION
The present invention relates to formulations and methods for delivering drug for SARS-CoV-2 in mammals (e.g., humans), including but not limited to SARS-CoV-2, SARS, MERS etc. The present invention relates to compositions intended for inhalation administration. More specifically, it concerns liquid and powder formulations targeting human respiratory system.

BACKGROUND OF THE INVENTION
An acute respiratory disease, caused by a novel coronavirus (SARS-CoV-2, previously known as 2019-nCoV), the coronavirus disease 2019 (COVID-19) has spread throughout China and received worldwide attention. On 11 March 2020, World Health Organization (WHO) officially declared the COVID-19 pandemic. More than 22,000 people died and 4,80,000 are infected as on current date.

Pneumonia is a common infection in the parenchyma of the lower respiratory tract that can affect all age populations. There is significant morbidity and mortality associated with pneumonia, especially in the very young and elderly populations. Pneumonia is the leading cause of death in children younger than 5 years of age worldwide. The average yearly incidence of pneumonia, specifically community-acquired pneumonia, is 5–11 per 1000, with most incident cases occurring in the winter months.

Pneumonia classification is based upon a variety of factors age, clinical presentation and comorbidities, as well as history of previous hospital admissions or residence in a nursing facility. The best approach is a good history and physical exam in combination with knowledge of the most common causes of pneumonia for the presenting age patient being seen. Community-acquired pneumonia (CAP) must be distinguished from hospital-acquired pneumonia (HAP), healthcare-associated pneumonia (HCAP), or ventilator-associated pneumonia (VAP) before treatment is started. In addition, the cause of the pneumonia must be determined to be bacterial, viral, or atypical in nature before treating.

Bacterial pneumonia, specifically Streptococcus pneumoniae, is the most common cause of pneumonia across all ages. Certain comorbidities or risk factors such as age greater than 65, alcohol abuse, recent antibiotic use (within the past 3 months), coexisting medical diagnoses of COPD or CHF, and exposure to day care/nursing home (child or adult) increase the likelihood that the patient may have illness caused by other bacterial causes or have a pneumonia that may require additional or different treatment. Acute respiratory distress syndrome (ARDS) is a type of respiratory failure characterized by rapid onset of widespread inflammation in the lungs. Symptoms include shortness of breath (dyspnea), rapid breathing (tachypnea), and bluish skin coloration (cyanosis). For those who survive, a decreased quality of life is common.

Causes may include sepsis, pancreatitis, trauma, pneumonia, and aspiration. The underlying mechanism involves diffuse injury to cells which form the barrier of the microscopic air sacs of the lungs, surfactant dysfunction, activation of the immune system, and dysfunction of the body's regulation of blood clotting. In effect, ARDS impairs the lungs' ability to exchange oxygen and carbon dioxide. Adult diagnosis is based on a PaO2/FiO2 ratio (ratio of partial pressure arterial oxygen and fraction of inspired oxygen) of less than 300 mm Hg despite a positive end-expiratory pressure (PEEP) of more than 5 cm H2O.

Corona Virus (CoVs) are positive-stranded RNA viruses with a crown-like appearance under an electron microscope (coronam is the Latin term for crown) due to the presence of spike glycoproteins on the envelope. The subfamily Orthocoronavirinae of the Coronaviridae family (order Nidovirales) classifies into four genera of CoVs: Alphacoronavirus (alphaCoV), Betacoronavirus (betaCoV), Deltacoronavirus (deltaCoV), and Gammacoronavirus (gammaCoV). Furthermore, the betaCoV genus divides into five sub-genera or lineages. Genomic characterization has shown that probably bats and rodents are the gene sources of alphaCoVs and betaCoVs.

Thus, SARS-CoV-2 belongs to the betaCoVs category. It has round or elliptic and often pleomorphic form, and a diameter of approximately 60–140 nm. Like other CoVs, it is sensitive to ultraviolet rays and heat. Furthermore, these viruses can be effectively inactivated by lipid solvents including ether (75%), ethanol, chlorine-containing disinfectant, peroxyacetic acid and chloroform except for chlorhexidine.

Hydroxychloroquine, is a medication used to prevent and treat malaria in areas where malaria remains sensitive to chloroquine. Other uses include treatment of rheumatoid arthritis, lupus, and porphyria cutanea tarda. It is taken by mouth, often in the form of hydroxychloroquine sulfate. Hydroxychloroquine increases lysosomal pH in antigen-presenting cells by two mechanisms: As a weak base, it is a proton acceptor and via this chemical interaction, its accumulation in lysozymes raises the intralysosomal pH, but this mechanism does not fully account for the effect of hydroxychloroquine on pH. Additionally, in parasites that are susceptible to hydroxychloroquine, it interferes with the endocytosis and proteolysis of hemoglobin and inhibits the activity of lysosomal enzymes, thereby raising the lysosomal pH by more than 2 orders of magnitude over the weak base effect alone. In 2003, a novel mechanism was described wherein hydroxychloroquine inhibits stimulation of the toll-like receptor (TLR) 9 family receptors. TLRs are cellular receptors for microbial products that induce inflammatory responses through activation of the innate immune system.

Studies have demonstrated that HCQ also confers its considerable broad-spectrum antiviral effects via interfering with the fusion process of these viruses by decreasing the pH and hence conferring antiviral effect. Also, chloroquine alters the glycosylation of the cellular receptors of coronaviruses and interferes with viral entry.

Azithromycin is a broad-spectrum antibiotic that acts by inhibiting protein synthesis. It is associated with side effects that might be avoided by aerosol delivery to the lungs. Azithromycin is an antibiotic medication used for the treatment of a number of bacterial infections. This includes middle ear infections, strep throat, pneumonia, traveler's diarrhea, and certain other intestinal infections. Along with other medications, it may also be used for malaria. It can be taken by mouth or intravenously with doses once per day.

Azithromycin prevents bacteria from growing by interfering with their protein synthesis. It binds to the 50S subunit of the bacterial ribosome, thus inhibiting translation of mRNA. Azithromycin a BCS class II drug, AZI demonstrates low oral bioavailability (37%) and requires high dose for therapy at which it causes detrimental effects on gastrointestinal and auditory systems.

HCQ and azithromycin on the core proteins of hyperinflammation. These proteins include major inflammatory response mediators such as the neutrophil chemoattractant CXCL8; immunoregulatory cytokines such as IL1B, IL2, IL4, IL6 and IL10; the proinflammatory protein TNF; the mediator of cellular response to viral infections IFNG; and the cytotoxicity signalling molecule IL12. The study results suggest that both hydroxychloroquine and azithromycin can exert an overall inhibitory effect on the core proteins of hyperinflammation. Despite the need for further validation, these results strongly indicate a major immunosuppressant role for both drugs on hyperinflammation.

In vitro, CQ, starting with 0.1 µM inhibited and, moreover, completely prevented SARS-CoV infections in cell cultures at 10 µM, suggesting a prophylactic effect and preventing the virus spread 5 hours after infection. HCQ is threefold more potent than CQ in SARS-CoV-2 infected cells, resulting in the clinical recommendation to treat orally with 800 mg at the first day and at 400 mg on the following four days.

Two major points of action are postulated for CQ/HCQ in the treatment of COVID-19: (1) they perturb the terminal glycosylation of the ACE2 protein and inhibit cell-binding of the virus, (2) the alkalizing medication elevates the pH value of the endosomes. Since Cathepsin L requires an acidic environment to crack the viral S protein, virus induced activities are reduced. Other possible explanations for the antiviral effect include a reduction in MAK kinase activation or a disruption of the maturation of the viral M protein. Further, CD8+ cells directed against the virus may be activated and thereby lead to a decrease of pro-inflammatory cytokines

Hydroxychloroquine impair the terminal glycosylation of ACE2 without significant change of cell-surface. ACE2 increases the local pH value, which reduces the activity of cathepsin L needed for hydrolysis of the viral S protein and might influence the generation of pro-inflammatory cytokines. Therefore, CQ might be a potent inhibitor of SARS-CoV infection.
Inhalation.

The local treatment of pulmonary infections with antibiotics has been suggested and, with tobramycin being the first antibiotic approved for this use, successfully introduced to the therapy of certain severe or even life-threatening types of infection. Tobramycin, which is supplied as TobiTM, is a sterile, clear, slightly yellow, non-pyrogenic, aqueous solution with the pH and salinity adjusted specifically for administration by a compressed air driven reusable nebuliser. It is approved for the treatment of cystic fibrosis patients infected with Pseudomonas aeruginosa.

Effective treatment of bacterial pneumonia requires the concentration of the antibiotic in the lung to exceed the minimum inhibitory concentration (MIC) of the infecting pathogen. However, while some antimicrobial drugs such as fluoroquinolones penetrate well into lung tissue when administered intravenously others (e.g., ß-lactams, colistin, aminoglycosides and glycopeptides such as vancomycin) have poor lung penetration and tis- sue distribution . Poor lung penetration of drugs can be overcome by dose increases, but this management approach is often limited by the associated risk of systemic adverse events; for example, high systemic concentrations of azithromycin are associated with Nausea, vomiting, diarrhea/loose stools, stomach pain. The effectiveness of IV antibiotic therapy may be further diminished by pharmacokinetic changes in critically ill patients, including changes in absorption, distribution and elimination. Such patient-specific variation makes adequate dosing of antibiotics challenging and may result in the delivery of drug concentrations that are either too low (and therefore sub-therapeutic) or too high (and therefore toxic).

In mechanically ventilated and intubated patients with pneumonia, targeting antibiotics to the lungs via aerosolization could offer a way to achieve high exposures of antibiotics directly at the site of infection, while minimizing systemic side effects.

Previous approaches to nebulized antibiotic therapy have had several clinical and technical limitations, including sub-optimal delivery and lack of drugs specifically formulated for aerosolization. While issues such as these have hampered aerosolized delivery techniques, several recent developments suggest that these short- comings could be eliminated. Nebulized antibiotics can achieve high drug concentrations in the lungs. The key advantage of administering antibiotics by inhalation rather than via IV infusion is the potential to deliver high concentrations of antibiotic directly to the site of lung infection. Nebulized antibiotics are associated with low systemic exposure. The high lung concentrations achieved with inhaled antibiotics are paired with low systemic absorption; indeed, administering antibiotics such as aminoglycosides by aerosolization generates significantly lower peak serum concentrations compared with intravenous administration.

One potential benefit of lower systemic concentrations is a reduced incidence of adverse events, such as nephrotoxicity. In addition, low systemic concentrations may also have the benefit of falling outside the mutant selection window, thus reducing the risk of systemic resistance development. Studies in patients with cystic fibrosis treated with aerosolized anti- biotics have not reported an increase in the emergence of resistance with inhaled therapy compared with standard therapy or placebo. This is supported by a recent double-blind placebo-controlled study of patients in the ICU, which demonstrated that in comparison with placebo, aerosolized antibiotics were not associated with the development of new antibiotic resistance.

Inhaled administration may reduce the need for systemic antibiotics
Aerosolized antibiotic therapy also provides the potential for a reduction in the overall use of systemic antibiotics, with clear benefits for antibiotic stewardship and management of emergent resistance. Increased resistance due to frequent or excessive use of systemic antimicrobials has been documented for several drug classes.

There is no existing ideal inhaled antibiotic therapy that should have a suitable formulation for aerosolization and consistently deliver high antibiotic concentrations to the site of infection via an efficient device; the drug should also have limited systemic penetration to prevent unwanted side effects.

Presently, oral administration of macrolides and fluoroquinolones active against typical and atypical pathogens are treatments of choice for CB. However, oral administration of macrolide antibiotics has adverse side effects. The most common side effects associated with the treatment of oral/parental macrolide antibiotics are diarrhoea/loose stools, nausea, abdominal pain and vomiting

In WO 00/35461, a method for the treatment of severe chronic bronchitis (bronchiectasis) using a concentrated aminoglycoside antibiotic formulation is disclosed. The method includes delivering the antibiotic to the lungs endobronchial space including alveoli in an aerosol or dry powder having a mass medium diameter predominately between 1 and 5 microns. The method comprises the administration of the antibiotic at a concentration one to ten thousand times higher than the minimal inhibitory concentration of the target organism. Preferably, the method comprises the endobronchial administration of aerosolized tobramycin to treat pseudomonal infections in severe chronic bronchitis patients.

EP0223831B1, a system for application of a water-soluble medicine in the respiratory tract is described, using liposomes for the encapsulation of the medicine and providing an appliance not described in more detail for the atomisation of a defined quantity of liposomes by means of ultrasound or pneumatically for inhalation.
There exists a significant need for efficient and stable formulation for inhalation that deliver Azithromycin and Hydroxychloroquine medicaments for prolonged time for individuals suffering from pulmonary disease specifically the community acquired pneumonia (CAP).

Hence to solve the existing gaps the present invention has found that the above problems can be overcome by the development of sustained release liposomal inhalation suspension formulation as described herein.

The present invention has developed is a formulation, which comprises Hydroxychloroquine or its pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof, and azithromycin, or its pharmaceutical acceptable salt, solvate or physiologically functional derivative thereof, preferably the product or formulation being in a form suitable for nasal or inhalation administration in the prophylaxis and/or treatment of SARS-CoV-2.

OBJECTIVE OF THE INVENTION
The primary objective of the present invention is related to the delivery of sustained release liposomal inhalation suspension formulation for lung infections, especially in cases where an individual suffers from Pneumonia and/or Acute lung injury and/or Acute respiratory distress syndrome.

Another objective of the present invention is to provide a topical drug delivery comprising a combination of drugs for pulmonary infectious diseases in the form of aerosols

SUMMARY OF INVENTION
The present invention has been accomplished under the circumstances in view. It is therefore the main object of the present invention to provide a shelving system for storing multiple discrete materials.

The invention provides a pharmaceutical aerosol for nasal, sinunasal or pulmonary administration comprising a dispersed liquid phase and a continuous gas phase. The dispersed liquid phase essentially consists of aqueous droplets comprising azithromycin and hydroxychloroquine antibiotics. The droplets of the dispersed phase have a mass median diameter from about 1.5 to about 4.5 µm, with the droplet size distribution exhibiting a geometrical standard deviation from about 1.2 to about 3.0.

In another aspect, the invention provides liquid and solid pharmaceutical compositions from which such aerosol can be prepared. The liquid composition comprises an effective dose of the active compound in a volume of not more than about 10 ml and more preferably less than 5 ml. In analogy, the solid composition is dissolvable or dispersible in an aqueous liquid solvent having a volume of not more than about 10 ml and more preferably less than 5 ml.

Furthermore, the invention provides a kit comprising a nebuliser and a liquid composition, wherein the nebuliser is adapted to aerosolise the liquid composition into an aerosol as described above.

The invention further discloses a method of preparing and delivering an aerosol to a person in need of nasal, sinunasal or pulmonary antibiotic treatment or prophylaxis. The method comprises the step of providing a liquid pharmaceutical composition comprising an effective dose of an active hydroxychloroquine and azithromycin antibiotics in a volume of not more than about 10 ml and more preferably less than 5 ml, and the step of providing a nebuliser capable of aerosolising said liquid pharmaceutical composition at a total output rate of at least 0.1 ml/min, the nebuliser further being adapted to emit an aerosol comprising a dispersed phase having a mass median diameter from about 1.5 to about 6 µm and a geometrical standard deviation from about 1.2 to about 3.0. In a subsequent step, the nebuliser is operated to aerosolise the liquid composition, thus providing a pharmaceutical aerosol which can be inhaled by a patient in need of antibiotic therapy.

BRIEF DESCRIPTION OF THE DRAWINGS
So that the manner in which the above-recited features of the present invention is understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

The features and advantages of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings, which form a part of this application and in which:

Fig. 1 depicts mass distribution of Liposomal Liposomal Hydroxychloroquine and azithromycin nebulizate collected on impactor stages as a function of cutoff diameter. The three Liposomal Hydroxychloroquine and azithromycin lots of Table 15 legend (designated as 1, 2, and 3) were used with the air-jet nebulizer and ACI system (solid symbols) and with NGI system (open symbols) in accordance with one or more embodiments of the present invention;

Fig. 2 depicts reduction in the Log10CFU/Lungs of Rats after Inhalation of Liposomal hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) 75 mg/mL. The symbols represent the Log10CFU/lungs of each rat 18 days after the instillation. Twenty-eight days after the initiation of treatment, all mice were euthanized and lungs were processed to determine the post-treatment MAH 104 burden. Dots resting on the x-axis are animals that had no culturable MAH in their lungs. Abbreviations: QID×28d?=?treatment every day for 28d; Q1D×14d?=?treatment every other day for 14d, then no treatment for 14d; Q2D×28d?=?treatment every other day for 28d. Horizontal lines represent the mean and SEM. Statistical comparisons: *?=?p<0.05 compared with inhaled saline control in accordance with one or more embodiments of the present invention;

Fig 3: Mechanism of action of Azxithromycin and Hydroxychloroquine in ARDS/ALI/Pnuemonia inflammation in accordance with one or more embodiments of the present invention;

Fig. 4 is a schematic diagram depicting an overview of the modelling strategy used in the present invention. Nominal dose: amount of Liposomal hydroxychloroquine and azithromycin filled in the nebuliser; delivered dose: amount of Liposomal hydroxychloroquine and azithromycin in aerosol particles generated by the air-jet nebuliser, and available in the face mask for inhalation; Inhaled dose: amount of Liposomal hydroxychloroquine and azithromycin in aerosol particles available at the upper respiratory tract (i.e., the dose which is inhaled); deposited dose: amount of Liposomal hydroxychloroquine and azithromycin in aerosol particles deposited in the lower respiratory tract; systemic dose: amount of Liposomal hydroxychloroquine and azithromycin absorbed via the alveolar lining fluid of the lower respiratory tract and released into circulation. in accordance with one or more embodiments of the present invention;

Fig 5 is a graphical representation of the lung organ in the model for pulmonary administration of the present invention. The extension of the model structure compared to a standard PK-SIM* model is marked by the bold box that represents the alveolar lining fluid (ALF compartment) in accordance with one or more embodiments of the present invention;

Fig 6 is a schematic diagram depicting PBPK model-building and scaling steps. IP: intrapulmonary; IV: intravenous; physic-chemical data: characterization of Liposomal hydroxychloroquine and azithromycin, in accordance with one or more embodiments of the present invention;

Fig 7 is a graph depicting the fraction of inhaled dose deposited in the different regions of the respiratory tract as calculated with the MPPD tool for quiet nasal inhalation. Age specific lung model: 3 months, 21 months, 23 months and 28 months, in accordance with one or more embodiments of the present invention;

Fig 8 is a graph depicting the age dependent fraction of inhaled dose deposited in the alveolar space as calculated with the MPPD tool for different distressed breathing scenarios compared to results from normal breathing, in accordance with one or more embodiments of the present invention;

Fig 10: Effect of free and liposomal hydroxychloroquine and azithromycin on S.Pneumonia biofilm by MBEC assay. Free and liposomal hydroxychloroquine and azithromycin formulations were injected into the biofilm at concentrations from 8 to 1024 mg/L. The controls were untreated biofilm; data represent three independent experiments in triplicate and are shown as means+SEM. P values were considered significant when compared with the control: ***P,0.001, in accordance with one or more embodiments of the present invention;

Fig 11: QS molecule production measured by b-galactosidase activity. In the presence of free and liposomal azithromycin at 1/16 and 1/32 the MIC, P. aeruginosa PAO1 strain was exposed to free and liposomal azithromycin. Then the supernatants were collected and incubated with the reporter strain A. tumefaciens (A136). Miller units were used to measure the b-galactosidase activities. Each bar represents the mean+SEM of three independent experiments. P values were considered significant when compared with the control and between groups: ***P,0.001 and **P,0.01, in accordance with one or more embodiments of the present invention;

Fig 12: Effects of subinhibitory concentrations (1/16 and 1/32 the MIC) of free and liposomal hydroxychloroquine and azithromycin on virulence factor production by PA01. (a) Lipase, (b) chitinase, (c) elastase and (d) protease. The results represent the mean+SEM in triplicate of three independent experiments. The results were normalized by dividing the average absorbance of the virulence factor assays over the OD600 (bacterial density) at 24 h for lipase, chitinase and elastase experiments. P values were considered significant when compared with the control: ***P,0.001, **P,0.01 and *P,0.05, in accordance with one or more embodiments of the present invention;

Fig 13: Effect of a subinhibitory concentration of liposomal azithromycin on P. aeruginosa motility. The motility was examined at free and liposomal azithromycin at 1/16 and 1/32 the MIC. Twitching [1% (w/v) agarose], swarming [0.5% (w/v) agarose] and swimming [0.3% (w/v) agarose]. P valueswere considered significant when compared with the control and between groups: ***P,0.001 and **P,0.01, in accordance with one or more embodiments of the present invention;

Fig 14: Flow cytometry histograms. Fusion (%) of labelled liposome-PKH2-GL with BAA-255 at 1, 5 and 10 h intervals, in accordance with one or more embodiments of the present invention;

DETAILED DESCRIPTION OF THE INVENTION
The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.

The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are construed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.

Accordingly, the present invention provides a method of preparing a liposome delivery system comprising a combination of drugs for individuals with pulmonary infections.

Preparation of liposomes
The dehydration – rehydration vesicle (DRV) method was used for the preparation of liposomes. DPPC and cholesterol in a molar ratio of 6: 1 (lipid to cholesterol) were used. DPPC (0.11382 g) and cholesterol (0.01 g) were added to a round-bottomed flask and then dissolved in a sufficient amount of chloroform – methanol (2: 1) mixture. The mixture in the round- bottomed flask was dried to a lipid film with a rotary evaporator (Rotavapor; Bu ¨chi Labortechnik AG) in a water bath at 410C under controlled vacuum (V-800, Brinkman). A 0.03 g aliquot of hydroxychloroquine sulphate and azithromycin dihydrate dissolved in PBS (pH 7.4) was added to rehydrate the lipid film. The lipid suspensions in PBS were vortexed for 5 min and then sonicated for 2 min (cycles of 45 s on and 10 s off) in an ultrasonic dismembrator bath (FS20H; Fisher Scientific, Ottawa, Ontario, Canada) with amplitude of 45 Hz . The lipid suspension was divided into aliquots of 1 mL. The tubes were frozen for 15 min then freeze dried in a freeze-dried system (model 77540, Labanco Corporation, Kansas City, MO, USA). The obtained powdered formulations were stored in a freezer at 80C until use. For rehydration, 100 mL of PBS was added and the mixture was vortexed and incubated for 5 min at 400C. This step was repeated three times and finally 700 mL of PBS was added to make it up to 1000 mL of suspension. The excess of unencapsulated drug was removed following three rounds of washing with PBS using a centrifuge (16 000 g for 15 min at 48 0C).
The stability of liposomes containing > or =3 mol% AZH and HCQ was observed to follow the following rank order: DPPC >DSPC > DMPC.

Penetration in bacterial biofilms BBFs
Treating infectious diseases has recently become extremely difficult and these diseases have become a threat worldwide. This is mainly be- cause bacteria, as a cause of infectious diseases, form bacterial biofilms (BBFs) which make antibiotics ineffective. BBF is a structure in which bacteria are scattered on a solid–liquid or gas–liquid interface formed on a hydrous substrate containing extracellular polymeric substances (EPS), which mainly contains polysaccharides, proteins, nucleic acids, and lipids produced by bacteria. The structure of BBF varies greatly in scale and form depending on conditions such as surface properties of adhesive sur- faces, nutritional state of bacterial cells, and bacterial species composition [3]. This is because the ratio of the constituent components of the BBF matrix varies depending on the bacterial species residing in the BBF. Within the BBF matrix, there are network gaps or cavities, which are several µm wide. This network can be used as a passage for water, nu- trients, and metabolites necessary for the growth of cells. The cells in the BBF are gathered between these gaps and cavities.

In one embodiment of the present invention the primary focus is on liposomes. Liposomes can enclose drugs or active ingredients of hydrophobic, hydrophilic, or amphoteric nature. A great advantage of liposomes is that they have excellent biocompatibility. Encapsulation of the antibiotics in liposomes allows for improved stability and controlled drug release, and safeguards the antibiotics against the degradative effect of the defense mechanisms of the body, preserving their therapeutic response. Further, their physical and surface properties can be easily controlled by selecting lipids and changing the membrane composition. To develop liposomes effective against BBFs, factors like particle size, bilayer morphology, surface properties, and encapsulation rate are considered important. In addition, the BBF extracellular matrix structure and antibiotic compatibility is considered. The liposomes have been reported to be applied via different routes of drug administration: dermal, vaginal, ocular, pulmonary route. It is considerably effective in suppressing nephrotoxicity, an adverse effect of amphotericin B that has been a concern in clinical settings. In addition, it maintains the same level of effect against mycosis. Several studies have evaluated the effects of liposomes on BBF-forming bacteria. However, there are few comprehensive reports on the surface properties of liposomes that enable the liposomes to pass through BBFs and effectively act on them. Further- more, for nanocarriers that act on BBFs, the properties that are beneficial for retention or permeability have not been reported.

In another embodiment of the present invention the characteristics of liposomes that are responsible for the ability of antibacterial agents to act on BBFs, in terms of improving both retention and permeation is identified and established.

The present invention evaluated four types of liposomes with different surface charges and PEG modification against a strong BBF-forming bacterial species, S.Pneumoniae BAA-255. We measured antimicrobial activity, retention, permeability, and the anti-biofilm effect of these liposomes against the S.Pneumoniae BAA-255 BBF.

Microbiological assay for the measurement of azithromycin
To measure the encapsulation efficiency of azithromycin into liposomal vesicles, laboratory-strain S.Pneumoniae (ATCC BAA-255) was used as an indicator organism as suggested by the CLSI. Agar diffusion assay was used to quantify the concentrations of hydroxychloroquine and azithromycin incorporated into liposomes. S.Pneumoniae (ATCC BAA-255) was cultured overnight in CAMHB, and a bacterial solution was prepared equivalent to 0.5 McFarland standards (1.5×l08 cfu/mL). The bacterial cells were then added to an autoclaved molten agar solution at 41.80C and immediately discharged into a sterile glass plate (440×340 mm) to form a thin layer of agar and bacteria. The liposomal azithromycin sample was centrifuged at 12000 g for 20 min at 48C and Triton X-100 in PBS (0.2%, v/v) was added to the obtained pellet in order to release the drug from the liposome. We must point out that this concentration of Triton X-100 (0.2%) has no effect on the bacterial growth. A well-puncher device was used to make wells of 5 mm in diameter, which were filled with 25 mL of liposome samples or standard solutions of azithromycin, and the glass plate was incubated for 18 h at 37OC. After the incubation period of 18 h, inhibition zones obtained on the plate were measured in triplicate. In order to quantify the encapsulation efficiency of the liposomal azithromycin formulations, average values of the triplicates were used. The sensitivity of the microbio-logical assay was 0.00390 mg/L and the quantifiable limit for azithromycin was 0.003 mg/L. For azithromycin, the standard curve linearity extended over the range 0.003 – 2 mg/L and gave a correlation coefficient .0.99. The concentrations of the obtained measurements are the means of at least three independent experiments measured in triplicate for each experiment.

Determination of encapsulation efficiency
The encapsulation efficiency of liposomal azithromycin was determined as the percentage of azithromycin entrapped in the liposomes (determined by microbiological assay as mentioned in the previous paragraph) with respect to the initial amount of the azithromycin in solution. The encapsulation efficiency was measured using the formula:
Encapsulation efficiency (%) = DRVs ×100
Csol
where CDRVs corresponds to the concentration of the antibiotic entrapped in DRVs and Csol corresponds to the initial concentration of the antibiotic added to the mixture.
The concentration of the entrapped hydroxychloroquine and azithromycin in the liposome was determined by the microbiological assay as described above.

Determination of particle size and polydispersity index
The mean diameter of liposomes and the polydispersity index were measured using a submicron particle sizer model 270 (Nicomp, Santa Barbara, CA, USA).63,66 The liposomal hydroxychloroquine and azithromycin samples obtained after rehydra- tion by adding 1000 mL of PBS were subjected to particle size analysis. Liposomal hydroxychloroquine and azithromycin was diluted in clear glass tubes with double- distilled water to get a sufficient reading in the range 250–350 kHz for the photo pulse to obtain the particle size data. The process was repeated two or three times with the liposomal hydroxychloroquine and azithromycin sample to take an average value of the particle size.

Differential scanning calorimetry (DSC) characterization
DSC analysis was performed using the TA Instruments Q100 differential scanning calorimeter (Grimsby, Ontario, Canada). A scan rate of 108C/min was employed with a temperature range of 25–2008C. An 5 – 7 mg sample was used for analysis, using an empty pan as reference. Pure DPPC, physical mixture-1 (DPPC, cholesterol), physical mixture-2 (DPPC, cholesterol and azithromycin) and azithromycin-loaded liposome samples were prepared for thermal analysis. The measurements of each sample were repeated three times. The main phase transition temperatures were determined using TA universal analysis 2000 program.

Stability studies of liposomal azithromycin
The stability study of liposomal azithromycin was evaluated in PBS at 4 and 370C. The stability of liposomal hydroxychloroquine and azithromycin was measured as the per- centage retention of the initial encapsulated hydroxychloroquine and azithromycin after the incu- bation period of time at 4 and 37OC. Briefly, liposomal hydroxychloroquine and azithromycin was suspended in PBS and incubated in a water bath shaker with mild agitation at 100 rpm (Julabo SW22 Incubator Shaker; Labortechnik, Seelbach,

Liposomal Hydroxychloroquine and Azithromycin formulations useful in the present disclosed methods can be prepared as described below. Generally, Hydroxychloroquine and Azithromycin is used in the form of a pharmaceutically acceptable salt, for example the sulfate salt of hydroxychloroquine and anhydrous form of Azithromycin.

The lipids used in the compositions of the present invention can be synthetic, semi-synthetic or naturally-occurring lipids, including phospholipids, tocopherols, steroids, fatty acids, glycoproteins such as albumin, anionic lipids and cationic lipids. The lipids may be anionic, cationic, or neutral. In one embodiment, the lipid formulation is substantially free of anionic lipids, substantially free of cationic lipids, or both. In one embodiment, the lipid formulation comprises only neutral lipids. In another embodiment, the lipid formulation is free of anionic lipids or cationic lipids or both. In another embodiment, the lipid is a phospholipid. Phospholipids include egg phosphatidyl choline (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); the soya counterparts, soy phosphatidyl choline (SPC); SPG, SPS, SPI, SPE, and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC), other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the 1 position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and the phospholipid can be made up of fatty acids of different chain lengths and different degrees of unsaturation. In particular, the compositions of the formulations can include dipalmitoylphosphatidylcholine (DPPC), a major constituent of naturally-occurring lung surfactant as well as dioleoylphosphatidylcholine (DOPC). Other examples include dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipids like palmitoylstearoylphosphatidylcholine (PSPC) and palmitoylstearoylphosphatidylglycerol (PSPG), driacylglycerol, diacylglycerol, seranide, sphingosine, sphingomyelin and single acylated phospholipids like mono-oleoyl-phosphatidylethanol amine (MOPE).

The lipids used can include ammonium salts of fatty acids, phospholipids and glycerides, phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphotidylcholines (PCs), phosphatidylinositols (PIs) and the phosphatidylserines (PSs). The fatty acids include fatty acids of carbon chain lengths of 12 to 26 carbon atoms that are either saturated or unsaturated. Some specific examples include: myristylamine, palmitylamine, laurylamine and stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2,3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). Examples of PGs, PAs, PIs, PCs and PSs include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI, DPPI, DSPI, DMPS, DPPS and DSPS, DSPC, DPPG, DMPC, DOPC, egg PC.

In another embodiment, the liposome comprises a lipid selected from the group consisting of phosphatidyl cholines (PCs), phosphatidyl-glycerols (PGs), phosphatidic acids (PAs), phosphatidylinositols (PIs), and phosphatidyl serines (PSs).

In another embodiment, the lipid is selected from the group consisting of: egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS), phosphatidylethanolamine (EPE), phosphatidic acid (EPA), soy phosphatidyl choline (SPC), soy phosphatidylglycerol (SPG), soy phosphatidylserine (SPS), soy phosphatidylinositol (SPI), soy phosphatidylethanolamine (SPE), soy phosphatidic acid (SPA), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated egg phosphatidylglycerol (HEPG), hydrogenated egg phosphatidylinositol (HEPI), hydrogenated egg phosphatidylserine (HEPS), hydrogenated phosphatidylethanolamine (HEPE), hydrogenated phosphatidic acid (HEPA), hydrogenated soy phosphatidyl choline (HSPC), hydrogenated soy phosphatidylglycerol (HSPG), hydrogenated soy phosphatidylserine (HSPS), hydrogenated soy phosphatidylinositol (HSPI), hydrogenated soy phosphatidylethanolamine (HSPE), hydrogenated soy phosphatidic acid (HSPA), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidyl-ethanolamine (DOPE), palmitoylstearoylphosphatidyl-choline (PSPC), palmitoylstearolphosphatidylglycerol (PSPG), mono-oleoyl-phosphatidylethanolamine (MOPE), tocopherol, ammonium salts of fatty acids, ammonium salts of phospholipids, ammonium salts of glycerides, myristylamine, palmitylamine, laurylamine, stearylamine, dilauroyl ethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP), dipalmitoyl ethylphosphocholine (DPEP) and distearoyl ethylphosphocholine (DSEP), N-(2,3-di-(9-(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylacid (DMPA), dipalmitoylphosphatidylacid (DPPA), distearoylphosphatidylacid (DSPA), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphospatidylinositol (DSPI), dimyristoylphosphatidylserine (DMPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), and mixtures thereof.
In another embodiment, the liposome comprises a phosphatidyl choline. The phosphatidyl choline may be unsaturated, such as DOPC or POPC, or saturated, such as DPPC. In another embodiment, the liposome does not include a sterol. In one embodiment, the liposome consists essentially of a phosphatidyl choline and a sterol. In another embodiment, the liposome consists essentially of DPPC and cholesterol.

Liposomes or lipid antiinfective formulations composed of phosphatidylcholines, such as DPPC, aid in the uptake by the cells in the lung such as the alveolar macrophages and helps to sustain release of the antiinfective agent in the lung (Gonzales-Rothi et al. (1991)). The negatively charged lipids such as the PGs, PAs, PSs and PIs, in addition to reducing particle aggregation, can play a role in the sustained release characteristics of the inhalation formulation as well as in the transport of the formulation across the lung (transcytosis) for systemic uptake.

The present invention is directed to methods of treating a pulmonary condition in a subject need thereof comprising administering to the subject and effective amount of any one of the aforementioned liposomal antibiotic formulations. In some embodiments, the pulmonary condition is an acute respiratory distress syndrome or Acute lung injury bacterial infection. In some embodiments, the method comprises administering to a patient in need thereof an effective amount of a liposomal hydroxychloroquine-azithromycin formulation (also referred to herein as “liposomal hydroxychloroquine-azithromycin”) by inhalation daily. In some embodiments, the administration by inhalation comprises nebulizing the liposomal formulation.
In some embodiments, the liposomal hydroxychloroquine-azithromycin formulation is administered daily for a period of time, followed by second period of time (an “off” period) wherein no liposomal formulation is administered.

For example, In another embodiment of the present invention, the method of treating a pulmonary disorder comprises administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine-azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 5 to 25 days, followed by an off period of 5 to 15 days;
and the effective dose comprises 10 to 2500 mg of hydroxychloroquine and 5 to 1500 mg of azithromycin daily during the administration period.

In another embodiment of the present invention the aforementioned treatment cycle is administered to the patient at least twice. In other embodiments, the treatment cycle may be administered 3, 4, 5, 6, or more times.

During the administration period, liposomal hydroxychloroquine-azithromycin is administered daily. In some embodiments, liposomal hydroxychloroquine-azithromycin can be administered every other day or every third day during the administration period. As explained above, the administration period can be 15 to 75 days. In some embodiments, the administration period is 15 to 35 days, or 20 to 35 days. In other embodiments, the administration period is 20 to 30 days, 25 to 35 days or 25 to 30 days. In other embodiments, the administration period is about 25, 26, 27, 28, 29 or 30 days. In another embodiment, the administration period is about 28 days.

During the off period the liposomal hydroxychloroquine-azithromycin formulation is not administered to the patient. In some embodiments, the off period is 15 days or longer, for example, 15 to 75 days, 15 to 35 days, or 20 to 35 days. In other embodiments, the off period is 20 to 30 days, 25 to 35 days or 25 to 30 days. In other embodiments, the off period is about 25, 26, 27, 28, 29 or 30 days. In other embodiments, the off period is about 28 days, while in still other embodiments, the off period is at least 29 days.

In another embodiment of the present invention, the off period is of 25 to 75 days, 35 to 75 days, or 45 to 75 days. In other embodiments, the off period is 50 to 75 days, 50 to 70 days, 50 to 65 days or 50 to 60 days. In other embodiments, the off period is about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days, while in other embodiments, the off period is about 56 days.

In another embodiment of the present invention, the administration period is about 28 days and the off period is about 28 days, while in other embodiments, the administration period is about 28 days and the off period is about 56 days.

In another embodiment of the present invention, the effective dose comprises 10 to 2,500 mg of hydroxychloroquine and 5 to 1500 of azithromycin. In other embodiments, the effective dose is about 150mg to about 400 mg of hydroxychloroquine and 100 to 350 of azithromycin. In other embodiments, the effective dose is about 230 mg to about 330 mg hydroxychloroquine and 220 to 340 of azithromycin. In other embodiments, the effective dose of hydroxychloroquine-azithromycin is about 100, 150, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700 or 750 mg of hydroxychloroquine and azithromycin daily. In other embodiments, the effective dose is about 280 or about 560 mg of hydroxychloroquine and 100 to 350 of azithromycin.

In another embodiment of the present invention, the administration period is about 15 days, and the dose is about 280 to about 560 mg of hydroxychloroquine-azithromycin. In other embodiments, the administration period is about 28 days, the off period is about 28 days, and the dose is about 280 to about 560 mg. In other embodiments, the administration period is about 28 days, the off period is about 56 days, and the dose is about 280 to about 560 mg.

Thus, in another embodiment of the present invention the pulmonary disorder is selected from the group consisting of chronic obstructive pulmonary disease, bronchiectasis, pulmonary infection, cystic fibrosis, alpha-1-antitrypsin enzyme deficiency and a combination thereof. In some embodiments, the pulmonary condition is cystic fibrosis. In other embodiments, the pulmonary condition is a bacterial pulmonary infection, Pseudomonas (e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P. acidovorans), staphylococcal, Methicillin-resistant Staphylococcus aureus (MRSA), streptococcal (including by Streptococcus pneumoniae), Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus, Yersinia pesos, Burkholderia pseudomallei, B. cepacia, B. gladioli, B. multivorans, B. vietnamiensis, Mycobacterium tuberculosis, M. avium complex (MAC) (M. avium and M. intracellulare), M. kansasii, M. xenopi, M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M. chelonei) infections. In another embodiment of the present invention, the infection is a P. aeruginosa infection, while in other embodiments, the infection is a non-tuberculous mycobacterial infection. The pulmonary infection may or may not be associated with cystic fibrosis.

Thus, in another embodiment of the present invention the pulmonary condition is both cystic fibrosis and a pulmonary infection such as P. aeruginosa. In other embodiment, the pulmonary condition is bronchiectasis. The bronchiectasis may or may not be associated with cystic fibrosis.

The present method provides advantageous levels of hydroxychloroquine and azithromycin at the site of the pulmonary disorder, while limiting systemic exposure to the drug, and also provides a sustained benefit to the subject for surprisingly extended periods of time. While not being bound by any particular theory, it is believed that administration of liposomal hydroxychloroquine-azithromycin in accordance the with methods described herein results a “depot” effect in the lungs of the subject. Specifically, it is believed that the liposome particles are small enough and contain an appropriate lipid formulation to penetrate and diffuse through pneumonia fluid and into the bacterial biofilm. The liposomes shield the entrapped cationic hydroxychloroquine and azithromycin in neutral liposomes to minimize electrostatic interaction with the negatively charged sputum/biofilm, which would otherwise reduce its bioavailability. In addition, there are Streptococcus pneumoniae derived virulence factors (rhamnolipids) (Davey et al. 2003), which release hydroxychloroquine and azithromycin from the liposomes. Therefore, in relatively high concentrations of drug can be delivered locally to the bacterial macro-colony environment.

Additionally, it is believed that inhalation of liposomal hydroxychloroquine and azithromycin leads to a dose dependent recruitment of macrophages as an adaptive response to inhalation of drug/lipid formulation. The presence of alveolar macrophages (which have been shown to be functionally normal in liposomal hydroxychloroquine-azithromycin treated rats) may be particularly beneficial in ARDS and Penumonia patients. ARDS and Penumonia patients are known to have reduced number of macrophages in their lungs and possibly with poor functionality, which may contribute to the chronicity of Streptococcus pneumoniae lung infection.

The dose dependent recruitment of macrophages may also contribute to the sustained effects observed using the methods of the present invention. Specifically, the macrophages in the lung may take up liposomal hydroxychloroquine-azithromycin, and then remain in the lung for a period of time, followed by release of the liposomal hydroxychloroquine-azithromycin by the macrophages. The present method thus provides, in some embodiments, advantageous levels of hydroxychloroquine and azithromycin in the blood and in the sputum. For example, the methods provide relatively low systemic exposure to hydroxychloroquine and azithromycin, while providing high, sustained levels of hydroxychloroquine and azithromycin at the site of the pulmonary condition.

When the pulmonary disorder includes a pulmonary infection, the present invention also provides a reduction in the colony forming units of the bacteria in the lung for a sustained period of time. For example, the CFU's are reduced compared to a baseline value. In some embodiments, the patient has a reduction in log10 CFU of the bacterial infection in the lungs of at least about 0.5 for at least 15 days after the administration period ends. In other embodiments, the reduction in the log10 CFU is at least by 1.0, 1.5, 2.0 or 2.5. Streptococcus infections, in particular, can form large colonies with several millimeters in diameter particularly in patients with ARDS and Pneumonia. In some embodiments, the CFU's are reduced as described above in a mucoid strain of a Pseudomonas infection.

In another embodiment of the present invention the patient experiences an improvement in lung function for at least 15 days after the administration period ends. For example, the patient may experience an increase in the forced expiratory volume in one second (FEV1), an increase in blood oxygen saturation, or both. In some embodiments, the patient has an FEV1 that is increased by at least 5% or at least 10% over the FEV1 prior to the treatment cycle. In other embodiments, FEV1 is increased by 5 to 50%, 5 to 25%, or 5 to 20%. In other embodiments, FEV1 is increased by 5 to 15% or 5 to 10%. In other embodiments, FEV1 is increased by 10 to 50%, 10 to 40%, 10 to 30% or 10 20%. FEV1 is frequently measured in mL. Accordingly, in some embodiments, FEV1 is increased by at least 25 mL when compared to FEV1 prior to the treatment. In some embodiments, FEV1 is increased by 25 to 500 mL, 25 to 400, 25 to 300 or 25 to mL. In other embodiments, FEV1 is increased by 50 to 500 mL, 50 to 400 mL, 50 to 300 mL, 50 to 200 mL or 50 to 100 mL.

In another embodiment of the present invention blood oxygen saturation is increased in the subject compared to the blood oxygen saturation levels prior to the administration. In some embodiments, blood oxygen saturation is increased by at least 1% or by at least 2% for at least 2 days after the administration period. In other embodiments, the blood oxygen saturation levels are increased by about 1 to 50%, 1 to 25%, 1 to 20%, 1 to 15%, 1 to 10% or 1 to 5%. In other embodiments, the blood oxygen saturation levels are increased by about 2 to 10% or 2 to 5%.

The aforementioned sustained periods of time may be at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 days after the administration period. In other embodiments, the sustained period of time is at least 28, 35, 42, 48 or 56 days after the administration period. In other embodiments, sustained period of 15 to 75 days, 15 to 35 days, or 20 to 35 days. In other embodiments, the sustained period of time is 20 to 30 days, 25 to 35 days or 25 to 30 days. In other embodiments, the sustained period of time is about 25, about 26, about 27, about 28, about 29 or about 30 days, or about 28 days, or at least 29 days. In other embodiments, the sustained period of time during is 25 to 75 days, 35 to 75 days, or 45 to 75 days. In other embodiments, the sustained period is 50 to 75 days, 50 to 70 days, 50 to 65 days or 50 to 60 days. In other embodiments, the sustain period is about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59 or about 60 days, while in other embodiments, the sustained period is about 56 days.

In another embodiment of the present invention the aforementioned methods advantageously provide a reduced incidence of pulmonary exacerbations in the patient. The method also advantageously increases the length of time to pulmonary exacerbation. For example, in some embodiments, the length of time to pulmonary exacerbation is at least about 20 days. In other embodiments, the length of time is 20 to 100 days. In other embodiments, the length of time is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100 days. In other embodiments, the length of time is 25 to 75 days, 30 to 75 days, 35 to 75 days or 40 to 75 days. In other embodiments, the length of time is 30 to 60 days.

In another embodiment of the present invention the incidence of rescue treatment is reduced. In other embodiments, the length of time to rescue treatment is reduced, for example when the patient has a pulmonary infection, the time to anti-infective rescue treatment is reduced. In some embodiments, the length of time is 20 to 100 days. In other embodiments, the length of time is 25 to 100 days, 30 to 100 days, 35 to 100 days or 40 to 100 days. In other embodiments, the length of time is 25 to 75 days, 30 to 75 days, 35 to 75 days or 40 to 75 days. In other embodiments, the length of time is 30 to 60 days.

In another embodiment of the present invention the liposomal hydroxychloroquine-azithromycin formulation used in the aforementioned methods comprises hydroxychloroquine-azithromycin and any of the lipids described above. In some embodiments, the liposomal hydroxychloroquine-azithromycin formulation comprises a phospholipid and a sterol, such as DPPC and cholesterol. In other embodiments, the liposomal hydroxychloroquine-azithromycin formulation comprises DPPC and cholesterol in about a 2 to 1 ratio by weight. In some embodiments, the liposomal hydroxychloroquine-azithromycin formulation has a lipid to drug ratio of about 0.5 to about 1.0, about 0.5 to 0.7, or about 0.6 by weight. In other embodiments, the liposomal hydroxychloroquine-azithromycin formulation has a lipid to drug ratio of about 0.3 to about 1.0 by weight, while in other embodiments, the lipid to drug ratio is about 0.5 to 0.7 by weight, or about 0.65 by weight. The liposomes in the formulation may have a amend diameter of 100 to 1000 nm, 100 to 500 nm, 200 to 500 nm, or about 300 nm. In some embodiments, the total concentration of hydroxychloroquine-azithromycin in the liposomal hydroxychloroquine-azithromycin formulation is about 20 to 100 mg/mL, 20 to 90 mg, mL, 30 to 90 mg/mL, 30 to 80 mg/mL, or 40 to 80 mg/mL. In other embodiments, the concentration is about 30, 40, 50, 60, 70, 80 or 90 mg/mL.

In another embodiment of the present invention, the aforementioned method comprises:
administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of about 15 days, followed by an off period of about 28 days based on the clinical functioning of the patient. The effective dose comprises about 230 mg to about 330 mg hydroxychloroquine and 220 to 340 of azithromycin daily during the administration period; and the liposomal hydroxychloroquine-azithromycin formulation comprises DPPC and cholesterol in about a 2:1 ratio, and a lipid to hydroxychloroquine-azithromycin ratio of about 0.5 to about 0.7.

In another embodiment of the present invention the method comprises administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein the treatment cycle comprises an administration period of about 15 days, followed by an off period of about 5 days; the effective dose comprises about 230 mg to about 330 mg hydroxychloroquine and 220 to 340 of azithromycin daily during the administration period; and the liposomal hydroxychloroquine-azithromycin formulation comprises DPPC and cholesterol in about a 2:1 ratio, and a lipid to hydroxychloroquine-azithromycin ratio of about 0.5 to about 0.7.

In another embodiment of the present invention relates to a method of providing a sustained treatment effect in a subject comprising: administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of hydroxychloroquine and 5 to 1500 of azithromycin daily during the administration period.
In another embodiment, the present invention relates to a method of improving oxygen saturation levels in a patient with a pulmonary condition comprising: administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of hydroxychloroquine and 5 to 1500 of azithromycin daily during the administration period.

In another embodiment, the present invention relates to a method of improving FEV1 in a patient with a pulmonary condition comprising: administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 100 to 2500 mg of hydroxychloroquine-azithromycin daily during the administration period.

In another embodiment, the present invention relates to a method of reducing bacterial density in the lung or sputum of a patient with a bacterial pulmonary infection comprising: administering to the patient an effective dose of a nebulized liposomal hydroxychloroquine and azithromycin formulation for at least one treatment cycle, wherein: the treatment cycle comprises an administration period of 15 to 75 days, followed by an off period of 15 to 75 days; and the effective dose comprises 10 to 2500 mg of hydroxychloroquine and 5 to 1500 of azithromycin daily during the administration period, and wherein the bacterial density remains reduced for at least 15 days after the last day of the administration.

EXEMPLIFICATION
Introduction to Materials and Methods
Lipid based or liposomal hydroxychloroquine and azithromycin, formulations for inhalation are sustained-release formulations of hydroxychloroquine and azithromycin encapsulated inside nanoscale liposomal carriers designed for administration via inhalation. Sustained-release targeting of high concentrations of hydroxychloroquine and azithromycin in the lungs and biofilm penetration properties of these formulations have several advantages over inhalation of the “free” antibiotic, e.g., inhaled hydroxychloroquine and azithromycin. Hydroxychloroquine and azithromycin can be encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cholesterol, at a targeted lipid-to-drug ratio of about 0.6-0.7:1 (w/w). An example of a 70 mg/mL liposomal hydroxychloroquine and azithromycin formulation useful in the aforementioned methods is presented below:
Table 1 liposomal hydroxychloroquine and azithromycin formulation
Component Concentration

hydroxychloroquine ~55 mg/mL
azithromycin ~75 mg/mL
Dipalmitoylphosphatidylcholine (DPPC) ~30 mg/mL
Cholesterol ~15 mg/mL
1.5% NaCl QS

1Added to the formulation as Hydroxychloroquine sulfate, USP.

These formulations have several advantages in treating pulmonary conditions, for example, pneumonia subjects with infection caused by S.Pneumonia, including:

1. The ability to attain a prolonged antibiotic effect of hydroxychloroquine and azithromycin in the lung by achieving high concentrations and a prolonged half life due to sustained release.
2. The ability to target and increase the effective concentration of hydroxychloroquine and azithromycin in the lung with low systemic levels of the medications.
3. The potential to better target bacteria growing in a biofilm as a result of unique properties of lipid based or liposomal anitibiotics.
4. Additional release of the drug at the site of infection in the lungs of pneumonia/ARDS patients, due to targeted action of secreted phospholipase C and rhamnolipids from bacteria and/or phospholipase A2 or defensins from activated polymorphonuclear leukocytes
5. Hydroxychloroquine is a 4-aminoquinoline with a unique activity against ARDS/ALI. Azithromycin is an azalide, a type of macrolide antibiotic. It works by decreasing the production of protein, thereby stopping bacterial growth. Consequently, some S.Pneumonia. strains which are resistant will likely remain susceptible to hydroxychloroquine and azithromycin.
6. Hydroxychloroquine and azithromycin has less binding affinity than other aminoglycosides for megalin, the transporter responsible for renal cortical aminoglycoside accumulation, and thus inherently has a lower potential for nephrotoxicity.
7. The increase in both the half life, and the area under the concentration curve (AUC) of lipid based or liposomal hydroxychloroquine and azithromycin, along with biofilm penetration should allow for less frequent administration, enhanced bactericidal activity and reduced potential for selection of resistant organisms.

Preclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of hydroxychloroquine and azithromycin in the lungs of rats that received a 60 mg/kg dose aerosol of Liposomal hydroxychloroquine and azithromycin was ten-fold higher than the AUC of free hydroxychloroquine and azithromycin in the lungs of rats that received an equal dose of free hydroxychloroquine and azithromycin by inhalation. Generally, 10% of the administered antibiotic is deposited in the lungs for rats. Conversely, the AUC of drug in the liver of rats that received an equal dose of free hydroxychloroquine and azithromycin was significantly higher than the liver AUC of rats that received aerosols of Liposomal Hydroxychloroquine and azithromycin. Additionally, data from 30-day inhalation toxicology studies in rats and dogs suggest that there will be no safety pharmacology issues with inhaled Liposomal hydroxychloroquine and azithromycin.

In 14 days rat model studies of streptococcus infection, it was noted that of Liposomal hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) administered every other day for 14 days (Q2D×7), which effectively delivered half the cumulative dose of antibiotic than the other groups, was as effective as of Liposomal hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) given once per day. With 28 day dosing in this model, there were equivalent reductions in CFUs in animals receiving Liposomal hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) dosed daily at or dosed every other day at hydroxychloroquine (40 mg/kg) and azithromycin ( 200 mg/mL). Liposomal hydroxychloroquine (40 mg/kg) and azithromycin ( 200 mg/mL) administered at once a day for 14 days was effective for 28 days, which suggests a higher AUC and possibly a prolonged post-antibiotic effect with Liposomal hydroxychloroquine (40 mg/kg) and azithromycin ( 200 mg/mL). dosed once per day.

The administration of Liposomal Hydroxychloroquine and azithromycin via inhalation in the animal model resulted in increased lung (AUC) above the MIC of the bacteria, and demonstrated sustained therapeutic effect, with a reduced frequency, and duration of dosing as compared to free hydroxychloroquine and azithromycin. Importantly, the preclinical data for Liposomal Hydroxychloroquine and azithromycin appear that this specific formulation may be advantageous over other inhalation products that are hindered by a rapid clearance from lung tissue, necessitating frequent dosing which poses a burden for patients and might limit patient compliance.

Additionally, clinical experience demonstrated that nebulized Liposomal hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) administered once per day for 14 days is well tolerated, and elicits a clinically relevant effect on pulmonary function and decrease in S.Pneumonea density in Pneumonia patients. Also, evaluation of the PK data indicates the systemic exposure to Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) , even at the, is very low. By either Cmax or AUC or mg of the antibiotics which is recovered in the urine, the observed systemic exposure to hydroxychloroquine and azithromycin, associated with Liposomal Hydroxychloroquine and azithromycin, given by inhalation is approximately 1/6th to 1/4th the exposure seen with oral and free hydroxychloroquine and azithromycin and is less than 1/200 compared to normal parenteral doses of Hydroxychloroquine and azithromycin. The data further indicate high levels of Hydroxychloroquine and azithromycin are achieved in the sputum. Median AUC values for sputum were 260 and 1230-fold greater than the median AUC values for serum on day 1 and day 14 respectively.

Inhaled liposomal hydroxychloroquine and azithromycin maintains prolonged targeted lung exposures and enhance the uptake of drug to the site of infection.

Preclinical Studies with Liposomal Hydroxychloroquine and azithromycin
Several preclinical studies were conducted with the 20 and 50 mg/mL formulations. Anti-Pneumonia activity of Liposomal Hydroxychloroquine and azithromycin in in vitro and in vivo models was demonstrated. Additionally, studies confirmed that virulence factors secreted by S.Pnuemonea facilitate the further release of hydroxychloroquine and azithromycin from the liposomes, and characterized the deposition and sustained release of hydroxychloroquine and azithromycin in the lungs of rats, and rabbits. The safety of a 30 day administration of Liposomal Hydroxychloroquine and azithromycin in two species was also established.

Nonclinical pharmacokinetics have demonstrated that the AUC (0-48 hr) of hydroxychloroquine and azithromycin in the lungs of rats that received dose of Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/kg) via nebulization, was five-fold higher than the AUC of Hydroxychloroquine and azithromycin in the lungs of rats that received an equal dose of free Hydroxychloroquine and azithromycin by inhalation. High levels of hydroxychloroquine and azithromycin were sustained in the lung (>320 µg/mL through 150 hr), suggesting a depot effect. Conversely, the AUC of drug in the liver of rats that received an equal dose of free Hydroxychloroquine and azithromycin was significantly higher than the AUC of rats that received aerosols of Liposomal Hydroxychloroquine and azithromycin. There were no significant differences in the AUC of the antibiotics in the serum and urine of the animals; serum levels were undetectable after 24 hr. This profile supports the intended sustained release and depot effect of hydroxychloroquine and azithromycin in the lung following administration of nebulized Liposomal Hydroxychloroquine and azithromycin, potentially representing an enhanced efficacy profile. These data for Liposomal Hydroxychloroquine and azithromycin appear supportive of the hypothesis that this specific formulation may be advantageous over other inhalation products that are hindered by a rapid clearance from lung tissue, necessitating frequent dosing (Geller, Pitlick et al. 2002), and placing a burden on patients.

Additionally, toxicokinetic data from 30-day inhalation GLP toxicology studies in rats and dogs showed that there is a 15 fold increase in lung deposition of hydroxychloroquine and azithromycin dogs as compared to the free hydroxychloroquine and azithromycin treated group, with comparable plasma and urine levels, indicating high lung concentrations with low systemic exposure.

The pharmacodynamic effect of Liposomal Hydroxychloroquine and azithromycin was evaluated in vivo in a rat model of chronic pulmonary infection with streptococcus (Glauser et al, 1987). In a 14 days Streptococcus infection model, of Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/kg) was administered every other day for 14 days (Q2D×7). This regimen was significaltly effective Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) (given once per day for 14 days). When dosing was extended to 28 days, there were equivalent reductions in CFUs for animals receiving Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) or dosed every other day at Hydroxychloroquine (40 mg/kg) and azithromycin ( 200 mg/mL). This indicated a higher AUC and a prolonged post-antibiotic effect with Liposomal Hydroxychloroquine (20 mg/kg) and azithromycin ( 100 mg/mL) dosed once per day. The preclinical pharmacodynamic data were thus consistent with a sustained antimicrobial benefit enhanced by the site-specific delivery of drug to the lungs via inhalation.

Thus, administration of Liposomal Hydroxychloroquine and azithromycin via inhalation resulted in increased lung concentrations (AUC) several fold above the MIC of the bacteria, with the potential to provide a sustained therapeutic effect with a reduced frequency and duration of dosing.

Results suggest that hydroxychloroquine is at least additive and often synergistic with Azithromycin for the reduction of bacteria growth and spread. Even if hydroxychloroquine and Azithromycin are only additive, this would still be of clinical importance since the use of both drugs together may allow reduction in the doses of both and thereby decrease their nonoverlapping toxicities.

Nebulization of Liposomal Amikacin:
The aerosol properties of Liposomal Amikacin produced from the eFlow 40L are shown in Table 15. When compared to nebulizate generated from the LC Star, the mass median aerodynamic diameter (MMAD) values for the eFlow are ˜0.5 µm larger. The actual size dependent mass distributions from both ACI (with eFlow) and NGI (with LC Star) cascade impactors for nebulized Liposomal Amikacin are shown in FIG. 1. Aerosol from the eFlow/ACI measurements was slightly narrower in size distribution than that from the LC Star/NGI. This difference is reflected in the lower mean geometric standard deviation (GSD) (1.66 versus 1.99) which is a measure of the width of the distribution around the MMAD, see values in Table 14. This narrower distribution offsets any potential effect of a larger MMAD and therefore, the amount of nebulized drug in the respirable range (<5 µm droplet size) is comparable for both eFlow and LC Star.

Liposome–bacterial interactions by flow cytometry
Labelled liposomes without antibiotics were utilized to avoid bacterial death due to antibiotics. The fusion of liposomal bilayer PKH2-GL with PAO1 reached 15.4 % within 1 h and a maximum of 24.8% after 5 h of contact, showing strengthening of liposome fusion with bacteria. The fusion signal reached 18.5% at 10 h of contact before the fluorescent signals began to decline. A negative panel in the figure indicates the peak of bacterial florescence without label, and positive panels were in the range 50%–65% for the duration of the experiments. The positive panel shows that PKH2-GL was compatible with the bacterial membrane.

Example 1:

Development of a model for dose determinations in a pediatric population
A PBPK model for the diseased children was developed in a multi-step scaling approach (Figure 3; Examples 2-10) using preclinical as well as predicted and measured clinical data (Table B-l). Dose selection was based on multiples of the IC90 value generated from typical in vitro Minimum Inhibitory Concentration assays in order to achieve efficacy. The average IC90 value of Hydroxychloroquine and Azithromycin for the least sensitive prototypic S.Pneumonia strain as determined in MIC assays was ~90 ng/mL (n=20). A value 100 fold over this IC90 (9 µg/ml) was taken as target concentration in order to account for possible differences in RSV clinical isolate sensitivity.

In the present approach, a model was developed that considers the anatomy and physiology of the young children, growth and development processes such as organ maturation, changes in blood flow, body composition, and ontogeny of elimination mechanisms, including changes in the respiratory system (see Figure 1, and the more detailed explanation further below).

PBPK modelling (Barrett et al. 2012, Clin. Pharmacol. Ther. 92: 40-9; Khalil and Laer 2011, J. Biomed. Biotechnol. Epub 2011 Jun 1) was used to bridge pediatric and adult pharmacology. This was done by establishing an inhalation PBPK model for adults, which was then scaled to children. The PBPK models was built using the software PK-Sim* (Bayer Technology Services, Leverkusen, Germany; www.pk-sim.com, version 5.1.3 for PBPK model building, and version 5.2.2 and 5.3.2 for population simulations,). PK-Sim* is a commercially available tool for PBPK modelling of drugs in laboratory animals and humans. PK-Sim* includes a generic PBPK model for protein therapeutics and macromolecules (Figure 2). For a detailed description about the general PBPK model structure implemented in PK-Sim* see Willmann et al. (2007, J. Pharmacokinet. Pharmacodyn. 34: 401-431; 2005, 1: 159-168; 2003, Biosilico 1: 121-124).

This model was used to build the PBPK model for intravenous (IV) administration and the base model for pulmonary administration. In order to describe the absorption from the alveolar space, an additional compartment representing the alveolar lining fluid (ALF) was inserted into the lung of the standard whole body PBPK model exported from PK-Sim* (Figure 2). The alveolar lining fluid contains the amount of dose deposited in alveolar space following inhalation. The volumes of the ALF compartment for the different species were calculated from literature values for the alveolar surface area and the thickness of the alveolar lining fluid (Tschumperlin and Margulies 1999, J. Appl. Physiol., 86: 2026-33; Patton 1996, Advanced Drug Delivery Reviews 19: 3-36; Bastacky et al. 1995, J. Appl. Physiol. 79: 1615-28). The volume of the ALF compartment was assumed to be constant after inhalation of aerosol due to fast reabsorption of inhaled water.
Also a diffusional exchange pathway connecting the alveolar space to the lung tissue (interstitium) was inserted into the model. The diffusion rate was calculated by the following first order equation:
dN/dt = Pa!v * Aa!v * (Calf - Qnt)
with N: amount of drug
Paiv: alveolar permeability (epithelial cell barrier). The parameter value was fitted to plasma concentration-time profiles following inhalation in rats.
Aa(v: alveolar surface areas from literature.
Ca!f: concentration of drug in ALF.
Cint: concentration of drug in lung interstitium.

Following inhalation, aerosol particles are deposited in various regions of the respiratory tract. To estimate the fraction deposited in the lower respiratory tract following inhalation of hydroxychloroquine and azithromycin, aerosol deposition for different paediatric age groups was scaled using a dedicated tool incorporated into the PBPK model, Multiple-Path Particle Dosimetry (MPPD) V2.ll (2002-2009, a detailed description can be found on http://www.ara.com/products/mppd.htm). The MPPD Model was developed by Applied Research Associates, Inc. and The Hamner Institutes for Health Sciences, USA, in collaboration with the National Institute of Public Health and the Environment (RIVM), The Netherlands, and the Ministry of Housing, Spatial Planning and the Environment, The Netherlands. It allows the description of the average regional depositions in the head, tracheobronchial and alveolar regions, and average deposition per airway generation, for different paediatric age groups, and for particles of different sizes. Overall, regional deposition depends on lung morphology (which is age specific), particle properties (size and density distribution) and breathing pattern (frequency, volume). As such, the MPPD tool calculates the deposition of aerosols in the respiratory tract of adults and children (ages: 3, 21, 23 and 28 months, 3, 8, 9, 14 and 18 years) for particles of different sizes. Deposition is calculated using theoretically derived efficiencies for deposition by diffusion, sedimentation and impaction within the airway or airway bifurcation. Filtration of aerosols by the head is determined using empirical efficiency functions.

Although only a few exemplary embodiments of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims. In the claims, means-plus-function clauses are intended to cover the structures described herein as performing the recited function and not only structural equivalents but also equivalent structures.

The invention as described hereinabove in the context of the preferred embodiments is not to be taken as limited to all of the provided details thereof, since modifications and variations thereof may be made without departing from the spirit and scope of the invention.
,CLAIMS:5. CLAIMS
I/WE Claim:
1. A stable sustained release liposomal inhalation suspension formulation for pulmonary delivery comprising liposomal particles characterized by a synergistic combination of;
Azithromycin and hydroxychloroquine;
lipid comprising mixture of anionic lipid and saturated lipid;
cholesterol; and
1.5% NaCl -QS.

2. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein the zeta potential of the liposomal dispersion is -40 to -10 mV, %. Hydroxychloroquine and azithromycin entrapment is more than 96%.

3. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein saturated lipid can be selected from the group consisting of Hydrogenated soy phosphatidylcholine (HSPC), Dipalmitoylphosphatidylcholine (DPPC), Distearyloylphosphatidylcholine, (DSPC) and Diarachidoylphosphatidylcholine (DAPC).

4. The stable prolonged release liposomal inhalation suspension formulation for pulmonary delivery as claimed in claim 1 wherein the liposomal inhalation suspension formulation is to be delivered through nebulization device.

5. The method for preparing the stable liposomal inhalation suspension formulation in an effective amount of a systemically active formulation comprising a synergistic combination of hydroxychloroquine and Azithromycin for pulmonary delivery comprises of;
dissolving Azithromycin, hydroxychloroquine, lipid mixture, cholesterol in solvent,
evaporating the above mixture in rotary vacuum evaporator to get lipid film,
hydrating the lipid film of step by distilled water,
sonicating the hydrated film of step,
centrifuging the sonicated film of step, and
decanting liposomal suspension.