WO2021232052A1 - Microencapsulated delivery system for release of anti-inflammatory agents into the lung - Google Patents

Abstract

Nanoparticle compositions disclosed here contain rapamycin, an antioxidant, and a pulmonary surfactant protein. Therapeutically effective amounts of these nanoparticle compositions can be used to treat viral infections or bacterial infections. Therapeutically effective amounts of these nanoparticle compositions can be used to improve lung function in certain patients.

Description

MICROENCAPSULATED DELIVERY SYSTEM FOR RELEASE OF ANTIINFLAMMATORY AGENTS INTO THE LUNG

Technical Field

[0001] The disclosure relates to microencapsulated compositions containing an antioxidant and a pulmonary surfactant protein and methods of delivery of such compositions to the lung.

Background

[0002] Increasing age is associated with a chronic state of oxidation and inflammation. These conditions are measured as a baseline increase of pro-inflammatory cytokines in the circulation and in some tissues, termed inflammaging and metainflammation, respectively. Inflammation is directly linked to increased oxidation. There is a need for compositions and methods to positively affect the oxidative and inflammatory status of the lung, and modify lung function sufficiently to increase resistance to respiratory infections.

Summary

[0003] Disclosed here are nanoparticle compositions containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The pulmonary surfactant protein can be a pulmonary surfactant protein A (SP-A) or a pulmonary surfactant protein D (SP-D). The antioxidant in the nanoparticle composition is one or more of melatonin, spermidine, and N-acetyl-L-cysteine. Disclosed here are methods of treating a viral infection by administering to a subject with the viral infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The viral infection can be severe acute respiratory syndrome. The viral infection can be influenza. [0004] Disclosed here are methods of treating a bacterial infection by administering to a subject with the bacterial infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The bacterial infection can be a Mycobacterium tuberculosis infection. The pulmonary surfactant protein can be a pulmonary surfactant protein A or pulmonary surfactant protein D or both. The antioxidant can be one or more of melatonin, spermidine, and N-acetyl-L-cysteine.

[0005] Disclosed here are methods of improving lung function by administering to an elderly subject a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The pulmonary surfactant protein can be a pulmonary surfactant protein A or pulmonary surfactant protein D or both. The antioxidant can be one or more of melatonin, spermidine, and N-acetyl-L-cysteine.

[0006] Still other aspects, embodiments, and advantages of these exemplary aspects and embodiments, are discussed in detail below. Moreover, it is to be understood that both the foregoing information and the following detailed description are merely illustrative examples of various aspects and embodiments, and are intended to provide an overview or framework for understanding the nature and character of the claimed aspects and embodiments. Accordingly, these and other objects, along with advantages and features of the present disclosure herein disclosed, will become apparent through reference to the following description and the accompanying drawings. Furthermore, it is to be understood that the features of the various embodiments described herein are not mutually exclusive and may exist in various combinations and permutations.

Brief Description of the Drawings

[0007] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. Embodiments are illustrated by way of example and not by way of limitation in the accompanying drawings. [0008] FIGS. 1A - II are graphical representations of the effects of delivery of nanoparticles in young and old animals, according to an embodiment.

[0009] FIG. 2 is a diagrammatic representation of the timeline and approach for SARS-CoV-2 and Mycobacterium challenges, according to an embodiment.

[0010] FIG. 3 is a graphical representation of rapamycin and an antioxidant as sustained release formulations in nanoparticles, according to an embodiment.

[0011] FIGS. 4A - 4C are graphical representations of the cytokine levels in the aging lung, according to an embodiment.

[0012] FIGS. 5A-5D are graphical representations of increased protein oxidation and reduced protein function associated with aging in humans, according to an embodiment.

[0013] FIGS. 6A - 6C demonstrate the state of lung macrophage in old age, according to an embodiment.

[0014] FIG. 7 is a graphical representation of the replenishment of elderly lung mucosa with functional surfactant protein D (SP-D), according to an embodiment.

[0015] FIGS. 8A and 8B are graphical representations of increased inflammation and pro oxidative signatures in lung ALF associated with aging.

[0016] Reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used here to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Alterations and further modifications of the inventive features illustrated herein, and additional applications of the principles of the disclosure as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the disclosure.

Detailed Description

[0017] Nanoparticle compositions described here contain rapamycin, an antioxidant, and a pulmonary surfactant protein. The pulmonary surfactant protein can be a pulmonary surfactant protein A or a pulmonary surfactant protein D. The antioxidant in the nanoparticle composition is one or more of melatonin, spermidine, and N-acetyl-L-cysteine. Therapeutically effective amounts of these nanoparticle compositions can be used to treat viral infections or bacterial infections. The infection can be caused by a severe acute respiratory syndrome. The infection can be caused by an influenza virus. Therapeutically effective amounts of these nanoparticle compositions can be used to improve lung function in certain patients. These microencapsulated compositions containing anti-inflammatory agents also affect the risk of viral infections, such as influenza and SARS, and bacterial infections, such as tuberculosis (TB). For example, the nanoparticle compositions can be used to treat an elderly subject. Various pharmaceutical nanotechnologies can be used to deliver the nanoparticle compositions, including polymeric nanoparticles, magnetic nanoparticles, liposomes, carbon nanotubes, quantum dots, dendrimers, metallic nanoparticles, and polymeric nanoparticles. Nanoparticle compositions are formulated to be delivered to a patient via any available route, including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, intradermal, mucosal, intranasal, inhalation, bronchial administration, and/or sublingual administration.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0019] The term “administered” or “administering” refers to any method of providing a nanoparticle composition containing one or more of rapamycin, an antioxidant, and a pulmonary surfactant protein to a patient such that the nanoparticle composition has its intended effect on the patient. The term “therapeutically effective amount” refers to an amount of a composition of the present disclosure that is effective when administered alone or in combination to prevent or treat the infections listed herein. When applied to a combination, the term refers to combined amounts of the active ingredients, including one or more of rapamycin, an antioxidant, and a pulmonary surfactant protein, that result in the preventive or therapeutic effect, whether administered in combination, serially, or simultaneously. The terms “treatment,” “treating,” and “treat” refer to any indicia of success in the treatment or amelioration of an infection, injury, disease, or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the infection, injury, disease, or condition more tolerable to the subject, or making the infection, injury, disease, or condition less debilitating; and/or improving a subject's physical or mental well-being.

[0020] Reversal of the oxidative status of the lung in vivo can reestablish innate function of the lung in the elderly. Intervention with microencapsulated compositions containing anti inflammatory agents restores the balance between pro-inflammatory agents and pathways and the activity of negative regulators of inflammation within the pulmonary alveolus (alveolar lining fluid and alveolar resident and compartment cells).

[0021] Disclosed here are nanoparticle compositions containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The pulmonary surfactant protein can be a pulmonary surfactant protein A (SP-A) or a pulmonary surfactant protein D (SP-D). The antioxidant in the nanoparticle composition is one or more of melatonin, spermidine, and N-acetyl-L-cysteine. Disclosed here are methods of treating a viral infection by administering to a subject with the viral infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The viral infection can be severe acute respiratory syndrome. The viral infection can be influenza. A SARS-CoV-2 infection presents a model of acute infection, based on its clinical and epidemiological relevance. The current COVID-19 pandemic has >160 million cases with over 3 million deaths. According to the U.S. Center for Disease Control and Prevention, 8 out 10 COVID-19 deaths reported in the U.S. have been in adults 65 years old and older. The elderly are a significant target and reservoir for both transmission of new SARS-CoV-2, in general, and particularly to those with compromised immunity. For example, surfactant protein functions are involved in both SARS and TB diseases outcomes. Elderly animal models were used to assess susceptibility to SARS-CoV-2 as model pathogen.

[0022] Oxidative stress pathways active in old age are targeted for reversal using an antioxidant/rapamycin slow release nanoparticle delivery system. Surfactant protein activities are restored in the lung using nanoparticles loaded with functional surfactant protein A (SP-A) and surfactant protein (SP-D). The impact of reducing oxidation on immune and metabolic pathways are also evaluated.

[0023] Disclosed here are methods of treating a bacterial infection by administering to a subject with the bacterial infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. In an embodiment, the bacterial infection is a Mycobacterium tuberculosis infection. The pulmonary surfactant protein can be a pulmonary surfactant protein A or pulmonary surfactant protein D or both. The antioxidant can be one or more of melatonin, spermidine, and N-acetyl-L-cysteine.

[0024] Disclosed here are methods of improving lung function by administering to an elderly subject a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein. The pulmonary surfactant protein can be a pulmonary surfactant protein A or pulmonary surfactant protein D or both. The antioxidant can be one or more of melatonin, spermidine, and N-acetyl-L-cysteine. The lung mucosa status in the elderly favors pro-inflammatory proteins. There are several parallels between human and rodent lung alveolar environment function during old age. Inflammation is increased in the lungs of elderly humans (66+) and old mice (18-months+). Increased levels of inflammatory proteins such as complement components are present in the lungs of old mice or elderly human subjects. The relative changes in complement components in the alveolar lining fluid (ALF) of old mice (j C2, † C3 and † C4) suggest that there are alterations in membrane attack complex (MAC) formation and antibody (Ab) complex mediated clearance associated with the classical arm of the complement cascade. This is supported by reports of reduced Ab function in elderly subjects and may contribute to their increased susceptibility to infections. Low C2 and high Factor B in the lung mucosa of old mice and elderly humans may favor the alternative pathway in lower respiratory tract secretions. This contrasts with what is seen in the healthy human adult lung. Importantly, lung mucosa in elderly humans constitutes a higher oxidized environment, a factor shown to contribute to local tissue inflammation of the lung alveolar space. High levels of oxidized proteins and oxidants in elderly human lungs result in the dysfunction of important innate soluble components, such as SP-A and SP-D. Importantly, replenishment with functional SP-D in the human elderly lung mucosa reestablished the capacity of human macrophages to control pathogens such as Mycobacterium tuberculosis infection.

[0025] Systems analyses of the human lung alveolar space environment are conducted. Lipid and proteomic assessments of adult and elderly human lung mucosa by LC-MS/MS identified a total of 1,154 proteins, with adults having 349 (30.2%) unique proteins, elderly 96 (8.3%), and 709 (61.4%) shared across both groups. For proteins of interest (anti/pro-oxidative, anti/pro- inflammatory), their fold change in the elderly lung mucosa was calculated relative to adult based on the total TIC. In general, elderly ALF contained higher levels of pro-oxidative/proinflammatory proteins and lower levels of anti-oxidative/anti-inflammatory proteins. Proteomics methods are used to determine the impact of the nanoparticle delivery system on the lung of old mice.

[0026] Encapsulated compositions of rapamycin alone (R), antioxidants alone (AOs, melatonin, spermidine, and N-acetyl-L-cysteine), SP-A/-D (SPs), or the combination of all three (R+AO+SPs) were prepared. PLGA particle preparation, size analysis, density, aerodynamic diameter, and flow properties were analyzed. Formulation pharmacokinetics after inhalation, as well as cytotoxicity and pathology indicate that these compounds (alone or in combination) were evaluated. These compositions were stable and safe in the lung alveolar environment. For example, in the mouse/hamster lung alveolar environment, these compositions were stable at 14 days-post- dosage, e.g. R and SP-A/-D encapsulated in 172.5 pg and AOs (each of them) encapsulated in 117.5 pg of nanoparticles that provided release of ~25 pg of each in the lung per dose. Hamsters receive < 12 mg/Kg of nanoparticles intratracheally per month following USDA guidelines.

[0027] The potential of certain nanoparticle compositions to restore oxidative balance in the elderly animals was tested. Elderly individuals present high degree of oxidative stress in their lungs driving ALF soluble components dysfunctionality. These nanoparticle compositions loaded with antioxidants were intratracheally delivered into the alveolar space of old mice (24-month). Nanoparticles contained the antioxidants melatonin, spermidine, and N-acetyl-L-cysteine (AOs, 25 pg each), rapamycin (R, 25 pg) and SP-D (25 pg). Nanoparticles were characterized and optimized for content-release kinetics prior to instillation (data not shown). With reference to FIGS. 1A - 1G, old mice were i.t. treated once with empty (vehicle, n=3) or AOs+R+SP-D nanoparticles (n=4). One-month post-treatment, soluble protein oxidation (3-nitrotyrosine modifications content) (FIG. 1A) and flow cytometry analysis of BAL samples were performed for total granulocytes (FIG. IB), lymphocytes (FIG. 1C) and CDllb⁺CDllC⁺ population (FIG. ID). Mitochondrial superoxide production was determined using MitoSOX by flow. Levels of IL- 12p70 (FIG. IE), IFN-g (FIG. IF) and MMP-9 (FIG. 1G) were measured by Luminex.. One month after a single nanoparticle treatment, a significant reduction in soluble protein oxidation (FIG. 1A) and cellular oxidative stress in granulocytes (FIG. IB) and lymphocytes (FIG. 1C) was observed (reduction of cellular superoxide measured by MitoSOX™ Red Mitochondrial Superoxide Indicator) in old mice treated with AOs+R+SPD. Moreover, the CDllb⁺ CDllc⁺ alveolar population also had a reduction in cellular oxidation (FIG. ID). Reduction of pro- inflammatory cytokines (FIG. IE and FIG. IF) and metalloproteases linked to tissue damage (FIG. 1G) was also observed. These results suggest that the nanoparticle intervention can reduce the oxidative status of the cellular alveolar compartment in vivo. Susceptibility of the elderly to M.tb infection may be linked to the presence of a CD1 lb+ CD1 lc+ alveolar subpopulation, which M.tb uses as a niche to replicate.

[0028] Oxidative stress of the lung environment is a determinant of the immune response against respiratory infections. Old mice were treated with empty nanoparticles (vehicle) or AOs+R+SP-D (O+T) nanoparticles once only, and infected with 100 M.tb CFUs one month later. These mice were then analyzed for CFU at 10-days post infection (DPI) (FIG. 1H) (n= 4-5/group), and at 40-DPI (FIG. II) (n=3/group). The M.tb infection was controlled significantly better (significantly by 40-days post-infection) when compared to untreated old mice (FIGS. 1H & II). For FIGS. 1A - II, Unpaired t test, M ± SD; Student’s t test vehicle (empty us. AOs+R+SPD nanoparticles in Old, **p< 0.005. Y: Young + Vehicle (empty nanoparticles); O: Old + Vehicle (empty nanoparticles); O+T: Old + Treatment [antioxidants (AOs) + Surfactant Protein D (SP-D) + Rapamycin (R) nanoparticles].

[0029] Embodiments include compositions and methods to improve the oxidative status of the lung by delivering antioxidants and soluble functional innate components or other commercial products, alone or in combination, loaded into nanoparticle delivery systems, or free in solution, to restore the lung mucosa homeostasis, and thus, driving resistance to respiratory infections. The impact of reduced oxidation on molecular and cellular pathways during aging was evaluated. [0030] Slow release of encapsulated antioxidants and rapamycin and/or functional SP-A/-D into the lungs of elderly animals can reduce the oxidative status and improve SP-A/-D functions. The elderly lung alveolar environment has high levels of oxidants and decreased levels of anti oxidants, antimicrobial peptides, and SPA/- D function. Thus, certain lung changes with increasing age can be reversed by delivering antioxidants and SP-A/-D directly to the lung. An intervention in the lung (e.g., inhalation of formulated compounds) can improve lung health and make elderly humans more resistant to respiratory infections. Poly(lactic co-glycolic acid)(PLGA) nanoparticles were optimized for influenza and the porcine RRS virus, in collaboration with the Southwest Research Institute microencapsulation unit.

[0031] Antioxidant containing nanoparticles were delivered to the lungs of old mice. Interventions start in 3 -month (young control) and 18-month (old) hamsters, equivalent to 30 and 70-year old humans, respectively, by intratracheal delivery of vehicle buffer [i.e. saline, control], empty vehicle (i.e. empty nanoparticles alone, control), or encapsulated R, AOs, SPs, or R+AOs+SPs on a biweekly basis for 1 month (n= 5/age group/treatment). At 1 month of treatment, the oxidative status of the lungs is determined in each treated group looking at levels (by Multiplex assays and mass spectrometry - lung proteomics looking at oxidation, pro-/anti-inflammatory markers)4 and functionality (binding assays, etc.) of specific immune components [cytokines, chemokines, SP-A/-D, complement, mannose binding lectin (MBL), Abs (IgG/IgA,/IgM), antimicrobial peptides, markers of oxidation (glutathione system - GSH levels, GSH/GSSG ratio), etc.) that are altered and/or dysfunctional during the aging process. Intervention induced cytotoxicity and tissue pathology (lung, spleen, liver) are assessed. Stage of systemic and tissue inflammation, cell phenotype, and function are measured.

[0032] The delivery of R, AOs, and SPs as sustained release formulations in nanoparticles with maximum release at 14 days that last for 28 days in the lung without apparent cytotoxicity and pathology have been demonstrated (FIG. 3). Over a 1 -month treatment period, a total of ~50 pg of each compound is delivered into the hamster lung alveolar environment (~25 pg/dose, biweekly for 1 -month). Oxidation and subsequent inflammation are reduced in R- and AOs- treated mice when compared to controls (vehicle and empty nanoparticles). The R/AOs-treated old mice can reestablish lung alveolar environment immune homeostasis at levels similar or closer to those observed in young mice. Other combinations which maximize compound release while minimizing tissue damage can also be used. The optimal intervention timing to maximize R+AOs+SPs concentrations in the lung alveolar space can be determined by experimentation.

[0033] Nanoparticle-formulations can improve health and make elderly animals more resistant to lung infections. This is demonstrated using SARS-CoV-2 (acute) and M.tb (chronic) infection models. To determine resistance to SARS-CoV-2 infection, 3-months and 18-months old hamsters are treated with saline, empty nanoparticles (vehicle) or with R+AOs+SPs nanoparticles identically as above (FIG. 2), and subsequently infected intranasally (i.n.) with a 0.5 to 1 hamster lethal dose 50 (MLD50) of SARS-CoV-2 strain USA-WA1/2020 (BEI Resources NR-52281) at the completion of 1- month treatment with nanoparticles. Nanoparticle treatment is expected to decrease the lethality expected in old hamsters. This SARS-CoV-2 USA-WA1/2020 strain was isolated from an oropharyngeal swab from a patient with a respiratory illness in January 2020 in Washington, US. The viral strain is currently in the ABSL3 labs, working stocks at passage 6, deep-sequencing confirmed). Syrian hamsters have been shown to be a good model for SARS- CoV-2 infection. There is currently no evidence that sex could influence the outcome of SARS- CoV-2 infection in hamsters. Hamster experiments are conducted under ABSL3 conditions. At day 0 (baseline) and days 3, 7, and 14 post-infection (FIG. 2), organs are harvested and lung inflammation, oxidation status, cellular phenotype, morphometric pathology, molecular histopathology, and SARS-CoV-2 viral load in lungs determined (n= 5/age group/treatment/time- point). Control groups, both young and old hamsters, are challenged without previous treatment, or after treatment with empty nanoparticles. The interventions are tracked using a combination of R, AOs, SPs or R+AOs+SPs to demonstrate that lung oxidative status and reduce inflammation during infection in old hamsters can be reversed, and SARS-CoV-2 infection can be managed by minimizing lung tissue damage.

[0034] Young and old hamsters receiving SP-A/-D will have improved control of SARS-CoV- 2 infection although this improvement will be significantly higher in older hamsters. Importantly, older hamsters receiving the combination of R+AOs+SPs pre-SARS CoV- 2 infection will have reduced oxidation and increased function of soluble innate components in their lungs, allowing them to significantly improve SARS-CoV-2 infection outcome by reducing viral load, as well as inflammation and tissue damage. Based on the preliminary data, 1 -month treatment with nanoparticles (only 1 dose at time zero, and assessment of the lung status 30 days later) is sufficient to observe a significant reduction in the oxidative status of the lung mucosa and alveolar macrophages and T cells (FIGS. 1A - II). Because the maximum release was observed at day 14 (FIG. 3), a second dose at day 15, and infection at 1 -month since the 1st dose can be incorporated. This would allow two waves of rapamycin, antioxidants and functional SP-A/-D to normalize the oxidative status of the lung prior to the challenges. Studies including nanoparticles as a pre- and post-infection therapy can be conducted where hamsters are treated twice with nanoparticles prior to infection, challenged with SARS-CoV-2 after 1 month, and retreated with nanoparticles once at 3 days post-infection (for SARS-CoV-2, exponential time-point of the infection) to assess the impact of the nanoparticles cargo on the lung environment and innate and adaptive immune responses. Reestablishment of the lung environment, including functional SP-D, will allow hamsters to significantly increase their resistance to acute (SARS-CoV-2) infection. While not expected, nanoparticles-content release may vary as the infection progresses. The delivery of the nanoparticles can be sequential; thus, ensuring the maximum presence of antioxidants and functional surfactant proteins in the lungs in a specific time point.

[0035] Interventions in elderly animals restore homeostasis in the lung alveolar environment making the elderly more resistant to respiratory infections. Interventions in vivo can alter the lung environment making the host more resistant to acute SARS-CoV-2 infection (and potentially to other viral/bacterial respiratory infections).

[0036] The lung mucosa status in the elderly favors pro-inflammatory cytokines. Inflammation is increased in the lungs of elderly humans (66+) (FIG. 4B) and old mice (18- months+) (FIG. 4A). In old age, inflammatory mediators such as IL-6 (and numerous others) are increased in the lung. IL- 6 and IL-10 shown as examples. The normal balance is disrupted, as alveolar macrophages (AM) continue to synthesize anti-inflammatory cytokines (IL-10), as shown in FIG. 4C. FIGS. 4A - 4C are graphical representations of the cytokine levels in the aging lung. Alveolar lining fluid (ALF) from young [Y] and old [O] mice in FIG. 4A or ALF from adult [A] and elderly [E] humans in FIG. 4B were obtained by BAL (mouse ALF, n=16/group, and human ALF (n=4/ group). ALF was normalized for well loading by protein content of 0.1 pg/mΐ (by BCA) adding 100 mΐ per well. Cytokines were measured by ELISA. Student’s t test old mouse vs. young mouse ALF or elderly vs. adult human ALF, *p<0.05; **p<0.005. FIG. 4C is a graphical representation of the cytokine mRNA levels in AMs from young and old mice. RNA was isolated from AMs after BAL and cytokine mRNA levels determined by qRT-PCR. Levels of mRNA were normalized to b-actin levels and cytokine expression levels determined relative to levels in AMs from young mice (n=3-5). Student’s t test, young mouse vs. old mouse or adult vs. elderly human; **p<0.005.

[0037] Circulating inflammatory proteins are evident in the lung in old age. Increased levels of inflammatory proteins such as complement components in the lungs of old mice or elderly human subjects. The 6 relative changes in complement components in the ALF of old mice ( I C2, † C3 and † C4) suggest that there are alterations in membrane attack complex (MAC) formation and/or antibody (Ab) complex clearance associated with the classical arm of the complement cascade. This is supported by reports of reduced Ab function in elderly subjects and may contribute to their increased susceptibility to infections Similarly, data here indicate that low 41 C2 and high Factor B in the ALF of old mice and elderly humans may favor the alternative pathway in lower respiratory tract secretions. This contrasts with what is seen in the healthy human adult lung. Importantly, ALF in elderly humans constitutes a higher oxidized environment (FIGS. 5A and 5B), a factor that was shown contributes to local tissue inflammation of the lung alveolar space. High levels of oxidized proteins (FIGS. 5A and 5B) and oxidants (FIGS. 8A and 8B) in elderly human lungs result in the dysfunction of important innate soluble components present such as SP- A and SP-D (FIGS. 5C and 5D).

[0038] FIGS. 5A-5D are graphical representations of increased protein oxidation and reduced protein function associated with aging in humans. FIG. 5A is a graphical representation of protein carbonyls as detected by ELISA in adult or elderly human ALF. FIG. 5B is a graphical representation of protein 3-nitrotyrosines detected by ELISA in adult or elderly ALF. FIGS. 5C and 5D are graphical representations of SP-A and SP-D, respectively, in adult and elderly subjects. M.tb was incubated in physiological concentrations and conditions of adult (A, white bars) or elderly (E, black bars) ALF. Exposed bacteria were immobilized onto a cell culture plate and probed with monoclonal antibodies directed against SP-A and SP-D. Relative quantities of bound protein were measured by standard ELISA by measuring the absorbance at OD450. Experiments for n=4 in each group are shown, M ± SEM; * Student’s /-test of human A- ALF vs. E-ALF; ns, not significant; *p<0.05; **p<0.005. [0039] AMs possess a unique inflammatory signature in old age. AMs from old mice have increased levels of MAP kinases and NF-kB activation (FIG. 6A), likely responding to the cumulative effects of oxidative stress that increase ROS production and trigger NF-kB activation, a critical transcription 34 regulator of inflammatory mediators. FIGS. 6A - 6C demonstrate the state of lung macrophage in old age. FIG. 6A is a set of confocal images of phospho-p38 (red) and NF-kb (green) in AMs. Blue represents nuclei. Mean fluorescence intensity (MFI) for phosphor- p38 in AMs from young mice is 12.6 ± 1.5 (FIG. 6A, top left panel) and 21.9 ± 2.5 in AMs from old mice (FIG. 6A, top right panel). MFI for NF-KB-p65 in AMs of young mice is 15.4 ± 1.5 (FIG. 6A, bottom left panel) and 67.9 ± 7.3 in AMs from old mice (FIG. 6A, bottom right panel). NF-KB p65 is seen in the cytoplasm in young mice, while several AMs in old mice have nuclear localized NF-KB. Representative of n=2.

[0040] Lung macrophages from young (Y) and old (O) mice were isolated and mRNA expression of selected genes was analyzed. FIG. 6B is a set of graphical representations of expression of selected genes. Higher mRNA and expression levels of iNOS (not shown), CCL2, IFN-B, IL- 10, IL-12p40, TNF a , and M-CSF were also elevated in AMs from old mice (as shown in FIG. 6B, top left, middle left, and bottom left panels). Some AMs from young (Y) and old (O) mice were isolated, fixed, and stained extracellularly with Abs against the mannose receptor (MR), CD86, or MHCII (as shown in FIG. 6B, top right panel, middle right panel, and bottom right panel, respectively). Interestingly, expression levels of classic M2-like markers such as arginase 1 and the mannose receptor (MR, CD206) were elevated in AMs from old mice, but with an unaltered expression of CD86 and MHCII activation markers (FIG. 6B).

[0041] Mean fluorescent intensity (MFI) of MR, CD86 or MHCII was measured on CDl lc+ cells. MFI values were normalized by subtracting isotype controls. Sorted AMs from old mice presented two distinct AM subpopulations: CDl lc+ CDl lb+ AMs (black bar) had a pro- inflammatory profile, whereas CDl lc+ CD l ib- AMs (white bars) had an immune-regulatory profile (as shown in FIG. 6C); n=3, Student’s t test, Y vs. O, *p<0.05; **p<0.005. Thus, the inflammatory profile of AMs is unique, which may result from ongoing efforts to reduce inflammation in the lung alveolar space even in the elderly. [0042] These analysis of AMs from old mice revealed two distinct AM subpopulations: a major CDllc+ CDllb- and a minor but significantly increased (4-fold) CDllc+ CDllb+ population, the latter with higher expression of CD206, TLR2, CD16/CD32, MHCII, and CD86 as well as monocytic markers Ly6C, CX3CR1, and CD115, suggesting that this population is of monocytic origin (data not shown, see¹¹). Sorted CDllc+ CDllb+ AMs from old mice expressed higher mRNA levels of CCL2, IL-Ib, IL-6, and COX2, whereas CD11C+ CD1 lb- AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-b and IL-10 (FIG. 6C).

[0043] Importantly, CD 11 c+ CD llb+ AMs are highly susceptible to M/L infection. As animals age, there is a tissue oxidation-inflammation axis, where lung alveolar space immune regulatory mechanisms are eventually influenced by systemic tissue oxidation that drives inflammation. This highly inflammatory tissue environment drives more oxidation of proteins and subsequently their dysfunction, which enhances the elderly susceptibility to acute and chronic respiratory viral and bacterial infections.

[0044] Soluble innate components unique to the lung were manipulated. Levels and function of surfactant proteins SP-A, SP-D, and complement in the lungs of adult or elderly humans were measured. Oxidation of lung specific SP-A/-D is detectable in lung mucosa from the elderly, which negatively impacts SP-A/-D binding to M.tb. Oxidized and dysfunctional SP-A/-D play a role in the susceptibility of the elderly to M.tb infection (FIGS. 5C-5D). Replenishment with functional SP-D in the human elderly lung mucosa can reestablish the capacity of human macrophages to control M.tb infection.

[0045] Replenishment of elderly lung mucosa with functional SP-D restores control of M. tb by human macrophages. Human macrophages were infected with adult or elderly ALF-exposedM/L, or elderly ALF-exposed M.tb that had been replenished with functional SP-D. Replenishment of elderly ALF with SP-D led to significant decreases in bacterial burden. In FIG. 7, representative experiment of n = 3 using 3 different human A- ALF and 3 different human E-ALFs were performed triplicate, M ± SD; Student’s t test human A- ALF vs. E-ALF at each time point, *p<0.05.

[0046] Systems analyses of the human lung alveolar space environment are evaluated. Lung proteomics are performed to study oxidation and pro-/anti-inflammatory markers, which require systematic analyses to identify the local and peripheral signature associated with oxidation. The elderly human (and mouse, data not shown) lung alveolar space environment exists in an inflammatory and pro-oxidative state. The lipid and proteomic assessments of adult and elderly human lung mucosa by .5,6 LC-MS/MS identified a total of 1,154 proteins, with adults having 349 (30.2%) unique proteins, elderly 96 (8.3%), and 709 (61.4%) shared across both groups. For proteins of interest (anti/pro-oxidative, anti/pro-inflammatory), their fold change in the elderly ALF relative to adult was calculated based on the total TIC (total ion current). In general, elderly ALF contained higher levels of pro oxidative/pro-inflammatory proteins and lower levels of anti- oxidative/anti-inflammatory proteins (FIGS. 8A and 8B). Proteomics methods are used to determine the impact of the nanoparticle delivery system on the lung of old mice.

[0047] FIGS. 8A and 8B are graphical representations of increased inflammation and pro oxidative signatures in lung ALF associated with aging. Fold change of elderly relative to adult ALF based on total TIC [total ion current; the sum of the total ion current values of all tandem (MS2) spectra assigned to a protein] for proteins classified as pro-oxidative or pro inflammatory found in human ALF. Values correspond to log2 (median total TIC elderly ALF vs. adult ALF) for n=4.

[0048] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present disclosure is not intended to be limited to the above described embodiments, but rather is as set forth in the following claims:

Claims

Claims What is claimed is:

1. A nanoparticle composition comprising rapamycin, an antioxidant, and a pulmonary surfactant protein.

2. The nanoparticle composition of Claim 1, wherein the pulmonary surfactant protein is a pulmonary surfactant protein A.

3. The nanoparticle composition of Claim 1, wherein the pulmonary surfactant protein is a pulmonary surfactant protein D.

4. The nanoparticle composition of Claim 1, wherein the antioxidant is one or more of melatonin, spermidine, and N-acetyl-L-cysteine.

5. A method of treating a viral infection, comprising: administering to a subject with the viral infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein.

6. The method of treating the viral infection of Claim 5, wherein the viral infection is severe acute respiratory syndrome.

7. The method of treating the viral infection of Claim 5, wherein the viral infection is influenza.

8. The method of treating the viral infection of Claim 5, wherein the pulmonary surfactant protein is a pulmonary surfactant protein A.

9. The method of treating the viral infection of Claim 5, wherein the pulmonary surfactant protein is a pulmonary surfactant protein D.

10. The method of treating the viral infection of Claim 5, wherein the antioxidant is one or more of melatonin, spermidine, and N-acetyl-L-cysteine.

11. A method of treating a bacterial infection, comprising: administering to a subject with the bacterial infection a therapeutically effective amount of a nanoparticle composition containing rapamycin, an antioxidant, and a pulmonary surfactant protein.

12. The method of treating the bacterial infection of Claim 11, wherein the bacterial infection is a Mycobacterium tuberculosis infection.

13. The method of treating the bacterial infection of Claim 11, wherein the pulmonary surfactant protein is a pulmonary surfactant protein A.

14. The method of treating the bacterial infection of Claim 11, wherein the pulmonary surfactant protein is a pulmonary surfactant protein D.

15. The method of treating the bacterial infection of Claim 11, wherein the antioxidant is one or more of melatonin, spermidine, and N-acetyl-L-cysteine.