ABSTRACT

Keywords: nanomedicine, acute lung injury, acute respiratory distress syndrome, TREM-1, triggering receptor expressed on myeloid cells GLP-1, glucagon-like peptide-1(7-36) amide 17-AAG, SSM, sterically stabilized phospholipid micelles 34.1 IntroductionThis chapter discusses the opportunities that nanomedicine offers to develop targeted therapies in diseases such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which have an abysmal prognosis. The mortality rate from ARDS remains unacceptably high, ranging from 34 to 64%. Hence, there is a

need for effective pharmacotherapies to treat ARDS. Although several promising therapies are currently being identified for the treatment of ARDS, the delivery of these potential targets, particularly peptides and proteins, to the lung is also an ongoing challenge. Nanoscience and nanotechnology are the basis of innovative techniques that offer great potential of delivery of drugs targeted to the site of inflamed lungs. Nanoscale drug delivery systems have the ability to improve the pharmacokinetics and increase the biodistribution of therapeutic agents to target organs which results in improved efficacy while reducing the drug toxicity. These systems are exploited for therapeutic purpose to carry the drug in the body in a controlled manner from the site of administration to the therapeutic target. To this end, we have identified several potential targets and proposed the delivery of these agents using nanomicelles to improve the drug delivery.In recent years, nanomedicine has become an attractive concept for the targeted delivery of therapeutic and diagnostic compounds to the lung [1-11]. Nanoscale drug delivery systems improve the pharmacokinetics, biodistribution and bioactivity of loaded drugs by prolonging circulation time, reducing the dose administered, and passively targeting to inflamed and injured organs through hyperpermeable (“leaky”) microcirculation.Among various drug delivery systems considered for pulmonary application, the use of biocompatible and biodegradable lipid-based and polymeric nanoparticles represents several advantages for the treatment of respiratory diseases. A number of different strategies have been proposed for modification of nanoparticle characteristics to control their behavior within biological environments, like cell-specific targeted drug delivery or modified biological distribution of drugs, both at the cellular and organ level. Thus, this method of targeted drug delivery to the lung is particularly attractive for inflammatory conditions such ALI and ARDS [4, 12-15].Despite recent advances in diagnostic and therapeutic modalities, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) still represents an unmet medical need because it is associated with appreciable morbidity and mortality (30-40%) and substantial medical expenditure [16-18]. Hence, there is an urgent need to develop and test new drugs to treat this devastating disorder [19]. Unfortunately, contemporary drug development

approaches to address this challenge that center on mono-metabolic pathway inhibitors are hindered by diverse mechanisms underlying ALI and ARDS pathogenesis and by non-selective drug effects that could predispose to serious adverse events [20].To overcome both barriers, we emulated the clinical success of combination therapy in cancer and HIV by devising an innovative nanopharmacotherapeutic strategy consisting of combination of 3 long-acting and safe nanomedicines that selectively target and inhibit three distinct key intracellular proinflammatory signaling cascades activated in ALI and ARDS [20]. Accordingly, we have harnessed unique attributes of three novel, long-acting, biocompatible, and biodegradable anti-inflammatory nanomedicines. They consist of two amphipathic peptide drugs, human glucagon-like peptide-1(7-36) amide (GLP-1) and triggering receptor expressed on myeloid cells (TREM-1) peptide, and 17-allylamino-17-demethoxygeldanamycin (17-AAG), a water-insoluble cytotoxic drug. This innovative approach consists of self-assembly of each drug with generally regarded as safe (GRAS) distearoylphosphatidylethanolamine covalently linked to polyethylene glycol of molecular weight 2000 (DSPE-PEG2000), a component of FDA-approved Doxil®, that forms long-acting, biocompatible and biodegradable sterically stabilized phospholipid micelles in aqueous milieu (SSM; size ~15 nm) [20]. 34.2 ALI and ARDSALI and ARDS arise from direct and indirect injury to the lungs and result in a life-threatening form of respiratory failure with diffuse, bilateral lung injury and severe hypoxemia caused by non-cardiogenic pulmonary edema, which affects approximately 1 million people worldwide annually [18]. As with other inflammatory processes, lung inflammation is accompanied by many cellular and biochemical processes; some of them are specific to the syndrome and include injury to both the pulmonary capillary endothelium and the alveolar epithelium [21, 22]. The importance of ALI and ARDS has been highlighted by the emergence of SARS (severe acute respiratory syndrome). ALI and ARDS are a leading cause of morbidity and mortality in the USA [16-18]. The major reason underlying the lag in improvement in outcome is the lack

of novel and specific therapies for ALI and ARDS. Thus, both ALI and ARDS represent an unmet medical need, and there is an urgent need to develop novel therapies for this condition.The molecular pathobiology of ALI and ARDS is being extensively defined and the role of several molecules, including pattern recognition receptors present on the immune cells such as Toll-like receptors and downstream signaling molecules such as NF-kB and effector molecules such as TNF-a and IL-1b, are being investigated in the pathogenesis and treatment of acute lung injury and ARDS [22]. Targeting central molecules such as NFkB attenuates lung inflammation but has major limitations because the inhibition of NF-kB is immunosuppressive and compromises host defense [22]. However, because of the complex nature of the disease, targeting single cytokine or chemokine has also failed to attenuate lung inflammation as these are not sufficient singly to attenuate lung inflammation in ALI and ARDS [17].Thus, we propose innovative approaches that involve (1) targeting multiple upstream molecules (triggering receptor expressed on myeloid cells (TREM-1), reactive oxygen species and Hsp90 that lead to activation of NF-κB ultimately leading to ALI and ARDS with poor outcomes in many cases; (2) developing a novel approach to deliver inhibitors of these molecules in vivo. We have previously shown that these individual nanoformulations are effective at attenuating lung inflammation; and (3) using combination therapeutic approach, which involves three distinct intracellular metabolic pathways likely to be successful as it is in patients with cancer [20]. 34.3 Nanomedicine for ALI and ARDS

Nanoparticles have potential application in medical field, including diagnostics and therapeutics. Nanoscale drug delivery systems have the ability to improve the pharmacokinetics and increase the biodistribution of therapeutic agents to target organs, which results in improved efficacy and reduces drug toxicity [7, 23-25]. Nanocarriers are particularly designed to target inflammation and cancer, which have permeable vasculature. Additionally, several nanocarriers have the desirable advantage of improving solubility

of hydrophobic compounds in the aqueous media to render them suitable for parenteral administration. In particular, delivery systems have shown to increase the stability of a wide variety of therapeutic agents such as hydrophobic molecules, peptides, and oligonucleotides [20, 23, 24].These systems are exploited for diagnostic and therapeutic purposes to carry the drug in the body in a controlled manner from the site of administration to the therapeutic target [2, 9, 11, 12, 15]. This implies the trafficking of drug molecules and drug delivery system across numerous physiological barriers, which represents the most challenging goal in drug targeting. Nanoparticles can be constructed by various methodologies so that effect can be targeted at the desired site [8, 15, 20, 23, 24, 25]. 34.3.2 How Do We Apply Nanomedicine to ALI and

ARDSTo begin to address the role of nanomedicine in treating ALI and ARDS, we developed novel long-acting biocompatible and biodegradable phospholipid micelles (hydrodynamic size, ~15 nm) to modulate key signaling molecules that are critical to the inflammatory response in ALI and ARDS [21, 23-25]. We selected molecules that initiate and propagate inflammatory response by distinct mechanisms so that multiple pathways can be targeted either singly or by a combinatorial approach. Among these, triggering receptor expressed on myeloid cells (TREM-1), reactive oxygen species and Hsp90 were initially selected to modulate the inflammatory response in the lung [20].Realizing short half-life of peptide drugs (minutes) hampers their clinical use, we invented micellar TREM-1 peptide and human glucagon-like peptide-1(7-36) amide (GLP-1), a 29-amino acid pleiotropic immunomodulator, where each peptide drug is stabilized in its active form (a-helix) and its bioactivity is prolonged for hours in vivo. Likewise, water-insolubility of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a selective Hps90 inhibitor, constrains its use in humans. Accordingly, self-association of 17-AAG with these micelles overcomes this limitation while at the same time increasing its stability and bioavailability. These long-acting micellar drugs provide significant advancement in the treatment of experimental of ALI, which could then be extended

to critically ill patients. Nanoparticles can be introduced by systemic administration, such as oral, dermal and intravenous, or directly introduced into the lung through inhalation, intranasal, or oropharyngeal aspiration. In a recent study, we tested the efficacy of GLP-1 nanomicelles in a rat model of lipopolysaccharide (LPS)-induced lung injury [24]. In vivo administration of GLP1-SSM to LPS-induced ALI rats

resulted in significant downregulation of lung inflammation, with dose-dependent anti-inflammatory activity observed. Similar therapeutic activity was not detected for GLP-1 in saline, indicating that these nanocarriers played a critical role in protecting the enzyme-labile GLP-1 and delivering it to inflamed tissues in vivo. This study showed that a lipid-based nanoformulation of GLP-1 is effective at attenuating inflammation in ALI and ARDS [23]. We have also tested the efficacy of TREM-1 nanomicellar peptide in a model of LPS-induced sepsis and lung injury and shown that TREM-1 nanomicelles are more efficacious than the naked peptide at abrogating inflammation [20]. Studies with other nanomicellar preparations such as 17 AAG for treatment of ALI and ARDS are currently ongoing in our laboratory [20]. Together, our studies demonstrate the feasibility of translating the use of these nanomicellar preparations for translational human studies to the clinics to treat this devastating disease. We posit that combinatorial administration of nanomicelles that modulate distinct signaling pathways will prove to be more potent but will need further studies to optimize the administration of the nanopreparations. 34.4 Nanomedicine for Drug Delivery to the

34.4.1 Nanomedicine Targeted to LungWe have used nanomicellar preparations for targeted delivery of drugs to the lung using systemic administration. Micelles are self-assemblies of amphiphiles that form supramolecular core-shell structures in the aqueous environment. Hydrophobic interactions are the predominant driving force in the assembly of the amphiphiles in the aqueous medium when their concentrations

exceed the critical micellar concentration. Sterically stabilized phospholipid micelles (SSM) are a novel, long-acting, biocompatible, and biodegradable phospholipid-based drug delivery platform that was developed and patented in our laboratory as versatile carrier for peptide and water-insoluble drugs [20, 23-25]. Our approach entails self-assembly of distearoylphosphatidyl-ethanolamine covalently linked to polyethylene glycol of molecular weight 2000 (DSPE-PEG2000) with drugs such as GLP-1 to form long-acting sterically stabilized phospholipid micelles in aqueous milieu (hydrodynamic size ~15 nm) [20, 23-25]. These micelles are composed of hydrophilic corona that houses amphipathic peptide drugs, such as TREM-1 peptide and GLP-1, and hydrophobic core that accommodates water-insoluble drugs, such as 17-AAG. They are simple to prepare and, unlike liposomes, can be stored in lyophilized form without lyo-or cryo-protectants for extended period of time. Lipid-based nanomicelles stabilize TREM-1 peptide and GLP-1 in active biological form (a-helix), which is preferred for ligand-receptor interactions, and prevents rapid peptide degradation in vivo thereby prolonging bioactivity. In addition, SSM solubilize high concentrations of 17-AAG [20]. Unlike surfactant micelles, low critical micellar concentration (~1 µM) of these nanoparticles prevents their disintegration upon dilution in biological fluids. Importantly, the PEG2000 moiety of SSM confers steric hindrance in the circulation while their nanosize mitigates renal clearance and extravasation from intact microvessels. This, in turn, prolongs circulation time of drug-loaded micelles and promotes preferential extravasation from hyperpermeable lung microcirculation, the hallmark of ALI and ARDS, into injured lung [23-25]. 34.4.2 Drug Delivery of NanoparticlesSystemic delivery of these agents is based on the principle of passive targeting. Passive targeting occurs due to extravasation of the nanoparticles at the diseased site where the microvasculature is leaky [6]. Examples of diseases where passive targeting of nanocarriers can be achieved are tumor and inflamed tissues. Microvascular leakiness in ALI and ARDS is the result of increased permeability and the presence of cytokines and other vasoactive factors that enhance permeability [21]. Thus, drugs used for

treatment of ALI and ARDS can be administered systemically and will localize to the lungs by passive targeting. We propose that this innovative passively targeted therapeutic strategy amplifies drug delivery to lung thereby maximizing efficacy and enhancing resolution of inflammation while reducing collateral damage to innocent bystander organs as occurs in patients with ALI and ARDS. Controlled drug delivery systems have also become increasingly attractive options for inhalation therapies [1, 2, 4, 7, 11-15]. The large surface area of the lungs and the minimal barriers impeding access to the lung periphery make this organ a suitable portal for a variety of therapeutic interventions. The blood barrier between the alveolar space and the pulmonary capillaries is very thin to allow for rapid gas exchange. Alveoli are small and there are approximately 300 million of them in each lung. Although alveoli are tiny structures, they have a very large surface area in total (~100 m2) for performing efficient gas exchange making it an attractive organ for direct drug delivery [6, 17, 21]. Among various drug delivery systems considered for pulmonary application, nanoparticles demonstrate several advantages for the treatment of respiratory diseases, like prolonged drug release, cell specific targeted drug delivery or modified biological distribution of drugs, both at the cellular and organ level [1, 6, 10, 11, 15]. Nanoparticles composed of biodegradable lipid-based nanomicelles and polymers fulfill many requirements placed on these delivery systems, such as ability to be transferred into an aerosol, stability against forces generated during aerosolization, biocompatibility, targeting of specific sites or cell populations in the lung, release of the drug in a predetermined manner, and degradation within an acceptable period of time [8, 15, 23, 24]. Clearly, further studies are warranted to establish the role of aerosolized nanopreparations for inhalational therapy. 34.5 ConclusionsNanomedicine, the medical application of nanobiotechnology, holds great promise in treatment of serious lung disorders that still represent unmet medical needs, such as ALI and ARDS. To this end, multifunctional engineered lipid-and polymer-based

nanoparticles are particularly advantageous because they could deliver simultaneously several drugs that selectively target distinct metabolic pathways that modulate ALI and ARDS without undue systemic toxicity. Undoubtedly, recent progress witnessed in this nascent field of biomedical research should be translated into clinical practice in the near future. Disclosures and Conflict of Interest

The authors declare that they have no conflict of interest and have no affiliations or financial involvement with any organization or entity discussed in this chapter. This includes employment, consultancies, honoraria, grants, stock ownership or options, expert testimony, patents (received or pending) or royalties. No writing assistance was utilized in the production of this chapter and the authors have received no payment for its preparation. Corresponding AuthorDr. Ruxana T. SadikotEmory University School of Medicine Section of Pulmonary and Critical Care Medicine 201 Dowman Drive, Atlanta, GA 30322, USAEmail: ruxana.sadikot@emory.edu About the Authors

Ruxana T. Sadikot is a professor of medicine at Emory University and is the chief of section of pulmonary and critical care medicine at the Atlanta VAMC. She received her medical training from the University of Bombay, India, and from Royal College of Physicians, London, UK. She received her Pulmonary and Critical Care training at Vanderbilt University in Nashville, TN, USA. Dr. Sadikot is funded by the US Department of Veterans Affairs and her research focuses on understanding the mechanisms of lung injury and immune response.