Liposomal Aerosols of Nitric Oxide (NO) Donor as a Long-Acting Substitute for the Ultra-Short-Acting Inhaled NO in the Treatment of PAH.

PURPOSE: This study seeks to develop a liposomal formulation of diethylenetriamine NONOate (DN), a long acting nitric oxide (NO) donor, with a goal to replace inhaled NO (iNO) in the treatment of pulmonary arterial hypertension (PAH). METHODS: Liposomal formulations were prepared by a lipid film hydration method and modified with a cell penetrating peptide, CAR. The particles were characterized for size, polydispersity index (PDI), zeta potential, entrapment efficiency, storage and nebulization stability, and in-vitro release profiles. The cellular uptake and transport were assessed in rat alveolar macrophages (NR8383) and transforming growth factor ß (TGF-ß) activated rat pulmonary arterial smooth muscle cells (PASMCs). The fraction of the formulation that enters the systemic circulation, after intratracheal administration, was determined in an Isolated Perfused Rat Lung (IPRL) model. The safety of the formulations were assessed using an MTT assay and by measuring injury markers in the bronchoalveolar lavage (BAL) fluid; the pharmacological efficacy was evaluated by monitoring the changes in the mean pulmonary arterial (mPAP) and systemic pressure (mSAP) in a monocrotaline (MCT) induced-PAH rat model RESULTS: Liposome size, zeta potential, and entrapment efficiency were 171 ± 4 nm, -37 ± 3 mV, and 46 ± 5%, respectively. The liposomes released 70 ± 5% of the drug in 8 h and were stable when stored at 4°C. CAR-conjugated-liposomes were taken up more efficiently by PASMCs than liposomes-without-CAR; the uptake of the formulations by rat alveolar macrophages was minimal. DN-liposomes did not increase lung weight, protein quantity, and levels of injury markers in the BAL fluid. Intratracheal CAR-liposomes reduced the entry of liposomes from the lung to blood; the formulations produced a 40% reduction in mPAP for 180 minutes. CONCLUSION: This study establishes the proof-of-concept that peptide modified liposomal formulations of long-acting NO donor can be an alternative to short-acting iNO.

Newer devices and improved formulations of inhaled insulin.

INTRODUCTION: Delivery of therapeutic insulin via the pulmonary route has been the most investigated non-invasive alternative to the commonly used subcutaneous (SC) route for diabetes management. Despite discontinuation of the first inhalable insulin, Exubera®, due to suboptimal market acceptance, development of orally inhaled insulin delivery systems has been galvanized by the recent approval of Afrezza® and several others awaiting approval. AREAS COVERED: The scope of this review article includes the prospects for and the challenges faced in developing inhaled insulin delivery systems; discussion of orally inhaled therapeutic insulin delivery systems that were discontinued, recently approved or are currently under active investigation; and formulation approaches that have the potential to deliver insulin via the pulmonary route. EXPERT OPINION: The pulmonary route is the most advantageous route for non-invasive insulin delivery. Inhalable insulin therapeutics have the potential to be successful, provided that the formulations can be made with modified release patterns to substitute for both prandial and basal insulin injections, the delivery devices are convenient and easy to use, and the long-term safety of inhaled insulin is documented through extensive studies.

Peptide-coated liposomal fasudil enhances site specific vasodilation in pulmonary arterial hypertension.

This study sought to develop a liposomal delivery system of fasudil--an investigational drug for the treatment of pulmonary arterial hypertension (PAH)--that will preferentially accumulate in the PAH lungs. Liposomal fasudil was prepared by film-hydration method, and the drug was encapsulated by active loading. The liposome surface was coated with a targeting moiety, CARSKNKDC, a cyclic peptide; the liposomes were characterized for size, polydispersity index, zeta potential, and storage and nebulization stability. The in vitro drug release profiles and uptake by TGF-ß activated pulmonary arterial smooth muscle cells (PASMC) and alveolar macrophages were evaluated. The pharmacokinetics were monitored in male Sprague-Dawley rats, and the pulmonary hemodynamics were studied in acute and chronic PAH rats. The size, polydispersity index (PDI), and zeta potential of the liposomes were 206-216 nm, 0.058-0.084, and -20-42.7 mV, respectively. The formulations showed minimal changes in structural integrity when nebulized with a commercial microsprayer. The optimized formulation was stable for >4 weeks when stored at 4 °C. Fasudil was released in a continuous fashion over 120 h with a cumulative release of 76%. Peptide-linked liposomes were taken up at a higher degree by TGF-ß activated PASMCs; but alveolar macrophages could not engulf peptide-coated liposomes. The formulations did not injure the lungs; the half-life of liposomal fasudil was 34-fold higher than that of plain fasudil after intravenous administration. Peptide-linked liposomal fasudil, as opposed to plain liposomes, reduced the mean pulmonary arterial pressure by 35-40%, without influencing the mean systemic arterial pressure. This study establishes that CAR-conjugated inhalable liposomal fasudil offers favorable pharmacokinetics and produces pulmonary vasculature specific dilatation.

Bio-responsive delivery of tissue plasminogen activator for localized thrombolysis.

In this study, we have developed an albumin-camouflaged/thrombin-triggered delivery system for site-specific delivery of tissue plasminogen activator (tPA). The camouflaged construct is expected to suppress tPA's enzymatic activity in the systemic circulation but regenerate its thrombolytic action upon contact with thrombin present on the thrombus. tPA was camouflaged with human serum albumin (HSA) via a thrombin-cleavable peptide (GFPRGFPAGGCtPA). The surface of the albumin molecule was decorated with a homing peptide (CQQHHLGGAKQAGDV) that binds with GPIIb/IIIa expressed on activated platelets. To avoid non-specific interactions, the unpaired cysteine-34 of HSA was permanently blocked by iodoacetamide and the primary amines were temporarily masked with citraconic anhydride. Shielding with HSA suppressed 75% of tPA's activity which, upon contact with 25 nM thrombin, was regenerated to ~90% of that of native tPA. The fibrin agar plate assay further confirmed the thrombin-mediated release of tPA from the camouflaged construct. The integrity of camouflaged construct was maintained in human plasma or blood. The fluorescence microscopic studies confirmed the binding affinity of camouflaged tPA with the activated platelets. Furthermore, when evaluated in a rat thrombosis model, the thrombolytic activity of camouflaged tPA was similar to that of native tPA. However, the degradation of circulating fibrinogen was reduced by 2-fold with HSA-decorated tPA compared with that of native tPA, which is an indication of reduced risk of hemorrhagic incidence. This proof-of-principle study suggests that the activity of tPA can be suppressed by HSA and regenerated by thrombin present at the thrombus.

Starch-coated magnetic liposomes as an inhalable carrier for accumulation of fasudil in the pulmonary vasculature.

In this study, we tested the feasibility of magnetic liposomes as a carrier for pulmonary preferential accumulation of fasudil, an investigational drug for the treatment of pulmonary arterial hypertension (PAH). To develop an optimal inhalable formulation, various magnetic liposomes were prepared and characterized for physicochemical properties, storage stability and in vitro release profiles. Select formulations were evaluated for uptake by pulmonary arterial smooth muscle cells (PASMCs) - target cells - using fluorescence microscopy and HPLC. The efficacy of the magnetic liposomes in reducing hyperplasia was tested in 5-HT-induced proliferated PASMCs. The drug absorption profiles upon intratracheal administration were monitored in healthy rats. Optimized spherical liposomes - with mean size of 170 nm, zeta potential of -35mV and entrapment efficiency of 85% - exhibited an 80% cumulative drug release over 120 h. Fluorescence microscopic study revealed an enhanced uptake of liposomes by PASMCs under an applied magnetic field: the uptake was 3-fold greater compared with that observed in the absence of magnetic field. PASMC proliferation was reduced by 40% under the influence of the magnetic field. Optimized liposomes appeared to be safe when incubated with PASMCs and bronchial epithelial cells. Compared with plain fasudil, intratracheal magnetic liposomes containing fasudil extended the half-life and area under the curve by 27- and 14-fold, respectively. Magnetic-liposomes could be a viable delivery system for site-specific treatment of PAH.

Serum albumin-protamine conjugate for biocompatible platform for targeted delivery of therapeutic macromolecules.

A well-defined, one-to-one conjugate between human serum albumin (HSA) and protamine was synthesized and characterized as a biocompatible carrier for macromolecules. In circulation, the conjugate will camouflage drug molecules upon complex formation, while liberating free drug at the desired location using a triggering mechanism. The N-terminus of protamine was thiolated and conjugated with the unpaired Cysteine-34 of HSA, and was purified by ion-exchange chromatography. The molecular weight of the conjugate was 70.8 kDa, confirming one-to-one conjugation between HSA (66.6 KDa) and protamine (4200 Da). Superimposed fluorescence spectra of native HSA and HSA-protamine conjugate indicated no conformational change around the Trp-214. The conjugate had marked reduction in hemolytic and cytotoxic properties compared to protamine. When therapeutic potential was tested using tissue plasminogen activator as a model drug, HSA-protamine conjugate suppressed the enzymatic activity by 65%, which was fully recovered by a triggering agent, heparin. The construct showed binding characteristics with activated platelets upon conjugation with a targeting peptide, demonstrating flexibility to introduce suitable homing moiety on the surface. The camouflaged construct retained triggered release property in human plasma condition. Overall, the conjugate has a good potential to serve as a biocompatible platform for macromolecular drugs.

In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals.

Delivery of therapeutic agents via the pulmonary route has gained significant attention over the past few decades because this route of administration offers multiple advantages over traditional routes that include localized action, non-invasive nature and favorable lung-to-plasma ratio. However, assessment of post administration behavior of inhaled pharmaceuticals-such as deposition of particles over the respiratory airways, interaction with the respiratory fluid and movement across the air-blood barrier-is challenging because the lung is a very complex organs that is composed of airways with thousands of bifurcations with variable diameters. Thus, much effort has been put forward to develop models that mimic human lungs and allow evaluation of various pharmaceutical and physiological factors that influence the deposition and absorption profiles of inhaled formulations. In this review, we sought to discuss in vitro, in vivo and ex vivo models that have been extensively used to study the behaviors of airborne particles in the lungs and determine the absorption of drugs after pulmonary administration. We have provided a summary of lung cast models, cascade impactors, noninvasive imaging, intact animals, cell culture and isolated perfused lung models as tools to evaluate the distribution and absorption of inhaled particles. We have also outlined the limitations of currently used models and proposed future studies to enhance the reproducibility of these models.

Thrombus-targeted nanocarrier attenuates bleeding complications associated with conventional thrombolytic therapy.

PURPOSE: To test the hypothesis that thrombus-specific tissue plasminogen activator (tPA)-loaded nanocarriers enhance thrombolytic efficacy and attenuate hemorrhagic complications. METHODS: A series of pegylated and non-pegylated tPA-loaded liposomes were prepared and their surfaces were decorated with the peptide sequence (CQQHHLGGAKQAGDV) of fibrinogen gamma-chain that binds with GPIIb/IIIa expressed on activated platelets. All formulations were characterized for physical properties, stability and in vitro release profile. The thrombolytic activities of tPA-loaded liposomes were tested by visual end-point detection, fibrin agar-plate and human blood clot-lysis assays. The thrombus-specificity of the peptide-modified-liposomes was evaluated by studying the binding of fluorescent peptide-liposomes with activated platelets. The pharmacokinetic profile and thrombolytic efficacy were evaluated in healthy rats and an inferior vena-cava rat model of thrombosis, respectively. RESULTS: Both pegylated and non-pegylated peptide-modified-liposomes showed favorable physical characteristics and colloidal stability. Formulations exhibited an initial burst release (40-50% in 30 min) followed by a continuous release of tPA (80-90% in 24 h) in vitro. Encapsulated tPA retained >90% fibrinolytic activity as compared to that of native tPA. Peptide-grafted-liposomes containing tPA demonstrated an affinity to bind with activated platelets. The half-life of tPA was extended from 7 to 103 and 141 min for non-pegylated and pegylated liposomes, respectively. Compared to native tPA, liposomal-tPA caused a 35% increase in clot-lysis, but produced a 4.3-fold less depletion of circulating fibrinogen. CONCLUSIONS: tPA-loaded homing-peptide-grafted-liposomes demonstrate enhanced thrombolytic activity with reduced hemorrhagic risk.

Preparation and characterization of anionic oligopeptide-modified tissue plasminogen activator for triggered delivery: an approach for localized thrombolysis.

PURPOSE: The study sought to synthesize anionic peptide-conjugated tissue plasminogen activator (tPA) for its targeted/triggered delivery, where tPA's activity would be masked in the circulation and regenerated at the thrombus site by a commonly used anticoagulant, heparin, to minimize tPA associated bleeding complications. METHODS: tPA was conjugated to Polyglutamate, and the activity of oligoanion-modified tPA was tested by fibrinolytic assay. Separately human serum albumin (HSA) was conjugated to protamine and the formation of its electrostatic complex with anionic peptide was monitored by Förster Resonance Energy Transfer (FRET). The masking of tPA-activity via steric hindrance created by albumin, and subsequent regeneration with therapeutic dose of heparin was tested by enzymatic assay. Stability of 'camouflaged-tPA' was determined in human plasma. Using fluorescence microscope, binding of camouflaged-tPA with activated platelets was monitored. Heparin modulated clot-lysis was evaluated in human blood clot. RESULTS: The anionic tPA retained ~97% activity of the unmodified-tPA. FRET experiments confirmed the electrostatic interaction between polyglutamate and protamine which was subsequently reversed by heparin. Complexation with HSA-protamine masked ~60% of tPA activity which was fully regenerated by heparin. The complex retained its prodrug character in human plasma after incubation at 37°C. Fluorescence microscopic study confirmed binding of the construct with activated platelets. In lysing human clot, the camouflage could mask tPA-activity until it was triggered at a heparin level of 0.4U/mL. CONCLUSION: Oligoanion-modified tPA could be used for targeted/triggered delivery where its enzymatic activity could be masked by HSA-protamine conjugate and successfully regenerated by therapeutic dose of heparin.

Computational and bioengineered lungs as alternatives to whole animal, isolated organ, and cell-based lung models.

Development of lung models for testing a drug substance or delivery system has been an intensive area of research. However, a model that mimics physiological and anatomical features of human lungs is yet to be established. Although in vitro lung models, developed and fine-tuned over the past few decades, were instrumental for the development of many commercially available drugs, they are suboptimal in reproducing the physiological microenvironment and complex anatomy of human lungs. Similarly, intersubject variability and high costs have been major limitations of using animals in the development and discovery of drugs used in the treatment of respiratory disorders. To address the complexity and limitations associated with in vivo and in vitro models, attempts have been made to develop in silico and tissue-engineered lung models that allow incorporation of various mechanical and biological factors that are otherwise difficult to reproduce in conventional cell or organ-based systems. The in silico models utilize the information obtained from in vitro and in vivo models and apply computational algorithms to incorporate multiple physiological parameters that can affect drug deposition, distribution, and disposition upon administration via the lungs. Bioengineered lungs, on the other hand, exhibit significant promise due to recent advances in stem or progenitor cell technologies. However, bioengineered approaches have met with limited success in terms of development of various components of the human respiratory system. In this review, we summarize the approaches used and advancements made toward the development of in silico and tissue-engineered lung models and discuss potential challenges associated with the development and efficacy of these models.

Heparin-triggered release of camouflaged tissue plasminogen activator for targeted thrombolysis.

A targetable, heparin-triggered release system for tissue plasminogen activator (tPA) was designed to prevent the excessive 'lytic' state associated with the current tPA therapy for acute thrombotic conditions, such as myocardial infarction (MI). The strategy is, upon target accumulation, to trigger tPA release from a prodrug construct by a usual heparin dose. A relatively inactive form of tPA was constructed by conjugating tPA with low-molecular weight heparin followed by complexation with albumin-protamine conjugate, termed 'camouflage'. The modified tPA was ~97% as active as native tPA. The prodrug construct of tPA significantly masked the enzymatic activity, which was fully recovered upon heparin addition. The camouflaged tPA was stable in human blood for at least 30min and was able to trigger enzyme activation in vitro at heparin level of 0.4U/mL. In vivo studies on jugular vein rat thrombosis model showed that the clot lysis of the heparin-triggered camouflaged tPA group was equivalent to the tPA+heparin group without prolongation of activated partial thromboplastin time (aPTT) before and after the treatment. This proof-of-principle study suggests that the activity of the tPA prodrug construct can be triggered at the thrombus site at therapeutic heparin concentration conjunctively used for MI with reduced bleeding risk.