Inhaled Medicines: Past, Present, and Future.

The purpose of this review is to summarize essential pharmacological, pharmaceutical, and clinical aspects in the field of orally inhaled therapies that may help scientists seeking to develop new products. After general comments on the rationale for inhaled therapies for respiratory disease, the focus is on products approved approximately over the last half a century. The organization of these sections reflects the key pharmacological categories. Products for asthma and chronic obstructive pulmonary disease include ß -2 receptor agonists, muscarinic acetylcholine receptor antagonists, glucocorticosteroids, and cromones as well as their combinations. The antiviral and antibacterial inhaled products to treat respiratory tract infections are then presented. Two "mucoactive" products-dornase α and mannitol, which are both approved for patients with cystic fibrosis-are reviewed. These are followed by sections on inhaled prostacyclins for pulmonary arterial hypertension and the challenging field of aerosol surfactant inhalation delivery, especially for prematurely born infants on ventilation support. The approved products for systemic delivery via the lungs for diseases of the central nervous system and insulin for diabetes are also discussed. New technologies for drug delivery by inhalation are analyzed, with the emphasis on those that would likely yield significant improvements over the technologies in current use or would expand the range of drugs and diseases treatable by this route of administration. SIGNIFICANCE STATEMENT: This review of the key aspects of approved orally inhaled drug products for a variety of respiratory diseases and for systemic administration should be helpful in making judicious decisions about the development of new or improved inhaled drugs. These aspects include the choices of the active ingredients, formulations, delivery systems suitable for the target patient populations, and, to some extent, meaningful safety and efficacy endpoints in clinical trials.

Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial.

BACKGROUND: Brensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG). In chronic inflammatory lung diseases such as non-cystic fibrosis bronchiectasis (NCFBE), neutrophils accumulate in the airways resulting in excess active NSPs that cause damaging inflammation and lung destruction. METHODS: The 24-week WILLOW trial (NCT03218917) was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with NCFBE conducted at 116 sites across 14 countries. In this trial, treatment with brensocatib was associated with improvements in clinical outcomes including time to first exacerbation, reduction in exacerbation frequency and a reduction in NE activity in sputum. An exploratory analysis of NE activity in white blood cell (WBC) extracts and NE, PR3 and CatG activity in sputum was conducted to further characterize brensocatib's effect and identify potential correlated effects. RESULTS: NE, PR3 and CatG activities were reduced in sputum and NE activity was reduced in WBC extracts in a dose-dependent manner after four weeks of brensocatib treatment, with a return to baseline four weeks after the end of treatment. Brensocatib produced the greatest reduction in the sputum activity of CatG, followed by NE and then PR3. Positive correlations among the sputum NSPs were observed both at baseline and in response to treatment, with the strongest correlation among the sputum NSPs for NE and CatG. CONCLUSIONS: These results suggest a broad anti-inflammatory effect of brensocatib underlying its clinical efficacy observed in NCFBE patients. TRIAL REGISTRATION: The study was approved by the corresponding ethical review boards of all participating centers. The trial was approved by the Food and Drug Administration and registered at clinicaltrials.gov (NCT03218917) on July 17, 2017 and approved by the European Medicines Agency and registered at the European Union Clinical trials Register (EudraCT No. 2017-002533-32). An independent, external data and safety monitoring committee (comprising physicians with pulmonary expertise, a statistician experienced in the evaluation of clinical safety, and experts in periodontal disease and dermatology) reviewed all adverse events.

Assessment of Inhaled Treprostinil Palmitil, Inhaled and Intravenous Treprostinil, and Oral Selexipag in a Sugen/Hypoxia Rat Model of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP), a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE), has beneficial effects in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension (PAH) that compare favorably to the oral phosphodiesterase 5 inhibitor (PDE5) sildenafil. In this study in male Sprague-Dawley rats, a dry powder formulation of TP (TPIP) was compared with inhaled and intravenous TRE and oral selexipag to evaluate inhibition of hemodynamic and pathologic changes in the lungs and heart induced by Su/Hx challenge. Su (20 mg/kg) was injected subcutaneously followed by 3 weeks of Hx (10% O2/balance N2) and then initiation of test article administration over 5 weeks with room air breathing. Hemodynamics and histopathology were measured at the end of the study. Su/Hx challenge approximately doubled the mean pulmonary arterial blood pressure (mPAP) and the Fulton index, decreased cardiac output (CO), doubled the wall thickness and muscularization of the small (10-50 µm) and medium (51-100 µm) sized pulmonary arteries, and increased the percentage of obliterated pulmonary blood vessels. Even though inhaled TRE (65 µg/kg, 4× daily), intravenous TRE (810 ng/kg/min), and oral selexipag (30 mg/kg, twice daily) provided some beneficial effects against the Su/Hx challenge, the overall benefit was generally greater with TPIP at high dose (117 µg/kg, once daily). These results demonstrate that TPIP compares favorably to inhaled and intravenous TRE and oral selexipag with respect to inhibition of the pathophysiological changes induced by Su/Hx challenge in rats. SIGNIFICANCE STATEMENT: Treprostinil palmitil (TP) is a long-acting pulmonary vasodilator prodrug of treprostinil (TRE) formulated for inhaled administration by dry powder [treprostinil palmitil inhalation powder (TPIP)]. Comparison of the activity of TPIP, inhaled and intravenous TRE, and oral selexipag in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension demonstrated that each of these drugs exert protection against the hemodynamic and histopathological changes induced by the Su/Hx challenge, with the greatest effect on these changes produced by TPIP.

Development and Preclinical Evaluation of New Inhaled Lipoglycopeptides for the Treatment of Persistent Pulmonary Methicillin-Resistant Staphylococcus aureus Infections.

Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.

Treprostinil palmitil, an inhaled long-acting pulmonary vasodilator, does not show tachyphylaxis with daily dosing in rats.

BACKGROUND: Treprostinil palmitil (TP) is an inhaled long-acting pulmonary vasodilator prodrug of treprostinil (TRE) that has been formulated for delivery as a suspension (treprostinil palmitil inhalation suspension; TPIS) and as a dry powder (treprostinil palmitil inhalation powder; TPIP). In humans, tachyphylaxis is frequently observed with continuous intravenous (IV) or subcutaneous (SC) infusion of TRE and requires dosage escalation to maintain activity. The aim of the present study was to determine whether tachyphylaxis occurs with repeat daily administration of inhaled TPIS. METHODS: Experiments were performed in male Sprague-Dawley rats prepared with a telemetry probe implanted into the right ventricle to measure the change in right ventricular pulse pressure (ΔRVPP) induced by exposure to a 10% oxygen gas mixture. TPIS (6 mL) at concentrations of 0.25, 0.5, and 1 mM was given by nose-only inhalation using an Aeroneb Pro nebulizer, either as a single administration or daily for 16 or 32 consecutive days. In studies involving consecutive daily administrations of TPIS, the delivered TP dosage was 140.3 µg/kg at 1 mM and ranged from 40.2 to 72.2 µg/kg at 0.5 mM. A separate cohort of telemetered rats received continuous IV infusion of TRE via an Alzet mini-pump at a dosage rate of 250 ng/kg/min for 16 days. Blood and lung tissue samples were obtained, and the concentration of TRE in the plasma and TRE and TP in the lungs were measured approximately 1 h after TPIS administration. RESULTS: Dose-response studies with TPIS administered as a single administration inhibited the hypoxia-induced increase in RVPP in both a concentration-dependent (0.25, 0.5, and 1 mM) and time-dependent (1-24 h) manner. TPIS, given QD or BID at inhaled doses ranging from 40.2 to 140.3 µg/kg for 16 or 32 consecutive days, produced statistically significant (P < .05) inhibition of the increase of RVPP due to hypoxia over the full duration of the dosing periods. By contrast, the inhibition of the hypoxia-induced increase in RVPP observed with IV TRE infusion (250 ng/kg/min) disappeared after 16 days of infusion. The plasma concentrations of TRE were significantly higher after IV TRE (range, 2.85-13.35 ng/mL) compared to inhaled TPIS (range, 0.22-0.73 ng/mL) CONCLUSIONS: There was no evidence of tachyphylaxis with repeat daily dosing of TPIS for a period of up to 32 days. The absence of tachyphylaxis with TPIS is likely related to its local vasodilatory effects within the lungs, combined with an absence of sustained high plasma concentrations of TRE.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts.

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01-100 µM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 µM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 µM), the DP1 receptor antagonist BW A868C (1 µM) also inhibited relaxation reaching significance above 10 µM. In contrast, the EP3 receptor antagonist L798106 (1 µM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 µM) blocked transforming growth factor ß1 (TGF-ß1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 µM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-ß1 in the presence of TRE.

Development and Characterization of Treprostinil Palmitil Inhalation Aerosol for the Investigational Treatment of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP) is a prodrug of treprostinil (TRE), a pulmonary vasodilator that has been previously formulated for inhaled administration via a nebulizer. TP demonstrates a sustained presence in the lungs with reduced systemic exposure and prolonged inhibition of hypoxia-induced pulmonary vasoconstriction in vivo. Here, we report on re-formulation efforts to develop a more convenient solution-based metered-dose inhaler (MDI) formulation of TP, a treprostinil palmitil inhalation aerosol (TPIA) that matches the pharmacokinetic (PK) and efficacy profile of a nebulized TP formulation, treprostinil palmitil inhalation suspension (TPIS). MDI canisters were manufactured using a two-stage filling method. Aerosol performance, formulation solubility, and chemical stability assays were utilized for in vitro evaluation. For in vivo studies, TPIA formulations were delivered to rodents using an inhalation tower modified for MDI delivery. Using an iterative process involving evaluation of formulation performance in vitro (TP and excipient solubility, chemical stability, physical stability, and aerosol properties) and confirmatory testing in vivo (rat PK and efficacy, guinea pig cough), a promising formulation was identified. The optimized formulation, TPIA-W, demonstrates uniform in vitro drug delivery, a PK profile suitable for a once-daily administration, efficacy lasting at least 12 h in a hypoxic challenge model, and a significantly higher cough threshold than the parent drug treprostinil.

Nanoscale Probing of Liposome Encapsulating Drug Nanocrystal Using Atomic Force Microscopy-Infrared Spectroscopy.

Use of liposomes encapsulating drug nanocrystals for the treatment of diseases like cancer and pulmonary infections is gaining attention. The potential therapeutic benefit of these engineered formulations relies on maintaining the physical integrity of the liposomes and the stability of the encapsulated drug. With the significant advancement in the microscopic and analytical techniques, analysis of the size and size distribution of these nanosized vesicles is possible. However, due to the limited spatial resolution of conventional vibrational spectroscopy techniques, the chemical composition of individual nanosized liposome cannot be resolved. To address this limitation, we applied atomic force microscopy infrared spectroscopy (AFM-IR) to assess the chemical composition of individual liposomes encapsulating ciprofloxacin in dissolved and nanocrystalline form. Spatially resolved AFM-IR spectra acquired from individual liposomes confirmed the presence of peaks related to N-H bending vibration, C-N stretching and symmetric, and asymmetric vibration of the carboxyl group present in the ciprofloxacin. Our results further demonstrated the effectiveness of AFM-IR in differentiating the liposome containing ciprofloxacin in dissolved or nanocrystalline form. Spectra acquired from dissolved ciprofloxacin had peaks related to the ionised carboxyl group, i.e., at 1576 and 1392 cm-1, which were either absent or far weaker in intensity in the spectra of liposomal sample containing ciprofloxacin nanocrystals. These findings are highly significant for pharmaceutical scientists to ascertain the stability and physicochemical composition of individual liposomes and will facilitate the design and development of liposomes with greater therapeutic benefits.

Changes in respiratory symptoms during 48-week treatment with ARD-3150 (inhaled liposomal ciprofloxacin) in bronchiectasis: results from the ORBIT-3 and -4 studies.

It is not known if inhaled antibiotics improve respiratory symptoms in patients with bronchiectasis. In the recent phase-3 ORBIT trials, 48 weeks' treatment with ARD-3150 (inhaled liposomal ciprofloxacin) did not significantly improve symptoms using the prespecified method of analysis comparing baseline symptoms to those after 48 weeks, when patients had been off treatment for 28 days. This method of analysis does not take account of possible improvements in symptoms while on active treatment.A post hoc analysis of two identical randomised trials of ARD-3150 (ORBIT-3 and -4) administered 28 days on and 28 days off in patients with bronchiectasis and chronic Pseudomonas aeruginosa infection. The quality-of-life bronchiectasis respiratory symptom scale (QOL-B-RSS), which has a one-week recall period, was administered every 28 days. We examined whether respiratory symptoms improved during on-treatment periods and the relationship of changes in QOL-B-RSS to changes in bacterial load using a mixed-model repeated measures approach.ARD-3150 treatment resulted in a significant improvement in respiratory symptoms during the on-treatment periods with concordant results between ORBIT-3 (estimate 1.4 points, se 0.49; p=0.004) and ORBIT-4 (estimate 1.1 point, se 0.41; p=0.006). The proportion of patients achieving a symptom improvement above the minimum clinically important difference was higher with ARD-3150 compared with placebo during on-treatment cycles (p=0.024). Changes in respiratory symptoms were correlated with changes in bacterial load in the treatment group (r=-0.89, p<0.0001). Individual estimates for decrements in the QOL-B RSS during exacerbation were -9.4 points (se 0.91) in ORBIT-3 and -10.8 points (0.74) in ORBIT-4 (both p<0.0001).Inhaled ARD-3150 resulted in significant improvements in respiratory symptoms during the on-treatment periods which were lost during off-treatment periods. These results supports the concept that reducing bacterial load can improve respiratory symptoms in patients with bronchiectasis.

Solid State Characterization of Ciprofloxacin Liposome Nanocrystals.

Liposomes have been widely researched as drug delivery systems; however, the solid state form of drug inside the liposome, whether it is in solution or in a solid state, is often not studied. The solid state properties of the drug inside the liposomes are important, as they dictate the drug release behavior when the liposomes come into contact with physiological fluid. Recently, a new approach of making liposomal ciprofloxacin nanocrystals was proposed by the use of an additional freeze-thawing step in the liposomal preparation method. This paper aims to determine the solid state properties of ciprofloxacin inside the liposomes after this additional freeze-thawing cycle using cryo-TEM, small-angle X-ray scattering (SAXS), and cross-polarized light microscopy (CPLM). Ciprofloxacin precipitated in the ciprofloxacin hydrate crystal form with a unit cell dimension of 16.7 Å. The nanocrystals also showed a phase transition at 93 °C, which represents dehydration of the hydrate crystals to the anhydrate form of ciprofloxacin, verified by temperature-dependent SAXS measurements. Furthermore, the dependence of the solid state form of the nanocrystals on pH was investigated in situ, and it was shown that the liposomal ciprofloxacin nanocrystals retained their crystalline form at pH 6-10. Understanding the solid state attributes of nanocrystals inside liposomes provides improved understanding of drug dissolution and release as well as opening avenues to new applications where the nanosized crystals can provide a dissolution benefit.

Direct Comparison of Standard Transmission Electron Microscopy and Cryogenic-TEM in Imaging Nanocrystals Inside Liposomes.

The use of electron microscopy techniques in the understanding of shape and size of nanoparticles are commonly applied to drug nanotechnology, but the type of microscopy and suitability for the particles of interest can have a significant impact on the result. The size and shape of the nanoparticles are crucial in clinical applications; however, direct comparison of the results from standard transmission electron microscopy (TEM) and cryo-TEM have rarely been reported. As a useful case for comparison, liposomal drug nanocrystals are studied here. In this study, the effect of thawing temperature on the size and shape of the ciprofloxacin nanocrystals was determined. A quantitative standard TEM assay was developed to allow for high-throughput particle size analysis. These results were compared to size and shape information obtained using the cryo-TEM method. The results showed broad agreement between the two TEM methods and that ciprofloxacin nanocrystals formed shorter and thinner crystals inside the liposomes at higher thawing temperatures. The results provide confidence in the use of standard TEM to determine the size and shape distribution of solid nanoparticles (in this case, encapsulated inside liposomes) from aqueous media without fear of sample preparation altering the conclusions.

Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin.

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

Sweetening Inhaled Antibiotic Treatment for Eradication of Chronic Respiratory Biofilm Infection.

PURPOSE: The failure of chronic therapy with antibiotics to clear persistent respiratory infection is the key morbidity and mortality factor for patients with chronic lung diseases, primarily due to the presence of biofilm in the lungs. It is hypothesised that carbon sources, such as mannitol, could stimulate the metabolic activity of persister cells within biofilms and restore their susceptibility to antibiotics. The aims of the current study are to: (1) establish a representative in vitro model of Pseudomonas aeruginosa biofilm lung infection, and (2) investigate the effects of nebulised mannitol on antibiotic efficacy, focusing on ciprofloxacin, in the eradication of biofilm. METHOD: Air interface biofilm was cultured onto Snapwell inserts incorporated into a modified pharmacopeia deposition apparatus, the Anderson Cascade Impactor (ACI). Three different formulations including mannitol only, ciprofloxacin only and combined ciprofloxacin and mannitol were nebulised onto the P. aeruginosa biofilm using the modified ACI. Antibacterial effectiveness was evaluated using colony-forming units counts, biofilm penetration and scanning electron microscopy. RESULTS: Nebulised mannitol promotes the dispersion of bacteria from the biofilm and demonstrated a synergistic enhancement of the antibacterial efficacy of ciprofloxacin compared to delivery of antibiotic alone. CONCLUSIONS: The combination of ciprofloxacin and mannitol may provide an important new strategy to improve antibiotic therapy for the treatment of chronic lung infections. Furthermore, the development of a representative lung model of bacterial biofilm could potentially be used as a platform for future new antimicrobial pre-clinical screening.

Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug.

PURPOSE: In order to attenuate the drug release rate, a single freeze-thaw step was previously shown to convert encapsulated drug into a single nanocrystal within each liposome vesicle. The goal of this study was to alter the nanocrystalline character, and thus the drug encapsulation state and release profile, by addition of surfactant prior to freeze-thaw. METHODS: A liposomal ciprofloxacin (CFI) formulation was modified by the addition of surfactant and frozen. After thawing, these formulations were characterized in terms of drug encapsulation by centrifugation-filtration, liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, and in vitro release (IVR) performance. RESULTS: The addition of increasing levels of polysorbate 20 (0.05 to 0.4%) or Brij 30 (0.05 to 0.3%) to the CFI preparations followed by subsequent freeze-thaw, resulted in a greater proportion of vesicles without drug nanocrystals and reduced the extent of growth of the nanocrystals thus leading to modified release rates including an increase in the ratio of non-encapsulated to sustained release of drug. CONCLUSIONS: This study provides another lever to achieve the desired release rate profile from a liposomal formulation by addition of surfactant and subsequent freeze-thaw, and thus may provide a personalized approach to treating patients.

Aerosol performance and long-term stability of surfactant-associated liposomal ciprofloxacin formulations with modified encapsulation and release properties.

Previously, we showed that the encapsulation and release properties of a liposomal ciprofloxacin formulation could be modified post manufacture, by addition of surfactant in concert with osmotic swelling of the liposomes. This strategy may provide more flexibility and convenience than the alternative of manufacturing multiple batches of liposomes differing in composition to cover a wide range of release profiles. The goal of this study was to develop a surfactant-associated liposomal ciprofloxacin (CFI) formulation possessing good long-term stability which could be delivered as an inhaled aerosol. Preparations of 12.5 mg/ml CFI containing 0.4% polysorbate 20 were formulated between pH 4.7 and 5.5. These formulations, before and after mesh nebulization, and after refrigerated storage for up to 2 years, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. Within the narrower pH range of 4.9 to 5.2, these formulations retained their physicochemical stability after 2-year refrigerated storage, were robust to mesh nebulization, and formed respirable aerosols with a volume mean diameter (VMD) of 3.7 µm and a geometric standard deviation (GSD) of 1.7. This study demonstrates that it may be possible to provide a range of release profiles by simple addition of surfactant to a liposomal formulation post manufacture, and that these formulations may retain their physicochemical properties after long-term refrigerated storage and following aerosolization by mesh nebulizer.

Pharmacologic inhibition of dipeptidyl peptidase 1 (cathepsin C) does not block in vitro granzyme-mediated target cell killing by CD8 T or NK cells.

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1), also known as cathepsin C (CatC), can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs), including neutrophil elastase, proteinase 3, and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore, it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein, we demonstrate that the processing of human granzymes A and B, transitioning from zymogen to active proteases, is not solely dependent on DPP1. Thus, the killing of target cells by primary human CD8+ T cells, NK cells, and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation, the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast, preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall, our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover, the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors, such as brensocatib, are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities.

Inhaled, dual release liposomal ciprofloxacin in non-cystic fibrosis bronchiectasis (ORBIT-2): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: The delivery of antipseudomonal antibiotics by inhalation to Pseudomonas aeruginosa-infected subjects with non-cystic fibrosis (CF) bronchiectasis is a logical extension of treatment strategies successfully developed in CF bronchiectasis. Dual release ciprofloxacin for inhalation (DRCFI) contains liposomal ciprofloxacin, formulated to optimise airway antibiotic delivery. METHODS: Phase II, 24-week Australian/New Zealand multicentre, randomised, double-blind, placebo-controlled trial in 42 adult bronchiectasis subjects with ≥2 pulmonary exacerbations in the prior 12 months and ciprofloxacin-sensitive P aeruginosa at screening. Subjects received DRCFI or placebo in three treatment cycles of 28 days on/28 days off. The primary outcome was change in sputum P aeruginosa bacterial density to the end of treatment cycle 1 (day 28), analysed by modified intention to treat (mITT). Key secondary outcomes included safety and time to first pulmonary exacerbation-after reaching the pulmonary exacerbation endpoint subjects discontinued study drug although remained in the study. RESULTS: DRCFI resulted in a mean (SD) 4.2 (3.7) log10 CFU/g reduction in P aeruginosa bacterial density at day 28 (vs -0.08 (3.8) with placebo, p=0.002). DRCFI treatment delayed time to first pulmonary exacerbation (median 134 vs 58 days, p=0.057 mITT, p=0.046 per protocol). DRCFI was well tolerated with a similar incidence of systemic adverse events to the placebo group, but fewer pulmonary adverse events. CONCLUSIONS: Once-daily inhaled DRCFI demonstrated potent antipseudomonal microbiological efficacy in adults with non-CF bronchiectasis and ciprofloxacin-sensitive P aeruginosa. In this modest-sized phase II study, DRCFI was also well tolerated and delayed time to first pulmonary exacerbation in the per protocol population.

Brensocatib (an oral, reversible inhibitor of dipeptidyl peptidase-1) attenuates disease progression in two animal models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a painful and incurable disease characterized by chronic joint inflammation and a progressive destruction of cartilage and bone. Although current treatments have improved clinical outcomes for some patients, the high relapse rates and sizeable proportion of non-responders emphasize the need for further research. Arthritic joints are massively infiltrated by neutrophils, which influence inflammatory and immune processes by releasing cytokines, chemokines, eicosanoids, and neutrophil serine proteases (NSPs) - all of which are known to contribute to RA initiation and progression. Active NSPs are generated from zymogens at the promyelocytic stage of neutrophil differentiation under the action of dipeptidyl peptidase 1 (DPP-1) and DPP-1 knockout mice are resistant to the development of arthritis. Thus, DPP-1 inhibition represents a promising therapeutic approach in RA. In this study, we assessed the efficacy of a potent and highly selective DPP-1 inhibitor, brensocatib, in two well established RA models - rat collagen-induced arthritis (CIA) and mouse collagen antibody-induced arthritis (CAIA). In both models, brensocatib at 3 and 30 mg/kg/day significantly reduced bone marrow NSP levels, in keeping with prior pharmacodynamic studies in rodents. More importantly, brensocatib treatment significantly improved disease score at both dosages in both rodent models. In the mouse CAIA model, brensocatib even proved at least as potent as anti-TNF antibodies in diminishing both the histopathological score and neutrophil infiltration into arthritic joints. Together, these results show that brensocatib alters RA disease progression in rodents and supports the need for its further evaluation as a potential therapeutic option, or to complement existing RA treatments.

Effect of Inhalation Profile on Delivery of Treprostinil Palmitil Inhalation Powder.

Treprostinil palmitil (TP), a prodrug of treprostinil, is being developed as an inhalation powder (TPIP) for the treatment of patients with pulmonary arterial hypertension (PAH) and pulmonary hypertension due to interstitial lung disease (PH-ILD). In ongoing human clinical trials, TPIP is administered via a commercially available high resistance (HR) RS01 capsule-based dry powder inhaler (DPI) device manufactured by Berry Global (formerly Plastiape), which utilizes the patient's inspiratory flow to provide the required energy to deagglomerate and disperse the powder for delivery to their lungs. In this study, we characterized the aerosol performance of TPIP in response to changes in inhalation profiles to model more realistic use scenarios, i.e., for reduced inspiratory volumes and with inhalation acceleration rates that differ from those described in the compendia. The emitted dose of TP for all combinations of inhalation profiles and volumes ranged narrowly between 79 and 89% for the 16 and 32 mg TPIP capsules at the 60 LPM inspiratory flow rate but was reduced to 72-76% for the 16 mg TPIP capsule under the scenarios at the 30 LPM peak inspiratory flow rate. There were no meaningful differences in the fine particle dose (FPD) at all conditions at 60 LPM with the 4 L inhalation volume. The FPD values for the 16 mg TPIP capsule ranged narrowly between 60 and 65% of the loaded dose for all inhalation ramp rates with a 4 L volume and at both extremes of ramp rates for inhalation volumes down to 1 L, while the FPD values for the 32 mg TPIP capsule ranged between 53 and 65% of the loaded dose for all inhalation ramp rates with a 4 L volume and at both extremes of ramp rates for inhalation volumes down to 1 L for the 60 LPM flow rate. At the 30 LPM peak flow rate, the FPD values for the 16 mg TPIP capsule ranged narrowly between 54 and 58% of the loaded dose at both extremes of the ramp rates for inhalation volumes down to 1 L. Based on these in vitro findings, the TPIP delivery system appears not to be affected by the changes in inspiratory flow profiles or inspiratory volumes that might be expected to occur in patients with PAH or PH associated with underlying lung conditions such as ILD.