Preclinical Investigation of a Lipoglycopeptide Dry Powder Inhalation Therapy for the Treatment of Pulmonary MRSA Infection.

The increased prevalence of pulmonary methicillin-resistant Staphylococcus aureus (MRSA) infection in patients living with cystic fibrosis (CF) is concerning due to a correlation with reduced life expectancy and lack of available treatment options. RV94 is a next generation lipoglycopeptide designed for pulmonary delivery that preclinically demonstrated high potency against MRSA in planktonic and protected colonies and improved pulmonary clearance relative to same class molecules. Here, RV94 was formulated into a dry powder for inhalation (DPI) to investigate the localized treatment of pulmonary MRSA presented in a potentially more convenient dosage form. RV94 DPI was generated using a spray-drying process with 12.5 wt% trileucine and demonstrated aerosol characteristics (2.0 µm MMAD and 73% FPF) predictive of efficient pulmonary deposition. In vivo PK from a single dose of RV94 DPI delivered by inhalation to rats yielded lung levels (127 µg/g) much greater than the MRSA minimum inhibitory concentration (0.063 µg/mL), low systemic levels (0.1 µg/mL), and a lung t1/2 equal to 3.5 days. In a rat acute pulmonary MRSA model, a single dose of RV94 DPI delivered by inhalation either up to seven days prior to or 24 h after infection resulted in a statistically significant reduction in lung MRSA titer.

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.

The pharmacokinetic profile of brensocatib and its effect on pharmacodynamic biomarkers including NE, PR3, and CatG in various rodent species.

Brensocatib is a novel, oral, selective, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), which activates several neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) in the bone marrow during the early stage of neutrophil maturation. These NSPs are associated with pathogen destruction and inflammatory mediation; their dysregulated activation can result in excess secretion of active NSPs causing damaging inflammation and contributing to neutrophil-mediated inflammatory and autoimmune diseases. Pharmacological inhibition of DPP1 in the bone marrow could therefore represent an attractive strategy for these neutrophil-driven diseases. A completed Phase 2 trial in non-cystic fibrosis bronchiectasis patients (ClinicalTrials.gov number NCT03218917; EudraCT number: 2017-002533-32) indeed demonstrated that administration of brensocatib attenuated the damaging effects of chronic inflammation by inhibiting the downstream activation of NSPs. To support a range of preclinical programs and further understand how rodent species and strains may affect brensocatib's pharmacokinetic (PK) profile and its pharmacodynamic (PD) effects on NE, PR3, and CatG, an extensive naïve dosing study with brensocatib at different dosing levels, frequencies, and durations was undertaken. Dose-dependent PK exposure responses (AUC and Cmax) were observed regardless of the rodent species and strain. Overall, mice showed greater reduction in NSP activities compared to rats. Both mice and rats dosed once daily (QD) had equivalent NSP activity reduction compared to BID (twice a day) dosing when the QD dose was 1.5-times the BID daily dose. For both mouse strains, CatG activity was reduced the most, followed by NE, then PR3; whereas, for both rat strains, PR3 activity was reduced the most, followed by CatG, and then NE. Maximum reduction in NSP activities was observed after ∼7 days and recoveries were nearly symmetrical. These results may facilitate future in vivo brensocatib study dosing considerations, such as the timing of prophylactic or therapeutic administration, choice of species, dosage and dosing frequency.

Brensocatib, an oral, reversible inhibitor of dipeptidyl peptidase 1, mitigates interferon-α-accelerated lupus nephritis in mice.

Neutrophils have been implicated in initiating and perpetuating systemic lupus erythematosus and the resultant kidney damage in lupus nephritis (LN) patients, in part through an excessive release of neutrophil serine proteases (NSPs). NSP zymogens are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation and released by mature neutrophils in response to inflammatory stimuli. Thus, a potential strategy to attenuate disease progression in LN would be to inhibit DPP1. We tested whether brensocatib, a highly selective and reversible DPP1 inhibitor, could mitigate LN progression in an interferon-alpha (IFNα)-accelerated NZB/W F1 mouse model. To confirm brensocatib's pharmacodynamic effect on NSPs in this mouse strain, repeated dose studies were conducted for 7 and 14 days in naïve NZB/W F1 mice via oral gavage twice a day. Brensocatib at 2 and 20 mg/kg/day achieved a significant reduction in bone marrow NSP activities after 7 days of daily administration. To initiate LN disease progression, the mice were injected with an IFNα-expressing adenovirus. After 2 weeks, three brensocatib doses (or vehicle) were administered for 6 more weeks. Throughout the 8-week study, brensocatib treatment (20 mg/kg/day) significantly reduced the occurrence of severe proteinuria compared to the vehicle control. Brensocatib treatment also entailed a significant reduction in the urine albumin-to-creatinine ratio, indicating decreased kidney damage, as well as a significant reduction in blood urea nitrogen level, suggesting improved renal function. Based on kidney histopathology analysis, brensocatib treatment significantly lowered both the renal tubular protein score and the nephropathy score compared to the vehicle group. A trend towards reduced glomerulonephritis score with brensocatib treatment was also observed. Lastly, brensocatib significantly reduced LN mouse kidney infiltration by various inflammatory cells. In conclusion, these results suggest that brensocatib alters disease progression in LN mice and warrant further evaluation of DPP1 inhibition in LN.

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.

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.

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.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PURPOSE: Neutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study. METHODS: Two NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3. RESULTS AND DISCUSSION: The zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method. CONCLUSION: The NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Strategies to Overcome Biological Barriers Associated with Pulmonary Drug Delivery.

While the inhalation route has been used for millennia for pharmacologic effect, the biological barriers to treating lung disease created real challenges for the pharmaceutical industry until sophisticated device and formulation technologies emerged over the past fifty years. There are now several inhaled device technologies that enable delivery of therapeutics at high efficiency to the lung and avoid excessive deposition in the oropharyngeal region. Chemistry and formulation technologies have also emerged to prolong retention of drug at the active site by overcoming degradation and clearance mechanisms, or by reducing the rate of systemic absorption. These technologies have also been utilized to improve tolerability or to facilitate uptake within cells when there are intracellular targets. This paper describes the biological barriers and provides recent examples utilizing formulation technologies or drug chemistry modifications to overcome those barriers.

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.

Treprostinil palmitil inhibits the hemodynamic and histopathological changes in the pulmonary vasculature and heart in an animal model of pulmonary arterial hypertension.

Treprostinil palmitil (TP) is a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE). In this study, TP was delivered by inhalation (treprostinil palmitil inhalation suspension, TPIS) in a rat Sugen 5416 (Su)/hypoxia (Hx) model of pulmonary arterial hypertension (PAH) to evaluate its effects on hemodynamics, pulmonary vascular remodeling, and cardiac performance and histopathology. Male Sprague-Dawley rats received Su (20 mg/kg, s.c), three weeks of Hx (10% O2) and 5 or 10 weeks of normoxia (Nx). TPIS was given during the 5-10 week Nx period after the Su/Hx challenge. Su/Hx increased the mean pulmonary arterial blood pressure (mPAP) and right heart size (Fulton index), reduced cardiac output (CO), stroke volume (SV) and heart rate (HR), and increased the thickness and muscularization of the pulmonary arteries along with obliteration of small pulmonary vessels. In both the 8- and 13-week experiments, TPIS at inhaled doses ranging from 39.6 to 134.1 µg/kg, QD, dose-dependently improved pulmonary vascular hemodynamics, reduced the increase in right heart size, enhanced cardiac performance, and attenuated most of the histological changes induced by the Su/Hx challenge. The PDE5 inhibitor sildenafil, administered at an oral dose of 50 mg/kg, BID for 10 weeks, was not as effective as TPIS. These results in Su/Hx challenged rats demonstrate that inhaled TPIS may have superior effects to oral sildenafil. We speculate that the improvement of the pathobiology in this PAH model induced by TPIS involves effects on pulmonary vascular remodeling due to the local effects of TRE in the lungs.

Robustness of aerosol delivery of amikacin liposome inhalation suspension using the eFlow® Technology.

The purpose of these studies was to understand the effect on product performance of batch-to-batch variability in both the amikacin liposome inhalation suspension (ALIS) formulation and its delivery device, the Lamira® nebulizer system, designed and manufactured by PARI (PARI Pharma GmbH, Munich, Germany). Three batches of ALIS spanning a range of lipid concentrations (43, 48 and 54 mg/mL) were tested with nine PARI inhalation devices that varied within the production process of the vibrating membrane with respect to hole geometry. Three hole geometry clusters were built including a geometry close to the mean geometry (median) and two geometries deviating from the mean geometry with smaller (smaller) and larger (larger) holes. The output parameters included the nebulization rate, the aerosol droplet size distribution, the liposome vesicle size post-nebulization, and the fraction of amikacin that remained encapsulated post-nebulization. Across the 27 experimental combinations of three formulation batches and nine devices, the nebulization time varied between 12 and 15 min with the fastest nebulization rate occurring with the combination of low lipid concentration and larger hole geometry (0.68 g/min) and the slowest nebulization rate occurring with the combination of high lipid concentration and the smaller hole geometry (0.59 g/min). The mean liposome vesicle size post-nebulization ranged from 269 to 296 nm across all experimental combinations which was unchanged from the control samples (276-292 nm). While all three batches contained > 99% encapsulated amikacin prior to nebulization, the nebulization process resulted in a consistent generation of ~ 35% unencapsulated amikacin (range: 33.8% to 37.6%). There was no statistically significant difference in the generated aerosol particle size distributions. The mass median aerodynamic diameters (MMAD) ranged from 4.78 µm to 4.98 µm, the geometric standard deviations (GSD) ranged from 1.61 to 1.66, and the aerosol fine particle fraction (FPF < 5 µm) ranged from 50.3 to 53.5%. The emitted dose (ED) of amikacin ranged from 473 to 523 mg (80.2 to 89.3% of loaded dose (LD)) and the fine particle dose (FPD < 5 µm) ranged from 244 to 278 mg (41.4 to 47.1% of label claim (LC)). In conclusion, while variations in the lipid concentration of the ALIS formulation and the device hole geometry had a small but significant impact on nebulization time, the critical aerosol performance parameters were maintained and remained within acceptable limits.

Storage stability of inhalable, controlled-release powder formulations of ciprofloxacin nanocrystal-containing liposomes.

Novel inhalable and controlled release powder formulations of ciprofloxacin nanocrystals inside liposomes (CNL) were recently developed. In the present study, the storage stability of CNL powders consisting of lyoprotectant (i.e. sucrose or lactose), lipids, ciprofloxacin (CIP), and magnesium stearate or isoleucine was investigated. These powders were produced by spray drying, collected in a dry box at <15% relative humidity (RH), then stored at room temperature and either 4 or 20 %RH. Liposomal integrity, CIP encapsulation efficiency (EE), in vitro drug release (IVR), aerosol performance, and solid-state properties were examined over six months. Sucrose CNL powder exhibited consistent liposomal integrity, aerosol performance, and controlled release of CIP over six months of storage at 4 %RH. However, storage of the powder at 20 %RH for the same period caused sucrose crystallization and consequently a significant drop in EE and aerosol performance (p-values < 0.05), along with the IVR of CIP becoming similar to that of the non-crystalline CIP liposomal dispersions (f2 > 50). Lactose CNL maintained superior aerosol performance over the six months irrespective of the storage RH. However, liposomal instability occurred at both RHs within the first month of storage with a significant drop in EE and an increase in liposome size (p-values < 0.05). Moreover, the IVR assay of CIP from lactose CNL showed a less controlled release and a substantial difference (f2 < 50) from its initial value after six months regardless of the storage RHs. In conclusion, dry powder inhalers of CNL were physiochemically stable in sucrose lyoprotectant when stored below 4 %RH at room temperature for six months.

Recent advances in prodrug-based nanoparticle therapeutics.

Extensive research into prodrug modification of active pharmaceutical ingredients and nanoparticle drug delivery systems has led to unprecedented levels of control over the pharmacological properties of drugs and resulted in the approval of many prodrug or nanoparticle-based therapies. In recent years, the combination of these two strategies into prodrug-based nanoparticle drug delivery systems (PNDDS) has been explored as a way to further advance nanomedicine and identify novel therapies for difficult-to-treat indications. Many of the PNDDS currently in the clinical development pipeline are expected to enter the market in the coming years, making the rapidly evolving field of PNDDS highly relevant to pharmaceutical scientists. This review paper is intended to introduce PNDDS to the novice reader while also updating those working in the field with a comprehensive summary of recent efforts. To that end, first, an overview of FDA-approved prodrugs is provided to familiarize the reader with their advantages over traditional small molecule drugs and to describe the chemistries that can be used to create them. Because this article is part of a themed issue on nanoparticles, only a brief introduction to nanoparticle-based drug delivery systems is provided summarizing their successful application and unfulfilled opportunities. Finally, the review's centerpiece is a detailed discussion of rationally designed PNDDS formulations in development that successfully leverage the strengths of prodrug and nanoparticle approaches to yield highly effective therapeutic options for the treatment of many diseases.

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.

Modeling of a spray drying method to produce ciprofloxacin nanocrystals inside the liposomes utilizing a response surface methodology: Box-Behnken experimental design.

Spray drying was previously used to modify the physical form of the encapsulated ciprofloxacin drug to produce ciprofloxacin nanocrystals inside the liposomes (CNL). The purpose of the present study was to optimize CNL powder production by evaluating the response surface via design of experiments (DoE). Using the Box-Behnken (BB) design, the study independent variables were the protectant type (sucrose, trehalose or lactose), protectant amount, drying temperature, and spray gas flow. Individual spray drying experiments were performed at various set points for each variable followed by characterization of the produced powders. Liposomal particle size, drug encapsulation efficiency (EE%), liposomal surface zeta potential, and nanocrystal dimensions were the design dependant variables. By applying the least square regression method on the experimental data, mathematical models were developed using the mathematical software package MATLAB R2018b. Model reliability and the significance of the model's factors were estimated using analysis of variance (ANOVA). The generated CNL powders showed spherical to elliptical liposomal vesicles with particle sizes ranging from 98 to 159 nm. The EE (%) ranged from 30 to 95% w/w while the zeta potential varied between -3.5 and -10.5 mV. The encapsulated ciprofloxacin nanocrystals were elongated cylindrical structures with an aspect ratio of 4.0-7.8. Coefficients of determination (R2 > 0.9) revealed a good agreement between the predicted and experimental values for all responses except for the nanocrystal dimensions. Sucrose and lactose were superior to trehalose in protecting the liposomes during spray drying. The amount of sugar significantly affected the characteristics of the CNL powders (p-value < 0.05). In conclusion, the DoE approach using BB design has efficiently modelled the generation of CNL by spray drying. The optimum processing conditions which produced high drug encapsulation (90%) after formation of nanocrystals and a vesicle size of ~125 nm utilized 57% (w/w) sucrose, an 80 °C inlet temperature, and an atomization rate of 742 L/hr.

Characterisation of cough evoked by inhaled treprostinil and treprostinil palmitil.

Cough is induced by inhaled prostacyclin analogues including treprostinil (TRE), and, at higher doses, treprostinil palmitil (TP), a prodrug of TRE. In this report, we have investigated mechanisms involved in TRE- and TP-induced cough, using a dry powder formulation of TP (TPIP) to supplement previous data obtained with an aqueous suspension formulation of TP (TPIS). Experiments in guinea pigs and rats investigated the prostanoid receptor subtype producing cough and whether it involved activation of sensory nerves in the airways and vasculature. Experiments involved treatment with prostanoid, tachykinin and bradykinin receptor antagonists, a cyclooxygenase inhibitor and TRE administration to the isolated larynx or intravenously. In guinea pigs, cough with inhaled TRE (1.23 µg·kg-1) was not observed with an equivalent dose of TPIP and required higher inhaled doses (12.8 and 35.8 µg·kg-1) to induce cough. TRE cough was blocked with IP and tachykinin NK1 receptor antagonists but not with EP1, EP2, EP3, DP1 or bradykinin B2 antagonists or a cyclooxygenase inhibitor. TRE administered to the isolated larynx or intravenously in rats produced no apnoea or swallowing, whereas citric acid, capsaicin and hypertonic saline had significant effects. The mechanisms inducing cough with inhaled TRE likely involves the activation of prostanoid IP receptors on jugular C-fibres in the tracheobronchial airways. Cough induced by inhaled dry powder and nebulised formulations of TP occurs at higher inhaled doses than TRE, presumably due to the slow, sustained release of TRE from the prodrug resulting in lower concentrations of TRE at the airway sensory nerves.

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.

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.