Differences in the coronal proteome acquired by particles depositing in the lungs of asthmatic versus healthy humans.

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

PURPOSE: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383. METHODS: For all drugs, uptake at 5 µM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 µM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated. RESULTS: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics. CONCLUSIONS: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

The discovery of long-acting saligenin ß2 adrenergic receptor agonists incorporating a urea group.

A series of novel, potent and selective human ß(2) adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.

The discovery of long-acting saligenin ß2 adrenergic receptor agonists incorporating hydantoin or uracil rings.

A series of novel, potent and selective human ß(2) adrenoceptor agonists incorporating a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long duration of action in vitro on guinea pig trachea, and 12h in guinea pigs in vivo at its EC(90) 25 µM. It had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in both rats and dogs (2% and 5%, respectively). An improved method for measuring the absorbed fraction of analogues dosed to rats, which considers the glucuronidated fraction is presented. Compound 12a was metabolised in human liver microsomes and hepatocytes to the active hydantoic acid 12m.

Challenges in inhaled product development and opportunities for open innovation.

Dosimetry, safety and the efficacy of drugs in the lungs are critical factors in the development of inhaled medicines. This article considers the challenges in each of these areas with reference to current industry practices for developing inhaled products, and suggests collaborative scientific approaches to address these challenges. The portfolio of molecules requiring delivery by inhalation has expanded rapidly to include novel drugs for lung disease, combination therapies, biopharmaceuticals and candidates for systemic delivery via the lung. For these drugs to be developed as inhaled medicines, a better understanding of their fate in the lungs and how this might be modified is required. Harmonized approaches based on 'best practice' are advocated for dosimetry and safety studies; this would provide coherent data to help product developers and regulatory agencies differentiate new inhaled drug products. To date, there are limited reports describing full temporal relationships between pharmacokinetic (PK) and pharmacodynamic (PD) measurements. A better understanding of pulmonary PK and PK/PD relationships would help mitigate the risk of not engaging successfully or persistently with the drug target as well as identifying the potential for drug accumulation in the lung or excessive systemic exposure. Recommendations are made for (i) better industry-academia-regulatory co-operation, (ii) sharing of pre-competitive data, and (iii) open innovation through collaborative research in key topics such as lung deposition, drug solubility and dissolution in lung fluid, adaptive responses in safety studies, biomarker development and validation, the role of transporters in pulmonary drug disposition, target localisation within the lung and the determinants of local efficacy following inhaled drug administration.

An electrochemical microfluidic platform for human P450 drug metabolism profiling.

This paper is the first report of a P450-electrode in a microfluidic format. A 30 µL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron > nifedipine > quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.

Aryl aminopyrazole benzamides as oral non-steroidal selective glucocorticoid receptor agonists.

Aryl aminopyrazole amides capped with N-alkylbenzamides 13-16 are selective glucocorticoid receptor agonists. 2,6-Disubstituted benzamides have prednisolone-like potency or better in vitro. Good oral exposure was demonstrated in the rat, with compounds with lower lipophilicity, for example N-hydroxyethyl benzamides (e.g., 16e).