Surfactant-Assisted Distal Pulmonary Distribution of Budesonide Revealed by Mass Spectrometry Imaging.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle (n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa (n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs (n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

Identification of the Nicotinamide Salvage Pathway as a New Toxification Route for Antimetabolites.

Interest in the modulation of nicotinamide adenine dinucleotide (NAD) metabolome is gaining great momentum because of its therapeutic potential in different human disorders. Suppression of nicotinamide salvage by nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, however, gave inconclusive results in neoplastic patients because several metabolic routes circumvent the enzymatic block converging directly on nicotinamide mononucleotide adenylyl transferases (NMNATs) for NAD synthesis. Unfortunately, NMNAT inhibitors have not been identified. Here, we report the identification of Vacor as a substrate metabolized by the consecutive action of NAMPT and NMNAT2 into the NAD analog Vacor adenine dinucleotide (VAD). This leads to inhibition of both enzymes, as well as NAD-dependent dehydrogenases, thereby causing unprecedented rapid NAD depletion, glycolytic block, energy failure, and necrotic death of NMNAT2-proficient cancer cells. Conversely, lack of NMNAT2 expression confers complete resistance to Vacor. Remarkably, Vacor prompts VAD formation and growth suppression in NMNAT2-positive neuroblastoma and melanoma xenografts. Our data show the first evidence of harnessing the entire nicotinamide salvage pathway for antimetabolic strategies.

Dexpramipexole improves bioenergetics and outcome in experimental stroke.

BACKGROUND AND PURPOSE: Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury. EXPERIMENTAL APPROACH: The effects of dexpramipexole on bioenergetics, Ca2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging. KEY RESULTS: Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro. CONCLUSION AND IMPLICATIONS: Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Electrospray MS and MALDI imaging show that non-specific lipid-transfer proteins (LTPs) in tomato are present as several isoforms and are concentrated in seeds.

Non-specific lipid-transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut-off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC-MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato.