From bench to bedside: in vitro and in vivo evaluation of a neonate-focused nebulized surfactant delivery strategy.

BACKGROUND: Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. METHODS: Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100-600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. RESULTS: Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 µm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). CONCLUSIONS: The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016-004547-36).

Phase I metabolism of ganstigmine. Rat, dog, monkey and human liver microsomal extracts investigated by liquid chromatography electrospray tandem mass spectrometry.

Ganstigmine, a new acetylcholinesterase inhibitor, was incubated with rat liver microsomes and the resulting metabolites were identified by high-performance liquid chromatographic/mass spectrometric (HPLC/MS) and HPLC/MS/MS analyses. The results showed the formation of eight main metabolites, among which geneseroline and molecules corresponding to mono-hydroxylated, demethylated and reduced ganstigmine. The metabolic profile drawn for humans, dog and monkey showed a pattern very similar to that of rat: only in the case of liver dog microsomes higher amounts of geneseroline and of a metabolite identified as demethylated and reduced drug were detected.

Crudes of synthesis of neuropeptide Y and beta-amyloid peptide examined by liquid chromatography/electrospray tandem mass spectrometry.

The two peptides, neuropeptide Y (M(r) = 4254) and beta (1-39) amyloid (M(r) = 4230) are attracting pharmaceutical and biomedical interest for different yet equally important reasons. The first is recognized for its various physiological functions in the peripheral and central nervous system, while the second has been identified as one of the components of the cerebral amyloid deposits characteristic of Alzheimer's disease. High level purity of both peptides is considered of prime importance for correct interpretation of data obtained by biological and pharmaceutical assays and binding tests. Solid-phase synthesis of both peptides resulted in crudes of reaction containing both the target peptides as well as a number of undesired side products. The use of liquid chromatography/electrospray-tandem mass spectrometry (LC/ESI-MS/MS) furnished reliable information on the target peptides and provided sequencing information on a number of side products. Furthermore, LC/MS/MS data of doubly and triply protonated sequences yielded a number of C-terminal fragment ions exhibiting the loss of NH3 considered to be an important process for the understanding of fragmentation mechanism(s) of multiply protonated peptides.

Fast-atom bombardment mass spectrometry for mapping of endogenous methylated purine bases in urine extracts.

Fast-atom bombardment (FAB) mass spectrometry, linked with tandem mass spectrometry (MS/MS), was employed for the identification of methylated purine bases in four urinary extracts of healthy subjects and fourteen urinary extracts of patients bearing colorectal tumors. In order to obtain an easy structural identification of the species present in urinary extracts, the MS/MS spectra of MH+ species of twenty nine diagnostically relevant purine bases were studied. Even if definitive quantitative data cannot be obtained by this approach, FAB mass spectra of urine extracts lead to a readily reproducible mapping of endogenous purine bases, allowing a distinction between healthy and sick subjects. Bases such as 9-ethyladenine, N6-2-isopentenyladenine and N6-benzyladenine were detected only in urine samples of colorectal tumor bearing patients. The detection in urine of compounds such as 7-methylguanine and 1-methylguanine, and their increase in the urine of colorectal tumor bearing patients, has been justified either by a more rapid turnover of nucleic acids in tumor tissue or by an increase in the extent of their methylation. The obtained results indicate that the method can be employed for diagnostic purposes.

Matrix-assisted laser desorption/ionization mass spectrometry in milk science.

The protein content of several cow milks was investigated by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In particular, milk from four different breeds of cow (Holstein Frisons, Brown Swiss, Jersey and Reggiana) at the same lactation stage and under the same feeding system were analysed and clear differences were found in their MALDI spectra. The various lactation stages were also investigated. A series of analyses were devoted to industrial milk treatment, by studying possible thermal damage occurring during milk pasteurization and sterilization.

Matrix-assisted laser desorption/ionization mass spectrometry: a valid analytical tool in the dairy industry.

Matrix-assisted laser desorption/ionization mass spectrometry has been employed to analyse the protein content of milk samples obtained with different pasteurization and sterilization methods. The data obtained indicate that the technique can be employed as a valid analytical tool in the dairy industry, allowing the monitoring of the functioning of the treatment plant in terms of physical parameters.

An electrospray investigation on in vitro glycation of ribonuclease.

The in vitro reactions of RNase with different concentrations of glucose or fructose have been studied by means of electrospray mass spectrometry coupled with microcolumn high performance liquid chromatography. The results obtained have shown that, subsequent to the protein glycation, a series of cross-linking products are generated. Their molecular weights demonstrate that severe degradation processes of the proteic substrate takes place after the cross-linking process.

A new effective method for the evaluation of glycated intact plasma proteins in diabetic subjects.

The molecular weights of plasma proteins from healthy subjects and from patients with well-or badly-controlled diabetes mellitus have been determined by use of a matrix-assisted laser desorption ionization method, representing a highly accurate technique for the determination of the molecular weight of large biomolecules. Using this approach, different molecular weights of human serum albumin have been found for healthy (66,572-66,694 dalton) and diabetic (66,785-68,959 dalton) subjects. Such differences can be rationalized as being due to the different number of glucose molecules condensed on the protein and/or their further oxidation products; in the case of our diabetic patients this number is in the range of 1.4-14.8. The data show the high validity and specificity of the technique, which allows us to evaluate, without any protein degradation procedure, the number of glucose molecules condensed on a specific protein and ascertain the relationship of this number to the physiopathogenetic conditions of the subjects studied.

Matrix-assisted laser desorption/ionization capabilities in the study of non-enzymatic protein glycation.

Matrix-assisted laser desorption/ionization mass spectrometry has been successfully applied in the study of non-enzymatic glycation of different proteins. In the case of bovine serum albumin, glycated by in vitro experiments performed under pseudophysiological conditions, a clear increase in molecular weight is observed with respect to both glucose concentrations and incubation time. The in vitro glycation of ribonuclease with glucose and fructose shows some peculiar differences either in terms of the number of condensed sugar molecules or in terms of the reaction kinetics. The same approach, applied to plasma proteins of healthy and diabetic subjects, provides evidence for the occurrence of glycation of human serum albumin for the latter subjects.

Different mass spectrometric approaches in the identification of endogenous methylated purine bases in urine extracts.

A preliminary identification of endogenous and exogenous methylated purine bases in urinary extracts of healthy and tumor-bearing subjects has been performed using high performance liquid chromatography, and tandem mass spectrometry (MS/MS). MS/MS gave particularly fast and sensitive analyses, allowing the simultaneous and rapid determination of 26 different urinary methylated purines. Both sets of data confirm that tumor-bearing patients show changed levels of methylated purine bases.

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry.

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the protein with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show the occurrence of a steady state, corresponding to the complete saturation of all the protein sites reactive against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed glucose moieties vs. free glucose.

The lysine glycation 1. A preliminary investigation on the products arising from the reaction of protected lysine and D-glucose.

The nature of the products arising from a 10 days, sterile incubation at 37°C and pH 7.2 of a 1:1 mixture of N-α-(p-tosyl)-lysine-methylesterhydrochloride and anhydrous D-glucose was investigated by fast atom bombardment mass spectrometry and(1)H and(13)C nuclear magnetic resonance spectroscopies. Differently to the reactivity usually described on the basis of other analytical techniques, FAB mass spectrometric measurements indicate the occurrence of the reaction of protected lysine with more than one D-glucose molecule.

Characterization of the molecular species of glycerophospholipids from rabbit kidney: an alternative approach to the determination of the fatty acyl chain position by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis.

An alternative approach to identifying fatty acid chain position in the molecular species of glycerophospholipids has been studied and developed. The fatty acyl groups esterified to the glycerol backbone in isomeric glycerophosphatidyl-choline, -serine and -ethanolamine as well as glycerophosphatidic acid can be detected by the presence of a pair of anions derived from phosphatidic acid parent ions (M minus the polar head groups in glycerophospholipids), designed to be [M--polar head--R2COOH]- and [M--polar head--R2CO--H]-, produced by negative ion fast atom bombardment combined with mass-analysed ion kinetic energy analysis. Because of the significant abundance of [M--polar head--R2COOH]- anion, fatty acid chains differing by 2 Da can be distinguished by accurate measurements of the electrostatic voltage related to this ion. Three-volt differences can be evidenced. Using this approach, the molecular species of glycerophosphatidyl-choline, -serine, -ethanolamine and -inositol from rabbit kidney were characterized after the separation of both class and species by normal and reversed-phase high-performance liquid chromatography, respectively. We identified 11 arachidonoyl-containing molecular species of glycerophospholipids and the other 17 lipid molecules in this biological material. A couple of 1- alkenyl-2-arachidonoyl-sn-glycerol-3-phosphoethanolamine species, identified as plasmalogen GPE 16:0-20:4 and plasmalogen GPE 18:0-20:4, were found for the first time in rabbit kidney.