Excretion of Urinary Metabolites of the Phthalate Esters DEP and DEHP in 16 Volunteers after Inhalation and Dermal Exposure.

Phthalate esters are suspected endocrine disruptors that are found in a wide range of applications. The aim of this study was to determine the excretion of urinary metabolites in 16 individuals after inhalation and/or dermal exposure to 100⁻300 µg/m³ of deuterium-labelled diethyl phthalate (D4-DEP) and bis(2-ethylhexyl) phthalate (D4-DEHP). Dermal exposure in this study represents a case with clean clothing acting as a barrier. After inhalation, D4-DEP and D4-DEHP metabolites were excreted rapidly, though inter-individual variation was high. D4-DEP excretion peaked 3.3 h (T½ of 2.1 h) after combined inhalation and dermal exposure, with total excreted metabolite levels ranging from 0.055 to 2.351 nmol/nmol/m³ (nmol of urinary metabolites per phthalates air concentration in (nmol/m³)). After dermal exposure to D4-DEP, metabolite excretion peaked 4.6 h (T½ of 2.7 h) after exposure, with excreted metabolite levels in between 0.017 and 0.223 nmol/nmol/m³. After combined inhalation and dermal exposure to D4-DEHP, the excretion of all five analysed metabolites peaked after 4.7 h on average (T½ of 4.8 h), and metabolite levels ranged from 0.072 to 1.105 nmol/nmol/m³ between participants. No dermal uptake of particle phase D4-DEHP was observed. In conclusion, the average excreted levels of metabolites after combined inhalation and dermal exposure to D4-DEP was three times higher than after combined exposure to D4-DEHP; and nine times higher than after dermal exposure of D4-DEP. This study was made possible due to the use of novel approaches, i.e., the use of labelled phthalate esters to avoid the background concentration, and innovative technique of phthalate generation, both in the particle and the gas phase.

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Lipid Peroxide-Mediated Oxidative Rearrangement of the Pyrazinone Carboxamide Core of Neutrophil Elastase Inhibitor AZD9819 in Blood Plasma Samples.

This study focused on the mechanistic interpretation of ex vivo oxidation of a candidate drug in blood plasma samples. An unexpected lipid peroxide-mediated epoxidation followed by a dramatic rearrangement led to production of a five-membered oxazole derivative from the original six-membered pyrazinone-carboxamide core of a human neutrophil elastase inhibitor, 6-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-N-ethyl-5-methyl-3-oxo-4-(3-(trifluoromethyl)phenyl)-3,4-dihydropyrazine-2-carboxamide (AZD9819). The rearranged oxidation product 2-(1-(4-cyanophenyl)-1H-pyrazol-5-yl)-5-(N-ethylacetamido)-N-(3-(trifluoromethyl)phenyl)oxazole-4-carboxamide was characterized by accurate-mass tandem mass spectrometry fragmentations, by two-dimensional NMR and X-ray crystallography of an authentic standard, and by incorporation of an (18)O atom from molecular (18)O2 to the location predicted by our proposed mechanism. The lipid peroxide-mediated oxidation was demonstrated by using human low-density lipoprotein (LDL) in pH 7.4 phosphate buffer and by inhibiting the oxidation with ascorbic acid or l-glutathione, two antioxidants effective in both plasma and the LDL incubation. A nucleophilic mechanism for the epoxidation of AZD9819 by lipid hydroperoxides explains the prevention of its ex vivo oxidation by acidification of the plasma samples. The discovery of the lipid peroxide-dependent oxidation of an analyte and the means of prevention could provide valuable information for biotransformation and bioanalysis.

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine.

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.

The streptococcal Blr and Slr proteins define a family of surface proteins with leucine-rich repeats: camouflaging by other surface structures.

Regions with tandemly arranged leucine-rich repeats (LRRs) have been found in many prokaryotic and eukaryotic proteins, in which they provide a remarkably versatile framework for the formation of ligand-binding sites. Bacterial LRR proteins include the recently described Slr protein of Streptococcus pyogenes, which is related to internalin A of Listeria monocytogenes. Here, we show that strains of the human pathogen Streptococcus agalactiae express a protein, designated Blr, which together with Slr defines a family of internalin A-related streptococcal LRR proteins. Analysis with specific antibodies demonstrated that Blr is largely inaccessible on S. agalactiae grown in vitro, but surface exposure was increased approximately 100-fold on mutants lacking polysaccharide capsule. In S. pyogenes, surface exposure of Slr was not affected in a mutant lacking hyaluronic acid capsule but was increased >20-fold in mutants lacking M protein or protein F. Thus, both Blr and Slr are efficiently camouflaged by other surface structures on bacteria grown in vitro. When Blr and Slr exposed on the bacterial surface were compared, they exhibited only little immunological cross-reactivity, in spite of extensive residue identity, suggesting that their surface-exposed parts have been under evolutionary pressure to diverge functionally and/or antigenically. These data identify a family of immunologically diverse streptococcal LRR proteins that show unexpected complexity in their interactions with other bacterial surface components.