Small vessel vasculitis is a life-threatening condition and patients typically present with renal and pulmonary injury. Disease pathogenesis is associated with neutrophil accumulation, activation, and oxidative damage, the latter being driven in large part by myeloperoxidase (MPO), which generates hypochlorous acid among other oxidants. MPO has been associated with vasculitis, disseminated vascular inflammation typically involving pulmonary and renal microvasculature and often resulting in critical consequences. MPO contributes to vascular injury by 1) catabolizing nitric oxide, impairing vasomotor function; 2) causing oxidative damage to lipoproteins and endothelial cells, leading to atherosclerosis; and 3) stimulating formation of neutrophil extracellular traps, resulting in vessel occlusion and thrombosis. Here we report a selective 2-thiouracil mechanism-based MPO inhibitor (PF-1355 [2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) and demonstrate that MPO is a critical mediator of vasculitis in mouse disease models. A pharmacokinetic/pharmacodynamic response model of PF-1355 exposure in relation with MPO activity was derived from mouse peritonitis. The contribution of MPO activity to vasculitis was then examined in an immune complex model of pulmonary disease. Oral administration of PF-1355 reduced plasma MPO activity, vascular edema, neutrophil recruitment, and elevated circulating cytokines. In a model of anti-glomerular basement membrane disease, formerly known as Goodpasture disease, albuminuria and chronic renal dysfunction were completely suppressed by PF-1355 treatment. This study shows that MPO activity is critical in driving immune complex vasculitis and provides confidence in testing the hypothesis that MPO inhibition will provide benefit in treating human vasculitic diseases.
ABSTRACT:The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PB-MEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r 2 of 0.60.Removal of the three system L substrates increased the r 2 to 0.89.In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.One major hurdle of successful central nervous system drug delivery is to penetrate the blood-brain barrier (BBB) to reach the therapeutic targets. The BBB is a continuous layer of endothelial cells that are connected to each other through tight junctions. In contrast to the endothelial cells of peripheral blood vessels, the brain microvessel endothelial cells are characterized by unique intercellular tight junctions, the absence of fenestrations, and minimal pinocytic activity. The BBB represents a physiological barrier that efficiently restricts free paracellular passage of most substances from the blood to the brain extracellular environment. Furthermore, the brain microvessel endothelial cells possess a variety of metabolic enzyme systems, which further limit the brain entry of compounds (Pardridge, 1983). Finally, a complex of membrane-bound transport systems, including active efflux transporters, such as P-glycoprotein (Pgp) (Schinkel et al., 1996;Miller et al., 2000) and multidrug resistanceassociated protein (MRP) Zhang et al., 2000), and active uptake transporters, such as the system L amino acid transporter (LAT) (Pardridge, 1983;Smith, 1991), further regulates brain penetration.Pharmaceutical companies have been actively pursuing various methods to accurately predict the brain penetrati...
Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin.
For general toxicity studies, a technique was designed to consistently sample the most important neuroanatomic regions of the brain, spinal cord, and peripheral nerve of cynomolgus monkeys using a limited number of blocks and slides. Using the most rostral portion of the pons as a landmark, the entire fixed brain was cut dorsoventrally into cross-sectional slabs 4 mm in thickness. For microscopic evaluation, six blocks of the brain at the levels of the frontal pole, anterior commissure, rostral thalamus, caudal thalamus, middle cerebellum with brainstem, and occipital lobe were trimmed to fit in standard tissue cassettes. Cross- and oblique sections of the spinal cord including the dorsal root ganglion and dorsal and ventral nerve roots were obtained at the levels of C1-C4, T10-T12, and L1-L4. Cross- and longitudinal sections of the sciatic nerve were also obtained. This technique offers a consistent and reliable method to routinely sample most of the important regions of the central and peripheral nervous system of monkeys using ten blocks. This method is readily adaptable to other species of nonhuman primates, dogs, and minipigs and can be quickly learned by the technicians performing the trimming procedures.
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