No abstract

Short-chain fatty acids (SCFAs) have a range of effects in metabolism and immune regulation. We have observed that delivery of SCFAs to lysosomes has potent immune regulatory effects, possibly as a surrogate signal for the presence of anaerobic organisms. To better understand the pharmacology of lysosomal SCFA donors, we investigated the distribution and metabolism of propionate and butyrate donors. Each analog (1 a and 2 a) can donate three SCFA equivalents via ester hydrolysis through six intermediate metabolites. The compounds are stabilized by low pH, and stability in cells is usually higher than in medium, but is cell-type specific. Butyrate derivatives were found to be more stable than propionates. Triesters were more stable than di-or mono-esters. The donors were surprisingly stable in vivo, and hydrolysis of each position was organ specific. Jejunum and liver caused rapid loss of 4'' esters. The gut metabolite pattern by i. v. differed from that of p.o. application, suggesting luminal and apical enzyme effects in the gut epithelium. Central organs could de-esterify the 11position. Levels in lung relative to other organs were higher by p.o. than via i. v., suggesting that delivery route can influence the observed pharmacology and that gut metabolites distribute differently. The donors were largely eliminated by 24 h, following near linear decline in organs. The observed levels and distribution were found to be consistent with pharmacodynamic effects, particularly in the gut.

A new phenolic glucoside gallate, vanillic acid 4-O-beta-D-(6'-O-galloyl) glucopyranoside (1) was isolated from the bark of Terminalia macroptera Guill.et Perr., together with 3,3',4'-tri-O-methylellagic acid (2) and two triterpene glucopyranosyl esters, 24-deoxysericoside (3) and chebuloside II (4). Compounds 2-4, not described previously for this plant, showed antimicrobial activities against Bacillus subtilis, while 3 and 4 possessed haemolytic properties. In both assays 1 was found to be inactive.

Macrolide antibiotics, notably azithromycin, have clinically useful effects in a range of inflammatory diseases and especially those of the lung. Effects include a reduction of inflammatory cytokines, reductions in neutrophil infiltration and potentially a polarisation of infiltrating cells to a pro-resolution phenotype. The mode of action behind this effect is unlikely to be a single interaction and may involve reductions in prostaglandin synthesis via phospholipase inhibition, modulation of NFκB translocation, reduction in IL-8 production and reduction in reflux aspiration to the airways. While some of the clinical effects can be rationalised through antibacterial actions leading to changes in normal flora and reducing Pseudomonads in particular, there is also evidence for effects unrelated to antibacterial actions that appear to relate to reductions in neutrophil activation, potentially related to high accumulation in neutrophil lysosomes. Concerted efforts to improve on these effects have focused on either generating non-antibacterial analogues, or in conjugating anti-inflammatory drugs to the macrolide backbone. Both approaches have provided strong pre-clinical data suggesting that the selective disposition of macrolides to inflamed tissue, as well as their pleiotropic effects on immune cells, contribute to their broad anti-inflammatory effects. The more recent observations of stronger macrolide effects in the context of neutrophil-mediated disease and corresponding effects on IL-17 positive cells in tissue suggest that it may be possible to select patients likely to respond to macrolide therapy. The discovery of non-antibacterial macrolides that preserve this anti-inflammatory effect provides a means to bring these effects more broadly to the clinic without selecting for large-scale resistance to antibacterial macrolides or to other anti-infectives via cross-resistance.

Topical imiquimod based creams are indicated as immune stimulants for papillomas and various skin neoplasms. Imiquimod is considered a TLR7 ligand. These creams are also used in research to induce skin inflammation in mice as a model for psoriasis. We observed that this inflammatory response was not strictly imiquimod dependent and we set out to establish which components drive the proinflammatory effects. To this end, we examined the induction response in a BALB/cJRj mouse model, in which 50 mg of cream is applied to 2 cm 2 of skin (125 mg/kg imiquimod -5% W/V, and/or 625 mg/kg isostearic acid -25% W/V). Comparing cream formulations containing isostearic acid, imiquimod and the combination, we observed that isostearic acid causes skin inflammation within 2 days, whereas imiquimod requires up to 5 days for initial signs. Isostearic acid activated an inflammasome response, stimulated release of proinflammatory cytokines and upregulated the IL-23/17 axis. Animals treated with isostearic acid had enlarged livers (+40% weight), which was not observed with imiquimod alone. Imiquimod was readily metabolized and cleared from plasma and liver, but was maintained at high levels in the skin throughout the body (200 mM at area of application; 200 µM in untreated skin). Imiquimod application was associated with splenomegaly, cytokine induction/release and initial body weight loss over 3 days. Despite high imiquimod skin levels throughout the animal, inflammation was only apparent in the treated areas and was less severe than in isostearic acid groups. As the concentrations in these areas are well above the 10 µM required for TLR7 responses in vitro, there is an implication that skin inflammation following imiquimod is due to effects other than TLR7 agonism (e.g., adenosine receptor agonism). In brain, isostearic caused no major changes in cytokine expression while imiquimod alone sightly stimulated expression of IL-1β and CCL9. However, the combination of both caused brain induction of CCL3, -9, CXCL10, -13, IL-1β and TNFα. The implication of these data is that isostearic acid facilitates the entry of imiquimod or peripherally secreted cytokines into the brain. Our data suggest that psoriaform skin responses in mice are more driven by isostearic acid, than generally reported and that the dose and route used in the model, leads to profound systemic effects, which may complicate the interpretation of drug effects in this model.