Enhanced Transferrin Receptor Expression by Proinflammatory Cytokines in Enterocytes as a Means for Local Delivery of Drugs to Inflamed Gut Mucosa

Therapeutic intervention in inflammatory bowel diseases (IBDs) is often associated with adverse effects related to drug distribution into non-diseased tissues, a situation which attracts a rational design of a targeted treatment confined to the inflamed mucosa. Upon activation of immune cells, transferrin receptor (TfR) expression increases at their surface. Because TfR is expressed in all cell types we hypothesized that its cell surface levels are regulated also in enterocytes. We, therefore, compared TfR expression in healthy and inflamed human colonic mucosa, as well as healthy and inflamed colonic mucosa of the DNBS-induced rat model. TfR expression was elevated in the colonic mucosa of IBD patients in both the basolateral and apical membranes of the enterocytes. Increased TfR expression was also observed in colonocytes of the induced colitis rats. To explore the underlying mechanism CaCo-2 cells were treated with various proinflammatory cytokines, which increased both TfR expression and transferrin cellular uptake in a mechanism that did not involve hyper proliferation. These findings were then exploited for the design of targetable carrier towards inflamed regions of the colon. Anti-TfR antibodies were conjugated to nano-liposomes. As expected, iron-starved Caco-2 cells internalized anti-TfR immunoliposomes better than controls. Ex vivo binding studies to inflamed mucosa showed that the anti-TfR immunoliposomes accumulated significantly better in the mucosa of DNBS-induced rats than the accumulation of non-specific immunoliposomes. It is concluded that targeting mucosal inflammation can be accomplished by nano-liposomes decorated with anti-TfR due to inflammation-dependent, apical, elevated expression of the receptor

Rivastigmine alleviates experimentally induced colitis in mice and rats by acting at central and peripheral sites to modulate immune responses.

The cholinergic anti-inflammatory system and α7 nicotinic receptors in macrophages have been proposed to play a role in neuroimmunomodulation and in the etiology of ulcerative colitis. We investigated the ability of a cholinesterase (ChE) inhibitor rivastigmine, to improve the pathology of ulcerative colitis by increasing the concentration of extracellular acetylcholine in the brain and periphery. In combination with carbachol (10 µM), rivastigmine (1 µM) significantly decreased the release of nitric oxide, TNF-α, IL-1β and IL-6 from lipopolysaccharide-activated RAW 264.7 macrophages and this effect was abolished by α7 nicotinic receptor blockade by bungarotoxin. Rivastigmine (1 mg/kg) but not (0.5 mg/kg), injected subcutaneously once daily in BALB/c mice with colitis induced by 4% dextran sodium sulphate (DSS), reduced the disease activity index (DAI) by 60% and damage to colon structure. Rivastigmine (1 mg/kg) also reduced myeloperoxidase activity and IL-6 by >60%, and the infiltration of CD11b expressing cells by 80%. These effects were accompanied by significantly greater ChE inhibition in cortex, brain stem, plasma and colon than that after 0.5 mg/kg. Co-administration of rivastigmine (1 mg/kg) with the muscarinic antagonist scopolamine significantly increased the number of CD11b expressing cells in the colon but did not change DAI compared to those treated with rivastigmine alone. Rivastigmine 1 and 2 mg given rectally to rats with colitis induced by rectal administration of 30 mg dintrobezene sulfonic acid (DNBS) also caused a dose related reduction in ChE activity in blood and colon, the number of ulcers and area of ulceration, levels of TNF-α and in MPO activity. The study revealed that the ChE inhibitor rivastigmine is able to reduce gastro-intestinal inflammation by actions at various sites at which it preserves ACh. These include ACh released from vagal nerve endings that activates alpha7 nicotinic receptors on circulating macrophages and in brainstem neurons

Effect of rivastigmine treatment on macroscopic parameters of acute colitis induced in rats by DNBS.

<p>DNBS = dinitrobenzene sulphonic acid. DW = distilled water. Riv = rivastigmine. Significantly different from DNBS, **p<0.01.</p

α-bungarotoxin inhibits effect of rivastigmine and carbachol on release of NO in LPS-activated macrophages.

<p>Data represents mean ± SEM of 2 independent experiments performed in eight replicates for each sample. Significantly different from LPS alone *<i>p</i><0.05, **<i>p</i><0.01; significantly different from macrophages pretreated with rivastigmine+carbachol; # <i>p</i><0.05.</p

mRNA of pro-inflammatory cytokines in colon of mice with DSS- induced colitis.

<p>Legend as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057668#pone-0057668-g003" target="_blank">Figure 3</a>. Data are mean ± SEM and represents pooled data from 9–10 mice per group. Significantly different from mice with DSS-induced colitis treated with PBS, ** <i>p</i><0.01.</p

Histopathological characterization of colon segments mice with DSS-induced colitis.

<p>Magnification×10. A: Distal colon sections of control mouse showing well organized crypts with goblet cells (1) and lamina propria (2). B: Colon of PBS-injected mouse with DSS-induced colitis showing crypt loss (1), mucosal erosions, sub-mucosal edema and massive infiltration of inflammatory cells (2). C: Colon of mouse treated with rivastigmine (0.5 mg/kg) that partially repaired the crypt dysplasia (1) but had little effect on cellular infiltrate and edema of the sub-mucosal layer (2). D: Colon of mouse treated with rivastigmine (1 mg/kg) that partially restored crypt damage (1), reduced sub-mucosal edema (2) and prevented cellular inflammatory infiltrate. E: Co administration of scopolamine (1 mg/kg) and rivastigmine (1 mg/kg) increased sub-mucosal edema and cellular infiltrate compared to that in colon of mice given rivastigmine (1 mg/kg) alone.</p

Effect of rivastigmine with and without scopolamine on CD11b+ cell infiltration in DSS-induced colitis.

<p>A: Immunofluorescence stained mouse tissue sections showing CD11b (red) cells. White arrows show cells which express with CD11b+ in colitis tissue (pink). B: Quantification of the pink CD11b+ expressing cells (ImageJ) in the samples of mice colon sections. Data represent the mean ± SEM from three slides per animal, 6 animals per group; scale bar 20 µm. Significantly different from mice with DSS-induced colitis treated with PBS, **<i>p</i><0.01; significantly different from mice treated with rivastigmine (0.5 mg/kg), # <i>p</i><0.05; significantly different from mice treated with rivastigmine (1 mg/kg), ^§<i>p</i><0.05.</p

Effect of rivastigmine treatment on macroscopic parameters of acute colitis in mice treated with DSS.

<p>DDS = dextran sulphate solution. PBS phosphate buffered saline. Riv 0.5 or 1 = mice treated with rivastigmine (0.5 or 1 mg/kg) once daily; Riv 1+ Scop 1 = mice treated with rivastigmine (1 mg/kg) once daily and scopolamine (1 mg/kg) bid; DAI = disease activity index. Significantly different from water+PBS ** p<0.01; significantly different from DSS+PBS,</p>‡<p>p<0.05.</p>‡‡<p>p<0.01; significantly different from DSS+Riv 0.5 mg,</p>#<p>p<0.05;</p>##<p>p<0.01; significantly different from DSS+Riv 1 mg/kg,</p>§<p>p<0.05;</p>§§<p>p<0.01.</p

Cholinesterase inhibition by rivastigmine in colon, plasma, and brain of DSS drinking treated mice.

<p>Rivastigmine 0.5 or 1 = mice treated with rivastigmine (0.5 mg/kg or 1 mg/kg) once daily. Data represent percent ChE inhibition induced by rivastigmine compared to that in mice treated with PBS and drinking DSS. Data represent the mean ± SEM from 10 animals. Significantly different from value in mice treated with rivastigmine (0.5 mg/kg), **<i>p</i><0.01.</p

Effect of rivastigmine on cytokines and MPO activity in colon of mice with DSS-induced colitis.

<p>PBS = phosphate buffered saline. DSS = dextran sodium sulphate. Data are mean ± SEM, Significantly different from mice treated with PBS and drinking DSS *<i>p</i><0.05, **<i>p</i><0.01.</p