5‑Aminosalicylic Acid Azo-Linked to Procainamide Acts as an Anticolitic Mutual Prodrug via Additive Inhibition of Nuclear Factor kappaB

To improve the anticolitic efficacy of 5-aminosalicylic acid (5-ASA), a colon-specific mutual prodrug of 5-ASA was designed. 5-ASA was coupled to procainamide (PA), a local anesthetic, via an azo bond to prepare 5-(4-{[2-(diethylamino)­ethyl]­carbamoyl}­phenylazo)­salicylic acid (5-ASA-azo-PA). 5-ASA-azo-PA was cleaved to 5-ASA and PA up to about 76% at 10 h in the cecal contents while remaining stable in the small intestinal contents. Oral gavage of 5-ASA-azo-PA and sulfasalazine, a colon-specific prodrug currently used in clinic, to rats showed similar efficiency in delivery of 5-ASA to the large intestine, and PA was not detectable in the blood after 5-ASA-azo-PA administration. Oral gavage of 5-ASA-azo-PA alleviated 2,4,6-trinitrobenzenesulfonic acid-induced rat colitis. Moreover, combined intracolonic treatment with 5-ASA and PA elicited an additive ameliorative effect. Furthermore, combined treatment with 5-ASA and PA additively inhibited nuclear factor-kappaB (NFκB) activity in human colon carcinoma cells and inflamed colonic tissues. Finally, 5-ASA-azo-PA administered orally was able to reduce inflammatory mediators, NFκB target gene products, in the inflamed colon. 5-ASA-azo-PA may be a colon-specific mutual prodrug acting against colitis, and the mutual anticolitic effects occurred at least partly through the cooperative inhibition of NFκB activity

Dextran-5-(4-ethoxycarbonylphenylazo)salicylic acid ester, a polymeric colon-specific prodrug releasing 5-aminosalicylic acid and benzocaine, ameliorates TNBS-induced rat colitis

<p>Local anesthetics have beneficial effects on colitis. Dextran-5-(4-ethoxycarbonylphenylazo)salicylic acid ester (Dex-5-ESA), designed as a polymeric colon-specific prodrug liberating 5-ASA and benzocaine in the large intestine, was prepared and its therapeutic activity against colitis was evaluated using a TNBS-induced rat colitis model. Dex-5-ESA liberated 5-ASA and benzocaine in the cecal contents while (bio)chemically stable in the small intestinal contents and mucosa. Oral administration of Dex-5-ESA (equivalent to 10 mg 5-ASA/kg, twice a day) alleviated colonic injury and reduced MPO activity in the inflamed colon. In parallel, pro-inflammatory mediators, COX-2, iNOS and CINC-3, elevated by TNBS-induced colitis, were substantially diminished in the inflamed colon. Dex-5-ESA was much more effective for the treatment of colitis than 5-(4-ethoxycarbonylphenylazo)salicylic acid (5-ESA) that may not deliver benzocaine to the large intestine. Our data suggest that Dex-5-ESA is a polymeric colon-specific prodrug, liberating 5-ASA and benzocaine in the target site (large intestine), probably exerting anti-colitic effects by combined action of 5-ASA and benzocaine.</p

Isolation of MRSA persisters.

<p>An MRSA overnight culture was treated with 10X MIC (20 μg/mL) gentamicin for 4 h and the titer of viable cells was determined. After the 4 h treatment with gentamicin, the culture was treated with additional antibiotics at the indicated concentrations (10X MIC) for an additional 4 h, followed by once again determining the titer of viable cells. Results are shown as means ± s.d.; n = 3. Gm: gentamicin, Cipro: ciprofloxacin, Van: vancomycin.</p

NH125 eradicates MRSA biofilms.

<p>(A) MRSA biofilms formed on 13 mm cellulose ester membranes for 24 hours were treated with 10X MIC of vancomycin (Van) or NH125 for 24 h. Survival was measured by comparing the number of viable cells in biofilms between non-treated and treated samples. (B) MRSA biofilms grown in a 96-well microtiter plate for 48 h were treated with the indicated concentration of vancomycin or NH125 for 24 h. The remaining biofilms were stained with 0.1% crystal violet dissolved with 95% ethanol and OD_{590 nm} was measured. Results are shown as means ± s.d.; n = 3.</p

Distinguishing properties of weak slice conditions

The slice condition and the more general weak slice conditions are geometric conditions on Euclidean space domains which have evolved over the last several years as a tool in various areas of analysis. This paper explores some of the ner distinctive properties of the various weak slice conditions

Minimal inhibitory concentration (MIC) against <i>S</i>. <i>aureus</i> MW2.

<p>Minimal inhibitory concentration (MIC) against <i>S</i>. <i>aureus</i> MW2.</p

Validation of SYTOX Green assay robustness.

<p>To test the robustness of the SYTOX Green assay, the Z’-factor was calculated from fluorescence intensity data from a 384-well plate where half of the wells contained 0.1% DMSO (negative control, open circles) and the remaining wells contained 10X MIC lysostaphin (A) or 10X MIC nisin (B) (positive controls, solid circles). Fluorescence was measured with an excitation wavelength of 485 nm and an emission wavelength of 525 nm after incubation in the dark for 1 h. The Z’-factors for each assay were 0.767 (A) and 0.712 (B).</p

Lysostaphin and nisin kill MRSA persisters by inducing membrane permeabilization.

<p>MRSA persisters were treated with 0.1% DMSO (A), 10X MIC lysostaphin (B), or 10X MIC nisin (C). Membrane permeabilization (open circles) was measured spectrophotometrically by monitoring the uptake of SYTOX Green (excitation wavelength of 485 nm and an emission wavelength of 525 nm). Colony forming unit counts of persisters (solid circles) were measured by serial dilution and plating on TSA plates. The data points on the x-axis are below the level of detection (2x10² CFU/mL). Results are shown as means ± s.d.; n = 3.</p

Antimicrobial activity of niclosamide and oxyclozanide against the ESKAPE pathogens.

<p>Disc diffusion assay to detect zone of growth inhibition due to bactericidal activity of niclosamide and oxyclozanide against <i>E</i>. <i>faecium</i>, <i>S</i>. <i>aureus</i> methicillin resistant strain (MW2), <i>K</i>. <i>pneumoniae</i>, <i>A</i>. <i>baumannii</i>, <i>P</i>. <i>aeruginosa</i> and <i>E</i>. <i>aerogenes</i>.</p

Permeabilization of MRSA cells by niclosamide and oxyclozanide.

<p>Time course of membrane permeabilization by oxyclozanide <b>(A)</b> and niclosamide <b>(B)</b> was measured by following the increase in Sytox Green fluorescence (λexc = 485 nm; λem = 530 nm). The drugs were tested at serially diluted concentrations between 1 to 64 μg/ml.</p