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Purpose: In our previous study, riluzole (RLZ) azo-linked to salicylic acid (RAS) was prepared as a colon-targeted RLZ prodrug against rat colitis. However, RAS was not a satisfactory colon-targeted prodrug because of its non-negligible systemic absorption, leading to low colonic delivery efficiency and the ability to limit the systemic absorption of RLZ. This study aimed to improve the colon specificity and anticolitic activity of RAS. Methods: Salicylic acid (SA) was conjugated with the acidic amino acids aspartic acid (Asp) and glutamic acid (Glu) and subsequently azo-coupled with RLZ to yield Asp-conjugated RAS (RAS-Asp) and Glu-conjugated RAS (RAS-Glu). Results: Amino acid-conjugated RAS lowered the distribution coefficient and cell permeability of RAS while exhibiting a release profile of RLZ similar to that of RAS in the cecal contents. Upon oral gavage, amino acid-conjugated RAS delivered a larger amount of RLZ to the cecum than RAS. The ability of amino acid-conjugated RAS to limit the systemic absorption of RLZ was greater than that of RAS. No significant differences were observed in the colon-specific performance between RAS-Asp and RAS-Glu. In a DNBS-induced rat colitis model, amino acid-conjugated RAS was more effective than RAS in ameliorating colonic damage and inflammation and modulating the anti-inflammatory GSK3β-IL-10 pathway in the inflamed colon, without a significant difference between RAS-Asp and RAS-Glu. Conclusion: Conjugation of acidic amino acids with RAS improved the colon specificity, anticolitic activity, and safety of RAS. N-Salicyloyl acidic amino acids may act as high-performance colon-specific promiety for a candidate drug modifiable to a colon-targeted prodrug with an azo bond as a colon-specific link.

Background: Chloroquine (CQ) is an effective and safe antimalarial drug that is also used as a disease-modifying antirheumatic drug. Recent studies have shown that CQ can sensitize cancer cells to anti-cancer therapies. Methods: In this study, we investigated the molecular mechanisms underlying CQ-mediated chemosensitization in human colon carcinoma cells. Results: CQ prevented hypoxia-inducible factor (HIF)-1α protein induction in human colon carcinoma cells. CQ also suppressed HIF-1 activity, as represented by CQ inhibition of HIF-1-dependent luciferase activity and reduced induction of vascular endothelial growth factor. Under hypoxia, CQ restricted HIF-1α synthesis but did not affect HIF-1α transcription and protein stability. The hypoxic state activated ataxia telangiectasia and Rad3-related (ATR) kinase and increased the level of phosphorylated checkpoint kinase 1, a substrate of ATR kinase; however, this was prevented by CQ. An ATR kinase inhibitor suppressed the hypoxic induction of HIF-1α protein and was as effective as CQ. The cytotoxicity of 5-fluorouracil (5-FU), the first choice for the treatment of colorectal cancer, was attenuated under hypoxia. CQ enhanced the cytotoxicity of 5-FU treatment, which was mimicked by the transient transfection with HIF-1α siRNA. Conclusions: Under hypoxia, CQ-mediated sensitization of colon carcinoma HCT116 cells to 5-FU involves HIF-1 inhibition via ATR kinase suppression. Graphical abstract:

Although CD44-targeted delivery of pure drug microcrystals of azathioprine (AZA) could be a desirable approach to treat ulcerative colitis (UC), premature drug release and systemic absorption before reaching the colitis region remain a major obstacle. In this study, to overcome these limitations, we developed on-demand reconstitutable HA-doped AZA microcrystals (EFS/HA-AZAs) viaincorporating hyaluronic acid (HA)-doped AZA microcrystals (HA-AZAs) into a Eudragit FS (EFS) microcomposite. Since EFS acts as a protective layer, the premature release of AZA in the simulated conditions of the stomach and small intestine was substantially reduced, while HA-AZAs were successfully reconstituted from the EFS/HA-AZAs in the colonic environment, resulting from the pH-triggered dissolution of EFS. After complete reconstitution of HA-AZAs in the colon, HA-AZAs selectively accumulated in the inflamed region viathe HA-CD44 interaction. Owing to successful colitis-targeted delivery, EFS/HA-AZAs showed potent anti-inflammatory effects in a dextran sulfate sodium-induced murine colitis model within 7 days without systemic toxicity. These results suggest that EFS/HA-AZAs could be a promising drug delivery system for UC treatment.

Although nitric oxide (NO) has been emerging as a novel local anticancer agent because of its potent cytotoxic effects and lack of off-target side effects, its clinical applications remain a challenge because of the short effective diffusion distance of NO that limits its anticancer activity. In this study, we synthesized albumin-coated poly(lactic-co-glycolic acid) (PLGA)-conjugated linear polyethylenimine diazeniumdiolate (LP/NO) nanoparticles (Alb-PLP/NO NPs) that possess tumor-penetrating and NO-releasing properties for an effective local treatment of melanoma. Sufficient NO-loading and prolonged NO-releasing characteristics of Alb-PLP/NO NPs were acquired through PLGA-conjugated LP/NO copolymer (PLP/NO) synthesis, followed by nanoparticle fabrication. In addition, tumor penetration ability was rendered by the electrostatic adsorption of the albumin on the surface of the nanoparticles. The Alb-PLP/NO NPs showed enhanced intracellular NO delivery efficiency and cytotoxicity to B16F10 murine melanoma cells. In B16F10-tumor-bearing mice, the Alb-PLP/NO NPs showed improved extracellular matrix penetration and spatial distribution in the tumor tissue after intratumoral injection, resulting in enhanced antitumor activity. Taken together, the results suggest that Alb-PLP/NO NPs represent a promising new modality for the local treatment of melanoma.

Glycogen synthase kinase-3β (GSK3β), a multi-functional kinase, is a promising therapeutic target for the treatment of inflammation. Inhibitory κB kinase (IKK)-activated GSK3β inhibitory peptide (IAGIP) was designed as an inflammation-responsive anti-colitic therapeutic. To optimize therapeutic efficiency, IAGIP was tested using two different drug delivery techniques: colon-targeted delivery and cell-permeable peptide modification. In cell-based experiments, in response to tumor necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated activation of IKK, cell-permeable IAGIP (CTP-IAGIP) inhibited GSK3β, leading to increased production of anti-inflammatory cytokine interleukin-10 (IL-10) and suppression of TNF- and LPS-induced NFκB activity. Oral gavage of CTP-IAGIP loaded in the colon-targeted capsule attenuated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and lowered the expression levels of NFκB-regulated proteins in the inflamed colons. CTP-IAGIP further induced IL-10 production in the inflamed colonic tissues; however, the levels of IL-10 were not affected in the normal colonic tissue or colonic tissue in which inflammation had subsided. Collectively, our data suggest that IAGIP administered using the aforementioned drug delivery techniques is an orally active anti-colitic drug selectively responding to inflammation.

In this study, we developed oral core–shell nanoparticles composed of curcumin nanocrystals in the core and chitosan/alginate multilayers in the shell for inflammation-targeted alleviation of ulcerative colitis (UC). The release rate of curcumin from the core–shell nanoparticles was low at a pH mimicking the stomach and small intestine, whereas it was higher at a pH mimicking the colon. Further, biodistribution studies in the gastrointestinal tract of mice showed that distribution of nanoparticles was significantly higher in the colon than that in the stomach and small intestine. Quantitative analysis of drugs in colonic tissues and confocal imaging of colons revealed preferential accumulation of nanoparticles in inflamed tissues than that in healthy tissues. In vivoanti-inflammatory studies revealed that nanoparticles exhibit enhanced efficacy in alleviating inflammation-related symptoms in a mouse colitis model. The results suggest that the core–shell nanoparticles presented here can be exploited as efficient colon-targeted drug delivery systems for UC therapy.

To develop a 5-aminosalicylic acid (5-ASA)-based anticolitic drug with enhanced therapeutic activity, a colon-targeted codrug constituting 5-ASA and a GPR109A agonist was designed. 5-ASA azo-coupled with nicotinic acid (ASA-azo-NA) was synthesized, and the colon specificity and anticolitic effects were evaluated. Approximately 89% of ASA-azo-NA was converted to 5-aminonicotinic acid (5-ANA) and 5-ASA after 24 h of incubation in the cecal contents. 5-ANA was identified as a GPR109A agonist (concentration that gives half-maximal response (EC50): 18 μM) in a cell-based assay. Upon oral gavage of ASA-azo-NA (oral ASA-azo-NA) and sulfasalazine (oral SSZ), a colon-targeted 5-ASA prodrug, cecal accumulation of 5-ASA was comparable, and 5-ANA was barely detectable in the blood, while it was detected up to 62.7 μM with oral 5-ANA. In parallel, oral ASA-azo-NA did not elicit an adverse skin response. In murine macrophage and human colon carcinoma cells, activation of GPR109A by 5-ANA elevated the level of the anti-inflammatory cytokine IL-10, suppressed NF-κB activation, and potentiated the inhibitory activity of 5-ASA on NF-κB. Oral ASA-azo-NA ameliorated rat colitis and was more effective than oral SSZ, which were substantially blunted following cotreatment with the GPR109A antagonist, mepenzolate. In conclusion, ASA-azo-NA is a colon-targeted anticolitic codrug with a reduced risk of skin toxicity induced by the GPR109A agonist, therapeutically surpassing a current 5-ASA-based anti-inflammatory bowel disease drug in a rat colitis model.

Febuxostat (FEB) is a selective xanthine oxidase (XO) inhibitor approved for chronic management of hyperuricemia (HU) in patients with gout. Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DIC) are the first choice for the prevention and treatment of gouty inflammation and flare. In this study, a sensitive and simple bioanalytical method using HPLC coupled with a fluorescence detector (HPLC-FL) was developed for the simultaneous determination of FEB and DIC. The linearity (1–1500 ng mL−1for FEB and 5–1500 ng mL−1for DIC), precision, accuracy, recovery, matrix effect, and stability of the newly developed method were validated as per the US Food and Drug Administration (FDA) guidelines. Next, the in vivoand in vitrometabolic interactions between FEB and DIC were comprehensively examined. The in vivopharmacokinetics of FEB were found to be significantly altered by the co-administration of DIC in rats. Further, the in vitromicrosomal metabolism study revealed that the altered pharmacokinetics of FEB could be attributed primarily to the competitive inhibition of FEB metabolism by DIC. To our knowledge, this is the first systematic study showing the in vivopharmacokinetic interaction of FEB with DIC following intravenous and oral administration in rats and the associated metabolic inhibition mechanism together with a new HPLC-FL method to simultaneously determine FEB and DIC in biological matrices.

We investigated if the therapeutic switching of sofalcone (SFC), a gastroprotective agent, to an anticolitic agent is feasible using colon-targeted drug delivery. SFC can activate the anti-inflammatory nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-hemeoxygenase-1 (HO-1) pathway in human colon epithelial cells and murine macrophages. For the efficient treatment of colitis, SFC was coupled with acidic amino acids to yield SFC-aspartic acid (SFC-AA) and SFC-glutamic acid, and their colon targetability and therapeutic effects were assessed as an anticolitic agent in a 2,4-dinitrobenezenesulfonic acid-induced rat colitis model. The SFC derivatives were decoupled up to 72% in the cecal contents but remained stable in the small intestinal contents. Oral gavage of SFC-AA (oral SFC-AA, equivalent to 1.67 mg/kg of SFC) delivered SFC (maximal cecal concentration: 57.36 μM) to the cecum, while no SFC was detected with oral gavage of SFC (oral SFC, 1.67 mg/kg). Moreover, oral SFC-AA (equivalent to 10 mg/kg of SFC) did not afford detectable concentration of SFC in the blood but detected up to 4.64 μM with oral SFC (10 mg/kg), indicating efficient colonic delivery and limited systemic absorption of SFC upon oral SFC-AA. Oral SFC-AA ameliorated colonic damage and inflammation in rat colitis with elevating colonic levels of HO-1 and nuclear Nrf2 protein, and the anticolitic effects of SFC-AA were significantly undermined by an HO-1 inhibitor. At an equivalent dose of SFC, oral SFC-AA but not oral SFC increased colonic HO-1 and nuclear Nrf2 levels, and oral SFC-AA was more effective than oral SFC in treating rat colitis. Moreover, oral SFC-AA was as effective against colitis as oral sulfasalazine being used for the treatment of inflammatory bowel disease. In conclusion, colon-targeted delivery of SFC facilitated the therapeutic switching of the drug to an anticolitic drug via Nrf2 activation.

To test whether sulpiride (SP), an anti-psychotic and prokinetic drug, shows beneficial effects on experimental murine colitis, a colon-targeted prodrug of SP, 5-(aminoethanoylsulfamoyl)-N-[(1-ethylpyrrolidin-2-yl)methyl]-2-methoxybenzamide (glycylsulpiride (GSP)), was synthesized and its colonic delivery and therapeutic activity against 2,4-dinitrobenzenesulfonic acid (DNBS)–induced rat colitis were assessed. Synthesis of GSP was verified by infrared and proton nuclear magnetic resonance spectroscopy. GSP was converted to SP when incubated with the cecal contents but not when incubated with the small intestinal contents. The percent conversion was about 50.5% at 6 h and 67.7% at 10 h. Colonic delivery of GSP was examined by comparison with sulfasalazine (SSZ), a colon-specific prodrug of 5-aminosalicylic acid currently used for the treatment of inflammatory bowel disease. The two prodrugs accumulated similar concentrations of the corresponding parent drugs in the cecum at 2, 4, and 6 h after oral gavage. Although oral gavage of GSP released millimolar level of SP in the large intestine, SP was hardly detected in the blood. GSP improved colonic damage score and reduced myeloperoxidase activity up to 80.5% in the inflamed colon in a dose-dependent manner. Moreover, GSP was able to reduce the levels of inflammatory mediators in the inflamed colon. Overall, the anti-colitic effectiveness of GSP and SSZ was similar. In conclusion, colonic delivery of SP ameliorates DNBS-induced colitis in rats with no significant systemic absorption of SP. Thus, colon-targeted SP may be therapeutically switched to an anti-colitic drug.

Objective: Mesalazine, 5-aminosalicylic acid (5-ASA), is an anti-inflammatory drug that is most widely used for the treatment of Inflammatory Bowel Disease (IBD). Despite extensive clinical use, the exact pharmacological mechanism underlying the anti-colitic effects of 5-ASA has not yet been elucidated. A potential molecular mechanism underlying 5-ASA-mediated anti-colitic activity was investigated. Methods: An anti-inflammatory pharmacology of 5-ASA was scrutinized in human colon carcinoma cells and murine macrophages and in a TNBS-induced rat colitis model. Results: 5-ASA induced phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and its substrate acetyl-CoA carboxylase in cells. 5-ASA activation of AMPK occurred regardless of the presence of the pro-inflammatory mediators, Tumor Necrosis Factor Alpha (TNF-α) and lipopolysaccharide. 5-ASA inhibits TNF-α-dependent Nuclear Factor-Kappa B (NF-κB) activation, which was dampened by AMPK inhibition. Oral gavage of sulfasalazine (a colon-specific prodrug of 5- ASA) or rectal administration of 5-ASA ameliorated 2,4,6-trinitrobenzene sulfonic acid (TNBS)- induced rat colitis and activated AMPK in the inflamed colonic tissues while markedly diminishing the levels of NF-κB-regulated pro-inflammatory mediators cyclooxygenase-2, inducible nitric oxide synthase, and cytokine-induced neutrophil chemoattractant-3, elevated by the induction of inflammation. Rectal co-administration of 5-ASA and an AMPK inhibitor undermined 5-ASA-mediated activation of AMPK and its anti-colitic effects. Conclusion: These findings suggest that the activation of AMPK is involved in 5-ASA-mediated anticolitic effects at least partly via interference with pro-inflammatory NF-κB signaling.

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.

To develop a colon specific prodrug of celecoxib with improved therapeutic potency and cardiovascular toxicity for chemoprevention of colorectal adenomas, we prepared glycyl celecoxib (GC), aspart-1-ylcelecoxib (A1C) and glutam-1-ylcelecoxib (G1C) and examined colon specific properties in vitro. The amino acid conjugation lowered the apparent partition coefficient of celecoxib (4.6) to 1.2 (GC), 0.6 (A1C) and 0.8 (G1C). The celecoxib-amino acid conjugates were stable in pH 1.2 and 6.8 buffer solutions. On incubation with the contents of small intestine, while GC was stable up to 10 h, A1C and G1C were degraded and liberated celecoxib up to 19–32 % of the dose at 10 h. In the cecal contents, the three conjugates were cleaved to release celecoxib and amounts of celecoxib released from GC, A1C and G1C were 20, 64 and 55 % at 10 h and 30, 75 and 60 % of the dose at 24 h, respectively. Taken together, while GC may deliver celecoxib to the large intestine without premature degradation in the upper intestine, A1C and G1C may have advantages over GC in that they are less absorbable in the upper intestine due to lower partition coefficient and are converted to celecoxib more efficiently at the target site.To develop a colon specific prodrug of celecoxib with improved therapeutic potency and cardiovascular toxicity for chemoprevention of colorectal adenomas, we prepared glycyl celecoxib (GC), aspart-1-ylcelecoxib (A1C) and glutam-1-ylcelecoxib (G1C) and examined colon specific properties in vitro. The amino acid conjugation lowered the apparent partition coefficient of celecoxib (4.6) to 1.2 (GC), 0.6 (A1C) and 0.8 (G1C). The celecoxib-amino acid conjugates were stable in pH 1.2 and 6.8 buffer solutions. On incubation with the contents of small intestine, while GC was stable up to 10 h, A1C and G1C were degraded and liberated celecoxib up to 19–32 % of the dose at 10 h. In the cecal contents, the three conjugates were cleaved to release celecoxib and amounts of celecoxib released from GC, A1C and G1C were 20, 64 and 55 % at 10 h and 30, 75 and 60 % of the dose at 24 h, respectively. Taken together, while GC may deliver celecoxib to the large intestine without premature degradation in the upper intestine, A1C and G1C may have advantages over GC in that they are less absorbable in the upper intestine due to lower partition coefficient and are converted to celecoxib more efficiently at the target site.

For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP–NIP). The most potent CPP–NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP–NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP–NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP–NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP–NIP (CPP–NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.

Importance of the field:Generally, a prodrug, a pharmacologically inactive derivative of an active drug, is designed to modulate pharmacokinetic properties of the parent drug. Targeted distribution of an orally administered drug at the large intestine confers therapeutic advantages on treatment of colonic diseases, peptide and protein therapy and chronotherapy.Areas covered in this review:To achieve such distribution control in the gastrointestinal tract, the adoption of the prodrug concept gives birth to a colon-specific prodrug. The requirement for a prodrug to be colon-specific is described along with the necessary and sufficient conditions of drugs for conversion to a colon-specific prodrug. The known and previously unnoticed factors that negatively influence therapeutic activity and reproducibility of a colon-specific prodrug are presented with suggestions to minimize the negative influence.What the reader will gain:This review provides tactics to satisfy the requirements for being colon-specific and the potential strategies to circumvent obstacles in developing an efficient colon-specific prodrug.Take home message:On design of a colon-specific prodrug, one should take into consideration not only delivery of a drug to the target site, but also the therapeutic effectiveness there.

We investigated a molecular mechanism underlying quercetin-mediated amelioration of colonic mucosal injury and analyzed chemical structure contributing to the quercetin's effect. Quercetin up-regulated vascular endothelial growth factor (VEGF), an ulcer healing factor, not only in colon epithelial cell lines but also in the inflamed colonic tissue. VEGF derived from quercetin-treated colon epithelial cells promoted tube formation. The VEGF induction was dependent on quercetin-mediated hypoxia-inducible factor-1 (HIF-1) activation. Quercetin delayed HIF-1α protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1α hydroxylation and subsequent von Hippel Lindau-dependent HIF-1α degradation. HPH inhibition by quercetin was neutralized significantly by an elevated dose of iron. Consistent with this, cellular induction of HIF-1α by quercetin was abolished by pretreatment with iron. Two iron-chelating moieties in quercetin, -OH at position 3 of the C ring and/or -OH at positions 3' and 4' of the B ring, enabled the flavonoid to inhibit HPH and subsequently induce HIF-1α. Our data suggest that the clinical effect of quercetin may be partly attributed to the activation of an angiogenic pathway HIF-1-VEGF via inhibiting HPH and the chelating moieties of quercetin were required for inhibiting HPH.

In previous reports, a novel colon-specific prodrug, 5-aminosalicyltaurine (5-ASA-Tau) administered orally, is successfully delivered to and liberates 5-aminosalicylic acid (5-ASA) and taurine in the inflamed large intestine of rats. Furthermore, the prodrug ameliorates the 2,4,6-trinitrobenzene-sulfonic acid-induced colitis, and taurine acts not only as a carrier but also as an active therapeutic agent. In this study, we investigated the anti-inflammatory properties of the prodrug at a molecular level. After rectal administration of taurine, formation of taurine chloramine (TauCl) in the inflamed colonic tissue was examined using high-performance liquid chromatography. In human colon epithelial cell lines, nuclear factor-κB (NF-κB) activity was accessed using an NF-κB-dependent luciferase reporter gene. Protein levels were monitored by Western blotting. DNA binding activity of the NF-κB subunit p65 was determined using a DNA binding assay kit. A millimolar level of TauCl was formed in the inflamed tissue. TauCl inhibited tumor necrosis factor (TNF)-dependent NF-κB activation by modifying thiol(s) on p65 and blocking DNA binding. In addition, 5-ASA inhibited phosphorylation of p65 at serine 536, which is critical for transcriptional activity of NF-κB. Furthermore, combined TauCl/5-ASA treatment additively inhibited TNF-dependent NF-κB activation. Together, our data suggest that the colon-specific carrier taurine contributes to the clinical effect of the prodrug by potentiating the inhibitory effect of the active ingredient 5-ASA on a major proinflammatory signal, TNF-dependent NF-κB activation in the inflamed large intestine.

In previous reports, a novel colon-specific prodrug, 5-aminosalicyltaurine (5-ASA-Tau) administered orally, is successfully delivered to and liberates 5-aminosalicylic acid (5-ASA) and taurine in the inflamed large intestine of rats. Furthermore, the prodrug ameliorates the 2,4,6-trinitrobenzene-sulfonic acid-induced colitis, and taurine acts not only as a carrier but also as an active therapeutic agent. In this study, we investigated the anti-inflammatory properties of the prodrug at a molecular level. After rectal administration of taurine, formation of taurine chloramine (TauCl) in the inflamed colonic tissue was examined using high-performance liquid chromatography. In human colon epithelial cell lines, nuclear factor-κB (NF-κB) activity was accessed using an NF-κB-dependent luciferase reporter gene. Protein levels were monitored by Western blotting. DNA binding activity of the NF-κB subunit p65 was determined using a DNA binding assay kit. A millimolar level of TauCl was formed in the inflamed tissue. TauCl inhibited tumor necrosis factor (TNF)-dependent NF-κB activation by modifying thiol(s) on p65 and blocking DNA binding. In addition, 5-ASA inhibited phosphorylation of p65 at serine 536, which is critical for transcriptional activity of NF-κB. Furthermore, combined TauCl/5-ASA treatment additively inhibited TNF-dependent NF-κB activation. Together, our data suggest that the colon-specific carrier taurine contributes to the clinical effect of the prodrug by potentiating the inhibitory effect of the active ingredient 5-ASA on a major proinflammatory signal, TNF-dependent NF-κB activation in the inflamed large intestine.

Tumour necrosis factor α (TNF-α) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor κB (NF-κB) and requires recruitment of receptor-interacting protein (RIP) to TNFR1. The indispensable role of RIP in TNF-induced NF-κB activation has been demonstrated in RIP-/- mice and in cell lines derived from such mice. In the present study, we show that the TNF-α-induced accumulation of hypoxia-inducible factor 1α (HIF-1α) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP-/- mice to either cobalt or PMA resulted in an equivalent HIF-1α induction to that seen in RIP+/+ fibroblasts. In contrast, RIP-/- cells were unable to induce HIF-1α in response to TNF-α. Further, transient transfection of NIH 3T3 cells with an NF-κB super-repressor plasmid (an inhibitor of NF-κB activation) also prevented HIF-1α induction by TNF-α. Surprisingly, although HIF-1α mRNA levels remained unchanged after induction by TNF, induction of HIF-1α protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF-1α, made resistant to von Hippel—Lindau (VHL), and wild-type HIF-1α transfected into VHL-/- cells. These results indicate that HIF-1α induction by TNF-α in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-κB-driven transcription. Thus the results describe a novel mechanism of HIF-1α up-regulation and they identify HIF-1α as a unique component of the NF-κB-mediated inflammatory/survival response.

A bacteria-infected wound can lead to being life-threatening and raises a great economic burden on the patient. Here, we developed polyethylenimine 1.8k (PEI1.8k) surface modified NO-releasing polyethylenimine 25k (PEI25k)-functionalized graphene oxide (GO) nanoparticles (GO-PEI25k/NO-PEI1.8kNPs) for enhanced antibacterial activity and infected wound healing via binding to the bacterial surface. In vitro antibacterial activity and in vivo wound healing efficacy in an infected wound model were evaluated compared with NO-releasing NPs (GO-PEI25k/NO NPs). Surface modification with PEI1.8kcan enhance the ability of nanoparticles to adhere to bacteria. GO-PEI25k/NO-PEI1.8kNPs released NO in a sustained manner for 48 h and exhibited the highest bactericidal activity (99.99% killing) against methicillin-resistant Staphylococcus aureus(MRSA) and multidrug-resistant Pseudomonas aeruginosa(MRPA) without cytotoxicity to L929 mouse fibroblast cells at 0.1 mg/mL. In the MRPA-infected wound model, GO-PEI25k/NO-PEI1.8kNPs showed 87% wound size reduction while GO-PEI25k/NO NPs showed 23% wound size reduction at 9 days postinjury. Masson trichrome and hematoxylin and eosin staining revealed that GO-PEI25k/NO-PEI1.8kNPs enhanced re-epithelialization and collagen deposition, which are comparable to healthy mouse skin tissue. GO-PEI25k/NO-PEI1.8kNPs hold promise as effective antibacterial and wound healing agents.

Riluzole (RLZ) is a neuroprotective drug indicated for amyotrophic lateral sclerosis. To examine the feasibility of RLZ for repositioning as an anti-inflammatory bowel disease (IBD) drug, RLZ (2, 5, and 10 mg/kg) was administered orally to rats with colitis induced by 2,4-dinitrobenzenesulfonic acid. Oral RLZ was effective against rat colitis in a dose-dependent manner, which was statistically significant at doses over 5 mg/kg. To address safety issues upon repositioning and further improve anti-colitic effectiveness, RLZ was coupled with salicylic acid (SA) via an azo-bond to yield RLZ-azo-SA (RAS) for the targeted colonic delivery of RLZ. Upon oral gavage, RAS (oral RAS) was efficiently delivered to and activated to RLZ in the large intestine, and systemic absorption of RLZ was substantially reduced. Oral RAS ameliorated colonic damage and inflammation in rat colitis and was more effective than oral RLZ and sulfasalazine, a current anti-IBD drug. Moreover, oral RAS potently inhibited glycogen synthase kinase 3β (GSK3β) in the inflamed distal colon, leading to the suppression of NFκB activity and an increase in the level of the anti-inflammatory cytokine interleukin-10. Taken together, RAS, which enables RLZ to be delivered to and inhibit GSK3β in the inflamed colon, may facilitate repositioning of RLZ as an anti-IBD drug.

Microbial metabolites play a critical role in mucosal homeostasis by mediating physiological communication between the host and colonic microbes, whose perturbation may lead to gut inflammation. The microbial metabolite 3-indolepropionic acid (3-IPA) is one such communication mediator with potent antioxidative and anti-inflammatory activity. To apply the metabolite for the treatment of colitis, 3-IPA was coupled with acidic amino acids to yield colon-targeted 3-IPA, 3-IPA-aspartic acid (IPA-AA) and 3-IPA-glutamic acid (IPA-GA). Both conjugates were activated to 3-IPA in the cecal contents, which occurred faster for IPA-AA. Oral gavage of IPA-AA (oral IPA-AA) delivered a millimolar concentration of IPA-AA to the cecum, liberating 3-IPA. In a 2,4-dinitrobenzene sulfonic acid (DNBS)-induced rat colitis model, oral IPA-AA ameliorated rat colitis and was less effective than sulfasalazine (SSZ), a current anti-inflammatory bowel disease drug. To enhance the anticolitic activity of 3-IPA, it was azo-linked with the GPR109 agonist 5-aminonicotinic acid (5-ANA) to yield IPA-azo-ANA, expecting a mutual anticolitic action. IPA-azo-ANA (activated to 5-ANA and 2-amino-3-IPA) exhibited colon specificity in in vitro and in vivo experiments. Oral IPA-azo-ANA mitigated colonic damage and inflammation and was more effective than SSZ. These results suggest that colon-targeted 3-IPA ameliorated rat colitis and its anticolitic activity could be enhanced by codelivery of the GPR109A agonist 5-ANA.

Upon microbial infection, host immune cells recognize bacterial cell envelope components through cognate receptors. Although bacterial cell envelope components function as innate immune molecules, the role of the physical state of the bacterial cell envelope (i.e., particulate versus soluble) in host immune activation has not been clearly defined.

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1a (HIF-1a) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1a was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1a protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1a and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1a and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1a and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.