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Nanoparticle (NP)-based drug delivery systems accumulate in the disrupted epithelium of inflamed colon tissue in ulcerative colitis. However, premature early drug release and uptake or degradation of NPs during their passage through the harsh gastric or intestinal environment compromise their therapeutic outcomes. This study aimed to develop an advanced colitis-targeted hybrid nanoparticles-in-microparticles (NPsinMPs) drug delivery system to overcome the aforementioned challenges. First, sustained drug releasing poly(lactic-co-glycolic acid) NPs were generated and further encapsulated in pH-sensitive Eudragit FS30D MPs to ensure complete drug protection in a gastric-like pH and for selective delivery of NPs to the colon. SEM and confocal microscopy for the NPsinMPs revealed successful NP encapsulation. NPsinMPs prevented drug release in an acidic gastric-like and intestinal-like pH and presented a sustained release thereafter at an ileal and colonic pH, indicating the degradation of the outer pH-sensitive MPs and release of NPs. Furthermore, in vivo imaging of gastrointestinal tract of a colitis mouse orally administered with fluorescent NPsinMPs revealed higher fluorescence intensities selectively in the colon, demonstrating the release of loaded NPs and their concomitant accumulation at the site of colon inflammation. NPsinMPs markedly mitigated experimental colitis in mice indicated by improved histopathological analysis, decreased myeloperoxidase activity, neutrophils and macrophage infiltration, and expression of proinflammatory cytokines in colonic tissues compared with NP-treated mice. The present results show the successful formulation of an NPsinMP-based drug delivery system and provide a platform to improve NP-based colon-targeted drug delivery through improved protection of encapsulated NPs and their payload in the early small intestine. Image, graphical abstract [ABSTRACT FROM AUTHOR]

In this study, we developed pH-triggered surface charge-reversal lipid nanoparticles (LNPs), loaded with budesonide, which could precisely deliver the drug to inflamed colon segments for the treatment of ulcerative colitis. Polyethyleneimine (PEI) was used to render LNPs cationic (PEI-LNPs), and Eudragit® S100 (ES) was coated on PEI-LNPs to obtain pH-triggered charge-reversal LNPs (ES-PEI-LNPs). ES coating avoided a burst drug release under acidic conditions mimicking the stomach and early small intestine environments and showed a sustained release in the colon. The surface charge of ES-PEI-LNPs switched from negative to positive under colonic conditions owing to pH-triggered removal of the ES coating. Bioimaging of the mouse gastrointestinal tract and confocal analysis of colon tissues revealed that ES-PEI-LNPs selectively accumulated in an inflamed colon. Furthermore, ES-PEI-LNPs mitigated experimental colitis in mice. These results suggest that the pH-triggered charge-reversal LNPs could be a promising drug carrier for ulcerative colitis therapy and other colon-targeted treatments. [ABSTRACT FROM AUTHOR]

Oral colon-targeted drug delivery has gained popularity as an effective strategy for treatment of inflammatory bowel disease (IBD). In this study, we prepared colon-targeted dexamethasone microcrystals (DXMCs) coated with multilayers of chitosan oligosaccharide (CH), alginate (AG), and finally Eudragit S 100 (ES) (ES 1 AG 4 CH 5 -DXMCs) using a layer-by-layer (LBL) coating technique. Particle size, surface charge, in vitro drug release, and in vivo anti-inflammatory activity of ES 1 AG 4 CH 5 -DXMCs were evaluated. ES 1 AG 4 CH 5 -DXMCs had an average particle size of 2.34 ± 0.19 μm and a negative surface charge of - 48 ± 9 mV. ES 1 AG 4 CH 5 -DXMCs demonstrated pH-dependent dexamethasone release, avoiding initial burst drug release in acidic pH conditions of the stomach and small intestine, and providing subsequent sustained drug release in the colonic pH. Importantly, ES 1 AG 4 CH 5 -DXMCs exhibited a significant therapeutic activity in a mouse model of colitis compared to other DXMCs. Overall, the LBL-coated DXMCs presented here could be a promising colon-targeted therapy for IBD. [ABSTRACT FROM AUTHOR]

Bilirubin (BR) is generated by the reduction of biliverdin (BV), a metabolite that results from the catalytic degradation of heme by the isoforms of heme oxygenase (HO). BV is nontoxic and water-soluble but BR is potentially toxic and lipophilic. Therefore, a further metabolic step is required for BR before excretion is possible. The reductive conversion of BV to BR costs energy and is evolutionarily conserved in human physiology. There must be a compelling reason for this apparently nonsensical evolutionary conservation. In addition to the differences between BR and BV—such as water solubility, antioxidant activity, and participation as a receptor ligand—in the present study, we focused on the chemistry of the two metabolites with regard to an electrophilic functional group called a Michael reaction acceptor (MRA). Our data reveal that the BR reacts with thiol compounds forming adducts, whereas no reaction occurs with BV. Furthermore, the binding of biotin-tagged BR to Kelch-like ECH-associated protein 1 (KEAP1)—a biological electrophile sensor—was prevented by pretreatment with BR or a thiol compound, but was not by pretreatment with BV. In cells, BR could bind to KEAP1 to release and activate nuclear factor-erythroid 2 (NF-E2) p45-related factor 2, a cytoprotective transcription factor, leading to the induction of HO-1. These findings may provide a physiological rationale for the energy-consuming conversion of BV to BR. [ABSTRACT FROM AUTHOR]

The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs ( Riptortus pedestris ) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila . Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila , we used an aprA -deficient P. entomophila mutant strain (Δ aprA ). When Δ aprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the Δ aprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses. [ABSTRACT FROM AUTHOR]

Mesalazine (5-aminosalicylic acid, 5-ASA), a currently used drug for anti-inflammatory bowel disease, is easily oxidized by HOCl, a strong oxidant generated in gut inflammation, to produce electrophilic quinones. We investigated whether this chemical feature has an implication in the anti-inflammatory pharmacology of 5-ASA. Human colon carcinoma HCT116 cells were treated with HOCl-reacted 5-ASA. Oxidized 5-ASA activated Nrf2 while 5-ASA itself was not effective. Activation of Nrf2 led to induction of hemeoxygenase (OH)-1, an anti-inflammatory enzyme. Western blot analysis of Keap1, a cytosolic repressor of Nrf2, following precipitation of biotin-labeled proteins in cell lysates treated with biotin-tagged 5-ASA, revealed a much greater amount of Keap1 when biotin-tagged 5-ASA was oxidized with HOCl. Precipitation of Keap1 was attenuated markedly by pretreatment with oxidized 5-ASA or a sulfhydryl donor. In addition, treatment with oxidized 5-ASA in cell lysates reduced the Keap1 amount that coimmunoprecipitated with Nrf2. In parallel, rectal administration of 5-ASA increased the level of HO-1 and nuclear Nrf2 in the inflamed colonic tissues, but not in normal colonic tissues. Moreover, oral gavage of sulfasalazine, a colon-specific prodrug of 5-ASA currently used clinically, activated the Nrf2-HO-1 pathway in the colonic tissues where inflammation was in progress, which was not observed when inflammation subsided. Collectively, our data suggest that Nrf2-HO-1 pathway is involved in the anti-inflammatory pharmacology of 5-ASA, which was likely being exerted exclusively in the inflammatory state. [ABSTRACT FROM AUTHOR]

[Display omitted] Activating NRF2-driven transcription with non-electrophilic small molecules represents an attractive strategy to therapeutically target disease states associated with oxidative stress and inflammation. In this study, we describe a campaign to optimize the potency and efficacy of a previously identified bis-sulfone based non-electrophilic ARE activator 2. This work identifies the efficacious analog 17 , a compound with a non-cytotoxic profile in IMR32 cells, as well as ARE activators 18 and 22, analogs with improved cellular potency. In silico drug-likeness prediction suggested the optimized bis-sulfones 17 , 18 , and 22 will likely be of pharmacological utility. [ABSTRACT FROM AUTHOR]

The objective of this study was to prepare Herceptin (HCT)-functionalized paclitaxel nanocrystals and evaluated their cell-specific interactions, cellular accumulation, and growth inhibition in HER2-positve breast cancer cells as a tumor-targeted delivery module. Paclitaxel (PTX) was fabricated in the form of nanocrystals (PNCs) by a sono-precipitation method, and HCT were coated using a facile non-covalent method (PNCs-HCT). Our results showed that the PNCs-HCT were stable for at least 1 month at 4 °C with no noticeable desorption of HCT. The release test showed that PNCs-HCT exhibited sustained drug release similar to only PNCs but with a higher release rate than only PTX powder. Cellular uptake, cytotoxicity, and cell cycle arrest studies revealed that PNCs-HCT exhibit greater binding affinity and higher cell-specific internalization to HER2-positive breast cancer cell lines as compared to PNCs, followed by enhanced cell growth inhibition. HCT-functionalized PNCs presented in this study offer a promising strategy for targeted pure drug nanocrystal delivery and enhancing the efficiency of anticancer therapy. [ABSTRACT FROM AUTHOR]

Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris , harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia . However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia . These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont. [ABSTRACT FROM AUTHOR]

We investigated a potential molecular target for anti-colitic effects of esculetin, 6,7-dihydroxycoumarin. Esculetin administered rectally effectively ameliorated TNBS-induced rat colitis and attenuated the expression of pro-inflammatory mediators in the inflamed colon. In human colon carcinoma HCT116 cells, esculetin induced hypoxia-inducible factor-1α (HIF-1α), leading to secretion of vascular endothelial growth factor, a HIF-1 target gene product involved in ulcer healing of the gastrointestinal mucosa. Esculetin directly inhibited HIF prolyl hydroxylase-2 (HPH-2), an enzyme playing a major role in negatively regulating HIF-1α protein stability. Esculetin inhibition of HPH and consequent induction of HIF-1α were attenuated by escalating dose of either ascorbate or 2-ketoglutarate, the required factors of the enzyme. Structurally, the catechol moiety in esculetin was required for HPH inhibition. Collectively, HPH may be a molecular target for esculetin-mediated anti-colitic effects and the catechol moiety in esculetin is the pharmacophore for HPH inhibition. [ABSTRACT FROM AUTHOR]

Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization—Pluronic F127 and chitosan—for inhibiting multi-drug resistant gene 1 ( MDR1 ) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (∼268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1 . In vitro cytotoxicity tests revealed that the 24 and 48 h IC 50 values of NPs-P/C 1 were 2.03 and 0.67 μ m , respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C 1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step-fabricated, dual-surface-functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy. [ABSTRACT FROM AUTHOR]

Resveratrol, a natural polyphenolic phytoalexin, is a dietary supplement that improves the outcomes of metabolic, cardiovascular, and other age-related diseases due to its diverse pharmacological activities. Although there have been several preclinical and clinical investigations of resveratrol, the contributions of gut phase-II metabolism and enterohepatic circulation to the oral bioavailability and pharmacokinetics of resveratrol remain unclear. Furthermore, a physiologically-based pharmacokinetic (PBPK) model that accurately describes and predicts the systemic exposure profiles of resveratrol in clinical settings has not been developed. Experimental data were acquired from several perspectives, including in vitro protein binding and blood distribution, in vitro tissue S9 metabolism, in situ intestinal perfusion, and in vivo pharmacokinetics and excretion studies. Using these datasets, an in-house whole-body PBPK model incorporating route-dependent phase-II (glucuronidation and sulfation) gut metabolism and enterohepatic circulation processes was constructed and optimized for chemical-specific parameters. The developed PBPK model aligned with the observed systemic exposure profiles of resveratrol in single and multiple dosing regimens with an acceptable accuracy of 0.538–0.999-fold errors. Furthermore, the model simulations elucidated the substantial contribution of gut first-pass metabolism to the oral bioavailability of resveratrol and suggested differential effects of enterohepatic circulation on the systemic exposure of resveratrol between rats and humans. After partial modification and verification, our proposed PBPK model would be valuable to optimize dosage regimens and predict food-drug interactions with resveratrol-based natural products in various clinical scenarios. [Display omitted] • New PBPK models for resveratrol that were first verified against rat and human data. • Phase-II metabolism and enterohepatic circulation were involved in the models. • The models incorporated the Q Gut model to capture route-dependent gut metabolism. • The models predicted blood resveratrol exposure at clinical multiple dosing regimens. • Substantial contributions of gut first-pass metabolism to the F oral of resveratrol. [ABSTRACT FROM AUTHOR]

Abstract: Senescence marker protein 30 (SMP30) was recently identified as gluconolactonase, which is involved in vitamin C (VC) biosynthesis. Therefore, the antioxidant property of SMP30 is thought to be mediated by its gluconolactonase function. However, pathologic effects of SMP30 deficiency independent of VC biosynthesis have not been studied in models of neurodegenerative diseases. In the present study, we evaluated the effect of SMP30 deficiency on Parkinson’s disease (PD) in SMP30 knockout (KO) mice. Wild type and SMP30 KO mice supplemented with VC were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our results showed that MPTP-induced dopaminergic neuronal loss and motor function impairment were more significant in the SMP30 KO mice. Reactive oxygen species generation and microglia activation were higher in MPTP-treated SMP30 KO mice. However, SMP30 deficiency mitigated MPTP-induced astrocyte activation and glia-derived neurotrophic factor production. Cultures of astrocytes recovered from wild type and SMP30 KO mice revealed that SMP30 deficiency abolished 1-methyl-4-phenyl-pyridinium-induced astroglial activation by blocking the extracellular signal-regulated kinase pathway. Taken together, our findings demonstrate for the first time that SMP30 deficiency increases the severity of PD and suggest a beneficial role of SMP30 in protective astrocyte activation in response to neurodegeneration. The present study shows that modulation of astrocytic SMP30 can be a promising target for treating PD. [Copyright &y& Elsevier]

Abstract: To develop a more potent NFκB inhibitor from salicylic acid which is known to inhibit activity of NFκB, a transcription factor regulating genes involved in immunity, inflammation and tumorigenesis, derivatives of salicylic acid (SA) where the 5 position, carboxyl or hydroxyl group was modified were treated in HCT116 cells transfected with an NFκB dependent luciferase gene and LPS-stimulated RAW264.7 cells. Amidation of the carboxylic group or substitution of chlorine at the 5 position increased the ability of SA to suppress the expression of NFκB dependent luciferase and inducible nitric oxide synthase, a product of an NFκB target gene. Moreover, simultaneous amidation and chlorination of SA (5-chlorosalicylamide; 5-CSAM) conferred an additive NFκB inhibitory activity on SA. To further enhance the inhibitory activity, N-modification was imposed on 5-CSAM. N-(5-chlorosalicyloyl)phenethylamine (5-CSPA), N-(5-chlorosalicyloyl)3-phenylpropylamine (5-CSPPA) and N-(5-chlorosalicyloyl)4-hydroxyphenylethylamine (5-CSHPA) showed greater potencies for inhibiting NFκB activity than other derivatives. Their IC50s’ in the luciferase assay measured 15μM (5-CSPA), 17μM (5-CSPPA) and 91μM (5-CSHPA). Rectal administration of 5-CSPA ameliorated TNBS-induced rat colitis, which was more effective than a conventional drug, 5-aminosalicylic acid. These data may provide useful information for development of a therapeutic agent for treatment of diseases where NFκB plays a critical role in the pathogenic progresses. [Copyright &y& Elsevier]

Abstract: Prolyl 4-hydroxylases (P4Hs), such as collagen prolyl-4-hydroxylases (CPHs) and hypoxia inducible factor prolyl-4-hydroxylases (HPHs), have recently been recognized as promising drug targets for the treatment of fibrotic and ischemic diseases. CPHs and HPHs catalyze identical metabolic reactions, yet lead to quite different physiological consequences, collagen synthesis and the regulation of oxygen homeostasis. Selective modulation of the two enzymes should provide a therapeutic benefit upon pharmacotherapy. In an in vitro VHL capture assay, hydroxylation of the 19mer HIF peptide (corresponding to HIF-1α residues 556–574) by HPH-2 was effectively prevented by nitric oxide (NO) donors, (±)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The NO donors also caused inhibition of HPHs and accumulation of nonhydroxylated HIF-1α protein in A549 human lung adenocarcinoma cells. Hyperoxia (100% O2) attenuated both NO donor-induced accumulation of HIF-1α and inhibition of HPH-mediated hydroxylation. In the presence of a proteasome inhibitor, MG132, the hyperoxia-mediated degradation of HIF-1α was deterred and hydroxylated HIF-1α was detected. SNAP, while being an effective inhibitor of proline 4-hydroxylation of HIF-1α by HPH-2, did not diminish proline hydroxylation of collagen by CPHs. Our data suggest that NO inhibits HPH-2 via competing with dioxygen and that the discriminative effect of NO on CPHs and HPH-2 is attributable to the difference in the affinity of the two enzymes toward dioxygen. [Copyright &y& Elsevier]

Abstract: Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) α protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1α protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel–Lindau-dependent HIF-1α degradation. CAPE inhibition of HPH and induction of HIF-1α protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (–C) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH. [ABSTRACT FROM AUTHOR]

In this study, we developed Lactobacillus rhamnosus GG (LGG)-encapsulating exfoliated bentonite/alginate nanocomposite hydrogels for protecting probiotics by delaying gastric fluid penetration into the nanocomposite and their on-demand release in the intestine. The pore size of the bentonite/alginate nanocomposite hydrogels (BA15) was two-fold smaller than that of alginate hydrogel (BA00). Following gastric pH challenge, the survival of LGG in BA15 decreased by only 1.43 log CFU/g as compared to the 6.25 log CFU/g decrease in alginate (BA00). Further, the internal pH of BA15 decreased more gradually than that of BA00. After oral administration in mice, BA15 maintained shape integrity during gastric passage, followed by appropriate disintegration within the target intestinal area. Additionally, a fecal recovery experiment in mice showed that the viable counts of LGG in BA15 were six-fold higher than those in BA00. The findings suggest the exfoliated bentonite/alginate nanocomposite hydrogel as a promising platform for intestinal delivery of probiotics. • LGG was encapsulated in exfoliated bentonite/alginate nanocomposite hydrogels. • Improved hydrogel pore size dramatically enhanced LGG survival at gastric pH. • Complete intestinal release of LGG was observed after hydrogel disintegration. • Fecal recovery of bentonite/alginate LGG was 6-fold greater than of alginate LGG. [ABSTRACT FROM AUTHOR]

Abstract This study demonstrates the development of a nitric oxide (NO)-releasing hydrogel wound dressing and its efficacy at accelerating methicillin-resistant Staphylococcus aureus (MRSA)-infected wound healing. A DETA/NONOate-doped alginate (Alg-DETA/NO) hydrogel was synthesized using alginate as a hydrogel-forming wound dressing material and diethylenetriamine/diazeniumdiolate (DETA/NONOate) as an NO donor. Alg-DETA/NO exhibited a prolonged NO release profile over a period of 4 days. The rheological properties of Alg-DETA/NO did not differ significantly from those of pure alginate. Importantly, Alg-DETA/NO showed potent antibacterial activity against MRSA, with minimal toxicity to mouse fibroblasts. The application of Alg-DETA/NO to MRSA-infected wounds in a mouse model showed a favorable wound healing with accelerated wound-size reduction and reduced skin bacterial infection. Additionally, histological examination revealed that Alg-DETA/NO reduced inflammation at the wound site and promoted re-epithelialization, angiogenesis, and collagen deposition. Thus, Alg-DETA/NO presented herein could serve as a safe and potent hydrogel dressing for the treatment of MRSA-infected wounds. [Display omitted] • We synthesized an alginate-diethylenetriamine/NONOate hydrogel (Alg-DETA/NO). • Alg-DETA/NO showed NO-releasing properties with low toxicity toward fibroblasts. • Alg-DETA/NO absorbed the wound exudate and transformed into a hydrogel. • Alg-DETA/NO had excellent therapeutic activities in mice with MRSA-infected wounds. • Alg-DETA/NO accelerated wound healing by targeting different wound-healing phases. [ABSTRACT FROM AUTHOR]