Anti-Ulcerative Effect of Curcumin-Galactomannoside Complex on Acetic Acid-Induced Experimental Model by Inhibiting Inflammation and Oxidative Stress.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects the mucosa and submucosa of colon. The pathogenesis of ulcerative colitis (UC) is related to reduced antioxidant capacity and increased inflammatory processes. Reactive oxygen metabolites are the potent inflammatory mediators that may be involved in tissue injury in inflammatory bowel disease. Conventional drug therapies for UC come with a myriad of side effects which further raise the need for natural bioactive agents. Curcumin has proven to be beneficial in the prevention and treatment of a number of inflammatory diseases, but due its poor bioavailability, the therapeutic applications are limited. Thus, to enhance its bioavailability, a new formulation - curcumin-galactomannoside (CGM)- was made by complexing curcumin with galactomannans derived from fenugreek. The present study aims to evaluate the effects of CGM on experimental UC model. Adult male Wistar rats were divided into 5 groups: normal control rats (NC); ulcerative colitis control rats (UC); UC + sulfasalazine (SS) treated; UC + curcumin (CM) treated; and UC + CGM supplemented for 21 days. The colonic mucosal injury was assessed by macroscopic and histological examination, along with evaluation of antioxidant status, inflammatory mediators, and gene expressions. Administration of CGM significantly enhanced antioxidant activities and decreased the level of inflammatory mediators and also suppressed the expression of inflammatory markers as compared with other groups. In conclusion, findings from these results reveal that CGM exerts marked curative effects on acute experimental colitis, possibly by regulating the antioxidant status and modulating inflammatory cascade.

Improvement of Aglycone π-Stacking Yields Nanomolar to Sub-nanomolar FimH Antagonists.

Antimicrobial resistance has become a serious concern for the treatment of urinary tract infections. In this context, an anti-adhesive approach targeting FimH, a bacterial lectin enabling the attachment of E. coli to host cells, has attracted considerable interest. FimH can adopt a low/medium-affinity state in the absence and a high-affinity state in the presence of shear forces. Until recently, mostly the high-affinity state has been investigated, despite the fact that a therapeutic antagonist should bind predominantly to the low-affinity state. In this communication, we demonstrate that fluorination of biphenyl α-d-mannosides leads to compounds with perfect π-π stacking interactions with the tyrosine gate of FimH, yielding low nanomolar to sub-nanomolar KD values for the low- and high-affinity states, respectively. The face-to-face alignment of the perfluorinated biphenyl group of FimH ligands and Tyr48 was confirmed by crystal structures as well as 1 H,15 N-HSQC NMR analysis. Finally, fluorination improves pharmacokinetic parameters predictive for oral availability.

Synthesis and evaluation of thiomannosides, potent and orally active FimH inhibitors.

FimH is a type I fimbrial lectin located at the tip of type-1 pili of Gram-negative uropathogenic Escherichia coli (UPEC) guiding its ability to adhere and infect urothelial cells. Accordingly, blocking FimH with small molecule inhibitor is considered as a promising new therapeutic alternative to treat urinary tract infections caused by UPEC. Herein, we report that compounds having the S-glycosidic bond (thiomannosides) had improved metabolic stability and plasma exposures when dosed orally. Especially compound 5h showed the potential to inhibit biofilm formation and also to disrupt the preformed biofilm. And compound 5h showed prophylactic effect in UTI model in mice.

The potential of FimH as a novel therapeutic target for the treatment of Crohn's disease.

INTRODUCTION: Crohn's disease (CD) is a life-long chronic disorder characterized by intestinal inflammation. Current treatments for CD are directed towards abnormal immune responses rather than the intestinal bacteria that trigger intestinal inflammation. Areas covered: Adherent-Invasive Escherichia coli (AIEC) bacteria abnormally colonize the ileal mucosa in a subgroup of CD patients. They can promote or perpetuate chronic inflammation and are therefore an interesting therapeutic target. Various strategies that target these E. coli strains have been developed to promote their intestinal clearance. Here, we review current AIEC-targeted strategies, especially anti-adhesive strategies, that are based on the development of FimH antagonists. We discuss their potential as personalized microbiota-targeted treatments for CD patients abnormally colonized by AIEC. Expert opinion: A large panel of mannose-derived FimH antagonists were tested for their ability to inhibit E. coli adhesion to host cells. Documented reports suggest that monovalent mannosides are promising candidates that could represent a complementary therapeutic strategy to prevent intestinal inflammation in the E. coli-colonized CD patient subgroup. Ongoing research continues to improve the pharmacokinetic properties of mannosides, and hopefully, clinical trials will be performed in CD patients in the near future.

Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn's disease.

INTRODUCTION: The bacterial adhesin FimH is a virulence factor and an attractive therapeutic target for urinary tract infection (UTI) and Crohn's Disease (CD). Located on type 1 pili of uropathogenic E. coli (UPEC), the FimH adhesin plays an integral role in the pathogenesis of UPEC. Recent efforts have culminated in the development of small-molecule mannoside FimH antagonists that target the mannose-binding lectin domain of FimH, inhibiting its function and preventing UPEC from binding mannosylated host cells in the bladder, thereby circumventing infection. Areas covered: The authors describe the structure-guided design of mannoside ligands, and review the structural biology of the FimH lectin domain. Additionally, they discuss the lead optimization of mannosides for therapeutic application in UTI and CD, and describe various assays used to measure mannoside potency in vitro and mouse models used to determine efficacy in vivo. Expert opinion: To date, mannoside optimization has led to a diverse set of small-molecule FimH antagonists with oral bioavailability. With clinical trials already initiated in CD and on the horizon for UTI, it is the authors, opinion that mannosides will be a 'first-in-class' treatment strategy for UTI and CD, and will pave the way for treatment of other Gram-negative bacterial infections.

Antivirulence C-Mannosides as Antibiotic-Sparing, Oral Therapeutics for Urinary Tract Infections.

Gram-negative uropathogenic Escherichia coli (UPEC) bacteria are a causative pathogen of urinary tract infections (UTIs). Previously developed antivirulence inhibitors of the type 1 pilus adhesin, FimH, demonstrated oral activity in animal models of UTI but were found to have limited compound exposure due to the metabolic instability of the O-glycosidic bond (O-mannosides). Herein, we disclose that compounds having the O-glycosidic bond replaced with carbon linkages had improved stability and inhibitory activity against FimH. We report on the design, synthesis, and in vivo evaluation of this promising new class of carbon-linked C-mannosides that show improved pharmacokinetic (PK) properties relative to O-mannosides. Interestingly, we found that FimH binding is stereospecifically modulated by hydroxyl substitution on the methylene linker, where the R-hydroxy isomer has a 60-fold increase in potency. This new class of C-mannoside antagonists have significantly increased compound exposure and, as a result, enhanced efficacy in mouse models of acute and chronic UTI.

Design and syntheses of mono and multivalent mannosyl-lipoconjugates for targeted liposomal drug delivery.

Multivalent mannosyl-lipoconjugates may be of interest for glycosylation of liposomes and targeted drug delivery because the mannose specifically binds to C-type lectin receptors on the particular cells. In this paper syntheses of two types of novel O-mannosides are presented. Conjugates 1 and 2 with a COOH- and NH2-functionalized spacer and the connection to a lysine and FmocNH-PEG-COOH, are described. The coupling reactions of prepared intermediates 6 and 4 with a PEGylated-DSPE or palmitic acid, respectively, are presented. Compounds 5, mono-, 8, di- and 12, tetravalent mannosyl-lipoconjugates, were synthesized. The synthesized compounds were incorporated into liposomes and liposomal preparations featuring exposed mannose units were characterized. Carbohydrate liposomal quartz crystal microbalance based assay has been established for studying carbohydrate-lectin binding. It was demonstrated that liposomes with incorporated mannosyl-lipoconjugates were effectively recognized by Con A and have great potential to be used for targeted liposomal drug delivery systems.

Pulmonary Delivery of Anti-Tubercular Drugs Using Ligand Anchored pH Sensitive Liposomes for the Treatment of Pulmonary Tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (M. TB) remains the prime cause of bacterial mortality and morbidity world-wide. Therefore, effective delivery and targeting of drug to the cellular tropics is essentially required to generate significant results for tuberculosis treatment. The aim of the present study was to develop and characterize ligand anchored pH sensitive liposomes (TPSL) as dry powder inhaler for the targeted delivery of drugs in the target site i.e. lungs. METHOD: Ligand anchored PSL (TPSL) was prepared by thin film hydration for the combined delivery of Isoniazid (INH) and Ciprofloxacin HCl (CIP HCl) using 4-aminophenyl-α-D mannopyranoside (Man) as surface functionalized ligand and characterized using different parameters. RESULTS: It was observed that size of the ligand anchored liposomes (TPSL) was slightly more than the non-ligand anchored liposomes (PSL). Drug release was studied at different pH for 24 hrs and it was observed that liposomes exhibited slow release at alkaline pH (58-64%) as compared to macrophage pH (81-87%) where it increased dramatically due to the destabilization of pH sensitive liposome (PSL). In vitro cellular uptake study showed that much higher concentration was achieved in the alveolar macrophage using ligand anchored liposomes as compared to its counterpart. In vivo study showed that maximum drug accumulation was achieved in the lung by delivering drug using ligand anchored PSL as compared to conventional PSL. CONCLUSION: It was concluded that ligand anchored pH sensitive liposome is one of the promising systems for the targeted drug therapy in pulmonary tuberculosis.

A novel mannoside-glycocluster adjuvant: Compared in vitro to CpG ODN and MPL and tested in vivo in mouse asthma model.

BACKGROUND: Allergen-specific immunotherapy balances the Th2-biased immunity towards Th1 and Treg responses. Adjuvants are used in allergen preparations to intensify the immune responses. The increased prevalence of allergies in developed societies has been associated with decreased microbial load during childhood. This has initiated a search for microbial structures to be used as adjuvants. Our study has shown that a synthetic triacedimannose (TADM) may suppress the Th2-type allergic inflammatory response. The aim of this study was to compare the properties of TADM with capacities of other adjuvants, CpG ODN and MPL, to modulate cytokine production in PBMC and regulate sensitisation in an OVA-sensitised mouse asthma model. METHODS: The effects of TADM were studied in vitro on birch stimulated PBMC cultures of birch allergic rhinitis patients with other known adjuvants. Cytokines in supernatants were measured by Luminex. Effects of TADM were analysed in vivo in a mouse model of OVA-induced allergic asthma by analysing BAL, cytokine mRNA and serum antibodies. RESULTS: TADM was the only adjuvant that significantly suppressed the production of all birch induced Th2-type cytokines. In a murine model, TADM significantly suppressed the specific IgE production and enhanced IFN-γ production. CONCLUSIONS: TADM suppresses the birch allergen induced Th2-type cytokine responses in allergic subjects more efficiently than the two other adjuvants, MPL and CpG ODN. TADM is immunomodulatory also in vivo and decreases the IgE levels and increases the IFN-γ responses in a murine model. These results suggest that TADM may be a promising candidate for novel adjuvants in immunotherapy.

Antiedematogenic Evaluation of Copaifera langsdorffii Leaves Hydroethanolic Extract and Its Major Compounds.

Inflammatory disorders affect many people worldwide, and medicinal plants are used to ameliorate these health problems. This paper reports the antiedematogenic and analgesic evaluation of Copaifera langsdorffii Desf. leaves hydroethanolic extract (Cop) and two of its isolated compounds: quercetin-3-O-α-l-rhamnopyranosyl (quercitrin) and kaempferol-3-O-α-l-rhamnopyranosyl (afzelin). For that, the following experimental protocols were undertaken locomotor performance, writhing induced by acetic acid, antinociceptivity induced by formalin, hot plate latency, paw oedema induced by carrageenan and dextran, and cell migration induced by lipopolysaccharide (LPS), as well as the measurement of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) in macrophages. Neither the extract nor the isolated compounds displayed analgesic activity. The obtained results showed that C. langsdorffii extract possesses antiedematogenic properties acting on peripheral sites, whereas quercitrin and afzelin are not involved. Moreover, these properties are not associated with cell migration inhibition, TNF-α, IL-6, or IL-10 regulation.

Afzelin attenuates asthma phenotypes by downregulation of GATA3 in a murine model of asthma.

Asthma is a serious health problem causing significant mortality and morbidity globally. Persistent airway inflammation, airway hyperresponsiveness, increased immunoglobulin E (IgE) levels and mucus hypersecretion are key characteristics of the condition. Asthma is mediated via a dominant T-helper 2 (Th2) immune response, causing enhanced expression of Th2 cytokines. These cytokines are responsible for the various pathological changes associated with allergic asthma. To investigate the anti-asthmatic potential of afzelin, as well as the underlying mechanisms involved, its anti-asthmatic potential were investigated in a murine model of asthma. In the present study, BALB/c mice were systemically sensitized using ovalbumin (OVA) followed by aerosol allergen challenges. The effect of afzelin on airway hyperresponsiveness, eosinophilic infiltration, Th2 cytokine and OVA­specific IgE production in a mouse model of asthma were investigated. It was found that afzelin­treated groups suppressed eosinophil infiltration, allergic airway inflammation, airway hyperresponsiveness, OVA-specific IgE and Th2 cytokine secretion. The results of the present study suggested that the therapeutic mechanism by which afzelin effectively treats asthma is based on reduction of Th2 cytokine via inhibition of GATA-binding protein 3 transcription factor, which is the master regulator of Th2 cytokine differentiation and production.

FimH antagonists: bioisosteres to improve the in vitro and in vivo PK/PD profile.

Urinary tract infections (UTIs), predominantly caused by uropathogenic Escherichia coli (UPEC), belong to the most prevalent infectious diseases worldwide. The attachment of UPEC to host cells is mediated by FimH, a mannose-binding adhesin at the tip of bacterial type 1 pili. To date, UTIs are mainly treated with antibiotics, leading to the ubiquitous problem of increasing resistance against most of the currently available antimicrobials. Therefore, new treatment strategies are urgently needed. Here, we describe the development of an orally available FimH antagonist. Starting from the carboxylate substituted biphenyl α-d-mannoside 9, affinity and the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) were substantially improved by a bioisosteric approach. With 3'-chloro-4'-(α-d-mannopyranosyloxy)biphenyl-4-carbonitrile (10j) a FimH antagonist with an optimal in vitro PK/PD profile was identified. Orally applied, 10j was effective in a mouse model of UTI by reducing the bacterial load in the bladder by about 1000-fold.

Enhanced bioavailability and safety of curcumagalactomannosides as a dietary ingredient.

In spite of the various bioavailable formulations of curcumin for pharma and dietary supplement applications, food grade formulations suitable as a dietary ingredient, and capable of providing significant levels of plasma curcumin, are limited. The present contribution describes the safety and oral bioavailability of a novel water soluble formulation of curcumin, curcumagalactomannosides (CGM), when used as a dietary ingredient in selected food items. CGM was prepared using a food grade hydrocolloid (galactomannans) derived from the kitchen spice fenugreek (Trigonella foenum graccum), without using any synthetic excipients. The safety of the formulation was assessed through acute and subchronic toxicity studies on Wistar rats and genotoxicity studies. The efficacy of CGM as a bioavailable dietary ingredient was assessed by successfully preparing various food items and by measuring the post-blood plasma curcumin levels at various time intervals after the consumption of food items. High performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) and electrospray ionization tandem mass spectrometer (ESI-MS/MS) was employed for the quantification of plasma curcuminoids. It was observed that CGM is safe and suitable for further development and clinical studies, with a no observable adverse effect level (NOAEL) up to 2.0 g kg⁻¹ per day b.wt. CGM was found to offer seven to ten times higher bioavailability of curcumin in humans, when incorporated into various food/beverage items at 100 mg CGM per serving size, as compared to the standard unformulated curcumin.

Targeting delivery of liposomes with conjugated p-aminophenyl-α-d-manno-pyranoside and apolipoprotein E for inhibiting neuronal degeneration insulted with ß-amyloid peptide.

Liposomes with conjugated p-aminophenyl-α-d-manno-pyranoside (APMP) and apolipoprotein E (ApoE) (APMP-ApoE-liposomes) were employed to carry neuron growth factor (NGF) across the blood-brain barrier (BBB) and enhance the survival of degenerated neurons. APMP-ApoE-liposomes were used to deliver NGF across a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes (HAs) for rescuing SK-N-MC cells from an insult of ß-amyloid peptide 1-42 (Aß1-42). An increase in the APMP concentration enhanced the particle size, HBMEC and HA viability, permeability for propidium iodide (PI), and permeability for NGF, however, reduced the absolute value of zeta potential, APMP conjugation efficiency and transendothelial electrical resistance (TEER). In addition, an increase in the ApoE concentration increased the particle size, absolute value of zeta potential, HBMEC and HA viability, permeability for PI, permeability for NGF and SK-N-MC cell viability, however, decreased the ApoE conjugation efficiency and TEER. APMP and ApoE on liposomes can be promising surface moieties to carry NGF across the BBB, target degenerated neurons and inhibit Aß1-42-induced neurotoxicity in Alzheimer's disease.

Prevention of Escherichia coli K1 bacteremia in newborn mice by using topical vaginal carbohydrates.

Pregnant Swiss-Webster mice were vaginally inoculated with 5 x 10(4) Escherichia coli K1 strain LH (O75:K1:H3) or C94 (O7:K1:H-). Inhibitor solutions were applied vaginally before delivery and the incidence of bacteremia and surface colonization determined in neonates at 3 days of age. E. coli K1 strain LH resulted in bacteremia in 77% and colonization in 74% of control newborn mice. After topical maternal vaginal D-mannose treatment, bacteremia and colonization were present in 25% of neonates. Topical vaginal application of subinhibitory concentration of gentamicin reduced bacteremia to 23% of neonates. Topical methyl-alpha-D-mannoside and p-nitrophenyl-D-mannoside, however, prevented bacteremia in 100% of newborn mice. A neonatal meningitis strain of E. coli K1 (C94) caused bacteremia in 100% of neonates and was also completely inhibited by methyl-alpha-D-mannoside. This technique of vaginal treatment before delivery may have applicability to human mothers and their infants.

Modified in vivo behavior of liposomes containing synthetic glycolipids.

Liposomes with synthetic saccharide determinants were prepared from synthetic cholesterol conjugates of D-mannose and 6-amino-6-deoxy-D-mannose and labeled with [51Cr]chromate. The kinetics and tissue distribution of label in mice were determined after footpad and subcutaneous injection. Liposomes bearing either of these saccharide determinants greatly increased retention of label at the injection sites compared to control liposomes, which contain no glycolipid, and to free [51Cr]chromate. Draining lymph nodes contained small fractions of the injected radioactivity but in some cases this retention was saccharide-dependent and highly concentrated. These results show that incorporation of synthetic glycolipids can substantially alter the in vivo lifetime and distribution of liposomes outside the bloodstream. Such surface-modified liposomes may be useful for sustained release or selective delivery of therapeutic or diagnostic agents.