The studies of drugs that could constitute a palliative to spinal cord injury (SCI) are a continuous and increasing demand in biomedicine field from developed societies. Recently we described the chemical synthesis and antiglioma activity of synthetic glycosides. A synthetic sulfated glycolipid (here IG20) has shown chemical stability, solubility in polar solvents, and high inhibitory capacity over glioma growth. We have used mass spectrometry (MS) to monitor IG20 m / z = 550.3 in cells and tissues of the central nervous system (CNS) that are involved in SCI recovery. IG20 was detected by MS in serum and homogenates from CNS tissue of rats, though in the latter a previous deproteinization step was required. The pharmacokinetic parameters of serum clearance at 24 h and half-life at 4 h were determined for synthetic glycoside in the adult rat using MS. A local administration of the drug near of spinal lesion site is proposed. © 2015 María Sánchez-Sierra et al.

We designed and synthesized two anomeric oleyl glucosaminides as anti-cancer agents where the presence of a trifluoroacetyl group close to the anomeric center makes them resistant to hydrolysis by hexosaminidases. The oleyl glycosides share key structural features with synthetic and natural oleyl derivatives that have been reported to exhibit anti-cancer properties. While both glycosides showed antiproliferative activity on cancer cell lines, only the α-anomer caused endoplasmic reticulum (ER) stress and cell death on C6 glioma cells. Analysis of sphingolipids and glycosphingolipds in cells treated with the glycosides showed that the α-anomer caused a drastic accumulation of ceramide and glucosylceramide and reduction of lactosylceramide and GM3 ganglioside at concentrations above a threshold of 20 μM. In order to understand how ceramide levels increase in response to α-glycoside treatment, further investigations were done using specific inhibitors of sphingolipid metabolic pathways. The pretreatment with 3-O-methylsphingomyelin (a neutral sphingomyelinase inhibitor) restored sphingomyelin levels together with the lactosylceramide and GM3 ganglioside levels and prevented the ER stress and cell death caused by the α-glycoside. The results indicated that the activation of neutral sphingomyelinase is the main cause of the alterations in sphingolipids that eventually lead to cell death. The new oleyl glycoside targets a key enzyme in sphingolipid metabolism with potential applications in cancer therapy. © 2015 Elsevier Inc. All rights reserved.

Herein, we report the design and preparation of multireversible smart porous surfaces combining two different abilities. On the one hand, either neutral or negatively charged surfaces can be formed by formation/disruption of host-guest complexes. On the other hand, these surfaces have the capability of alternating negatively and positively charge upon complexation of a polycation. Moreover, these two functionalization steps were demonstrated to be reversible so that the initial surface can be recovered and employed again. For this purpose, first, a copolymer was prepared by polymerization of two different monomers, i.e. styrene (S) and a styrene modified with cyclodextrin (SCD) by click chemistry. Blends of this copolymer and polystyrene were employed to fabricate porous surfaces with controlled pore sizes and chemical distribution by the breath figures technique. More precisely, the cyclodextrin (CD) moieties, specifically located inside the holes of the surface, interact reversibly with adamantane end-terminated poly(acrylic acid) chain (Ada-PAA85). The latter served to establish electrostatic interaction with a polycation (poly-l-lysine, PLL), leading to positively charged surface. These interactions, both host-guest and electrostatic, can be inverted obtaining again the original surface, proving the full reversibility of the system. © 2015 Elsevier Inc..

Objective: Preparation of functionalized micro- and submicrofibers by electrospinning of polyesters with glycosides which are structural elements of proteoglycans, for application to the repair of spinal cord lesions.Material and methods: Solutions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with synthetic glycosides were prepared varying systematically the processing conditions. Fiber morphology assessed by scanning electron microscopy. The stability of the interaction between the glycoside and the polymer fiber was evaluated in aqueous medium, and their toxicity in cultures of neural cells.Results: The fiber morphology was altered mainly by the solution parameters. In aqueous medium, the glycoside with a sulfate group was released from fibers at slower rate than the non-sulfated glycoside. The viability of neural cells was not affected by the glycosides.Conclusion: It is possible to fabricate aligned polyester micro fibers with glycosides. Most of the glycoside present in the fibers remains in the substrate after extraction in water for several days. Electrospinning is a very accessible and versatile technique for application to strategies of cellular therapy in spinal cord injuries.

GOALS AND BACKGROUND: Hypolactasia affects over half of the world population. Diagnosis remains problematic as currently available tests, such as the hydrogen breath test, have low reliability and lactose intolerance symptoms are unspecific. We evaluated the diagnostic performance and safety of a new noninvasive diagnostic test based on urine or serum measurement of D-xylose after lactase cleavage of orally administered 4-galactosylxylose (gaxilose). STUDY: In a multicentre, open-label, nonrandomized, phase IIb-III study, consecutive patients with symptoms suggestive of lactose intolerance sequentially underwent intestinal biopsy for direct measurement of lactase activity (reference standard), hydrogen breath test, and blood glucose test after lactose challenge, 4-and 5-hour urine-based gaxilose test, and blood-based gaxilose test. For the gaxilose tests, 0 to 4 and 4 to 5 hours urine samples were taken after a 0.45 g gaxilose dose, whereas serum samples were taken 90 minutes after a 2.7 g dose for D-xylose determination. Genetic testing of hypolactasia was also assessed. RESULTS: Of the 222 patients enrolled, 203 completed all diagnostic tests; 108 were hypolactasic according to biopsy. The sensitivities and specificities and positive and negative predictive values of the gaxilose tests were all >90\% versus 69\% to 85\% for the hydrogen breath test and the blood glucose test. The area under the ROC curve was significantly higher for the gaxilose tests (>0.9, P≤0.007). These tests also had higher sensitivity than genetic testing for hypolactasia and were well tolerated. CONCLUSIONS: The diagnostic performance of the gaxilose tests is excellent and can substantially improve the diagnosis of hypolactasia. Copyright © 2013 by Lippincott Williams &Wilkins.

Background: The phloroglucinol assay is the current method for d-xylose determination in urine/plasma/serum. However, its sensitivity is limited when low amounts of d-xylose are to be measured, such as in the noninvasive evaluation of intestinal lactase with 4-galactosylxylose (gaxilose). An improved assay was therefore needed. Methods: We developed and validated a modified version of the phloroglucinol-based assay for quantification of d-xylose in urine/serum samples. A method for gaxilose determination by gas chromatography (GC) was also optimized. Results: Linearity ranged from 0.125 to 5.0 mg/l (5–200 mg/l in original sample). Accuracy at LOQ (0.125 mg/l) was 0.97/2.49\% in spiked urine/serum; for other quality controls (QC), it was <1.27\%. Intra- and interassay precision at LOQ were 6.02\% and 6.45\% for urine, and 8.86\% and 10.00\%, respectively, for serum; for other QC, precision was <2.15\%. Linearity of gaxilose determination by GC was 3.90–195.17 for urine and 9.75–195.17 mg/l for serum with acceptable sensitivity and reproducibility. The method proved adequate for the d-xylose determination in healthy and hypolactasic subjects after oral administration of gaxilose. Conclusions: The modified method provides high sensitivity and robustness for d-xylose quantification in urine/serum for routine clinical use especially in the noninvasive diagnosis of intestinal lactase deficiency with the gaxilose test. © 2014 Wiley Periodicals, Inc.

GOALS AND BACKGROUND:: Hypolactasia is widespread, yet reliable diagnostic tests are lacking. A new test based on oral administration of 4-galactosylxylose (gaxilose) and urine or serum measurement of D-xylose after cleavage by intestinal lactase is under clinical development. We investigated the optimal dose of gaxilose and calculate cutoff values of D-xylose for that dose. STUDY:: In the randomized, dose-finding, phase I study, urine and serum pharmacokinetics of D-xylose were determined after oral administration of 6 ascending doses of gaxilose (and placebo) to 12 healthy adult volunteers. In the open, parallel, phase Ib study, 30 volunteers received the doses established for the urine and blood tests and D-xylose was measured. Cutoff values were calculated as 1.96×SD below the mean value. Safety was assessed through reporting of adverse events. RESULTS:: Gaxilose administration showed a progressive, dose-dependent increase in D-xylose in urine and serum. An optimal gaxilose dose of 0.45 g and urine collection periods of 4 and 5 hours were selected for further studies. For the blood test, a 2.7 g dose was selected and C max measured at 90 minutes. The calculated cutoff values of D-xylose for normal lactase activity were 27.58 and 37.87 mg for the 4- and 5-hour urine tests, respectively, and 0.97 mg/dL for the blood test. There were no treatment-related adverse events. CONCLUSIONS:: The methodology described provides a simple, safe test for the evaluation of lactase activity in vivo. Further evaluation of the test as a noninvasive diagnosis of hypolactasia is ongoing in patients with lactose intolerance. Copyright © 2013 by Lippincott Williams & Wilkins.

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 μM), significant increases in choline containing metabolites were observed in the 1H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death. © 2013 García-Álvarez et al.

Neurostatin, a natural glycosphingolipid, and NF115, a synthetic glycolipid, are inhibitors of glioma growth. While neurostatin shows high inhibitory activity on gliomas its abundance is low in mammalian brain. On the contrary NF115 exhibits less inhibitory activity on gliomas, but could be prepared by chemical synthesis. In this study we describe synthetic compounds, structurally related to NF115, capable of inhibiting glioma growth at low micromolar range. We used DNA microarray technology to compare the genes inhibited in U373-MG human glioma cells after treatment with the natural or synthetic inhibitor. New synthetic compounds were developed to interact with the product of Rho GDP dissociation inhibitor alpha gene, which was repressed in both treatments. Compounds that were inhibitors of glioma cell growth in assays for [3H]-thymidine incorporation were then injected in C6 tumor bearing rats and the tumor size in each animal group were measured. The GC-17, GC-4 and IG-5 are new compounds derived from NF115 and showed high antiproliferative activity on tumor cell lines. The GC-17 compound inhibited U373-MG glioblastoma cells (3.2 μM), the effects was fifty times more potent than NF115, and caused a significant reduction of tumor volume (P < 0.05) when tested in Wistar rats allotransplanted with C6 glioma cells. © 2012 Elsevier Ltd. All rights reserved.

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides' outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients. © 2012 Romero-Ramírez et al.

The synthesis of six conjugates of l-proline and β-cyclodextrin and their evaluation as catalysts of aldol reaction in water are described. The results indicated that the nature of the linker between proline and β-cyclodextrin is important for catalytic activity; the one with the most flexible linker gave the best results. Inhibition experiments showed that the cavity of β-cyclodextrin plays a role in the catalysis. Permethylation of the cyclodextrin hydroxyl groups led to higher conversion rates. © 2012 Elsevier Ltd. All rights reserved.

To achieve efficient proline-based catalysis in water, proline has been supported in the past to porous and hydrophobic solid resins leading to heterogeneous systems. These solid resins provide a hydrophobic environment to the active centers, mimicking what happens in natural enzymes. However, a more realistic mimetic approach would be to carry out the aldol reaction in a homogeneous way, maintaining the hydrophobic environment, using for example properly designed noncross-linked polymer carriers. In this work, we report the synthesis and aqueous catalytic evaluation of a linear copolymer bearing both pendant proline and permethylated β-cyclodextrin (β-CD) groups. It was designed on the basis that the presence of the hydrophobic cavity of the β-CD could bring aromatic substrates into close proximity to the surrounding catalytic proline residues through host-guest interactions. The compound is water-soluble and catalyzes aldol reactions in this medium without the need for any extra organic solvent. We employed a model reaction between cylohexanone and p-nitrobenzaldehyde, and we observed a decrease of the reaction rate when a competing aromatic compound, known to form a strong inclusion complex with β-CD, was added. The copolymeric catalyst showed a pH-dependent behavior. At pH 7, the copolymer is found in solution as extended single chains with negative charge, catalyzing the reaction in a fast and nonstereoselective mode. At the isoelectric point (pH 3.8) where the positive and negative charges of the zwitterionic proline are canceled by forming charge complexes, the copolymer forms multichain hydrophobic nanoaggregates most probably stabilized by the permethylated β-CD. Although the reaction inside these nanoreactors is slower, it exhibits high stereoselectivity. It is proposed that the observed stereoselectivity is caused by the exclusion of water from the core of these homogeneous entities. © 2012 American Chemical Society.

[No abstract available]

In this article, the application of high resolution NMR spectroscopy to study the effect of therapeutic compounds on cells, tissues and organisms is reviewed. To illustrate how these NMR methods can provide useful information for a better understanding about the mechanism of action of drugs and their interactions with metabolic pathways, the emphasis is placed on most recent work about drug therapeutic intervention in biological models of diseases and in humans. Specifically, the application of NMR spectroscopy to investigate the effect of drugs on the treatment of neurological disorders, cancer, infectious diseases and diabetes is illustrated. In addition, NMR studies of drug-induced toxicity and multinuclear NMR for monitoring drug delivery and catabolism are described. Current progress in NMR instrumentation and methods will continue to improve the sensitivity and maintain this very versatile technique as powerful tool for research in the field of medicinal chemistry. © 2011 Bentham Science Publishers Ltd.

The synthesis and biological activity of oleylN-acetyl-α- and β-d-glucosaminides (1 and 2, respectively) and their thioglycosyl analogues (3 and 4, respectively) are reported. The compounds exhibited antimitotic activity on rat glioma (C6) and human lung carcinoma (A549) cell cultures in the micromolar range. Analysis of cell extracts using ultra performance liquid chromatography-mass spectrometry showed that the synthetic glycosides produce alterations in glycosphingolipid metabolism, with variable effect on the level of glucosylceramide depending on the configuration of the antimitotic used. In vivo experiments in nude mice bearing an implanted C6 glioma showed that the α-thioglycoside 3 reduced tumor volume, while the O-glycosyl derivative was inactive, highlighting the importance of using enzyme resistant glycosides. © 2011 American Chemical Society.

The synthesis and catalytic activity of linear copolymers of proline methacrylate and styrene with different molar ratios are reported. The compounds are soluble catalysts for aldol reactions between aldehydes and ketones in water-without the need of any extra organic solvent. They were designed on the basis that the presence of hydrophobic groups around the proline moiety is beneficial for reactions to proceed in water. The copolymers were tested as catalyst in a model reaction between cylohexanone and p-nitrobenzaldehyde. Compared to the inactive proline-methacrylate homopolymers, the presence of styrene was essential for the reaction to proceed. The catalytic efficiency was dependent on the molar ratio proline methacrylate/styrene and the comonomer distribution along the polymer chain. The catalyst with the highest styrene content, which has the largest amount of alternating styrene-proline sequences, gave the highest conversion, showing the effect of the microstructure of the copolymer on the catalytic properties. A significant improvement of stereoselectivity was achieved when the reactions were performed in the presence of divalent salts, with magnesium dichloride giving the best results. In order to understand the effect of the salt on the stereoselectivity of the reaction, dynamic light scattering and 1H HR MAS NMR studies of the copolymers were performed. The results suggested a relationship between the stereoselectivity and the formation of large aggregates leading to the formation of a heterogeneous media during the reaction. The polymers were recovered and reused by precipitation in water at their isoelectric point. © 2011 American Chemical Society.

Oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside (1) was previously shown to exhibit antimitotic activity on glioma (C6) and melanoma (A375) cell lines. Preliminary studies about its mechanism of action using 1H MAS NMR suggested that 1 may be altering the metabolism of lipids. We have now studied the effect of 1 on the fatty acid, sphingolipid and ganglioside content in a line of carcinomic human alveolar epithelial cells (A549) using UPLC-MS. Oleic acid and NB-DNJ were used as positive controls for inhibition of fatty acid and ganglioside synthesis, respectively. Compound 1 (10 μM) was more efficient than oleic acid in reducing fatty acid levels of A549 cells, producing a decrease in the range of 40-15\%, depending on the acyl chain length and the number of insaturations. In addition, glycoside 1 caused a reduction on ganglioside content of A549 tumor cell line and accumulation of lactosylceramide, the common metabolic precursor for ganglioside biosynthesis. Alteration of ganglioside metabolism was also observed with two galactosylated derivatives of 1, which caused a more pronounced increase in lactosylceramide levels. Compound 1 at higher concentrations (above 30 μM) produced drastic alterations in glycosphingolipid metabolism, leading to cell metabolic profiles very different from those obtained at 10 μM. These biochemical changes were ascribed to activation of endoplasmic reticulum stress pathways. © The Royal Society of Chemistry 2011.

Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled post-translationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosidation of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as decoy acceptors of xylosylprotein 4-β-galactosyltransferase, we have heterologously expressed the catalytic domain of the human β-1,4-GalT 7 and studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme. © The Royal Society of Chemistry.

Treated glioblastoma patients survive from 6 to 14 months. In the first part of this review, we describe glioma origins, cancer stem cells and the genomic alterations that generate dysregulated cell division, with enhanced proliferation and diverse response to radiation and chemotherapy. We review the pathways that mediate tumour cell proliferation, neo-angiogenesis, tumor cell invasion, as well as necrotic and apoptotic cell death. Then, we examine the ability of gliomas to evade and suppress the host immune system, exhibited at the levels of antigen recognition and immune activation, limiting the effective signaling between glioma and host immune cells. The second part of the review presents current therapies and their drawbacks. This is followed by a summary of the work of our laboratory during the past 20 years, on oligosaccharide and glycosphingolipid inhibitors of astroblast and astrocytoma division. Neurostatins, the O-acetylated forms of gangliosides GD1b and GT1b naturally present in mammalian brain, are cytostatic for normal astroblasts, but cytotoxic for rat C6 glioma cells and human astrocytoma grades III and IV, with ID50 values ranging from 200 to 450 nM. The inhibitors do not affect neurons or fibroblasts up to concentrations of 4 μM or higher. At least four different neurostatin-activated, cell-mediated antitumoral processes, lead to tumor destruction: (i) inhibition of tumor neovascularization; (ii) activation of microglia; (iii) activation of natural killer (NK) cells; (iv) activation of cytotoxic lymphocytes (CTL). The enhanced antigenicity of neurostatin-treated glioma cells, could be related to their increased expression of connexin 43. Because neurostatins and their analogues show specific activity and no toxicity for normal cells, a clinical trial would be the logical next step. © the author(s), publisher and licensee Libertas Academica Ltd.

This paper describes a study on the preparation and characterisation of partially biodegradable microparticles of poly(ε-caprolactone)/poly(ethyl methacrylate) (PCL/PEMA) as carriers of synthetic glycolipids with antimitotic activity against brain tumour cells. Microparticles prepared by suspension polymerisation of methacrylate in the presence of already polymerised PCL showed a predominantly spherical but complex morphology, with segregation of PCL micro/nano-domains towards the surface. Small diameter discs were prepared by compression moulding of blends of microparticles and the active principle under mild conditions. The in vitro behaviour of the discs and release of the glycolipid were studied in different simulated fluid models. Ingress of fluids increased with increasing hydrophobicity of the medium. Release of the glycolipid was sustained in all fluids, the most prolonged profile being in human synovial fluid and phosphate-buffered saline modified with 20 vol.\% dioxane. Slow disintegration of the discs and partial degradation of the microparticles was evident in accelerated studies. The antimitotic activity of glycolipid released from the discs was proved against a human glioblastoma line. This activity, along with selectivity against human fibroblasts, could be controlled by the amount of drug charged in the disc. © 2009 Acta Materialia Inc.