Evaluation of Anti-Neuroinflammatory Activity of Isatin Derivatives in Activated Microglia.

Neuroinflammation plays a crucial role in the progression of Alzheimer's disease and other neurodegenerative disorders. Overactivated microglia cause neurotoxicity and prolong the inflammatory response in many neuropathologies. In this study, we have synthesised a series of isatin derivatives to evaluate their anti-neuroinflammatory potential using lipopolysaccharide activated microglia as a cell model. We explored four different substitutions of the isatin moiety by testing their anti-neuroinflammatory activity on BV2 microglia cells. Based on the low cytotoxicity and the activity in reducing the release of nitric oxide, pro-inflammatory interleukin 6 and tumour necrosis factor α by microglial cells, the N1-alkylated compound 10 and the chlorinated 20 showed the best results at 25 µM. Taken together, the data suggest that 10 and 20 are promising lead compounds for developing new neuroprotective agents.

Functional Heterogeneity of Mouse and Human Brain OPCs: Relevance for Preclinical Studies in Multiple Sclerosis.

Besides giving rise to oligodendrocytes (the only myelin-forming cell in the Central Nervous System (CNS) in physiological conditions), Oligodendrocyte Precursor Cells (OPCs) are responsible for spontaneous remyelination after a demyelinating lesion. They are present along the mouse and human CNS, both during development and in adulthood, yet how OPC physiological behavior is modified throughout life is not fully understood. The activity of adult human OPCs is still particularly unexplored. Significantly, most of the molecules involved in OPC-mediated remyelination are also involved in their development, a phenomenon that may be clinically relevant. In the present article, we have compared the intrinsic properties of OPCs isolated from the cerebral cortex of neonatal, postnatal and adult mice, as well as those recovered from neurosurgical adult human cerebral cortex tissue. By analyzing intact OPCs for the first time with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, we show that these cells behave distinctly and that they have different metabolic patterns in function for their stage of maturity. Moreover, their response to Fibroblast Growth Gactor-2 (FGF-2) and anosmin-1 (two molecules that have known effects on OPC biology during development and that are overexpressed in individuals with Multiple Sclerosis (MS)) differs in relation to their developmental stage and in the function of the species. Our data reveal that the behavior of adult human and mouse OPCs differs in a very dynamic way that should be very relevant when testing drugs and for the proper design of effective pharmacological and/or cell therapies for MS.

Neurotransmitter-Responsive Nanosensors for T2-Weighted Magnetic Resonance Imaging.

Neurotransmitter-sensitive contrast agents for magnetic resonance imaging (MRI) have recently been used for mapping signaling dynamics in live animal brains, but paramagnetic sensors for T1-weighted MRI are usually effective only at micromolar concentrations that themselves perturb neurochemistry. Here we present an alternative molecular architecture for detecting neurotransmitters, using superparamagnetic iron oxide nanoparticles conjugated to tethered neurotransmitter analogs and engineered neurotransmitter binding proteins. Interactions between the nanoparticle conjugates result in clustering that is reversibly disrupted in the presence of neurotransmitter analytes, thus altering T2-weighted MRI signals. We demonstrate this principle using tethered dopamine and serotonin analogs, together with proteins selected for their ability to competitively bind either the analogs or the neurotransmitters themselves. Corresponding sensors for dopamine and serotonin exhibit target-selective relaxivity changes of up to 20%, while also operating below endogenous neurotransmitter concentrations. Semisynthetic magnetic particle sensors thus represent a promising path for minimally perturbative studies of neurochemical analytes.

Micellar Iron Oxide Nanoparticles Coated with Anti-Tumor Glycosides.

The synthesis procedure of nanoparticles based on thermal degradation produces organic solvent dispersible iron oxide nanoparticles (OA-IONP) with oleic acid coating and unique physicochemical properties of the core. Some glycosides with hydrophilic sugar moieties bound to oleyl hydrophobic chains have antimitotic activity on cancer cells but reduced in vivo applications because of the intrinsic low solubility in physiological media, and are prone to enzymatic hydrolysis. In this manuscript, we have synthetized and characterized OA-IONP-based micelles encapsulated within amphiphilic bioactive glycosides. The glycoside-coated IONP micelles were tested as Magnetic Resonance Imaging (MRI) contrast agents as well as antimitotics on rat glioma (C6) and human lung carcinoma (A549) cell lines. Micelle antimitotic activity was compared with the activity of the corresponding free glycosides. In general, all OA-IONP-based micellar formulations of these glycosides maintained their anti-tumor effects, and, in one case, showed an unusual therapeutic improvement. Finally, the micelles presented optimal relaxometric properties for their use as T2-weighed MRI contrast agents. Our results suggest that these bioactive hydrophilic nano-formulations are theranostic agents with synergistic properties obtained from two entities, which separately are not ready for in vivo applications, and strengthen the possibility of using biomolecules as both a coating for OA-IONP micellar stabilization and as drugs for therapy.

Novel sulfoglycolipid IG20 causes neuroprotection by activating the phase II antioxidant response in rat hippocampal slices.

Compound IG20 is a newly synthesised sulphated glycolipid that promotes neuritic outgrowth and myelinisation, at the time it causes the inhibition of glial proliferation and facilitates exocytosis in chromaffin cells. Here we have shown that IG20 at 0.3-10 µM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). Excess production of reactive oxygen species (ROS) elicited by glutamate or ODG/reox was prevented by IG20 that also restored the depressed tissue levels of GSH and ATP in hippocampal slices subjected to OGD/reox. Furthermore, the augmented iNOS expression produced upon OGD/reox exposure was also counteracted by IG20. Additionally, the IG20 elicited neuroprotection was prevented by the presence of inhibitors of the signalling pathways Jak2/STAT3, MEK/ERK1/2, and PI3K/Akt, consistent with the ability of the compound to increase the phosphorylation of Jak2, ERK1/2, and Akt. Thus, the activation of phase II response and the Nrf2/ARE pathway could explain the antioxidant and anti-inflammatory effects and the ensuing neuroprotective actions of IG20.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

PURPOSE: After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. METHODS: Glial cells were inhibited with IG20 (IC50≈10 µM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. RESULTS: The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P <  0.05). CONCLUSIONS: We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

Novel synthetic sulfoglycolipid IG20 facilitates exocytosis in chromaffin cells through the regulation of sodium channels.

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy.

New oleyl glycoside as anti-cancer agent that targets on neutral sphingomyelinase.

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.

Mass Spectrometry in Pharmacokinetic Studies of a Synthetic Compound for Spinal Cord Injury Treatment.

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.

The effect of antitumor glycosides on glioma cells and tissues as studied by proton HR-MAS NMR spectroscopy.

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 ((1)H 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 (1)H 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.

Synthetic glycolipids for glioma growth inhibition developed from neurostatin and NF115 compound.

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.

Specific synthesis of neurostatin and gangliosides O-acetylated in the outer sialic acids using a sialate transferase.

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.

Synthesis of antimitotic thioglycosides: in vitro and in vivo evaluation of their anticancer activity.

The synthesis and biological activity of oleylN-acetyl-α- and ß-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.

Effect of drugs in cells and tissues by NMR spectroscopy.

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.

Lipid and ganglioside alterations in tumor cells treated with antimitotic oleyl glycoside.

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 (1)H 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.

Detection of metabolite changes in C6 glioma cells cultured with antimitotic oleyl glycoside by 1H MAS NMR.

The synthetic glycoside, oleyl N-acetyl-alpha-D-glucosaminide (1), was previously shown to exhibit antimitotic activity on rat (C6) and human (U-373) glioma lines. To obtain information about its mechanism of action, metabolite changes in C6 glioma cells were analyzed after treatment with 1 using high-resolution magic angle spinning (1)H NMR. Compound 1 caused either a decrease or an increase in the intensity of the signal assigned to coenzyme A (CoA) metabolites depending on the concentration used. The data obtained from the (1)H NMR spectra of cells cultured with 1, combined with those obtained after treatment with oleic acid (an inhibitor of acetyl-CoA carboxylase) and phenyl butyrate (a known antineoplastic agent), suggest that 1 may be altering the metabolism of fatty acids and induce apoptosis of C6 glioma cells. These results point to NMR spectroscopy as an efficient technique for monitoring the response of the cells to therapeutic agents.

Studies on the uptake of glucose derivatives by red blood cells.

Erythrocytes express the same glucose transporter (GLUT-1) as is present in the blood-brain barrier. With the aim of testing the viability of using this transport system to deliver glucosyl drug derivatives to the brain, the uptake of several dopamine-glucose conjugates and a few structurally related analogues by erythrocytes was studied with HPLC and (1)H MAS NMR spectroscopy. The results showed that slight structural changes determine the uptake of glycoconjugates by red blood cells. However, experiments in the presence of glucose transport inhibitors showed that none of the conjugates that efficiently crossed the cell membrane were transported by GLUT-1.

Design and synthesis of glycoside inhibitors of glioma and melanoma growth.

An N-acetylglucosaminide derivative with a pentaerythritol substituent at position C-6 was previously synthesized and shown to inhibit neural tumor growth. Now, we report the preparation of a series of new synthetic compounds introducing systematic changes in the nature, polarity, and size of the sugar substituents. The antimitotic activity of the new compounds was tested on cultured rat (C6) and human (U-373) glioma lines and on a human melanoma line (A-375). The antimitotic and antitumoral activity of the new compounds on glioma cell lines increased up to 2 orders of magnitude with respect to the parent compound or was abolished, permitting a detailed structure-function analysis of the new antitumorals. One of the glycosides inhibited melanoma division with an ID50 below the micromolar range.