M01 as a novel drug enhancer for specifically targeting the blood-brain barrier.

Drug delivery to the brain is limited for most pharmaceuticals by the blood-brain barrier (BBB) where claudin-5 dominates the paraendothelial tightening. For circumventing the BBB, we identified the compound M01 as a claudin-5 interaction inhibitor. M01 causes transient permeabilisation of the BBB depending on the concentration of small molecules in different cell culture models within 3 to 48 h. In mice, brain uptake of fluorescein peaked within the first 3 h after M01 injection and normalised within 48 h. Compared to the cytostatic paclitaxel alone, M01 improved delivery of paclitaxel to mouse brain and reduced orthotopic glioblastoma growth. Results on interactions of M01 with claudin-5 were incorporated into a binding model which suggests association of its aromatic parts with highly conserved residues of the extracellular domain of claudin-5 and adjacent transmembrane segments. Our results indicate the following mode of action: M01 preferentially binds to the extracellular claudin-5 domain, which weakens trans-interactions between adhering cells. Further decrease in membranous claudin-5 levels due to internalization and transcriptional downregulation enables the paracellular passage of small molecules. In summary, the first small molecule is introduced here as a drug enhancer, which specifically permeabilises the BBB for a sufficient interval for allowing neuropharmaceuticals to enter the brain.

Author Correction: pKa of opioid ligands as a discriminating factor for side effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

pKa of opioid ligands as a discriminating factor for side effects.

The non-selective activation of central and peripheral opioid receptors is a major shortcoming of currently available opioids. Targeting peripheral opioid receptors is a promising strategy to preclude side effects. Recently, we showed that fentanyl-derived µ-opioid receptor (MOR) agonists with reduced acid dissociation constants (pKa) due to introducing single fluorine atoms produced injury-restricted antinociception in rat models of inflammatory, postoperative and neuropathic pain. Here, we report that a new double-fluorinated compound (FF6) and fentanyl show similar pKa, MOR affinity and [35S]-GTPγS binding at low and physiological pH values. In vivo, FF6 produced antinociception in injured and non-injured tissue, and induced sedation and constipation. The comparison of several fentanyl derivatives revealed a correlation between pKa values and pH-dependent MOR activation, antinociception and side effects. An opioid ligand's pKa value may be used as discriminating factor to design safer analgesics.

Modulation of µ-opioid receptor activation by acidic pH is dependent on ligand structure and an ionizable amino acid residue.

BACKGROUND AND PURPOSE: Adverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on µ-opioid receptor binding and signalling induced by the µ-opioid receptor ligands fentanyl, DAMGO, and naloxone. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with µ-opioid receptors were used to study opioid ligand binding, [35 S]-GTPγS binding, and cAMP reduction at physiological and acidic pH. We used µ-opioid receptors mutated at H2976.52 to A (MOR-H2976.52 A) to delineate ligand-specific interactions with H2976.52 . KEY RESULTS: Low pH and the mutant receptor MOR-H2976.52 A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G-protein activation were decreased under these conditions. Fentanyl-induced signalling was not influenced by pH and largely independent of H2976.52 . CONCLUSIONS AND IMPLICATIONS: Our investigations indicate that low pH selectively impairs µ-opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52 . We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52 .

Opioid receptor signaling, analgesic and side effects induced by a computationally designed pH-dependent agonist.

Novel pain killers without adverse effects are urgently needed. Opioids induce central and intestinal side effects such as respiratory depression, sedation, addiction, and constipation. We have recently shown that a newly designed agonist with a reduced acid dissociation constant (pKa) abolished pain by selectively activating peripheral µ-opioid receptors (MOR) in inflamed (acidic) tissues without eliciting side effects. Here, we extended this concept in that pKa reduction to 7.22 was achieved by placing a fluorine atom at the ethylidene bridge in the parental molecule fentanyl. The new compound (FF3) showed pH-sensitive MOR affinity, [35S]-GTPγS binding, and G protein dissociation by fluorescence resonance energy transfer. It produced injury-restricted analgesia in rat models of inflammatory, postoperative, abdominal, and neuropathic pain. At high dosages, FF3 induced sedation, motor disturbance, reward, constipation, and respiratory depression. These results support our hypothesis that a ligand's pKa should be close to the pH of injured tissue to obtain analgesia without side effects.

Novel Opioid Analgesics and Side Effects.

Conventional opioids mediate analgesia as well as severe adverse effects via G-protein coupled opioid receptors (OR) in both inflamed (peripheral injured tissue) and healthy (brain, intestinal wall) environments. To exclude side effects, OR activation can be selectively achieved in damaged tissue by lowering the pKa of an opioid ligand to the acidic pH of inflammation. As a result, protonation of the ligand and consequent OR binding and activation of G-proteins is pH- and injury-specific. A novel compound (NFEPP) demonstrates the feasibility of this approach and displays blockade of pain transmission only at the peripheral site of injury, but with lack of central and gastrointestinal adverse effects. These findings suggest disease-specific receptor activation as a new strategy in drug design.

Impairment of ghrelin synthesis in Helicobacter pylori-colonized stomach: new clues for the pathogenesis of H. pylori-related gastric inflammation.

Ghrelin, the ligand of growth hormone secretagogue receptor 1a, takes part in several functions of the digestive system, including regulation of appetite, energy homeostasis, gastric acid secretion and motility. Ghrelin has also immunoregulatory properties and is supposed to inhibit some inflammatory pathways that can mediate gastric damage. Interestingly, ghrelin synthesis is reduced in the gastric mucosa of patients with Helicobacter pylori (H. pylori) infection, a worldwide condition inducing a T helper (Th)1/Th17 cell response-driven gastritis, which may evolve towards gastric atrophy and cancer. In this article, we review the available data on the expression of ghrelin in H. pylori infection and discuss how the defective ghrelin synthesis may contribute to sustain the ongoing inflammatory response in this disease.

Neurogenesis in temporal lobe epilepsy: relationship between histological findings and changes in dentate gyrus proliferative properties.

OBJECTIVE: The relationship between hippocampal histopathological abnormalities, epileptogenesis and neurogenesis remains rather unclear. METHODS: Tissue samples including the subgranular zone of dentate gyrus (DG) were freshly collected for tissue culture for neurospheres generation in 16 patients who underwent surgery for drug-resistant temporal lobe epilepsy. Remaining tissues were histologically examined to assess the presence of mesial temporal sclerosis (MTS) and focal cortical dysplasia. RESULTS: MTS was detected in 8 cases. Neurospheres were formed in 10/16 cases. Only three out of these 10 cases exhibited MTS; on the contrary 5/6 cases lacking neurosphere proliferation presented MTS. There was a significant correlation between presence of MTS and absence of proliferation (p = 0.0389). We also observed a correlation between history of febrile seizures (FS) and presence of MTS (p = 0.0004) and among the 6 cases lacking neurosphere proliferation, 4 cases (66.6%) had experienced prolonged FS. Among "proliferating" cases the percentage of granular cells pathology (GCP) was lower (20% vs 50%) compared to "non proliferating" cases. CONCLUSION: A decreased potential to generate neurosphere from the SGZ is related to MTS and to alterations of dentate gyrus granule cells, especially in MTS type 1b and GCP type 1. These histological findings may have different prognostic implications, regarding seizure and neuropsychological outcome, compared to patients with other epileptogenic lesions (such as FCD, glioneuronal tumours, vascular lesions).

Highly sensitive carbon nanotube-based sensing for lactate and glucose monitoring in cell culture.

Monitoring of metabolic compounds in cell cultures can provide real-time information of cell line status. This is particularly important in those lines not fully known, as the case of embryonic and mesenchymal cells. On the other hand, such approach can pave the way to fully automated systems for growing cell cultures, when integrated in Petri dishes. To date, the main efforts emphasize the monitoring of few process variables, like pH, pO(2), electronic impedance, and temperature in bioreactors. Among different presented strategies to develop biosensors, carbon nanotubes exhibit great properties, particularly suitable for high-sensitive detection. In this work, nanostructured electrodes by using multiwalled carbon nanotubes are presented for the detection of lactate and glucose. Some results from simulations are illustrated in order to foresee the behavior of carbon nanotubes depending on their orientation, when they are randomly dispersed onto the electrode surface. A comparison between nonnanostructured and nanostructured electrodes is considered, showing that direct electron-transfer between the protein and the electrode is not possible without nanostructuration. Such developed biosensors are characterized in terms of sensitivity and detection limit, and are compared to previously published results. Lactate production is monitored in a cell culture by using the developed biosensor, and glucose detection is also performed to validate lactate behavior.

Occludin protein family: oxidative stress and reducing conditions.

The occludin-like proteins belong to a family of tetraspan transmembrane proteins carrying a marvel domain. The intrinsic function of the occludin family is not yet clear. Occludin is a unique marker of any tight junction and is found in polarized endothelial and epithelial tissue barriers, at least in the adult vertebrate organism. Occludin is able to oligomerize and to form tight junction strands by homologous and heterologous interactions, but has no direct tightening function. Its oligomerization is affected by pro- and antioxidative agents or processes. Phosphorylation of occludin has been described at multiple sites and is proposed to play a regulatory role in tight junction assembly and maintenance and, hence, to influence tissue barrier characteristics. Redox-dependent signal transduction mechanisms are among the pathways modulating occludin phosphorylation and function. This review discusses the novel concept that occludin plays a key role in the redox regulation of tight junctions, which has a major impact in pathologies related to oxidative stress and corresponding pharmacologic interventions.

Effect of radiofrequency electromagnetic field exposure on in vitro models of neurodegenerative disease.

In this work we tested viability, proliferation, and vulnerability of neural cells, after continuous radiofrequency (RF) electromagnetic fields exposure (global system for mobile telecommunications (GSM) modulated 900 MHz signal at a specific absorption rate (SAR) of 1 W/kg and maximum duration 144 h) generated by transverse electromagnetic cells. We used two cellular systems, SN56 cholinergic for example, SN56 cholinergic cell line and rat primary cortical neurons, and well-known neurotoxic challenges, such as glutamate, 25-35AA beta-amyloid, and hydrogen peroxide. Exposure to RF did not change viability/proliferation rate of the SN56 cholinergic cells or viability of cortical neurons. Co-exposure to RF exacerbated neurotoxic effect of hydrogen peroxide in SN56, but not in primary cortical neurons, whereas no cooperative effects of RF with glutamate and 25-35AA beta-amyloid were found. These data suggest that only under particular circumstances exposure to GSM modulated, 900 MHz signal act as a co-stressor for oxidative damage of neural cells.

Continuous exposure to 900MHz GSM-modulated EMF alters morphological maturation of neural cells.

The effects of radiofrequency electromagnetic field (RF-EMF) exposure on neuronal phenotype maturation have been studied in two different in vitro models: murine SN56 cholinergic cell line and rat primary cortical neurons. The samples were exposed at a dose of 1W/kg at 900 MHz GSM modulated. The phenotype analysis was carried out at 48 and 72 h (24 and 48 h of SN56 cell line differentiation) or at 24, 72, 120 h (2, 4 and 6 days in vitro for cortical neurons) of exposure, on live and immunolabeled neurons, and included the morphological study of neurite emission, outgrowth and branching. Moreover, cortical neurons were studied to detect alterations in the expression pattern of cytoskeleton regulating factors, e.g. beta-thymosin, and of early genes, e.g. c-Fos and c-Jun through real-time PCR on mRNA extracted after 24h exposure to EMF. We found that RF-EMF exposure reduced the number of neurites generated by both cell systems, and this alteration correlates to increased expression of beta-thymosin mRNA.