Jörg Huwyler

The effect of the mGlu5 receptor antagonist MPEP in rodent tests of anxiety and cognition: a comparison

RationaleAntagonists at the metabotropic glutamate 5 (mGlu5) receptor produce robust anxiolytic effects in a number of rat tests. However, there is evidence that mGlu5 receptor antagonists may also impair working memory and spatial learning following intracerebroventricular administration.ObjectivesThe aim of this study is to compare the effect of the potent and selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-piperidine (MPEP), administered systemically on rodent tests of cognition and anxiety.MethodsMPEP was assessed in the following rodent tests, 60xa0min following oral administration: Geller–Seifter conflict, conditioned emotional response (CER), Vogel conflict, delayed match to position (DMTP) and Morris water maze. Diazepam was also tested as a comparator.ResultsMPEP had a significant anxiolytic effect, comparable in magnitude to diazepam, at 10–30xa0mg/kg in the two conflict and CER tasks. There was no effect of MPEP up to 30xa0mg/kg on working memory in the DMTP task, but at 100xa0mg/kg, there was a significant reduction in choice accuracy at the longest delay interval (24xa0s). MPEP (3–30xa0mg/kg) did not significantly impair spatial learning in the Morris water maze, although during the last probe trial, 30-mg/kg-treated rats were significantly less accurate than controls. In contrast, diazepam significantly impaired performance in both the DMTP and Morris water maze tests. Assessment of plasma and brain concentration of MPEP ∼75xa0min following oral administration showed a dose linearity from 3 to 30xa0mg/kg and good brain penetration, i.e. a brain/plasma ratio of 3.1.ConclusionsOral administration of the selective mGlu5 receptor antagonist MPEP induces a robust anxiolytic-like effect in rat conflict tests comparable to that seen with diazepam, but in contrast to diazepam, MPEP does not impair working memory or spatial learning at anxiolytic doses.

Targeting of the photocytotoxic compound AlPcS4 to hela cells by transferrin conjugated peg-liposomes

Photodynamic therapy has attracted increasing interest over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species (ROS), such as singlet oxygen, which are cytotoxic. The goal of our study was to enhance the photodynamic activity of the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) through its specific delivery to tumor cells. Since many tumor cells, among which are HeLa cells, overexpress the transferrin receptor, we synthesized transferrin conjugated PEG‐liposomes that contained AlPcS4 that could be internalized by receptor mediated endocytosis. The antiproliferative activity of the targeted liposomes was evaluated and compared to the native AlPcS4 and the non‐targeted liposome. These findings were supplemented with data on intracellular concentration of the photo‐active compounds. The accumulation together with ROS production after irradiation was visualized by using confocal microscopy to confirm the data found in the antiproliferative and accumulation assay. Tf‐Lip‐AlPcS4 was 10 times more photocytotoxic (IC50, 0.63 μM) than free AlPcS4 at a light dose of 45 kJ/m whereas Lip‐AlPcS4 displayed no photocytotoxicity at all. The high photocytotoxicity of Tf‐Lip‐AlPcS4 was shown to be the result of a high intracellular concentration (136.5 μM) in HeLa cells, which could be lowered dramatically by incubating the conjugate with a competing transferrin concentration. The images of intracellular accumulation and ROS production matched the accumulation and photocytotoxicity profile of the different photo‐active compounds. The photodynamic activity of the Tf‐Lip‐AlPcS4 conjugate on HeLa cells is much more potent than free AlPcS4 as a result of selective transferrin receptor mediated uptake.

Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood–brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1( MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture conditions.

Improved oral bioavailability of cyclosporin A in male Wistar rats. Comparison of a Solutol HS 15 containing self-dispersing formulation and a microsuspension.

Oral bioavailability of the highly lipophilic and poorly water-soluble immunosuppressive agent cyclosporin A (CyA) in two different formulations was investigated in male Wistar rats. An aqueous microsuspension and a self-dispersing formulation composed of the surface-active ingredients Solutol HS 15:Labrafil M2125CS:oleic acid=7:2:1 (v/v/v) were administered to the animals at a dose level of 20 mg/kg. In order to calculate the absolute oral bioavailability, CyA was additionally administered intravenously at 10 mg/kg as microsuspension. It was found that the oral bioavailability of CyA in the Solutol HS 15-based formulation was twofold higher as compared to the microsuspension (69.9+/-2.8 vs. 35.7+/-3.3%, P=0.001). By contrast, the time to reach maximum plasma concentration (t(max)) and the terminal half-life (t(1/2)) did not differ significantly with the different formulations (t(max): 7.0+/-1.0 vs. 6.3+/-1.7 h; t(1/2): 20.5+/-2.9 vs. 16.7+/-4.7 h). In vitro solubility experiments demonstrated a marked increase in the aqueous solubility of CyA in the presence of the self-dispersing formulation as compared to the micronized powder alone (solubility after 120 min at 37 degrees C: 136 vs. 23.2 microg/ml in human gastric juice; 133 vs. 10.8 microg/ml in simulated intestinal juice). Most likely, the enhanced systemic exposure of CyA in the self-dispersing formulation was caused by improved solubility of CyA in the gastrointestinal fluids in the presence of the surface-active ingredients. Additional factors that may have contributed to increased oral bioavailability are inhibition of metabolism and/or transport processes as well as permeability enhancement by the co-administered excipients.

Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides as potent, orally available mGlu1 receptor enhancers.

Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides with improved pharmacokinetic properties have been designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists.

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. These compounds were able to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus and in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration.