Gianluca Lozza

Blockade of adenosine A2A receptors by SCH 58261 results in neuroprotective effects in cerebral ischaemia in rats.

BLOCKADE of adenosine receptors can reduce cerebral infarct size in the model of global ischaemia. Using the potent and selective A2A adenosine receptor antagonist, SCH 58261, we assessed whether A2A receptors are involved in the neuronal damage following focal cerebral ischaemia as induced by occluding the left middle cerebral artery. SCH 58261 (0.01 mg/kg either i.p. or i.v.) administered to normotensive rats 10min after ischaemia markedly reduced cortical infarct volume as measured 24 h later (30% vs controls, p < 0.05). Similar effects were observed when SCH 58261 (0.01 mg/kg, i.p.) was administered to hypertensive rats (28% infarct volume reduction vs controls, p < 0.05). Neuroprotective properties of SCH 58261 administered after ischaemia indicate that blockade of A2A adenosine receptors is a potentially useful biological target for the reduction of brain injury.

Interleukin‐10 modulates neuronal threshold of vulnerability to ischaemic damage

Interleukin‐10 (IL‐10) is a powerful suppressor of cellular immune responses, with a postulated role in brain inflammation. First, we have evaluated the role of this cytokine in ischaemic brain damage using IL‐10 knockout (IL‐10–/–) mice. The middle cerebral artery (MCA) was occluded in either IL‐10–/– or wild‐type animals of corresponding strain (C57Bl/6) and age. Infarct volume was assessed 24 h later in serial brain sections. Brain infarct produced by MCA occlusion was 30% larger in the IL‐10–/– than in wild‐type mice (21.8 ± 1.2 vs. 16.9 ± 1.0 mm3, respectively; P < 0.01; Students t‐test). To further characterize these findings, studies were extended to in vitro models. Primary neuronal cortical cultures derived from IL‐10–/– animals were more susceptible to both excitotoxicity and combined oxygen–glucose deprivation compared with cell cultures from wild‐type mice. Moreover, when added to the culture medium, recombinant murine IL‐10 (0.1–100 ng/mL) exerted a concentration‐dependent prevention of neuronal damage induced by excitotoxicity in both cortical and cerebellar granule cell cultures taken from either strain. The accordance of in vivo and in vitro data allows us to suggest a potential neuroprotective role of IL‐10 against cerebral ischaemia when administered exogenously or made available from endogenous sources.

Age-progressing cognitive impairments and neuropathology in transgenic CRND8 mice

Patients with Alzheimers disease suffer from progressive cognitive impairments and show distinct post-mortem neuropathology, including beta-amyloid plaques. Transgenic (Tg) CRND8 mice carry a mutated human amyloid precursor protein gene and show age-related increases in beta-amyloid production and plaque deposition. It was previously reported that during the early stages of plaque deposition, Tg CRND8 mice demonstrated Morris maze impairments. However, it is unknown if Tg mice would be impaired at an earlier age prior to plaque deposition or more impaired at a later age with more extensive plaque deposition. In the current study, we describe Tg CRND8 age-progressing beta-amyloid neuropathology and cognitive abilities in greater detail. At all ages, Tg mice showed normal short-term memory in the Y-maze. Pre-plaque Tg and age-matched Non-Tg mice did not differ in learning the spatial Morris water maze. However, both early and late plaque Tg mice showed impairments during acquisition. In addition, although early plaque Tg mice performed well in the probe trial, late plaque Tg mice demonstrated impaired probe trial performance. Therefore compared to their Non-Tg littermates, Tg CRND8 mice demonstrate cognitive impairments that progressed with age and seemed to coincide with the onset of beta-amyloid plaque deposition.

Lack of tolerance to motor stimulant effects of a selective adenosine A2A receptor antagonist

It is well known that tolerance develops to the actions of caffeine, which acts as an antagonist on adenosine A(1) and A(2A) receptors. Since selective adenosine A(2A) antagonists have been proposed as adjuncts to 3,4-dihydroxyphenylalanine (L-DOPA) therapy in Parkinsons disease we wanted to examine if tolerance also develops to the selective A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo [1,5-c]pyrimidine (SCH 58261). SCH 58261 (0.1 and 7.5 mg/kg) increased basal locomotion and the motor stimulation afforded by apomorphine. Neither effect was subject to tolerance following long-term treatment with the same doses given intraperitoneally twice daily. There were no adaptive changes in A(1) and A(2A) adenosine receptors or their corresponding messenger RNA or in dopamine D(1) or D(2) receptors. These results demonstrate that the tolerance that develops to caffeine is not secondary to its inhibition of adenosine A(2A) receptors. The results also offer hope that long-term treatment with an adenosine A(2A) receptor antagonist may be possible in man.

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression.

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Nonsteroidal anti-inflammatory drugs increase tumor necrosis factor production in the periphery but not in the central nervous system in mice and rats

Abstract: Nonsteroidal anti‐inflammatory drugs (NSAIDs), which inhibit prostaglandin (PG) synthesis, augment production of tumor necrosis factor (TNF) in most experimental models. We investigated the effect of two NSAIDs, indomethacin and ibuprofen, on the production of TNF in the CNS induced by intracerebroventricular injection of lipopolysaccharide (LPS). Indomethacin and ibuprofen, administered intraperitoneally, augmented (three‐ to ninefold) the levels of TNF in serum and peripheral organs of mice injected intraperitoneally with LPS and in rats with adjuvant arthritis (up to a sevenfold increase). However, NSAIDs (intraperitoneally or intracerebroventricularly) did not increase brain TNF production induced by intravenous LPS. In fact, indomethacin decreased (1.4–1.8‐fold) TNF levels in the spinal cord of rats with experimental autoimmune encephalomyelitis and in the cortex of rats with focal cerebral ischemia. Systemic administration of iloprost inhibited serum TNF levels after intraperitoneal LPS, whereas intracerebroventricular injection of iloprost or PGE2 did not inhibit brain TNF induced by intracerebroventricular LPS. Both peripheral and central TNF productions were inhibited by cyclic AMP level‐elevating agents or dexamethasone. Thus, a PG‐driven negative feedback controls TNF production in the periphery but not in the CNS.

Ecdysone-based system for controlled inducible expression of metabotropic glutamate receptor subtypes 2, 5, and 8

Stable and inducible expression of human metabotropic glutamate receptor types 2, 5, and 8 was achieved in HEK293 cells using the ecdysone inducible system. Treatment of the respective cell lines with ponasterone A resulted in time and concentration-dependent induction of receptor expression. In all cases, the functional activation of receptorswas determined bymeasuring increases in intracellular calcium. The physiologically G• i-coupled receptors mGluR2 andm GluR8 were successfully coupled to phospholipase Cactivation using the chimeric Gprotein G• q/o. The pharmacological properties of recombinant receptors were characterized and proved to be similar to native receptors. Our data suggest that the ecdysone system has a number of characteristics thatmake itwell suited for expressing mGluRs and that the combined use of this systemand chimeric G proteins allows receptors to be characterized using a rapid and straightforward Ca 2+assay.

Tricyclic thienopyridine-pyrimidones/thienopyrimidine-pyrimidones as orally efficacious mGluR1 antagonists for neuropathic pain.

Introduction of small unsaturated alkylamino groups at the 4-position of the A-ring of the tricyclic framework (triazafluorenone) afforded extremely potent and selective mGluR1 antagonists with desirable properties. Compounds 11q and 11s are active in the SNL pain model with ED(50)s 3.3 and 6.4 mg/kg respectively. Metabolic outcome of propargyl amino moiety was studied.

Fused tricyclic mGluR1 antagonists for the treatment of neuropathic pain

A series of fused tricyclic mGluR1 antagonists containing a pyridone ring were synthesized. In vitro, these antagonists were potent against both human and rat isozymes, as well as selective for inhibiting mGluR1 over mGluR5. When dosed orally, several examples were active in vivo in a rat SNL test.

Evaluation of Cannabinoid Receptor 2 and Metabotropic Glutamate Receptor 1 Functional Responses Using a Cell Impedance–Based Technology

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca2+ levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca2+ assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance–based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.