Objective-Conventional anticonvulsants reduce neuronal excitability through effects on ion channels and synaptic function. Anticonvulsant mechanisms of the ketogenic diet remain incompletely understood. Since carbohydrates are restricted in patients on the ketogenic diet, we evaluated the effects of limiting carbohydrate availability by reducing glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in experimental models of seizures and epilepsy.Methods-Acute anticonvulsant actions of 2DG were assessed in vitro in rat hippocampal slices perfused with 7.5mM [K + ] o , 4-aminopyridine (4-AP), or bicuculline and in vivo against seizures evoked by 6 Hz stimulation in mice, audiogenic stimulation in Fring's mice, and maximal electroshock and subcutaneous Metrazol in rats. Chronic antiepileptic effects of 2DG were evaluated in rats kindled from olfactory bulb or perforant path. Interpretation-The glycolytic inhibitor 2DG exerts acute anticonvulsant and chronic antiepileptic actions and has a novel pattern of effectiveness in preclinical screening models. These results identify metabolic regulation as a potential therapeutic target for seizure suppression and modification of epileptogenesis. Results-2DG

The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism initiated through NF-kappaB to terminate the activation of human and mouse macrophages.

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A(1) adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A(1)AR (hA(1)AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH(2))(n)()- bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA(1)AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.