Use of a primary rat astrocyte Ca2+ mobilisation assay to profile positive allosteric modulator (PAM) compounds at the mGluR5 receptor

Roberta Milone, Peter Lovell, Melanie Munro, Alison Muir, Alan Wise, Ben Powney, Kim Winborn, Sharon Butler

GlaxoSmithKline, Harlow, Essex, UK

The mGluR5 receptor belongs to the family C subgroup of GPCRs and modulates glutamate neurotransmission in the CNS (Krivoy et al, 2007). We have identified and characterised PAMs at mGluR5, which we anticipate will be useful in the treatment of schizophrenia. Preclinically, compounds such as ADX47273, a characterised mGluR5 PAM, are efficacious in a number of rat behavioural models relevant to this disease (Lui et al, 2008; Le Poul et al, 2005).

In this study, we have developed a 96-well plate based assay to identify candidate compounds for in vivo profiling in schizophrenia rat behavioural models. Rat cortical astrocytes were isolated as described (Mehberg and Miller, 2003) . Fluorescent imaging of calcium responses was measured on a FLuorescent Imaging Plate Reader (FLIPR TETRA) using a calcium-4 dye. Using this system, we assessed the profiles of a standard, ADX47273, and a range of GSK compounds. The increase in intracellular calcium levels upon activation by an EC20 of glutamate was plotted as a measure of modulation. Efficacy values were measured relative to an EC100 of glutamate.

In this work, in line with previous studies, we have shown that ADX47273 modulates glutamate activity at the rat mGluR5 receptor with pEC50 6.7 (+/-0.27, n=6) and efficacy of 61% (n=6). Proprietary compounds showed a range of potencies and efficacies. The efficacy values correlate well with alpha factor data generated, in a CHO cell line expressing the human mGluR5 receptor, from the leftward shift of glutamate concentration-response curves in response to an increasing concentration of the positive modulator compound (Cato et al, 2007).

In conclusion, we have developed an assay which can be used to define efficacy and potency values for mGluR5 PAM. These data can be use to select a range of compounds for further profiling to determine the level of potency and potentiation which is required for in vivo activity in disease models.

Cato et al (2007) Poster presented at Montreal SBS meeting

Krivoy et al (2007) European Neuropsychopharmacology 18, 395-405

Le Poul et al (2005) Poster presented at Taormina mGluR meeting, Sicily

Liu et al (2008) JPET, 327(3):827-39

Meberg and Miller (2003) Methods in Cell Biology 71: 111-127