Synergistic effect of 5-HT2C and 5-HT1B receptor agonists in the reduction of food intake

Barbora Doslikova1,2, Alastair S Garfield1, Jill Shaw1, Mark L Evans2, Denis Burdakov1, Brian Billups1, Lora K Heisler1. 1Department of Pharmacology, University of Cambridge, Tennis Court Road, CB2 1P, Cambridge, UK, 2Metabolic Research Laboratories, Institute of Metabolic Science, University of Cambridge, Addenbrooke\'s Hospital, CB2 0QQ, Cambridge, UK

Obesity is associated with dysregulated energy balance, whereby food intake exceeds energy expenditure. The brain serotonin pathway is an essential component of the neural network regulating appetite. Serotonin’s effects on energy balance are primarily mediated by 5-HT2C and 5-HT1B receptors (5-HT2CR; 5-HT1BR) in the arcuate nucleus of the hypothalamus (ARC). We previously reported that serotonin stimulates ARC neurons expressing the anorectic melanocortin-receptor agonist pro-opiomelanocortin (POMC) via Gq-coupled 5-HT2CRs and reduces an inhibitory postsynaptic input onto POMC neurons via Gi-coupled 5-HT1BRs.

We hypothesised that combined stimulation of 5-HT2CRs and 5-HT1BRs would be significantly more potent in mediating serotonin-induced appetite suppression than either alone.

The study objectives were to assess the effect of 5-HT2CR agonist WAY-161,503 and 5-HT1BR agonist CP-94,253 on (1) food intake in 24-hour fasted adult male C57BL6 mice (27±1 g); (2) ARC in vivo neuronal activation in adult male C57BL6 mice (27±1 g) using c-FOS immunohistochemistry; and (3) intracellular calcium concentrations of ARC POMC neurons in male and female POMC-GFP mice (18±3 g) by calcium imaging.

(1) Our findings confirmed that the 5-HT2CR agonist WAY-161,503 (5, 10, 20 mg/kg, i.p.) and 5-HT1BR agonist CP-94,253 (5, 10, 20 mg/kg, i.p.) dose-dependently reduced 6-hour food intake in C57BL6 mice (n=10; RM ANOVA for treatment effect: F(3,26)=2.372, p=0.09 and F(3,25)=3.024, p<0.05 respectively). Moreover, combined administration of subanorectic doses of both agonists (10 mg/kg WAY-161,503 and 20 mg/kg CP-94,253, i.p., dissolved in autoclaved distilled water) produced a substantial reduction in feeding (approximately 45%, n=10; RM ANOVA for treatment effect: F(3,81)=7.381, p<0.001), suggesting the presence of synergy.

(2) Analysis of c-FOS immunoreactivity in brains collected 2 hours post drug administration revealed significantly higher activation of the ARC neurons in animals treated with the combination of the drugs (10 mg/kg WAY-161,503 and 20 mg/kg CP-94,253; 75 neurons) compared to animals treated with the individual drugs or vehicle (50 neurons; n=10; one-way ANOVA: F(3,22)=5.502, p<0.01).

(3) Finally, we monitored changes in intracellular fura-2-bound calcium concentrations in ARC POMC neurons from brain slices of POMC-GFP mice in response to a four-minute drug infusion. A higher proportion (59%) of ARC POMC neurons were activated by the combination of the drugs (20 uM WAY-161,503 and 200 nM CP-94,253 administered in artificial cerebrospinal fluid) compared to ARC POMC neurons stimulated with either of the drugs alone (WAY-161,503 alone = 36%, CP-94253 alone 30%; n=165).

Collectively, these data suggest that combinatorial 5-HT1BR and 5-HT2CR agonism augments their anorectic potency and is in part mediated via activation of ARC POMC neurons. Exploring the synergy between 5‐HT2CR and 5‐HT1BR agonists further could allow development of more efficacious obesity pharmacotherapies.