Sumatriptan

Cell lines

HEK 293 cells

Preparation Method

HEK 293, a human transformed embryonic kidney cell line, was grown in Dulbecco's modi®ed Eagle's medium containing 1mM sodium pyruvate, 2mM L-glutamine, antibiotics and 10% foetal calf serum. The selection and culture medium for HEK 293 cells expressing the 5-HT 1B receptor contained an extra 800μg/ml geneticin. Geneticin was left out at least 2 days before assay. Cells were grown at 37℃ in humidi®ed air with 5% CO₂. Cells were plated in Multi-well 24 plates. The next day, cells were washed and incubated for 20min in the presence of 10pM-10μM Sumatriptan with controlled salt solution (composition in mM : NaCl=120, KCl=5, MgCl₂=0.8, CaCl₂=1.8, glucose=15 and phenol red=0.04 in 25mM Tris-HCl, pH 7.4) containing either 100μM forskolin or solvent. The incubation was stopped with ice-cold HClO₄. The extract was neutralized to pH 7.5 with K₃PO₄/KOH solution. After precipitation of KClO₄ at 4℃, plates were centrifuged, and the supernatant was assayed for cyclic AMP content with a commercial [ 125 I]-cyclic AMP radioimmunoassay kit according to the procedure recommended by the manufacturer. The cyclic AMP levels were expressed as percentage of forskolin-stimulated cyclic AMP production (set at 100%), and plotted against the drug concentration on a logarithmic scale. Sigmoidal curves of best ®t were calculated by non linear regression analysis. The pIC₅₀ value referred to the concentration of a compound producing half the maximum reduction in cyclic AMP seen with 5-HT.

Reaction Conditions

10pM-10μM; 20min

Applications

Sumatriptan inhibited forskolin-stimulated cAMP accumulation in HEK-293 cells expressing h5-HT1B receptors (IC₅₀=20nM).

Animal models

C57BL6 male mice

Preparation Method

All experiments were conducted on C57BL6 male mice weighing 20-30g. Animals were housed in a 12-h light–dark cycle. Experiments were conducted between 09.00 and 14.00h at a temperature of 22°C. A stock of 5.0mg/ml NTG dissolved in 30% alcohol, 30% propylene glycol, and water was freshly diluted each day in 0.9% saline in a polypropylene tube. Doses of 0.5, 1, 5 or 10mg/kg were administered intraperitoneally. Control mice received an intraperitoneal injection of 0.9% saline. Five minutes after the administration of 10mg/kg NTG, each animal was treated with i.p. Sumatriptan(600μg/kg) or saline. Alternatively, an identical set of NTG-injected mice was treated with a single injection of intrathecal Sumatriptan (0.06μg in 5μl) or saline at 5min after NTG administration. Animals were habituated to the testing apparatus for 60min on the day prior to and again immediately before determination of baseline nociceptive thresholds. Each group of animals underwent one of two different tests before and following NTG injection. Hargreaves assay was used to determine thermal nociceptive thresholds. Mechanical nociceptive thresholds were determined with von Frey monofilaments.

Dosage form

600μg/kg; i.p.

Applications

Sumatriptan reversed nitroglycerin (NTG)-induced thermal and mechanical allodynia in male C57BL/6 mice.

References:
[1] Lesage AS, Wouters R, Van Gompel P, et al. Agonistic properties of alniditan, sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines. Br J Pharmacol. 1998;123(8):1655-1665.
[2] Bates EA, Nikai T, Brennan KC, et al. Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice. Cephalalgia. 2010;30(2):170-178.