Background
Opicapone is a peripherally selective inhibitor of catechol-O-methyltransferase (COMT).1 It inhibits COMT activity in rat liver, but not brain, homogenates (ED50 = 1.05 mg/kg). Opicapone (3 mg/kg) increases plasma levels of L-DOPA and reduces plasma levels of 3-O-methyl-DOPA (3-OMD) when administered in combination with benserazide and L-DOPA.2 Formulations containing opicapone have been used as adjuvants in the treatment of “off” episodes associated with Parkinson’s disease.
1.Kiss, L.E., Ferriera, H.S., Torr?o, L., et al.Discovery of a long-acting, peripherally selective inhibitor of catechol-O-methyltransferaseJ. Med. Chem.53(8)3396-3411(2010)
2.Bonifácio, M.J., Torr?o, L., Loureiro, A.I., et al.Pharmacological profile of opicapone, a third generation nitrocatechol catechol-O-methyl transferase inhibitor, in the ratBr. J. Pharmacol.172(7)1739-1752(2014)
Protocol
Kinase experiment: | ATP content of human primary hepatocytes is determined using the ATP Lite assay system, which is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. Twenty-four hours after being seeded, cell cultures are washed with Hank's balanced salt solution (HBSS) and are then incubated with test compounds prepared in culture media without fetal bovine serum (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 μM) for 24 h at 37°C in humidified 5% CO2-95% air. Positive controls (cells incubated with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone-FCCP, 10 and 50 μM) are run in parallel. After incubation, media are removed from the wells and substituted with 100 μL HBSS plus 50 μL cell lysis solution. Plates are shaken for 5 min at 400 r.p.m. at room temperature. Substrate solution (50 μL) is then added to each well and plates are again shook for 5 min at 400 r.p.m. at room temperature in subdued light. Three standard concentrations of ATP (1, 10 and 100 μM) and blanks are run in parallel in plate wells without cells. Plates are dark adapted for 10 min and luminescence is determined on a MicrobetaTriLux scintillation counter[1]. |
Animal experiment: | Rats[1] Male Wistar rats (240) are used. In experiments designed to evaluate the efficacy of the compound at inhibiting COMT, animals are administered Opicapone (0.03, 0.1, 0.3, 0.6, 1, 3 and 10 mg/kg) and are killed at 2 and 6 h post-administration. In experiments designed to evaluate COMT time-activity profile, animals are given Opicapone (3 mg/kg) and are killed at different post-administration periods (15 and 30 min, and 1, 2, 4, 8, 18, 24, and 48 h). In experiments designed to evaluate the effects of the compounds on central catecholamines, animals are given 3 mg/kg Opicapone or Tolcapone and 1 h before being killed, animals are administered levodopa/benserazide (levodopa 12 mg/kg and benserazide 3 mg/kg). |
参考文献: [1]. Bonifácio MJ, et al. Pharmacological profile of Opicapone, a third-generation nitrocatechol catechol-O-methyl transferase inhibitor, in the rat. Br J Pharmacol. 2015 Apr;172(7):1739-52.
[2]. Ferreira JJ, et al. Opicapone as an adjunct to levodopa in patients with Parkinson's disease and end-of-dose motor fluctuations: a randomised, double-blind, controlled trial. Lancet Neurol. 2016 Feb;15(2):154-165. |
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