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Research Area
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Description
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Cancer |
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Biological Activity
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Description
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Nepicastat hydrochloride is a novel, potent and selective inhibitor of both bovine and human dopamine-β-hydroxylase with IC50 of 8.5 nM and 9 nM, respectively. |
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Targets
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Bovine dopamine-beta-hydroxylase |
Human dopamine-beta-hydroxylase |
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IC50 |
8.5 nM [1] |
9 nM [1] |
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In Vitro
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In vitro, Nepicastat hydrochloride shows the selective and concentration-dependent inhibition effects on bovine and human dopamine-beta-hydroxylase activity with IC50 of 8.5 nM and 9.0 nM, respectively. While Nepicastat hydrochloride has negligible affinity for twelve other enzymes and thirteen neurotransmitter receptors. [1] |
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In Vivo
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In the artery, left ventricle and cerebral cortex of spontaneously hypertensive rats (SHRs), Nepicastat hydrochloride reduces noradrenaline content, and increases dopamine content and dopamine/noradrenaline ratio in a dose-dependent manner. In addition, Nepicastat hydrochloride also produces the similar effects on noradrenaline, dopamine and dopamine/noradrenaline ratio in tissues and plasma of beagle dogs. [1] In inactin-anesthetized SHRs, Nepicastat hydrochloride (3 mg/kg, i.v.) produces the antihypertensive effects and causes a significant decrease in renal vascular resistance (38%) and an increase in renal blood flow (22%). [2] In dogs with chronic heart failure, low-dose Nepicastat hydrochloride (0.5 mg/kg) prevents left ventricular (LV) dysfunction and remodeling, and combination therapy of Nepicastat hydrochloride and enalapril results in additional improvements in all morphological features. [3] In rat brain, Nepicastat hydrochloride at a dose of 50 mg/kg ( i.p.) leads to the reduction of norepinephrine (NE) and blocks cocaine-primed reinstatement of cocaine seeking. [4] |
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Clinical Trials
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Nepicastat hydrochloride is currently in Phase II clinical trials in patients with Posttraumatic Stress Disorder. |
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Features
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Protocol
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Kinase Assay
[1]
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| In vitro studies |
Bovine and human dopamine-beta-hydroxylase activity are assayed by measuring the conversion of tyramine to octopamine. Human dopamine-beta-hydroxylase is purified from the culture medium of the neuroblastoma cell line SK-N-SH. The assay is performed at pH 5.2 and 32°C in a medium containing 0.125 M sodium acetate, 10 mM fumarate, 0.5 ± 2 μM CuSO4, 0.1 mg/mL catalase, 0.1 mM tyramine and 4 mM ascorbate. In a typical assay, 0.5 ± 1 mu of enzyme are added to the reaction mixture and, subsequently, a substrate mixture containing catalase, tyramine and ascorbate is added to initiate the reaction (final volume of 0.2 mL). Samples are incubated with or without the appropriate concentration of nepicastat (RS-25560-197, S-enantiomer) or RS-25560-198 (R-enantiomer) at 37 °C for 30-40 minutes. The reaction is quenched by the stop solution containing 25 mM EDTA and 240 μM 3-hydroxytyramine (internal standard). The samples are analysed for octopamine by reverse phase high pressure liquid chromatography (h.p.l.c.) with ultraviolet (u.v.)-detection at 280 nM. The h.p.l.c. run is carried out at a flow rate of 1 mL/min with a LiChroCART 125-4 RP-18 column and isocratic elution with 10 mM acetic acid, 10 mM 1-heptane sulphonic acid, 12 mM tetrabutyl ammonium phosphate and 10% methanol. The remaining % activity is calculated based on controls (without RS 25560), corrected with internal standards and fitted to a non-linear four-parameter concentration-response curve. The activity of nepicastat at twelve selected enzymes and receptors is determined by use of established assays. Binding data are analysed by iterative curve-fitting to a four parameter logistic equation. Ki values are calculated from IC50 values by the Cheng-Pruso? equation. Enzyme inhibitory activity is expressed as IC50 (concentration required to produce 50% inhibition of enzyme activity). |
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Animal Study
[1]
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| Animal Models |
Spontaneously hypertensive rats (SHRs). |
| Formulation |
Nepicastat hydrochloride is dissolved in distilled water. |
| Doses |
≤100 mg/kg |
| Administration |
Administered via p.o. |
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References |
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[1] Stanley WC, et al. Br J Pharmacol. 1997, 121(8), 1803-1809.
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[2] Stanley WC, et al. J Cardiovasc Pharmacol. 1998, 31(6), 963-970.
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[3] Sabbah HN, et al. Circulation. 2000, 102(16), 1990-1995.
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[4] Schroeder JP, et al. Neuropsychopharmacology. 2010, 35(12), 2440-2449.
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