Catechol-O-methyltransferase (COMT),ED50 < 1.4 mg⋅kg(-1)

Opicapone prolongs the bioavailability of L-DOPA without being harmful since it inhibits peripheral COMT over an extended period of time. Opicapone's IC50 value of 98 μM indicates a reduction in cellular ATP level. After subjecting human primary hepatocytes to increasing concentrations of Ro 40-7592, OR-611, or Opicapone for a full day, the ratio of JC-1 aggregates to JC-1 Evaluate the monomer (ratio of λex 544 λem 590 to λex 485 λem 538) revealed a concentration-dependent decrease in cellular mitochondrial membrane potential. Opicapone has an IC50 of 181 μM, which lowers the potential of the cell's mitochondrial membrane[1].

Rats' peripheral COMT is inhibited by opticapartone, which has an ED50 value of less than 1.4 mg/kg six hours after injection. Within the first eight hours, the effect remained, and by the twenty-four-hour mark, COMT had not returned to control levels. While Ro 40-7592 showed a significant effect only two hours after administration, a single injection of Opicapone resulted in a sustained increase in plasma L-DOPA levels and a concurrent drop in 3-OMD from two to twenty-four hours after administration. Opicapone's effects on brain catecholamines following the administration of L-DOPA can persist for up to 24 hours. After a 24-hour incubation period, opicapone is also the least efficient drug in decreasing the ATP level and mitochondrial membrane potential in human primary hepatocytes [1].

COMT activity
On the day of the experiment, erythrocytes were thawed in ice and haemolysed by the addition of four volumes of MilliQ water. After vigorous mixing, the tubes were kept on ice for 10 min, after which they were centrifuged at 20 000× g at 4°C for 20 min. The supernatant collected was used for the COMT activity assay. Tissues were thawed on ice. Liver fragments were homogenized in a Precellys 24 Dual Tissue Homogenizer (Bertin Corporation, Washington DC, USA) for two cycles of 5 s with an interval of 5 min on ice. Kidney and brains were homogenized with Silent Crusher M homogenizer (Heidolph, Schwabach, Germany) with probe 8F/M for about 45 s at maximum velocity. Homogenates were used for COMT activity determination. Total protein in homogenates and haemolysed samples was determined with the BioRad Protein Assay (BioRad, Hercules, CA, USA) using a standard curve of BSA (50–250 μg⋅mL−1).[1]
COMT activity was determined using adrenaline as a substrate and measuring the metanephrine formed as described previously (Bonifácio et al., 2003). In brief, the reaction mix (total volume of 1000 μL) contained 500 μL sample (2 mg total protein), pargyline (100 μM), magnesium chloride (100 μM), EGTA (1 mM), S-adenosylmethionine (500 μM for liver and erythrocytes; 250 μM for kidney and 100 μM for brain) and adrenaline (1000 μM for liver, erythrocytes, kidney and 100 μM for brain) in 5 mM phosphate buffer pH 7.8. Reactions were started with the substrate and were then carried out for 5 min (liver), 10 min (erythrocytes and kidney) or 15 min (brain) at 37°C. Reactions were stopped by the addition of 200 μL 2 M PCA and, after deproteinization, samples were injected onto HPLC-ED.[1]

ATP assay
ATP content of human primary hepatocytes was determined using the ATP Lite assay system (Perkin Elmer, Waltham, MA, USA), 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 were washed with Hank's balanced salt solution (HBSS) and were 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) were run in parallel. After incubation, media were removed from the wells and substituted with 100 μL HBSS plus 50 μL cell lysis solution. Plates were shaken for 5 min at 400 r.p.m. at room temperature. Substrate solution (50 μL) was then added to each well and plates were 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 were run in parallel in plate wells without cells. Plates were dark adapted for 10 min and luminescence was determined on a MicrobetaTriLux (Perkin Elmer) scintillation counter.[1]
Mitochondrial membrane potential assay
Mitochondrial membrane potential evaluation in human primary hepatocytes was performed using the fluorescent dye JC-1. JC-1 selectively enters into the mitochondria and, while in healthy cells with high mitochondrial membrane potential, JC-1 spontaneously forms complexes with intense red fluorescence, in apoptotic or unhealthy cells with low membrane potential, JC-1 remains in the monomeric form showing green fluorescence. Twenty-four hours after being seeded, cell cultures were washed with HBSS and 100 μL 15 μM JC-1 prepared in HBSS were added. After 1 h incubation at 37°C in the dark, the solution was removed from the wells and cells were washed once with 100 μL HBSS. Cells were then incubated with test compounds prepared in culture media without FBS (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. Negative controls (no compound) and positive controls (cells incubated with FCCP, 10 and 50 μM) were run in parallel. After the incubation, media were removed from the cells and substituted with 100 μL HBSS. Fluorescence was measured using a fluorescence microplate reader (Spectramax Gemini, Molecular Devices, Sunnyvale, CA, USA) at λex485 nm, λem538 nm (green) and λex544 nm, λem590 nm (red). Mitochondrial membrane potential was calculated as: ratio = λex544λem590/λex485λem538.[1]

Using 240 male Wistar rats. In the experiment designed to evaluate the inhibitory effect of this compound on COMT, animals were given Opicapone (0.03, 0.1, 0.3, 0.6, 1, 3, and 10mg/kg) and killed at 2 and 6 hours after administration. In the experiment aimed at evaluating the COMT time activity curve, animals were administered Opicapone (3 mg/kg) and euthanized at different post administration time periods (15 and 30 minutes, as well as 1, 2, 4, 8, 18, 24, and 48 hours). In the experiment aimed at evaluating the effect of compounds on central catecholamines, animals were given 3 mg/kg of Opicapone or Ro 40-7592, and one hour before euthanasia, the animals were given L-DOPA/benzalkoxyl (L-DOPA 12 mg/kg and benzalkoxyl 3 mg/kg).[1]

Absorption, Distribution and Excretion
Orally administered opicapone demonstrates a linear, dose-dependent absorption profile. Opicapone is rapidly absorbed, with an oral bioavailability of about 20%. Following administration of a single 50 mg dose of opicapone, the median Tmax was two hours, ranging from one to four hours. A moderate fat or moderate calorie meal was shown to decrease the Cmax by 62%, the mean overall plasma exposure (AUC) by 31%, and the Tmax by 4 hours.
Following administration of a single 100 mg dose of radiolabeled opicapone in healthy subjects, about 70% of the total dose was recovered in feces, where 22% of the recovered dose was excreted as an unchanged parent drug. About 20% of the total dose was recovered in exhaled air and about 5% was recovered in the urine, where less than 1% of the recovered dose was in an unchanged form. The primary detectable metabolite in the urine was the glucuronide metabolite.
Following oral administration, the apparent V_d of opicapone at a dose of 50 mg was 29 L with an inter-subject variability of 36%. One study showed small systemic accumulation after multiple-dosing.
Following oral administration of 50 mg opicapone, the apparent total body clearance was 22 L/h, with an inter-subject variability of 45%.

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Metabolism / Metabolites
According to clinical and _in vitro_ studies, sulphation is the primary metabolic pathway of opicapone, forming the inactive metabolite. Opicapone can also undergo glucuronidation, COMT-mediated methylation, reduction, and glutathione conjugation. As two major circulating metabolites, BIA 9-1103 (3-O-sulphated opicapone) accounts for 67.1% of the total radioactivity and BIA 9-1104 (4-O-methylated opicapone) accounts for 20.5% of the total radioactivity. Other metabolites are generally unquantifiable in plasma samples. Opicapone can undergo N-oxide reduction to form BIA 9-1079, which was shown to be an active metabolite in non-clinical studies; however, it is generally undetectable in humans. Other inactive metabolites include BIA 9-1100, BIA 9-1101, and BIA 9-1106.

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Biological Half-Life
The mean elimination half-life of opicapone is one to two hours. Despite the short half-life, the observed half-life of opicapone-induced COMT inhibition in human red blood cells was 61.6 hours with a standard deviation of 37.6 hours.

Hepatotoxicity
In prelicensure controlled trials, serum ALT elevations occurred uncommonly in opicapone-treated subjects and in rates similar to that of placebo controls. In studies of more than 1000 patients treated with opicapone there were no instances of serious hepatic events and no relevant changes in serum enzymes. After its approval and more widespread use, there have been no reports clinically apparent liver injury attributable to opicapone. There has, however, been limited clinical experience with its use.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Opicapone is >99% bound to plasma proteins, which is independent of the drug concentration.

[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 L-DOPA 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

Opicapone is a ring assembly and an oxadiazole.
Opicapone is a potent, reversible, and peripherally-acting third-generation inhibitor of catechol-o-methyltransferase (COMT), an enzyme involved in the breakdown of various catecholamines including dopamine. Many patients with Parkinson’s disease treated with levodopa plus a dopa decarboxylase (DDC) inhibitor (eg carbidopa) experience motor complications over time, which calls for the management of these symptoms through the use of a dopamine agonist, a monoamine oxidase B inhibitor (selegiline, rasagiline), a catechol-O-methyl transferase (COMT) inhibitor, or amantadine, or using a modified-release formulation of levodopa. Opicapone is used for adjunct therapy to levodopa and carbidopa in adult patients with Parkinson's disease and end-of-dose motor fluctuations. Opicapone was approved for use by the European Commission in June 2016 and the FDA in April 2020. It is marketed under the brand name Ongentys as once-daily oral capsules. Exhibiting a long duration of action that exceeds 24 hours, opicapone can be administered once-daily and demonstrates the lowest risk for cytotoxicity compared to other catechol-O-methyltransferase inhibitors.
Opicapone is a Catechol-O-Methyltransferase Inhibitor. The mechanism of action of opicapone is as a Catechol O-Methyltransferase Inhibitor.
Opicapone is a catechol-O-methyltransferase inhibitor that is used as adjunctive therapy to levodopa/carbidopa in patients with Parkinson disease experiencing difficulty with “off” episodes when motor symptoms breakthrough on treatment. Opicapone has been associated with a minimal rate of serum enzyme elevations during therapy and has not been linked to cases of clinically apparent liver injury with jaundice.

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Drug Indication
Opicapone is indicated as adjunctive therapy in adults with Parkinson’s disease and end-of-dose motor fluctuations or “off” episodes whose symptoms cannot be stabilized on the combination therapy of levodopa and DOPA decarboxylase inhibitor (e.g., carbidopa).
Ontilyv is indicated as adjunctive therapy to preparations of levodopa/ DOPA decarboxylase inhibitors (DDCI) in adult patients with Parkinson's disease and end-of-dose motor fluctuations who cannot be stabilised on those combinations.
Ongentys is indicated as adjunctive therapy to preparations of levodopa/ DOPA decarboxylase inhibitors (DDCI) in adult patients with Parkinson's disease and end-of-dose motor fluctuations who cannot be stabilised on those combinations. Ongentys is indicated as adjunctive therapy to preparations of levodopa/ DOPA decarboxylase inhibitors (DDCI) in adult patients with Parkinson's disease and end-of-dose motor fluctuations who cannot be stabilised on those combinations.

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Mechanism of Action
Levodopa (L-Dopa) is the gold standard for managing motor and some non-motor symptoms associated with Parkinson's Disease; however, only a small fraction of administered L-Dopa actually crosses the blood-brain barrier to exert its therapeutic action and patients face the risk of developing end-of-dose motor fluctuations, which reflects the rapid peripheral metabolism of L-dopa by aromatic L-amino acid decarboxylase and catechol-O-methyltransferase (COMT). Opicapone is a peripheral, selective, and reversible catechol-O-methyltransferase (COMT) inhibitor. It displays a high binding affinity that is in sub-picomolar ranges, resulting in a slow complex dissociation rate constant and long duration of action _in vivo_. When opicapone is added to the treatment regimen that contains L-Dopa and DOPA decarboxylase inhibitor, opicapone helps to increase the plasma levels and enhance the therapeutic efficacy of L-Dopa.

C15H10CL2N4O6

413.167

411.998

C, 43.61; H, 2.44; Cl, 17.16; N, 13.56; O, 23.23

923287-50-7

135565903

Light yellow to yellow solid powder

1.80±0.1 g/cm3 (20 °C, 760 mmHg)

701.1±70.0 °C (760 mmHg)

4.598

2

8

2

27

557

0

[O-][N+](C1C(O)=C(O)C=C(C2ON=C(C3C(C)=C(Cl)C(C)=[N+]([O-])C=3Cl)N=2)C=1)=O

ASOADIZOVZTJSR-UHFFFAOYSA-N

InChI=1S/C15H10Cl2N4O6/c1-5-10(13(17)20(24)6(2)11(5)16)14-18-15(27-19-14)7-3-8(21(25)26)12(23)9(22)4-7/h3-4,22-23H,1-2H3

2,5-dichloro-3-(5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl)-4,6-dimethylpyridine 1-oxide

BIA-91067; BIA 91067; BIA 9-1067

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~242.03 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.1 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.5 mg/mL (6.1 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)

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 (Please use freshly prepared in vivo formulations for optimal results.)