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
Oral absorption of octopaine follows a linear dose-dependent pattern. Opicapone is rapidly absorbed, with an oral bioavailability of approximately 20%. After a single 50 mg dose of octopaine, the median time to peak concentration (Tmax) is 2 hours, ranging from 1 to 4 hours. A moderate-fat or moderate-calorie diet can reduce Cmax by 62%, mean total plasma exposure (AUC) by 31%, and Tmax by 4 hours. In healthy subjects, after a single 100 mg dose of radiolabeled octopaine, approximately 70% of the total dose is recovered in feces, of which 22% is excreted unchanged. Approximately 20% of the total dose is recovered in exhaled breath, and approximately 5% is recovered in urine, of which less than 1% is excreted unchanged. The major metabolite detected in urine is a glucuronide metabolite. Following oral administration, the apparent volume of distribution (Vd) of 50 mg octopaine is 29 L, with an inter-individual coefficient of variation of 36%. One study showed that a small amount of systemic accumulation occurred after multiple doses. Following oral administration of 50 mg octopaine, the apparent systemic clearance was 22 L/h, with an inter-individual coefficient of variation of 45%. Metabolites/Metabolites: According to clinical and in vitro studies, sulfation is the main metabolic pathway of octopaine, producing inactive metabolites. Opicarone can also undergo glucuronidation, COMT-mediated methylation, reduction, and glutathione conjugation. As two major circulating metabolites, BIA 9-1103 (3-O-sulfated octopaine) accounts for 67.1% of total radioactivity, and BIA 9-1104 (4-O-methylated octopaine) accounts for 20.5%. Other metabolites are generally not quantifiable in plasma samples. Opipcapone undergoes an N-oxide reduction reaction to generate BIA 9-1079, which non-clinical studies have shown to be an active metabolite; however, BIA 9-1079 is generally undetectable in humans. Other inactive metabolites include BIA 9-1100, BIA 9-1101, and BIA 9-1106.
Biological Half-Life
The mean elimination half-life of opipcapone is 1 to 2 hours.
Despite the short half-life, the observed half-life of opipcapone-induced COMT inhibition in human erythrocytes is 61.6 hours with a standard deviation of 37.6 hours.

Hepatotoxicity
In pre-market controlled trials, elevated serum ALT levels were uncommon in the ocpicappon treatment group, with an incidence similar to that in the placebo control group. In studies of over 1000 patients treated with ocpicappon, no serious liver events occurred, and no related changes in serum enzymes were observed. Since ocpicappon's approval and widespread use, no clinically significant cases of liver injury have been reported. However, its clinical application experience is limited. Probability Score: E (Unlikely to be a cause of clinically significant liver injury). Protein Binding Opicappon binds to plasma proteins at a rate >99%, regardless of drug concentration.

[1]. 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]. 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

Opipcapone is a cyclic compound belonging to the oxadiazole class. It is a potent, reversible, peripherally acting third-generation catechol-O-methyltransferase (COMT) inhibitor. COMT is an enzyme involved in the breakdown of various catecholamines, including dopamine. Many Parkinson's disease patients treated with levodopa in combination with dopa decarboxylase (DDC) inhibitors (such as carbidopa) develop motor complications over time, requiring the use of dopamine agonists, monoamine oxidase B inhibitors (selegiline, rasagiline), catechol-O-methyltransferase (COMT) inhibitors, or amantadine, or extended-release levodopa formulations to control these symptoms. Opipcapone is used to treat adult Parkinson's disease patients as adjunctive therapy to levodopa and carbidopa, particularly for end-of-dose motor fluctuations. Octipacone was approved by the European Commission in June 2016 and by the U.S. Food and Drug Administration (FDA) in April 2020. Marketed under the brand name Ongentys, it is a once-daily oral capsule. Octipacone has a duration of action exceeding 24 hours and can be administered once daily. Compared to other catechol-O-methyltransferase inhibitors, it has the lowest risk of cytotoxicity. Octipacone is a catechol-O-methyltransferase inhibitor. Its mechanism of action is the inhibition of catechol-O-methyltransferase. Octipacone is a catechol-O-methyltransferase inhibitor that can be used as adjunctive therapy to levodopa/carbidopa in the treatment of Parkinson's disease, particularly in cases of "off-peak" (relapse of motor symptoms) during treatment. The incidence of serum enzyme elevation during ocipacone treatment is extremely low, and it has not been found to be associated with clinically significant liver damage with jaundice.

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Drug Indications
Opicapone is indicated for adult patients with Parkinson's disease who have persistently unstable symptoms and end-dose motor fluctuations or "off-period" symptoms after treatment with a combination of levodopa and a dopa decarboxylase inhibitor (e.g., carbidopa).
Antiliv is indicated for adult patients with Parkinson's disease who have persistently unstable symptoms and end-dose motor fluctuations after treatment with a combination of levodopa and a dopa decarboxylase inhibitor (DDCI).
Ongentys is indicated for adult patients with Parkinson's disease who have persistently unstable symptoms and end-dose motor fluctuations after treatment with a combination of levodopa and a dopa decarboxylase inhibitor (DDCI). Patients with stable disease in these combination therapies. Ongentys is indicated for adult patients with Parkinson's disease as adjunctive therapy to levodopa and a dopa decarboxylase inhibitor (DDCI) who have persistently unstable end-dose motor fluctuations and whose disease has not been stabilized after treatment with these combination therapies.

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Mechanism of Action
Levodopa (L-Dopa) is the gold standard for treating motor symptoms and some non-motor symptoms of Parkinson's disease; however, only a small fraction of administered levodopa crosses the blood-brain barrier to exert its therapeutic effect, and patients face the risk of end-dose motor fluctuations, reflecting the rapid peripheral metabolism of levodopa by aromatic L-amino acid decarboxylases and catechol-O-methyltransferases (COMTs). Opipcapone is a peripherally selective, reversible catechol-O-methyltransferase (COMT) inhibitor. It has a subpicomolar binding affinity, resulting in a slow complex dissociation rate constant and a long duration of action in vivo. When opipcapone is added to a treatment regimen containing levodopa and a dopa decarboxylase inhibitor, opipcapone helps increase levodopa plasma concentrations, enhancing its therapeutic effect.

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.)