Benserazide

We measured H₂O₂ production with the ROS-Glo™ H₂O₂ Assay system. All compounds were tested at 10 µM in PBS or MEM plus or minus 1 mM sodium pyruvate. Compounds were tested in triplicate and each experiment was performed a minimum of three separate times. Stock solutions were prepared as follows: 10 mM menadione in DMSO, 70 mM pyrogallol in DMSO, 40 mM benserazide in DMSO, 10 mM eseroline in DMSO, and 10.2 mM alamethicin in ethanol. Vehicle was present at 0.1% in these experiments. The H₂O₂ assay was performed as directed by the manufacturer as follows. The compounds were added to an opaque white 96 well plate in the desired media or PBS. The H₂O₂ substrate solution was then added, bringing the final volume to 100 µl. The plate was incubated at 37°C in a 5% CO₂ incubator for 60 min. 100 µl of the ROS-Glo detection solution was added to each well at the end of the incubation. After an additional 20 min incubation at room temperature, luminescence was recorded using a GloMax^® Multi Detection System luminometer.
To confirm the selectivity of ROS-Glo for H₂O₂, 35 U of catalase was included in some of the 100 μl reactions. In order to estimate the amount of H₂O₂ generated by these compounds, a standard curve was also performed at the same time as the corresponding experiment. Concentrations between 0 and 30 μM H₂O₂ were used to construct the standard curve. A standard curve in MEM media was used to quantitate the data using MEM media or MEM with pyruvate and a standard curve in PBS was used to quantitate the data in PBS. Due to the large difference in background between samples in MEM, samples in MEM with pyruvate, and those containing catalase, all data points and the standard curve were background corrected using the appropriate controls. The equation of the linear line of the curve was then used to determine the amount of H₂O₂ generated in each sample.
Assays to determine the production of H₂O₂ in the presence of cells were performed as described above with the following modifications. The day before the experiment, cells were plated in clear, tissue culture treated 96-well plates at a density of 10,000 MDA-MB-231 cells per well in a total volume of 70 µl. Wells with cells were paired with corresponding wells without cells (medium alone). After overnight incubation at 37°C in a 5% CO₂ incubator, 10 µl of compounds or their vehicle in the appropriate medium were added, followed by 20 µl of the H₂O₂ substrate solution for a total volume of 100 µl. After the 1 h incubation at 37°C in a 5% CO₂ incubator, 50 µl of each reaction mixture was transferred to a white 96-well plate and mixed with 50 µl of the ROS-Glo detection solution. The plate was then incubated 20 min at room temperature before reading luminescence. The standard curve for these experiments was performed analogously to the experiment. Briefly, the incubation of the H₂O₂ samples occurred in a cell culture treated plate, after which 50 µl was moved to a white luminometer plate and mixed with 50 µl of detection reagent.

Participants ingested in one testing session levodopa, in another naltrexone, and in another a placebo (microcrystalline cellulose), following a double-blind, cross-over design with counterbalanced order. Levodopa is an amino acid precursor of dopamine leading to a transient systemic increase of dopamine availability. To inhibit peripheral synthesis of dopamine from levodopa, the single dose of 150 mg levodopa (p.o.) was combined with 62.5 mg of a benserazide to prevent peripheral side effects such as nausea (Rinne et al., 1975 (link)). Naltrexone is an opioid receptor antagonist with predominant receptor binding affinity at µ-opioid receptors together with a lower binding affinity at κ-opioid receptors and a much lower affinity at δ-opioid receptors (Raynor et al., 1994 (link)). Participants received a single dose of 50 mg naltrexone (p.o.) that has been shown to induce more than 90% receptor blockade (Weerts et al., 2013 (link)).
After drug intake, a waiting period of one hour started. This waiting time was chosen based on peak plasma concentrations of levodopa and naltrexone at approximately 1 hr to 1.5 hr after ingestion (Nyholm et al., 2012 (link); Wall et al., 1981 (link)). At the end of each testing session, participants indicated whether they thought that they had received the placebo or one of the drugs (response alternatives: ‘placebo’, ‘levodopa’, ‘naltrexone’, or ‘don’t know’) to test for potential unblinding.

l-DOPA methyl ester (10 mg/kg) was administered in 0.9% saline s.c. with 15 mg/kg benserazide HCl, at 1 ml/kg. BRC was administered i.p. at 2.5 mg/kg (2 ml/kg) (solubilised as described in^{11 (link)}). All drugs were obtained from Sigma Aldrich, UK. Duration and severity of AIMs were evaluated to obtain a global AIMS score as described in^{10 (link)}. In the meta-analysis we defined “non-dyskinetic” rats as those achieving an individual element score (amplitude × duration) of 1 or less at any timepoint, while any rat exceeding 1 in any given element was allocated as “dyskinetic”.

The control group received only levodopa and benserazide hydrochloride tablets: oral, 0.125 g/dose for the first dose, 3 times/d. After 1 week of continuous dosing, the dose was changed to 0.125 g 2. Per week (depending on the patient’s condition), and the maximum dose should not exceed 1.5 g in 1 day. The observation group was given the combination treatment of Resagiline + levodopa and benserazide hydrochloride tablets, in which the dose and regimen of levodopa and benserazide hydrochloride tablets were the same as above, and Resagiline was administered orally at 1 mg once daily. The duration of treatment for both groups was 1 year. In addition, patients in both groups were given the same routine rehabilitation training.