Edaravone

Essential oil content was determined gravimetrically after hydrodistillation in a Clevenger apparatus [57 (link)]. The total flavonoid content was estimated as rutin equivalents by a spectrophotometric procedure after 5% AlCl₃ addition [58 (link)]. The total caffeoylquinic acid content was determined by the colorimetric Arnow method using 3-O-caffeoylquinic acid as the standard [59 (link)]. Total content of carbohydrate polymers (water-soluble polysaccharides and pectic substances) was determined with spectrophotometric phenol–sulphuric acid method [60 (link)]. Essential oil component was analysed by GC/MS method on a 6890N gas chromatograph (Agilent Technologies, city, state abbrev if USA, country) coupled to a Agilent Technologies 5973 N mass selective/quadrupole detector using a fused capillary column HP-5MS (30 m × 0.25 mm, film thickness 0.50 μm, 5% diphenyl- and 95% dimethylpolysiloxane stationary phase) [61 (link)]. The monosaccharide composition of polysaccharides was determined after acidic hydrolysis with trifluoroacetic acid (TFA) following by 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling and microcolumn HPLC with ultraviolet detection separation (HPLC-UV) of PMP-labeled hydrolyzates [57 (link),62 (link)].

The COSMO-RS (Conductor-like Screening Model for Real Solvents) [63 (link),64 (link),65 (link),66 (link)] was applied for the theoretical characteristics of solid–liquid multi-component systems. This commonly used framework takes advantage of the first principle quantum chemistry computations augmented with statistical thermodynamics for assessments of thermodynamic properties including chemical activities. Although it was designed for liquid systems, the solid–liquid equilibria (SLE) can also be treated, if only fusion data are provided either from direct measurements or external computations. In the case of edaravone, both the melting temperature, T_m = 403.15 K, and heat of fusion H_fus = 29.91 kJ/mol are known [14 (link)] and as such were used for solubility computations. The fusion data are indispensable since the values of the chemical potential of the solute in the saturated conditions are determined by the activity of the pure solid phase, according to the fundamental formula: $$\ln \left({\mathrm{a}}_{\mathrm{i}}^{\mathrm{s}}\right)=\ln \left({\mathsf{\gamma }}_{\mathrm{i}}^{\mathrm{sat}}{\mathrm{x}}_{\mathrm{i}}^{\mathrm{sat}}\right)=-\frac{{\Delta }_{\mathrm{fus}}{G}_i^m\left(p,T\right)}{\mathrm{RT}}$$
where ${\Delta }_{\mathrm{fus}}{G}_i^m\left(p,T\right)$ is the partial molar Gibbs free energy of fusion at the solubility measurement conditions. Practically, solubility is commutated by iteratively solving the following equation: $$ln\left({\gamma }_i^{sat,i+1}{x}_i^{sat,i+1}\right)=\frac{1}{RT}\left({\mu }_i^{o,liq}-{\mu }_i^{\left(i\right)}\left({\gamma }_i^{sat,i}{x}_i^{sat,i}\right)+max\left(0,{\Delta }_{fus}{G}_i^m\left(p,T\right)\right)\right)$$
In the above equation superscripts i and i + 1 denote the values obtained in two subsequent iterations. The iterative cycle is repeated until convergence is achieved, which means that the computation is supposed to be interrupted if the difference in the computed solubility drops below a defined threshold value. The bulk phase used for solubility computations has the same composition as the solute-free solvent used in experimental measurements.

Glycosaminoglycan analysis was performed by adapting a protocol established by Fuller et al (2004 (link)). MSD primary fibroblasts and control fibroblasts were grown in T75 cell culture flasks (CellStar, Greiner bio‐one, Kremsmünster, Austria) with tazarotene/bexarotene 10/20 μM or DMSO as the control for 21 days. Cells from confluent flasks were harvested, and protein concentration was measured by BCA assay after lysis of 1/5^th of the cells. 4/5^th of the cells were frozen at −20°C and stored until further processing.
After thawing, cell pellets were resuspended in 50 μl PBS per 120 μg total protein. Fifty microliter of each sample was dried using a centrifugal concentrator under vacuum and reconstituted in 100 μl of 0.25 M PMP solution (0.25 M 1‐phenyl‐3‐methyl‐5‐pyrazolone (PMP)) in 0.4 M ammonia solution (11.95 ml of MeOH and 2.59 ml of ammonium hydroxide (28–30% ammonia) added to 35.5 ml MilliQ water (pH 9.5–10) containing 1 μM of internal standard (chondroitin disaccharide di‐4 S [CAS 136144‐56‐4], Carbosynth Ref: OC28898)). Samples were vortexed, sonicated, and mixed prior to 90 min incubation on a PCR thermocycler at 70°C and cooling for 10 min. Samples were acidified with 500 μl of 0.2 M formic acid, and PMP was extracted from the acidified samples by adding 500 μl chloroform and shaking for 1 min. Samples were centrifuged for 5 min at 13,000 g to separate the layers and the bottom organic layer was discarded. The procedure was repeated four times for each sample to completely remove PMP. The remaining aqueous layer (600 μl for each sample) was concentrated to 80 μl using a centrifugal concentrator under vacuum. After centrifugation for an additional 5 min at 13,000 g the supernatant (at least 60 μl) of every sample was referred to LC–MS/MS analysis on an Agilent UPLC system (Agilent Pursuit 3 PFP 2.0 ×100 mm 3 μm Column (Agilent, Santa Clara, USA)) and AB Sciex 6500 TQ Mass Spec System (Sciex, Framingham, USA).

The outcome of the study was the first recorded diagnosis of IHD (including angina pectoris) or stroke event, defined by the presence of diagnosis, hospitalization, and treatment or rehabilitation records. The ICD-10 codes for each outcome are presented in Supplementary Table 1. IHD was considered an event when record of the focal diagnosis (Supplementary Table 1), hospitalization, and treatment occurred in the same month. IHD treatment included percutaneous coronary intervention and coronary artery bypass operations (Supplementary Table 2). Stroke was considered as an event when record of the focal diagnosis (Supplementary Table 1), hospitalization, and treatment occurred in the same month, and any kind of rehabilitation occurred within two months from the diagnosis. Stroke treatment included computed tomography or magnetic resonance imaging followed by medication (antithrombotic agents, ATC code: B01 or edaravone, ATC code: N07X0) or surgical procedures expected to be performed as a part of stroke treatment. In both of the study outcome events, death caused by the focal disease before hospitalization was excluded from the events.