2.5. Animals and Pharmacokinetic Study Design

The Sprague Dawley (SD) rat study was approved by CHA University Animal Experimental Ethics Committee, Republic of Korea (approval number: IACUC200007). Male SD rats (7 weeks, 222–271 g) were purchased from Orient Bio Inc. (Seongnam, Republic of Korea). All rats were separately housed in cages in a ventilated animal room with a temperature of 23 ± 1 °C, a relative humidity of 50 ± 5%, and a 12-h/12-h of light/dark cycle. Rats were freely accessible to food and water, but they were fasted overnight before the drug administration. For pharmacokinetic studies, rats were randomly allocated into nine groups. The pharmacokinetic study design is summarized in Table 1. In R1-R9 groups, blood samples (200 μL) were drawn from the jugular vein into tubes treated with heparin and 10% TCA. The 10% TCA was used as an antioxidant stabilizer that improves the plasma stability of edaravone [10]. Blood samples were centrifuged at 13,000 rpm for 10 min followed by being transferred into new tubes and stored at −80 °C until the analysis. Except for the tosylate salt and other moieties from the prodrug, the edaravone equivalent doses were calculated and are summarized in Table 1 and Table 2.

The pharmacokinetic study design using Sprague Dawley rats.

The pharmacokinetic study design using beagle dogs (n = 5).

Rats were randomly allocated into three groups (18 rats/group) for tissue distribution studies (R7, R8, and R9). These rats were sacrificed at 15, 30, 45 min and 1, 2, 4 h after orally administering TEJ-1704. Six major organs including brain, heart, lung, kidney, liver, and gastrointestinal tract were immediately removed, washed in normal saline, and dried with filter papers. Organs were accurately weighed and individually homogenized with distilled water (organ: distilled water = 1:4 w/v). The tissue homogenate was stored at −80 °C until analysis.

The beagle dog study was approved by Knotus Co., Ltd. Animal Experiment Ethics Committee (approval number: 20-KE-318), Korea. All dogs were separately housed in cages in a ventilated animal room with a temperature of 23 ± 3 °C, a relative humidity of 55 ± 15%, and a 12-h/12-h of light/dark cycle. Dogs were freely accessible to food and water, but they were fasted overnight before the drug administration. Male beagle dogs (13 months, average 9.4 kg) were randomly divided into six groups (5 dogs/group) for the pharmacokinetic study (Table 2). In group B1, the administration route of edaravone was set as an IV infusion in the same way as the clinical route. Blood and CSF samples were collected into tubes treated with heparin and 10% TCA. Urine and fecal samples were collected using metabolic cages for the excretion study. After oral administration of edaravone or TEJ-1704, urine and feces were collected at 0–2, 2–6, 6–12, 12–24 h. Urine volumes were recorded and transferred to centrifuge tubes (1 mL each) for use as urine samples. Fecal weights were recorded after thoroughly mixing feces collected at each sampling period. Then 1 g of feces was accurately weighed, mixed with 4 mL of distilled water, and homogenized to be used as a fecal sample. All samples were stored at −80 °C until analysis.