Ketoprofen

The P-gp inhibitors selected for this study were obtained as reported in Ref. [27 (link)]. Acetonitrile (Chromasolv), formic acid, ammonium formate (MS grade), NaCl, KCl, Na₂HPO₄ 2H₂O, KH₂PO₄ (reagent grade), verapamil hydrochloride and ketoprofen (racemic analytical standard) were purchased by Merck (Milan, Italy). ketoprofen ethyl ester (KEE) was obtained by Fisher’s reaction from ketoprofen and ethanol. Ultrapure water or mQ water (resistivity 18 MΩ cm) was obtained from Millipore’s Simplicity system (Milan, Italy). Phosphate buffer solution (PBS) was prepared by being dissolved in mQ water and reported salts at the following concentrations: 8.01 g L⁻¹ of NaCl, 0.2 g L⁻¹ of KCl, 1.78 g L⁻¹ of Na₂HPO₄ 2H₂O, and 0.27 g L⁻¹ of KH₂PO₄. The samples of human plasma were collected from healthy volunteers, pooled and kept at −80 °C until use. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethics Committee (Comitato Etico Regionale per la Sperimentazione Clinica della Regione Toscana, Sezione AREA VASTA CENTRO) Institutional Review Board (CE: 11166_spe, 11 September 2018 and CE: 10443_oss, 14 February 2017).

Antipyrine, cimetidine, clotrimazole, N-desmethyl-tamoxifen hydrochloride, furosemide, hydrochlorothiazide, ketoprofen, maleic acid, (+/−)-metoprolol-(+)-tartrate, (+/−)-norverapamil hydrochloride, piroxicam, tamoxifen, terbutaline hemisulfate and (+/−)-verapamil hydrochloride 99% were purchased from Sigma-Aldrich (St. Louis, MO, USA); (+/−)-propranolol hydrochloride and ranitidine hydrochloride were obtained from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany), and carbamazepine was purchased from Acros Organics (New Jersey, USA). Midazolam and α-hydroxyMidazolam were purchased from Lipomed AG (Arlesheim, Switzerland), and agar, calcium chloride dihydrate, glucose hydrate, magnesium chloride hexahydrate, potassium chloride, sodium chloride, sodium hydroxide, sodium phosphate monobasic and sodium hydrogencarbonate were obtained from Hänseler AG (Herisau, Switzerland). Sodium taurocholate was purchased from Prodotti Chimici e Alimentari S.p.A., (Basaluzzo, Italy) and lecithin (grade EPCS > 98% phospholipids) was obtained from Lipoid GmbH (Ludwigshafen, Germany).

Adult, male BALB/c mice (20–30 g; 7–8 week old), obtained from the animal rearing facilities at the Faculty of Medicine, University of São Paulo, Brazil, were used in the experiments in accordance with the Ethical Principles for Animal Research established by the ICB/USP Ethics Committees for Animal Use in Research, according to the National Council for Animal Experimentation Control principles, consistent with the Animal Welfare Act. According to the internal laboratory rules, euthanasia was to be performed if severe distress related to the protocol developed during the experiment. The mice were group-housed in a temperature-controlled room at 22°C with a 12-h light/12-h dark cycle and allowed free access to food (Nuvilab CR-1; Quimtia S/A, Brazil) and filtered water.
ATB-352 was provided by Antibe Therapeutics, Inc., while ketoprofen was purchased from Sigma-Aldrich (Oakville, ON, Canada). Suspensions were prepared in 0.5% carboxymethylcellulose (CMC; Cromoline Química Fina Ltd., Diadema, SP, Brazil) with the aid of a vortex mixer.
The mice were orally treated with vehicle (4 mL/kg of 0.5% CMC), ketoprofen (3, 10, or 30 mg/kg), or equimolar doses of ATB-352 (4.6, 15, or 46 mg/kg).

Lactic acid (Sigma-Aldrich, St Louis, MO) was diluted in sterile water, and ketoprofen (Sigma-Aldrich, St Louis, MO) was dissolved in a solution of sterile water, ethanol, and kolliphor (92.5, 5, 2.5%; Sigma-Aldrich, St Louis, MO). Lactic acid and ketoprofen were administered by intraperitoneal injection with a 27 G needle, at a volume of 10 ml/kg. Morphine (Sigma-Aldrich, St Louis, MO) was dissolved in saline and administered subcutaneously with a 27 G needle, at a volume of 10 ml/kg. U69,593 (Sigma-Aldrich, St Louis, MO) was dissolved in a small amount of Lactic acid and then diluted with saline to a final concentration of 0.056% Lactic acid.