Verapamil hydrochloride

The experimental procedure was performed as previously reported. Briefly, the mean length of the proximal jejunum (approximately 2 cm distal to the ligament of Treitz) was 10 ± 1 cm [48 (link)]. An oxygenated Krebs–Ringer bicarbonate buffer (KRB) solution (pH 7.4) containing 0.5 mM MgCl₂, 4.5 mM KCl, 120 mM NaCl, 0.7 mM Na₂HPO₄, 1.5 mM NaH₂PO₄, 1.2 mM CaCl₂, 15 mM NaHCO₃, and 10 mM glucose was perfused at a flow rate of 0.4 mL/min using a peristaltic pump through an inlet tube kept at 37 °C. After a 30-min equilibration period, the following drugs (dissolved in KRB) were administered: (1) 10 µM OCT (99% purity, Chengdu Xinlinbang Bio-pharmaceutical Co.) (Group A, control group, n = 4); (2) 10 µM OCT plus 1 mM verapamil hydrochloride (Sigma-Aldrich, St. Louis, MO) (Group B, n = 4); (3) 10 µM OCT plus 1 mM probenecid (Sigma-Aldrich, St. Louis, MO) (Group C, n = 4); and (4) 10 µM OCT plus 1 mM verapamil hydrochloride plus 1 mM probenecid (Group D, n = 4). Portal vein blood (200–300 µl) was collected at 5, 10, 15, 30, 45, and 60 min for OCT determination via LC–MS/MS. The AUC was calculated using the integral method. These experiments were performed both in healthy and PH rats; the latter was called Group A1, B1, C1, and D1 (n = 4 per group).

hESCs were first seeded at 50% confluency onto six-well-plate coated with Matrigel. To induce differentiation, adherent cells were treated with 50 ng ml⁻¹ BMP4 and BMP8a for 1 h in differentiation medium and followed by transduction of lentivirus expressing DAZL-IRES-eGFP and BOULE-P2A-mCherry for 1 day, recovery for 1 day, selection with blasticidin at 2 μg ml⁻¹ for 3 days, and recovery for another day. Thereafter, cells were treated with differentiation medium until day 8. Differentiated cells were collected into a 15 ml tube, washed once with 1 ml chilled PBS, and suspended in 5 μg ml⁻¹ Hoechst 33342 (Beyotime) and 20 μM verapamil hydrochloride (Sigma). Cell suspension was incubated at 37 °C for 40 min. During the incubation, tubes were mixed several times. The cells were resuspended in 20 μM verapamil hydrochloride (Sigma) and analysed by LSRFortessa SORP (BD). A total of >500,000 cells from each sample were subjected to DNA content analysis using programs in FlowJo 7.6 software package.

The isomers ELF94 and ELF96 used in this study were synthesized, as reported by Dei et al. [23 (link)]. Acetonitrile (Chromasolv), formic acid, ammonium formate (MS grade), verapamil hydrochloride, and ketoprofen (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). The human plasma was 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 following 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, 11/09/2018 and CE: 10443_oss, 14/02/2017).

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).

The chemicals β-caryophyllene (≥98.5% purity), β-caryophyllene oxide (95% purity), and verapamil hydrochloride (≥98.0% purity) were purchased from Merck Life Science S.r.l. (Milan, Italy), while sorafenib tosylate (≥99.0% purity) and MK571 (≥96.0% purity) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Dulbecco’s Modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 culture medium, fetal bovine serum, buffer, and cofactors were from Aurogene S.r.l. (Rome, Italy). EtOH (100% v/v) was used to dissolve β-caryophyllene and β-caryophyllene oxide, while DMSO (100% v/v) was used for sorafenib tosylate, verapamil hydrochloride, and MK571. Solvents were used up to 1% v/v nontoxic concentration.

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).

The MRP transporter inhibitors mometasone furoate, lasalocid A sodium, verapamil hydrochloride were purchased from Sigma-Aldrich, Saint Louis, MO. Compounds were dissolved in DMSO and spread onto NGM plates in different concentration ranges: 25–100 μM (mometasone and verapamil) and 125–500 nM (lasalocid). DMSO (0,5% w/v) was used as control. Subsequently, Bcc strains (grown in standard conditions) were spotted onto the plates that were incubated overnight at 37°C. After the incubation 30–40 WT L4 worms were spotted onto the bacterial lawn. The plates were then incubated at 20°C for 3 days and scored for living worms every 24 h. The experiments were performed in triplicate, and the data reported are mean values.