Probenecid

DNDI-6148 and DNDI-5421 (structures as published in [9 (link)]) were provided by DNDi (Geneva, Switzerland), and stock solutions for in vitro assays (20 mM) were prepared in 100% DMSO. Potassium antimonyl tartrate (Sb^III) was purchased from Sigma-Aldrich (Diegem, Belgium) and stock solutions were made in phosphate-buffered saline (PBS) at 5.12 mg/mL. The efflux pump inhibitors verapamil, cyclosporine and probenecid were purchased from Sigma-Aldrich and were formulated in 100% DMSO at 20 mM, except for probenecid, which was diluted up to 50 mM in PBS after the addition of ethanol (2%) and NaOH. Dilution series from the stock solutions were prepared in demineralized water to ascertain a < 1% final in-test concentration of DMSO. For the in vivo experiments, DNDI-6148 was prepared at 12.5 mg/mL in 2% ethanol, followed by the addition of 1N NaOH (1.0 eq.) and then further diluted in 5% dextrose in water.

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).

MPTP and probenecid (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 0.9% sterile saline and dimethylsulfoxide, respectively. The mice received a subcutaneous (s.c.) injection of MPTP (25 mg/kg) and an intraperitoneal (i.p.) injection of probenecid (250 mg/kg) for 10 times with intervals of 3.5 days (Figure 1).^{47 (link)} N-methyl-D-aspartic acid (NMDA), R-(R*, S*)-α-(4-hydroxyphenyl)-beta-methyl-4-(phenyl methyl)-1-piperidinepropanol (Ro25-6981) and 2-(4-morpholineylethyl) 1-phenylcyclohexane-1-carboxylate (PRE084) were purchased from Sigma-Aldrich and dissolved in 0.9% sterile saline. N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy) phenyl] ethylamine hydrochloride (NE100) was kindly supplied by Taisho Pharmaceutical Co. Ltd (Tokyo, Japan) and was dissolved in distilled water. Ro25-6981 (6 mg/kg), NMDA (30 mg/kg), PRE084 (1 mg/kg) and NE100 (1 mg/kg) were daily administered (i.p.)^{13 (link)} for 5 weeks. Control mice were given an equal volume of vehicle.

Sixteen‐week‐old male AQP4^+/+ and AQP4^−/− mice received a total of 10 doses of MPTP‐HCl (20 mg/kg in saline, subcutaneously [sc]) in combination with an adjuvant, Probenecid (250 mg/kg in dimethyl sulfoxide [DMSO], ip). Mice were treated in a similar manner with Probenecid and saline as controls. The 10 doses were administered on a 5‐week schedule, such that injections were given with an interval of 3.5 days between consecutive doses. Animals were killed 7 days after the last injection. AQP4^−/− and AQP4^+/+mice after chronic MPTP treatment showed no mortality. Probenecid was purchased from Sigma Chemical Co. Probenecid was used to inhibit the rapid clearance and excretion of MPTP from the brain and kidney following each injection. Neither Probenecid nor DMSO at the concentrations used in this study produced any significant effect on striatal dopamine (DA) contents.30

TmDOTP⁵⁻ for BIRDS was purchased from Macrocyclics Inc. (Plano, TX, USA), while SPIO-NPs (Molday ION) were purchased from BioPAL Inc. (Worcester, MA, USA). The Molday ION (10 mg Fe/mL, dextran-coated, hydrodynamic diameter 30 nm, zeta potential −4.8 mV) were used without further modification or dilution to avoid altering their physical properties. Probenecid (used for temporary inhibition of renal clearance) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fischer 344 rats (male, 200–250 g) were obtained from Yale University vendors. RG2 and 9L tumor cell lines were purchased from American Type Culture Collections (Manassas, VA, USA). All animal experiments were conducted in accordance with Yale University's approved institutional animal care and use committee (IACUC) protocols. Tumor inoculation, animal preparation, and handling were conducted as described in our previous work [57 (link), 58 (link)]. In vivo magnetic resonance (MR) scans were conducted on a 9.4T Agilent (Santa Clara, CA, USA) or Bruker (Billerica, MA, USA) horizontal-bore spectrometer with a 1.4-cm ¹H surface RF coil.

The animal experiments were conducted in accordance with the guidelines and were approved by the National Taiwan Normal University (NTNU) Research Committee. Male C57BL/6 mice (8 weeks old, 18–22 g) were purchased from the National Laboratory Animal Center (Tainan City, Taiwan). The mice were housed in individually ventilated cages under controlled temperature (25 ± 2 °C), relative humidity (50%), and 12 h on/off light cycle with ad libitum access to food and water at the Animal House Facility of NTNU.
After one-week habituation, mice were randomly divided into 4 groups (n = 7). NC009-1 (40 mg/kg) or vehicle (DMSO:Cremophor EL:0.9% saline = 1:2:7) was intraperitoneally (i.p.) administrated 5 times per week for 6 weeks. Experimental parkinsonism was established by i.p. injections of 20 total doses of MPTP (25 mg/kg in 0.9% saline; Toronto Research Chemicals, Toronto, Ontario, Canada) along with probenecid (250 mg/kg in 0.1 M NaOH; Sigma-Aldrich), while control group received injections of saline. probenecid was administered 1 h prior to MPTP administration as it decreases the clearance of MPTP and intensifies its neurotoxicity [80 (link)]. The dosage regimen was administered over 4 weeks with 5 doses per week (once daily for five consecutive days). Appropriate guidelines were abided in handling MPTP.