Alamethicin

Oxazepam, R,S-Oxazepam glucuronide, ketoconazole, β-glucuronidase, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cannabinoids and their metabolites (THC, 11-OH-THC, THC-COOH, and CBD) were purchased from Cayman Chemicals (Ann Arbor, MI, USA) or Sigma-Aldrich after obtaining the appropriate Drug Enforcement Administration (DEA) licenses (state and federal). UDP-glucuronic acid (UDPGA) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Ultra-low binding microcentrifuge tubes, alamethicin, and magnesium chloride (MgCl₂) were purchased from VWR (Radnor, PA, USA). Sekisui Xenotech, LLC (Lenexa, KS, USA) supplied pooled human liver microsomes (HLM; n = 50 subjects, mixed gender) and human kidney microsomes (HKM; n = 8 subjects, mixed gender). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), and fetal bovine serum (FBS) were all purchased from Gibco (Grand Island, NY, USA), while geneticin (G418) was purchased from Seradigm (Radnor, PA, USA). BCA protein assays were purchased from Pierce (Rockford, IL, USA). LC–MS-grade methanol, acetonitrile, and ammonium formate were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Acquity UHPLC BEH C18 columns (1.7 µm 2.1 × 100 mm) were purchased from Waters (Milford, MA, USA).

NHDF were seeded at 1.5 x 10⁶ in T75 flasks and infected with MOI of 10 for 18 h. The assay was conducted as above using 10 μCi of [³H]-2-DG and 83.3 μM unlabeled 2-DG to start transport. To inhibit mammalian cell glucose transport, cytochalasin B was added with 2-DG to a final concentration of 15 μM. The reaction was stopped as above and monolayers were washed 5 times rapidly to remove exogenous [³H]-2-DG. Amastigotes were isolated from host cell monolayers in Krebs-Henseleit Buffer (KHB; 111 mM sodium chloride, 4.7 mM potassium chloride, 1.25 mM calcium chloride, 2 mM magnesium sulfate, and 1.2 mM sodium phosphate dibasic) then lysed for scintillation counts and protein normalization. To verify that the intracellular parasites had taken up [³H]-2-DG label from host cell, amastigotes were isolated following radiolabeling of infected monolayers as above, split equally into two tubes and incubated with vehicle alone (1% DMSO) or 0.05 mg/mL alamethicin (VWR) in 500 μL for 15 minutes at room temperature while shaking to permeabilize the parasite plasma membrane. An additional 500 μL of buffer was added, and amastigotes were pelleted at 4000 g for 10 minutes at 4°C and lysed in sodium hydroxide for scintillation counts.