Therefore, the unbound inhibition constants ( em K /em i,u) are approximately equal to the respective total inhibition constants for both CYP2C8 and CYP2C9 under current incubation conditions. Michaelis\Menten constant (Km) and ataluren protein binding but had a minimal effect on maximum velocity (Vmax) of glucuronidation. Due to the decreased unbound Michaelis\Menten constant (Km,u), the ataluren unbound intrinsic clearance (CLint,u) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7\fold more active than human liver microsomes, in terms of CLint,u/mg?protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes. metabolismfor 10?minutes at 10C). The supernatant fractions were analyzed by LC/RAM/MS to determine percent loss of Rabbit Polyclonal to USP30 substrate in incubations. Detailed LC/MS conditions are summarized in LC\MS/MS Analysis section. After chromatographic separation, the detected NVP-BGT226 radioactive peaks were integrated, and their identity was confirmed using mass spectrometry analysis. [14C]\ataluren remaining at specific timepoint after incubation was compared with the amount of [14C]\ataluren at 0?minutes incubation sample in the same matrix (eg, with or without cofactors etc) to calculate the percentage of ataluren remaining. The percentage of [14C]\ataluren acyl glucuronide formation at specific timepoint after incubation was calculated using the amount of metabolite formed over the total radioactivity in each sample. 3.1.2. Plasma protein binding [14C]\Ataluren, at 50, 500, 5000, and 50?000?ng/mL or 0.175, 1.75, 17.5, and 175?M, in male mouse, rat, dog, monkey, and human plasma, was incubated (n?=?3) at 37C for approximately 15?minutes, transferred into NVP-BGT226 Centrifree? Micropartition Units (30?000?Da molecular weight cutoff, 1?mL capacity; Amicon Inc), and centrifuged at 37C and 2000?for approximately 10?minutes so that the amount of the sample filtered was approximately??20% of the total volume. After centrifugation, the entire ultrafiltrate was weighed and analyzed for radioactivity using liquid scintillation counting. The percent bound = (1\Cu/Cm)100, where Cu is the concentration of radioactivity in the ultrafiltrate NVP-BGT226 and Cm is the concentration of radioactivity in the plasma before centrifugation. 3.2. Reaction phenotyping and kinetics for the formation of ataluren acyl glucuronide 3.2.1. Recombinant human UGT enzymes Ataluren at 10?mol/L, was incubated (n?=?2) with recombinant human uridine diphosphate glucuronosyltransferase (rUGT) enzymes: rUGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 at 0.25?mg protein/mL. Incubations NVP-BGT226 were conducted in duplicate at 37C. The incubation mixture (0.2?mL, final volume with 1% methanol) consisted of Tris\HCl buffer (100?mmol/L, pH 7.7 at 37C), MgCl2 (10?mmol/L), EDTA (1?mmol/L), saccharic acid 1,4\lactone (0.1?mmol/L), and UDPGA (8?mmol/L). Alamethicin was prepared in 20?mmol/L EDTA and the activation of the recombinant UGT enzymes was performed by adding an equal volume of the protein and alamethicin solution to achieve 25?g alamethicin/mg protein. The protein/alamethicin mixture was preincubated for 15?minutes on ice, prior to the addition of ataluren to the mixture. Ataluren was added in methanol (1% v/v). Reactions were initiated by the addition of UDPGA and were terminated at 0 and 60?minutes by the addition of 2% v/v formic acid in acetonitrile containing ataluren\D4 as an internal standard. Samples were subjected to centrifugation (920?for 10?minutes at 10C) and the supernatant fractions were analyzed by LC\MS/MS to quantify the formation of ataluren\valuevalue(L/min/mg)22227733434440.33264213133249521563128847.3164177164 em CLint,u,UGT /em (mL/min/g of tissue) NANANANA1.6113.016.912.54.1512.1820.016.50.9743.383.643.39 Open in a separate window Abbreviations: BSA, bovine serum albumin; CLint,u,UGT, unbound intrinsic clearance by glucuronidation; fu,inc, fraction unbound from protein in the incubation; HIM, human intestinal microsomes; HKM: human kidney microsomes; HLM, human liver microsomes; Km, Michaelis\Menten constant; Km,u, unbound Michaelis\Menten constant; NA, Not applicable; UGT, uridine diphosphate glucuronosyltransferase; Vmax, maximum velocity. aMean??standard error (n?=?3). bMean??standard deviation (n?=?3). Open in a separate window Figure 3 Enzyme kinetic (Michaelis\Menten) plots for ataluren glucuronidation with or without the presence of BSA in (A) recombinant human UGT1A9, (B) human kidney microsomes, (C) human liver microsomes, and (D) human intestinal microsomes (n?=?3 for each data point) Open in a separate window Figure 4 Eadie\Hofstee plots for ataluren glucuronidation kinetics with or without the presence of BSA in (A) recombinant human UGT1A9, (B) human kidney microsomes, (C).