Rapid equilibrium dialysis device

Compound
binding to the cell homogenate or microsomal fraction was determined
using dialysis in the cassette mode, as previously described.^{21 (link)} Briefly, the cell homogenate or microsome fraction
was spiked with the compounds and dialyzed for 4 h at 37 °C using
a Rapid Equilibrium Dialysis device (Thermo Fisher Scientific Inc.).
Protein was precipitated with acetonitrile/water (60:40) spiked with
50 nM warfarin, and samples were analyzed using UPLC–MS/MS,
as described below. The fraction of unbound compound in the cell homogenate
(f_u,hom) or microsomal fraction (f_u,mic) was calculated, as previously described^{21 (link)} where PA_buffer is the
peak area
of compound in the buffer chamber and PA_hom or PA_mic is the peak area of compound in the homogenate or microsomal chamber,
respectively, all corrected for the peak area of the internal standard.
The fraction of unbound compound in hepatocytes (f_u,cell) was calculated according to where D was estimated for
each homogenate preparation based on a cellular volume of 6.5 μL/mg
protein,^{34 (link)} and on the protein concentration
measured using the BCA protein assay reagent kit. The f_u,hep used for CL_int,u,hep was calculated as f_u,cell but with D being 0.1
corresponding to 10 times higher cell concentration in the binding
experiment (10 × 10⁶ hepatocytes/mL) than that in
the intrinsic clearance measurement.

The unbound MM-129 concentration analysis was conducted using the rapid equilibrium dialysis device (Thermo Scientific, Waltham, MA, USA) according to the manufacturer instructions. Sodium citrate-anticoagulated pooled plasma was obtained from healthy rats by centrifugation of whole blood at 3500× g for 20 min at 4 °C. Spiked MM-129 rat pooled plasma samples (0.5 mL) at the concentration of 50 μM were dialyzed against phosphate buffered saline (0.75 mL) at pH 7.4 for 4 h. Total and unbound MM-129 concentration was measured using the HPLC method described above.

The fraction unbound in microsomes and rCYP (fu_mic and fu_CYP respectively) of CCT241736 was measured using a Rapid Equilibrium Dialysis device (Thermo Fisher Scientific, Loughborough. UK). HLM (1 mg/ml) and control rCYP (100 pmol/ml) containing 1 μM CCT241736 were dialysed against 100 mM KPB for 4 h at 37 °C. Samples were removed, matrix matched and quenched with methanol containing internal standard; further processing is as described in Section 2.6.

The fractions of unbound acacetin in plasma (fu_P), microsomes (fu_MIC), SGF (fu_SGF), and SIF(fu_SIF) were measured using a rapid equilibrium dialysis device (Thermo Fisher Scientific, Inc., Waltham, MA, USA) as previously described with slight modifications [25 (link)]. Briefly, 0.2 mL of the matrix spiked with acacetin (10 μM) was placed into the “sample” chamber, and 0.4 mL of PBS was placed into the adjacent “buffer” chamber. The fractions of unbound acacetin were calculated as the concentration ratio between the two chambers. The blood-to-plasma concentration ratio (R_B) of acacetin was determined as described previously [25 (link)]. Briefly, 1-mL blood spiked with acacetin (1 μM) was incubated at 37 °C for 30 min. The blood sample was centrifuged at 2000× g for 5 min to prepare the plasma sample. The concentrations of acacetin in the aforementioned plasma and buffer samples were measured using the LC-MS/MS method.

The antimicrobial activities of the compounds were measured using the microdilution method⁴³ . Measurement of the bactericidal activity of lysocin E (0.25 µg ml⁻¹) and nisin (2 µg ml⁻¹) with or without ApoA-I was performed according to the National Committee for Clinical Laboratory Standards method⁴⁴ . We chose the concentration of 25 µg ml⁻¹ ApoA-I as the enhancing effect of ApoA-1 on lysocin E was observed in a linear range. For the ∆menA and ∆menB strains, TSB medium was used instead of cation-adjusted-Mueller Hinton broth (CA-MHB) in the microdilution method. Sera and ApoA-I, II were added to the bacterial culture before distributing them into 96-well plates. ApoA-I and ApoA-II antimicrobial activity were evaluated using the RN4220 strain. The protein binding rate of lysocin E against human plasma was determined by a rapid equilibrium dialysis device (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.