Gentest pre coated pampa plate system

Distribution coefficients at physiological pH (D_7.4) of the complexes were determined by the traditional shake-flask method in n-octanol/buffered aqueous solution at pH 7.40 (20 mM phosphate buffer) at various chloride concentrations and at 25.0 ± 0.2 °C using UV-vis detection as described in our former works [37 (link),38 (link),42 (link)]. PAMPA was applied for the complexes with a Gentest pre-coated PAMPA Plate System (Corning, Corning, NY, USA) [56 (link)]. Briefly, a 96-well filter plate was used as the permeation acceptor and the 96-well bottom plate was used as the permeation donor. For simplicity, PBS was used both as donor and acceptor buffer throughout this study. The initial donor solutions were prepared by diluting stock solutions in PBS (300 μM (complexes 1 and 3) or 65 μM (complexes 2 and 4). Donor plate was filled with 300 μL of the donor solutions (containing the test compounds). Each well of the filter plate contained 200 μL buffered solution as acceptor phase. The resulting ‘sandwich’ was protected with parafilm to prevent evaporation and incubated at room temperature for 5 h. Then, solutions from the donor and acceptor wells were transferred to Eppendorf tubes and their UV-vis spectra were recorded to determine the concentration of the components. P_eff values were calculated according to the equation reported by Yu et al. [57 (link)].

The ADME-Tox parameters including permeability, metabolic stability, DDIs, and hepatotoxicity were carried out as described previously [9 (link)–11 (link), 13 (link), 47 (link)]. The ability of AS-1 to passively penetrate through the biological membranes was estimated by Gentest Pre-coated PAMPA Plate System (Corning, Tewksbury, MA) and expressed as the permeability coefficient Pe. The human metabolism of the compound AS-1 was studied using human liver microsomes (HLMs) provided by Sigma-Aldrich. The potential DDIs were predicted by luminescent CYP3A4, CYP2D6, and CYP2C9 P450-Glo assays (Promega, Madison, WI). The respective strong CYP’s inhibitors, ketoconazole (KE, half maximal inhibitory concentration (IC₅₀) = 0.14 μM), quinidine (QD, IC₅₀ = 0.01 μM), and sulfaphenazole (SE, IC₅₀ = 0.08 μM), were used as the references. The hepatic safety of AS-1 was estimated here using hepatoma HepG2 cell growth. The cells were seeded in a 96-well plate and incubated in the presence of AS-1 at the concentration range 0.1–100 μM. One micromolar of cytostatic drug doxorubicin (DX) and 10 μM of mitochondrial toxin carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were used as the references.

Distribution coefficient (D₇.₄) values of COTI-NH₂ were determined by the shake-flask method in n-octanol/buffered aqueous solution at pH 7.4 in 20 mM phosphate buffer (0.10 M KCl) at 25.0 ± 0.2 °C following a similar approach to that reported in our previous works [8 (link),20 (link)]. The other compounds and all of their Cu(II) complexes were too lipophilic to obtain experimental data. Thus, the logP values were calculated for COTI-NMeCy, COTI-Nme₂, and COTI-2 (MarvinSketch,version 16.12.12.0, calculation module developed by ChemAxon (Budapest, Hungary), MarvinSketch/">http://www.chemaxon.com/products/marvin/MarvinSketch/ (accessed on 20 August 2022)).
The thermodynamic solubility of COTI-NMeCy, COTI-Nme₂, COTI-NH₂, and COTI-2 was measured for the saturated solutions in water at pH 7.4 (20 mM HEPES buffer) at 25.0 ± 0.1 °C. The concentrations of the compounds were determined by UV-vis spectrophotometry using stock solutions of the compounds with known concentrations dissolved in pure DMSO, and 50% and 1% (v/v) DMSO/buffered aqueous solution for the calibration. The parallel artificial membrane permeability assay (PAMPA) was applied for the ligands with a Corning Gentest pre-coated PAMPA Plate System [24 (link)]. P_eff values were calculated according to the equation reported by Yu et al. [25 (link)].

The reference compounds, well-permeable caffeine and low-permeable norfloxacin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Used for this study, pre-coated PAMPA Plate System Gentest™ was provided by Corning (Tewksbury, MA, USA). The system consisted of 96-well receiver filter plate pre-coated with structured layers of phospholipids and a donor microplate. The assay was performed as described previously [17 (link),45 (link)]. In brief, the tested compound KSK-59 and the reference compounds were diluted in the PBS buffer (pH 7.4) and applied into the plate at the final concentration of 200 μM. After 5 h of incubation at RT° the UPLC-MS spectrometry (Waters ACQUITY™ TQD system with the TQ Detector, Waters, Milford, MA, USA) method was used to estimate the quantity of compounds that penetrated from donor to acceptor wells. The permeability coefficients (Pe, cm/s) were calculated using the formulas provided by Corning.

Precoated PAMPA Plate System Gentest was
provided by Corning, (Tewksbury, MA). Compound (R)-7 [(R)-AS-1] was tested in
a similar way to racemate I.^{29 (link)} The detailed procedure and proper formulas were described previously.^{73 (link)}