The very high lipophilicity of DEHTP resulted in the formation of an insoluble film on the surface of the reaction medium which precluded the measurement of in vitro clearance which is consistent with previous studies (McNally et al., 2019 (link); McNally et al., 2021 (link)). Therefore, only the measurement of in vitro clearance of MEHTP was possible (Figure 1 ). In vitro incubations, the determination of in vitro half-life, in vitro intrinsic clearance and the calculation of in vivo clearance were identical to previous studies and are described therein (McNally et al., 2019 (link); McNally et al., 2021 (link)).
The NADPH regenerating system consisted of the following final concentrations: 1.3 mM NADP+; 3.3 mM glucose-6-phosphate; 5 mM magnesium chloride; 0.4 U/ml glucose-6-phosphate dehydrogenase; 50 mM phosphate buffer (pH 7.4). Final microsomal protein concentration was 0.5 mg/ml. Incubations were performed in polypropylene tubes and pre-warmed reaction mixtures were started by addition of substrate dissolved in acetonitrile. The final acetonitrile concentration was less than 1% and, typically, a substrate concentration of 10 µM was used (initial investigations were performed to check solubility in the reaction mixture). Incubations were conducted in a water bath at 37°C. At the time points chosen for measurement, tubes were mixed by inversion and an aliquot removed and quenched by adding to an equal volume of ice-cold methanol followed by centrifugation to precipitate the protein as a pellet. The supernatant was removed for analysis. Three replicates were sampled at each time point. Control incubations consisted of a reaction mix excluding glucose-6-phosphate dehydrogenase (for evaluation of non-specific binding) and reaction mix excluding microsomes (for evaluation of substrate stability).
The method of Jones and Houston (2004) (link) was used to determine the in vitro half-life of substrate depletion. At least three independent incubations were performed, and results were assessed visually for reproducibility. However, due to differences in sampling time points between experiments, results from individual incubations were not combined.
The NADPH regenerating system consisted of the following final concentrations: 1.3 mM NADP+; 3.3 mM glucose-6-phosphate; 5 mM magnesium chloride; 0.4 U/ml glucose-6-phosphate dehydrogenase; 50 mM phosphate buffer (pH 7.4). Final microsomal protein concentration was 0.5 mg/ml. Incubations were performed in polypropylene tubes and pre-warmed reaction mixtures were started by addition of substrate dissolved in acetonitrile. The final acetonitrile concentration was less than 1% and, typically, a substrate concentration of 10 µM was used (initial investigations were performed to check solubility in the reaction mixture). Incubations were conducted in a water bath at 37°C. At the time points chosen for measurement, tubes were mixed by inversion and an aliquot removed and quenched by adding to an equal volume of ice-cold methanol followed by centrifugation to precipitate the protein as a pellet. The supernatant was removed for analysis. Three replicates were sampled at each time point. Control incubations consisted of a reaction mix excluding glucose-6-phosphate dehydrogenase (for evaluation of non-specific binding) and reaction mix excluding microsomes (for evaluation of substrate stability).
The method of Jones and Houston (2004) (link) was used to determine the in vitro half-life of substrate depletion. At least three independent incubations were performed, and results were assessed visually for reproducibility. However, due to differences in sampling time points between experiments, results from individual incubations were not combined.
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