The lopinavir half-life and intrinsic clearance were determined for the neonatal HLMs as 52.1 min and 133 L/minmg, respectively (Table 1). (HLMs). We identified ritonavir as a potent inhibitor of CYP3A7 oxidation of DHEA-S (IC50 = 0.0514 M), while lopinavir is a much weaker inhibitor (IC50 = 5.88 M). Furthermore, ritonavir is a time-dependent inhibitor of CYP3A7 with a and 4 C in an Eppendorf 5910R centrifuge to remove unbroken cells, nuclei, mitochondria, and lysosomes. The supernatants, corresponding to the cytosolic and microsomal fractions, were then centrifuged for 1 h at 105,000 and 4 C in a Beckman L-80 ultracentrifuge with a Sorvall 50.2 Ti rotor (Sorvall, Newton, CT). The upper lipid bilayer was removed with a cotton swab, and the cytosolic supernatants were collected. The microsomal pellets were resuspended in wash buffer (150 mM potassium chloride and 10 mM EDTA) and homogenized with a Teflon-glass homogenizer. The microsomal suspension samples were further centrifuged for 1 h at 105,000 and 4 C in a Beckman ultracentrifuge with a Sorvall 50.2 Ti rotor. The resulting microsomal pellets were homogenized in 250 mM sucrose and stored at ?80 C. The neonatal HLM protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham MA). A pool of three neonatal HLMs was prepared by combining microsomes based on an equal protein amount for each. The neonatal HLMs were assessed for CYP3A and CYP4A11 enzyme activities using midazolam and lauric acid as probe substrates, respectively, and metabolite formation was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (data not shown). Recombinant CYP3A7 Activity and Inhibition Assays for DHEA-S The kinetic reactions (250 L) evaluating DHEA-S metabolism by the recombinant CYP3A7 Supersomes contained various concentrations of DHEA-S (2.5C120 M) dissolved in methanol (0.5% v/v), CYP3A7 Supersomes (10 pmol/mL), 100 mM potassium phosphate buffer (pH 7.4), and 3 mM MgCl2. After an equilibration at 37 C for 3 min, the reactions, prepared in triplicate, were initiated by the addition of a NADPH-regenerating system mix consisting of NADP+ (1 mM), glucose-6-phosphate (10 mM), and glucose-6-phosphate dehydrogenase (2 IU/mL). The reactions were incubated for 10 min at 37 C under agitation and were stopped by the addition of ice-cold methanol (250 L) containing 100 ng/mL DHEA-S-and 4 C. Supernatants were transferred to high performance liquid chromatography (HPLC) vials, and aliquots of 5 L were analyzed by LC-MS/MS. The 16-hydroxy DHEA-S metabolite was quantified based on a calibration curve ranging from 0.025 to 10 M. Similar assay conditions were used for the inhibition reactions with the HIV protease inhibitors and the recombinant CYP3A7 Supersomes except that the concentration of DHEA-S was set to 5 M, and the reaction volume was 200 L. The concentration ranges tested for lopinavir and ritonavir, dissolved in methanol, were 0.05 to 100 M and 0.0005 to 1 1 M, respectively. Solvent control (methanol) was used as the 100% control activity. Methanol concentration in the final incubation reactions was 1% (v/v). For ritonavir time-dependent inhibition, recombinant CYP3A7 Supersomes (100 pmol/mL) were preincubated in triplicate with ritonavir at 0.01, 0.05, 0.1, 0.25, 0.5, and 1 M in 100 mM potassium phosphate buffer (pH 7.4) and 3 mM MgCl2. Ritonavir was dissolved in methanol (0.4%, v/v), and solvent control (methanol) was used as the 100% control activity. The preincubation reactions (final volume of 240 L) were started by the addition of the NADPH-regenerating system mix. After 0, 8, 16, 24, and 32 min at 37 C, a 20 L aliquot of the recombinant CYP3A7/ritonavir preincubation was transferred to a 50 Colistin Sulfate M DHEA-S reaction (180 L) containing 100 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl2, and the NADPH-regenerating system mix. The DHEA-S incubations were stopped after 8 min at 37 C by the addition of ice-cold methanol (200 L) containing 100 ng/mL DHEA-S-and 4 C. Supernatants were transferred to HPLC vials, and aliquots of 5 L were analyzed by LC-MS/MS for formation of.Adult HLM incubations containing ritonavir at 62.5 nM were carried out to assess the effect on lopinavir clearance. and lysosomes. The supernatants, corresponding to the cytosolic and microsomal Colistin Sulfate fractions, were then centrifuged for 1 h at 105,000 and 4 C in a Beckman L-80 ultracentrifuge with a Sorvall 50.2 Ti rotor (Sorvall, Newton, CT). The upper lipid bilayer was removed with a cotton swab, and the cytosolic supernatants were collected. The microsomal pellets were resuspended in wash buffer (150 mM potassium chloride and 10 mM EDTA) and homogenized with a Teflon-glass homogenizer. The microsomal suspension samples were further centrifuged for 1 h at 105,000 and 4 C in a Beckman ultracentrifuge with a Sorvall 50.2 Ti rotor. The resulting microsomal pellets were homogenized in 250 mM sucrose and stored at ?80 C. The neonatal HLM protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham MA). A pool of three neonatal HLMs was prepared by combining microsomes based on an equal protein amount for each. The neonatal HLMs were assessed for CYP3A and CYP4A11 enzyme activities using midazolam and lauric acid as probe substrates, respectively, and metabolite formation Colistin Sulfate was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis (data not shown). Recombinant CYP3A7 Colistin Sulfate Activity and Inhibition Assays for DHEA-S The kinetic reactions (250 L) evaluating DHEA-S metabolism by the recombinant CYP3A7 Supersomes contained various concentrations of DHEA-S (2.5C120 M) dissolved in methanol (0.5% v/v), CYP3A7 Supersomes (10 pmol/mL), 100 mM potassium phosphate buffer (pH 7.4), and 3 mM MgCl2. After an equilibration at 37 C for 3 min, the reactions, prepared in triplicate, were initiated by the addition of a NADPH-regenerating system mix consisting of NADP+ (1 mM), glucose-6-phosphate (10 mM), and glucose-6-phosphate dehydrogenase (2 IU/mL). The reactions were incubated for 10 min at 37 C under agitation and were stopped by the addition of ice-cold methanol (250 L) containing 100 ng/mL DHEA-S-and 4 C. Supernatants were transferred to high performance liquid chromatography (HPLC) vials, and aliquots of 5 L were analyzed by LC-MS/MS. The 16-hydroxy DHEA-S metabolite was quantified based on a calibration curve ranging from 0.025 to 10 M. Similar assay conditions were used for the inhibition reactions with the HIV protease inhibitors and the recombinant CYP3A7 Supersomes except that the concentration of DHEA-S was set to 5 M, and the reaction volume was 200 L. The concentration ranges tested for lopinavir and ritonavir, dissolved in methanol, were 0.05 to 100 M and 0.0005 to 1 1 M, respectively. Solvent control (methanol) was used as the 100% control activity. Methanol concentration in the final incubation reactions was 1% (v/v). For ritonavir time-dependent inhibition, recombinant CYP3A7 Supersomes (100 pmol/mL) were preincubated in triplicate with ritonavir at 0.01, 0.05, 0.1, 0.25, 0.5, and 1 M in 100 mM potassium phosphate buffer (pH 7.4) and 3 mM MgCl2. Colistin Sulfate Ritonavir was dissolved in methanol (0.4%, v/v), and solvent control (methanol) was used as the 100% control activity. The preincubation reactions (final volume of 240 L) were started by the Rabbit polyclonal to Caspase 1 addition of the NADPH-regenerating system blend. After 0, 8, 16, 24, and 32 min at 37 C, a 20 L aliquot of the recombinant CYP3A7/ritonavir preincubation was transferred to a 50 M DHEA-S reaction (180 L) comprising 100 mM potassium phosphate buffer (pH 7.4), 3 mM MgCl2, and the NADPH-regenerating system blend. The DHEA-S incubations were halted after 8 min at 37 C by the addition of ice-cold methanol (200 L) comprising 100 ng/mL DHEA-S-and 4.
