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FIG. 4.Nonlinear regression analysis of the degradation of the whole acyl glucuronide (top) and appearance of aglycone (bottom) during incubation at 37°C with HSA, pH 7.4. , tolmetin; f, zomepirac; Œ, ketoprofen; ‚, fenoprofen; F, suprofen; E, furosemide; , diclofenac; ✳, ibuprofen. cannot be assessed in a screening process. Furthermore, HSA remains the protein most extensively studied. It is widely distributed in the plasma compartment and easily related to the immune system. The knowledge gathered on covalent binding of acyl glucuronide and HSA allowed the comparison and validation of the results achieved with this model. A product showing a significant covalent binding to HSA in our model will require specific attention during the development process. The constant values calculated for the disappearance of acyl glucuronides were lower than those previously published, especially for zomepirac and tolmetin (Hasegawa et al., 1982; Ojingwa et al., 1994). In fact, those reported values represented the global degradation of 1-O-acyl glucuronide (hydrolysis isomerization), whereas the values presented in this study only represent the degradation of isomeric forms. In the conditions of analysis described here, the exact IN VITRO ASSESSMENT OF ACYL GLUCURONIDE REACTIVITY TABLE 2 Apparent first-order degradation of various acyl glucuronides and aglycone appearance constants measured during incubation phases with 0.5 mM HSA, pH 7.4, at 37°C Mean S.D. (n 3). Compound Name Acyl Glucuronide Degradation Rate Aglycone Appearance Rate Tolmetin 0.055 0.020 0.106 0.049 Zomepirac 0.025 0.008 0.103 0.027 Suprofen 0.046 0.021 0.035 0.018 Diclofenac 0.050 0.016 0.081 0.023 Fenoprofen 0.021 0.014 0.044 0.011 Ibuprofen 0.028 0.003 0.037 0.013 Ketoprofen 0.037 0.010 0.016 0.005 Furosemide 0.006 0.011 0.000 0.001 h 1 409 Downloaded from dmd.aspetjournals.org by guest on May 29, 2013
410 BOLZE ET AL. FIG. 5.Time-dependent irreversible binding of several aglycone after incubation of their respective acyl glucuronide (produced by human liver microsomes; incubation of 400 M aglycone) in human serum albumin solution 0.5 mM at 37°C, pH 7.4. Data are the average of triplicate measurements. , tolmetin; f, zomepirac; Œ, ketoprofen; ‚, fenoprofen; F, suprofen; E, furosemide; , diclofenac; ✳, ibuprofen. FIG. 6.The ranking of compounds according to their extent of covalent binding expressed in millimoles irreversibly bound per mole of protein and normalized by protein content (A) and expressed as the percentage of total acyl glucuronide present at the beginning of the reactivity phase (B). Data are the average of triplicate measurements; bars indicate S.D. Downloaded from dmd.aspetjournals.org by guest on May 29, 2013
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