ABSTRACT:The active forms of all marketed hydroxymethylglutaryl (HMG)-CoA reductase inhibitors share a common dihydroxy heptanoic or heptenoic acid side chain. In this study, we present evidence for the formation of acyl glucuronide conjugates of the hydroxy acid forms of simvastatin (SVA), atorvastatin (AVA), and cerivastatin (CVA) in rat, dog, and human liver preparations in vitro and for the excretion of the acyl glucuronide of SVA in dog bile and urine. Upon incubation of each statin (SVA, CVA or AVA) with liver microsomal preparations supplemented with UDP-glucuronic acid, two major products were detected. Based on analysis by high-pressure liquid chromatography, UV spectroscopy, and/or liquid chromatography (LC)-mass spectrometry analysis, these metabolites were identified as a glucuronide conjugate of the hydroxy acid form of the statin and the corresponding ␦-lactone.

The formation constants of the 1:1 and 1:2 complexes of Hg(II) with glutathione and their protonated species have been determined by using a competitive potentiometric titration with the competing ligand diethylenetriaminepentaacetic acid (DTPA). The formation constants of the 1:1 complex and its protonated species have not been reported previously. The formation constant of the 1:2 complex of Hg(II) and glutathione is substantially smaller than the accepted values that has been reported in the literature. These results have important implications in the models that have been employed to explain the mobilization and distribution of Hg(II) in biological systems.

Recombinant P450 2B1 wild-type and the active-site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7-hydroxycoumarin formation was decreased for all the mutants except II 14V. Uncoupling to H_»0 was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for II 14V. Uncoupling to H 2 C>2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.

A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.

The formation constants of lead chelates of the stereoisomers of 2,3-dimercaptosuccinic acid (DMSA) were determined from potentiometric titrations in the presence of the competing ligand, EDTA. The lead chelates formed at pH 7.4 with the stereoisomers of DMSA are the monomeric complexes PbL and HPbL. Formation of PbL and HPbL at pH 7.4 is independent of total concentrations of lead and DMSA present, and so is the concentration ratio of PbL:HPbL. Lead is completely chelated at pH 7.4 when the total concentration of ligand is equal to or greater than the total concentration of lead present. Lead tends to bind to a greater extent with rac- than with meso-DMSA, and the relative extent increases with an increase in the concentration ratio of ligand to lead and finally reaches a constant value of 45. The binding sites in the chelates, PbL, of the stereoisomers of DMSA are the two thiolate groups and one carboxylate group. rac-DMSA also forms a dimeric complex Pb2L2 in which both carboxylate groups of the ligands participate in binding with lead ions. The formation constants of the lead chelates of rac-DMSA were invariably found to be larger than those of the corresponding of meso-DMSA chelates, because in all the lead chelates of the stereoisomers of DMSA formed in solution, rac-DMSA existed in staggered anti conformations, whereas meso-DMSA preferred a staggered gauche conformation with respect to carboxylate groups in the ligands. The potential of using ZnL2 of rac-DMSA as a therapeutical lead chelator was assessed by considering its lead-mobilizing ability and its ability to deplete endogenous zinc; on this basis it is predicted that ZnL2 of rac-DMSA is a better chelator than meso-DMSA for the treatment of lead poisoning.