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Leslie J Dickmann

Publications and source records attributed to Leslie J Dickmann.

4 recordsLinked to original sources
PubMed2004

Differential activation of CYP2C9 variants by dapsone.

Studies have shown that CYP2C9.1 mediated metabolism of flurbiprofen or naproxen is activated by co-incubation with dapsone. However, dapsone activation has not been examined in the known variant forms of CYP2C9. Six concentrations of flurbiprofen (2-300microM) or naproxen (10-1800 microM) were co-incubated with six concentrations of dapsone (0-100 microM) and with reconstituted, purified CYP2C9.1, CYP2C9.2 (R144C), CYP2C9.3 (I359L), or CYP2C9.5 (D360E), in order to assess degrees of activation. Dapsone increased the efficiency (V(m)/K(m)) of flurbiprofen 4'-hydroxylation by CYP2C9.1, CYP2C9.2, CYP2C9.3, and CYP2C9.5 by 8-, 31-, 47-, and 22-fold, respectively. In similar experiments using the substrate naproxen, dapsone increased the efficiency of naproxen demethylation 7-, 15-, 13-, and 22-fold, in CYP2C9.1, CYP2C9.2, CYP2C9.3, and CYP2C9.5, respectively. Also, dapsone normalized naproxen's kinetic profile from biphasic (CYP2C9.1 and CYP2C9.2) or linear (CYP2C9.3 and CYP2C9.5) to hyperbolic for all variant forms. Thus, amino acid substitutions of CYP2C9 variants affect the degree of dapsone activation in a genotype-dependent fashion. Furthermore, the degree of effect noted across variants appeared to be dependent on the substrate studied.

Alleles↗
PubMed2004

Differential roles of Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of CYP2C9.

CYP2C9 metabolizes a wide range of drugs, many of which are negatively charged at physiological pH. Therefore, it has been thought that complementarily charged amino acid(s) are critically involved in substrate binding. Previous studies have implicated arginine residues at positions 97, 105, and 108 and aspartate at position 293 in the normal catalytic function of the enzyme. To elucidate the role of these amino acids in the substrate specificity of CYP2C9, a series of mutants were constructed and analyzed for functional activity, thermal stability, and ligand binding. Charge-modifying mutations at positions 97, 105, and 293 decreased catalytic activity toward diclofenac, (S)-warfarin, and pyrene in a substrate-independent manner with Arg105 the least, and Arg97 the most, sensitive amino acids in this regard. Decreases in functional activity paralleled thermal instability of the mutants, suggesting that loss of function reflects more generalized structural changes rather than the absence of a specifically charged amino acid at these three positions. The R108H mutant was inactive toward all three substrates because of unexpected nitrogen ligation to the heme. Conversely, the R108F mutant exhibited substrate-dependent catalytic behavior, with almost complete loss of activity toward (S)-warfarin and diclofenac, but preservation of pyrene metabolism. In addition, the R108F mutation abrogated the Type I difference spectra induced by flurbiprofen and benzbromarone, obligate anions at physiological pH. These data identify critical roles for Arg97 and Asp293 in the structural stability of the enzyme and demonstrate a selective role for Arg108 in the binding and metabolism of negatively charged substrates of CYP2C9.

Amino Acid Substitution↗
PubMed2004

CYP2C9 genetic polymorphisms and warfarin.

The objective of this study was to report 2 cases of CYP2C9 genetic polymorphism and elevated warfarin S:R ratios in patients taking low doses of warfarin, and compare the observed characteristics with those in published reports. Two patients of different age groups and races were evaluated for CYP2C9 genotype and warfarin S:R ratios. The patients had been stabilized on weekly warfarin doses of 10.5 mg and 10 mg, respectively. Each patient was found to have at least 1 variant CYP2C9 allele. Elevated warfarin S:R ratios in both patients provided evidence for impaired metabolism of S-warfarin. This report of a CYP2C9*3 heterozygous individual taking a low dose of warfarin is consistent with previous reports in the literature. This summary of a CYP2C9*6 homozygous individual taking a low dose of warfarin is the first such published report. CYP2C9 genotyping in these patients provided a likely explanation for their continued low warfarin dosage requirements. Awareness of a patient's CYP2C9 genotype may provide an explanation for low warfarin dosage requirements in stable patients and may help in determining the optimal dose in patients being initiated on warfarin.

Adult↗
PubMed2002

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.

Alleles↗