Abstract Aus dem Lacton (I) erhält man mit den Arylmagnesiumbromiden (IIa) und (IIb) verschiedene Addukte: Mit (IIa) entsteht lediglich (IIIa), mit (IIb) werden Gemische aus (IIIb), (IV) und (V) erhalten.
Several human immunodeficiency virus (HIV) protease inhibitors, including atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, were tested for their potential to inhibit uridine 5′-diphospho-glucuronosyltransferase (UGT) activity. Experiments were performed with human cDNA-expressed enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) as well as human liver microsomes. All of the protease inhibitors tested were inhibitors of UGT1A1, UGT1A3, and UGT1A4 with IC50 values that ranged from 2 to 87 μM. The IC50 values found for all compounds for UGT1A6, 1A9, and 2B7 were >100 μM. The inhibition (IC50) of UGT1A1 was similar when tested against the human cDNA-expressed enzyme or human liver microsomes for atazanavir, indinavir, and saquinavir (2.4, 87, and 7.3 μM versus 2.5, 68, and 5.0 μM, respectively). By analysis of the double-reciprocal plots of bilirubin glucuronidation activities at different bilirubin concentrations in the presence of fixed concentrations of inhibitors, the UGT1A1 inhibition by atazanavir and indinavir was demonstrated to follow a linear mixed-type inhibition mechanism (Ki = 1.9 and 47.9 μM, respectively). These results suggest that a direct inhibition of UGT1A1-mediated bilirubin glucuronidation may provide a mechanism for the reversible hyperbilirubinemia associated with administration of atazanavir as well as indinavir. In vitro-in vivo scaling with [I]/Ki predicts that atazanavir and indinavir are more likely to induce hyperbilirubinemia than other HIV protease inhibitors studied when a free Cmax drug concentration was used. Our current study provides a unique example of in vitro-in vivo correlation for an endogenous UGT-mediated metabolic pathway.
1. 4-Tritiated-tamoxifen (4- [3 H]-tamoxifen) and 4-deuterated-tamoxifen (4- [2 H]-tamoxifen) were synthesized to examine tamoxifen metabolism by human P450 (CYP) forms and also for the possibility of determining tamoxifen-4-hydroxylation in humans in vivo. 2. Liver microsomes from several species and cDNA-expressed human P450s were incubated with 4- [3 H]-tamoxifen and the reaction monitored by assaying 4-hydroxytamoxifen (4-OH-tam) and 3 H 2 O formed. However, tamoxifen-4-hydroxylation did not generate stoichiometric amounts of 3 H 2 O and the expected unlabelled 4-OH-tam but instead yielded radiolabelled 4-OH-tam, apparently from [3 H]-migration to the ortho -position, referred to as the NIH shift. 3. CYP2D6 was the prime catalyst of tam-4-hydroxylation, whereas CYP2B6, 2C9 and 2C19 yielded only low levels of 4-OH-tam; nevertheless, in all cases the 4-OH-tam was radioactive, apparently resulting from reactions involving an NIH shift. 4. Chicken liver microsomal preparation, being catalytically the most active in tamoxifen-4-hydroxylation, was incubated with deuterated tamoxifen (4- [2 H]-tamoxifen) in order to determine whether an NIH shift occurs. Ion-trap mass-spectrometry of the HPLC-purified 4-OH-tam, from that incubation, indicated about 60% of [2 H]-retention in 4-OH-tam, signifying an NIH shift. These findings indicate that the aromatic hydroxylation of tamoxifen does not entail hydroxyl insertion with an Sn2-displacement of hydrogen or a hydrogen isotope (2 H or 3 H), but apparently involves epoxidation followed by migration of the 3 H, 2 H or 1 H to the ortho -position, and dissociation of the 1 H in preference to 3 H or 2 H, i.e. retention of the hydrogen isotope appears to be related to the bond strengths: C- 3 H>C- 2 H>C- 1 H.
Fluoxetine [+/--N-methyl-3-phenyl-3-[(alpha, alpha, (-trifluoro-p-tolyl)oxy]-propylamine)] a selective serotonin reuptake inhibitor, is widely used in treating depression and other serotonin-dependent disease conditions. Racemic, (R)- and (S)-fluoxetine are potent reversible inhibitors of CYP2D6, and the racemate has been shown to be a mechanism-based inhibitor of CYP3A4. Racemic fluoxetine also demonstrates time- and concentration-dependent inhibition of CYP2C19 catalytic activity in vitro. In this study, we compared fluoxetine, its (R)- and (S)-enantiomers, ticlopidine, and S-benzylnirvanol as potential time-dependent inhibitors of human liver microsomal CYP2C19. In a reversible inhibition protocol (30 min preincubation with liver microsomes without NADPH), we found (R)-, (S)- and racemic fluoxetine to be moderate inhibitors with IC(50) values of 21, 93, and 27 microM, respectively. However, when the preincubation was supplemented with NADPH, IC(50) values shifted to 4.0, 3.4, and 3.0 microM, respectively resulting in IC(50) shifts of 5.2-, 28-, and 9.3-fold. Ticlopidine showed a 1.8-fold shift in IC(50) value, and S-benzylnirvanol shifted right (0.41-fold shift). Follow-up K(I) and k(inact) determinations with fluoxetine confirmed time-dependent inhibition [K(I) values of 6.5, 47, and 14 microM; k(inact) values of 0.023, 0.085, 0.030 min(-1) for (R)-, (S)-, and racemate, respectively]. Although the (S)-isomer exhibits a much lower affinity for CYP2C19 inactivation relative to the (R)-enantiomer, it exhibits a more rapid rate of inactivation. Racemic norfluoxetine exhibited an 11-fold shift (18-1.5 microM) in IC(50) value, suggesting that conversion of fluoxetine to this metabolite represents a metabolic pathway leading to time-dependent inhibition. These data provide an improved understanding of the drug-interaction potential of fluoxetine.
We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) with estrogen receptor α (ERα), ERβ, and the androgen receptor (AR). In this study, we characterize the ERα, ERβ, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERα, ERβ, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17β-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERα agonist activity and ERβ and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERα agonist activity and did not alter ERβ or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERα and ERβ agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERα and ERβ activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.
The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the ..beta..-D-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-(G-/sup 3/H)camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess ..beta..-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2(U-/sup 14/C)campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C/sub 14/ to C/sub 20/) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid andmore » isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.« less
