Michael H. Court

Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes.

BackgroundOxidation of propofol to 4-hydroxypropofol represents a significant pathway in the metabolism of this anesthetic agent in humans. The aim of this study was to identify the principal cytochrome P-450 (CYP) isoforms mediating this biotransformation. MethodsPropofol hydroxylation activities and enzyme kinetics were determined using human liver microsomes and cDNA-expressed CYPs. CYP-specific marker activities and CYP2B6 protein content were also quantified in hepatic microsomes for correlational analyses. Finally, inhibitory antibodies were used to ascertain the relative contribution of CYPs to propofol hydroxylation by hepatic microsomes. ResultsPropofol hydroxylation by hepatic microsomes showed more than 19-fold variability and was most closely correlated to CYP2B6 protein content (r = 0.904), and the CYP2B6 marker activities, S-mephenytoin N-demethylation (r = 0.919) and bupropion hydroxylation (r = 0.854). High- and intermediate-activity livers demonstrated high-affinity enzyme kinetics (Km < 8 &mgr;m), whereas low-activity livers displayed low-affinity kinetics (Km > 80 &mgr;m). All of the CYPs evaluated were capable of hydroxylating propofol; however, CYP2B6 and CYP2C9 were most active. Kinetic analysis indicated that CYP2B6 is a high-affinity (Km = 10 ± 2 &mgr;m; mean ± SE of the estimate), high-capacity enzyme, whereas CYP2C9 is a low-affinity (Km = 41 ± 8 &mgr;m), high-capacity enzyme. Furthermore, immunoinhibition showed a greater contribution of CYP2B6 (56 ± 22% inhibition; mean ± SD) compared with CYP2C isoforms (16 ± 7% inhibition) to hepatic microsomal activity. ConclusionsCytochrome P-450 2B6, and to a lesser extent CYP2C9, contribute to the oxidative metabolism of propofol. However, CYP2B6 is the principal determinant of interindividual variability in the hydroxylation of this drug by human liver microsomes.

Pharmacogenetic determinants of interindividual variability in bupropion hydroxylation by cytochrome P450 2B6 in human liver microsomes.

Bupropion is primarily metabolized in human liver by cytochrome P450 (CYP) 2B6, an isoform that shows high interindividual variability in expression and catalysis. The aim of this study was to identify mechanisms underlying this variability through comprehensive phenotype-genotype analysis of a well-characterized human liver bank (n = 54). There was substantial variability in microsomal bupropion hydroxylation activities (over 45-fold) and CYP2B6 protein content (over 288-fold), with excellent correlation between protein and activity values (rs = 0.88). CYP2B6 mRNA levels showed less variability (13-fold) and poorer correlation (rs = 0.44) to CYP2B6 protein resulting from 20-30% of livers that contained substantial CYP2B6 mRNA, but low CYP2B6 protein. Livers were genotyped for the common coding polymorphisms (Q172H, K262R and R487C) and 14 additional variations identified by sequencing of the gene promoter to -3000 bp. Of 14 haplotypes that were inferred, *1A (reference), *1H (-2320t>c; -750t>c) and *6B (-1456t>c; -750t>c; Q172H; K262R) were most common with frequencies of 0.28, 0.20 and 0.26, respectively. Alcohol use history (P = 0.011) and *6B haplotype (P = 0.011) were identified as significant predictors of bupropion hydroxylation. A consideration of the effects of these variables on CYP2B6 mRNA and protein levels suggests that alcohol use is associated with enhanced CYP2B6 gene transcription, but the presence of at least one *6B allele reduces this effect on bupropion hydroxylation at the post-transcriptional level. In conclusion, the results of this study indicate that interindividual variability in bupropion hydroxylation is a consequence of interactions between environmental and genetic influences on CYP2B6 gene function.

Inhibition of alprazolam and desipramine hydroxylation in vitro by paroxetine and fluvoxamine : comparison with other selective serotonin reuptake inhibitor antidepressants

In vitro preparations of human liver microsomes were used to study the inhibiting effects of two selective serotonin reuptake inhibitor (SSRI) antidepressants, paroxetine and fluvoxamine, on metabolism via hydroxylation of alprazolam and of desipramine. These reactions are mediated by Cytochromes P450-3A4 and P450-2D6, respectively. Paroxetine was a highly potent inhibitor of desipramine hydroxylation; the inhibition constant (Ki) value of 2.0 microM indicated greater inhibiting potency than fluoxetine or norfluoxetine. The in vitro data predicted in vivo impairment of desipramine clearance by coadministration of paroxetine which was in the same range as observed in a clinical study. Fluvoxamine, by contrast, was a much weaker inhibitor of desipramine hydroxylation, having a Ki value (16.6 microM) similar to those of sertraline and desmethylsertraline. For hydroxylation of alprazolam, paroxetine was a relatively weak inhibitor, approximately comparable to fluoxetine, whereas fluvoxamine showed inhibiting capacity similar to that of norfluoxetine. The in vitro data predicted the degree of impairment of alprazolam clearance observed in vitro model can therefore provide clinically relevant data on prediction of potential drug interactions with SSRIs.

Molecular genetic basis for deficient acetaminophen glucuronidation by cats: UGT1A6 is a pseudogene, and evidence for reduced diversity of expressed hepatic UGT1A isoforms.

The domestic cat has a significantly lower capacity to glucuronidate planar phenolic xenobiotics compared with most other mammalian species. The aim of this study was to determine the mechanistic basis for this anomaly. Current knowledge of the substrate specificity of UDP-glucuronosyltransferase (UGT) isoforms indicates that the cat may either lack or poorly express UGT1A6. Initially, a novel cloning technique was used to identify UGT1A genes expressed in cat liver. Only two unique UGT1A isoforms could be discriminated. The first (28%, of clones) was most homologous to UGT1A1 (the bilirubin-UGT), while the second (72% of clones) showed homology to several isoforms, but could not be unambiguously identified, and was designated cat UGT1A02. Southern blot analysis confirmed the presence of a single UGT1A6-homologous region in the cat genome. Subsequent cloning and sequencing of the entire UGT1A6 exon 1 coding region revealed five deleterious genetic mutations. Identical mutations were found by sequencing of UGT1A6 exon 1 from five other unrelated cats. Four of these five genetic lesions were also identified in the UGT1A6 exon 1 region of a margay (Leopardus wiedii). Finally, RT-PCR of liver mRNA from four different cats confirmed the presence of UGT1A1 and UGT1A02, but not UGT1A6. In conclusion, UGT1A6 is a pseudogene in the domestic cat and in at least one other phylogenetically related species. Furthermore, cats appear to have a less diverse pattern of UGT1A isoform expression compared with other species. Such differences most likely reflect the highly carnivorous diet of Feliform species and resultant minimal exposure to phytoalexins.

Interindividual variability in hepatic drug glucuronidation: studies into the role of age, sex, enzyme inducers, and genetic polymorphism using the human liver bank as a model system

The human liver bank has provided an invaluable model system for the study of interindividual variability in expression and activity of the major hepatic UGTs, including UGT1A1, 1A4, 1A6, 1A9, 2B7, and 2B15. Based on studies using UGT-isoform–selective probes, the rank order of activity variability is UGT 1A1>1A6>2B15>1A4 = 1A9>2B7, with coefficient of variation values ranging from 92 to 45%. Liver donor age, sex, enzyme inducers, and genetic polymorphism are factors that have been implicated as sources of this variability in UGT activity. The expression of UGTs prior to, and immediately following, birth is quite limited, explaining the susceptibility of neonates to certain drug toxicities. Old age appears to have minimal effect on UGT function. Sex differences in UGT activity are relatively small and are confined to several UGTs, including UGT2B15, which shows higher activity in males, compared with females. Enzyme inducers, including coadministered drugs, smoking, and alcohol, may increase hepatic UGT levels. Human liver bank phenotype-genotype studies, using UGT-isoform–selective probes have identified common genetic polymorphisms that are predictive of glucuronidation activity in vitro and that were subsequently verified as predictors of probe-drug clearance by glucuronidation in vivo.

Genotype‐phenotype Associations of Cytochrome P450 3A4 and 3A5 Polymorphism with Midazolam Clearance in Vivo

The molecular basis for the wide interindividual variability of cytochrome P450 (CYP) 3A metabolic activity was studied in vivo at a genetic level. A single oral dose of midazolam was administered to 26 healthy subjects. The variability in midazolam oral clearance was 11‐fold. No differences in midazolam oral clearance related to gender or ethnicity were observed. Selective sequencing of CYP3A4 and CYP3A5 genes revealed 18 single nucleotide polymorphisms (SNPs), including 8 novel CYP3A4 SNPs. Thirteen novel CYP3A4 haplotypes, 2 novel CYP3A5 haplotypes, and 1 major novel multigene haplotype (CYP3A4*VI‐CYP3A5*3A) were also identified. No significant genotype‐phenotype or haplotype‐phenotype associations were found for any of the SNPs or haplotypes studied, including CYP3A4*1B, CYP3A5*3, and CYP3A5*6, even when ethnicity was considered. The only exceptions were the haplotype CYP3A4*VI and the multigene haplotype CYP3A4*VI‐CYP3A5*3A. The carriers of the haplotype CYP3A4*VI had a 1.8‐fold higher clearance of midazolam in black subjects (ANOVA on ranks, P = .028) compared with other individuals, and the carriers of the multigene haplotype CYP3A4*VI‐CYP3A5*3A had a 1.7‐fold higher clearance in the entire population (ANOVA on ranks, P = .012). In conclusion, these results indicate that the genetic variants identified so far in the CYP3A4 and CYP3A5 genes have only a limited impact on CYP3A‐mediated drug metabolism in vivo.

Molecular basis for deficient acetaminophen glucuronidation in cats. An interspecies comparison of enzyme kinetics in liver microsomes.

Cats are highly susceptible to acetaminophen toxicity because of deficient glucuronidation of this drug in vivo. The enzyme kinetic basis for this defect is unknown. Therefore, the kinetic properties of acetaminophen UDP-glucuronosyltransferase (acetaminophen-UGT) were investigated, using hepatic microsomes from cats (N = 4) compared with those of species that are less sensitive to acetaminophen intoxication including dogs (N = 4), humans (N = 4), and six other mammalian species (one liver from each). Gunn rats were also studied, since they express defective UGT family 1 isoenzymes and are also prone to acetaminophen toxicity. Acetaminophen kinetics were biphasic in all instances with distinct high and low affinity components. Km values for the high affinity activity in cat microsomes (0.31 +/- 0.1 mM; mean +/- SEM) were intermediate between those of dogs (0.11 +/- 0.02 mM) and humans (0.60 +/- 0.06 mM) and other species (0.22 to 6.7 mM; range). On the other hand, high affinity Vmax values were over 10-fold less in cat microsomes (0.025 +/- 0.006 nmol/min/mg) than in dogs (0.92 +/- 0.09 nmol/min/mg) and humans (0.27 +/- 0.09 nmol/min/mg); and over 5-fold less compared with microsomes from other species (range 0.13 to 7.63 nmol/min/mg). Gunn rat microsomes showed a similar 10-fold difference in high affinity Vmax values between the homozygous mutant (0.67 nmol/min/mg) and homozygous normal (6.75 nmol/min/mg) animals. These results demonstrate that, relative to a number of other species, cats have remarkably low hepatic levels of a high affinity acetaminophen-UGT. This difference is sufficient enough to explain poor glucuronidation of acetaminophen in vivo and susceptibility to acetaminophen intoxication.

Quantitative distribution of mRNAs encoding the 19 human UDP-glucuronosyltransferase enzymes in 26 adult and 3 fetal tissues

The purpose of this study was to generate, by real-time PCR, a quantitative expression level profile of the 19 human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A and 2B, in 26 adult and 3 fetal tissues, for better understanding of their roles in xenobiotic and endobiotic metabolism. Adult liver contained the highest level of combined UGTs mRNA, and UGT2B4 was the most abundant isoform in this tissue (40% of total). Other well expressed hepatic UGTs, in decreasing order of mRNA level, were 1A9, 2B7, 1A4, 2B10, 1A1, 1A6, 2B11, 2B15, 1A3, 2A3, 2B17 and 2B28. UGT2B4 was by far the most abundant isoform in the fetal liver (90% of total). The combined UGT mRNA expression in both adult and fetal olfactory epithelium was high, about 20% the adult hepatic level. Interestingly, a large developmental change was found in this tissue from high UGT2A1 and UGT2A2 expression in the fetus to UGT1A6 in the adult. The most abundantly expressed UGTs in the small intestine were 2A3, 1A10, 1A1, 1A6 and 2B7, while 1A10 and 2A3 predominated in the colon. The results provide the most comprehensive data to date on the tissue distribution of the human UGTs.

Biotransformation of Chlorzoxazone by Heptatic Microsomes from Humans and Ten Other Mammalian Species

The 6‐hydroxylation of chlorzoxazone (CLZ) is currently being used in both in vivo and in vitro studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Comparatively little is known with regard to the biotransformation of this drug in other species. The NADPH‐dependent biotransformation of CLZ was therefore studied using hepatic microsomes derived from humans and ten other mammalian species. In all species, 6‐hydroxychlorzoxazone (6OH‐CLZ) was the only metabolic product that could be identified by HPLC with ultraviolet detection. Enzyme kinetic analysis was used to characterize this CLZ 6‐hydroxylase activity. Although the majority of kinetic data conformed to a single‐enzyme Michaelis–Menten model, a two‐enzyme (high and low affinity) model was required for four species (ferret, monkey, pig, and rat). Apparent Km values for the high‐affinity component ranged from 12 μM (pig) to 95 μM (rabbit). The rank order of Vmax/Km, an index of intrinsic clearance, was: mouse>horse>monkey>rabbit>cow>ferret>pig>human1>rat>human2>cat>dog. Diethyldithiocarbamate (DDC), a CYP2E1 inhibitor in humans, was a potent mechanism‐based inhibitor of 6OH‐CLZ formation in microsomes from all species examined. Preincubation of microsomes for 15 min in the presence of DDC and NADPH significantly enhanced the maximum degree of inhibition but had no effect on inhibitor potency. Inhibitor concentrations at 50% of maximum inhibition (IC50max) for DDC with preincubation ranged from 9 μM (human) to 45 μM (cow).

Curcuminoids Inhibit Multiple Human Cytochromes P450, UDP-Glucuronosyltransferase, and Sulfotransferase Enzymes, whereas Piperine Is a Relatively Selective CYP3A4 Inhibitor

Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimers disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC50 values ranging from 0.99 ± 0.04 to 25.3 ± 1.3 μM, whereas CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC50 values >60 μM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (Ki = 11.0 ± 1.3 μM), whereas inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 ± 1.1 and 7.8 ± 0.9 μM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC50 5.5 ± 0.7 μM, Ki = 5.4 ± 0.3 μM) with less effect on other enzymes evaluated (IC50 > 29 μM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC50 = 8.8 ± 1.2 μM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC50 > 50 μM). Based on these data and expected tissue concentrations of inhibitors, we predict that a p.o. administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa.