Kenneth E. Thummel

First-pass metabolism of midazolam by the human intestine

The in vivo intestinal metabolism of the CYP3A probe midazolam to its principal metabolite, 1′‐hydroxymidazolam, was investigated during surgery in 10 liver transplant recipients. After removal of the diseased liver, five subjects received 2 mg midazolam intraduodenally, and the other five received 1 mg midazolam intravenously. Simultaneous arterial and hepatic portal venous blood samples were collected during the anhepatic phase; collection of arterial samples continued after reperfusion of the donor liver. Midazolam, 1′‐hydroxymidazolam, and 1′‐hydroxymidazolam glucuronide were measured in plasma. A mass balance approach that considered the net change in midazolam (intravenously) or midazolam and 1′‐hydroxymidazolam (intraduodenally) concentrations across the splanchnic vascular bed during the anhepatic phase was used to quantitate the intestinal extraction of midazolam after each route of administration. For the intraduodenal group, the mean fraction of the absorbed midazolam dose that was metabolized on transit through the intestinal mucosa was 0.43 ± 0.18. For the intravenous group, the mean fraction of midazolam extracted from arterial blood and metabolized during each passage through the splanchnic vascular bed was 0.08 ± 0.11. Although there was significant intersubject variability, the mean intravenous and intraduodenal extraction fractions were statistically different (p = 0.009). Collectively, these results show that the small intestine contributes significantly to the first‐pass oxidative metabolism of midazolam catalyzed by mucosal CYP3A4 and suggest that significant first‐pass metabolism may be a general phenomenon for all high‐turnover CYP3A4 substrates.

Human liver carbamazepine metabolism: Role of CYP3A4 and CYP2C8 in 10,11-epoxide formation

A number of drugs inhibit the metabolism of carbamazepine catalyzed by cytochrome P450, sometimes resulting in carbamazepine intoxication. However, there is little information available concerning the identity of the specific isoforms of P450 responsible for the metabolism of this drug. This study addressed the role of CYP3A4 in the formation of carbamazepine-10,11-epoxide, the major metabolite of carbamazepine. Results of the study showed that: (1) purified CYP3A4 catalyzed 10,11-epoxidation; (2) cDNA-expressed CYP3A4 catalyzed 10,11-epoxidation (Vmax = 1730 pmol/min/nmol P450, Km = 442 microM); (3) the rate of 10,11-epoxidation correlated with CYP3A4 content in microsomes from sixteen human livers (r2 = 0.57, P < 0.001); (4) triacetyloleandomycin and anti-CYP3A4 IgG reduced 10,11-epoxidation to 31 +/- 6% (sixteen livers) and 43 +/- 2% (four livers) of control rates, respectively; and (5) microsomal 10,11-epoxidation but not phenol formation was activated 2- to 3-fold by alpha-naphthoflavone and progesterone and by carbamazepine itself (substrate activation). These findings indicate that CYP3A4 is the principal catalyst of 10,11-epoxide formation in human liver. Experiments utilizing a panel of P450 isoform selective inhibitors also suggested a minor involvement of CYP2C8 in liver microsomal 10,11-epoxidation. Epoxidation by CYP2C8 was confirmed in incubations of carbamazepine with cDNA-expressed CYP2C8. The role of CYP3A4 in the major pathway of carbamazepine elimination is consistent with the number of inhibitory drug interactions associated with its clinical use, interactions that result from a perturbation of CYP3A4 catalytic activity.

Mutual repression between steroid and xenobiotic receptor and NF-κB signaling pathways links xenobiotic metabolism and inflammation

While it has long been known that inflammation and infection reduce expression of hepatic cytochrome P450 (CYP) genes involved in xenobiotic metabolism and that exposure to xenobiotic chemicals can impair immune function, the molecular mechanisms underlying both of these phenomena have remained largely unknown. Here we show that activation of the nuclear steroid and xenobiotic receptor (SXR) by commonly used drugs in humans inhibits the activity of NF-kappaB, a key regulator of inflammation and the immune response. NF-kappaB target genes are upregulated and small bowel inflammation is significantly increased in mice lacking the SXR ortholog pregnane X receptor (PXR), thereby demonstrating a direct link between SXR and drug-mediated antagonism of NF-kappaB. Interestingly, NF-kappaB activation reciprocally inhibits SXR and its target genes whereas inhibition of NF-kappaB enhances SXR activity. This SXR/PXR-NF-kappaB axis provides a molecular explanation for the suppression of hepatic CYP mRNAs by inflammatory stimuli as well as the immunosuppressant effects of xenobiotics and SXR-responsive drugs. This mechanistic relationship has clinical consequences for individuals undergoing therapeutic exposure to the wide variety of drugs that are also SXR agonists.

Natural allelic variants of breast cancer resistance protein (bcrp) and their relationship to Bcrp expression in human intestine

The aim of this study was to identify the extent of genetic variability in breast cancer resistance protein (BCRP) in humans. We first analysed the sequence of BCRP cDNA from human livers and from human intestines phenotyped for expression of intestinal BCRP. We then determined the frequency of all known coding single nucleotide polymorphisms (cSNPs) using DNA from individuals representing 11 different ethnic populations. Nine SNPs including four non-synonymous and three synonymous cSNPs and two intronic SNPs were identified. Of the missense mutations, exon 2 SNP (G34A) resulted in a V12M change; exon 5 SNP (C421A) resulted in a Q141K substitution; exon 6 SNP (A616C) resulted in an I206L amino acid substitution; and exon 15 SNP (A1768T) resulted in a N590Y change in the BCRP protein. The two most frequent polymorphisms identified in the human population studied were the G34A and C421A transitions. There was marked variation in BCRP genotypes and allele frequencies in the different populations. BCRP mRNA was phenotyped in human small bowel intestinal samples by real-time polymerase chain reaction and BCRP protein was analysed on immunoblots of tissue from the same individuals. There was a 78-fold variation in expression of BCRP mRNA and significant variation in BCRP protein expression in human intestine. Expression of intestinal BCRP mRNA and protein was not different between persons expressing the common Gln141 allele compared to the Lys141 allele. Thus, common natural allelic variants of BCRP have been identified, and did not influence interindividual variation in expression of BCRP mRNA in human intestine, but remain to be tested for their effect on BCRP function.

Oxidation of acetaminophen to N-acetyl-p-aminobenzoquinone imine by Human CYP3A4

We have investigated: (a) the formation of N-acetyl-p-aminobenzoquinone imine (NAPQI) from acetaminophen (APAP) by reconstituted human liver CYP3A4, (b) the kinetics of NAPQI formation in microsomes prepared from four human livers varying in CYP1A2, 2E1 and 3A4 content determined by Western blot analysis, (c) the contribution of CYP3A4 to the total formation of NAPQI from 0.1 mM APAP in human liver microsomes using troleandomycin as a specific inhibitor, and (d) the relationship between the contribution of CYP3A4 to NAPQI formation and relative CYP3A4 content. The Km of CYP3A4 for APAP was found to be approximately 0.15 mM, similar to concentrations observed in humans after therapeutic doses of the drug. The kinetics of formation of NAPQI in human liver microsomes were complex; the lower Km was similar to that found for reconstituted CYP3A4. The contribution of CYP3A4 to total NAPQI formation varied from 1 to 20% among livers, and correlated with the relative CYP3A4 content, r2 = 0.88, P < 0.05. Our findings indicate that CYP3A4, the major P450 isoform in human liver and enterocytes, contributes appreciably to the formation of the cytotoxic metabolite NAPQI at therapeutically relevant concentrations of APAP and suggest that APAP may be a previously unrecognized inhibitor of this enzyme.

Identification of Cytochrome P450 2E1 as the Predominant Enzyme Catalyzing Human Liver Microsomal Defluorination of Sevoflurane, Isoflurane, and Methoxyflurane

BackgroundRenal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. MethodsMicrosomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (paranitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective Inhibitors. ResultsThe rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > O. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either paranitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0.66). Sevoflurane defluorination was also highly correlated with that of enflurane (r = 0.93), which is known to be metabolized by human P450 2E1. Diethyldithiocarbamate, a selective inhibitor of P450 2E1, produced a concentration-dependent inhibition of sevoflurane, methoxyflurane, and isoflurane defluorination. No other isoform-selective inhibitor diminished the defluorination of sevoflurane, whereas methoxyflurane defluorination was inhibited by the selective P450 inhibitors furafylline (P45O 1A2), sulfaphenazole (P450 2C9/10), and quinidine (P450 2D6) but to a much lesser extent than by diethyldithiocarbamate. ConclusionsThese results demonstrate that cytochrome P450 2E1 is the principal, if not sole human liver microsomal enzyme catalyzing the defluorination of sevoflurane. P450 2E1 is the principal, but not exclusive enzyme responsible for the metabolism of methoxyflurane, which also appears to be catalyzed by P450s 1A2, 2C9/10, and 2D6. The data also suggest that P450 2E1 is responsible for a significant fraction of isoflurane metabolism. Identification of P450 2E1 as the major anesthetic metabolizing enzyme in humans provides a mechanistic understanding of clinical fluorinated ether anesthetic metabolism and toxicity.

Enzyme-catalyzed processes of first-pass hepatic and intestinal drug extraction.

Oral bioavailability of pharmacologically effective drugs is often limited by first-pass biotransformation. In humans, both hepatic and intestinal enzymes can catalyze the metabolism of a drug as it transits between the gastrointestinal lumen and systemic blood for the first time. Although a spectrum of drug biotransformations can occur during first-pass, the most common are oxidations catalyzed by cytochromes P450. It is the isozymes CYP2D6, CYP3A4, CYP1A2, CYP2C9 and CYP2C19 that are most often implicated in first-pass drug elimination. For any given substrate, enzyme specificity, enzyme content, substrate binding affinity and sensitivity to irreversible catalytic events all play a role in determining the overall efficiency, or intrinsic clearance, of elimination. Several models have been proposed over the past twenty-five years that mathematically describe the process of drug extraction across the liver. The most widely used, the well-stirred model, has also been considered for depiction of first-pass drug elimination across the intestinal wall. With these models it has been possible to examine sources of interindividual variability in drug bioavailability including, variable constitutive enzyme expression (both genetic and environmentally determined), enzyme induction by drugs, disease and diet, and intrinsic or acquired differences in plasma protein binding and organ blood flow (food and drug effects). In recent years, the most common application of hepatic clearance models has been the determination of maximum organ availability of a drug from in vitro derived estimates of intrinsic metabolic clearance. The relative success of the in vitro-in vivo approach for both low and highly extracted drugs has led to a broader use by the drug industry for a priori predictions as part of the drug selection process. A considerable degree of effort has also been focused on gut wall first-pass metabolism. Important pathways of intestinal Phase II first-pass metabolism include the sulfation of terbutaline and isoproterenol and glucuronidation of morphine and labetalol. It is also clear that some of the substrates for CYP3A4 (e.g., cyclosporine, midazolam, nifedipine, verapamil and saquinavir) undergo significant metabolic extraction by the gut wall. For example, the first-pass extraction of midazolam by the intestinal mucosa appears, on average, to be comparable to extraction by the liver. However, many other CYP3A substrates do not appear susceptible to a gut wall first-pass, possibly because of enzyme saturation during first-pass or a limited intrinsic metabolic clearance. Both direct biochemical and indirect in vivo clearance data suggest significant inter-individual variability in gut wall CYP3A-dependent metabolism. The source of this constitutive variability is largely unknown. Because of their unique anatomical location, enzymes of the gut wall may represent an important and highly sensitive site of metabolically-based interactions for orally administered drugs. Again, interindividual variability may make it impossible to predict the likelihood of an interaction in any given patient. Hopefully, though, newer models for studying human gut wall metabolic extraction will provide the means to predict the average extraction ratio and maximum first-pass availability of a putative substrate, or the range of possible inhibitory or inductive changes for a putative inhibitor/inducer.

New Era in Drug Interaction Evaluation: US Food and Drug Administration Update on CYP Enzymes, Transporters, and the Guidance Process

Predicting clinically significant drug interactions during drug development is a challenge for the pharmaceutical industry and regulatory agencies. Since the publication of the US Food and Drug Administrations (FDAs) first in vitro and in vivo drug interaction guidance documents in 1997 and 1999, researchers and clinicians have gained a better understanding of drug interactions. This knowledge has enabled the FDA and the industry to progress and begin to overcome these challenges. The FDA has continued its efforts to evaluate methodologies to study drug interactions and communicate recommendations regarding the conduct of drug interaction studies, particularly for CYP‐based and transporter‐based drug interactions, to the pharmaceutical industry. A drug interaction Web site was established to document the FDAs current understanding of drug interactions (http:www.fda.govcderdrugdrugInteractionsdefault.htm). This report provides an overview of the evolution of the drug interaction guidances, includes a synopsis of the steps taken by the FDA to revise the original drug interaction guidance documents, and summarizes and highlights updated sections in the current guidance document, Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling.

Drug metabolizing enzymes

D h n a p F s i ( t m s p t n t n d f r C i i l rom Clinical Pharmacology, AstraZeneca, Mölndal; Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis; Department of Biology (Galton Lab), University College London, London; Center for Drug Evaluation and Research, Food and Drug Administration, Rockville; School of Pharmacy, University of California San Francisco, San Francisco; Pharmacogenomics, Pfizer Global Research and Development, Groton; University of Chicago, Chicago; and University of Washington, Seattle. his commentary was based on presentations made at a Food and Drug Administration/Johns Hopkins University/Pharmaceutical Research and Manufacturers of America educational workshop, September 13, 2004, Rockville, Md. he views presented in this article do not necessarily reflect those of the Food and Drug Administration. eceived for publication June 6, 2005; accepted Aug 12, 2005. eprint requests: Shiew-Mei Huang, PhD, FCP, Deputy Office Director for Science, Office of Clinical Pharmacology and Biopharmaceutics, Center for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave, Silver Spring, MD 20993-0002. -mail: huangs@cder.fda.gov lin Pharmacol Ther 2005;78:559-81. 009-9236/

Human Kidney Methoxyflurane and Sevoflurane Metabolism Intrarenal Fluoride Production as a Possible Mechanism of Methoxyflurane Nephrotoxicity

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