Mary F. Paine

Oral first‐pass elimination of midazolam involves both gastrointestinal and hepatic CYP3A‐mediated metabolism

To determine in humans the relative roles of intestinal and hepatic metabolism in the oral first‐pass elimination of a CYP3A substrate using midazolam as a model compound.

THE HUMAN INTESTINAL CYTOCHROME P450 “PIE”

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major “pieces” of the human liver P450 “pie” and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414–423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies. P450s 3A4, 2C9, 2C19, and 2J2 were detected in all individuals and ranged from 8.8 to 150, 2.9 to 27, <0.6 to 3.9, and <0.2 to 3.1 pmol/mg, respectively. CYP2D6 was detected in 29 individuals and ranged from <0.2 to 1.4 pmol/mg. CYP3A5 was detected readily in 11 individuals, with a range (average) of 4.9 to 25 (16) pmol/mg that represented from 3 to 50% of total CYP3A (CYP3A4 + CYP3A5) content. CYP1A1 was detected readily in three individuals, with a range (average) of 3.6 to 7.7 (5.6) pmol/mg. P450s 1A2, 2A6, 2B6, 2C8, and 2E1 were not or only faintly detected. As anticipated, average CYP3A content (50 pmol/mg) was the highest. Excluding CYP1A1, the remaining enzymes had the following rank order: 2C9 > 2C19 > 2J2 > 2D6 (8.4, 1.1, 0.9, and 0.5 pmol/mg, respectively). Analysis of a pooled preparation of the 31 donor specimens substantiated these results. In summary, as in the liver, large interindividual variation exists in the expression levels of individual P450s. On average, CYP3A and CYP2C9 represents the major pieces of the intestinal P450 pie, accounting for 80 and 15%, respectively, of total immunoquantified P450s.

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.

THE ROLE OF HEPATIC AND EXTRAHEPATIC UDP-GLUCURONOSYLTRANSFERASES IN HUMAN DRUG METABOLISM *,†

At present, the methods and enzymology of the UDP-glucuronosyltransferases (UGTs) lag behind that of the cytochromes P450 (CYPs). About 15 human UGTs have been identified, and knowledge about their regulation, substrate selectivity, and tissue distribution has progressed recently. Alamethicin has been characterized as a treatment to remove the latency of microsomal glucuronidations. Most UGT isoforms appear to have a distinct hepatic and/or extrahepatic expression, resulting in significant expression in kidney, intestine, and steroid target tissues. The gastrointestinal tract possesses a complex expression pattern largely containing members of the UGT1A subfamily. Thus, these forms are poised to participate in the first pass metabolism of oral drugs. The authors and others have identified a significant expression of UGT1A1 in human small intestine, an enzyme possessing considerable allelic variability and a polymorphic expression pattern in intestine. Intestinal glucuronidation therefore plays a major role not only in first pass metabolism, but also in the degree of interindividual variation in overall oral bioavailability. Due to issues such as significant genetic variability and tissue localization in first-pass organs, clearance due to UGT1A1 should be minimized for new drugs.

P-glycoprotein increases from proximal to distal regions of human small intestine.

AbstractPurpose. The contribution of the efflux transporter P-glycoprotein (P-gp) as a barrier to drug absorption may depend on its level of expression at the site of absorption. Accordingly, the distribution of P-gp was examined along the entire length of the human small intestine. Methods. Homogenates prepared from mucosal scrapings from every other 30-cm segment of four unrelated human donor small intestines were analyzed for P-gp and the control protein villin by Western blot. Results. In each donor intestine, relative P-gp expression (P-gp/villin integrated optical density ratio) progressively increased from proximal to distal regions. Among individuals, relative P-gp levels varied 2.1-fold in the duodenal/proximal jejunal region, 1.5- to 2.0-fold in the middle/distal jejunal region, and 1.2- to 1.9-fold in the ileal region. Within-donor variation was somewhat greater, from 1.5- to 3.0-fold. Conclusions. These results provide further evidence that the site of absorption can represent another source for the interindividual variation in the oral bioavailability of drugs.

Seville orange juice‐felodipine interaction: Comparison with dilute grapefruit juice and involvement of furocoumarins

Our objective was to determine whether Seville orange juice produces a grapefruit juice–like interaction with felodipine and whether bergamottin, 6′,7′‐dihydroxybergamottin, or other furocoumarins are involved.

Effects of a Chargrilled Meat Diet on Expression of CYP3A, CYP1A, and P-Glycoprotein Levels in Healthy Volunteers

BACKGROUND & AIMS Carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons present in chargrilled meat are substrates for inducible CYP1A and CYP3A enzymes and for P-glycoprotein. We examined whether consumption of a chargrilled meat diet results in induction of these proteins. METHODS Ten healthy adults were fed a diet enriched with chargrilled meat for 7 days. Duodenal biopsy specimens were obtained on days 1, 5, and 12 and analyzed for CYP1A, CYP3A, and P-glycoprotein messenger RNA (mRNA) and protein. On days 5 and 12, hepatic CYP3A4 and CYP1A2 activities were measured and colon biopsies were performed. The levels of polycyclic aromatic hydrocarbon DNA adducts in peripheral blood mononuclear cells were measured on days 1, 4, 11, and 26. RESULTS There was no detectable induction of CYP3A4, CYP3A5, or P-glycoprotein mRNAs or protein in small intestine or colon and no induction of hepatic CYP3A4 enzyme activity. In contrast, the chargrilled meat diet resulted in unequivocal induction of CYP1A enzymes in the liver and small intestine of each subject. There was an inverse correlation between the level of peripheral polycyclic aromatic hydrocarbon DNA adducts measured on day 11 and both liver CYP1A2 activity (P = 0.027) and enterocyte CYP1A1 protein concentration (P = 0.046). CONCLUSIONS Ingestion of chargrilled meat results in induction of CYP1A enzymes but not CYP3A4 or P-glycoprotein. This observation, combined with the correlation between adduct levels and CYP1A expression, supports an adaptive role for CYP1A but not CYP3A4 or P-glycoprotein.

Variation in oral clearance of saquinavir is predicted by CYP3A5*1 genotype but not by enterocyte content of cytochrome P450 3A5

Saquinavir, a widely prescribed human immunodeficiency virus 1 protease inhibitor, has a low and variable oral bioavailability that has been attributed to extensive first‐pass extraction mediated by hepatic or intestinal cytochrome P450 (CYP) 3A4 and intestinal P‐glycoprotein (P‐gp). The polymorphic CYP3A5 has also been shown to influence the saquinavir metabolite/parent urinary ratio, suggesting a role for CYP3A5.

Comparison of CYP2D6 content and metoprolol oxidation between microsomes isolated from human livers and small intestines.

AbstractPurpose. To assess the role of intestinal CYP2D6 in oral first-pass drug clearance by comparing the enzyme content and catalytic activity of a prototype CYP2D6 substrate, metoprolol, between microsomes prepared from human intestinal mucosa and from human livers. Methods. Microsomes were prepared from a panel of 31 human livers and 19 human intestinal jejunal mucosa. Microsomes were also obtained from the jejunum, duodenum and ileum of four other human intestines to assess regional distribution of intestinal CYP2D6. CYP2D6 content (pmole/mg microsomal protein) was determined by Western blot. CYP2D6 activity was measured by α-hydroxylation and O-demethylation of metoprolol. Results. Kinetic studies with microsomes from select livers (n = 6) and jejunal mucosa (n = 5) yielded KM estimates of 26 ± 9 μM and 44 ± 17 μM, respectively. The mean Vmax (per mg protein) for total formation of α-OH-M and ODM was 14-fold higher for the liver microsomes compared to the jejunal microsomes. Comparisons across intestinal regions showed that CYP2D6 protein content and catalytic activity were in the order of jejunum > duodenum > ileum. Excluding the poor metabolizer genotype donors, CYP2D6 content varied 13-and 100-fold across the panels of human livers (n = 31) and jejunal mucosa (n = 19), respectively. Metoprolol α-hydroxylation activity and CYP2D6 content were highly correlated in the liver microsomes (r = 0.84, p < 0.001) and jejunal microsomes (r = 0.75, p < 0.05). Using the well-stirred model, the mean microsomal intrinsic clearance (i.e., Vmax/KM) for the livers and jejunum were scaled to predict their respective in vivo organ intrinsic clearance and first-pass extraction ratio. Hepatic and intestinal first-pass extractions of metoprolol were predicted to be 48% and 0.85%, respectively. Conclusions. A much lower abundance and activity of CYP2D6 are present in human intestinal mucosa than in human liver. Intestinal mucosal metabolism contributes minimally to the first-pass effect of orally administered CYP2D6 substrates, unless they have exceptionally high microsomal intrinsic clearances and/or long residence time in the intestinal epithelium.

TWO MAJOR GRAPEFRUIT JUICE COMPONENTS DIFFER IN INTESTINAL CYP3A4 INHIBITION KINETIC AND BINDING PROPERTIES

Bergamottin (BG) and 6′,7′-dihydroxybergamottin (DHB) are the most abundant furanocoumarins present in grapefruit juice and have been proposed as major intestinal CYP3A4 inhibitors contributing to grapefruit juice-drug interactions. The relative contribution of BG versus DHB to the interaction potential is unclear, in part due to inconsistencies in the literature regarding inhibitory potency. To resolve these inconsistencies, the inhibitory kinetics of each furanocoumarin toward CYP3A4 catalytic activity were systematically characterized using representative probes from two distinct CYP3A4 substrate subgroups (testosterone and midazolam). With human intestinal microsomes, DHB was a substrate-independent reversible (Ki, ∼0.8 μM) and mechanism-based (KI, ∼3 μM; kinact, 0.3-0.4 min-1) inhibitor of CYP3A4. In contrast, BG was a substrate-dependent reversible inhibitor, with a Ki (13 μM) using midazolam that was 8-fold greater than that using testosterone, but a substrate-independent mechanism-based inhibitor (KI, ∼25 μM; kinact, ∼0.35 min-1). Similar trends resulted with cDNA-expressed CYP3A4, only the KI values for BG were ∼10-fold lower than with microsomes. This seemed to reflect a much greater degree of microsomal protein binding by BG compared with DHB. Differential inhibition kinetics and binding properties between BG and DHB could account in part for the apparent in vitro inconsistencies in the literature. Results also emphasize the importance of appropriate substrate selection when designing inhibition studies involving dietary constituents.