REGULATORY

Drug metabolism in the intestine

Esther van de Kerkhof, Notox

The intestine is armed with a broad spectrum of drug metabolising enzymes and drug transporters to strengthen its function as first barrier of defence against orally ingested xenobiotics.

As oral administration is the most convenient and therefore most frequently used route of drug administration, this gastrointestinal barrier determines to a large extent the bioavailability of drugs. In the following, a short introduction to the intestine and its drug metabolising capacities will be given with special reference to species differences and available methods to study intestinal drug metabolism.

Why is the intestine of interest?

Although the liver has long been thought to play the major role in drug metabolism, the metabolic capacity of the intestine has been increasingly recognised1. In the 1970’s, intestinal metabolism was already reported2. Nonetheless, until recently the clinical significance of intestinal drug metabolism remained under debate3. Although basic knowledge concerning human intestinal drug enzyme and transporters expression has been collected over the last decade4, the ultimate proof for their significance has been demonstrated by in vivo studies. For several compounds, such as cyclosporine A and verapamil among others, in vivo studies have shown significant first-pass metabolism by the intestinal wall1,5. Recent studies with intestinal precision-cut slices (intact tissue slices) have shown that the metabolic rates in enterocytes (drug metabolising cells of the intestine) are comparable with metabolic rates in hepatocytes (drug metabolising cells of the liver) for several metabolic reactions in both rat and human (see table I)6,7.

For example, the glucuronidation and sulphation rates of 7-hydroxycoumarin in rat enterocytes are comparable to those in hepatocytes. Another example, CYP3A conversion rates (6ß-hydroxylation of testosterone) in human enterocytes are higher than in human hepatocytes.

*SI: small intestine, pmol/min/mg enterocyte (25% of tissue is enterocytes); #pmol/min/mg colonocyte (17% of tissue is colonocyte); pmol/min/mg hepatocyte (80% of tissue is hepatocyte of which 50% of the hepatocytes contributes to drug metabolism9; ND is not detectable; NM: not measured

Drug metabolising enzymes and transporters in the intestinal tract

Drug metabolising enzymes and drug transporters are broadly expressed along the intestinal tract and can contribute together to all three phases of drug metabolism as depicted in figure 1: introduction of functional groups in the drug molecule (Phase I), conjugation of drug molecules or phase I products (Phase II), excretion by efflux transporters (commonly referred to as Phase III) and deconjugation in the lumen (Phase III of drug metabolism).

In the proximal part of the intestinal lumen (the part that is the closest to the stomach), the concentrations of orally taken drugs, but also of dietary components, are highest. To form an optimal barrier preventing xenobiotics from entering the body, the highest density of drug metabolising enzymes should also be present in the proximal part of the intestine. This is indeed the case for phase I enzymes: CYP3A4, CYP2C8-10 and CYP2D610-12.

Apart from drug metabolising enzymes, drug uptake and efflux transporters, such as PEPT113 or MRP2/ABCC2 and MDR1/ABCB1 (Pgp)14, form another obstacle to drug absorption. For example, concomitant administration of cefadroxil and cephalexin in humans has been shown to decrease the exposure of cefadroxil after oral administration presumably due to the competitive inhibition of intestinal PEPT1 mediated transport of cefadroxil by cephalexin13.

Species differences: Rat versus man

Animal models are commonly used for drug metabolism studies, but drug metabolism and especially drug-drug interactions are highly species specific. A comparison of drug metabolising rates in human and rat intestine is depicted in table I. In general, the metabolic rates (obtained with saturating concentrations of substrate) are surprisingly similar in rat and human tissue. Only for the metabolic rates of CYP3A conversions, human tissue showed higher maximal rate of metabolism compared with rat tissue at the same substrate concentration. Of note, the 2ß-hydroxytestosterone is only detected in human tissue and not in rat tissue. The relative metabolic rates in small intestine and liver, however, are not similar in rat and man.

For example, androstenedione, 6ß-hydroxytestosterone and 16α-hydroxytestosterone formations are higher in rat liver than in intestine in contrast to human tissue, where the metabolic rates are higher in small intestine than liver. Glucuronidation is the highest in small intestine in rat, but in human tissue the formation rate is higher in liver. Furthermore, sulphation rates are similar in rat intestine and liver, but are 2.5-fold higher in human small intestine compared with human liver. On the other hand, the 7EC O-deethylation rate is the highest in liver in both species. This suggests that species differences in metabolic rates in liver are not necessarily correlated with those in intestine. Therefore, organ-specific metabolism should be studied in human tissue, since it cannot be extrapolated from rat to human.

In addition, several species differences have been described with respect to induction of drug metabolising enzymes. For instance, a compound like rifampicin is a ligand for both human and rabbit PXR (Pregnane X Receptor involved in the induction of CYP3A among others), but not of rat PXR, whereas pregnenolone 16α-carbonitrile, is a known activator of rat and mouse PXR15.

How to measure intestinal metabolism

Only a few methods are available to study intestinal drug metabolism in vivo or in situ. These techniques are indirect and ethically complex when performed in man. Above all, they often do not provide adequate intestinal drug metabolism data because of the difficulty to discriminate between liver and intestinal contributions. Several in vitro methods are available to study drug metabolism, such as intact cell systems, subcellular fractions and cell lines.

*Only applicable to animal tissue, * * human cell lines

In table 2, these methods are evaluated with respect to four parameters:

• Presence of intact cells (allowing investigation of drug metabolism and transporter interactions);
• Viability up to 24 h (allowing investigation of induction processes);
• Applicability to both human and animal tissue (allowing investigation of species differences) ;
• Amount of tissue needed for experimentation (in view of the limited access to human small intestinal tissue).

Every in vitro method has its own advantages and limitations. The choice of the method should therefore depend on the question of interest. Subcellular fractions and cell lines are rather efficient methods to study drug metabolism at the individual enzyme level, but the isolated intestinal perfusion, everted sac and Ussing chamber preparations are particularly useful for studying overall drug metabolism and interactions with transporters. Tissue biopsies, precision-cut intestinal slices and primary cells seem most appropriate to study metabolism of slowly metabolized drugs as well as the induction processes via various pathways16.

The significance of the intestine to drug metabolism

Metabolic rates alone do not reveal whether the intestinal wall significantly contributes to total body drug disposition. Factors such as dissolution of the drug from the formulation in the gastrointestinal tract, transit time, location of absorption, mucosal blood flow, may all influence the intestinal first-pass metabolism of drugs, but the combined outcome of the factors is hard to predict. Nonetheless, the intestine has the machinery and capacity to metabolise drugs.

The significance of the intestine in determining the fate of drugs within the body is not only expressed by its high capacity to metabolise drugs, but also by its sensitivity to induction and inhibition of drug metabolising enzymes. It was suggested by Lin et al that intestinal enzymes respond to a greater extent than hepatic enzymes to orally administered inducers like drugs and food components because of exposure to their relatively high concentrations in the intestine 3.

A well-known example is the induction of CYP3A4 and MDR1 (Pgp/ ABCB1) by St John’s Wort in transplantation patients, causing a serious decrease in cyclosporine A plasma concentration, which in several cases has lead to organ rejection after transplantation17. Furthermore, significant inhibition of CYP3A4 by grapefruit juice has been shown to increase the felodipine AUC three-fold in hypertensive patients18, supporting the significance of intestinal metabolism in vivo.

To conclude

The intestine is able to metabolise drugs, thereby strengthening its function as first line of defence against xenobiotics. In vivo studies have proven that significant first-pass metabolism by the intestinal wall has implications for the bioavailability of several compounds and the relevance of drug transporters in this process has also been proven. For several metabolic reactions, the metabolic rates are comparable in enterocytes and hepatocytes. Furthermore, species differences in metabolic rates are noted and imply that, for a good prediction of drug metabolism in man, studies with human tissue are required.

Esther van de Kerkhof

Esther van de Kerkhof worked as a PhD candidate at the department of Pharmacokinetics and Drug Delivery, University of Groningen, the Netherlands under supervision of prof. dr. G.M.M. Groothuis, prof. dr. D.K.F. Meijer and dr. ir. I.A.M. de Graaf. June 2007, she successfully defended her thesis: ‘Drug metabolism in human and rat intestine: An in vitro approach’. The aim of these studies was to evaluate the application of rat and human intestinal precision- cut slices for drug metabolism studies. Since April 2007, she has supervised ADME and Kinetics studies at NOTOX in ‘s Hertogenbosch, The Netherlands.

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NOTES :
1 Glaeser H, Drescher S, Hofmann U, Heinkele G, Somogyi AA, Eichelbaum M, Fromm MF. (2004) Impact of concentration and rate of intraluminal drug delivery on absorption and gut wall metabolism of verapamil in humans. Clin Pharmacol Ther 76: 230-8
2 Stohs SJ, Grafstrom RC, Burke MD, Orrenius S. (1976) Xenobiotic metabolism and enzyme induction in isolated rat intestinal microsomes. Drug Metab Dispos 4: 517-21
3 Lin JH, Chiba M, Baillie TA. (1999) Is the role of the small intestine in first-pass metabolism overemphasized? Pharmacol Rev 51: 135-58
4 Obach RS, Zhang QY, Dunbar D, Kaminsky LS. (2001) Metabolic characterization of the major human small intestinal cytochrome p450s. Drug Metab Dispos 29: 347-52
5 Kolars JC, Awni WM, Merion RM, Watkins PB. (1991) First-pass metabolism of cyclosporin by the gut. Lancet 338: 1488-90
6 Van de Kerkhof EG, De Graaf IAM, De Jager MH, Meijer DKF, Groothuis GMM. (2005) Characterization of rat small intestine and colon precision-cut slices as an in vitro system for drug metabolism and induction studies. Drug Metab Dispos 33: 1613-20
7 Van de Kerkhof EG, Ungell ALB, Sjoberg AK, De Jager MH, Hilgendorf C, De Graaf IAM, Groothuis GMM. (2006) Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with using chamber preparations Drug Metabol Dispos 24: 1893-902
8 De Kanter R, De Jager MH, Draaisma AL, Jurva JU, Olinga P, Meijer DK, Groothuis GM. (2002) Drug-metabolising activity of human and rat liver, lung, kidney and intestine slices. Xenobiotica 32: 349-62
9 De Graaf IA, de Kanter R, de Jager MH, Camacho R, Langenkamp E, van de Kerkhof EG, Groothuis GM. (2006) Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction. Drug Metab Dispos 34: 591-9
10 Thorn M, Finnstrom N, Lundgren S, Rane A, Loof L. (2005) Cytochromes P450 and MDR1 mRNA expression along the human gastrointestinal tract. Br J Clin Pharmacol 60: 54-60
11 Krishna DR, Klotz U. (1994) Extrahepatic metabolism of drugs in humans. Clin Pharmacokinet 26: 144-60
12 Shen DD, Kunze KL, Thummel KE. (1997) Enzyme-catalyzed processes of first-pass hepatic and intestinal drug extraction. Adv Drug Deliv Rev 27: 99-127
13 Tsuji A. (2006) Impact of transporter-mediated drug absorption, distribution, elimination and drug interactions in antimicrobial chemotherapy. J Infect Chemother 12: 241-50
14 Fricker G, Miller DS. (2002) Relevance of multidrug resistance proteins for intestinal drug absorption in vitro and in vivo. Pharmacol Toxicol 90: 5-13
15 LeCluyse EL. (2001) Pregnane X receptor: molecular basis for species differences in CYP3A induction by xenobiotics. Chem Biol Interact 134: 283-9
16 Van de Kerkhof EG, De Graaf IAM, Groothuis GMM (2007): In vitro methods to study intestinal drug metabolism, Curr Drug Metab, in press
17 Ruschitzka F, Meier PJ, Turina M, Luscher TF, Noll G. (2000) Acute heart transplant rejection due to St John’s wort. Lancet 355: 548-9
18 Bailey DG, Malcolm J, Arnold O, Spence JD. (1998) Grapefruit juice-drug interactions. Br J Clin Pharmacol 46: 101-10
19 Montrose M, Keely S, Barrett K. 1999. In Textbook of Gastroenterology, ed. Yamada T, Alpers D, Laine L, Owyang C, Powell D, pp. 320-34: Lippincott Williams & wilkins

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