![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Preclinical Development, Nerviano Medical Sciences, Nerviano, Milan, Italy (M.M., R.d.K.); and Groningen University Institute for Drug Exploration, Department of Pharmacokinetics & Drug Delivery, Groningen, the Netherlands (G.G.)
(Received December 21, 2005; accepted March 21, 2006)
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). Second, anatomically, the small intestine has a serial relationship, with the liver in the process of absorption being the anterior organ. Third, in addition to drug-metabolizing enzymes such as cytochrome P450 (P450) isoenzymes, efflux proteins, such as P-glycoprotein and multidrug resistance-associated proteins, present in the apical membrane of the enterocytes, influence the metabolism process by recycling drugs between enterocytes and lumen, with the consequent higher drug exposure to intestinal metabolic enzymes (Doherty and Charman, 2002). Thus, the amount of an orally administered drug that reaches the systemic circulation can be reduced by both intestinal and hepatic metabolism. For some substrates, it has even been suggested that the role of intestinal metabolism is quantitatively greater than that of hepatic metabolism in the overall first-pass effect (Wu et al., 1995; Paine et al., 1996). Therefore, the important question is raised as to which factors determine the role of intestinal metabolism in the first-pass effect.
The contribution of the small intestine to overall drug metabolism is difficult to evaluate clinically because the organs are perfused in series. Therefore, to obtain a better understanding of the role of the intestine, especially in human drug metabolism, an in vitro technology is very much needed. Intestinal microsomes have been used to study intestinal metabolism, but their major drawback is that metabolic activity is at least partly lost during preparation because of the presence of proteases, with the consequent underestimation of the role of the intestine (Emoto et al., 2000a). Other laboratory techniques available to study first pass-metabolism include everted sacs, which also lose their tissue integrity because they require, for metabolic activity, an artificial NADPH generation system to be present (Emoto et al., 2000b). Furthermore, some (but not all) single drug-metabolizing isoenzymes are commercially available, but up-scaling to the organ activity is not straightforward because of nonphysiological cosubstrate concentrations, lack of interaction with other enzymes, and lack of intercellular communication. Therefore, to overcome these problems, we selected intestinal slices as in vitro tool, taking advantage of the maintenance of the tissue integrity and the relatively simple and straightforward preparation technique suitable to different species (Martignoni et al., 2004; de Kanter et al., 2005). Rat and mouse were selected because they are extensively used in toxicology and pharmacology studies. In particular, athymic nude mice (nu/nu) were chosen because they are commonly used in tumor growth inhibition research (Kelland, 2004). In humans, but also in laboratory animal species, CYP3A appears to be the major drug-metabolizing enzyme subfamily in intestine (Emoto et al., 2000a; McKinnon et al., 1995). Therefore, a set of human CYP3A substrates was selected and incubated with intestinal and liver slices of rat and mouse and with rat intestinal microsomes to compare their metabolic rates between different organs and between different in vitro models. Inhibition by ketoconazole, a P450 inhibitor, was included to confirm the role of P450-mediated metabolism. A scaling factor was determined, in order to be able to compare the data of microsomes and slices. Slice viability during incubation was assessed by measuring ATP content in both liver and intestinal slices. In addition, we compared the relative mRNA expression levels of CYP3A11, CYP3A13, CYP3A25, and CYP3A41 in mouse liver and intestine, by using real-time RT-PCR, to better understand the role of these organs in CYP3A-mediated metabolism. Knowledge about CYP3A mRNA expression in intestine has been described for the rat (Matsubara et al., 2004), but is still lacking for the mouse.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Animals. Sprague-Dawley male rats and male nude mice were obtained from Charles River (Como, Italy) and were maintained under a 12-h light/dark cycle, with free access to drinking water. Nude mice were fed with 4RFN food pellets, which are enriched in protein and lipid content and sterilized by 
-irradiation, whereas rats received standard 4RF21 pellets (Mucedola, Settimo Milanese, MI, Italy). Animals were housed in standard cages with bedding, but for nude mice, the air supply was filtered using EPA filters to protect the nude mice against infections.
Liver and Intestinal Slice Preparation. After i.p. anesthesia with 100 mg/kg sodium thiopental (rat) and a cocktail of ketamine (67 mg/kg), xylazine (15 mg/kg), and acepromazine (1 mg/kg) (mouse), the livers and the first 25 to 30 cm of intestine (thus, mainly duodenum of the small intestine) were excised and stored in ice-cold Williams' Medium E until use (maximum 0.5 h). Liver slices (diameter 8 mm) were prepared in ice-cold Williams' Medium E that was oxygenated with 95% O2/5% CO2 and supplemented with extra glucose (25 mM), using a Krumdieck tissue slicer as described elsewhere (Martignoni et al., 2004). The slices obtained were subsequently stored in ice-cold Williams' Medium E until use (within 0.5 h after the preparation).
Agarose-filled and -embedded slices were prepared as described elsewhere (de Kanter et al., 2005). Shortly thereafter, the excised 25 cm of the small intestine was first cut into two parts that were subsequently ligated on one side. These parts were then filled with 3% (w/v) low-melting agarose solution in 0.9% (w/v) NaCl at 37°C and allowed to gel in ice-cold Williams' Medium E. The agarose-filled intestine was cut into 1-cm parts and these were embedded in the agarose solution at 37°C, using the Tissue Embedding Unit from Alabama R&D (Munford, AL), and allowed to gel so that agarose gel cylinders with a diameter of 16 mm were formed. These cylinders were used to prepare precision-cut intestinal slices, with a diameter of 16 mm and a thickness of approximately 0.25 mm, using a Krumdieck tissue slicer as described above for liver slices. When the slices were transferred to the incubation plates, the agarose surrounding the slices was separated from the slice, so that only the ring of intestinal tissue (diameter approximately 3-5 mm) was used.
Culture of Liver Slices. Slices were individually incubated in 6-well culture plates (3.2 ml of Williams' Medium E, liver slices) or in 12-well culture plates (1.3 ml of Williams' Medium E, intestinal slices) under 95% O2/5% CO2 at 37°C and supplemented with glucose (25 mM) and gentamicin (50 µg/ml). For intestinal slices, amphotericin B (2.5 µg/ml) also was added.
Liver and intestinal slices were incubated in triplicate with 100 µM concentrations of the following substrates: testosterone, triazolam, quinidine, lidocaine, carbamazepine, verapamil, and midazolam. The compounds were dissolved in dimethyl sulfoxide (100 mM) so that the final concentration of organic solvent was 0.1%. Test compounds were also coincubated in the presence of 10 µM ketoconazole, to further characterize the role of P450. The incubation period was 3 h, and at different time points (0, 10, 20, 30, 60, 90, and 180 min) a medium aliquot (80 µl) was removed and added to an equal volume of ice-cold acetonitrile. At the end, the slices were disrupted using an MSE Ultrasonic disintegrator (Fisons, Loughborough, UK) to check the amount of metabolites trapped inside the slices. For all the substrates, apart from testosterone, no differences between slice homogenate and slice medium was observed (data not shown).
LC-MS/MS Analysis. After centrifugation at 5000g for 20 min, the samples were analyzed by LC-MS/MS. For testosterone only, 1 ml of homogenate was extracted with 6 ml of dichloromethane. After removal of the water phase and protein interphase, the organic solvent was evaporated, and testosterone and its metabolites were dissolved in 1 ml of organic phase. The supernatants of centrifuged samples were analyzed by LC-MS/MS, using a Turbo Ion Spray source and a Triple Quadrupole API 4000 instrument (PerkinElmer, Woodbridge, Canada). A 4.6 (inner diameter) x 12.5 mm C8 column (Zorbax; Agilent Technologies) was applied. A mobile phase containing 10 mM ammonium formate (pH 4.0) and acetonitrile was used; for all compounds but testosterone, acetonitrile was increased from 5% to 95% within 0.4 min and then back to 5% in 1.4 min. The flow rate was 1.5 ml/min for the first 0.2 min to equilibrate the column quickly, and 0.2 min after injection, the flow rate was reduced to 0.6 ml/min. For testosterone, acetonitrile in the eluents was increased from 5% to 50% during the first 6 min using a flow of 0.5 ml/min and then back to 5% in 2 min. After injection of 20 µl of the sample, ion spectra were acquired in positive mode, and the quantification was performed by comparing the peak areas with authentic standards of each metabolite. The results are expressed as pmol/min/mg protein using data obtained in the time span in which metabolite formation rates were linear in time (30 min for triazolam, quinidine, lidocaine, carbamazepine, and verapamil, 20 min for midazolam in both liver and intestine slices; and 20 min and 180 min for testosterone in liver slices and intestine slices, respectively). The protein content was determined for each intestinal slice using the Bio-Rad Protein Assay Kit.
Rat Intestinal Microsome Incubation. Male intestinal rat microsomes (Xenotech) were incubated at a concentration of 0.5 mg protein/ml in 100 mM phosphate buffer at pH 7.4 at 37°C, in the presence of a 100 µM concentration of the substrates as described for slices at 37°C. The reactions were started by the addition of the cofactor NADPH (final concentration, 1 mM). At different time points (0, 10, 20, 30, and 45 min), an aliquot (80 µl) was removed and the reaction was terminated by the addition of an equal volume of ice-cold acetonitrile. All incubations were performed in duplicate. Analysis was performed using LC-MS/MS, as described above.
|
1 g) from rat intestine were weighed and homogenized in a known volume (5 ml) of 100 mM phosphate containing 150 mM KCl and 1 mM EDTA, pH 7.4, using a Potter homogenizer. An aliquot of the homogenate was used for the determination of the total amount of the protein. Microsomes were prepared from the homogenate by centrifugation (100,000g for 90 min) of the postmitochondrial supernatant (9000g for 20 min). The microsomal pellet was resuspended in buffer and centrifuged again at 100,000g for 90 min. Microsomes were resuspended in about 1 ml of 100 mM phosphate buffer containing 150 mM KCl and stored in aliquots of 0.1 ml at -80°C. The protein concentration of the microsomes and of the homogenate was determined using the Bio-Rad Protein Assay Kit.
ATP Content. The ATP content of slices incubated in parallel was determined as described before (de Kanter et al., 2005), using the ATPLite-M kit from PerkinElmer (Boston, MA) and a TopCount NXT Luminescence Instrument from PerkinElmer (Boston, MA).
RNA Preparation from Liver and Intestinal Samples. Tissue samples (30 mg) of liver, duodenum, ileum, and colon were taken from three male nude mice after anesthesia, as described above, and stored in RNAlater at 4°C. Total RNA was extracted from the tissue using the QIAGEN RNeasy mini kit. The quality of the isolated RNA was assessed using an RNA 6000 Nano Assay and the Agilent 2100 bioanalyzer. RNA concentration was determined using a RiboGreen RNA quantitation kit.
Reverse Transcription. The mixture was prepared as follows: 1x first strand buffer, 64 units of RNaseOUT, 200 units of SuperScript, 0.6 mM 2'-deoxynucleoside 5'-triphosphate (dATP, dGTP, dCTP, and dTTP), 0.75 µg of random hexamer primers, 10 mM dithiothreitol, and 16 ng of bovine serum albumin. To this mixture, 1 µg of total extract RNA was added. The reverse transcription reaction was performed for 10 min at 25°C, 60 min at 42°C, and 30 min at 37°C.
Design of Primers and Probes. The cDNA sequences of mouse CYP3A11, CYP3A13, CYP3A25, CYP3A41, and ß-actin were obtained from GenBank accession numbers NM 007818.2, NM 007819.1, NM 019792.1, NM 017396.1, and NM 007393. PCR primers and probe sequences were designed using PrimerExpress software (Applied Biosystems) and are shown in Table 1. Nucleotide primers and probe sequences were checked against the National Center for Biotechnology Information BLAST database to ensure specificity for the selected gene.
|
Real-Time Quantitative PCR. Real-time quantitative PCR was performed, using an iCycler iQ real-time PCR detector system (Bio-Rad). The PCR was performed in a 96-well plate. The reaction mixture (13.5 µl) was added in each well to give the following concentrations: 1x master mix reagents, 200 to 900 nM concentrations of each primer, and 200 nM probe for each P450 mRNA assay. cDNA (1.5 µl) was added to each well, and the final volume was 15 µl. The thermal cycle condition was 50°C for 2 min, 95°C for 10 min to activate Amplitaq Gold DNA polymerase, denaturation at 95°C for 15 s, and annealing/extension at 59°C for 1 min (40 cycles). Quantitative PCR for ß-actin mRNA was also performed to normalize for RNA loading.
Statistical Analysis. All data are given as means ± S.E.M. and are average values from three values per experiment; experiments were repeated either two or three times. Statistical evaluation among groups was carried out using a two-tailed Student's t test, and p < 0.05 was considered significant.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Metabolite Formation by Liver and Intestinal Slices from Rats. Both rat liver and intestinal slices showed extensive metabolism of testosterone toward several metabolites. The metabolite formation expressed per milligram of protein per minute by liver slices was, respectively, 2-fold (androstenedione), 3-fold (6ß-TOH), and 6-fold (16
-TOH) higher in comparison to intestinal slices (Fig. 2A). 7-TOH and 2+2ß-TOH were detected in rat liver slice incubations, but not in intestinal slices. In contrast, 16ß-TOH formation appeared to be higher in intestinal slices than in liver slices (7-fold). Also 3-OH-quinidine formation was 3-fold higher in intestinal slices than in liver slices, but the formation of carbamazepine epoxide was higher in liver slices in comparison to intestinal slices (7-fold). The formation of metabolites of lidocaine, verapamil, midazolam, and triazolam was not significantly different between slices from liver and intestine when expressed per milligram of protein (Fig. 2A). The ratio of activity in liver/intestine is given in Table 2.
|
Incubation of intestinal slices with 10 µM ketoconazole inhibited the formation of the metabolites from all compounds tested for more than 80%, apart from androstenedione formation, which was inhibited 50%. In liver slices, the percentage of inhibition varied from 0% to 100%, depending on the substrate (Fig. 3A).
|
Drug Metabolism by Rat Intestinal Microsomes. The metabolic activities by intestinal slices and intestinal rat microsomes calculated per milligram of intestinal protein using the scaling factor of 0.05 are shown in Table 3. The metabolic rates in intestinal slices were significantly higher for all substrates studied (3- to 29-fold) compared with the metabolic rates in microsomes.
|
Metabolite Formation by Liver and Intestinal Slices from Mice. Also using slices from mouse liver and intestine, testosterone gave rise to the formation of several metabolites such as 6ß-TOH, 16-TOH, 16ß-TOH, and androstenedione in both liver and intestine, but in contrast to rat, no 7-TOH was found. 2+2ß-TOH was found in liver slices, but not in intestine slices. Metabolite formation was, respectively, 8-fold (androstenedione), 11-fold (16-TOH), and 5-fold (16ß-TOH) higher in liver slices (Fig. 2B). There were no significant differences observed in the metabolite formation of 6ß-TOH, 1-OH-triazolam, 3-OH-quinidine, and Nor-verapamil between liver and intestine. Significantly higher was the formation of MEGX (4-fold formation), carbamazepine epoxide (6-fold formation), and 1-OH- and 4-OH-midazolam (2-fold formation for both 1-OH- and 4-OH-hydroxylation) in mouse liver slices (Fig. 2B, Table 2).
As was shown for rat, incubation of mouse intestinal slices with 10 µM ketoconazole inhibited the formation of the metabolites of all compounds tested more than 80%, apart from androstenedione formation, which was inhibited 50%. In mouse liver slices, the percentage of inhibition varied from 0% to 100%, depending on the substrate (Fig. 3B).
Detection of CYP3A mRNA Levels in Mouse Liver and Intestine. In rats, the expression of CYP3A isoforms in liver and intestine has been described previously (Matsubara et al., 2004), whereas in mice, this information is still lacking. Therefore, to be able to better interpret differences in metabolic rates between liver and intestine, we investigated their relative mRNA expression in mouse liver and intestine. Due to the absence of RNA standards in the current study, expression of the investigated isoforms can be compared among tissues (liver versus intestine of the same species), but not between the different isoforms. In both mouse liver and intestine, the major mouse CYP3A isoforms (CYP3A11, CYP3A13, CYP3A25, and CYP3A41) were detected. CYP3A11 (6%), CYP3A25 (10%), and CYP3A41 (5%) were less expressed in duodenum compared with liver (Table 4). CYP3A11 (2%), CYP3A25 (4%), and CYP3A41 (2%) were detected in ileum at a lower level than in liver. On the contrary, CYP3A13 was expressed more in the intestine (202% in duodenum and 103% in ileum) than in liver. For each of the CYP3A isoforms, the expression in the ileum was 30 to 50% lower than in duodenum (CYP3A11, 38%; CYP3A13, 51%; CYP3A25, 35%; and CYP3A41, 34%). In colon, CYP3A13 was the only CYP3A isoform detected at an appreciable level (46% of liver value), whereas the other isoforms were less than 0.1% compared with liver (Table 4).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) and that CYP3A4 is responsible for the oxidative metabolism of 50% of drugs currently on the market (de Wildt et al., 1999), we selected a set of marker substrates that are known to be metabolized by human CYP3A.
The feasibility of liver and intestinal slices for metabolism studies was shown before (Martignoni et al., 2004; de Kanter et al., 2005; van de Kerkhof et al., 2005). In both liver and intestinal slices, ATP was maintained at a satisfactory level during incubation (Fig. 1), although a drop of ATP content was measured after the first 30 min of incubation in intestinal slices. We have also observed this phenomenon in earlier studies (de Kanter et al., 2005), where we showed that ATP levels in vivo (approximately 2 nmol ATP/mg protein) and the ATP content in intestinal slices during incubation were comparable. The increased ATP level at time 0 is still unexplained but may be ascribed to ATP synthesis at 4°C in the oxygenated media (de Kanter et al., 2005). The superiority of intestinal slices over intestinal microsomes as a model for metabolic studies was demonstrated when the same substrates were incubated with both in vitro preparations. The metabolic rate expressed per milligram of total intestinal protein was significantly higher using slices for all metabolic conversions studied (Table 3). The difference in metabolite rates between slices and microsomes ranged from 3- to 29-fold between substrates. This dissimilarity may be explained assuming the involvement of different isoforms with different stability. Also, the involvement of transporters that may take up or extrude the compounds into and out of the cells at a different rate may help to explain those differences. We conclude from the higher metabolic capacity that intestinal slices are a more appropriate in vitro model to study gut metabolism than intestinal microsomes, although only a proper in vitro-in vivo comparison can give a definitive answer on whether the higher metabolic rates as observed with intestinal slices are closer to the in vivo situation.
Incubation of liver and intestinal slices with the selected human CYP3A substrates shows that both liver and intestine participate in metabolism of these substrates in both species, although to a different extent, depending on the substrate (Fig. 2A for rat and Fig. 2B for mouse). In general, the metabolite formation rate is quite similar for both species. Differences were observed for the formation of androstenedione (5-fold), 6ß-TOH (2-fold), 16-TOH (2-fold), and 2+2ß-TOH (4-fold) being higher in mouse liver than rat liver. In addition, 7-TOH testosterone was not detected in mouse liver slices.
With respect to the ratio of liver to intestine activities, considerable species differences were found: 16ß-TOH and 3-OH-quinidine formation rates were significantly higher in intestine slices in comparison to liver slices in rat, but not in mouse. This was caused by the lower metabolic activity in rat liver compared with mouse liver. For most of the metabolites, the formation rate was higher in liver than in intestine, but the liver/intestine ratio varied between the species (Table 2). This indicates that this ratio cannot be simply extrapolated between species.
To confirm the role of P450 in both the intestinal and the hepatic metabolism, slices were incubated with ketoconazole, a broad rat P450 inhibitor (Kobayashi et al., 2003). In the clinic, ketoconazole affects largely the pharmacokinetics of drugs that are primarily metabolized by CYP3A4, resulting in a substantial decrease of first-pass metabolism (Moody et al., 2004). In intestinal slices from both mouse and rat, ketoconazole strongly inhibited the metabolism of all tested compounds, whereas in liver, the inhibitory effect was more variable. This means that in vivo, drug-drug interactions such as that by ketoconazole may occur both in liver and intestine, which results in higher exposure to the parent compound. In addition, we hypothesize that metabolite formation of the tested substrates in intestinal slices is mainly mediated by CYP1A and CYP3A isoforms in rat, because those two isoforms are mostly expressed in rat intestine (Kaminsky and Zhang, 2003), and by CYP3A in mouse, because until now, this isoform has been the only P450 found to be present in mouse intestine (Emoto et al., 2000a). In liver slices, however, no complete inhibition was reached with ketoconazole, suggesting that metabolism can be attributed not only to CYP1A and CYP3A but also to other P450s enzymes, which are apparently little expressed in rat and mouse intestine. For example, it is known that CYP2D participates in lidocaine N-deethylation in rats (Wan et al., 1997), but CYP2D is little expressed in rat intestine (Aiba et al., 2003). Also, hepatic metabolism of midazolam in mouse, yielding 1-OH and 4-OH metabolites, has, apart from CYP3A, a significant CYP2C component (Perloff et al., 2000), and mouse and rat intestine appear to have very low CYP2C expression (Emoto et al., 2000b; Kaminsky and Zhang, 2003). In rats, apart from CYP3A1, CYP1A1 and CYP2B1 also were detected in enterocytes, whereas CYP2C11, CYP2A1, CYP2B2, CYP2E1, CYP3A2, and CYP4A1, which are expressed in liver, were not detectable in intestine by using RT-PCR and immunoblot analysis (Kaminsky and Zhang, 2003). On the other hand, it was reported that in rats, CYP2C6, CYP2C11, CYP2B1, CYP2D1, and CYP1A1 are expressed in duodenum, jejunum, and ileum, at least at the mRNA level (Lindell et al., 2003). In addition, previous studies revealed that rat CYP3A isoforms are expressed differently between liver and intestine and that some CYP3A substrates are metabolized by different isoforms in those two organs (Takara et al., 2003; Matsubara et al., 2004). Rat CYP3A1 and CYP3A2 are expressed predominantly in liver and are not detectable in the intestinal tract, whereas CYP3A9 and CYP3A18 are detectable in both liver and intestine. Earlier, it was found that, in rat, CYP3A9 mRNA was 3-fold more expressed in duodenum than in liver and CYP3A18 mRNA was 16-fold more expressed in duodenum than in liver (Matsubara et al., 2004). In contrast, CYP3A62 mRNA has been reported to be the predominant form in rat intestinal tract, being 9-fold more expressed in duodenum in comparison to liver (Matsubara et al., 2004). This different expression in CYP3A isoforms between liver and intestine in rats may explain the differences in formation rates of CYP3A metabolites between liver and intestine, as was found in this study (Fig. 2A). However, whether, for example, the higher expression of CYP3A62 in rat intestine is responsible for the observed higher 3-OH-quinidine and 16ß-OHT formation in rat (Fig. 2A) can only be speculated at this moment. Another explanation for this difference is that the biotransformation reactions studied are not mainly catalyzed by CYP3A isoenzymes, as is suggested by different inhibition profiles by ketoconazole in liver slice incubations, compared with intestinal slice incubations (Fig. 3, A and B). Here, we show that, similar to rats (Matsubara et al., 2004), mouse CYP3A isoenzymes are expressed differently between liver and intestine. Mouse CYP3A11, CYP3A25, and CYP3A41 are predominantly expressed in the liver, whereas CYP3A13 is detected more in the intestine than in liver (Table 4). In humans, CYP3A4/5 is the most expressed P450 isoform in the small intestine (McKinnon et al., 1995), whereas CYP1A1, CYP2C19, and CYP2D6 are less expressed isoforms (Doherty and Charman, 2002). Together, these observations indicate that in human, mouse, and rats, the intestine, expressing a different P450 profile than liver (including different CYP3A isoforms), may play a unique role in the metabolism of xenobiotics and may contribute to first-pass metabolism to a different extent than the liver. In addition, species differences in liver/intestine ratios of enzyme activities make interspecies extrapolation hazardous.
Another observation was that, in mice, the mRNA expression of P450 isoforms decreased along the mouse intestinal tract, from duodenum to colon, which is in accordance with earlier findings in rat (Matsubara et al., 2004) and humans (de Waziers et al., 1990). Also, P450-mediated enzyme activities were recently shown to decrease along the intestinal tract, using rat intestinal slices (van de Kerkhof et al., 2005).
The current study underlines the potential of intestinal slices to study intestine metabolism in vitro and, consequently, to predict drug-drug interactions (Kanazu et al., 2005). Because the preparation of slices is basically the same for each species, we expect that slices from other species including human can also be used to predict intestinal metabolism. This in vitro model to study (human) gut metabolism is of interest, considering that no other relatively simple models that maintain tissue architecture are available. More investigations are needed to further characterize the role of the intestine in first-pass metabolism, such as the investigation of a broader range of marker substrates covering different P450 isoenzymes, and the effect of different inhibitors. Besides, a comparison between in vitro and in vivo data would allow a better estimation and prediction of the role of the intestine.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: P450, cytochrome P450; RT-PCR, quantitative reverse transcription-polymerase chain reaction; PCR, polymerase chain reaction; MEGX, monoethylglycinexylidide; Nor-verapamil, normethyl-verapamil; OH, hydroxy; TOH, hydroxytestosterone; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry.
1 Current affiliation: Solvay Pharmaceuticals, Weesp, the Netherlands. 
Address correspondence to: Marcella Martignoni, Preclinical Development, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano (MI), Italy. E-mail: marcella.martignoni{at}nervianoms.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  V. M. Lakshmi, F. F. Hsu, and T. V. Zenser N-Demethylation Is a Major Route of 2-Amino-3-Methylimidazo[4,5-f]quinoline Metabolism in Mouse Drug Metab. Dispos., June 1, 2008; 36(6): 1143 - 1152. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
, the formation of 16