Intestinal and Hepatic Metabolic Activity of Five Cytochrome P450 Enzymes: Impact on Prediction of First-Pass Metabolism

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266

233

ABSTRACT:Intestinal first-pass metabolism may contribute to low oral drug bioavailability and drug-drug interactions, particularly for CYP3A substrates. The current analysis predicted intestinal availability (F G ) from in vitro metabolic clearance and permeability data of 25 drugs using the Q Gut model. The drug selection included a wide range of physicochemical properties and in vivo F G values (0.07-0.94). In vitro clearance data (CLu int ) were determined in human intestinal (HIM) and three liver (HLM) microsomal pools (n ‫؍‬ 105 donors) using the substrate depletion method. Apparent drug permeability (P app ) was determined in Caco-2 and Madin-Darby canine kidney cells transfected with human MDR1 gene (MDCK-MDR1 cells) under isotonic conditions (pH ‫؍‬ 7.4). In addition, effective permeability (P eff ) data, estimated from regression analyses to P app or physicochemical properties were used in the F G predictions. Determined CLu int values ranged from 0.022 to 76.7 l/min/pmol of CYP3A (zolpidem and nisoldipine, respectively). Differences in CLu int values obtained in HIM and HLM were not significant after normalization for tissue-specific CYP3A abundance, supporting their interchangeable usability. The F G predictions were most successful when P app data from Caco-2/MDCK-MDR1 cells were used directly; in contrast, the use of physicochemical parameters resulted in significant F G underpredictions. Good agreement between predicted and in vivo F G was noted for drugs with low to medium intestinal extraction (e.g., midazolam predicted F G value 0.54 and in vivo value 0.51). In contrast, low prediction accuracy was observed for drugs with in vivo F G <0.5, resulting in considerable underprediction in some instances, as for saquinavir (predicted F G is 6% of the observed value). Implications of the findings are discussed.

Section: Discussionsupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Prediction of Human Intestinal First-Pass Metabolism of 25 CYP3A Substrates from In Vitro Clearance and Permeability Data

Gertz

Harrison²,

Houston³

et al. 2010

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266

233

“…In HLMs, this reaction is also NADPH-dependent and mediated almost exclusively by CYP2C19, as shown by its sensitivity to inhibitors of this enzyme, including inhibitory antibodies, and the lack of inhibition by neutralizing monoclonal antibodies against CYP2A6, CYP2D6, and (Walsky and Obach, 2004) and 30 M and 40 pmol/min/mg (Galetin and Houston, 2006), respectively]. The somewhat lower V max value obtained in the present study may be due to enzyme inhibition by the organic solvent present in our assay.…”

Section: Discussioncontrasting

confidence: 50%

High-Throughput Radiometric CYP2C19 Inhibition Assay Using Tritiated (S)-Mephenytoin

Marco

Cellucci²,

Chaudhary³

et al. 2007

ABSTRACT:A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4-hydroxylation of (S)-mephenytoin labeled with tritium in the 4 position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3-and 5-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC 50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.

“…5, A-F, Ⅺ); however, in the ileum, simulated permeability of quinidine in the lower concentration range deviated slightly from the measured permeability. One of the plausible explanations for this inconsistency is that, because several cytochrome P-450 enzymes were reported to be expressed in the intestinal epithelial layer, cytochrome P-450-mediated metabolism of drugs during the intestinal absorption process might affect the permeability measured by the in situ single-pass perfusion method (Ching et al, 1995;Tracy et al, 1999;Galetin and Houston, 2006). In addition, the effects of other ABC transporters, such as breast cancer resistance protein and multidrug resistanceassociated protein 2, are possible factors in the deviation (Englund et al, 2006;Han and Sugiyama, 2006).…”

Section: Downloaded Frommentioning

confidence: 99%

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Shirasaka¹,

Masaoka²,

Kataoka³

et al. 2008

ABSTRACT:In a previous study, the concentration-dependent permeability of P-glycoprotein (P-gp) substrate drugs, quinidine, verapamil, and vinblastine, in several cell monolayers with different levels of P-gp expression was analyzed kinetically to obtain fundamental parameters for P-gp-mediated transport, V max and K m(app) values. Both V max and K m(app) values of each drug were found to show linear correlations with the expression level of P-gp. These findings imply the possibility of estimating the V max and K m(app) values of P-gp substrate drugs in the in vivo intestinal membrane on the basis of the P-gp expression level. In the present study, concentrationdependent drug permeability to the rat small intestines (upper jejunum and ileum) was simulated on the basis of V max and K m(app) values of each drug estimated from the P-gp expression level in the rat small intestines. To validate the predictability of these procedures, drug permeability in the rat small intestines was measured by the in situ single-pass perfusion method. It was confirmed that simulated permeability of each drug in the rat jejunum and ileum corresponded well with permeability measured by the in situ single-pass perfusion method. This study clearly demonstrated the potential to estimate the permeability of P-gp substrate drugs in the human intestine from its P-gp expression level and thus the possibility to predict the oral absorption of those drugs.P-glycoprotein (P-gp), one of the most important efflux transporters in the field of biopharmacy, is an ATP-binding cassette (ABC) transporter encoded by the multidrug resistance 1 gene (MDR1/ABCB1). P-gp is expressed not only in cancer cells but also in many normal tissues (Loo and Clarke, 1999). So far, many reports have described the effects of P-gp on the pharmacokinetic profiles of clinically important drugs, by denaturing absorption, distribution, and excretion. For example, P-gp located in the apical domain of the enterocytes of the gastrointestinal tract limits the uptake and absorption of its substrate drugs after oral administration (Hunter and Hirst, 1997;Meijer et al., 1997;Jonker et al., 1999). Because of the great impact of P-gp on the pharmacokinetic profiles of a variety of drugs, various in vitro experimental techniques are now used to identify the compounds subjected to P-gp-mediated efflux (Kim et al., 1998;Polli et al., 1999Polli et al., , 2001Susanto and Benet, 2002; Shirasaka et al., 2006a,b). However, it is still difficult to quantitatively predict the effect of P-gp on in vivo intestinal absorption of such drugs.In a previous study, we measured the apical (AP) to basal (BL) absorptive permeability of P-gp substrate drugs in several cultured cell monolayers with different expression levels of P-gp (Shirasaka et al., 2008). In all cell monolayers, AP to BL permeability of P-gp substrate drugs showed a sigmoid-type relation to their donor (AP) concentration and reached a maximal value at the higher concentration range because of the saturation of P-gp-mediated efflux. Con...