In Vitro Methods to Study Intestinal Drug Metabolism

Kerkhof, Esther G. van de; Graaf, Inge A. M. de; Groothuis, Geny M. M.

doi:10.2174/138920007782109742

Cited by 109 publications

(73 citation statements)

References 183 publications

(492 reference statements)

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“…The mucosal scrapings were mixed with 5 volumes (w/v) of 10 mM phosphate buffer containing 5 mM EDTA, pH 7.4, and homogenized to prepare 20% homogenates. The homogenates were subjected to centrifugation to isolate cellular S9 fractions as described elsewhere (van de Kerkhof et al, 2007). The protein concentrations of the cellular fractions S9 were determined using a commercially available kit (BCA protein assay; Pierce Chemical, Rockford, IL) following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Extensive Intestinal First-Pass Elimination and Predominant Hepatic Distribution of Berberine Explain Its Low Plasma Levels in Rats

Liu¹,

Hao²,

Xie³

et al. 2010

Drug Metab Dispos

282

235

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ABSTRACT:Berberine, one of the most commonly used natural products, exhibits a poor plasma concentration-effect relationship whose underlying mechanisms remain largely unclear. This study was designed to test the hypothesis that extensive first-pass elimination and abundant tissue distribution of berberine may be its specific pharmacokinetic properties. For that, four different dosing routes, intragastric, intraduodenal, intraportal, and intravenous, were used to investigate the gastric, intestinal, and hepatic first-pass elimination of berberine. After intragastric dosing, approximately half of berberine ran intact through the gastrointestinal tract and another half was disposed of by the small intestine, leading to an extremely low extent of absolute oral bioavailability in rats (0.36%). Moreover, the major berberine metabolites were identified and quantified in rat enterocyte S9 fractions, portal vein plasma, and intestinal perfusates; plasma concentrations and tissue distribution of berberine and its major metabolites were determined as well. Data indicated that M1, M2 glucuronide, and M3 were the major metabolites generated from the small intestine and that there was a 70-fold increase in the ratio of the area under the concentration-time curve value for berberine (liver versus plasma). We conclude that intestinal first-pass elimination of berberine is the major barrier of its oral bioavailability and that its high extraction and distribution in the liver could be other important factors that lead to its low plasma levels in rats.

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Section: Methodsmentioning

confidence: 99%

Extensive Intestinal First-Pass Elimination and Predominant Hepatic Distribution of Berberine Explain Its Low Plasma Levels in Rats

Liu¹,

Hao²,

Xie³

et al. 2010

Drug Metab Dispos

282

235

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“…It is known that several uptake and efflux transporter and P450 isoforms are expressed in the human and rat intestine (van de Kerkhof et al, 2007). If a drug is a substrate of efflux transporters, it may enter and exit the enterocytes several times, and, with each cycle, small quantities may be metabolized by cytochrome P450 (P450) enzymes localized in the endoplasmic reticulum.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Cytochrome P450 Protein Expression along the Entire Length of the Intestine of Male and Female Rats

Mitschke

Reichel²,

Fricker³

et al. 2008

Drug Metab Dispos

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ABSTRACT:Intestinal cytochrome P450 (P450) proteins play an important role in the biotransformation of drugs and may significantly limit their oral absorption and bioavailability. Therefore, we have investigated the amount of P450 proteins via Western blot analysis along the entire intestine of male and female rats. Despite of the use of an inbred rat strain, controlled housing conditions for the animals, and a timed sample preparation, high interindividual differences in the expression of all P450 proteins was observed. CYP3A (135-243 fmol/mg of protein) and CYP2B1 (107-645 fmol/mg of protein) were the most abundant P450 isoforms in the duodenum and jejunum of rat intestine but were present in neither the ileum nor the colon. Compared with CYP2B1 and CYP3A, CYP2D1 (25-71 fmol/mg of protein) and CYP2C6 (3-10 fmol/mg of protein) were only expressed in minor amounts. CYP2C11 could not be identified in the entire rat intestine. In conclusion, this is the first systematic evaluation and quantification of the expression of P450 proteins along the entire length of the intestine in both male and female rats. These data will provide a basis for a better understanding of the extent of intestinal metabolism along the gastrointestinal tract.Absorption through the gut is a key step in the oral delivery of drugs. The main interface between gut lumen and the bloodstream is an epithelial cell layer consisting of polarized enterocytes controlling the passage of exogenous substances into the portal circulation. Enterocytes have a variety of structural features, including tight junctions reducing paracellular permeability, numerous drug transporters, and a set of metabolic enzymes that may all affect the entry of drugs into the body. The extent to which a drug is absorbed also depends on the intrinsic properties of the compound such as solubility, permeability, efflux or uptake transport properties, and susceptibility to metabolic degradation (Martinez and Amidon, 2002;Benet et al., 2004).Although the liver is known as the major site of first-pass extraction, recent studies have indicated that the small intestine also contributes significantly to the first-pass metabolism of many drugs, e.g., cyclosporine (Wu et al., 1995), nifedipine (Iwao et al., 2002), midazolam (Paine et al., 1996, and diltiazem (Iwao et al., 2004). It is known that several uptake and efflux transporter and P450 isoforms are expressed in the human and rat intestine (van de Kerkhof et al., 2007). If a drug is a substrate of efflux transporters, it may enter and exit the enterocytes several times, and, with each cycle, small quantities may be metabolized by cytochrome P450 (P450) enzymes localized in the endoplasmic reticulum. The P450 enzymes belong to a superfamily of heme proteins that show a broad substrate specificity, with substrates ranging in size from ethylene (M r 28) to cyclosporine (M r 1201) (Isin and Guengerich, 2007). Although the liver is regarded as the main organ of drug metabolism, P450 proteins are also expressed in other tissues, e.g., kidn...

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“…Various methods have been utilized to assess the intestinal drug metabolism. 30) Komura and Iwaki have reported the possibility of in vitro stability assay with intestinal microsomes to evaluate the species differences in the intestinal first-pass metabolism of CYP3A substrates. 31) Furthermore, kinetic models to predict the intestinal first-pass metabolism from in vitro experiments have been reported.…”

Section: Discussionmentioning

confidence: 99%

Investigation of the Intestinal Permeability and First-Pass Metabolism of Drugs in Cynomolgus Monkeys Using Single-Pass Intestinal Perfusion

Takahashi

Washio

Suzuki

et al. 2010

Biological & Pharmaceutical Bulletin

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where Fa is the fraction of dose absorbed from the gastrointestinal (GI) tract, Fg is the intestinal availability, that correspondent to the fraction of dose not metabolized in the intestinal wall during the absorption process and Fh is the fraction of dose not metabolized in the liver before reaching the systemic circulation. Therefore, the values of (1ϪFg) and (1ϪFh) mean the first-pass metabolism of orally administered drugs in GI tract and the liver, respectively. In order to select the appropriate candidates for oral drug product, quantitative assessments on each process are highly required.Recently, not only the small animals such as mice and rats, but also cynomolgus monkeys are often used as an animal model for PK studies in the drug discovery stage. However, lower oral BAs of test compounds were often observed in cynomolgus monkeys compared with those in other animals.1,2) Also in our previous study, various marketed drugs showed low oral BAs in cynomolgus monkeys than those in humans. From the kinetic analysis of the plasma concentrations of drugs after intravenous and oral administration, it was indicated that the low oral BAs in cynomolgus monkeys are mostly attributed to the low value of Fa*Fg.3,4) However, only from the in vivo PK analysis, it is theoretically impossible to evaluate Fa and Fg separately, resulting the confusion in the contribution of intestinal absorption on the oral BA of various drugs.Tamura et al. investigated the mechanisms of intestinal absorption of tacrolims by using the simultaneous perfusion method of intestinal lumen and mesenteric veins in rats. 5)They have calculated the fraction of metabolized in the intestinal cells from the difference between eliminated amount of the drug from the intestinal lumen and absorbed amount into the blood perfusate. Also, Mihara et al. evaluated quantitatively the first-pass metabolism of eperison in rats by using the similar perfusion method. 6) Although the simultaneous perfusion method is useful to assess the intestinal metabolism of drugs, this method requires complicated operations of the mesenteric vein and is difficult to perform in cynomolgus monkeys. Moreover, perfusion of the mesenteric vein might affect the absorption and metabolism of drugs in the intestine.The present study is performed 1) to establish the experimental method to evaluate the drug permeability and the metabolism in the intestine of cynomolgus monkeys and then 2) to clarify the causes of low BA of drugs in cynomolgus monkeys. For these purposes, an in situ single-pass intestinal perfusion method was performed with blood sampling from both portal and peripheral veins, instead of the perfusion of the mesenteric vein. The intestinal permeability of drugs (P eff ) is calculated from the decrease in drug concentration in the perfusate after single pass perfusion through the intestinal tract. The intestinal metabolism during the absorption process (1ϪFg) is calculated from the difference between drug amounts that disappeared from the perfusate and absorbed into the por...

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In Vitro Methods to Study Intestinal Drug Metabolism

Cited by 109 publications

References 183 publications

Extensive Intestinal First-Pass Elimination and Predominant Hepatic Distribution of Berberine Explain Its Low Plasma Levels in Rats

Extensive Intestinal First-Pass Elimination and Predominant Hepatic Distribution of Berberine Explain Its Low Plasma Levels in Rats

Characterization of Cytochrome P450 Protein Expression along the Entire Length of the Intestine of Male and Female Rats

Investigation of the Intestinal Permeability and First-Pass Metabolism of Drugs in Cynomolgus Monkeys Using Single-Pass Intestinal Perfusion

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