Intestinal Drug-metabolizing Enzymes Research Articles

Usefulness of Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells for the Prediction of Intestinal Drug Absorption in Humans.

This study aimed to demonstrate the usefulness of human jejunal spheroid-derived differentiated intestinal epithelial cells as a novel in vitro model for clarifying the impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans. Three-dimensional human intestinal spheroids were successfully established from surgical human jejunal specimens and expanded for a long period using L-WRN-conditioned medium, which contains Wnt3a, R-spondin 3, and noggin. The mRNA expression levels of intestinal pharmacokinetics-related genes in the human jejunal spheroid-derived differentiated intestinal epithelial cells were drastically increased over a 5-day period after seeding compared with those in human jejunal spheroids and were approximately the same as those in human jejunal tissue over a culture period of at least 13 days. Activities of typical drug-metabolizing enzymes [cytochrome P450 (CYP) 3A, CYP2C9, uridine 5'-diphospho-glucuronosyltransferase 1A, and carboxylesterase 2] and uptake/efflux transporters [peptide transporter 1/solute carrier 15A1], P-glycoprotein, and breast cancer resistance protein) in the differentiated cells were confirmed. Furthermore, intestinal availability (Fg) values estimated from the apical-to-basolateral permeation clearance across cell monolayer showed a good correlation with the in vivo Fg values in humans for five CYP3A substrate drugs (Fg range, 0.35-0.98). In conclusion, the functions of major intestinal drug-metabolizing enzymes and transporters could be maintained in human jejunal spheroid-derived differentiated intestinal epithelial cells. This model would be useful for the quantitative evaluation of the impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans. SIGNIFICANCE STATEMENT: Limited information is available regarding the quantitative prediction of the impact of drug-metabolizing enzymes and transporters on the human intestinal absorption of substrates using in vitro assays with differentiated cells derived from human intestinal spheroids/organoids. This study confirmed the functions of typical drug-metabolizing enzymes and transporters in human jejunal spheroid-derived differentiated intestinal epithelial cells and demonstrated that intestinal availability (Fg) estimated from apical-to-basolateral permeation clearance across cell monolayers showed a good correlation with in vivo human Fg for CYP3A substrates.

Ontogeny of Small Intestinal Drug Transporters and Metabolizing Enzymes Based on Targeted Quantitative Proteomics.

Most drugs are administered to children orally. An information gap remains on the protein abundance of small intestinal drug-metabolizing enzymes (DMEs) and drug transporters (DTs) across the pediatric age range, which hinders precision dosing in children. To explore age-related differences in DMEs and DTs, surgical leftover intestinal tissues from pediatric and adult jejunum and ileum were collected and analyzed by targeted quantitative proteomics for apical sodium-bile acid transporter, breast cancer resistance protein (BCRP), monocarboxylate transporter 1 (MCT1), multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein (MRP) 2, MRP3, organic anion-transporting polypeptide 2B1, organic cation transporter 1, peptide transporter 1 (PEPT1), CYP2C19, CYP3A4, CYP3A5, UDP glucuronosyltransferase (UGT) 1A1, UGT1A10, and UGT2B7. Samples from 58 children (48 ileums, 10 jejunums, age range: 8 weeks to 17 years) and 16 adults (8 ileums, 8 jejunums) were analyzed. When comparing age groups, BCRP, MDR1, PEPT1, and UGT1A1 abundance was significantly higher in adult ileum as compared with the pediatric ileum. Jejunal BCRP, MRP2, UGT1A1, and CYP3A4 abundance was higher in the adults compared with children 0-2 years of age. Examining the data on a continuous age scale showed that PEPT1 and UGT1A1 abundance was significantly higher, whereas MCT1 and UGT2B7 abundance was lower in adult ileum as compared with the pediatric ileum. Our data contribute to the deeper understanding of the ontogeny of small intestinal drug-metabolizing enzymes and drug transporters and shows DME-, DT-, and intestinal location-specific, age-related changes. SIGNIFICANCE STATEMENT: This is the first study that describes the ontogeny of small intestinal DTs and DMEs in human using liquid chromatography with tandem mass spectrometry-based targeted quantitative proteomics. The current analysis provides a detailed picture about the maturation of DT and DME abundances in the human jejunum and ileum. The presented results supply age-related DT and DME abundance data for building more accurate PBPK models that serve to support safer and more efficient drug dosing regimens for the pediatric population.

Effects of Shengmai San on key enzymes involved in hepatic and intestinal drug metabolism in rats

Ethnopharmacological relevanceShengmai San (SMS) has been commonly used as a traditional Chinese medicine for the treatment of cardiovascular disorders, of which drug interactions need to be assessed for the safety concern. There is little evidence for the alterations of hepatic and intestinal drug-metabolizing enzymes after repeated SMS treatments to assess drug interactions. Aim of the studyThe studies aim to illustrate the effects of repeated treatments with SMS on cytochrome P450s (CYPs), reduced nicotinamide adenine dinucleotide (phosphate)-quinone oxidoreductase (NQO), uridine diphosphate-glucuronosyltransferase (UGT), and glutathione S-transferase (GST) using in vivo rat model. Materials and methodsThe SMS was prepared using Schisandrae Fructus, Ginseng Radix, and Ophiopogonis Radix (OR) (1:2:2). Chromatographic analyses of decoctions were performed using ultra-performance liquid chromatography (UPLC) and LC-mass spectrometry. Sprague-Dawley rats were orally treated with the SMS and its component herbal decoctions for 2 or 3 weeks. Hepatic and intestinal enzyme activities were determined. CYP3A expression and the kinetics of intestinal nifedipine oxidation (NFO, a CYP3A marker reaction) were determined. ResultsSchisandrol A, schisandrin B, ginsenoside Rb1 and ophiopogonin D were identified in SMS. SMS selectively suppressed intestinal, but not hepatic, NFO activity in a dose- and time-dependent manner. Hepatic and intestinal UGT, NQO and GST activities were not affected. A 3-week SMS treatment decreased the maximal velocity of intestinal NFO by 50%, while the CYP3A protein level remained unchanged. Among SMS component herbs, the decoction of OR decreased intestinal NFO activity. ConclusionsThese findings demonstrate that 3-week treatment with SMS and OR suppress intestinal, but not hepatic CYP3A function. It suggested that the potential interactions of SMS with CYP 3A drug substrates should be noticed, especially the drugs whose bioavailability depends heavily on intestinal CYP3A.

Investigation on Intestinal Proteins and Drug Metabolizing Enzymes in Simulated Microgravity Rats by a Proteomics Method.

The present study aimed to investigate the change of intestinal mucosa proteins, especially the alteration of intestinal drug metabolizing enzymes (IDMEs) following 14-day simulated microgravity. Morey–Holton tail-suspension analog was used to simulate microgravity. Intestinal mucosa proteins of rats were determined by label-free quantitative proteomic strategy. A total of 335 differentially expressed proteins (DEPs) were identified, 190 DEPs were upregulated, and 145 DEPs were downregulated. According to bioinformatic analysis, most of DEPs exhibited hydrolase, oxidoreductase, transferase, ligase, or lyase catalytic activity. DEPs were mainly enriched in metabolic pathways, including metabolism of amino acid, glucose, and carbon. Moreover, 11 of DEPs were involved in exogenous drug and xenobiotics metabolism. Owing to the importance of IDMEs for the efficacy and safety of oral drugs, the expression of cytochrome P450 1A2 (CYP1A2), CYP2D1, CYP3A2, CYP2E1, alcohol dehydrogenase 1 (ADH1), and glutathione S-transferase mu 5 (GSTM5) in rat intestine mucosa was determined by Western-blot. The activity of ADH, aldehyde dehydrogenase (ALDH) and GST was evaluated. Compared with control rats, the expression of CYP1A2, CYP2D1, CYP3A2, and ADH1 in the simulated microgravity (SMG) group of rats were dramatically decreased by 33.16%, 21.93%, 48.49%, and 22.83%, respectively. GSTM5 was significantly upregulated by 53.14% and CYP2E1 expression did not show a dramatical change in SMG group rats. Moreover, 14-day SMG reduced ADH activity, while ALDH and GST activities was not altered remarkably. It could be concluded that SMG dramatically affected the expression and activity of some IDMEs, which might alter the efficacy or safety of their substrate drugs under microgravity. The present study provided some preliminary information on IDMEs under microgravity. It revealed the potential effect of SMG on intestinal metabolism, which may be helpful to understand the intestinal health of astronauts and medication use.

P-003: Detection of bacterial and host proteins from a single colonic biopsy sample by a new mass spectrometry-based assay in pediatric ulcerative colitis

P-003: Detection of bacterial and host proteins from a single colonic biopsy sample by a new mass spectrometry-based assay in pediatric ulcerative colitis

Toxic effects of dietary exposure to T-2 toxin on intestinal and hepatic biotransformation enzymes and drug transporter systems in broiler chickens

The effects of the mycotoxin T-2 on hepatic and intestinal drug-metabolizing enzymes (cytochrome P450) and drug transporter systems (MDR1 and MRP2) in poultry were investigated during this study. Broiler chickens received either uncontaminated feed, feed contaminated with 68μg/kg or 752μg/kg T-2 toxin. After 3weeks, the animals were euthanized and MDR1, MRP2, CYP1A4, CYP1A5 and CYP3A37 mRNA expression were analyzed using qRT-PCR. Along the entire length of the small intestine no significant differences were observed. In the liver, genes coding for CYP1A4, CYP1A5 and CYP3A37 were significantly down-regulated in the group exposed to 752μg/kg T-2. For CYP1A4, even a contamination level of 68μg/kg T-2 caused a significant decrease in mRNA expression. Expression of MDR1 was not significantly decreased in the liver. In contrast, hepatic MRP2 expression was significantly down-regulated after exposure to 752μg/kg T-2. Hepatic and intestinal microsomes were prepared to test the enzymatic activity of CYP3A. In the ileum and liver CYP3A activity was significantly increased in the group receiving 752μg/kg T-2 compared to the control group. The results of this study show that drug metabolizing enzymes and drug transporter mechanisms can be influenced due to prolonged exposure to relevant doses of T-2.

Shed human enterocytes as a tool for the study of expression and function of intestinal drug‐metabolizing enzymes and transporters

Intestinal metabolism and transport are now recognized as protective barriers against orally ingested xenobiotics, including drugs. However, in vitro studies of the expression and function of intestinal proteins are hampered by the limited availability of human intestinal tissues. Because enterocytes are constantly shed in large numbers into the gut lumen, this study investigated whether these cells could be collected with a multilumen perfusion catheter and whether they are functionally active. In healthy volunteers, a 20-cm isolated jejunal segment was generated with the perfusion catheter by inflating 2 balloons with air. Shed cells were characterized by fluorescence-activated cell sorting analysis for leukocyte-specific CD45 and enterocyte-specific villin, as well as for apoptosis. Homogenates of the cells were used for reverse transcriptase polymerase chain reaction and Western blotting. Cytochrome P450 enzyme activity was determined with the calcium channel blocker verapamil as a substrate. On average, 4.83 mg protein and 56.23 million cells were collected from a 20-cm segment during 2 hours. A total of 84.2% of the cells were positive for enterocyte-specific villin, and only 1.6% of the collected cells were positive for CD45. The majority of cells (65.3%) were not in early or late apoptosis or necrosis. In all volunteers, drug-metabolizing enzymes (such as members of the cytochrome P450 family) could be detected as both messenger ribonucleic acid and proteins. Consistent with expression data, formation of verapamil metabolites catalyzed by CYP3A4 and CYP2C was shown. The majority of shed human enterocytes collected with a multilumen perfusion catheter were still functionally active and not apoptotic. Harvesting of spontaneously shed enterocytes provides a new tool for studies on expression and function of intestinal proteins.

Expression of intestinal drug-metabolizing enzymes in patients with chronic inflammatory bowel disease

Abstract The cause of chronic inflammatory bowel disease (IBD) remains unclear. A recent hypothesis suggests that ulcerative colitis may be caused by one or more reactive xenobiotic metabolites. If so, the presence, distribution, and activity of drug-metabolizing enzymes in the gut could be important, because those enzymes represent a first defense against ingested xenobiotics. To assess whether patients with IBD express certain enzymes differently in the gastrointestinal tract than do subjects without IBD, biopsy samples of the terminal ileum and various regions of the colon were stained immunohistochemically for cytochrome P-450 (CYP) isoenzymes (CYP1A1, CYP2D6, CYP2E1, CYP3A), epoxide hydrolase, α and π forms of glutathione- S -transferase (GST), and metallothionein (MTTH). One hundred fifty biopsy samples from 21 patients with irritable colon (no clinical or histologic signs of inflammation; control group), 25 patients with Crohn's disease (CD), and 37 patients with ulcerative colitis (UC) were evaluated. The patients' sex, age, smoking habits, and disease state were not related significantly to the frequency of positive versus negative staining of the samples. All enzymes were expressed less frequently in the colon than in the terminal ileum. With the exception of CYP1A1, all enzymes were detectable with similar frequency in the three study groups: CYP2E1, 77.4% to 84.6% positive stainings; CYP3A, 53.8% to 58.5%; CYP2D6, 23.1% to 36.9%; GST α , 14.1% to 20.8%; GST π , 19.2% to 24.5%; epoxide hydrolase, 1.9% to 3.8%; and MTTH, 19.2% to 25.4%. However, CYP1A1 staining was positive significantly more often in patients with CD (39.6%) and UC (39.4%) than in patients in the control group (19.2%). Because cells with CYP1A1 were found in the intestines of patients with IBD significantly more frequently than in patients in the control group (ie, with an irritable colon), this may suggest a function of this enzyme in the etiology or pathogenesis of inflammation in these disorders. Alternatively, the more frequent expression of CYP1A1 could be due secondarily to the presence of xenobiotics that then lead to inflammation.

Differential effects of dietary diallyl sulfide and diallyl disulfide on rat intestinal and hepatic drug‐metabolizing enzymes

The chemopreventive properties of allyl sulfides on carcinogenesis may be related to the modulation of drug-metabolizing enzymes involved in carcinogen activation or detoxication. In order to investigate the effects of diallyl sulfide (DAS) and diallyl disulfide (DADS) on intestinal and hepatic drug-metabolizing enzymes, rats were fed a diet containing 0.2% of either allyl sulfide. The DADS enhanced intestinal epoxide hydrolase (EH) and cytochrome P-450 (P-450) 2B1/2 protein levels and the activities of pentoxy- and benzyl-oxyresorufin O-dealkylases, arylhydrocarbon hydroxylase, microsomal epoxide hydrolase, p-nitrophenol UDP-glucuronyl transferase and glutathione S-transferase, and decreased nitrosodimethylamine demethylase activity. In liver, DADS produced similar effects and, in addition, increased P-450 1A1/2 protein level and phenoxazone metabolizing activities (ethoxy- and methoxyresorufin O-dealkylases), p-hydroxybiphenyl UDP-glucuronyl transferase, and decreased P-450 2E1 level. The DAS enhanced only EH activity in the small intestine and induced P-450 2B1/2 and epoxide hydrolase protein levels. In liver, DAS produced similar effects as DADS. The different effects of DAS on intestinal drug-metabolizing enzymes, compared to liver, could be ascribed to less metabolism of this compound in small intestine. It is also suggested that DAS and DADS may not yield the same metabolites and therefore would have different effects on intestinal drug-metabolizing enzymes.

Hepatic and Intestinal Drug-Metabolizing Enzymes and the Tissue Distribution and Excretion of 14C-7,12-Dimethylbenz[a]anthracene in Rats Fed Diets Varying in Fat Concentration

Diets containing 12 or 48% of calories from corn oil were fed to weanling Sprague-Dawley female rats for 28 days. Hepatic cytochrome P-450 content was higher with the high-fat diet, but the activities of cytochrome P-450 reductase, cytochrome c reductase, benzo[a]pyrene hydroxylase (BPH), and uridine-5'-diphosphoglucuronic acid (UDPGA) transferase were unchanged. There were no differences in small intestinal cytochrome P-450 or BPH attributable to diet. Additional rats given an oral dose of 14C-7,12-dimethylbenz[a]anthracene (14C-DMBA) after 28 days of feeding showed no effects of diet on the cumulative daily excretion of radioactivity from 14C-DMBA in the urine or feces over 72 h. However, rats fed the high-fat diet showed greater concentrations of radioactivity in the liver, kidney, adrenal, pituitary, breast, and adipose tissue at 4 h after dosing when compared to rats fed low-fat diets. The transiently higher tissue concentrations of 14C-DMBA in rats fed a high-fat diet prior to DMBA administration correlate with the enhancement of mammary cancer induction seen when high-fat diets are fed prior to administration of this carcinogen.

Comparison of the effects of feeding quercetin or flavone on hepatic and intestinal drug-metabolizing enzymes of the rat

The influence of dietary flavone and quercetin on the components of the drug metabolizing enzyme system was examined in the liver and small intestine of the rat. Quercetin given at a concentration of 1% in the diet for 14 days produced no significant changes on phase I or phase II enzyme activities. In contrast, 0.25% flavone caused significant increases in relative liver weight, microsomal and cytoplasmic proteins, and cytochrome P-450 content. The activities of hepatic ethoxyresorufin, pentoxyresorufin and ethoxycoumarin deethylases were significantly increased (by 20, 30 and 2.5-fold, respectively) over control levels. Hepatic UDP-glucuronyl transferase and glutathione transferase activities were increased 3–4-fold. In contrast, flavone induced no changes in these two intestinal enzyme activities. It is concluded that flavone produces an induction that shows both phenobarbital- and methylcholanthrene-type characteristics and that its effect on conjugating enzymes is tissue specific.

Effect of administration route of 3-methylcholanthrene on the inducibility of intestinal drug-metabolizing enzymes.

The inducibility of the mucosal drug-metabolizing enzymes of rat small intestine was studied by administering 3-methylcholanthrene either intragastrically or intraperitoneally. The aryl hydrocarbon hydroxylase activity was 4.1 times higher after intragastric than after intraperitoneal administration of methylcholanthrene. The ethoxycoumarin-O-diethylase activity was 11 times and UDP-glucuronosyltransferase (with p-nitrophenol as substrate) activity was 3 times higher after intragastric administration than after intraperitoneal administration. The epoxide hydratase activity was, on the other hand, 38% lower after intragastric administration of 3-methylcholanthrene than after intraperitoneal administration. The results suggest that compounds entering the body intragastrically, e.e. in the diet, might have profound enzyme-specific effects on the intestinal metabolic rates of drugs.