Rat Intestinal Perfusion Model Research Articles

Role of Intestinal Hydrolase in the Absorption of Prenylated Flavonoids Present in Yinyanghuo

Purpose: Yinyanghuo (Herba Epimdii) is a traditional Chinese herb containing prenylated flavonoids as its active constituents. The aim of this study was to examine the significance of the intestinal hydrolysis of prenylated flavonoids by lactase phlorizin hydrolase (LPH), an enzyme at the brush border membrane of intestinal cells. Methods: A four-site perfused rat intestinal model was used. The concentration of the flavonoids of interest and their metabolites in different intestinal segements were analyzed by HPLC, and the apparent permeabilities were calculated. A lactase phlorizin hydrolase inhibitor (gluconolactone) was employed to investigate the mechanism of the intestinal absorption, and the metabolites of the four flavonoids were identified using LC/MS/MS. Results: Diglycosides (icariin) or triglycosides (epimedin A, epimedin B, and epimedin C) were hydrolyzed rapidly in duodenum and jejunum producing one or two metabolites, while a monoglycoside (baohuoside I) was absorbed directly. When co-perfused with glucono-lactone, both the hydrolysis of diglycosides and triglycosides were significantly inhibited, with inhibition rates for icariin (62%, 50%, 40%, 46%), epimedin A, (55%, 26%, 21%, 14%); epimedin B (42%, 40%, 74%, 22%), and epimedin C (42%, 40%, 52%, 35%) in duodenum, jejunum, ileum, and colon, respectively. Also the metabolites of icariin, epimedin A, epimedin B, and epimedin C were identified as baohuoside I (one of two), sagittatoside A, sagittatoside B, and 2″-O-rhamnosylicariside II, respectively. Conclusions: The results showed that lactase phlorizin hydrolase was a major determinant of the intestinal absorption of prenylated flavonoids present in Yinyanghuo.

Absorption of CH330331, a novel 4‐anilinoquinazoline inhibitor of epidermal growth factor receptor tyrosine kinase: comparative studies using in vitro, in situ and in vivo models

CH330331 is a prototype of a new class of synthetic small molecule tyrosine kinase inhibitors (TKIs). In vitro Caco-2 cell monolayers, the in situ single-pass rat intestinal perfusion (SPIP) technique with mesenteric vein cannulated and an in vivo animal model were employed to investigate its permeability and transepithelial transport mechanisms. The Caco-2 model showed that the transport of CH330331 across the monolayers from the apical (AP) to basolateral (BL) side was 6- to 10-fold higher than that from the BL to AP side. The apparent permeability coefficient (P(app) ) values of CH330331 at 5-20 µg/ml from the AP to BL and from BL to AP side were 5.30-2.21 × 10(-6) cm/s, with a decrease in P(app) values from the AP to BL side at increased CH330331 concentrations. In the perfused rat intestinal model, a concentration dependent change in permeability was detected where P(blood) at 5 µg/ml (1.66 ± 0.69 × 10(-6) cm/s) and 10 µg/ml (1.80 ± 0.45 × 10(-6) cm/s) was significantly different from P(blood) at 20 µg/ml (0.98 ± 0.31 × 10(-6) cm/s, p<0.05). Some inhibitors could also change the transepithelial transport of CH330331. Moreover, the in vivo study showed that the oral bioavailability of CH330331 was 82.7% in the rat. All the results confirmed that the transepithelial transport of CH330331 was rapid and saturable, which might involve an active mechanism. The oral bioavailability of CH330331 was relatively high in vivo.

Intestinal absorption properties of flavones and extract of Fructus Aurantii Immaturus in rats

To investigate the intestinal absorption of naringin, hesperidin, neohesperidin and the extract of Fructus Aurantii Immaturus in rats. The rat intestinal perfusion and enzymes incubation models were used, together with the determination of the n-octanol/water partition coefficient of the components (P). In perfusion model, the P(eff) of all components were low, and the P(eff) of naringin, hesperidin and neohesperidin were 0.140-0.252, 0.156-0.268 and 0.154-0.285, respectively. In four different regions of intestine of rat and with different concentration, the P(eff) of the components both had no significant difference, whereas the P(eff) of the extract were higher than the P(eff) of the single component. The metabolite of components was not detected in intestine. The P of naringin, hesperidin and neohesperidin were 0.36, 0.40 and 0.48, respectively, and the pH of buffer solution had no influence to its distribution coefficient. Poor permeation contributed to the poor intestinal absorption of naringin, hesperidin and neohesperidin. The absorption of components in extract were increased, and the results suggest that the extract may enhance the intestinal absorption of the components.

Species and Gender Differences Affect the Metabolism of Emodin via Glucuronidation

The aim of the present study was to define the mechanisms responsible for poor bioavailability of emodin by determining its metabolism using in vitro and in situ disposition models of the intestine and liver. Liver microsomes of mice, rats, guinea pigs, dogs, and humans were used along with the rat intestinal perfusion model and the rat intestinal microsomes. In the rat intestine, excretion rates of emodin-3-O-glucuronide were significantly different (p < 0.05) in four regions of the intestine and were higher in males than in females (p < 0.01). Emodin glucuronidation in liver microsomes was species-dependent, and K (m) values varied 5.7-fold (3.2-18.2 microM) in males and 2.8-fold (4.6-13.0 microM) in females. The male intrinsic clearance (CL(int)) values differed by 5-fold (27.6-138.3 mL h(-1) mg(-1) protein), and female CL(int) values differed by 4.3-fold (24.3-103.5 mL h(-1) mg(-1) protein). Since CL(int) values of emodin glucuronidation were 10-fold higher than that of isoflavones, emodin was considered rapidly glucuronidated. In contrast to the large species-dependent effects on K (m) and CL(int) values, gender had a smaller effect on these kinetic parameters (2-fold, p < 0.05). Lastly, glucuronidation rates obtained using liver microsomes from various experimental animals of the same gender correlated well with those in human liver microsomes. In conclusion, Rapid metabolism by UDP-glucuronosyltransferase is the major reason why emodin has poor bioavailability. Species and gender affected emodin metabolism to a different degree, and experimental animals are expected to be useful in predicting emodin glucuronidation in humans.

Intestinal Absorption of γ-Tocotrienol Is Mediated by Niemann-Pick C1-Like 1: In Situ Rat Intestinal Perfusion Studies

gamma-Tocotrienol (gamma-T3) is a member of the vitamin E family that displays potent anticancer activity and other therapeutic benefits. The objective of this study was to evaluate gamma-T3 intestinal uptake and metabolism using the in situ rat intestinal perfusion model. Isolated segments of rat jejunum and ileum were perfused with gamma-T3 solution, and measurements were made as a function of concentration (5-150 microM). Intestinal permeability (P(eff)) and metabolism were studied by measuring total compound disappearance and major metabolite, 2,7,8-trimethyl-2-(beta-carboxy-ethyl)-6-hydroxychroman, appearance in the intestinal lumen. gamma-T3 and metabolite levels were also determined in mesenteric blood. The P(eff) of gamma-T3 was similar in both intestinal segments and significantly decreased at concentrations > or =25 microM in jejunum and ileum (p < 0.05), whereas metabolite formation was minimal and mesenteric blood concentrations of gamma-T3 and metabolite remained very low. These results indicate that gamma-T3 intestinal uptake is a saturable carrier-mediated process and metabolism is minimal. Results from subsequent in situ inhibition studies with ezetimibe, a potent and selective inhibitor of Niemann-Pick C1-like 1 (NPC1L1) transporter, suggested gamma-T3 intestinal uptake is mediated by NPC1L1. Comparable findings were obtained when Madin-Darby canine kidney II cells that express endogenous NPC1L1 were incubated with increasing concentrations of gamma-T3 or gamma-T3 with increasing concentrations of ezetimibe. The present data show for the first time that gamma-T3 intestinal absorption is partly mediated by NPC1L1.

Disposition of Naringenin via Glucuronidation Pathway Is Affected by Compensating Efflux Transporters of Hydrophilic Glucuronides

The purposes of this study were to investigate how efflux transporters and UDP-glucuronosyltransferases (UGT) affect the disposition of naringenin. A rat intestinal perfusion model with bile duct cannulation was used along with rat intestinal and liver microsomes. In the intestinal perfusion model, both absorption and subsequent excretion of naringenin metabolites were rapid and site-dependent (p < 0.05). Naringenin was absorbed the most in colon, and its glucuronides were excreted the most in duodenum. In metabolism studies, the intrinsic clearance value of naringenin glucuronidation was the highest in jejunum microsomes, followed by liver, ileal and colonic microsomes. The rapid metabolism in microsomes did not always translate into more efficient excretion in the rat perfusion model, however, because of presence of rate-limiting efflux transporters. When used separately, MK-571 (an inhibitor of multidrug resistance-related protein 2 or Mrp2) or dipyridamole (an inhibitor of breast cancer resistance protein or Bcrp1) did not affect excretion of naringenin glucuronides, but when used together, they significantly (p < 0.05) decreased intestinal and biliary excretion of naringenin glucuronides. In conclusion, efflux transporters Mrp2 and Bcrp1 are shown to compensate for each other and enable the intestinal excretion of flavonoid (i.e., naringenin) glucuronides.

Disposition of Flavonoids via Enteric Recycling: UDP-Glucuronosyltransferase (UGT) 1As Deficiency in Gunn Rats Is Compensated by Increases in UGT2Bs Activities

Flavonoids have poor bioavailabilities largely because of metabolism via UDP-glucuronosyltransferases (UGTs). This study aims to further understand the functions of UGT in metabolizing genistein and apigenin, two compounds metabolized more extensively in the gut than in the liver. Because Gunn rats are deficient in UGT1As, we determined whether this deficiency would result in less flavonoid glucuronidation, using rat intestinal perfusion model and microsomes prepared from rat liver and intestine. In yeast-expressed rat UGT isoforms, rat UGT1A isoforms (especially UGT1A7) were mainly responsible for flavonoid metabolism. In perfusion studies, the two flavonoids were rapidly absorbed at comparable rates, but the intestinal excretions of glucuronides in Gunn rats compared with Wistar rats were not only comparable for genistein but also were higher (p < 0.05) for apigenin, suggesting up-regulation of UGT isoforms in Gunn rats. To determine the possible compensatory UGT isoforms, we first verified that UGT1A activities were significantly lower (p < 0.05) in Gunn rats by using UGT1A-specific probes 7-ethyl-10-hydroxycamptothecin (SN-38) and prunetin. We then demonstrated using UGT2B probes testosterone, ezetimibe, and indomethacin that UGT2B activities were usually significantly higher in Gunn rats. In addition, testosterone was metabolized much faster in liver microsomes than in intestinal microsomes, and in microsomes prepared from Gunn rats compared with Wistar rats. In conclusion, flavonoids are efficiently metabolized by UGT1A-deficient Gunn rats because of compensatory up-regulation of intestinal UGT2Bs and hepatic anion efflux transporters, which increases their disposition and limits their oral bioavailabilities.

Pharmacokinetics of bicyclol in rats with acute hepatic failure

The aim of present study is to evaluate the pharmacokinetics of bicyclol in carbon tetrachloride (CCl4)-intoxicated rats. The plasma concentration of bicyclol was detected in rats after a single oral or intravenous administration by high-performance liquid chromatography (HPLC) analysis. Rat intestinal and hepatic perfusion models were employed to clarify the respective effect of gut and liver on the pharmacokinetics of bicyclol in acute hepatic failure (AHF) rats. Rat in vitro microsomal incubation was also conducted. The bioavailability of bicyclol was increased 3.1-fold after CCl4 intoxication in rats. The area under the curve (AUC)(0–∞), Cmax, and clearance (CL) of bicyclol after intravenous administration were 13.4 mg h l−1, 18.8 mg l−1, and 1.8 l h−1 kg−1 in control rats, and 130 mg h l−1, 33.1 mg l−1, and 0.15 l h−1 kg−1 in AHF rats, respectively. In the present study we investigated the pharmacokinetics of bicyclol in CCl4-intoxicated rats and differentiated the respective role of intestine and liver by using in situ intestinal and hepatic perfusion in rats, and in vitro rat microsomes incubation. The studies are expected to provide a better understanding related to the alteration of pharmacokinetics of bicyclol in pathological situation.

Intestinal Absorption Mechanisms of Prenylated Flavonoids Present in the Heat-Processed Epimedium koreanum Nakai (Yin Yanghuo)

The purpose is to determine absorption mechanism of five bioactive prenylated flavonoids (baohuoside I, icariin, epimedine A, B, and C) present in heat-processed Epimedium koreanum Nakai (Yin Yanghuo). Transport of five prenylated flavonoids present in heat-processed herbs were studied in the human intestinal Caco-2 model and the perfused rat intestinal model. In the perfused rat intestinal model, prenylated flavonoids with a monoglucosidic bond (e.g., icariin) was rapidly hydrolyzed into corresponding metabolites (e.g., baohuoside I). In the Caco-2 model, apical to basolateral permeability of a monoglycoside baohuoside I (1.46 x 10(-6) cm/sec) was more than 2 folds greater than four prenylated flavonoids with 2 or more sugar moieties (<0.6 x 10(-6) cm/sec). The slow apical to basolateral transport of baohuoside I was the result of efflux. This efflux was carrier-mediated and active since its transport was vectorial, concentration- and temperature-dependent with activation energies greater than 15 kcal/mol. Efflux of baohuoside I was significantly suppressed by inhibitors of BCRP and MRP2, whereas efflux of icariin was significantly inhibited only by p-glycoprotein inhibitor verapamil. Because YHH is often heat-processed for better efficacy, we determined and found the optimal condition for increasing contents of more bioavailable flavonoids (i.e., baohuoside I) to be 160-170 degrees C for 5-7 min. Poor bioavailability of prenylated flavonoids results from their poor intrinsic permeation and transporter-mediated efflux. Heat processing parameters may be optimized to preserve the herb's bioavailable flavonoids, which help retain and improve its efficacy during processing.

Role of P-Glycoprotein in the Intestinal Absorption of Tanshinone IIA, a Major Active Ingredient in the Root of Salvia miltiorrhiza Bunge

The extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9-3.4% in rats, with non-linear pharmacokinetics when its dosage increased. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSA disappearance from the luminal perfusates (P(lumen)) were 6.2- to 7.2-fold higher (P < 0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The P(blood), but not P(lumen), was significantly increased when co-perfused with verapamil, or quinidine (both P-gp inhibitors). The uptake and efflux of TSA in confluent Caco-2 cells were significantly altered in the presence of verapamil, quinidine, MK-571, or probenecid. The transport of TSA across Caco-2 monolayers was pH-, temperature- and ATP-dependent. Furthermore, the transport from the apical (AP) to basolateral (BL) side of the Caco-2 monolayers was 3.3- to 8.5-fold lower than that from the BL to AP side, but such a polarized transport was attenuated by co-incubated verapamil or quinidine. A polarized transport was also observed in the control MDCKII cells and more apparent in MDR1-MDCKII monolayers, with the P(app) values of TSA in the BL-AP direction being 7- to 9-fold higher in MDR1-MDCKII monolayers than those in the control MDCKII cells. Moreover, TSA significantly inhibited P-gp-mediated transport of digoxin in P-gp-overexpressing membrane vesicles with an IC(50) of 2.6 microM, but stimulated vanadate-sensitive P-gp ATPase activity with estimated K(m) and V(max) values of 10.70 +/- 0.69 microM and 67.65 +/- 1.31 nmol/min/mg protein, respectively. TSA was extensively metabolized to tanshinone IIB (TSB), and two other oxidative metabolites in rat liver microsomes, but the formation rate of TSB in rat intestinal microsomes was only about 1/10 of that in liver microsomes. These findings indicate that TSA is a substrate and reversing agent for P-gp; and P-gp-mediated efflux of TSA into the gut lumen and the first-pass metabolism contribute to the low oral bioavailability. Further studies are needed to explore the role of other drug transporters and first-pass metabolism in the low bioavailability of TSA.

Role of ATP-binding cassette drug transporters in the intestinal absorption of tanshinone IIB, one of the major active diterpenoids from the root of Salvia miltiorrhiza

There is an increasing use of herbal medicines worldwide, and the extracts from the root of Salvia miltiorrhiza are widely used in the treatment of angina and stroke. In this study, we investigated the mechanism for the intestinal absorption of tanshinone IIB (TSB), a major constituent of S. miltiorrhiza. The oral bioavailability of TSB was about 3% in rats with less proportional increase in its maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC) with increasing dosage. The time to Cmax (Tmax) was prolonged at higher oral dosage. In a single pass rat intestinal perfusion model, the permeability coefficients (Papp) based on TSB disappearance from the lumen (Plumen) were 6.2- to 7.2-fold higher (p < 0.01) than those based on drug appearance in mesenteric venous blood (Pblood). The uptake and efflux of TSB in Caco-2 cells were also significantly altered in the presence of an inhibitor for P-glycoprotein (PgP) or for multi-drug resistance associated protein (MRP1/2). TSB transport from the apical (AP) to basolateral (BL) side in Caco-2 monolayers was 3.3- to 5.7-fold lower than that from BL to AP side, but this polarized transport was attenuated by co-incubation of PgP or MRP1/2 inhibitors. The Papp values of TSB in the BL-AP direction were significantly higher in MDCKII cells over-expressing MDR1 or MRP1, but not in cells over-expressing MRP2-5, as compared with the wild-type cells. The plasma AUC0–24hr in mdr1a and mrp1 gene-deficient mice was 10.2- to 1.7-fold higher than that in the wild-type mice. Furthermore, TSB significantly inhibited the uptake of digoxin and vinblastine in membrane vesicles containing PgP or MRP1. TSB also moderately stimulated PgP ATPase activity. Taken collectively, our findings indicate that TSB is a substrate for PgP and MRP1 and that drug resistance to TSB therapy and drug interactions may occur through PgP and MRP1 modulation.

PEPT1 Enhances the Uptake of Gabapentin via Trans-Stimulation of b0,+ Exchange

The aims of this study were (1) to determine whether amino acid and dipeptide loading can improve the effective permeability of gabapentin and (2) to characterize the underlying mechanism that is responsible for this interaction. An in situ single-pass rat intestinal perfusion model was used to assess the effective permeability of gabapentin in rat, in the absence and presence of cellular loading by amino acid and dipeptide mixtures. Compared to gabapentin alone, cellular loading with amino acid and dipeptide mixtures significantly improved the effective permeability of gabapentin by 46-79% in jejunum and by 67-72% in ileum (p < or = 0.01). However, coperfusion of glycylsarcosine (i.e., PEPT1 substrate), methionine sulfoximine (i.e., glutamine synthase inhibitor), or lysine and arginine (i.e., b(0,+) substrates) with the amino acid and dipeptide mixtures compromised the intestinal uptake of gabapentin. These findings demonstrate, for the first time, a direct relationship between the PEPT1-mediated uptake of a dipeptide and the trans-stimulated uptake of gabapentin (an amino acid-like drug) through the transport system b(0,+).

Mechanisms Responsible for Poor Oral Bioavailability of Paeoniflorin: Role of Intestinal Disposition and Interactions with Sinomenine

To determine the intestinal disposition mechanisms of paeoniflorin, a bioactive glucoside, and to investigate the mechanisms by which sinomenine increases paeoniflorin bioavailability. A single-pass "four-site" rat intestinal perfusion model and a cultured Caco-2 cell model were employed. In both model systems, paeoniflorin permeability was poor. In the perfusion model, maximal absorption and metabolism of paeoniflorin occurred in duodenum and jejunum, which were significantly decreased by a glucosidase inhibitor gluconolactone (20 mM). On the other hand, paeoniflorin absorption in terminal ileum increased significantly but its metabolism did not in the presence of sinomenine and cyclosporine A. In the Caco-2 cell model, paeoniflorin was transported 48-fold slower than its aglycone (paeoniflorigenin). Absorptive transport of paeoniflorin was significantly (p < 0.05) increased by sinomenine (38%), verapamil (27%), and cyclosporine A (41%), whereas its secretory transport was significantly (p < 0.01) decreased by sinomenine (50%), verapamil (35%) and cyclosporine A (37%). In contrast, MRP inhibitors MK-571 and leukotriene C4 did not affect transport of paeoniflorin. Lastly, sinomenine was also shown to significantly increase the absorptive transport of digoxin (a prototypical p-glycoprotein substrate) and to significantly decrease its secretory transport. Poor permeation, p-gp-mediated efflux, and hydrolysis via a glucosidase contributed to the poor bioavailability of paeoniflorin. Sinomenine (an inhibitor of the p-gp-mediated digoxin efflux) increased paeoniflorin's bioavailability via the inhibition of p-gp-mediated paeoniflorin efflux in the intestine.

Transport Characteristics of Wheat Germ Agglutinin-Modified Insulin-Liposomes and Solid Lipid Nanoparticles in a Perfused Rat Intestinal Model

Wheat germ agglutinin (WGA) modified liposomes and solid lipid nanoparticles (SLNs) were evaluated for improving intestinal absorption of insulin. In an in situ local intestinal perfusion experiment, formulations containing 100 IU/kg insulin were administered to the duodenum, jejunum, and ileum of fasted rats. As hypothesized, ileum was the best intestinal location for the absorption of insulin-containing liposomes. Serum insulin concentrations decreased for the various formulations in different absorption sites according to the following trends: Duodenum > ileum > jejunum for WGA-modified insulin-containing liposomes; duodenum > jejunum > ileum for WGA-modified insulin-containing SLNs; ileum > jejunum > duodenum for insulin-containing liposomes; ileum > duodenum > jejunum for insulin-containing SLNs; and duodenum > or = ileum > jejunum for aqueous solution of insulin. These results imply that the nanoparticle type and delivery site were important factors with respect to increasing the bioavailability of insulin following oral administration. The proteolytic degradation as well as the epithelial permeability were primary determinants influcing insulin mucosal absorption.

Disposition of Flavonoids via Enteric Recycling: Structural Effects and Lack of Correlations between in Vitro and in Situ Metabolic Properties

The purpose of this study is to determine the importance of coupling of efflux transporters and metabolic enzymes in the intestinal disposition of six isoflavones (genistein, daidzein, formononetin, glycitein, biochanin A, and prunetin), and to determine how isoflavone structural differences affect the intestinal disposition. A rat intestinal perfusion model was used, together with rat intestinal and liver microsomes. In the intestinal perfusion model, significant absorption and excretion differences were found between isoflavones and their respective glucuronides (p <0.05), with prunetin being the most rapidly absorbed and formononetin glucuronides being the most excreted in the small intestine. In contrast, glucuronides were excreted very little in the colon. In an attempt to account for the differences, we measured the glucuronidation rates of six isoflavones in microsomes prepared from rat intestine and liver. Using multiple regression analysis, intrinsic clearance (CL(int)) and other enzyme kinetic parameters (V(max) and K(m)) were determined using appropriate kinetic models based on Akaike's information criterion. The kinetic parameters were dependent on the isoflavone used and the types of microsomes. To determine how metabolite excretion rates are controlled, we plotted excretion rates versus calculated microsomal rates (at 10 microM), CL(int) values, K(m) values, or V(max) values, and the results indicated that excretion rates were not controlled by any of the kinetic parameters. In conclusion, coupling of intestinal metabolic enzymes and efflux transporters affects the intestinal disposition of isoflavones, and structural differences of isoflavones, such as having methoxyl groups, significantly influenced their intestinal disposition.

A Mechanistic Study of the Intestinal Absorption of Cryptotanshinone, the Major Active Constituent ofSalvia miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models: 1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein (PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (Plumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (Pblood). Pblood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 microM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

Disposition of Flavonoids via Recycling: Comparison of Intestinal versus Hepatic Disposition

The purpose of this study was to compare intestinal versus hepatic disposition of six flavonoids to fully characterize their first-pass metabolism. The perfused rat intestinal model and microsomes prepared from rat liver, duodenum, jejunum, ileum, and colon were used. The results indicated that isoflavone (12.5 microM) glucuronidation was highly variable among different microsomes prepared from liver or intestine. Comparing to liver metabolism, the intestinal metabolism had higher K(m) values (>2-fold). Likewise, the hepatic intrinsic clearance (IC, or a ratio of V(max)/K(m)) values of isoflavones were generally higher than their intestinal IC values (200-2000% higher), except for prunetin, for which the jejunal IC value was 50% higher than its hepatic IC. When comparing intestinal metabolism, the results showed that intestinal metabolism rates and V(max) values of isoflavones were less when an additional A-ring electron-donating group was absent (i.e., daidzein and formononetin). In the rat perfusion model using the whole small intestine, genistein (10 microM) was well absorbed (77% or 352 nmol/120 min). The first-pass metabolism of genistein was extensive, with 40% of absorbed genistein excreted as conjugated metabolites into the intestinal lumen. In contrast, the bile excretion of genistein conjugates was much less (6.4% of absorbed genistein). In conclusion, intestinal glucuronidation is slower in isoflavones without an additional A-ring substitution. Perfusion studies suggest that intestine is the main organ for genistein glucuronide formation and excretion in rats and may serve as its main first-pass metabolism organ.

Role of Intestinal First-Pass Metabolism of Baicalein in its Absorption Process

The aim of the present study was to investigate the role of intestinal first-pass metabolism of baicalein (B) in its absorption process. The intestinal absorption of B was characterized using Caco-2 cell monolayer model and rat in situ single-pass intestinal perfusion model. In addition, preliminary metabolic kinetics of B was evaluated in both rat and human intestinal S9 fractions. B was well absorbed and extensively metabolized to baicalin (BG), baicalein-7-O-beta-glucuronide, in rat intestinal perfusion model, whereas less extent of metabolism was observed in the Caco-2 cell monolayer model. Moreover, BG generated in the intestinal epithelium during the absorption of B also rapidly transported to both the apical side (the apical chamber of Caco-2 model and the perfusate of the intestinal perfusion model) as well as the basolateral side of the small intestine (the basal chamber of Caco-2 model and the mesenteric vein of the intestinal perfusion model). From the preliminary metabolic studies, it was found that a higher loading dose of B resulted in a less extent of metabolism in intestine. In addition, the extent of metabolism of B was similar in jejunum and ileum when 50 microM of B was perfused through different sections of rat small intestine. The first-pass metabolism of B in small intestine may play an important role in its low oral bioavailability.

Disposition of Formononetin via Enteric Recycling: Metabolism and Excretion in Mouse Intestinal Perfusion and Caco-2 Cell Models

The purpose of this study was to determine the absorption and metabolism of formononetin using the mouse intestinal perfusion model, mouse intestinal homogenate, and the Caco-2 cell culture model. In the perfusion model where upper and lower small intestine were perfused simultaneously, absorption of formononetin was rapid and dimensionless effective permeabilities of formononetin (2.53-2.90) were similar to those for rats. Moreover, the amounts of sulfates excreted in mouse intestine (8-11 nmol/30 min/10 cm) were significantly higher than those for rats whereas the amounts of glucuronides excreted (7-10 nmol/30 min/10 cm) were comparable. Small amounts of formononetin glucuronide but not sulfate were found in mouse bile, but the total amounts were 120 times less than those for rats. Multidrug-resistance-related protein (MRP) inhibitors (leukotriene C(4) plus MK-571, C(26)H(26)ClN(2)O(3)S(2)) significantly decreased the excretion of glucuronide and sulfate in mouse intestine (52-74% for glucuronide, 13-26% for sulfate) and in Caco-2 cells (92% for glucuronide, 37% for sulfate). They also inhibited formation of conjugates in intestinal homogenate (approximately 60% for glucuronide, approximately 30% for sulfate) and Caco-2 cell lysate (approximately 92% for glucuronide, approximately 37% for sulfate). Organic anion transporter (OAT) inhibitors (estrone sulfate plus dihydroepiandrosterone sulfate) did not significantly change the excretion of formononetin conjugates in either model, even though they significantly decreased the formation of both. In conclusion, our study showed that formononetin has similar absorption in rat and mouse intestine, but metabolism was species-dependent. The mouse perfusion model may have an advantage over the rat intestinal perfusion model for flavonoid disposition studies in that both sulfates and glucuronides are excreted, as shown in humans.

Transport and Bioavailability Studies of Astragaloside IV, an Active Ingredient in Radix Astragali

Astragaloside IV is an important constituent of Radix Astragali, a herbal remedy widely used in traditional Chinese medicine. Radix Astragali is administered orally but little is known about the transport properties and bioavailability of astragaloside IV. In this paper we report studies of the absorption of astragaloside IV in the perfused rat intestinal model, transport and uptake in Caco-2 cell monolayers and in vivo bioavailability in rat after an oral dose. In the perfused rat intestinal model, absorption of astragaloside IV was low from an aqueous solution but was significantly higher from a solution of Radix Astragali. Absorption was not affected by bile duct ligation. Transport through Caco-2 cells gave a very low permeability value (mean P(app) of 6.7+/-1.0 x 10(-8) cm/sec.) and uptake was unaffected by P-glycoprotein inhibitors. The absolute bioavailability of astragaloside IV in rat was 2.2%. The correlation between low permeability in vitro and poor bioavailability in vivo indicates in vitro absorption studies are useful in the evaluation of traditional Chinese medicines.