Mesenteric Blood Samples Research Articles

A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments

A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A-B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10(-6) cm/s) was determined for atenolol (10.7 ± 1.2), caffeine (17.6 ± 3.1), cimetidine (6.9 ± 0.1), metoprolol (12.6 ± 0.7), theophylline (15.3 ± 1.6) and, ranitidine (3.8 ± 0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r = 0.89). These findings showed a high correlation with human data (r = 0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules.

Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: In vitro, rat in situ and human in vivo studies

The aim of this study was to evaluate the intestinal disposition of abiraterone acetate, an ester prodrug of the anticancer agent abiraterone. Stability of the prodrug and solubility and dissolution characteristics of both abiraterone and abiraterone acetate were monitored in vitro. Moreover, the in vivo intraluminal concentrations of abiraterone and abiraterone acetate upon intake of one tablet of 250mg abiraterone acetate were assessed in healthy volunteers. The intestinal absorption resulting from the intraluminal behavior of the ester prodrug was determined using the rat in situ intestinal perfusion technique with mesenteric blood sampling. Simulated and aspirated human intestinal fluids of the fasted state were used as solvent systems.Upon incubation of abiraterone acetate in human intestinal fluids in vitro, rapid hydrolysis of the prodrug was observed, generating abiraterone concentrations largely exceeding the apparent solubility of abiraterone, suggesting the existence of intestinal supersaturation. These findings were confirmed in vivo, by intraluminal sampling of duodenal fluids upon oral intake of an abiraterone acetate tablet by healthy volunteers.Rat in situ intestinal perfusion experiments performed with suspensions of abiraterone and abiraterone acetate in human intestinal fluids of the fasted state revealed significantly higher flux values upon perfusion with the prodrug than with abiraterone. Moreover, rat in situ intestinal perfusion with abiraterone acetate suspensions in simulated fluids of the fasted state in presence or absence of esterases demonstrated that increased hydrolytic activity of the perfusion medium was beneficial to the intestinal absorption of abiraterone.In conclusion, the rapid hydrolysis of abiraterone acetate in the intraluminal environment appears to result in fast and extensive generation of abiraterone supersaturation, creating a strong driving force for abiraterone absorption.

Human and simulated intestinal fluids as solvent systems to explore food effects on intestinal solubility and permeability

The mixed micelles and vesicles present in the intraluminal environment of the postprandial state exhibit suitable solubilizing capacities for lipophilic drugs. This increase in solubility, however, is accompanied by a decrease in the free fraction caused by micellar entrapment of these lipophilic compounds. In this study, both simulated and aspirated human intestinal fluids of fasted and fed state conditions were used to evaluate the influence of food on the intestinal disposition of a series of structurally related β-blockers, with varying logP values.Using the in situ intestinal perfusion technique with mesenteric blood sampling in rats, it was demonstrated that fed state conditions significantly decreased the absorptive flux of the more lipophilic compounds metoprolol, propranolol and carvedilol, whereas the influence on the flux of the hydrophilic β-blocker atenolol was limited.The solubility of BCS class II compound carvedilol was found to increase significantly in simulated and aspirated media of the fed state. Intestinal perfusions using intestinal media saturated with carvedilol, revealed a higher flux in the fasted state compared to the fed state, despite the higher solubility in the fed state. This study underscores the importance of addressing the complex nature of the behavior of compounds in the intraluminal environment in fasted and fed state conditions. Moreover, our data point out the value of studying the effect of food on both solubility and permeability using biorelevant experimental conditions.

Capture of Viable Circulating Tumor Cells in the Liver of Colorectal Cancer Patients

The incidence and number of circulating tumor cells (CTCs) in the peripheral blood of colorectal cancer patients are lower than in other cancer types, which may point to a particular biology of colorectal cancer affecting CTC detection. We detected CTCs in the peripheral and mesenteric blood of colorectal cancer patients by use of 2 independent technologies on the basis of different biological properties of colon cancer cells. Seventy-five patients diagnosed with localized (M0, n = 60) and metastatic (M1, n = 15) colorectal cancer were included. Peripheral and mesenteric blood samples were collected before tumor resection. We performed CTC enumeration with an EpCAM-independent enrichment method followed by the Epispot assay that detected only viable CK19-releasing CTCs. In parallel, we used the FDA-cleared EpCAM-dependent CellSearch® as the reference method. The enumeration of CK19-releasing cells by the CK19-Epispot assay revealed viable CTCs in 27 of 41 (65.9%) and 41 of 74 (55.4%) (P = 0.04) patients in mesenteric and peripheral blood, respectively, whereas CellSearch detected CTCs in 19 of 34 (55.9%) and 20 of 69 (29.0%) (P = 0.0046) patients. In mesenteric blood, medians of 4 (range 0-247) and 2.7 CTCs (range 0-286) were found with Epispot and CellSearch (P = 0.2), respectively, whereas in peripheral blood, Epispot and CellSearch detected a median of 1.2 (range 0-92) and 0 CTCs (range 0-147) (P = 0.002). A considerable portion of viable CTCs detectable by the Epispot assay are trapped in the liver as the first filter organ in CRC patients.

PXR/CYP3A4-Humanized Mice for Studying Drug–Drug Interactions Involving Intestinal P-Glycoprotein

Rodent models are less suitable for predicting drug-drug interactions at the level of the human intestinal mucosa, especially when nuclear receptors such as pregnane X receptor (PXR) are involved. Recently, a transgenic mouse model, expressing both human PXR and CYP3A4, was developed and shown to be a better predictor of CYP3A4 induction by xenobiotics in humans as compared to wild-type mice. In the present study, we tested the hypothesis that this mouse model can also predict PXR-mediated induction of intestinal P-gp in humans. By use of the in situ intestinal perfusion technique with mesenteric blood sampling, the effect of oral rifampicin treatment on intestinal permeability for the HIV protease inhibitor darunavir, a dual CYP3A4/P-gp substrate, was investigated. Rifampicin treatment lowered the intestinal permeability for darunavir by 50% compared to that in nontreated mice. The P-gp inhibitor GF120918 increased the permeability for darunavir by 400% in rifampicin-treated mice, whereas this was only 56% in mice that were not treated, thus indicating P-gp induction by rifampicin. The nonspecific P450 inhibitor aminobenzotriazole (100 μM) did not affect the permeability for darunavir. Quantitative Western blot analysis of the intestinal tissue showed that rifampicin treatment induced intestinal P-gp levels 4-fold, while CYP3A4 levels remained unchanged. Oral co-administration of rifampicin with the phytochemical sulforaphane for 3 days increased the permeability for darunavir by 50% compared to that with rifampicin treatment alone. These data show that PXR/CYP3A4-humanized mice can be used to study the inducing effects of xenobiotics on intestinal P-gp.

Site dependent intestinal absorption of darunavir and its interaction with ketoconazole

The expression of P-gp increases from proximal to distal parts of the small intestine, whereas for P450 enzymes the expression is reported to be highest in duodenum and jejunum, decreasing to more distal sites. To evaluate to what extent the regional differences in expression of P-gp and P450 enzymes affect the absorption of a dual substrate, we investigated the transport of darunavir across different small intestinal segments (duodenum, proximal jejunum and ileum). Moreover, the effect of ketoconazole on the intestinal absorption of darunavir was explored, since these drugs are commonly co-administered. Performing the rat in situ intestinal perfusion technique with mesenteric blood sampling, we found no significant differences in the transport of darunavir at the different intestinal segments. The involvement of P-gp in the absorption of darunavir was clearly shown by coperfusion of darunavir with the P-gp inhibitor zosuquidar. In presence of zosuquidar, a 2.2-, 4.2- and 5.7-fold increase in Papp values were measured for duodenum, proximal jejunum and ileum, respectively. Involvement of P450 mediated metabolism in the absorption of darunavir could not be demonstrated in this rat model. Upon studying the drug–drug interaction of darunavir with ketoconazole, data were indicative for an inhibitory effect of ketoconazole on P-gp as the main mechanism for the increased transport of darunavir across the small intestine.

Relationship between the levels of oxidative stress in mesenteric and peripheral serum and clinicopathological variables in colorectal cancer.

To explore the differences existing between the levels of oxidative stress in peripheral and mesenteric serum in patients with colorectal cancer. One hundred fifty patients with colorectal cancer who underwent surgery between May 2005 and March 2010 were prospectively analyzed. The differences between oxidative stress parameters in their peripheral and mesenteric blood were measured. The associations between peripheral and mesenteric levels and the staging and clinicopathological variables were investigated. Oxidative stress parameters were higher in patients with advanced tumor staging (p<0.01), lymph node invasion (p<0.01), and venous invasion (p<0.01). Differences between oxidative stress parameters in peripheral and mesenteric blood samples were also observed. The mesenteric levels of the oxidative stress markers were higher than the peripheral levels in these colorectal cancer patients. Higher levels of these oxidative stress markers are associated with an advanced state of cancer.

Boosting of HIV Protease Inhibitors by Ritonavir in the Intestine: The Relative Role of Cytochrome P450 and P-Glycoprotein Inhibition Based on Caco-2 Monolayers versus In Situ Intestinal Perfusion in Mice

HIV protease inhibitors are essential components of most recommended treatment regimens for HIV infection. They are always coadministered with ritonavir as a pharmacokinetic booster. Their bioavailability may be impaired because they are substrates of CYP3A4 and several transporters, including P-glycoprotein. The aim of this study was to explore the impact of ritonavir on the intestinal absorption of HIV protease inhibitors in two models: the Caco-2 system and the in situ intestinal perfusion model with mesenteric blood sampling in mice. Using the Caco-2 system, the effect of ritonavir on the permeability of the other HIV protease inhibitors was significant for saquinavir (2-fold increase) and indinavir (3-fold increase), negligible for darunavir and amprenavir, and nonexistent for nelfinavir, lopinavir, tipranavir, and atazanavir. However, performing the in situ intestinal perfusion technique in mice for three selected HIV protease inhibitors showed a significant increase in the intestinal permeability for all: indinavir (3.2-fold), lopinavir (2.3-fold), and darunavir (3-fold). The effect of aminobenzotriazole (a nonspecific cytochrome P450 inhibitor) on lopinavir permeability was comparable with using ritonavir, whereas there was no effect for indinavir and darunavir. We conclude that ritonavir can boost drug absorption by inhibiting P-glycoprotein and/or metabolism, in a compound-specific manner. The results of this study illustrate that a combination of absorption models needs to be considered to elucidate drug-drug interactions at the level of the intestinal mucosa.

Validation of a differential in situ perfusion method with mesenteric blood sampling in rats for intestinal drug interaction profiling

The present study explored the feasibility of a differential setup for the in situ perfusion technique with mesenteric cannulation in rats to assess drug interactions at the level of intestinal absorption. In contrast to the classic, parallel in situ perfusion setup, the differential approach aims to identify intestinal drug interactions in individual animals by exposing the perfused segment to a sequence of multiple conditions. First, the setup was validated by assessing the interaction between the P-glycoprotein (P-gp) inhibitor verapamil and the transport probes atenolol (paracellular transport), propranolol (transcellular) and talinolol (P-gp mediated efflux). While transport of atenolol and propranolol remained constant for the total perfusion time (2 h), a verapamil-induced increase in talinolol transport was observed within individual rats (between 3.2- and 5.2-fold). In comparison with the parallel setup, the differential in situ perfusion approach enhances the power to detect drug interactions with compounds that exhibit strong subject-dependent permeability. This was demonstrated by identifying an interaction between amprenavir and ketoconazole (P-gp and CYP3A inhibitor) in five out of seven rats (permeability increase between 1.9- and 4.2-fold), despite high inter-individual differences in intrinsic permeability for amprenavir. In combination with an increased throughput (up to 300%) and a reduced animal use (up to 50%), the enhanced power of the differential approach improves the utility of the biorelevant in situ perfusion technique with mesenteric blood sampling to elucidate the intestinal interaction profile of drugs and drug candidates.

INTESTINAL PERFUSION WITH MESENTERIC BLOOD SAMPLING IN WILD-TYPE AND KNOCKOUT MICE

In the present study, we successfully downscaled, for the first time, the in situ intestinal perfusion technique with mesenteric blood sampling from rat to mouse. To evaluate the feasibility of this approach, we assessed the apparent permeability (P(app)) of mouse intestine for a set of marker compounds [atenolol, paracellular transport; metoprolol, transcellular transport; talinolol, P-glycoprotein (P-gp)-mediated efflux] in both wild-type and P-gp-deficient mice. In wild-type mice, the observed P(app) values for atenolol (1.8 +/- 0.3 x 10(-6) cm/s) and metoprolol (50.2 +/- 20.1 x 10(-6) cm/s) were not significantly affected by inclusion of the P-gp inhibitor verapamil. In contrast, the P(app) value for talinolol (0.9 +/- 0.3 x 10(-6) cm/s) increased 5-fold in the presence of verapamil. The similarity between these values and previously determined P(app) values in rats indicates comparable passive barrier functions and P-gp-mediated efflux transport between mice and rats. In comparison with wild-type mice, the apparent permeability in P-gp-deficient mdr1a/1b(-/-) mice was significantly altered for talinolol (7-fold increase) but not for atenolol or metoprolol. Because of the availability of knockout mice, the intestinal perfusion technique with mesenteric blood sampling in mice may become an important tool to elucidate the role of intestinal metabolism and active transport in drug absorption during preclinical drug evaluation.

Detection of cancer cells in mesenteric vein and peripheral vessels by measuring telomerase activity in patients with colorectal cancer

Background Liver metastasis is an important factor determining prognosis in colorectal cancer. The objective of this study was to assess whether colorectal cancer cells in the drainage veins can be detected by measuring telomerase activity and its detection is correlated with liver metastasis. Methods Telomeric repeat amplification protocol assay in combination with an immunomagnetic sorting was used for measuring telomerase activity of epithelial cells in blood samples collected from mesenteric (tumor-drainage) vein and peripheral vessels of 41 colorectal cancer patients. Telomerase activity was calculated as relative telomerase activity (RTA) against a control template and analyzed in terms of liver metastasis. Results RTA of mesenteric blood samples was significantly higher in patients with liver metastasis (60.8%; n = 7) than in those without metastasis (19.7%; n = 34; P = .019). The RTA of peripheral blood sample was also higher in patients with liver metastasis (26.8%) than in those without metastasis (11.1%; p = .17). Moreover, 57% of cases with liver metastasis exhibited a positive telomerase activity in mesenteric blood sample, whereas it was 18% in cases without metastasis. Conclusions Our assay was proven to be a feasible method for detecting cancer cells in tumor-drainage veins. High telomerase activity of mesenteric blood samples reflected the existence of liver metastasis of colorectal cancer.

Detection of cytokeratin 19 and 20 mRNA in peripheral and mesenteric blood from colorectal cancer patients and their prognosis.

To detect the presence of cancer cells in peripheral and mesenteric blood and to examine their relationship to prognosis in colorectal cancer patients undergoing curative surgery, we examined cytokeratin 19 and 20 mRNA in peripheral and mesenteric venous blood. Using reverse transcriptase polymerase chain reaction, cytokeratin 19 and 20 mRNA was amplified in peripheral and mesenteric blood samples obtained from 35 colorectal cancer patients who underwent curative surgery. Cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood samples was detected in 18 of 35 cases (51%). There was no significant difference in Dukes' staging between the positive and negative groups. The median follow-up period was 56 months. In the positive group six patients (33%) showed recurrences. One patient (6%) showed recurrence in the negative group. The recurrence rate was significantly higher in the positive group than in the negative group. Five-year disease-free survival was significantly better in the negative group than in the positive group (94% vs. 65%). The detection of cytokeratin 19 or 20 mRNA in peripheral or mesenteric blood is thus associated with the prognosis for colorectal cancer patients undergoing curative surgery. Although the presence of these mRNAs in blood samples is a prognostic marker, the clinical utility of this assay is questionable because of the low recurrence rate in the positive group.

The effect of acute ligation of the rabbit appendix on antioxidant enzymes.

The role of oxygen free radicals (OFR) in the pathogenesis of many diseases is known. This experimental study was planned to investigate the role of OFR in acute abdominal inflammation. In this study, 20 adult rabbits were used. They were divided into two groups with 10 rabbits in each. In the study group, blood samples were taken from peripheral and mesenteric veins and then their appendices were ligated; after 24 h, blood samples were taken again and the appendices were resected. The same procedures were carried out in the control group, except for ligation of the appendices. Superoxide dismutase,catalase and glutathione peroxidase (GPX) activities were measured in all blood samples. The activities of catalase and GPX were increased postoperatively in peripheral and mesenteric blood samples in the experimental group. The catalase activity was increased in the control group. These results suggest that OFR increased sufficiently to activate the enzymatic defense system in acute appendicitis.

Bile acid active and passive ileal transport in the rabbit: effect of luminal stirring.

The intestinal absorption of bile acids (BA) with different chemical structure has been evaluated in the rabbit, after intestinal infusion of different concentrations (0.25-30 mM) of BA, by mesenteric blood sampling. Cholic (CA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA) acid, free and taurine (T-) conjugated, together with glycocholic (GCA) acid and deoxycholic acid (DCA) were studied. The apparent uptake parameters were calculated. All conjugated BA showed active transport (T max, nmol min-1 cm-1 int.), with Tmax values in the following order: TCA > TUDCA > TCDCA; unconjugated BA showed passive uptake, with values in the following order: DCA > CDCA > UDCA > CA. GCA and CA showed both passive uptake and active transport. For all BA studied the % uptake in the ileal segment considered was less than 10%, BA uptake being thus limited by transport and/or diffusion kinetics, rather than by flow velocity. The liquid resistance to BA radial diffusion inside the lumen was evaluated, and the infusate-to-blood uptake parameters corrected for it, in order to get the uptake parameters from the epithelium-to-liquid interface to mesenteric blood: the apparent Km decreased, passive uptake coefficient increased, while Tmax was unchanged. The passive component of the uptake, corrected for the luminal resistance, correlated with the BA hydrophobicity (r = 0.963; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)