Rat Intestinal Homogenates Research Articles

Evaluation of Selenomethionine Entrapped in Nanoparticles for Oral Supplementation Using In Vitro, Ex Vivo and In Vivo Models

Selenium methionine (SeMet) is an essential micronutrient required for normal body function and is associated with additional health benefits. However, oral administration of SeMet can be challenging due to its purported narrow therapeutic index, low oral bioavailability, and high susceptibility to oxidation. To address these issues, SeMet was entrapped in zein-coated nanoparticles made from chitosan using an ionic gelation formulation. The high stability of both the SeMet and selenomethionine nanoparticles (SeMet-NPs) was established using cultured human intestinal and liver epithelial cells, rat liver homogenates, and rat intestinal homogenates and lumen washes. Minimal cytotoxicity to Caco-2 and HepG2 cells was observed for SeMet and SeMet-NPs. Antioxidant properties of SeMet were revealed using a Reactive Oxygen Species (ROS) assay, based on the observation of a concentration-dependent reduction in the build-up of peroxides, hydroxides and hydroxyl radicals in Caco-2 cells exposed to SeMet (6.25-100 μM). The basal apparent permeability coefficient (Papp) of SeMet across isolated rat jejunal mucosae mounted in Ussing chambers was low, but the Papp was increased when presented in NP. SeMet had minimal effects on the electrogenic ion secretion of rat jejunal and colonic mucosae in Ussing chambers. Intra-jejunal injections of SeMet-NPs to rats yielded increased plasma levels of SeMet after 3 h for the SeMet-NPs compared to free SeMet. Overall, there is potential to further develop SeMet-NPs for oral supplementation due to the increased intestinal permeability, versus free SeMet, and the low potential for toxicity.

Factors Affecting Endogenous Oxalate Synthesis and Its Excretion in Feces and Urine in Rats

It has been observed that the feces as well as urine of rats fed diets supplemented with 3% glycine and 5.2% hydroxyproline contain unexpectedly high amounts of endogenously formed oxalate. That intestinal microorganisms do not synthesize significant amounts of oxalate was indicated by the findings that oral tetracycline had no effect on oxalate excretion and that germ-free rats excreted more oxalate than conventional rats. Since little intraperitoneally injected [14C]oxalate appeared in the feces, and rat intestinal mucosa homogenates were found to produce oxalate from a variety of precursors of which glyoxylic acid was by far the most important, it is probable that the intestinal mucosa may be an important source of fecal oxalate observed in these studies. Ninety percent of weanling rats fed complete diets supplemented with glycine and hydroxyproline developed urinary stones in 38 days. It has been concluded that in the treatment of patients with histories of calcium oxalate urolithiasis, more concern than is commonly shown should be directed towards the feeding of diets high in precursors of endogenous oxalate synthesis.oxalate hydroxyproline calculi

Rat intestinal homogenate and pancreatic juice can induce the Z-isomerization of all-E-lycopene in vitro

Lycopene is one of the carotenoids often consumed by humans in their diet. Although lycopene exists mainly in the form of the all E-isomer in foods, the considerable quantity of its Z-isomers is found in the human plasma and liver. This observation suggested that the lycopene all-E-isomer was converted into Z-isomers in the human body. In this study, the Z-isomerization of the all-E-isomer was induced in vitro by the pancreatic juice and small intestinal homogenate of male rats under the conditions of 37 °C, pH = 7.5, nitrogen and darkness, as well as shaking. After 2 hours, the proportion of the all E-isomer decreased to 25% and Z-isomer amounts increased relatively. The converted products were identified as 5, 9, and 11 Z-isomers by electronic absorption spectroscopy and mass spectrometry (MS). The observations from this experiment suggested that the Z-isomerization site of the lycopene all E-isomer was located in the small intestinal wall of the rat.

Synthesis, Biological Evaluation and Pharmacokinetic Studies of Mefenamic Acid - N-Hydroxymethylsuccinimide Ester Prodrug as Safer NSAID.

Non steroidal anti-inflammatory drugs are the most widely prescribed drugs to manage pain and inflammatory conditions, but their long term use is associated with gastrointestinal toxicity. The study aimed to synthesize an ester-based prodrug of a non steroidal anti-inflammatory agent, mefenamic acid in order to improve the therapeutic index vis a vis to overcome the side effects such as gastrointestinal irritation and bleeding associated with the use of mefenamic acid. The ester prodrug (MA-NH) was prepared by condensing mefenamic acid with N-hydroxymethylsuccinimide in the presence of Phosphorus oxychloride. The pharmacokinetic profile, including stability and release of mefenamic acid and N-hydroxymethylsuccinimide from the ester prodrug (MA-NH) was studied by RP- HPLC in acidic medium (pH 1.2), basic medium (pH 7.4), 80 % v/v human plasma, 10 % w/v rat intestinal homogenate and 10 % w/v rat liver homogenate (pH 7.4). The chemical structure of the title compound was characterized by using modern spectroscopic techniques. The prodrug was found to be stable in acid medium, but it hydrolyzed and released sufficient quantities of the drug in alkaline medium. The prodrug produced lesser number of ulcers and showed improved analgesic and anti-inflammatory activity as compared to the parent drug. The results indicate that the synthesized prodrug (MA-NH) is better in terms of analgesic and antiinflammatory activities and with less GI toxicity than the parent drug.

Enzymatic Formation of Novel Ginsenoside Rg1-α-Glucosides by Rat Intestinal Homogenates.

The variation of linkage positions in ginsenosides leads to diverse pharmacological efficiencies. The hydrolysis and transglycosylation properties of glycosyl hydrolase family enzymes have a great impact on the synthesis of novel and structurally diversified compounds. In this study, six ginsenoside Rg1-α-glucosides were found to be synthesized from the reaction mixture of maltose as a donor and ginsenoside Rg1 as a sugar acceptor in the presence of rat small intestinal homogenates, which exhibit high α-glucosidase activities. The individual compounds were purified and were identified by spectroscopy (HPLC-MS, (1)H-NMR, and (13)C-NMR) as 6-O-[α-D-glcp-(1→4)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→6)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-β-D-glcp-20-O-[α-D-glcp-(1→4)-(β-D-glcp)]-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→6)-β-D-glcp]-20-O-(β-glcp)-20(S)-protopanaxatriol, 6-O-[α-D-glcp-(1→3)-β-D-glcp]-20-O-(β-D-glcp)-20(S)-protopanaxatriol, and 6-O-β-D-glcp-20-O-[α-D-glcp-(1→3)-(β-D-glcp)]-20(S)-protopanaxatriol. Among these six, 6-O-β-D-glcp-20-O-α-D-glcp-(1→6)-(β-D-glcp)-20(S)-protopanaxatriol and 6-O-α-D-glcp-(1→6)-β-D-glcp-20-O-(β-D-glcp)-20(S)-protopanaxatriol are considered to be novel compounds of alpha-ginsenosidal saponins which pharmacological activities should be further characterized. This is the first report on the enzymatic elaboration of ginsenoside Rg1 derivatives using rat intestinal homogenates. To the best of our knowledge, it is also the first to reveal the sixth and 20th positions of an unusual α-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl sugar chain with 20(S)-protopanaxatriol saponins in Panax ginseng Mayer.

Absorption and cleavage of enalapril, a carboxyl ester prodrug, in the rat intestine: in vitro, in situ intestinal perfusion and portal vein cannulation models.

In recent years prodrug strategy has been used extensively to improve the pharmacokinetic properties of compounds exhibiting poor bioavailability. Mechanistic understanding of the absorption and the role of intestine and liver in the activation of oral prodrugs is crucial. Enalapril, a carboxyl ester prodrug, is reported to be metabolized by human carboxylesterase-1 (CES1) but not by carboxylesterase-2 (CES2) to its active metabolite enalaprilat. Further, it has been reported that the small intestines of both rat and human contain mainly CES2. The objective of this work was to understand whether enalapril remains unchanged as it is absorbed through the intestine into the portal circulation. This was evaluated using different intestinal preparations, an in situ intestinal perfusion experiment and a portal vein cannulated rat model. No turnover of enalapril was seen with commercial rat intestinal S9 and microsomes, but reasonable turnover was observed with freshly prepared rat intestinal and mucosal homogenate and S9. In the intestinal perfusion study, both enalapril and enalaprilat were observed in the mesenteric plasma with the data suggesting 32% hydrolysis of enalapril in the intestine. In the portal vein cannulated rat, about 51% of enalapril absorbed into intestine was converted to enalaprilat. Overall, it was demonstrated that even though enalapril has been shown to be a specific substrate for CES1, it is converted to enalaprilat to a significant extent in the intestine. Such experimental techniques can be applied by other scientific groups who are working on prodrugs to determine the region and extent of activation. Copyright © 2015 John Wiley & Sons, Ltd.

Synthesis, biological activities, and pharmacokinetics studies of a mutual prodrug of aceclofenac and paracetamol

An ester-based mutual prodrug (aceclofenac–paracetamol; AC-PR) was synthesized (one-pot method) with an aim of improving the therapeutic index through prevention of gastrointestinal irritation and bleeding that is associated with aceclofenac. The release of aceclofenac and paracetamol from the ester prodrug (AC-PR) was studied by reverse phase HPLC in hydrochloric acid buffer (pH 1.2), phosphate buffer (pH 7.4), 80 % v/v human plasma, 10 % w/v rat intestinal homogenate and 10 % w/v rat liver homogenate (pH 7.4). The prodrug showed negligible hydrolysis at pH 1.2 as compared to pH 7.4, suggesting that very less of the prodrug would hydrolyze in stomach, but would release the parent drugs at pH 7.4 in adequate amounts. The prodrug showed enhanced anti-inflammatory activity and significant protection against acetic acid-induced writhings (analgesic activity) as compared to that of aceclofenac. Further, the prodrug produced reduced number of ulcers as compared to that of the parent drug. These results suggest that the synthesized mutual prodrug (AC-PR) is better in terms of activity and GIT toxicity than the parent drug.

Stereoselective Evasion of P-glycoprotein, Cytochrome P450 3A, and Hydrolases by Peptide Prodrug Modification of Saquinavir

In an approach to overcome biological barriers mediated by P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A), a series of stereoisomeric valine-valine prodrugs of saquinavir (SQV) were synthesized and investigated with respect to affinity for efflux pump P-gp, and resistance to oxidative and hydrolytic enzymes. Cellular uptake and bidirectional transport in Caco-2 cells indicated that all peptide SQV conjugates can bypass P-gp-mediated efflux significantly, regardless of stereochemistry in promoieties. In comparison with D-configuration, L-configuration was favored for the interaction between prodrugs and rat hepatic CYP3A enzymes, and resulted in a relatively rapid clearance by CYP3A. Elimination half-life of SQV in rat liver microsomes was prolonged dramatically by sevenfold to 40-fold because of the prodrug modification with the rank order of D-valine-D-valine-SQV > D-valine-L-valine-SQV > L-valine-D-valine-SQV > L-valine-L-valine-SQV > D-valine-SQV > L-valine-SQV > SQV. Results of hydrolysis studies performed in rat intestinal homogenates and plasma indicated that prodrugs attached with D-valine exhibited significantly reduced biodegradation. In conclusion, the enhanced transepithelial accumulation and enzymatic stability observed by SQV peptide prodrug modification are found to be stereoselective. Specific stereoisomeric dipeptide prodrugs with optimized metabolic stability can be employed to improve oral bioavailability of SQV.

Tyrosine-Based 1-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]cytosine and -adenine ((S)-HPMPC and (S)-HPMPA) Prodrugs: Synthesis, Stability, Antiviral Activity, and in Vivo Transport Studies

Eight novel single amino acid (6-11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPC, 4) and its cyclic adenine analogue ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC(50) values for the prodrugs (<0.1-50 μM) vs vaccinia, cowpox, human cytomegalovirus, and herpes simplex type 1 virus were compared to those for the parent drugs ((S)-HPMPC, 2; (S)-HPMPA, 1; IC(50) 0.3-35 μM); there was no cytoxicity with KB or HFF cells at ≤100 μM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30-1732 min) with differences of 3-10× between phostonate diastereomers. The tyrosine alkylamide derivatives of 3 and 4 were the most stable. (l)-Tyr-NH-i-Bu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39% vs <5%).

Absorption enhancement of adefovir dipivoxil by incorporating MCT and ethyl oleate complex oil phase in emulsion

To improve the oral absorption of adefovir dipivoxil (ADV) by employing MCT and the esterase inhibitor ethyl oleate (EO) as a complex oil phase in emulsion. EO was used as the esterase inhibitor, and its inhibitory effect on esterase activity was assessed in rat intestinal homogenates. ADV emulsions with or without EO were prepared. The emulsions' protective effect against intestinal metabolism was evaluated in rat luminal contents, ex vivo, as well as in vivo. The IC(50) of EO in intestinal mucosal homogenates was 2.2 mg/mL. The emulsions exhibited significant protective effects in rat luminal contents compared to a simple suspension (98.7%, 96.3%, 95.7% vs 74.7%, P<0.01). The permeability calculated from the emulsion containing EO was significantly different (11.4 x 10(-6) vs 7.4/8.0 x 10(-6), P<0.05) from the simple suspension or the emulsion without EO in an ex vivo assay. A bioavailability study in vivo revealed that emulsions containing both EO and MCT as a complex oil phase demonstrated 1.6- and 1.5-fold enhancements in area under the curve (AUC(0-12)) values (5358 vs 3386/3618, P<0.05), respectively, when compared with emulsions containing EO or MCT as a single oil phase. Heterotic lipid formulations (emulsions) with an esterase inhibitor (ie, EO) may be useful in protecting ester prodrugs from intestinal metabolism and increasing their oral bioavailability.

Reversible Inhibitory Effects of Saturated and Unsaturated Alkyl Esters on the Carboxylesterases Activity in Rat Intestine

This study was conducted to investigate the relationship between the carbon chain length/double bonds of alkyl esters and their inhibitory potency/mechanism on carboxylesterases (CESs). CESs activity was evaluated by inhibition of adefovir dipivoxil (ADV) metabolism in rat intestinal homogenates. Furthermore, the inhibitory effect of BNPP and ethyl (E)-hex-2-enoate (C8:1) on drug absorption was evaluated in situ intestinal perfusion model. The results showed that the rank order of the inhibitory potency on CESs was C10:0 > C8:0 > C6:0 > C4:0 > C12:0, C8:1 > C8:0, C6:1 > C6:0, while the esters (C14:0, C13:1, C16:0, C18:0, C17:1, C20:0) were found to have no inhibitory effect at investigated concentrations. However, the unsaturated esters (C20:1, C20:2, C20:3) displayed the inhibitory effect on CESs. Moreover, the double reciprocal plots indicated that alky esters inhibited the CESs in competitive and mixed competitive ways which were reversible. In addition, the result of most effective CESs inhibitor C8:1 from in situ experiment showed that C8:1 can inhibit the CESs-mediated intestinal metabolism and improve the drug absorption. And the inhibition had no time-dependent effect, compared with that of BNPP groups. The study suggested that alkyl esters can be served as effective and reversible CESs inhibitors, besides that their inhibitory potency/mechanism can be affected by their carbon chain length/double bonds.

Safety evaluation of highly-branched cyclic dextrin and a 1,4-α-glucan branching enzyme from Bacillus stearothermophilus

Highly-branched cyclic dextrin (HBCD), a dextrin food ingredient presently only used in Japan, was investigated for digestibility and potential toxicity. HBCD was readily hydrolyzed in vitro to maltose and maltotriose by human salivary and porcine pancreatic α-amylases. Incubation of HBCD with a rat intestinal homogenate, containing digestive enzymes, resulted in the formation of maltose, maltotriose, and maltotetraose, and with longer incubation times, resulted in the formation of glucose. In an acute toxicity study, Wistar rats orally administered a single-dose of 2000 mg/kg body weight of HBCD did not display mortality or any signs or symptoms of toxicity or abnormalities upon necropsy. Transient loose stools were observed, but were resolved within 24 h of HBCD administration, and therefore, were not considered as compound-specific adverse effects. In the Ames assay, HBCD was non-mutagenic with or without metabolic activation. Toxicity testing of the branching enzyme (BE) involved in the synthesis of HBCD showed that the BE also was not acutely toxic when orally administered to rats and was non-mutagenic in the mouse lymphoma assay. The results of this study demonstrate that HBCD is digested to normal and safe products of carbohydrate digestion, and therefore, support the safety of HBCD for human consumption.

Bioavailability of 2- O-α- d-glucopyranosyl- l-ascorbic acid as ascorbic acid in healthy humans

Objective 2- O-α- d-glucopyranosyl-L-ascorbic acid (AA2G) is a stable glycoside, but its conversion to bioavailable ascorbic acid (AsA) in humans remains unknown. The aim of this study was to clarify that AA2G is hydrolyzed by human intestinal maltase and AA2G by oral ingestion is physiologically utilized the same as AsA in human subjects. Methods The hydrolyzing activities to AA2G by human or rat intestinal homogenates were measured by high-performance liquid chromatography. In a human experiment eight healthy female subjects (23.5 ± 0.5 y old, body mass index 20.1 ± 0.7 kg/m 2) ingested 3.84 g of AA2G (equivalent to 2 g of AsA) and 2 g of AsA. Blood was collected 0, 1, 2, 3, and 4 h after ingestion. The concentrations of serum AsA were compared with those of rats administered 76.8 mg of AA2G (equivalent to 40 mg of AsA). Results AA2G was hydrolyzed by maltase using human intestinal homogenate the same as that of rat. When AA2G was orally administered to human subjects, the changed value of the serum concentration of AsA was 1.6 mg/100 mL from baseline at 2 h and then maintained until 4 h after administration. These concentrations were not significantly different from those after ingestion of AsA. In the case of rat, the AsA concentrations in serum were linearly increased to 1.7 mg/100 mL until 3 h after administration. Conclusion AA2G is hydrolyzed by intestinal maltase and acts as AsA in humans. The present results will contribute to the development of functional food with health claims to supply AsA.

Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir

The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine- l-valine-acyclovir (LLACV), l-valine- d-valine-acyclovir (LDACV), d-valine- l-valine-acyclovir (DLACV) and d-valine- d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC–MS/MS and NMR spectroscopy. Uptake and transport of [ 3H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV > LLACV > DDACV > DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB).

Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: Design, preparation, and biological evaluation

Peptide oral delivery is still a significant challenge because of two major impediments, low absorption efficiency and instability in gastrointestinal tract. The aim of this study was to design, prepare, and evaluate an intestine enzyme-resistant, superior intestine absorptive, and biologically preserved glucagon-like peptide-1 (GLP-1) derivative using the site-specific modification of biotin, which can take advantage of the carrier-mediated active intestine transport. Two series of site-specific Lys 34- and Lys 26,34–biotin–GLP-1 derivatives were prepared, and their intestine membrane permeabilities, proteolytic stabilities against the intestine enzymes, and bioactivities were then evaluated. Especially, Lys 26,34–biotin–GLP-1 was found to have the most promising scores: (i) it displayed a 5.6-fold higher Caco-2 cell monolayer permeability than GLP-1; (ii) it showed an 8.5- and 3.5-fold longer half-life than GLP-1 in rat intestine fluid and homogenate, respectively; and interestingly (iii) it had a well-preserved insulinotropic activity (94.5% vs. GLP-1) in the rat islets. Finally, Lys 26,34–biotin–GLP-1 showed a 9-fold higher oral hypoglycemic efficacy (25.3%) than native GLP-1 (2.7%) ( P < 0.005) after direct peroral administration into type 2 diabetic db/db mice. This study highlights the oral hypoglycemic potential of site-specific Lys 26,34-biotinylated GLP-1, and this orally available analogue would find a role in the treatment of type 2 diabetes.

Effect of enalapril on the in vitro and in vivo peptidyl cleavage of a potent VLA-4 antagonist

BIO1211 is a small peptidyl potent antagonist of the activated form of α4β1 integrin. The effect of enalapril on the in vitro and in vivo cleavage of BIO1211 was investigated. In heparinized blood, plasma and rat liver, lung and intestinal homogenates, BIO1211 was converted rapidly to BIO1588 by hydrolytic cleavage of the terminal dipeptide moiety. This cleavage could be inhibited by EDTA and the ACE inhibitor, enalaprilat, the de-esterified acid derivative of enalapril. Enalaprilat inhibited the hydrolysis of BIO1211 in a concentration-dependent manner with IC50 values of 2 nM in human and sheep plasma and 10 nM in rat plasma. In rat lung homogenate supernatant, the maximum inhibition of the conversion of BIO1211 to BIO1588 was ∼80% at 1 µM with no further effect up to 100 µM of enalaprilat. Following a concomitant IV administration of enalapril and BIO1211 at 3 mg/kg each, the AUC and the half-life values of BIO1211 increased 18- and 10-fold, respectively. The AUC of BIO1588 decreased ∼2-fold with no change in its plasma half-life. When rats were dosed intravenously with enalapril followed by an intratracheal dose of BIO1211, there was ∼2.5-fold decrease in the AUC of BIO1588 and a 2.4-fold increase in its plasma half-life.

Identification of a Novel Intestinal First Pass Metabolic Pathway: NQO1 Mediated Quinone Reduction and Subsequent Glucuronidation

Quinones represent a very important class of compounds found in nature and for the chemically synthesized drugs. The present study was designed to elucidate the intestinal first pass metabolic pathways in vivo and in vitro, of tanshinone IIA (TS), a derivative of phenanthrene-quinone isolated from Salvia miltiorrhiza. Five metabolites, proposed to be TS catechol glucuronides (two position isomers), dehydrotanshinone IIA and its two catechol glucuronides, were identified from the rat intestinal homogenates after oral administration of TS. TS metabolism was further conducted in the subcellular system including cytosol, microsomes, mitochondrial and S9 under both phase I and phase II metabolic conditions. TS underwent negligible metabolism in all of the subcellular systems under phase I metabolic condition using NADPH as the cofactor. However, significant and substantial metabolic elimination of TS was observed in the cytosol and S9 fractions, while not in the microsomes fractions, when both NADPH and UDPGA were added. Two TS catechol glucuronides were identified from such an in vitro metabolic medium. Dicoumarol, a specific inhibitor of the NAD(P)H dependent quinone oxidoreductase (NQO1), significantly inhibited the metabolic elimination of TS in a noncompetitive way, suggesting that NQO1 was responsible for the quinone reduction of TS to form the catechol intermediate. The catechol intermediate failed to be detected directly was proved to be highly unstable and autoxidized back to TS accompanied with hydrogen peroxide generation. Dicoumarol exhibited a significant inhibitory effect on the hydrogen peroxide generation, further supporting that the reduction of TS was catalyzed by NQO1. The absolute bioavailability of TS was significantly enhanced by oral dicoumarol pretreatment. In conclusion, a novel intestinal metabolic pathway for quinones, NQO1 mediated reduction and subsequent glucuronidation, was determined using TS as a model compound. This study should be helpful for the general understanding of quinones absorption and intestinal first pass metabolism.

P0064 PP STRUCTURAL AND FUNCTIONAL RELATIONSHIP BETWEEN ZONULIN AND THE MUCOSAL MAST CELLS-DERIVED MAST CELL PROTEASE II

Introduction: Zonulin is a molecule involved in the regulation of intestinal tight junctions both in health and disease. While zonulin mechanism of action has been partially defined, its biochemical characteristics are still largely unknown. Aim: To purify and structurally characterize the zonulin protein. Methods: Rat small intestinal tissues were analyzed by a combination of gel filtration chromatography and zonulin sandwich ELISA. Rat intestine homogenates were loaded on a sephacryl column and fractions collected and assayed for their zonulin content. Each fraction was also resolved by SDS-PAGE, transferred, and immunoblotted with zonulin-immunoreactive, anti-Zot antibodies. Both wild type and WBB6/F1-W/Wv mice intestinal tissues were tested in the microsnapwell assay and trans-epithelial electrical resistance (TEER) and zonulin release from stimulated tissues determined. Results: Of the 6 fractions (F1-F6) collected and tested with zonulin ELISA, F5 contained the highest zonulin concentration. Western analysis revealed two major bands that migrated with approximate apparent Mr of 24,000 and 23,000 in the zonulin-positive fraction, F5, while the zonulin-negative fractions, F1–4, and 6, each revealed only one immunoreactive band (~24 kDa). Therefore, the ~23 kDa band from F5 was excised from a Comassie blue-stained gel and subjected to Matrix Assisted Laser Desorption Ionization mass spectrometry. Search using the Profound search engine for protein matches revealed a high similarity of this protein (estimate Z score 1.58) with the rat mast cell protease (MCP)-II. To establish whether zonulin and MCP-II are the same moiety, microsnapwell experiments were conducted in WBB6/F1-W/Wv mice characterized by pleiotropic defects in mucosal mast cells and, therefore, lack MCP-II. Tissues mounted in the microsnapwell system and exposed at increasing time intervals (up to 3 h) to zonulin-releasing stimuli showed a TEER decrease (D TEER= −170±15.8 Omhs/cm2 vs -43±11 in untreated tissues, p<0.001) and a parallel increase in zonulin release (10.0±0.8 ng/mg protein vs 0.2±0.7 in untreated tissues, p<0001) similar to that observed in stimulated wild type animals (−120±20 and 15.1±3.1, respectively). Conclusion: Zonulin is structurally and functionally similar, but not identical, to MCP-II and may represent one of the possible intestinal luminal PAR-2 activators involved in the pathophysiological regulation of intercellular tight junctions.

Quercetin-4‘-glucoside Is More Potent than Quercetin-3-glucoside in Protection of Rat Intestinal Mucosa Homogenates against Iron Ion-Induced Lipid Peroxidation

Quercetin is a typical antioxidative flavonoid found in vegetables, which is more commonly present as its glucosides, quercetin-3-glucoside (Q3G) and quercetin-4'-glucoside (Q4'G). The main aim of this study was to estimate the antioxidant activity of Q3G and Q4'G on iron ion-driven lipid peroxidation of the gastrointestinal mucosa. Q4'G markedly suppressed the lipid peroxidation when rat gastrointestinal mucosa homogenates were incubated with Fe(NO3)3 and ascorbic acid. Its effectiveness was greater as compared to that of Q3G and comparable to that of quercetin aglycone. Furthermore, Q4'G yielded higher amounts of quercetin aglycone than Q3G on incubation with the homogenates. However, Q4'G showed a lower chelating activity in comparison to Q3G. These results indicate that Q4'G, even though it has a low chelating activity, because of its efficient conversion to antioxidative aglycone on exposure to the mucosa, can act as a powerful antioxidant on iron ion driven lipid peroxidation in the intestinal mucosa. Thus, vegetables rich in Q4'G, such as onion, are likely to serve as favorable antioxidant sources for suppressing iron-induced oxidative stress in the intestinal tract.

Isolation and characterization of metabolites of centpropazine in rat liver, intestine, and red blood cell homogenates

The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague‐Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high‐performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37°C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2067–2075, 2002