Jejunal Perfusion Research Articles

Intestinal absorption pathways of lisinopril: Mechanistic investigations.

Lisinopril is an antihypertensive drug with poor intestinal permeability. Enhancement of intestinal absorption depends on a clear understanding of the permeation pathways and absorption mechanisms. Unfortunately, these are not fully elucidated for lisinopril. Accordingly, the aim was to determine lisinopril permeation pathways and obstacles limiting membrane transport with subsequent nomination of appropriate permeation enhancers. This employed an in situ rabbit intestinal perfusion technique, which revealed site-dependent absorptive clearance (PeA/L) from a lisinopril simple solution (5μg/ml), with paracellular absorption playing a role. Regional drug permeability ranked as colon>duodenum>jejunum>ileum opposing intestinal expression rank of P-glycoprotein (P-gp) efflux transporters. Duodenal and jejunal perfusion of a higher lisinopril concentration (50μg/ml) reflected saturable absorption, suggesting carrier-mediated transport. The effect of piperine and verapamil as P-gp inhibitors on intestinal absorption of lisinopril was investigated. Coperfusion with either piperine or verapamil significantly enhanced lisinopril absorption, with enhancement being dominant in the ileum segment. This supported the contribution of P-gp transporters to poor lisinopril permeability. On the other hand, coperfusion of lisinopril with zinc acetate dihydrate significantly multiplied lisinopril PeA/L by 2.3- and 6.6-fold in duodenum and ileum segments, respectively, through magnifying intestinal water flux. The study explored the barriers limiting lisinopril intestinal absorption. Moreover, the study exposed clinically relevant lisinopril interactions with common coadministered cargos that should be considered for an appropriate lisinopril regimen. However, this requires further in vivo verification.

Sodium Caprate Enhances Transcellular LPS Transport via Clathrin‐Mediated Endocytosis in Rat Jejunum

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways. We examined the effects of medium‐chain fatty acids (MCFA) on LPS transport in rat jejunum.FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal and distal colonic mucosa‐submucosa preparations, in which short‐circuit current (Isc) and tissue electrical resistance (TER) were measured. Serosal appearance of FITC‐LPS was measured with or without luminal application of sodium caprate (C10, 10 mM). FITC‐dextran 4kDa (FD4) m‐to‐s movement was also separately measured.Luminal application of caprate increased FITC‐LPS m‐to‐s transport without any changes in FD4 m‐to‐s movement and TER in the jejunum, whereas caprate remarkably increased FITC‐LPS transport with increased FD4 movement and decreased TER in the distal colon, suggesting that caprate enhances transcellular LPS transport in the jejunum, but increases paracellular permeability in the distal colon. In the jejunum, C10‐mediated FITC‐LPS m‐to‐s transport was inhibited by pretreatment with luminal Pitstop2 (30 μM), a selective inhibitor of clathrin‐mediated endocytosis (CME), whereas the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM), the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 1 mM), or the dynamin inhibitor Dynasore (50 μM) had no effect. In contrast, luminal OA (30 mM) with taurocholate (TCA, 0.1 mM) also increased FITC‐LPS transport with SSO, MβCD and Dynasore‐sensitive, but Pitstop2‐insensitive manner. In vivo, jejunal luminal perfusion of C10 with FITC‐LPS increased portal venous FITC‐LPS appearance 15 min after perfusion, higher than by OA/TCA, which was then inhibited by co‐perfusion of Pitstop2.These results suggest that luminal MCFA and LCFA differentially induce transcellular LPS transport in the jejunum; MCFA via CME, but LCFA via the CD36/lipid raft/caveolin pathway. Dietary fatty acids may impact LPS‐associated diseases, including metabolic disease and multiple organ failure.Support or Funding InformationVA Merit Review and Shire Pharmaceuticals

Molecular pharmacokinetic mechanism of the drug-drug interaction between genistein and repaglinide mediated by P-gp

This study was devised to investigate if P-glycoprotein (P-gp) mediated the drug–drug interaction (DDI) between genistein and repaglinide. When genistein was added, the plasma concentrations of repaglinide in rats were increased. The maximum plasma concentration (Cmax) of repaglinide increased from 70.80 ± 7.98 ng/mL to 124.71 ± 9.02 ng/mL and the area under the plasma concentration-time curve (AUC) increased from 134.89 ± 13.65 μg·h/L to 245.95 ± 7.24 μg·h/L. Intestinal absorption of repaglinide was markedly enhanced by genistein or P-gp inhibitor verapamil (Ver), both in situ rat jejunal perfusion studies and in vitro transport assays using everted rat intestinal sac preparations. Furthermore, the accumulation of repaglinide in both Caco-2 cells and IEC-6 cells also increased significantly in the presence of genistein and Ver. The transepithelial transport rate of repaglinide from basolateral-to-apical in MDR1-MDCK cells was 3.6-fold higher than the apical-to-basolateral rate with a net efflux ratio of 1.92 compared with mock-MDCK cells, which was significantly decreased following co-administration with genistein or Ver. In an intracellular accumulation experiment using Rhodamine 123 as a P-gp substrate, genistein significantly increased the intracellular fluorescence of Rhodamine 123. These results indicated that genistein had an inhibitory effect on the efflux function of P-gp. Through molecular docking assays we further found that genistein could bind to the nucleotide-binding domains (NBD) in the cytoplasm of P-gp, thus affecting the functions of P-gp. In conclusion, genistein inhibited the efflux of repaglinide mediated by P-gp in rats and in vitro. The findings suggested that the DDI between genistein and repaglinide is mediated by P-gp, and a dosage adjustment may be needed when they are co-administered in a clinical setting.

Intraluminal fluid infusion in a rat jejunum ischemia/reperfusion model is associated with improved tissue perfusion and less mucosal damage

This study used an experimental model mimicking early postoperative enteral feeding after the transfer of free jejunal flap and tested the hypothesis that jejunal infusion with dextrose or saline is associated with improved tissue perfusion and/or less mucosal damage after ischemia/reperfusion (IR) injury. Thirty-five male Sprague Dawley rats were randomly divided into five groups: sham group (no IR and no intraluminal infusion); IR control group (IR but not intraluminal infusion); IR plus intraluminal 0.9% NaCl infusion or 5% dextrose or 10% dextrose infusion groups. A jejunal segment of each rat was isolated. The animals had jejunal ischemia for 40min, reperfusion, and intestinal infusion on the basis of their allocation. Jejunal tissue perfusion was measured with laser Doppler flowmetry at one hour and two hours after reperfusion, after which the animals were sacrificed and tissue samples were obtained for the scoring of histological damage at superficial and cryptic epithelium, villus structure, and inflammatory cell infiltration and tissue nitric oxide (NO), interleukin (IL)-1, IL-6, and matrix metalloproteinase-1 (MMP) level measurements. At 1h of reperfusion, IR plus 5% dextrose and 10% dextrose groups both had significantly higher perfusion rates than the IR control group (384.8±26.7 and 462.4±44.7 versus 270.3±34.2 PU, respectively, p<0.05 for both). These differences were maintained at 2h of reperfusion (p<0.05 for both). Saline infusion, however, resulted in improved tissue perfusion only at the early phase of reperfusion. Intraluminal infusion with dextrose solution, either 5% or 10%, was associated with higher tissue NO, IL-1, and IL-6 levels than that in the sham group (p<0.05 for all). In addition, intraluminal infusion of any fluid resulted in less severe histological damage (8.1±0.9 versus 5.8±1.0, 5.4±0.9, and 5.2±1.9, for IR plus saline, 5% dextrose and 10% dextrose groups, respectively, p<0.05 for all). Intraluminal infusion of fluids, particularly dextrose solutions, may be protective against IR injury as demonstrated by improved tissue perfusion and less histological damage. In addition, increases in tissue NO, IL-1, and IL-6 levels in association with dextrose infusion may be explained by the activation of pro-inflammatory and anti-inflammatory protective pathways. These support early enteral feeding after free jejunum flap transfers; however, further studies are warranted.

Dobutamine Versus Vasopressin After Mesenteric Ischemia

BackgroundGastrointestinal blood flow may be compromised during and after vasopressor support. Endothelin expression may lead to microcirculatory dysfunction. The aim of this study was to analyze the effect of vasopressin and dobutamine after mesenteric ischemia on the gastrointestinal mucosal microcirculation, endothelin expression, and morphologic injury. Materials and methodsPigs were studied in four groups (six pigs in each group): 1, sham; 2-4 ischemia (1 h superior mesenteric artery occlusion with 30 min reperfusion and 30 min of vehicle [2], dobutamine [3], or vasopressin [4] administration, followed by 30-min break and thiopental-induced hypotension [3, 4]). Blood flow of the gastric, jejunal, and rectosigmoidal mucosa was measured. At the end of the experiment, the mucosal expression of endothelin-1 (ET-1) and its receptor subtypes A (ETA) and B were determined by polymerase chain reaction. Mucosal injury, apoptotic cell death, and leukocytic infiltration were determined by histology and immunohistochemical analysis of cleaved caspase-3 and myeloperoxidase. ResultsMesenteric ischemia increased jejunal mucosal ET-1 gene expression, arterial ET-1, intestinal fatty acid binding protein, and jejunal mucosal injury compared with sham. Dobutamine increased arteriovenous shunting at the cost of the jejunal mucosal blood perfusion. This was associated with an increased expression of ET-1 and ETA and mucosal leukocytic infiltration. In contrast, vasopressin increased postischemic capillary density and tissue blood flow. This was associated with a lower ET-1 gene expression. Vasopressin did not induce jejunal mucosal leukocytic infiltration. ConclusionsVasopressin reduces mesenteric ischemia-associated alterations of the microcirculation and tissue integrity, whereas dobutamine does not.

Anion and fluid secretory response of the murine jejunum to the heat‐stable Eschericia coli enterotoxin (STa) analogue linaclotide: Involvement of NHE3, Slc26a6, CFTR, proteinkinase GII (cGKII) and NHERF1‐3.

The different PDZ‐adaptors of the NHERF family differentially associate with intestinal electrolyte transporters and modulate their activity in an agonist‐specific fashion. STa induces secretory diarrhea via binding to the guanylate cyclase C, eliciting cGMP production, resulting both in a stimulation of CFTR‐mediated anion secretion and an inhibition of NHE3‐mediated fluid absorption, and these effects are modulated by different NHERF adaptor proteins in cell lines.AimWe studied the STa‐mediated short circuit current response in isolated jejunal mucosa as well as the STa action on in vivo fluid transport in anethstized mice, deficiet for NHE3, Slc26a6, CFTR and NHERF1‐3, to study how well in vitro and in vivo modulation of fluid transport by the above named transporters and NHERF proteins may correlate.Results10−7M of the STa analogue linacolide was applied to the luminal bath of chambered isolated jejunal mucosa of CFTR‐, NHE3‐, Slc26a6‐, cGKII, and NHERF1–3 deficient and WT mice, and short circuit current (Isc) response was measured. The same concentration of linaclotide was added to the jejunal perfusate during single pass perfusion of a jejunal segment in isoflurane‐anesthetized, acid/base‐ and blood pressure controlled KO and WT mice, and net fluid balance before and after linaclotide application assessed gravimetrically. The peak Isc response to linaclotide in chambered jejunal mucosa was absent in CFTR KO, reduced by 88% in cGKII KO, by 40% in NHERF1 KO, and not significantly in NHERF2, NHERF (PDZK1) and SLC26a6 KO isolated jejunal mucosa. In contrast, the magnitude of change from jejunal fluid absorption to secretion in vivo was reduced by 51% in cGKII KO, 49% in CFTR KO, 45% in NHERF1 KO, by 34% in NHERF2 KO, and not significantly in NHERF3 KO and Slc26a6 KO anesthetized mice. NHE3 deletion (48%), pharmacological NHE3 inhibition (50%) or Slc26a6 deletion (44%) also reduced the STa‐elicited change in fluid movement.ConclusionsThe results demonstrate that Isc measurements in isolated small intestinal mucosa are excellent models to selectively study agonist‐mediated CFTR activation, but do not accurately predict in vivo “secretory responses”, i.e. a relative decrease in the basal fluid absorptive flux. Activating the GCC receptor elicited a decrease in fluid absorption in CFTR KO mice, making this substance an interesting drug to improve gut fluidity in CF patients. Apart from direct interference with guanylate cyclase C activation, potential pharmacological strategies to prevent STa‐mediated fluid loss may be targeting both NHERF1 and NHERF2.Support or Funding InformationFunded by SFB621/C1, DFG grants SE460/13‐4, 19‐1 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal Transport of Model Compounds and the Mucosal Barrier Marker 51Cr-EDTA.

There is a renewed interest from the pharmaceutical field to develop oral formulations of compounds, such as peptides, oligonucleotides, and polar drugs. However, these often suffer from insufficient absorption across the intestinal mucosal barrier. One approach to circumvent this problem is the use of absorption modifying excipient(s) (AME). This study determined the absorption enhancing effect of four AMEs (sodium dodecyl sulfate, caprate, chitosan, N-acetylcysteine) on five model compounds in a rat jejunal perfusion model. The aim was to correlate the model compound absorption to the blood-to-lumen clearance of the mucosal marker for barrier integrity, 51Cr-EDTA. Sodium dodecyl sulfate and chitosan increased the absorption of the low permeation compounds but had no effect on the high permeation compound, ketoprofen. Caprate and N-acetylcysteine did not affect the absorption of any of the model compounds. The increase in absorption of the model compounds was highly correlated to an increased blood-to-lumen clearance of 51Cr-EDTA, independent of the AME. Thus, 51Cr-EDTA could be used as a general, sensitive, and validated marker molecule for absorption enhancement when developing novel formulations.

An investigation into the relationship between small intestinal fluid secretion and systemic arterial blood pressure in the anesthetized rat.

The effects of changes in the steady level of diastolic blood pressure on fluid flux across the jejunum has been investigated in the anesthetized rat during perfusion with a nutrient-free and Na+-free solution. Diastolic blood pressure was manipulated by intravenous infusions, during the jejunal perfusions, of vasodilators (vasoactive intestinal polypeptide, acetyl-β-methylcholine, and phentolamine) and a vasoconstrictor (arginine vasopressin), each of which acts through a different cellular mechanism. The outcome was that fluid flux was related by a parabolic relationship with diastolic blood pressure in which net secretion occurred over the range 40–100 mmHg, whereas net absorption was recorded at diastolic pressures exceeding 100 mmHg and below 40 mmHg. Against a background of normal absorption promoted by perfusion with 145 mmol L−1 Na+/5 mmol L−1 glucose solution, reductions in diastolic blood pressure markedly reduced the mean rate of fluid absorption by 58% overall, whereas the rate of glucose absorption remained unchanged. Our results were explained on the basis that vasodilatation led to increased capillary pressure and then to net filtration of fluid from the mesenteric capillary bed. Experiments in which Escherichia coli heat-stable toxin was added to the jejunal perfusate confirmed the absence of a secretory response, which was consistent with the absence of effect of the toxin on diastolic blood pressure.

Evaluation of Frog as an Animal Model to Study the Fraction of Oral Dose Absorbed in Humans

To evaluate single pass intestinal perfusion method in frog for the prediction of absorption in man and also to evaluate the predictability of this model to classify drugs correctly under biopharmaceutical classification system (BCS). Single Pass Intestinal Perfusion (SPIP) studies were performed in Frogs of the species Rana tigrina using established method for rats with some modifications. Permeability was determined for each selected drug in 6 frogs and the results were presented as mean ± SD. Effective permeability coefficient (Peff) of 12 drugs was calculated. Permeability assessment of some carrier transported drugs which are also substrates for CYP enzymes was also done. Correlation of frog Peff with fraction of dose absorbed (FA) in man was done. Rank order comparison of permeabilities obtained in this study with two other absorption models (human jejunal perfusion, rat intestinal perfusion) was made. The calculated Peff values correlated well with reported Peff values of probe drugs. Good correlation was found between Peff of humans and frogs (r2=0.942). The permeability classification based on frog intestinal permeability of drugs is in high agreement with previously introduced classification, and all the compounds are placed in correct categories as they belong to. The results of this study provided a basis that, as a permeability model in the early drug discovery stage, the single pass intestinal perfusion frog model can be used for the biopharmaceutics classification system. This model may represent a valuable alternative to the low speed and high cost of conventional animal models (typically rodents) for the assessment of intestinal permeability.

Quantitative assessment of the free jejunal graft perfusion

BackgroundReconstruction with free jejunal graft (FJG) is often performed for patients with hypopharyngeal or cervical esophageal cancer. During reconstruction with an FJG after pharyngoesophagectomy, it is critical to intraoperatively detect venous anastomotic failure and subsequent venous malperfusion to avoid postoperative FJG necrosis. This study introduces a novel method for assessing blood perfusion in FJGs by using indocyanine green (ICG) fluorescence angiography. MethodsWe used ICG fluorescence angiography to quantitatively assess FJG blood perfusion in archived fluorescence video files from 26 patients who had undergone FJG transfer. A software program “ROIs”, was used to create a time-fluorescence intensity curve. We retrospectively measured the maximum fluorescence intensity at the terminal ileum and the duration (T1/2max) between when the intensity began rising and when it reached half of the maximum. ResultsAmong the 26 patients, 5 patients suffered venous anastomotic failure. In three of these cases, anastomosis was corrected intraoperatively; the other two patients underwent a second FJG transfer. Retrospective assessment showed that the mean T1/2max at the FJG serosae was significantly longer in these five patients than that in FJGs with good blood perfusion. Our analysis revealed that a T1/2max >9.6 s may be a good indicator of FJG venous malperfusion. ConclusionsQuantitative analysis of ICG fluorescence angiography proved useful for detecting venous anastomotic failure of FJG, and may help to reduce vascular problems in FJG reconstruction.

Effect of β-Cyclodextrin Derivatives on the Diosgenin Absorption in Caco-2 Cell Monolayer and Rats

Orally administrated diosgenin, a steroidal saponin found in the roots of Dioscorea villosa, improves reduced skin thickness in ovariectomized mice, and plays an important role in the treatment of hyperlipidemia. Diosgenin has been noticed as an active element in cosmeceutical and dietary supplements. We have already elucidated that the absolute oral bioavailability of diosgenin is very low; however, a high skin distribution of diosgenin was also observed. The aim of the present study was to examine and compare the effects of β-cyclodextrin (β-CD) and 3 kinds of its derivatives such as hydroxypropyl β-CD on the diosgenin permeability using a Caco-2 model and rat jejunal perfusion. These derivatives of β-CD greatly improved the low solubility of diosgenin. No significant increase was observed in the lactate dehydrogenase leakage from Caco-2 cell, while a slight decrease was found on the transepithelial electrical resistance by diosgenin and β-CD derivatives. However, β-CD derivatives, especially hydroxyethyl β-CD and hydroxypropyl β-CD, markedly enhanced diosgenin permeability across the Caco-2 monolayer and rat jejunum. The bioavailability of diosgenin in the presence of β-CD derivatives were about 4 to 11 fold higher than diosgenin suspension. The mechanisms of these enhancement effects may be due to improvements in solubility and tight junction opening.

Quantitation of small intestinal permeability during normal human drug absorption.

BackgroundUnderstanding the quantitative relationship between a drug’s physical chemical properties and its rate of intestinal absorption (QSAR) is critical for selecting candidate drugs. Because of limited experimental human small intestinal permeability data, approximate surrogates such as the fraction absorbed or Caco-2 permeability are used, both of which have limitations.MethodsGiven the blood concentration following an oral and intravenous dose, the time course of intestinal absorption in humans was determined by deconvolution and related to the intestinal permeability by the use of a new 3 parameter model function (“Averaged Model” (AM)). The theoretical validity of this AM model was evaluated by comparing it to the standard diffusion-convection model (DC). This analysis was applied to 90 drugs using previously published data. Only drugs that were administered in oral solution form to fasting subjects were considered so that the rate of gastric emptying was approximately known. All the calculations are carried out using the freely available routine PKQuest Java (http://www.pkquest.com) which has an easy to use, simple interface.ResultsTheoretically, the AM permeability provides an accurate estimate of the intestinal DC permeability for solutes whose absorption ranges from 1% to 99%. The experimental human AM permeabilities determined by deconvolution are similar to those determined by direct human jejunal perfusion. The small intestinal pH varies with position and the results are interpreted in terms of the pH dependent octanol partition. The permeability versus partition relations are presented separately for the uncharged, basic, acidic and charged solutes. The small uncharged solutes caffeine, acetaminophen and antipyrine have very high permeabilities (about 20 x 10-4 cm/sec) corresponding to an unstirred layer of only 45 μm. The weak acid aspirin also has a large AM permeability despite its low octanol partition at pH 7.4, suggesting that it is nearly completely absorbed in the first part of the intestine where the pH is about 5.4.ConclusionsThe AM deconvolution method provides an accurate estimate of the human intestinal permeability. The results for these 90 drugs should provide a useful benchmark for evaluating QSAR models.

Lipid Absorption Triggers Drug Supersaturation at the Intestinal Unstirred Water Layer and Promotes Drug Absorption from Mixed Micelles

To evaluate the potential for the acidic intestinal unstirred water layer (UWL) to induce drug supersaturation and enhance drug absorption from intestinal mixed micelles, via the promotion of fatty acid absorption. Using a single-pass rat jejunal perfusion model, the absorptive-flux of cinnarizine and (3)H-oleic acid from oleic acid-containing intestinal mixed micelles was assessed under normal acidic microclimate conditions and conditions where the acidic microclimate was attenuated via the co-administration of amiloride. As a control, the absorptive-flux of cinnarizine from micelles of Brij® 97 (a non-ionizable, non-absorbable surfactant) was assessed in the absence and presence of amiloride. Cinnarizine solubility was evaluated under conditions of decreasing pH and decreasing micellar lipid content to assess likely changes in solubilization and thermodynamic activity during micellar passage across the UWL. In the presence of amiloride, the absorptive-flux of cinnarizine and (3)H-oleic acid from mixed micelles decreased 6.5-fold and 3.0-fold, respectively. In contrast, the absorptive-flux of cinnarizine from Brij® 97 micelles remained unchanged by amiloride, and was significantly lower than from the long-chain micelles. Cinnarizine solubility in long-chain micelles decreased under conditions where pH and micellar lipid content decreased simultaneously. The acidic microclimate of the intestinal UWL promotes drug absorption from intestinal mixed micelles via the promotion of fatty acid absorption which subsequently stimulates drug supersaturation. The observations suggest that formulations (or food) containing absorbable lipids (or their digestive precursors) may outperform formulations that lack absorbable components since the latter do not benefit from lipid absorption-induced drug supersaturation.

The Potential for Drug Supersaturation during Intestinal Processing of Lipid-Based Formulations May Be Enhanced for Basic Drugs

Co-administration of poorly water-soluble drugs (PWSD) with dietary or formulation lipids stimulates the formation of lipid colloidal phases such as vesicular and micellar species, and significantly expands the drug solubilization capacity of the small intestine. The mechanism of drug absorption from the solubilizing phases, however, has not been fully elucidated. Recently, we observed that drug supersaturation may be triggered during endogenous processing of lipid colloidal phases containing medium-chain lipid digestion products and that this may represent a mechanism to reverse the reduction in thermodynamic activity inherent in drug solubilization and thereby enhance absorption. The current studies expand these preliminary findings and explore the supersaturation tendency of five model PWSD during endogenous processing of intestinal colloidal phases containing long-chain lipid digestion products. Bile-lipid concentration ratios progressively increase during colloid transit through the gastrointestinal tract due to biliary dispersion of lipid digestion products and lipid absorption. The supersaturation potential was therefore evaluated under conditions of increasing bile and decreasing lipid concentrations and was found to be greater for the basic drugs cinnarizine (CIN) and halofantrine (HF), than the neutral drugs fenofibrate (FF) and danazol (DAN), and acidic drug meclofenamic acid (MFA). Assessment of intestinal absorptive flux using rat jejunal perfusion experiments subsequently showed that the absorption enhancement afforded by bile dilution of lipid colloidal phases was greater for CIN than DAN. The results confirm that bile plays a significantly greater role in the absorption of CIN (a weak base) from long-chain intestinal colloids when compared to DAN (an uncharged molecule) and that the difference reflects a greater propensity for supersaturation as intestinal colloids are dispersed and diluted by bile. The data suggest that coadministered digestible lipids may be particularly suited to enhance the absorption of poorly water-soluble weak bases.

Increasing Oral Absorption of Polar Neuraminidase Inhibitors: A Prodrug Transporter Approach Applied to Oseltamivir Analogue

Poor oral absorption is one of the limiting factors in utilizing the full potential of polar antiviral agents. The neuraminidase target site requires a polar chemical structure for high affinity binding, thus limiting oral efficacy of many high affinity ligands. The aim of this study was to overcome this poor oral absorption barrier, utilizing prodrug to target the apical brush border peptide transporter 1 (PEPT1). Guanidine oseltamivir carboxylate (GOCarb) is a highly active polar antiviral agent with insufficient oral bioavailability (4%) to be an effective therapeutic agent. In this report we utilize a carrier-mediated targeted prodrug approach to improve the oral absorption of GOCarb. Acyloxy(alkyl) ester based amino acid linked prodrugs were synthesized and evaluated as potential substrates of mucosal transporters, e.g., PEPT1. Prodrugs were also evaluated for their chemical and enzymatic stability. PEPT1 transport studies included [(3)H]Gly-Sar uptake inhibition in Caco-2 cells and cellular uptake experiments using HeLa cells overexpressing PEPT1. The intestinal membrane permeabilities of the selected prodrugs and the parent drug were then evaluated for epithelial cell transport across Caco-2 monolayers, and in the in situ rat intestinal jejunal perfusion model. Prodrugs exhibited a pH dependent stability with higher stability at acidic pHs. Significant inhibition of uptake (IC(50) <1 mM) was observed for l-valyl and l-isoleucyl amino acid prodrugs in competition experiments with [(3)H]Gly-Sar, indicating a 3-6 times higher affinity for PEPT1 compared to valacyclovir, a well-known PEPT1 substrate and >30-fold increase in affinity compared to GOCarb. The l-valyl prodrug exhibited significant enhancement of uptake in PEPT1/HeLa cells and compared favorably with the well-absorbed valacyclovir. Transepithelial permeability across Caco-2 monolayers showed that these amino acid prodrugs have a 2-5-fold increase in permeability as compared to the parent drug and showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.

Double-balloon jejunal perfusion to compare absorption of vitamin E and vitamin E acetate in healthy volunteers under maldigestion conditions

The vitamin E derivative, α-tocopheryl acetate, is often included in formulations used in enteral nutrition. In this respect, we compared α-tocopherol and α-tocopheryl acetate absorption under 'maldigestion' conditions, such as occurring during enteral tube feeding, using differentially labeled RRR-[5,7-methyl-((2)H(6))]-α-tocopherol and RRR-[5-methyl-(2)H(3)]-α-tocopheryl acetate allowing direct comparison between free and esterified forms. The two derivatives were given together in a single dose to six volunteers directly into the jejunum using a double-balloon perfusion system. Perfusion lasted for 1 h, and the collected blood and effluent samples were analyzed by liquid chromatography-mass spectrometry. In the isolated 20-cm length of exposed jejunum, on average ~ 6% of the two vitamin E forms were absorbed >1 h based on subtraction of effluent from influent. There was substantial difference in the absolute absorbed quantity between individuals, but no significant differences were observed in the absorption between the two labeled forms as assessed in the plasma. (2)H(3)-α-tocopherol was not present in the influent, but appeared in the effluent, indicating that the acetylated form of vitamin E is cleaved by brush border enzymes in the small intestine. This study shows that even in the absence of digestive enzymes and bile salts, the appropriately solubilized acetylated form of α-tocopherol exhibits the same bioavailability as free α-tocopherol. This suggests that both forms can be absorbed equally under maldigestion conditions such as present clinically during enteral tube feeding.

A Novel Local Recycling Mechanism That Enhances Enteric Bioavailability of Flavonoids and Prolongs Their Residence Time in the Gut

Recycling in the gastrointestinal tract is important for endogenous substances such as bile acids and for xenobiotics such as flavonoids. Although both enterohepatic and enteric recycling mechanisms are well recognized, no one has discussed the third recycling mechanism for glucuronides: local recycling. The intestinal absorption and metabolism of wogonin and wogonoside (wogonin-7-glucuronide) was characterized by using a four-site perfused rat intestinal model, and hydrolysis of wogonoside was measured in various enzyme preparations. In the perfusion model, the wogonoside and wogonin were interconverted in all four perfused segments. Absorption of wogonoside and conversion to its aglycon at the upper small intestine was inhibited in the presence of a glucuronidase inhibitor (saccharolactone) but was not inhibited by lactase phlorizin hydrolase (LPH) inhibitor gluconolactone or antibiotics. Further investigation indicated that hydrolysis of wogonoside in the blank intestinal perfusate was not correlated with bacterial counts. Kinetic studies indicated that K(m) values from blank duodenal and jejunal perfusate were essentially identical to the K(m) values from intestinal S9 fraction but were much higher (>2-fold) than those from the microbial enzyme extract. Lastly, jejunal perfusate and S9 fraction share the same optimal pH, which was different from those of fecal extract. In conclusion, local recycling of wogonin and wogonoside is the first demonstrated example that this novel mechanism is functional in the upper small intestine without significant contribution from bacteria β-glucuronidase.

Effect of Intestinal First-Pass Hydrolysis on the Oral Bioavailability of an Ester Prodrug of Fexofenadine

The contribution of intestinal first-pass hydrolysis to oral bioavailability was evaluated in rats using a model prodrug of fexofenadine (FXD), which has poor oral bioavailability. The prodrug, ethyl-FXD, has high membrane permeability but the oral bioavailability of FXD derived from ethyl-FXD was only 6.2%. Ethyl-FXD was not detected in the plasma, whereas FXD was detected, indicating complete first-pass hydrolysis. In in vitro experiments, hydrolase activity for ethyl-FXD was higher in the liver and blood than that in the intestine. However, the high blood protein binding of ethyl-FXD resulted in a high hepatic availability (F(h) = 88%). The complete bioconversion of ethyl-FXD in the in vivo oral administration is difficult to explain by first-pass hydrolysis in the liver and blood. Interestingly, in an in situ rat jejunal single-pass perfusion experiment, 84% of the ethyl-FXD taken up into enterocytes was hydrolyzed. Furthermore, only one-fifth of the FXD formed in mucosa reached the mesenteric vein because of its P-glycoprotein-mediated efflux into the intestinal lumen. These findings indicate that the intestinal bioconversion of ester prodrugs to their parent drugs is a key factor in determining their oral bioavailability.

Increases in Bioavailability of Poorly Absorbed Drug by Acylcarnitine

We examined the effect of acylcarnitines on the in situ bioavailability of lucifer yellow (LY) from the loops of small and large intestines of rats. The area under the blood concentration of LY versus time curve (AUC) from the jejunum was significantly increased by the treatments of the loop with 100 µM lauroylcarnitine (LC) or 100 µM palmitoylcarnitine (PC) (fourfold and 17-fold, respectively). No marked change in the expression of claudin-4 protein was observed by the treatments. On the contrary, the expression of P-glycoprotein (P-gp) was decreased by the treatment, more greatly by PC than by LC, suggesting that increases in the bioavailability of LY by LC and PC are associated with the decreased expression of P-gp in jejunum. The increase in the bioavailability was also observed for colon by the treatment of LC, but not that of PC. LC decreased the expression of claudin-4 protein, whereas PC decreased the expression of P-gp in colon. Therefore, LC and PC appear to have different impact on the intestinal transporters depending on the site (i.e., jejunum and colon).

Accounting for the solubility–permeability interplay in oral formulation development for poor water solubility drugs: The effect of PEG-400 on carbamazepine absorption

The purpose of this paper was to study the solubility–permeability interplay in formulation development for oral administration of poor aqueous solubility drugs. The apparent solubility of the lipophilic drug carbamazepine was measured in systems containing various levels of the co-solvent PEG-400. The corresponding permeability was then measured in the PAMPA assay and in the rat jejunal perfusion model. Thermodynamic activity was maintained equivalent in all permeability studies (50% saturation). PEG-400 increased carbamazepine solubility in a concentration-dependent fashion. Decreased carbamazepine intestinal permeability with increased apparent solubility was observed in both PAMPA and rat perfusion models. Additionally, we have shown that the intestinal absorption of carbamazepine is membrane-controlled, with essentially no effective barrier function of the unstirred water layer. A mass transport analysis was employed to describe the solubility–permeability interplay. It was shown that the increased solubility in the aqueous GI milieu reduced the apparent membrane/aqueous partitioning, thereby reducing the driving force for membrane permeability. The model enabled excellent quantitative prediction of the effective permeability as a function of the solubility. In conclusion, a direct tradeoff between solubility increase and permeability decrease has been shown, which has to be accounted for when developing oral formulation for lipophilic drugs.