Efflux Ratio Cannot Assess P-Glycoprotein-Mediated Attenuation of Absorptive Transport: Asymmetric Effect of P-Glycoprotein on Absorptive and Secretory Transport Across Caco-2 Cell Monolayers

Purpose. The purpose of this work was to determine whether P-glycoprotein (P-gp) modulates absorptive and secretory transport equally across polarized epithelium (i.e., Caco-2 cell monolayers) for structurally diverse P-gp substrates, a requirement for the use of the efflux ratio to quantify P-gp-mediated attenuation of absorption across intestinal epithelium. Methods. Studies were performed in Caco-2 cell monolayers. Apparent permeability (P _app) in absorptive (P _app,AB) and secretory (P _app,BA) directions as well as efflux ratios (P _app,BA / P _app,AB) were determined for substrates as a function of concentration. Transport of these compounds (10 M) was measured under normal conditions and in the presence of the P-gp inhibitor, GW918 (1 M), to dissect the effect of P-gp on absorptive and secretory transport. Apparent biochemical constants of P-gp-mediated efflux activity were calculated for both transport directions. Results. Efflux ratios for rhodamine 123 and digoxin were comparable (approx. 10). However, transport studies in the presence of GW918 revealed that P-gp attenuated absorptive transport of digoxin by approx. 8-fold but had no effect on absorptive transport of rhodamine 123 (presumably because absorptive transport of rhodamine 123 occurs via paracellular route). The apparent K _m for P-gp-mediated efflux of digoxin was >6-fold larger in absorptive vs. secretory direction. For structurally diverse P-gp substrates (acebutolol, colchicine, digoxin, etoposide, methylprednisolone, prednisolone, quinidine, and talinolol) apparent K _m was approximately 3 to 8-fold greater in absorptive vs. secretory transport direction, whereas apparent J _max was somewhat similar in both transport directions. Conclusions. P-gp-mediated efflux activity observed during absorptive and secretory transport was asymmetric for all substrates tested. For substrates that crossed polarized epithelium via transcellular pathway in both directions, this difference appears to be caused by greater apparent K _m of P-gp-mediated efflux activity in absorptive vs. secretory direction. These results clearly suggest that use of efflux ratios could be misleading in predicting the extent to which P-gp attenuates the absorptive transport of substrates.     

Effects of Grapefruit Juice and Orange Juice on the Intestinal Efflux of P-Glycoprotein Substrates

Purpose. The aim of this study is to investigate the effects of 50% ethyl acetate extracts of grapefruit juice (GFJ) and orange juice (OJ) on the transport activity of P-glycoprotein (P-gp) in the rat small intestine. Methods.The efflux of P-gp substrates from rat everted sac in the absence or presence of verapamil, GFJ, OJ or erythromycin was measured. Rhodamine123, fexofenadine and saquinavir were used as P-gp substrates. P-gp expression levels in the rat jejunum and ileum were determined by Western blot analysis. Results. The efflux of rhodamine123 from the everted sac increased from the apex of the jejunum to the low ileum and the expression of P-gp in the ileum was 2.31-fold higher than that in the jejunum. Verapamil and the 50% GFJ and OJ extracts inhibited the efflux from the intestine of all three drugs tested. Erythromycin decreased the efflux of rhodamine123 and fexofenadine, but did not affect the efflux of saquinavir in the intestine. Conclusions. GFJ and OJ extracts inhibited the efflux of P-gp substrates from the small intestine. Therefore, they may enhance the oral bioavailability of P-gp substrates by increasing absorption in the small intestine.     

Grapefruit Juice and its Constituents Augment Colchicine Intestinal Absorption: Potential Hazardous Interaction and the Role of P-Glycoprotein

Purpose  To investigate the potential interaction between grapefruit juice (GFJ) and the oral microtubule polymerization inhibitor colchicine, a P-gp and CYP3A4 substrate. Methods  Colchicine intestinal epithelial transport was investigated across Caco-2 cell monolayers in both AP–BL and BL–AP directions, in the absence/presence of known P-gp inhibitors (verapamil and quinidine). The concentration-dependent effects of GFJ and its major constituents (6′-7′-dihydroxybergamottin, naringin and naringenin) on colchicine Caco-2 mucosal secretion were examined. The effect of GFJ on colchicine intestinal-permeability was then investigated in-situ in the rat perfusion model, in both jejunum and ileum. Results  Colchicine exhibited 20-fold higher BL–AP than AP–BL Caco-2 permeability, indicative of net mucosal secretion, which was reduced by verapamil/quinidine. Colchicine AP–BL permeability was increased and BL–AP was decreased by GFJ in a concentration-dependent manner (IC₅₀ values of 0.75% and 0.46% respectively), suggesting inhibition of efflux transport, rather than metabolizing enzyme. Similar effects obtained following pre-experiment incubation with GFJ, even though the juice was not present throughout the transepithelial study. 6′-7′-Dihydroxybergamottin, naringin and naringenin displayed concentration-dependent inhibition on colchicine BL–AP secretion (IC₅₀ values of 90, 592 and 11.6 μM respectively). Ten percent GFJ doubled colchicine rat in-situ ileal permeability, and increased 1.5-fold jejunal permeability. Conclusion  The data suggest that GFJ may augment colchicine oral bioavailability. Due to colchicine narrow therapeutic-index and severely toxic side-effects, awareness of this interaction is prudent.     

Intestinal permeability and its relevance for absorption and elimination

Human jejunal permeability (P_eff) is determined in the intestinal region with the highest expression of carrier proteins and largest surface area. Intestinal P_eff are often based on multiple parallel transport processes. Site-specific jejunal P_eff cannot reflect the permeability along the intestinal tract, but they are useful for approximating the fraction oral dose absorbed. It seems like drugs with a jejunal P_eff > 1.5 x 10^–4 cm s^–1 will be completely absorbed no matter which transport mechanism(s) are utilized. Many drugs that are significantly effluxed in vitro have a rapid and complete intestinal absorption (i.e. >85%) mediated by passive transcellular diffusion. The determined jejunal P_eff for drugs transported mainly by absorptive carriers (such as peptide and amino acid transporters) will accurately predict the fraction of the dose absorbed as a consequence of the regional expression. The data also show that: (1) the human intestinal epithelium has a large resistance towards large and hydrophilic compounds; and (2) the paracellular route has a low contribution for compounds larger than approximately molecular weight 200. There is a need for more exploratory in vivo studies to clarify drug absorption and first-pass extraction along the intestine. One is encouraged to develop in vivo perfusion techniques for more distal parts of the gastrointestinal tract in humans. This would stimulate the development of more relevant and complex in vitro absorption models and form the basis for an accurate physiologically based pharmacokinetic modelling of oral drug absorption.     

Quantitative Evaluation of the Function of Small Intestinal P-Glycoprotein: Comparative Studies Between in Situ and in Vitro

Purpose. The extent of intestinal absorption of MDR1 P-glycoprotein (P-gp) substrate drugs may be affected by interindividual differences in the expression level of P-gp, and/or by simultaneously administered P-gp substrates/inhibitors. The purpose of the present study is to examine whether the extent to which the intestinal absorption is affected by P-gp can be predicted from in vitro experiments. Methods. The in situ intestinal perfusion experiments were performed for 12 compounds in mdr1a/1b (–/–) and normal mice to determine the permeability-surface area (PS) product. Thus determined intestinal P-gp function was compared with the in vitro P-gp function, which was determined by comparing the transcellular transport across human P-gp expressing and parental LLC-PK1 monolayers. Results. In situ experimental results revealed that the extent to which the intestinal absorption is affected by P-gp was in the following order; quinidine > ritonavir > loperamide, verapamil, daunomycin > digoxin, cyclosporin A > dexamethasone, and vinblastine. A significant correlation was observed between P-gp function determined in the intestinal perfusion and that in LLC-PK1 monolayers. Conclusion. The in vitro transcellular transport across P-gp expressing monolayers may be used to predict the extent to which the intestinal absorption is affected by P-gp.     

Intestinal Transport of Gentamicin with a Novel,Glycosteroid Drug Transport Agent

Purpose. The objective was to investigate the ability of a glycosteroid (TC002) to increase the oral bioavailability of gentamicin. Methods. Admixtures of gentamicin and TC002 were administered to the rat ileum by injection and to dogs by ileal or jejunal externalized ports, or PO. Bioavailability of gentamicin was determined by HPLC. ³H-TC002 was injected via externalized cannulas into rat ileum or jejunum, or PO and its distribution and elimination was determined. The metabolism of TC002 in rats was evaluated by solid phase extraction and HPLC analysis of plasma, urine and feces following oral or intestinal administration. Results. The bioavailability of gentamicin was substantially increased in the presence of TC002 in both rats and dogs. The level of absorption was dependent on the concentration of TC002 and site of administration. Greatest absorption occurred following ileal or jejunal administration. TC002 was significantly more efficacious than sodium taurocholate, but similar in cytotoxicity. TC002 remained primarily in the GI tract following oral or intestinal administration and cleared rapidly from the body. It was only partly metabolized in the GI tract, but was rapidly and completely converted to its metabolite in plasma and urine. Conclusions. TC002 shows promise as a new drug transport agent for promoting intestinal absorption of polar molecules such as gentamicin.     

Novel Experimental Parameters to Quantify the Modulation of Absorptive and Secretory Transport of Compounds by P-Glycoprotein in Cell Culture Models of Intestinal Epithelium

Purpose. The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression. Methods. The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal. Results. P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (P _PD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity. Conclusions. The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.     

Dissolution of Hydrocortisone in Human and Simulated Intestinal Fluids

Purpose. To compare solubility and dissolution rate of hydrocortisonein aspirated human intestinal fluids (HIFs) with simulated intestinalfluids (SIFs) and buffer. Methods. Solubility and flux from a rotating disk of hydrocortisonewere measured. The bile salt content, pH and osmotic pressure weredetermined in HIFs. Results. In fasted state the solubility of hydrocortisone was higher inHIFs than in the buffer and SIFs. The flux of hydrocortisone in HIFswas similar to the flux in the buffer but lower than the flux in SIFs atfasted state. Addition of intestinal surfactants in SIFs increasedsolubility and flux at both fasted and fed state. The increase in solubility wascaused by micelle formation in SIFs. The increase in flux may partlybe explained by increased solubility. The bile salt content of the HIFsdid not correlate with the solubility or the flux but pH in the HIFsseems to have some effect on the components of the HIFs resultingin increased solubility. Conclusions. It is possible to perform comparable dissolution tests inHIFs and SIFs. The lack of correlation between the results in HIFs andthe bile salt content may be explained by the relatively low lipofilicity ofthe model drug.     

Apparent Lack of Effect of P-Glycoprotein on the Gastrointestinal Absorption of a Substrate,Tacrolimus, in Normal Mice

Purpose. To study the contribution of P-glycoprotein (P-gp) to the oralabsorption of a substrate, tacrolimus, by comparing the extent and rateof bioavailability in normal and mdr1a knockout mice. Methods. Intravenous and oral (2 mg/kg) blood concentration data oftacrolimus in normal and knockout mice were obtained from a studyby K. Yokogawa et al. in Pharm. Res. 16:1213-1218 (1999). Meanbioavailability (F), mean hepatic first-pass extraction ratio (F_h), meanbioavailability rates, mean oral clearance, and mean total hepaticintrinsic clearance were calculated using standard pharmacokinetic methods. Results. The mean F of tacrolimus (an apparently highly permeablecompound) was increased from 0.22 in normal mice to 0.72 in knockoutmice. These values were consistent with mean predicted E_h (based onintravenous data) of 0.77 and 0.27 in normal and knockout mice,respectively. Great similarity in the relative bioavailability profile (suchas short T_max) between normal and knockout mice was also found. Meanoral clearance and mean total or unbound hepatic intrinsic clearance oftacrolimus in knockout mice were found to be about 10 times lowercompared to those in normal mice. Conclusions. The above results suggest an apparent lack of effect ofP-gp on the gastrointestinal absorption of tacrolimus in normal miceunder the study condition. It is postulated that the effect of P-gp onthe rate and extent of oral absorption should be more pronounced forthose more slowly or incompletely absorbed drugs (i.e., drugs withrelatively low permeabilities) as illustrated by talinolol in humans. Theclearance data also suggest a very dominant role of P-glycoprotein incontrolling the rate of hepatic metabolism of tacrolimus in normalmice, and P-glycoprotein may serve as an effective efflux pump fordirect transport of metabolites formed in hepatocytes into the bloodcirculation.     

Rhodamine 123 Requires Carrier-Mediated Influx for Its Activity as a P-Glycoprotein Substrate in Caco-2 Cells

Purpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity). Results. R123 secretory P _app (P _app,BA) showed concentration dependence, whereas R123 absorptive P _app (P _app,AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P _app,AB, but enhanced R123 and doxorubicin P _app,BA. In calcium-free medium, R123 P _app,AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.     

A Clinical Single-Pass Perfusion Investigation of the Dynamic in Vivo Secretory Response to a Dietary Meal in Human Proximal Small Intestine

Purpose To investigate the gastrointestinal secretory and enzymatic responses to a liquid meal during in vivo perfusion of the proximal human jejunum. Methods Human intestinal fluid was collected from the proximal jejunum by single-pass in vivo perfusion (Loc-I-Gut). The fluid was quantitatively collected at 10-min intervals during 90 min while perfusing a nutritional drink at 2 mL/min. Quantification of lipids in the fluid leaving the segment was performed by using novel chromatographic methods. Results The overall bile acid concentration varied between 0.5 and 8.6 mM with a peak level 40 min after the start of the liquid meal perfusion. The total concentration of phospholipids was between 0.1 and 3.9 mM and there was a rapid degradation of phosphatidylcholine to lysophosphatidylcholine. The tri-, di-, monoglycerides and free fatty acid levels increased sharply in the beginning and reached steady-state levels between 7 and 9.5 mM. Conclusions There is a rapid secretion of bile in response to food. Most of the dietary lipids are found in the form of their degradation products in vivo in human jejunum. This novel in vivo characterization, based on direct and high-recovery sampling of intestinal fluids, forms a basis for further development of improved in vitro drug dissolution test media.     

P-Glycoprotein Increases from Proximal to Distal Regions of Human Small Intestine

Purpose. The contribution of the efflux transporter P-glycoprotein (P-gp) as a barrier to drug absorption may depend on its level of expression at the site of absorption. Accordingly, the distribution of P-gp was examined along the entire length of the human small intestine. Methods. Homogenates prepared from mucosal scrapings from every other 30-cm segment of four unrelated human donor small intestines were analyzed for P-gp and the control protein villin by Western blot. Results. In each donor intestine, relative P-gp expression (P-gp/villin integrated optical density ratio) progressively increased from proximal to distal regions. Among individuals, relative P-gp levels varied 2.1-fold in the duodenal/proximal jejunal region, 1.5- to 2.0-fold in the middle/distal jejunal region, and 1.2- to 1.9-fold in the ileal region. Within-donor variation was somewhat greater, from 1.5- to 3.0-fold. Conclusions. These results provide further evidence that the site of absorption can represent another source for the interindividual variation in the oral bioavailability of drugs.     

Effect of the Flavonoids Biochanin A and Silymarin on the P-Glycoprotein-Mediated Transport of Digoxin and Vinblastine in Human Intestinal Caco-2 Cells

Purpose. The purpose of this study was to investigate the effects of flavonoids biochanin A and silymarin on intestinal absorption of P-gp substrates by determining their effects on P-gp-mediated efflux in Caco-2 cells. Methods. The cellular accumulation and bidirectional transport of digoxin and vinblastine in Caco-2 cells were determined in the presence and absence of flavonoids. Results. The 1.5-h accumulation of digoxin and vinblastine in Caco-2 cells was significantly increased by 50 M biochanin A or silymarin, and this effect was flavonoid-concentration dependent. The AP-to-BL transport of digoxin was significantly increased, whereas the BL-to-AP transport was significantly decreased by 50 M biochanin A or 75 M silymarin. At 150 M concentrations of biochanin A or silymarin, mean transport ratios (P _app,B-A/P _app,A-B) of 1.62 and 4.48, respectively, compared with the control ratio of 43.4, were obtained. Conclusion. These results indicate that biochanin A and silymarin can inhibit P-gp-mediated efflux in Caco-2 cells, suggesting they could potentially increase the absorption/bioavailability of coadministered drugs that are P-gp substrates.     

Role of P-Glycoprotein in Restricting Propranolol Transport in Cultured Rabbit Conjunctival Epithelial Cell Layers

Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC). Methods. The localization of P-gp in the cultured RCEC as well asin the excised conjunctiva was determined by immunofluorescencetechnique. The role of P-gp in transepithelial transport and uptake ofpropranolol in conjunctival epithelial cells cultured on Transwell filterswas evaluated in the presence and absence of P-gp competing substrates, ananti-P-gp monoclonal antibody (4E3 mAb), or a metabolicinhibitor, 2, 4-dinitrophenol (2,4-DNP). Results. Immunofluorescence studies revealed positive staining in theapical membrane of cultured RCEC and in the apical surface of thesuperficial cell layers in the excised conjunctiva, but not the basolateralmembrane of cultured RCEC. Transport of propranolol showed preferencein the basolateral-to-apical direction. The net secretory flux wassaturable with a K_m of 71.5 ± 24.0 nM and a J_max of 1.45 ± 0.17pmol/cm²/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil,4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptakeby 43% to 66%. On the other hand, neither -blockers (atenolol,metoprolol, and alprenolol) nor organic cation transporter substrates(tetraethylammonium (TEA) and guanidine), affected apical 50 nMpropranolol uptake. Conclusions. The energy-dependent efflux pump P-gp appears to bepredominantly located on the apical plasma membrane of the conjunctivalepithelium. It may play an important role in restricting the conjunctivalabsorption of some lipophilic drugs.     

Intestinal Permeability Enhancement: Efficacy,Acute Local Toxicity,and Reversibility

The absorption of the polar drug phenol red was assessed in a rat intestinal perfusion model, in the presence of a variety of potential intestinal permeability enhancers. Both the absorption rate constant K_A and the plasma phenol red concentration were measured. Perfusates were also assayed for the presence of lactate dehydrogenase (LDH) and lipid phosphate, as biochemical markers of intestinal wall damage. Histological evaluation of surfactant-perfused intestines was also carried out. The potential permeability enhancers studied were the surfactants sodium dodecyl sulfate (SDS), sodium taurocholate (TC), sodium taurodeoxycholate (TDC), polysorbate-80 (PS-80), and nonylphenoxypolyoxyethylene (NP-POE) with an average polar group size of 10.5 POE units. Among these, SDS and NP-POE-10.5 were the most potent permeability enhancers. The bile salt TDC was a more effective enhancer than the more polar TC. The polar non-ionic surfactant PS-80 was an ineffective enhancer. Phenol red K_A and plasma level were generally correlated with biochemical and histological measures of intestinal damage. These observations indicate that permeability enhancement and local damage are closely related sequelae of the interaction of surfactants with the intestinal wall, and suggest that local wall damage may be involved in the mechanism of permeability enhancement. The reversibility of permeability enhancement and acute local damage was assessed for the surfactants TDC and NP-POE-10.5. Enhancement of phenol red permeability was reversed within 1-2 hr of the cessation of enhancer treatment. Biochemical markers of local damage also fell to control values within 1-2 hr of removal of enhancer from the perfusate. Histological evaluation of perfused intestines revealed that morphological damage was reversed within 3 hr. These results demonstrate that surfactant-induced acute intestinal wall damage is rapidly repaired.     

Interrelationship Between Substrates and Inhibitors of Human CYP3A and P-Glycoprotein

Purpose. CYP3A and P-gp both function to reduce the intracellular concentration of drug substrates, one by metabolism and the other by transmembrane efflux. Moreover, it has been serendipitously noted that the two proteins have many common substrates and inhibitors. In order to test this notion more fully, systematic studies were undertaken to determine the P-gp-mediated transport and inhibitory characteristics of prototypical CYP substrates. Methods. L-MDR1, LLC-PK1, and Caco-2 cells were used to evaluate established CYP substrates as potential P-gp substrates and inhibitors in vitro, and mdrla deficient mice were used to assess the in vivo relevance of P-gp-mediated transport. Results. Some (terfenadine, erythromycin and lovastatin) but not all (nifedipine and midazolam) CYP3A substrates were found to be P-gp substrates. Except for debrisoquine, none of the prototypical substrates of other common human CYP isoforms were transported by P-gp. Studies in mdrla disrupted mice confirmed that erythromycin was a P-gp substrate but the CYP3A inhibitor ketoconazole was not. In addition, CYP3A substrates and inhibitors varied widely in their ability to inhibit the P-gp-mediated transport of digoxin. Conclusions. These results indicate that the overlap in substrate specificities of CYP3A and P-gp appears to be fortuitous rather than indicative of a more fundamental relationship.     

Selective Paracellular Permeability in Two Models of Intestinal Absorption: Cultured Monolayers of Human Intestinal Epithelial Cells and Rat Intestinal Segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.     

Surface Activity and Concentration Dependent Intestinal Permeability in the Rat

Purpose. To investigate the relation between intestinal effective permeability (P_eff) and surface activity of fluvastatin and verapamil. Methods. P_eff values were determined for fluvastatin, antipyrine and D-glucose following colon perfusions in the rat in situ. The perfusion solutions differed regarding concentrations of fluvastatin (0-2500 M) and surface tension (58.9-43.7 mN/m). A cellulose derivative, ethyl-(hydroxyethyl) cellulose (EHEC), was added to lower the surface tension of one of the perfusion solutions. The surface tension of perfusion solutions containing R/S-verapamil (8-814 M) and R/S-verapamil + chlorpromazine (814 M + 10 mM) were related to the corresponding P_eff values from the literature. Results. The P_eff of fluvastatin correlated inversely (r² = 0.985, p < 0.05) with the surface tension of the perfusion solutions below the critical micelle concentration (CMC, 1 mM). Decreasing the surface tension with EHEC increased the P_eff of fluvastatin by 36% (p < 0.001), but not to the extent anticipated from the correlation between the P_eff and the surface tension. EHEC also increased the P_eff of antipyrine by 49% (p < 0.01) but not for D-glucose. The P_eff of R/S-verapamil correlated inversely with the surface tension (r² = 0.980, p < 0.001). Conclusions. The ability of fluvastatin to decrease the surface tension at the membrane surface can partly explain the concentration dependent colonic P_eff of fluvastatin. This study shows that the surface activity of the drug molecule itself is an important physicochemical factor that should be taken into consideration when evaluating drug absorption studies performed in vitro or in situ.     

Influence of bile salts and lipids on intestinal absorption of griseofulvin in man

Summary The influence of bile salts and lipids on the intestinal absorption of griseofulvin has been studied in 11 healthy male volunteers by the intestinal perfusion technique. The drug in a nutrient solution (Realmentyl) was perfused into the second part of duodenum at 5 ml/min. Intestinal samples were taken continuously at 1 ml/min, 20 cm (at the angle of Treitz) and 45 cm distal to the perfusion point. To study the effect of lipids on griseofulvin absorption, the drug was perfused with solutions A and B, of which B contained a total lipid and caloric load three times that of A. The influence of bile salts on griseofulvin absorption was examined by perfusing the drug on Day 1 with bile salts and again on the following day after bile salt depletion. Bile salts and a varying quantity of lipid perfusate had no significant influence on the duodeno-jejunal griseofulvin absorption rate per cm of intestine. Lipids, however, may still play a role in griseofulvin absorption along the entire intestine.