Localisation of drug permeability along the rat small intestine, using markers of the paracellular, transcellular and some transporter routes

The small intestine is the major site of drug absorption. Some reports in the literature have evoked the concept of “absorption windows” in the small intestine: are there specific regions where drug absorption is significantly higher than others? To investigate this question, we used an everted gut sac method to study the permeability of drugs and markers every 3–4 cm down the entire small intestine in rat. These markers were chosen to be representative of the mechanisms by which drugs cross the small intestinal mucosa: paracellular and transcellular passive diffusion, via influx transporters, and a drug (digoxin) that is effluxed from cells by P-glycoprotein (P-gp). The passive diffusion and influx transporter markers gave similar profiles with a plateau of permeability along the jejunum, and with the exception of L-Dopa, lower permeability in the ileum. Digoxin showed a linear decrease in the profile from the proximal jejunum to the ileum. Permeability in the duodenum was two to three times lower than the jejunum for all compounds. There were no narrow specific regions of high permeability and so the concept of discrete “absorption windows” along the small intestine as suggested from some pharmacokinetic studies may be related to other effects such as pH and/or solubility.     

pH-dependent functional activity of P-glycoprotein in limiting intestinal absorption of protic drugs: kinetic analysis of quinidine efflux in situ

The purpose of this investigation is to evaluate the quantitative contribution of pH-dependent passive permeability on the functional activity of P-glycoprotein (P-gp) in limiting intestinal absorption of weakly basic drugs, in order to include this effect in prediction models. pH-dependent octanol/buffer partition coefficient, artificial membrane permeability and in situ rat intestinal permeability of quinidine were determined in the physiological pH range of gastrointestinal tract. In situ permeability, as a function of luminal pH, was also determined in the presence of P-gp inhibitor, verapamil (500 microM). Octanol/buffer partition coefficient, transport across artificial membrane, and rat in situ permeability showed high pH-dependency. Absorption quotient (AQ), calculated from in situ permeability to express the functional activity of P-gp, declined with increase in luminal pH or increase in luminal quinidine concentration because of the increased passive permeability or saturation of P-gp. AQ was 0.57 +/- 0.02 and 0.41 +/- 0.05, while passive permeability was 0.32 +/- 0.01 x 10(-4) cm/sec and 0.43 +/- 0.02 x 10(-4) cm/sec, in jejunum and ileum, respectively, at pH 7.4. Further, apparent Michaelis-Menten constants (K(M), J(P-gp,max)) for the quinidine efflux in jejunum indicated that efflux activity was more at luminal pH 4.5 over pH 7.4. K(M) values for jejunum quinidine efflux at pH 4.5 and pH 7.4 were determined to be 77.63 +/- 10.90 and 22.86 +/- 5.22 microM, with J(P-gp,max) values of 1.47 +/- 0.08 and 0.62 +/- 0.04 nM/cm2/sec, respectively. AQ vs passive permeability showed significant relationship indicating dependency of P-gp-mediated efflux on pH-dependent passive permeability, which is dictated by ionization status for a protic or ampholytic drug. In conclusion, an orally administered drug is absorbed from various segments of intestine, which inherit difference in luminal pH, transcellular permeability and P-gp expression. In situ data suggests that pH-dependency and regional variability in passive permeability of protic substrates significantly influence their P-gp-mediated efflux and may have implications on predictions of the in vivo drug absorption.     

Determination of site of absorption of propranolol in rat gut using in situ single-pass intestinal perfusion

Previously, permeability and site of intestinal absorption of propranolol have been reported using the Ussing chamber. In the present study, the utility of Single-Pass Intestinal Perfusion to study permeability and site of intestinal absorption of propranolol was evaluated in rats. Drug permeability in different regions of rat intestine viz. duodenum, jejunum, ileum and colon was measured. Propranolol (30 μg/ml) solution was perfused in situ in each intestinal segment of rats. Effective permeability (Peff) of propranolol in each segment was calculated and site of absorption was determined. The Peff of propranolol in rat duodenum, jejunum, ileum and colon was calculated to be 0.3316×10(-4) cm/s, 0.4035×10(-4)cm/s, 0.5092×10(-4) cm/s and 0.7167×10(-4) cm/s, respectively. The above results suggest that permeability of propranolol was highest through colon compared to other intestinal sites, which is in close agreement to that reported previously. In conclusion, in situ single pass intestinal perfusion can be used effectively to study intestinal permeability as well as site of intestinal absorption of compounds in rats.     

What kinds of substrates show P-glycoprotein-dependent intestinal absorption? Comparison of verapamil with vinblastine

The influence of P-glycoprotein (P-gp) on intestinal absorption of drugs was investigated by comparison of the uptakes of two P-gp substrates, verapamil and vinblastine, using intestinal segments of wild-type and mdr1a/1b gene-deficient (mdr1a/1b(-/-)) mice, and Caco-2 cells. When [(3)H]vinblastine was injected into intestinal segments of wild-type mice, vinblastine was absorbed from duodenum and ileum, but not from jejunum. This difference among intestinal regions could not be explained by segmental differences of mdr1a mRNA expression. In Caco-2 cells, it was found that vinblastine had a high value of efflux/influx ratio (an index of affinity for P-gp) of 12.1, and a low permeability of less than 1 x 10(-6) cm/sec. The corresponding values for verapamil were 4.9 and 10.6 x 10(-6) cm/sec, respectively. After oral administration of [(3)H]vinblastine to mice, the maximum concentration (C(max)) and the area under the plasma concentration time-curve from time 0 to 24 hr (AUC(0-24 hr)) for mdr1a/1b(-/-) mice were 1.5 times greater than those for wild-type mice, while these parameters were not significantly different between the two strains in the case of [(3)H]verapamil. Therefore, P-gp substrates may be classified into at least two types, i.e., verapamil-type, for which the intestinal absorption is unaffected by P-gp, and vinblastine-type, for which the intestinal absorption is influenced by P-gp. Vinblastine-type P-gp substrates, with low permeability and high affinity for P-gp, would be unfavorable candidates for oral drugs.     

3种药物转运体抑制剂对沙奎那韦在大鼠肠道吸收影响的体外试验

目的:体外研究药物转运体P糖蛋白(P-gp)和多药耐药相关蛋白2(Mrp2)特异性抑制剂对沙奎那韦在大鼠肠道吸收的影响。方法:取大鼠用乌拉坦麻醉后,分别取十二指肠、空肠、回肠、结肠各8cm,制备离体肠外翻模型,检测不同肠段在P-gp抑制剂地高辛(10μmol.L-1)和维拉帕米(100μmol.L-1)、Mrp2抑制剂丙磺舒(600μmol.L-1)分别与沙奎那韦(12.5μg.mL-1)的混合Krebs-Ringer缓冲液(K-R液)中孵育5、10、20、30、45、60、90min后对沙奎那韦的累积吸收量;另设含沙奎那韦的K-R液为对照组。结果:沙奎那韦在大鼠十二指肠、空肠、回肠、结肠K-R液中的累积吸收量分别为(7.25±1.23)、(4.96±1.58)、(3.89±0.95)、(5.85±1.21)μg,吸收速率为十二指肠>结肠>空肠>回肠。维拉帕米((10.03±3.56)、(7.52±2.21)、(7.45±1.8)μg)和地高辛((8.76±2.25)、(5.98±1.89)、(6.04±1.92)μg)可显著提高沙奎那韦在结肠、空肠、回肠K-R液中的累积吸收量(P<0.05),对十二指肠无显著影响(P>0.05);丙磺舒对沙奎那韦在各肠段K-R液中的累积吸收量均无显著影响(P>0.05)。结论:P-gp可显著影响沙奎那韦的肠道吸收,Mrp2对沙奎那韦的肠道吸收无影响。     

Tacrolimus is a class II low-solubility high-permeability drug: the effect of P-glycoprotein efflux on regional permeability of tacrolimus in rats

The objective of this study is to investigate the role of P-glycoprotein (P-gp), a membrane efflux pump associated with multidrug resistance (MDR) and a known substrate for tacrolimus, in determining the regional intestinal permeability of tacrolimus in rats. Thus, isolated segments of rat jejunum, ileum, or colon were perfused with tacrolimus solutions containing polyethoxylated hydrogenated castor oil 60 surfactant, and with or without verapamil, a P-gp substrate used to reverse the MDR phenotype. The results indicated that the intrinsic permeability of tacrolimus in the jejunum, calculated on the basis of the concentration of non-micellized free tacrolimus, was quite high ( approximately 1.4 x 10(-4) cm/s). The apparent permeability (P(app)) in the jejunum was unaffected by the presence of verapamil; however, the P(app) in the ileum and the colon increased significantly in the presence of verapamil and were similar to the values observed in the jejunum. The results suggest that systemic absorption of tacrolimus from the gastrointestinal tract could be significantly affected by P-gp efflux mechanisms. It is also possible that differences in P-gp function at various intestinal sites in a subject or at a given intestinal site in various subjects could lead to large intra- and interindividual variability in bioavailability of tacrolimus following oral administration.     

In situ kinetic modelling of intestinal efflux in rats: functional characterization of segmental differences and correlation with in vitro results

The objective was to devise and apply a novel modelling approach to combine segmental in situ rat perfusion data and in vitro cell culture data, in order to elucidate the contribution of efflux in drug absorption kinetics. The fluoroquinolone CNV97100 was used as a model P-gp substrate. In situ intestinal perfusion was performed in rat duodenum, jejunum, ileum and colon to measure the influence of P-gp expression on efflux. Inhibition studies of CNV97100 were performed in the presence of verapamil, quinidine, cyclosporin A and p-aminohippuric acid. Absorption/efflux parameters were modelled simultaneously, using data from both in situ studies as well as in vitro studies. The maximal efflux velocity was modelled as a baseline value, corrected for each segment based on the expression level. CNV97100 passive diffusional permeability (P(diff)) and its affinity for the efflux carrier (K(m)) were assumed to be the same in all segments. The results indicate the new approach to combine in situ data and in vitro data succeed in yielding a unified, quantitative model for absorption/efflux. The model incorporated a quantitative relationship between P-gp expression level and the efflux functionality, both across in situ and in vitro systems, as well across different intestinal segments in the in situ studies. Permeability values decreased from duodenum to ileum in accordance with the increasing P-gp expression levels in rat intestine. The developed model reflects a strong correlation between in vitro and in situ results, including intrinsic differences in surface area. The successful application of a model approach to combine absorption data from two different experimental systems holds promise for future efforts to predict absorption results from one system to a second system.     

Regional difference in P-glycoprotein function in rat intestine

It has been reported that inhibition of the P-glycoprotein (P-gp) results in the improved absorption of P-gp substrate in the intestinal tract. In fact, the increased permeability of P-gp substrate across the intestinal epithelium was observed following inhibition of P-gp in in vitro experiments. To develop the formulation containing P-gp inhibitor and P-gp substrate for practical use, it is necessary to know whether the results obtained in the in vitro experiments are reproducible at whole body level. It is also important to find out the regional difference of the P-gp activity in the intestinal tract. In this study, we examined whether verapamil, a specific inhibitor of P-gp, improves the absorption of rhodamine123 (Rho123), a substrate of P-gp, from the jejunum, ileum, and colon of rats using the in situ loop method. The water content in the loop decreased during the experiment, resulting in a significant change of the Rho123 concentration in the loop. Thus, to accurately determine the absorption rate of Rho123, it was necessary to measure the water movement. It was found that there was a regional difference in the water movement, i.e., greatest in colon, followed by ileum. Verapamil did not change the water movement in any intestinal regions. When the concentration of Rho123 in the loop was corrected by water movement, the Rho123 clearance was in the order of ileum (1.15 microL/min/cm), colon (0.83 microL/min/cm) and jejunum (0.47 microL/min/cm). In the presence of verapamil, the Rho123 clearance was significantly increased at jejunum and ileum but not in colon (ileum: 2.08 microL/min/cm, colon: 1.14 microL/min/cm, jejunum: 1.28 microL/min/cm). These results suggest that P-gp inhibits the drug absorption in jejunum and ileum. From these results, it is possible to evaluate the role of P-gp and its regional difference in the in situ experiments. In particular, the inhibition of P-gp results in an increase in absorption of the P-gp substrate limited to jejunum and ileum.     

溴吡斯的明在大鼠离体肠的吸收动力学

目的观察溴吡斯的明在各肠段的吸收动力学特征。方法采用大鼠外翻肠囊模型,反相离子对色谱法测定不同浓度(25、50、100 mg.L-1)的溴吡斯的明在各肠段的吸收量,计算吸收速率常数(Ka)和表观渗透系数(Papp),并考察P-糖蛋白抑制剂(环孢素和维拉帕米)对药物吸收的影响。结果在25、50、100 mg.L-1溴吡斯的明条件下,Ka按十二指肠、空肠、回肠、结肠依次减小,不同浓度溴吡斯的明对同一肠段的Ka无显著影响(P>0.05),十二指肠、空肠、回肠间的Ka无显著差异(P>0.05),在50、100 mg.L-1溴吡斯的明条件下,结肠的Ka与十二指肠、空肠、回肠比较有显著差异(P<0.05,P<0.01)。随着溴吡斯的明浓度增加,各肠段Papp显著降低,不同肠段间的Papp有显著差异(P<0.05,P<0.01)。P-糖蛋白抑制剂对溴吡斯的明吸收无影响(P>0.05)。结论溴吡斯的明在十二指肠有较好吸收,在空肠和回肠有一定吸收,在结肠中吸收较少。     

Role of P-glycoprotein in pharmacokinetics and drug interactions of digoxin and beta-methyldigoxin in rats

Digoxin and beta-methyldigoxin were evaluated pharmacokinetically in terms of P-glycoprotein (P-gp)-mediated drug interactions in rats. Evaluation was made by measuring the effects of a potent P-gp inhibitor (verapamil, cyclosporin A) on in vitro efflux transport of these compounds across the everted small intestine, on in situ absorption from the small intestine, and on in vivo total plasma clearance (CL(total)) as well as biliary and urinary excretions after intravenous administration. Both the intestinal efflux transport and absorption of beta-methyldigoxin were approximately 1.5-fold greater than those of digoxin, probably due to its higher lipophilicity. Addition of verapamil (300 microM) significantly decreased the intestinal efflux transport and increased the intestinal absorption of digoxin. In contrast, the influence of verapamil on beta-methyldigoxin was small. Intravenous cyclosporin A (30 mg/kg) significantly decreased in vivo CL(total) and biliary excretion of digoxin, but affected little on beta-methyldigoxin clearances. These results suggest that P-gp-mediated drug interactions can easily occur in digoxin, but hardly in beta-methyldigoxin. These findings may give a clue in selecting these digitalis compounds in clinical use, towards escape from P-gp-mediated drug interactions or reduction of interindividual variations.     

Intestinal secretion is a major route for parent ivermectin elimination in the rat.

The transepithelial intestinal elimination of ivermectin was studied using the intestinal closed-loop model in the rat. The common bile duct was cannulated, and duodenum, jejunum, and ileum were isolated in situ with their intact blood supplies. Following administration of 100, 200, or 400 microg/kg b.wt. ivermectin via the carotid artery, the elimination of parent ivermectin into the small intestinal lumen over 90 min was approximately 5-fold higher than in bile. The major amount of secreted ivermectin was recovered in the jejunum, but the duodenum showed a higher intestinal elimination capacity than the other intestinal segments with respect to the intestinal length. Systemic coadministration of the P-glycoprotein blocker verapamil significantly reduced the elimination capacity of jejunum by 50%, which resulted in a 30% decrease of ivermectin overall elimination by the small intestine. In contrast, verapamil did not significantly affect ivermectin secretion in duodenum, ileum, or bile in the same animals. Ivermectin small intestinal and biliary clearances were estimated to account for 27 and 5.5% of the total drug clearance, which was evaluated from a parallel in vivo experiment in which rats were given 200 microg/kg b.wt. ivermectin intra-arterially. In conclusion, intestinal secretion plays a greater role than biliary secretion in the overall elimination of ivermectin in the rat, providing major amounts of active drug to the intestinal lumen and to feces. This is discussed in terms of therapeutic efficacy against intestinal parasites in humans and animals and of ecotoxicity resulting from the contamination of livestock dung with parent drug.     

Why is it Challenging to Predict Intestinal Drug Absorption and Oral Bioavailability in Human Using Rat Model

Purpose To study the correlation of intestinal absorption for drugs with various absorption routes between human and rat, and to explore the underlying molecular mechanisms for the similarity in drug intestinal absorption and the differences in oral bioavailability between human and rat.Materials and Methods The intestinal permeabilities of 14 drugs and three drug-like compounds with different absorption mechanisms in rat and human jejunum were determined by in situ intestinal perfusion. A total of 48 drugs were selected for oral bioavailability comparison. Expression profiles of transporters and metabolizing enzymes in both rat and human intestines (duodenum and colon) were measured using GeneChip analysis.Results No correlation (r ² = 0.29) was found in oral drug bioavailability between rat and human, while a correlation (r ² = 0.8) was observed for drug intestinal permeability with both carrier-mediated absorption and passive diffusion mechanisms between human and rat small intestine. Moderate correlation (with r ² > 0.56) was also found for the expression levels of transporters in the duodenum of human and rat, which provides the molecular mechanisms for the similarity and correlation of drug absorption between two species. In contrast, no correlation was found for the expressions of metabolizing enzymes between rat and human intestine, which indicates the difference in drug metabolism and oral bioavailability in two species. Detailed analysis indicates that many transporters (such as PepT1, SGLT-1, GLUT5, MRP2, NT2, and high affinity glutamate transporter) share similar expression levels in both human and rat with regional dependent expression patterns, which have high expression in the small intestine and low expression in the colon. However, discrepancy was also observed for several other transporters (such as MDR1, MRP3, GLUT1, and GLUT3) in both the duodenum and colon of human and rat. In addition, the expressions of metabolizing enzymes (CYP3A4/CYP3A9 and UDPG) showed 12 to 193-fold difference between human and rat intestine with distinct regional dependent expression patterns.Conclusions The data indicate that rat and human show similar drug intestinal absorption profiles and similar transporter expression patterns in the small intestine, while the two species exhibit distinct expression levels and patterns for metabolizing enzymes in the intestine. Therefore, a rat model can be used to predict oral drug absorption in the small intestine of human, but not to predict drug metabolism or oral bioavailability in human.     

Different absorption behaviors among steroid hormones due to possible interaction with P-glycoprotein in the rat small intestine.

The intestinal absorption of ten steroid hormones was evaluated in the rat small intestine, especially focusing on the interaction with intestinal P-glycoprotein (P-gp). Hydrocortisone, prednisolone, 6alpha-methylprednisolone, and dexamethasone (adrenocortical steroid hormones) all disappeared in a regional-dependent manner (duodenum>jejunum>ileum). The decreased rate of disappearance in the lower small intestine seemed to be due to the involvement of absorption barriers like P-gp. In contrast, all sex hormones including progesterone exhibited very high absorbability in the entire small intestine (duodenum=jejunum=ileum), possibly demonstrating the absence of restricted absorption by intestinal P-gp. Progesterone enhanced the rate of disappearance of vinblastine but did not affect 6alpha-methylprednisolone. In the presence of vinblastine and verapamil, on the other hand, the rate of disappearance of 6alpha-methylprednisolone increased significantly. It was demonstrated that there was a plural P-gp family, which had different substrate specificities, in the rat intestine and that steroid hormones interacted with them as substrates or inhibitors in a very complex manner.     

Evidence of efflux-mediated and saturable absorption of rifampicin in rat intestine using the ligated loop and everted gut sac techniques

This study was carried out to explore whether efflux-mediated and saturable mechanisms play any role toward poor and variable intestinal absorption of rifampicin. In situ segmental permeability of rifampicin at various residence times was determined in rat gastrointestinal tract using the ligated loop technique. The involvement of efflux-mediated and saturable absorption of rifampicin was studied in rat intestine using the everted sac method. The samples were analyzed by a validated HPLC method. Rifampicin showed decreased permeability in jejunum and ileum with an increase in residence time. The permeation of rifampicin from the serosal to the mucosal side (secretion) was significantly higher than permeation from the mucosal to the serosal side (absorption) of jejunum and ileum. This indicated the involvement of efflux-mediated transport. Addition of verapamil, an inhibitor for P-glycoprotein (Pgp), multidrug resistance associated protein-2 (MRP-2), and other related transporters, increased absorption of rifampicin in jejunum and ileum by 2-3-fold and decreased secretion by almost 4-fold. The permeation rate (flux) of rifampicin through duodenum increased with concentration up to 300 microg/mL, becoming constant thereafter, indicating the existence of saturable absorption. There was no saturable permeation in jejunum and ileum. Thus the present study indicates the involvement of efflux-mediated and saturable absorption mechanisms of rifampicin in rat intestine, which act as barriers to the absorption of the drug. This explains the drug's poor absolute bioavailability. As Pgp varies from person to person to an extent of 2-8-fold, it can be one direct reason for the interindividual variable bioavailability shown by rifampicin.     

Factors influencing regional differences in intestinal absorption of UK-343,664 in rat: possible role in dose-dependent pharmacokinetics

The objective of this study was to evaluate potential contributions of intestinal export and metabolism to the oral dose-dependent pharmacokinetics of the human cGMP-specific phosphodiesterase type 5 inhibitor, UK-343,664. Differences between jejunal and ileal handling of this CYP3A and P-gp substrate were investigated. CYP3A and P-gp display differing activities in the upper and lower mammalian small intestine and their impact on variable drug absorption can be mechanistically assessed for individual compounds with in situ perfusion of rat's small intestine. Isolated segments of rat jejunum and ileum were perfused with UK-343,664 solution and measurements were made as a function of drug concentration for dose dependence and in the presence of CYP3A and P-gp inhibitors. Intestinal permeability and metabolism were measured by total drug disappearance and major metabolite, UK-347,334 (N-desethyl metabolite), appearance in the intestinal lumen. Intestinal tissue and mesenteric blood measurements of drug and metabolite were also determined. The effective permeability (P(eff)) of UK-343,664 and metabolite formation (F(met)) increased as a function of concentration. Regional differences in P(eff) and F(met) were observed with low-intestinal metabolism of UK-343,664 in both regions (<10%). P-gp inhibition caused significant increase in P(eff) and F(met) in jejunum and ileum while ketoconazole, a P-gp and CYP3A inhibitor, has only limited effect on metabolism. In conclusion, UK-343,664 absorption is mainly regulated by P-gp in jejunum and ileum while CYP3A intestinal metabolism has minimal effect. This role of P-gp could explain the dose-dependent pharmacokinetics of UK-343,664 and its unusual behavior of t(max) as a function of dose.     

Role of intestinal efflux transporters in the intestinal absorption of methotrexate in rats

The role of intestinal efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in intestinal absorption of methotrexate was examined in rats. In everted intestine, the mucosal efflux of methotrexate after application to serosal side was higher in jejunum than ileum, and the efflux in jejunum was suppressed by pantoprazole, a BCRP inhibitor, and probenecid, an MRP inhibitor, but not by verapamil, a P-gp inhibitor. The mucosal methotrexate efflux in ileum was suppressed by pantoprazole, but not by other inhibitors. On the other hand, the serosal efflux of methotrexate after application to mucosal side was greater in ileum than jejunum, and was suppressed by probenecid. In in-vivo rat studies, the intestinal absorption of methotrexate was significantly higher when methotrexate was administered to ileum than jejunum. Pantoprazole increased methotrexate absorption from jejunum and ileum. Probenecid increased the absorption of methotrexate from jejunum but decreased the absorption from ileum, as evaluated by peak plasma methotrexate levels. In conclusion, BCRP and MRPs are involved in the regional difference in absorption of methotrexate along the intestine, depending on their expression sites.     

Intestinal permeability of forskolin by in situ single pass perfusion in rats

The intestinal permeability of forskolin was investigated using a single pass intestinal perfusion (SPIP) technique in rats. SPIP was performed in different intestinal segments (duodenum, jejunum, ileum, and colon) with three concentrations of forskolin (11.90, 29.75, and 59.90 μg/mL). The investigations of adsorption and stability were performed to ensure that the disappearance of forskolin from the perfusate was due to intestinal absorption. The results of the SPIP study indicated that forskolin could be absorbed in all segments of the intestine. The effective permeability (P (eff)) of forskolin was in the range of drugs with high intestinal permeability. The P (eff) was highest in the duodenum as compared to other intestinal segments. The decreases of P (eff) in the duodenum and ileum at the highest forskolin concentration suggested a saturable transport process. The addition of verapamil, a P-glycoprotein inhibitor, significantly enhanced the permeability of forskolin across the rat jejunum. The absorbed fraction of dissolved forskolin after oral administration in humans was estimated to be 100 % calculated from rat P (eff). In conclusion, dissolved forskolin can be absorbed readily in the intestine. The low aqueous solubility of forskolin might be a crucial factor for its poor oral bioavailability.     

Prediction of in vivo intestinal absorption enhancement on P-glycoprotein inhibition, from rat in situ permeability

The purpose of this study is to determine the functional role of P-glycoprotein (P-gp) in intestinal absorption of drugs and to quantitatively predict the in vivo absorption enhancement on P-gp inhibition. In situ single-pass rat ileum permeability and aqueous solubility were measured for a set of 16 compounds. Permeability studies were also carried out in the presence of P-gp inhibitor to estimate the permeability enhancement on P-gp inhibition. A significant correlation was obtained between rat ileum permeability and the literature human intestinal absorption (HIA), F(a,human) (r = 0.891; p < 0.01). Compounds with permeability >0.2 x 10(-4) cm/s are completely absorbed; however, few practically insoluble compounds were overestimated with this relationship. Inhibition of P-gp increased the permeability (p < 0.05) of three moderately and three highly permeable compounds. Efflux inhibition ratio (EIR), the ratio of permeability due to P-gp-mediated efflux activity and passive permeability only, for these compounds was in the order of digoxin > paclitaxel > fexofenadine > quinidine > verapamil > cyclosporine. Integration of EIR with permeability versus F(a,human) predicted that modulation of P-gp has no significant effect on the absorption of highly permeable compounds (quinidine, verapamil, and cyclosporine A), while for moderately permeable compounds (digoxin, paclitaxel, and fexofenadine), P-gp profoundly influences the intestinal permeability. The in situ permeability in rat ileum may be used to predict the in vivo P-gp function and its quantitative contribution to intestinal drug absorption. Integration of the functional activity of P-gp with the characteristics of BCS may explain drug interactions and explore the possible pharmacokinetic advantage on P-gp inhibition.     

Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats

The expression of P-glycoprotein is induced in cell cultures upon exposure to various inducers. Therefore, the aim of the present study was to evaluate the in-vivo relevance of this observation, i.e. the influence of chronic pretreatments with selected drugs -- all of which are ligands to P-glycoprotein (P-gp) as demonstrated in radioligand binding studies and all of which have some or a considerable effect on P-gp expression in Caco-2 cells -- on the effective intestinal permeabilities of the model compound talinolol in rats employing in-situ single-pass intestinal perfusion of three different gut segments. Talinolol was selected, because it shows high selectivity for one of the exsorptive transporters (P-gp) and its intestinal permeability is very sensitive to changes in exsorption when the perfusate concentration is low. Prior to the induction study the perfusion model was optimized regarding the type and concentration of a competitive inhibitor which may be used to block the exsorption-related permeability reduction (through intestinal exsorption) during an ongoing perfusion and would permit an intra-individual comparison of the effective permeability without and with blockade of exsorption. While repetitive verapamil and talinolol dosing had no statistically significant exsorption-inducing effect, vinblastine and rifampicin pretreatments resulted in decreased intestinal talinolol permeabilities in the three tested gut segments, duodenum, jejunum, and colon [e.g., S-talinolol in jejunum: control, 2.50 x 10(-4) cm/s; vinblastine induction, 1.48 x 10(-4) cm/s (P<0.05); rifampicin induction, 1.51 x 10(-4) cm/s (P<0.05)]. Addition of an efficient secretion inhibitor (vinblastine) to the perfusate permitted the determination of the impact of inhibitable secretory processes on the total effective permeabilities and an estimation of passive permeability in the respective individual. The inhibitable permeability fractions were higher for vinblastine than for any other pretreatment and the difference from control pretreatment was statistically significant for all intestinal segments (duodenum, 61.8%; jejunum, 63.1%; colon, 43,7%; S-talinolol). Statistically significant differences were also detected for rifampicin in the perfused duodenum and jejunum (33.1 and 27.5% increase in inhibitable fraction, respectively, for S-talinolol). These differences are explained by a significant induction of outside-directed transport in the intestinal enterocytes by vinblastine and rifampicin.     

Application of compartmental modeling to an examination of in vitro intestinal permeability data: assessing the impact of tissue uptake, P-glycoprotein, and CYP3A.

P-glycoprotein (P-gp)-mediated drug efflux and cytochrome p450 3A (CYP3A) metabolism within the enterocyte have been implicated as potential biochemical barriers to intestinal drug permeability. The current studies examined the in vitro intestinal permeability of verapamil, a common P-gp and CYP3A substrate, using both disappearance and appearance measurements, and investigated the possible impact of P-gp efflux on the intestinal extraction of verapamil. Bidirectional permeability and metabolism studies were conducted across rat jejunal tissue in side-by-side diffusion chambers and data were modeled using compartmental kinetics. Substantial tissue uptake of verapamil was evident in the in vitro model and resulted in a disappearance permeability coefficient that was approximately 10-fold greater than that determined from verapamil appearance in the receptor chamber. Polarization of the bidirectional transport of verapamil was evident due to P-gp efflux (efflux ratio of 2.5), and significant intestinal extraction of verapamil on passage across the tissue was observed (mucosal to serosal extraction ratio of 0.31 +/- 0.04). Surprisingly, the selective P-gp inhibitor, valspodar (PSC833), had an insignificant impact on P-gp-mediated efflux of verapamil; however, selective CYP3A inhibition (afforded by midazolam) increased mucosal to serosal verapamil transport 1.6-fold, presumably through a reduction in intestinal metabolism. Using a four-compartment model, simulations of the impact of P-gp on the intestinal extraction ratio of verapamil demonstrated that for efflux to increase intestinal extraction, a nonlinear relationship must exist between the extent of drug metabolism and the extent of drug transport; the origin of this "nonlinearity" may include saturable drug metabolism, accumulation, and/or distribution.