Evaluation of drug permeability calculation based on luminal disappearance and plasma appearance in the rat single-pass intestinal perfusion model.

The rat single-pass intestinal perfusion (SPIP) model is commonly used to investigate gastrointestinal physiology and membrane drug transport. The SPIP model can be used with the intestinal segment inside or outside the abdomen. The rats can also be treated with parecoxib, a selective cycloxygenase-2 inhibitor that has been shown to affect some intestinal functions following abdominal surgery, such as motility, epithelial permeability, fluid flux and ion transport. However, the impact of extra-abdominal placement of the intestinal segment in combination with parecoxib on intestinal drug transport has not been investigated. There is also uncertainty how well intestinal permeability determinations based on luminal drug disappearance and plasma appearance correlate in the rat SPIP model. The main objective of this rat in vivo study was to investigate the effect of intra- vs. extra-abdominal SPIP, with and without, pretreatment with parecoxib. The effect was evaluated by determining the difference in blood-to-lumen 51Cr-EDTA clearance, lumen-to-blood permeability of a cassette-dose of four model compounds (atenolol, enalaprilat, ketoprofen, and metoprolol), and water flux. The second objective was to compare the jejunal permeability values of the model drugs when determined based on luminal disappearance or plasma appearance. The study showed that the placement of the perfused jejunal segment, or the treatment with parecoxib, had minimal effects on membrane permeability and water flux. It was also shown that intestinal permeability of low permeability compounds should be determined on the basis of data from plasma appearance rather than luminal disappearance. If permeability is calculated on the basis of luminal disappearance, it should preferably include negative values to increase the accuracy in the determinations.

Time-dependent effects on small intestinal transport by absorption-modifying excipients.

The relevance of the rat single-pass intestinal perfusion model for investigating in vivo time-dependent effects of absorption-modifying excipients (AMEs) is not fully established. Therefore, the dynamic effect and recovery of the intestinal mucosa was evaluated based on the lumen-to-blood flux (Jabs) of six model compounds, and the blood-to-lumen clearance of 51Cr-EDTA (CLCr), during and after 15- and 60-min mucosal exposure of the AMEs, sodium dodecyl sulfate (SDS) and chitosan, in separate experiments. The contribution of enteric neurons on the effect of SDS and chitosan was also evaluated by luminal coadministration of the nicotinic receptor antagonist, mecamylamine. The increases in Jabs and CLCr (maximum and total) during the perfusion experiments were dependent on exposure time (15 and 60 min), and the concentration of SDS, but not chitosan. The increases in Jabs and CLCr following the 15-min intestinal exposure of both SDS and chitosan were greater than those reported from an in vivo rat intraintestinal bolus model. However, the effect in the bolus model could be predicted from the increase of Jabs at the end of the 15-min exposure period, where a six-fold increase in Jabs was required for a corresponding effect in the in vivo bolus model. This illustrates that a rapid and robust effect of the AME is crucial to increase the in vivo intestinal absorption rate before the yet unabsorbed drug in lumen has been transported distally in the intestine. Further, the recovery of the intestinal mucosa was complete following 15-min exposures of SDS and chitosan, but it only recovered 50% after the 60-min intestinal exposures. Our study also showed that the luminal exposure of AMEs affected the absorptive model drug transport more than the excretion of 51Cr-EDTA, as Jabs for the drugs was more sensitive than CLCr at detecting dynamic mucosal AME effects, such as response rate and recovery. Finally, there appears to be no nicotinergic neural contribution to the absorption-enhancing effect of SDS and chitosan, as luminal administration of 0.1 mM mecamylamine had no effect.

Effect of absorption-modifying excipients, hypotonicity, and enteric neural activity in an in vivo model for small intestinal transport.

The small intestine mucosal barrier is physiologically regulated by the luminal conditions, where intestinal factors, such as diet and luminal tonicity, can affect mucosal permeability. The intestinal barrier may also be affected by absorption-modifying excipients (AME) in oral drug delivery systems. Currently, there is a gap in the understanding of how AMEs interact with the physiological regulation of intestinal electrolyte transport and fluid flux, and epithelial permeability. Therefore, the objective of this single-pass perfusion study in rat was to investigate the effect of three AMEs on the intestinal mucosal permeability at different luminal tonicities (100, 170, and 290 mOsm). The effect was also evaluated following luminal administration of a nicotinic receptor antagonist, mecamylamine, and after intravenous administration of a COX-2 inhibitor, parecoxib, both of which affect the enteric neural activity involved in physiological regulation of intestinal functions. The effect was evaluated by changes in intestinal lumen-to-blood transport of six model compounds, and blood-to-lumen clearance of 51Cr-EDTA (a mucosal barrier marker). Luminal hypotonicity alone increased the intestinal epithelial transport of 51Cr-EDTA. This effect was potentiated by two AMEs (SDS and caprate) and by parecoxib, while it was reduced by mecamylamine. Consequently, the impact of enteric neural activity and luminal conditions may affect nonclinical determinations of intestinal permeability. In vivo predictions based on animal intestinal perfusion models can be improved by considering these effects. The in vivo relevance can be increased by treating rats with a COX-2 inhibitor prior to surgery. This decreases the risk of surgery-induced ileus, which may affect the physiological regulation of mucosal permeability.

The effects of three absorption-modifying critical excipients on the in vivo intestinal absorption of six model compounds in rats and dogs.

Pharmaceutical excipients that may affect gastrointestinal (GI) drug absorption are called critical pharmaceutical excipients, or absorption-modifying excipients (AMEs) if they act by altering the integrity of the intestinal epithelial cell membrane. Some of these excipients increase intestinal permeability, and subsequently the absorption and bioavailability of the drug. This could have implications for both the assessment of bioequivalence and the efficacy of the absorption-enhancing drug delivery system. The absorption-enhancing effects of AMEs with different mechanisms (chitosan, sodium caprate, sodium dodecyl sulfate (SDS)) have previously been evaluated in the rat single-pass intestinal perfusion (SPIP) model. However, it remains unclear whether these SPIP data are predictive in a more in vivo like model. The same excipients were in this study evaluated in rat and dog intraintestinal bolus models. SDS and chitosan did exert an absorption-enhancing effect in both bolus models, but the effect was substantially lower than those observed in the rat SPIP model. This illustrates the complexity of the AME effects, and indicates that additional GI physiological factors need to be considered in their evaluation. We therefore recommend that AME evaluations obtained in transit-independent, preclinical permeability models (e.g. Ussing, SPIP) should be verified in animal models better able to predict in vivo relevant GI effects, at multiple excipient concentrations.

Increased understanding of intestinal drug permeability determined by the LOC-I-GUT approach using multislice computed tomography.

This study further evaluated the in vivo single-pass perfusion technique (LOC-I-GUT) in three different ways. First, the intestinal radius of the human small intestinal segment was measured on plain X-ray films; second, evaluation was performed by applying multislice computed tomography investigations; and third, furosemide was used as model drug in a transport study. In total 17 (6 + 4 +7) intubation/perfusion studies were performed in healthy volunteers. Mixobar was used as a positive radiographic contrast agent in the first six volunteers when plain film examination was made, followed by four studies using multislice computed tomography. Mantel area calculations of the perfused segment after X-ray investigations using barium as contrast were determined to be 101.0 +/- 2.9 cm2. Maximal dilatation of the closed segment with room air as contrast and using MSCT revealed a mantel area of 121.30 +/- 7.0 cm2 (P < 0.01). Thus, the mantle area increased a further 20% when the bowel was fully distended, reflecting different physiologic distention patterns for air and fluid. A jejunal single-pass perfusion study was performed in a further seven volunteers. In each experiment furosemide was perfused during 200 min, and in the treatment period (100-200 min), fexofenadine was added to the perfusion solution. The mean (+/-SD) P (eff) for furosemide was 0.17 +/- 0.07 and 0.12 +/- 0.09 x 10-4 cm/s in the control and treatment period, respectively. This study showed that the calculation of human in vivo permeability is based on physiological values, which are important for the wide application of these in vivo permeability data in physiologically based pharmacokinetic modeling.

Compound mixtures in Caco-2 cell permeability screens as a means to increase screening capacity.

The purpose of this investigation was a study of simultaneous permeability measurement using compound mixtures (cassette dosing) as an alternative to single compound evaluation in order to increase the capacity of screens for intestinal drug permeability. Drug transport across Caco-2 monolayers was studied, both in the apical to basolateral and the basolateral to apical direction. The apparent permeability coefficients for ten compounds displaying different intestinal transport mechanisms were determined, first as single compounds and then as components of a mixture. Seven beta-adrenoceptor antagonists and baclofen were analysed simultaneously using reversed phase HPLC with UV detection, D-glucose and mannitol were measured by scintillation counting. The results indicated that the Papp from the mixture as donor phase correlated well with that of the single compounds and merely small changes in the Papp of each compound were observed between the single compound and mixture experiments. This minor variation resulted in a change in rank-order of the poorly permeable compounds in the mixture, however, without affecting their association with the permeability class according to the biopharmaceutics classification system (BCS). It can be concluded that the use of compound mixtures is a suitable method for improving the capacity in permeability screens. Further improvement of the throughput may be expected upon automatisation of permeability measurements using robotics combined with increased selectivity using LC-MS analysis.