Search

Despite no broad, direct evidence in humans, there is a potential concern that surfactants alter active or passive drug intestinal permeation to modulate oral drug absorption. The purpose of this study was to investigate the impact of the surfactant polysorbate 80 on active and passive intestinal drug absorption in humans.The human (n = 12) pharmacokinetics (PK) of three probe substrates of intestinal absorption, valacyclovir, chenodeoxycholic acid (CDCA), and enalaprilat, were assessed. Endogenous bile acid levels were assessed as a secondary measure of transporter and microbiota impact.Polysorbate 80 did not inhibit peptide transporter 1 (PepT1)- or apical sodium bile acid transporter (ASBT)-mediated PK of valacyclovir and CDCA, respectively. Polysorbate 80 did not increase enalaprilat absorption. Modest increases in unconjugated secondary bile acid CPolysorbate 80 did not alter intestinal membrane fluidity or cause intestinal membrane disruption. This finding supports regulatory relief of excipient restrictions for Biopharmaceutics Classification System-based biowaivers.

PURPOSE: To demonstrate that oral drug absorption is terminated in finite time. To develop models based on biopharmaceutical/physiological and finite absorption time concepts.The models are based on i) the passive drug diffusion mechanism under the sink conditions principle ii) the rate limiting role of the drug's properties solubility and permeability and iii) the relevant restrictions associated with the gastrointestinal transit times of drug in the stomach, the small intestines and the colon. Two input functions of constant rate are considered for the absorption of drug from i) the stomach/small intestines with an upper limit of 5 h and ii) the colon with an upper limit of 30 h. Branched differential equations were written for the time course of drug in the body.Simulations were performed using different scenarios, assuming a variety of drug properties and limited or non-existent absorption from the colon. Literature oral data of cephradine, ibuprofen, flurbiprofen and itraconazole were analyzed. For all drugs examined, nice fittings of the branched differential equations to the experimental data were observed.For all drugs the absorption process was terminated in the small intestine. The meaning of partial AUCs, Cmax, tmax are questioned. Applications of these models to IVIVC are anticipated.

PURPOSE: Here, first experiences with a prototype tool for high throughput (passive) permeability profiling, a 96-well plate comprising the Permeapad® membrane, are reported. The permeabilities of a set of drugs were determined and compared to published measures of oral absorption, such as human fraction absorbed (Fa) and in vitro permeability values obtained using other tools. METHODS: The tool consists of a 96-well bottom and screen plate with the artificial, phospholipid-based barrier (Permeapad®) mounted between the plates' lower and upper compartments. The permeability of 14 model compounds including high- and low-absorption drugs, cationic, anionic, zwitterionic and neutral molecules, was determined by quantifying the compounds' transport over time, deriving the steady-state flux from the linear part of the cumulative curves and calculating the apparent permeability (Papp). The membrane structure was investigated in a high-resolution digital light microscope. RESULTS: The Permeapad® 96-well plate was found suited to distinguish high and low absorption drugs and yielded a hyperbolic correlation to Fa. The Papp values obtained were congruent with those determined with in-house prepared Permeapad® in the Franz cell set-up. Furthermore, good to excellent correlations were seen with Caco-2 permeability (R2 = 0.70) and PAMPA permeability (R2 = 0.89). Microscopic investigation of the Permeapad® barrier revealed the formation of phospholipid vesicles and myelin figures in aqueous environment. CONCLUSION: The Permeapad® 96-well plate permeation set-up is a promising new tool for rapid and reproducible passive permeability profiling.

The poor permeability of new drug candidates across intestinal epithelial membranes complicates their development in oral form. This study investigated the potential of cell-penetrating peptides (CPPs) to improve the intestinal permeation and absorption of low-permeable low-molecular-weight (low-MW) drugs. The in vitro epithelial permeation of six different drugs (metformin, risedronate, zanamivir, methotrexate [MTX], tacrolimus, and vincristine [VCR]) across Caco-2 cell monolayers was examined in the presence and absence of L- or D-penetratin, and the correlation between permeation enhancement efficiency and the properties of tested drugs was analyzed. In addition, a rat closed ileal loop absorption study was conducted to determine the in vivo effects of penetratin. MTX and VCR efficiently permeated Caco-2 monolayers in the presence of L- and D-penetratin, suggesting that CPPs enhanced the epithelial permeation of drugs with relatively high molecular weight and resultant limited intrinsic permeability. The in vivo rat closed ileal loop absorption study revealed the stimulatory effect of L- and D-penetratin on the intestinal absorption of MTX and VCR. CPPs are useful as oral absorption enhancers for low-permeable drugs.

During non-clinical and clinical development of a new molecular entity (NME), modeling and simulation (M&S) are routinely used to predict the exposure and pharmacokinetics (PK) of the drug compound in humans. The basic methodology and output are generally understood across all functional disciplines. However, this understanding is mostly restricted to traditional methods such as those in simplified kinetic models and void of adequate mechanistic foundation to address questions beyond the observed clinical data. In the past two decades, alternative and more mechanistic methods, particularly for describing absorption, distribution, excretion and metabolism (ADME) of drugs have been developed and applied under the general umbrella of physiologically-based pharmacokinetic (PBPK) methods. Their mechanistic nature gives the ability to ask many other questions which were not traditionally asked and provide some logically and evidenced-based potential answers. Whilst traditional PK methods are mainstream and understood by most scientists, mechanistic absorption models alongside other PBPK approaches are still deemed eclectic, despite making significant strides in the fundamental science as well as regulatory acceptance. On November 3rd, a short course was held at the annual American Association of Pharmaceutical Scientists (AAPS) meeting in San Antonio, Texas. The different talks were tailored to provide a basis or rationale for the subject, introduction to fundamental principles with historical perspective, a critique of the state-of-the-art, examples of successful application of the methods across different phases of the drug development process and the specific standards these mechanistic models should meet to be fully reliable from a regulatory perspective.

This report describes the effect of rhamnolipids (RLs) on the tight junctions (TJ) of the intestinal epithelium using the rat in-situ closed loop model.We investigated the transport of 5 (6)-carboxyfluorescein (CF) and fluorescein isothiocyanate-labeled dextrans with average molecular weights of 4.4 and 10 kDa (FD-4 and FD-10) when co-administered with different concentrations of RLs. Lactate dehydrogenase (LDH) leakage assay and histopathological examination of treated intestinal loops were used to assess potential toxicity of RLs. Further, the effect of kaempferol on accelerating the resealing of the tight junctions in vivo was also investigated RESULTS: Data shows that administration of different RLs concentrations (1.0-5.0% v/v) increased CF absorption through rat intestine by 2.84- and 15.82-folds with RLs concentrations of 1.0% and 5.0% v/v, respectively. RLs exhibited size-dependent increase on FD-4 and FD-10 absorption. Dosing RLs at 1.0% v/v didn't cause a significant LDH leakage or histopathological changes to intestinal mucosa compared to higher concentrations, which showed a progressive damaging effect. Using kaempferol, a natural flavonoid that stimulates the assembly of the TJs, proved to enhance the recovery of barrier properties of the intestinal mucosa treated with high concentrations of RLs (2.5% and 5% v/v).These results collectively illustrate the ability of RLs to enhance oral bioavailability of different molecules across the intestinal epithelial membrane in a concentration- and time-dependent fashion.

Attempts to formulate acyclovir to improve its bioavailability and reduce the frequency of dosing from the present q4h have not materialized.It was thought that an approach using permeability enhancer such as chitosan may impart improved absorption profile to acyclovir; however, the recently published pharmacokinetic data suggested otherwise. The lack of promise of chitosan formulation was attributed to the muco-adhesive properties of chitosan to hold off acyclovir and preventing its transport across the gastrointestinal tract. However, the above hypothesis was refuted by another published human pharmacokinetic study of fexofenadine formulated with chitosan formulation - in this work it was unambiguously shown that chitosan helped in enhanced absorption of fexofenadine which is a well-known Pgp substrate. If one examines the pharmacokinetic disposition of acyclovir, it is clear that renal elimination is so rapid necessitating frequent dosing of acyclovir. In summary, the ability of chitosan based formulations to aid in the oral absorption of drugs may be drug dependent as enumerated by data obtained from acyclovir and fexofenadine. While chitosan favourably improved the pharmacokinetics of fexofenadine, acyclovir may not be ideal for chitosan type of formulation.The choice of the drug and the formulation type intended to deliver the drug need to be made in a diligent and pragmatic fashion.

Purpose To examine the utility of human plasma as an assay medium in Caco-2 permeability studies to overcome poor mass balance and inadequate sink conditions frequently encountered with lipophilic compounds. Methods Caco-2 permeability was assessed for reference compounds with known transport mechanisms using either pH 7.4 buffer or human plasma as the assay medium in both the apical and basolateral chambers. When using plasma, Papp values were corrected for the unbound fraction in the donor chamber. The utility of the approach was assessed by measuring the permeability of selected antimalarial compounds using the two assay media. Results Caco-2 cell monolayer integrity and P-gp transporter function were unaffected by the presence of human plasma in the donor and acceptor chambers. For many of the reference compounds having good mass balance with buffer as the medium, higher Papp values were observed with plasma, likely due to improved acceptor sink conditions. The lipophilic antimalarial compounds exhibited low mass balance with buffer, however the use of plasma markedly improved mass balance allowing the determination of more reliable Papp values. Conclusions The results support the utility of human plasma as an alternate Caco-2 assay medium to improve mass balance and permeability measurements for lipophilic compounds. Electronic supplementary material The online version of this article (10.1007/s11095-018-2493-3) contains supplementary material, which is available to authorized users.

To develop novel bioactive-chylomicrons to solve oral delivery obstacles of Berberine chloride and target the lymphatic system. Berberine-loaded bioactive-chylomicrons were prepared and underwent full in vitro characterization. Intestinal permeability was appraised via both non-everted gut sac model and Caco-2 cell model. Furthermore, Bioactive-chylomicrons’ cellular uptake and distribution were examined by laser scanning confocal microscopy. Finally, a novel chylomicron flow-blockage assay on tissue and cellular levels were elaborated to assess the lymphatic targeting ability. Berberine-loaded chylomicrons showed spherical vesicles of size (175.6 nm), PDI (0.229), zeta potential (−16 .6 mV) and entrapment efficiency (95.5%). Ex-vivo intestinal permeability studies demonstrated 10.5 fold enhancement in permeability of Berberine-loaded chylomicrons over free Berberine. Moreover, Caco-2 studies revealed significant improvement in chylomicrons’ permeability and cellular uptake. Furthermore, confocal microscopy analyses revealed 2 fold increase in berberine-loaded chylomicrons’ intracellular fluorescence. Lymphatic targeting models were successfully elaborated using cycloheximide protein synthesis inhibitor. Such models demonstrated 47 and 27.5% reduction in ex-vivo and Caco-2 permeability respectively. Finally, a good rank order correlation was established between different permeability assessment techniques. The findings shed the light on the underlying mechanisms of Berberine bioavailability improvement. Consequently, berberine-loaded chylomicrons could be considered as promising bioactive-nanocarriers for Berberine lymphatic targeting and bioavailability improvement.

The effective rat intestinal permeability (P eff ) was deconvolved using a biophysical model based on parameterized paracellular, aqueous boundary layer, transcellular permeabilities, and the villus-fold surface area expansion factor. Four types of rat intestinal perfusion data were considered: single-pass intestinal perfusion (SPIP) in the jejunum (n = 40), and colon (n = 15), closed-loop (Doluisio type) in the small intestine (n = 78), and colon (n = 74). Moreover, in vitro Caco-2 permeability values were used to predict rat in vivo values in the rat data studied. Comparable number of molecules permeate via paracellular water channels as by the lipoidal transcellular route in the SPIP method, although in the closed-loop method, the paracellular route appears dominant in the colon. The aqueous boundary layer thickness in the small intestine is comparable to that found in unstirred in vitro monolayer assays; it is thinner in the colon. The mucosal surface area in anaesthetized rats is 0.96–1.4 times the smooth cylinder calculated value in the colon, and it is 3.1–3.6 times in the small intestine. The paracellular permeability of the intestine appeared to be greater in rat than human, with the colon showing more leakiness (higher P para ) than the small intestine. Based on log intrinsic permeability values, the correlations between the in vitro and in vivo models ranged from r2 0.82 to 0.92. The SPIP-Doluisio method comparison indicated identical log permeability selectivity trend with negligible bias.

Purpose Currently, the FDA allows biowaivers for Class I (high solubility and high permeability) and Class III (high solubility and low permeability) compounds of the Biopharmaceutics Classification System (BCS). Scientific evidence should be provided to support biowaivers for BCS Class I and Class III (high solubility and low permeability) compounds. Methods Data on the effects of excipients on drug permeability are needed to demonstrate that commonly used excipients do not affect the permeability of BCS Class III compounds, which would support the application of biowaivers to Class III compounds. This study was designed to generate such data by assessing the permeability of four BCS Class III compounds and one Class I compound in the presence and absence of five commonly used excipients. Results The permeability of each of the compounds was assessed, at three to five concentrations, with each excipient in two different models: Caco-2 cell monolayers, and in situ rat intestinal perfusion. No substantial increases in the permeability of any of the compounds were observed in the presence of any of the tested excipients in either of the models, with the exception of disruption of Caco-2 cell monolayer integrity by sodium lauryl sulfate at 0.1 mg/ml and higher. Conclusion The results suggest that the absorption of these four BCS Class III compounds would not be greatly affected by the tested excipients. This may have implications in supporting biowaivers for BCS Class III compounds in general.

A poorly water soluble acidic active pharmaceutical ingredient (API) was transformed into an ionic liquid (IL) aiming at faster and higher oral availability in comparison to a prodrug.API preparations were characterized in solid state by single crystal and powder diffraction, NMR, DSC, IR and in solution by NMR and ESI-MS. Dissolution and precipitation kinetics were detailed as was the role of the counterion on API supersaturation. Transepithelial API transport through Caco-2 monolayers and counterion cytotoxicity were assessed.The mechanism leading to a 700 fold faster dissolution rate and longer duration of API supersaturation of the ionic liquid in comparison to the free acid was deciphered. Transepithelial transport was about three times higher for the IL in comparison to the prodrug when substances were applied as suspensions with the higher solubility of the IL outpacing the higher permeability of the prodrug. The counterion was nontoxic with IC50 values in the upper μM / lower mM range in cell lines of hepatic and renal origin as well as in macrophages.The IL approach was instrumental for tuning physico-chemical API properties, while avoiding the inherent need for structural changes as required for prodrugs.

Exenatide, a potent insulinotropic agent, can be used for the treatment of non-insulin-dependent diabetes mellitus. However, the need for frequent injections seriously limits its therapeutic utility. The aim of present report was to develop an orally available exenatide formulation using goblet cell-targeting nanoparticles (NPs). The exenatide-loaded nanoparticles were prepared with modified chitosan which was conjugated with a goblet cell-target peptide, CSKSSDYQC (CSK) peptide. The CSK-chitosan nanoparticles shown reduced chitosan toxicity and enhanced the permeation of drugs across the Caco-2/HT-29 co-cultured cell monolayer, which simulated the intestinal epithelium. Following the oral administration of near-infrared fluorescent probe Cy-7-loaded NPs to mice, the distribution of the drugs was investigated with a near-infrared in vivo image system (FX Pro, Bruker, USA). The results showed that Cy-7 fluorescence disseminated from the oesophagus, then to stomach and small intestine and then was absorbed into hepatic, finally into the bladder; over time, Cy-7 was metabolized and excreted. The bioavailability of the modified nanoparticles was found to be 1.7-fold higher compared with the unmodified ones, and the hypoglycemic effect was also better. CSK peptide-modified chitosan nanoparticles could be a potential therapeutics for Type II diabetes patients.

We developed simulation and modeling methods to predict the in vivo pharmacokinetic profiles of acyclovir, following escalating oral doses of valacyclovir, in wildtype and Pept1 knockout mice. We also quantitated the contribution of specific intestinal segments in the absorption of valacyclovir in these mice. Simulations were conducted using a mechanistic advanced compartmental absorption and transit (ACAT) model implemented in GastroPlus™. Simulations were performed for 3 h post-dose in wildtype and Pept1 knockout mice following single oral doses of 10, 25, 50 and 100 nmol/g valacyclovir, and compared to experimentally observed plasma concentration-time profiles of acyclovir. Good fits were obtained in wildtype and Pept1 knockout mice. Valacyclovir was primarily absorbed from duodenum (42%) and jejunum (24%) of wildtype mice, with reduced uptake from ileum (3%) and caecum/colon (1%), for a total of 70% absorption. In contrast, the absorption of valacyclovir in Pept1 knockout mice was slow and sustained throughout the entire intestinal tract in which duodenum (4%), jejunum (14%), ileum (10%) and caecum/colon (12%) accounted for a total of 40% absorption. The ACAT model bridged the gap between in situ and in vivo experimental findings, and facilitated our understanding of the complicated intestinal absorption processes of valacyclovir.

A major obstacle preventing oral administration of macromolecular therapeutics is poor absorption across the intestinal epithelium into the bloodstream. One strategy to improve transport across this barrier is the use of chemical permeation enhancers. Several molecular families with permeation enhancing potential have been identified previously, including piperazines. In particular, 1-phenylpiperazine has been shown to enhance transepithelial transport with minimal cytotoxicity compared to similarly effective molecules. To better understand how the chemistry of 1-phenylpiperazine affects its utility as an intestinal permeation enhancer, this study examined a small library of 13 derivatives of 1-phenylpiperazine. The efficacy and cytotoxicity of 13 phenylpiperazine compounds were assessed in a Caco-2 model of the intestinal epithelium. Efficacy was measured using the paracellular diffusion marker calcein as well as by immunostaining and confocal imaging of Caco-2 monolayers. Of the 13 derivatives, two enhanced the permeability of the fluorescent marker calcein over 100-fold. It was found that hydroxyl or primary amine substitutions on the phenyl ring significantly increased toxicity, while aliphatic substitutions resulted in efficacy and toxicity profiles comparable to 1-phenylpiperazine. Several potent derivatives, including 1-methyl-4-phenylpiperazine and 1-(4-methylphenyl)piperazine, displayed lower toxicity than 1-phenylpiperazine, suggesting promise in future applications.

Evaluate the impact of luminal micellar phase on passive permeability of five lipophilic (1.9 ≤ clogP ≤ 9.0) small molecules using biorelevant media and evaluate the impact of luminal coarse lipid particles on danazol permeability after oral administration of a triglyceride solution to fed adults using PAMPA. Permeability of carbamazepine, furosemide, danazol, and Compound A was evaluated using Prisma™ HT, FaSSIF-V2, and FeSSIF-V2 in the donor compartment. Compound B could not be tested using Prisma™ HT, due to negligible solubility. Individual intestinal aspirates collected after administration of danazol solution in the olive oil portion of a meal and corresponding micellar phases were subjected to PAMPA. Commercially available Acceptor Sink Buffer was used in all cases. Unlike with furosemide (under constant pH) and Compound B, permeability of carbamazepine, danazol, and Compound A steadily decreased in the presence of increasing micelle concentration of media. Danazol permeability from aspirates was reduced compared to that from micellar phases; fluxes were similar. Using PAMPA, the impact of luminal micellar phase on passive permeability of lipophilic molecules varies with the molecule. After administration of a triglyceride solution of danazol, high danazol concentrations in coarse lipid particles balance in terms of drug flux the reduced permeability.

To develop an in vitro methodology for prediction of concentrations and potential precipitation of highly permeable, lipophilic weak bases in fasted upper small intestine based on ketoconazole and dipyridamole luminal data. Evaluate usefulness of methodology in predicting luminal precipitation of AZD0865 and SB705498 based on plasma data.A three-compartment in vitro setup was used. Depending on the dosage form administered in in vivo studies, a solution or a suspension was placed in the gastric compartment. A medium simulating the luminal environment (FaSSIF-V2plus) was initially placed in the duodenal compartment. Concentrated FaSSIF-V2plus was placed in the reservoir compartment.In vitro ketoconazole and dipyridamole concentrations and precipitated fractions adequately reflected luminal data. Unlike luminal precipitates, in vitro ketoconazole precipitates were crystalline. In vitro AZD0865 data confirmed previously published human pharmacokinetic data suggesting that absorption rates are not affected by luminal precipitation. In vitro SB705498 data predicted that significant luminal precipitation occurs after a 100 mg or 400 mg but not after a 10 mg dose, consistent with human pharmacokinetic data.An in vitro methodology for predicting concentrations and potential precipitation in fasted upper small intestine, after administration of highly permeable, lipophilic weak bases in fasted upper small intestine was developed and evaluated for its predictability in regard to luminal precipitation.

The aim of this study was to evaluate the oral exposure predictions obtained early in drug discovery with a generic GastroPlus Advanced Compartmental And Transit (ACAT) model based on the in vivo intravenous blood concentration-time profile, in silico properties (lipophilicity, pKa) and in vitro high-throughput absorption-distribution-metabolism-excretion (ADME) data (as determined by PAMPA, solubility, liver microsomal stability assays). The model was applied to a total of 623 discovery molecules and their oral exposure was predicted in rats and/or dogs. The predictions of Cmax, AUClast and Tmax were compared against the observations. The generic model proved to make predictions of oral Cmax, AUClast and Tmax within 3-fold of the observations for rats in respectively 65%, 68% and 57% of the 537 cases. For dogs, it was respectively 77%, 79% and 85% of the 124 cases. Statistically, the model was most successful at predicting oral exposure of Biopharmaceutical Classification System (BCS) class 1 compounds compared to classes 2 and 3, and was worst at predicting class 4 compounds oral exposure. The generic GastroPlus ACAT model provided reasonable predictions especially for BCS class 1 compounds. For compounds of other classes, the model may be refined by obtaining more information on solubility and permeability in secondary assays. This increases confidence that such a model can be used in discovery projects to understand the parameters limiting absorption and extrapolate predictions across species. Also, when predictions disagree with the observations, the model can be updated to test hypotheses and understand oral absorption.

The aim of the present study is to evaluate the formulation effect on the oral absorption of poorly water-soluble drugs using a dissolution/permeation system (D/P system). This D/P system, consisting of apical and basal chambers and a Caco-2 cell monolayer mounted between chambers, can be used to perform simultaneous analysis of drug dissolution and permeation process of drugs applied as various dosage forms. Oral administration study with rats was also performed for both drugs as the same dosage forms. When danazol, a low-soluble and high-permeable drug, was applied to the D/P system as various formulations, dissolved and permeated amounts were significantly high compared with those from a suspension form. On the other hand, whereas the dissolved amount of pranlukast, a low-soluble and low-permeable drug, was significantly increased by formulations, there were no significant changes observed in the permeated amount between suspension and formulation. The oral availability of danazol was significantly increased by formulations but not pranlukast, which corresponded well to in vitro evaluations. These results indicated that the D/P system might be applicable for selection of formulation on the basis of physicochemical drug properties.

To investigate the potential interaction between grapefruit juice (GFJ) and the oral microtubule polymerization inhibitor colchicine, a P-gp and CYP3A4 substrate.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.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 (IC(50) 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 (IC(50) values of 90, 592 and 11.6 microM respectively). Ten percent GFJ doubled colchicine rat in-situ ileal permeability, and increased 1.5-fold jejunal permeability.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.

Nonlinear oral absorption due to poor solubility often impedes drug development. The purpose of this study was to elucidate the rate-limiting process in oral absorption of Biopharmaceutical Classification System (BCS) class II (low solubility–high permeability) drugs in order to predict nonlinear absorption of dose caused by solubility-limited absorption. Oral absorption of danazol, griseofulvin, and aprepitant was predicted from a miniscale dissolution test and a physiologically-based model. The effect of particle size reduction and dose increase on absorption was investigated in vitro and in vivo to clarify the rate-limiting steps of dissolution-rate-limited and solubility-limited absorption. The rate-limiting steps of oral absorption were simulated and increase in the dissolution rate and administration dose showed a shift from dissolution rate-limited to solubility-limited absorption. In the study in dogs, particle size reduction improved the oral absorption of large particle drugs but had little effect on small particle drugs. Dose nonlinearity was observed with small particles at a high dose. Our model quantitatively predicted results observed in vivo, including but not exclusively, dissolution-rate-limited and solubility-limited absorption. The present study provides a powerful tool to predict dose nonlinearity and will aid in the success of BCS class II drug development.

Melittin has shown potential as a non-cytotoxic absorption enhancer in Caco-2 monolayers. Our objectives were to assess in vitro efficacy and cytotoxicity of melittin in two intestinal permeability models and investigate the potential mechanism by which melittin might enhance gastrointestinal absorption.The effects of melittin were examined in the mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12), using transepithelial electrical resistance (TER), transmission electron microscopy (TEM) and the MTT viability assay. The effects of melittin on TER, permeability and short circuit current (Isc) were also investigated in rat colon mucosae mounted in Ussing chambers. Ion transporting capacity of tissue was measured in response to secretagogues as surrogate markers of cytotoxicity. Melittin stability was examined by a means of a hemolytic assay. The mechanism by which melittin decreases TER across the rat mucosa was examined with a range of enzymatic inhibitors.Apical addition of melittin resulted in a reversible non-cytotoxic concentration-dependent decrease in TER across E12 monolayers, which was independent of the presence of mucus. Apical addition of melittin reduced TER and increased the permeability of [(14)C]-mannitol across rat colonic mucosae. The melittin-induced drop in TER in rat colon was significantly attenuated by W7 suggesting partial mediation by calmodulin.The rapid and reversible nature of melittin's permeation enhancing properties and its limited cytotoxicity in polarized intestinal epithelia, suggests a potential drug delivery role for the peptide in oral formulations of poorly absorbed drugs.

Melittin is an amphipathic antimicrobial peptide which has been shown to enhance the permeability of mannitol and reduce transepithelial electrical resistance (TER) across Caco-2 monolayers. The aim of this work was to further examine the potential of melittin as a paracellular permeability enhancer and to investigate the mechanism of interaction with tight junction proteins in Caco-2.The permeability of a range of fluorescent markers of differing molecular weights across monolayers was examined and immunofluorescence and western blotting analysis of tight junction proteins were also carried out. The mechanism of TER reduction was also examined using cell signalling inhibitors.Apical but not basolateral addition of melittin increased the permeability of a range FITC-dextrans (4-70 kDa) across monolayers. Melittin effects were reversible and no cytotoxicity was evident in polarized Caco-2 epithelia at the concentrations used. Altered expression of ZO-1, E-cadherin and F-actin was also detected. The phospholipase A2 inhibitors, aristolochic acid and indomethacin and the cyclooxygenase inhibitor, piroxicam, partially attenuated melittin-induced TER reduction, suggesting that part of the mechanism by which melittin opens tight junctions involves prostaglandin signalling.Apically-added melittin opens tight junctions, causing dramatic TER reductions with significant increases in flux of dextrans. These effects appear mediated in part via PLA2 and involve alterations in specific tight junction proteins.

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.

This study was conducted to develop and validate a dog colon model that predicts colon permeability in humans.The following compounds were studied: Class 1 highly soluble (HS)/highly permeable (HP): aminophylline, propranolol, CP-409092; Class 2 LS/HP: nifedipine; trovafloxacin, sertraline; Class 3 HS/LP: azithromycin, atenolol, CP-331684, CP-424391; Class 4 LS/LP: CJ-13610. Administration to dogs was made 30 cm cranial to the anal sphincter with a lubricated Schott Model VFS-5 flexible endoscope. The bioavailability of the compound following the colon administration in dogs, relative to the same formulation administered orally (relative bioavailability), was determined.Except for atenolol, a small hydrophillic molecule, the relative bioavailability from administration to the colon of the dog correlated well with the following compound properties: high solubility and high, passive permeabilityhigh solubility, low permeabilitylow solubility, high, passive permeability approximately low solubility, low permeability.The dog colon model is proposed as a surrogate for human intubation studies when the controlled release candidate falls in BCS Classes 2 (LS/HP), 3 (HS/LP), and 4 (LS/LP). However, no human intubation or dog colon studies are required for Class 1 (HS/HP), as these compounds are likely to be well absorbed from the colon.

The aim of this study was to investigate transepithelial ionic permeation via the paracellular pathway of human Caco-2 epithelial monolayers and its contribution to absorption of the base guanidine.Confluent monolayers of Caco-2 epithelial cells were mounted in Ussing chambers and the transepithelial conductance and electrical potential difference (p.d.) determined after NaCl dilution or medium Na substitution (bi-ionic conditions). Guanidine absorption (Ja-b) was measured +/- transepithelial potential gradients using bi-ionic p.d.'s.Basal NaCl replacement with mannitol gives a transepithelial dilution p.d. of 28.0 +/- 3.1 mV basal solution electropositive (PCl/PNa = 0.34). Bi-ionic p.d.'s (basal replacements) indicate a cation selectivity of NH4+K+ approximately Cs+Na+Li+tetraethylammonium+N-methyl-D: -glucamine+ approximately choline+. Transepithelial conductances show good correspondence with bi-ionic potential data. Guanidine Ja-b was markedly sensitive to imposed transepithelial potential difference. The ratio of guanidine to mannitol permeability (measured simultaneously) increased from 3.6 in the absence of an imposed p.d. to 13.8 (basolateral negative p.d.).Hydrated monovalent ions preferentially permeate the paracellular pathway (Eisenman sequence 2 or 3). Guanidine may access the paracellular pathway because absorptive flux is sensitive to the transepithelial potential difference. An alternative method to assess paracellular-mediated flux of charged organic molecules is suggested.

P-glycoprotein (P-gp) is made responsible for the limited oral bioavailability of P-gp substrates like peptidic HIV protease inhibitors (PIs). With respect to combined application of two PIs in antiretroviral regimes, we first investigated the influences on intestinal saquinavir uptake using different PIs in in situ perfusion studies.Perfusion experiments were carried out in three intestinal segments with P-gp substrates talinolol and saquinavir using fixed concentrations of PIs and with each varying concentrations in the jejunum and ileum. Furthermore, cellular uptake of fluorescent P-gp substrate rhodamine-123 and MRP-substrate carboxyfluorescein has each been quantified in P-gp and MRP-expressing cells by flow cytometry under co-administration of PIs.Increase of calculated permeabilities of P-gp-specific substrate talinolol was found under co-administration of both PIs, ritonavir and H17, with highest absorption rates in the ileal and colon segment. H17 proved to be a better P-gp inhibitor than ritonavir by resulting IC50 values and also in the cellular uptake of rhodamine. Similar increases of permeabilities in ileum and colon have also been found for saquinavir as P-gp as well as MRP-substrate with differences in the jejunal uptake, which was found higher for H17. Additional MRP-inhibitory activities of H17 were proved by increasing cellular uptake rates of carboxyfluorescein in MRP-expressing cells.The investigated PIs were characterized as effective P-gp inhibitors in the intestinal absorption of P-gp substrates. H17 showed MRP-inhibitory effects that also favor intestinal drug absorption of corresponding substrates. With respect to combined therapeutic application of PIs, compounds like H17 raise hopes for improved bioavailability of poorly absorbed compounds.

Purpose. To set up a theoretical basis for identifying biowaivers among Class II drugs and apply the methodology developed to nonsteroidal anti-inflammatory drugs (NSAIDs). Methods. The dynamics of the two consecutive drug processes dissolution and wall permeation are considered in the time domain of the physiologic transit time using a tube model of the intestinal lumen. The model considers constant permeability along the intestines, a plug flow fluid with the suspended particles moving with the fluid, and dissolution in the small particle limit. The fundamental differential equation of drug dissolution-uptake in the intestines is expressed in terms of the fraction of dose dissolved. Results. The fundamental parameters, which define oral drug absorption in humans resulting from this analysis, are i) the formulation-related factors, dose, particle radius size, and ii) the drug-related properties, dimensionless solubility/dose ratio (1/q), and effective permeability. Plots of dose as a function of (1/q) for various particle sizes unveil the specific values of these meaningful parameters, which ensure complete absorption for Class II drugs [(1/q) < 1]. A set of NSAIDs were used to illustrate the application of the approach in identifying biowaivers among the NSAIDs. Conclusions. The underlying reason for a region of fully absorbed drugs in Class II originates from the dynamic character of the dissolution-uptake processes. The dynamic character of the approach developed allows identification of biowaivers among Class II drugs. Several biowaivers among the NSAIDs were identified using solubility data at pH 5.0 and in fed-state-simulated intestinal fluid at pH 5.0. The relationships of formulation parameters, dose, particle radius, and the drug properties, dimensionless solubility/dose ratio (1/q), and permeability with the fraction of dose absorbed for drugs with low 1/q values [(1/q) < 1] can be used as guidance for the formulation scientist in the development phase.

We describe here a new method to estimate the oral drug permeability from the small intestine using surface plasmon resonance (SPR) technology. The interaction between drugs and brush border membrane (BBM) surfaces immobilized on biosensor chip were evaluated by measuring the SPR response signal.BBM vesicles, isolated from Sprague-Dawley (SD) rats, were immobilized onto the L1 chip composed of dextran derivatives with modified lipophilic residues. A SPR (BIAcore 3000) was used with L1 chip, and it was carried out in a running buffer, HEPES-buffered saline containing 0.1% DMSO. Fourteen drugs for the SPR experiments were flowed over the BBM immobilized L1 chip, and the response levels according to the BBM surfaces were evaluated directly in a continuous flow system.The immobilized BBM surface on L1 chip was very stable, and it was regenerated by injecting a new BBM vesicle solution. It was evident that drug binding events, using BBM surfaces, directly provides information that predicts the Fa value in human for transcellularly absorbed drugs. The throughput to assay each drug at a single concentration is 100 drugs for 24 h.The interaction between drugs and small intestinal surfaces was successfully assayed using SPR technology, and this SPR analysis exhibited advantages: lack of labeling requirements, the real-time acquirement of various results, and the repeated use for various drugs.

Previous attempts to ascertain the role of uptake and efflux transporters in the oral absorption of anti-HIV dideoxypurine nucleosides have been inconclusive. A novel in situ intestinal perfusion technique with complete mesenteric arterial/venous cannulation was used to examine the asymmetry of ileal dideoxynucleoside permeability under near in vivo conditions.Intestinal perfusions were performed in the rat ileum, with cannulation of the artery and vein immediately entering and leaving the segment. Urea and mannitol were used as passive permeability markers, and the directional transport of 2',3'-dideoxyinosine (ddI), 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), and 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA) were examined.Urea and mannitol exhibited symmetric permeability (PLtoB = PBtoL), whereas F-ddI and ddI showed significantly higher permeability in blood-to-lumen transport (PBtoLPLtoB). PBtoL for F-ddA exceeded PLtoB, but the difference did not reach significance at p0.05. PBtoL for ddI was demonstrated to be saturable with increasing ddI concentrations, but PLtoB was independent of ddI concentration. PBtoL for ddI was shown to be dependent on sodium concentration and inhibited by probenecid.Symmetric transport was demonstrated for urea and mannitol as expected for these passive permeability markers. F-ddI and ddI were shown to be preferentially transported from blood to lumen. The basolateral to luminal transport of ddI is saturable, inhibited by probenecid, and sodium ion dependent. These results are consistent with carrier-mediated uptake on the basolateral membrane.

To develop a quantitative biopharmaceutics drug classification system (QBCS) based on fundamental parameters controlling rate and extent of absorption.A simple absorption model that considers transit flow, dissolution, and permeation processes stochastically was used to illustrate the primary importance of dose/solubility ratio and permeability on drug absorption. Simple mean time considerations for dissolution, uptake, and transit were used to identify relationships between the extent of absorption and a drug's dissolution and permeability characteristics.The QBCS developed relies on a (permeability, dose/ solubility ratio) plane with cutoff points 2 x 10(-6)-10(-5) cm/s for the permeability and 0.5-1 (unitless) for the dose/solubility ratio axes. Permeability estimates, P(app) are derived from Caco-2 studies, and a constant intestinal volume content of 250 ml is used to express the dose/solubility ratio as a dimensionless quantity, q. A physiologic range of 250-500 ml was used to account for variability in the intestinal volume. Drugs are classified into the four quadrants of the plane around the cutoff points according to their P(app), q values, establishing four drug categories. i.e., I (P(app)10(-5) cm/s, qor = 0.5), II (P(app)10(-5) cm/s, q1), III (P(app)2 x 10(-6) cm/s. qor = 0.5), and IV (P(app)2 x 10(-6) cm/s, q1). A region for borderline drugs (2 x 10(-6)P(app)10(-5) cm/s, 0.5q1) was defined too. For category I, complete absorption is anticipated, whereas categories II and III exhibit dose/ solubility ratio-limited and permeability-limited absorption, respectively. For category IV, both permeability and dose/solubility ratio are controlling drug absorption. Semiquantitative predictions of the extent of absorption were pointed out on the basis of mean time considerations for dissolution, uptake, and transit in conjunction with drug's dose/solubility ratio and permeability characteristics. A set of 42 drugs were classified into the four categories, and the predictions of intestinal drug absorption were in accord with the experimental observations.The QBCS provides a basis for compound classification into four explicitly defined drug categories using the fundamental biopharmaceutical properties, permeability, and dose/solubility ratio. Semiquantitative predictions for the extent of absorption are essentially based on these drug properties, which either determine or are strongly related to the in vivo kinetics of drug dissolution and intestinal wall permeation.

The objectives of this study were to evaluate the effects of intestinal lumen pH, food intake, and acid-reducing agents on the intestinal permeability of atazanavir, an HIV-1 protease inhibitor.Atazanavir permeability across Caco-2 cell monolayers (P app) and in situ steady-state permeability across rat jejunum and ileum (P eff) were evaluated in buffers of varied pH (4.5-8.5), in fasted- or fed-state simulated intestinal fluid, or in presence of acid-reducing drugs (e.g., omeprazole).In vitro accumulation and apical-to-basolateral P app of atazanavir increased with decreasing pH. This effect appeared to be associated with lower atazanavir efflux by P-glycoprotein at acidic pH (5.5) compared to neutral pH. In situ atazanavir P eff across rat jejunum and ileum also decreased 2.7 and 2.3-fold, respectively, when pH was increased from 4.5 to 8.5. Several acid-reducing agents (e.g., omeprazole) moderately inhibited atazanavir efflux in Caco-2 monolayers; however, this effect was not observed in situ. Fed-state buffer significantly increased atazanavir apical-to-basolateral P app (p 0.001) and in situ P eff (p 0.05) compared to fasted-state buffer.Atazanavir permeability is sensitive to changes in intestinal lumen pH. This pH-sensitivity may contribute to atazanavir clinical interactions with acid-reducing agents and variable oral bioavailability.

Existing PBPK models incorporating intestinal first-pass metabolism account for effect of drug permeability on accessible absorption surface area by use of “effective” permeability, P eff , without adjusting number of enterocytes involved in absorption or proportion of intestinal CYP3A involved in metabolism. The current model expands on existing models by accounting for these factors. The PBPK model was developed using SAAM II. Midazolam clinical data was generated at GlaxoSmithKline. The model simultaneously captures human midazolam blood concentration profile and previously reported intestinal availability, using values for CYP3A CLu int , permeability and accessible surface area comparable to literature data. Simulations show: (1) failure to distinguish absorbing from non-absorbing enterocytes results in overestimation of intestinal metabolism of highly permeable drugs absorbed across the top portion of the villous surface only; (2) first-pass extraction of poorly permeable drugs occurs primarily in enterocytes, drugs with higher permeability are extracted by enterocytes and hepatocytes; (3) CYP3A distribution along crypt-villous axes does not significantly impact intestinal metabolism; (4) differences in permeability of perpetrator and victim drugs results in their spatial separation along the villous axis and intestinal length, diminishing drug-drug interaction magnitude. The model provides a useful tool to interrogate intestinal absorption/metabolism of candidate drugs.

To investigate the role of membrane-associated drug transporters in regulating the intestinal absorption of the HIV-1 protease inhibitor, atazanavir, and assess the potential contribution of these transporters in clinical interactions of atazanavir with other protease inhibitors and tenofovir disoproxil fumarate (TDF). Intestinal permeability of atazanavir was investigated in vitro, using the Caco-2 cell line system grown on Transwell inserts, and in situ, by single-pass perfusion of rat intestinal segments, jejunum and ileum, in the absence or presence of standard transporter inhibitors or antiretroviral drugs. Atazanavir accumulation by Caco-2 cells was susceptible to inhibition by P-glycoprotein and organic anion transporting polypeptide (OATP) family inhibitors and several antiretroviral drugs (protease inhibitors, TDF). The secretory flux of atazanavir (basolateral-to-apical Papp) was 11.7-fold higher than its absorptive flux. This efflux ratio was reduced to 1.5–1.7 in the presence of P-glycoprotein inhibitors or ritonavir. P-glycoprotein inhibition also resulted in 1.5–2.5-fold increase in atazanavir absorption in situ. Co-administration of TDF, however, reduced atazanavir intestinal permeability by 13–49%, similar to the effect observed clinically. Drug transporters such as P-glycoprotein and OATPs regulate intestinal permeability of atazanavir and may contribute to its poor oral bioavailability and drug-drug interactions with other protease inhibitors and TDF.

To investigate the hypothesis that intestinal bicarbonate secretions precipitate calcium as the carbonate salt, thereby resulting in poor absorption (20–40%) from calcium supplements. The in vitro effect of calcium dose and bicarbonate secretion rate on soluble calcium was determined by neutralizing elemental Ca2+(250, 475, and 630 mg) in 0.1 N HCl to pH 7 with a bicarbonate secretion rate of 0.12 or 1.2 mEq/min. P CO2 and pH of the solutions were monitored. Soluble calcium was analyzed using atomic absorption spectrometry. Additionally, the transport of calcium across Caco-2 cell monolayers was determined. Calcium from a 250 mg dose remained soluble during bicarbonate secretion, regardless of rate. Once the dose increased, the calcium remaining in solution decreased during neutralization with bicarbonate. The Ca2+/CaHCO3 + ratio had no effect on calcium permeation across Caco-2 cell monolayers. The physicochemical mechanism of intestinal calcium precipitation supports published clinical data by suggesting that once the solubility product of calcium carbonate is reached, increasing the calcium dose results in significant precipitation at intestinal pH values.

Traditionally, the oral route cannot be employed for the delivery of macromolecular drugs such as proteins and peptides due, in large part, to limited transport across the epithelial membrane. This particular challenge can potentially be addressed through the use of chemical permeation enhancers, which affect transcellular and/or paracellular transport routes. Although certain permeation enhancers have been proposed for use in oral delivery, potential for application is often unclear when the route of enhancer action is unknown. A combination of theory and experiments was developed for determining mechanism of enhancer action. The effect of 51 enhancers on Caco-2 cells was studied using TEER, MTT, and LDH assays. The mechanistic details of intestinal permeability enhancement were uncovered for a broad set of enhancers in vitro. Understanding gained from enhancer mechanisms enabled the deduction of structure–function relationships for hydrophilic and hydrophobic permeation enhancers as well as the identification of a transcellular enhancer, 0.01% (w/v) palmityldimethyl ammonio propane sulfonate, which enabled the non-cytotoxic intracellular delivery of a model drug. The results presented here emphasize the importance of understanding enhancer mechanism and uncover a zwitterionic surfactant capable of safely and effectively achieving intraepithelial drug delivery in vitro.

We recommend that regulatory agencies add the extent of drug metabolism (i.e., >or=90% metabolized) as an alternate method in defining Class 1 marketed drugs suitable for a waiver of in vivo studies of bioequivalence. That is, >or=90% metabolized is an additional methodology that may be substituted for >or=90% absorbed. We propose that the following criteria be used to define>or=90% metabolized for marketed drugs: Following a single oral dose to humans, administered at the highest dose strength, mass balance of the Phase 1 oxidative and Phase 2 conjugative drug metabolites in the urine and feces, measured either as unlabeled, radioactive labeled or nonradioactive labeled substances, account for >or=90% of the drug dosed. This is the strictest definition for a waiver based on metabolism. For an orally administered drug to be >or=90% metabolized by Phase 1 oxidative and Phase 2 conjugative processes, it is obvious that the drug must be absorbed. This proposal, which strictly conforms to the present>or=90% criteria, is a suggested modification to facilitate a number of marketed drugs being appropriately assigned to Class 1.

The use of intestinal permeation enhancers to overcome the absorption challenges associated with oral drug delivery has been hampered by the notion that enhancer efficacy is directly linked to toxicity. This study attempts to gain insight into the principles governing the potency and toxicity behavior of enhancers. Fifty-one enhancers were selected from 11 chemical categories and their potency and toxicity were analyzed in Caco-2 monolayers at concentrations spanning three orders of magnitude. A small but significant fraction of the 153 enhancer formulations studied demonstrated unexpected but desired behavior, that is, substantial efficacy without marked toxicity. Our results revealed that both chemical category and concentration proved critical in determining the usefulness of many enhancers, and the concept of an enhancer’s ‘therapeutic window’ is discussed. Several of the most promising enhancers identified by the study were tested for their effect on the transport of the marker molecules mannitol and 70 kDa dextran across Caco-2 cells and were capable of increasing permeability more than 10-fold. The results presented here underscore the potential of chemical permeation enhancers while providing valuable direction as to what classes and concentrations of compounds are of interest when searching for safe and effective additions to oral formulations.

The objective was to establish in vitro passive permeability (P e) vs in vivo fraction absorbed (f a)-relationships for each passage through the human intestine, liver, renal tubuli and brain, and develop a P e-based ADME/PK classification system (PCS). P e- and intestinal f a-data were taken from an available data set. Hepatic f a was calculated based on extraction ratios of the unbound fraction of drugs (with support from animal in vivo uptake data). Renal f a (reabsorption) was estimated using renal pharmacokinetic data, and brain f a was predicted using animal in vitro and in vivo brain P e-data. Hepatic and intestinal f a-data were used to predict bile excretion potential. Relationships were established, including predicted curves for bile excretion potential and minimum oral bioavailability, and a 4-Class PCS was developed: I (very high P e; elimination mainly by metabolism); II (high P e) and III (intermediate P e and incomplete f a); IV (low P e and f a). The system enables assessment of potential drug–drug transport interactions, and drug and metabolite organ trapping. The PCS and high quality P e-data (with and without active transport) are believed to be useful for predictions and understanding of ADME/PK, elimination routes, and potential interactions and organ trapping/toxicity in humans.

The purpose of this study was to develop a new system for computer simulation to predict fraction absorbed (F a ) of Biopharmaceutical Classification System (BCS) class II (low solubility–high permeability) drugs after oral administration to humans, from a miniscale dissolution test. Human oral absorption of 12 lipophilic drugs was simulated theoretically by using the dissolution and permeation parameters of the drugs. A miniscale dissolution test and a solubility study were carried out in a conventional buffer and a biorelevant medium (pH 6.5). A dissolution parameter, which can simulate in vivo dissolution, was obtained from the in vitro dissolution curve. Human intestinal permeability was estimated assuming that the permeation was limited by diffusion through the unstirred water layer. The F a in humans was predicted and then compared with clinical data. The dissolution and solubility of most model drugs were faster and higher in a biorelevant medium than in a conventional buffer. The simulated absorption was limited by the drug dissolution rate and/or solubility. Predicted F a was significantly correlated with clinical data (correlation coefficient r 2 = 0.82, p < 0.001) when the dissolution profiles in biorelevant medium were used for the simulation. This new system quantitatively simulated human absorption and would be beneficial for the prediction of human F a values for BCS class II drugs.

PURPOSE: The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism. METHODS: The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis. RESULTS: The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel. CONCLUSIONS: Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.

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.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.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 (PPD). 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.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.

It was the purpose of this study to develop a new oral drug delivery system for low molecular weight heparin (LMWH) providing an improved bioavailability and a prolonged therapeutic effect.The permeation enhancing polycarbophil-cysteine conjugate (PCP-Cys) used in this study displayed 111.4 +/- 6.4 microM thiol groups per gram polymer. Permeation studies on freshly excised intestinal mucosa were performed in Ussing chambers demonstrating a 2-fold improved uptake of heparin as a result of the addition of 0.5% (w/v) PCP-Cys and the permeation mediator glutathione (GSH).Tablets containing PCP-Cys, GSH, and 279 IU of LMWH showed a sustained drug release over 4 h. To guarantee the swelling of the polymeric carrier matrix in the small intestine tablets were enteric coated. They were orally given to rats. For tablets being based on the thiomer/GSH system an absolute bioavailability of 19.9 +/- 9.3% (means +/- SD; n = 5) vs. intravenous injection could be achieved. whereas tablets comprising unmodified PCP did not lead to a significant (p0.01) heparin concentration in plasma. The permeation enhancing effect and subsequently a therapeutic heparin level was maintained for 24 h after a single dose.Because of the strong and prolonged lasting permeation enhancing effect of the thiomer/GSH system, the oral bioavailability of LMWH could be significantly improved. This new delivery system represents therefore a promising tool for the oral administration of heparin.

To evaluate the potential of phosphate ester prodrugs to significantly improve the absorptive flux of poorly soluble parent drugs.Absorptive transport studies of parent drugs and their prodrugs were carried out in Caco-2 cells. Prodrugs of parent drugs with variable aqueous solubilities were tested: Hydrocortisone-phosphate/Hydrocortisone, Fosphenytoin/phenytoin, TAT-59/DP-TAT-59, and Entacapone phosphate/Entacapone. Additional absorption studies were carried out in rats.Absorptive fluxes of DP-TAT-59 and phenytoin increased 9.8 or 3.3-fold after dosing TAT-59 and 500 microM fosphenytoin, respectively. Hydrocortisone's flux did not increase with hydrocortisone-phosphate at 100 microM. Permeability of the highly lipophilic and protein bound compound, DP-TAT-59, was significantly increased with serosal albumin. No permeability increase was observed for the other drugs with albumin. Entacapone phosphate failed to improve the flux of entacapone compared to an entacapone solution, but the prodrug solution did yield higher entacapone plasma levels in rats when compared with an entacapone suspension.Ideal phosphate prodrug candidates are characterized by high permeability and low solubility (BCS Class II drugs). For low dose BCS Class II drug candidates, however, no biopharmaceutical advantage may be gained. Phosphate prodrugs of parent drugs with limited permeability may fail. When screening highly lipophilic parent drugs transport studies should be done with albumin.

To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line.Two approaches were taken to decrease apoptosis. First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2. Second. normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis. The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy. The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers.Normal 2/4/A1 cells died by apoptosis at 39 degrees C. Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells. Optimization of the culture conditions for 2/4/A1 cells inhibited cell death. The integrity was comparable to that of the human jejunum (50 omega x cm2), making this approach preferable to Bcl-2 overexpression. Transcriptional analysis showed that some (e.g., MDRI). but not all (e.g., PepT1), transporters were found in 2/4/A1 cells. Studies using substrates for PepT1, P-gp. MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1.The improved culture procedure will facilitate the use of 2/4/A1 cells. 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport.